Search Results (1,007)

Adenoviral vectors are widely used for gene therapy and vaccine development. To circumvent pre-existing immunity against commonly used human adenovirus type 5, vectors based on rare human serotype or animal adenoviruses have attracted increasing interest. Previously, we constructed vectors based on fowl adenovirus 4 (FAdV-4) and replaced the knob of FAdV-4 fiber2 with that of FAdV-1 fiber1 to generate FAdV4-CF1K vectors with enhanced transduction efficiency in human cells. In this study, we aimed to modify the packaging system to efficiently produce FAdV-4 vectors carrying transgenes toxic to viral replication. Chicken LMH cells failed to form colonies at low seeding densities. We collected used medium from LMH cell cultures and used it as a supplement to adapt LMH cells, generating the colony-competent subclone LMH-C3532. A lentiviral vector encoding a codon-optimized tetracycline repressor (tetR) was transduced into LMH-C3532 to establish a tetR-integrated cell line, LMH-tetR24. An adenoviral plasmid, pKFAV4-CF1K-CtG, was constructed in which a tetracycline operator (tetO)-bearing CMV promoter controlled GFP expression. The SwaI-flanked GFP in this plasmid was replaced with the HA gene from an H5N1 influenza virus to generate pKFAV4-CF1K-CtHA. Linearized adenoviral plasmids were transfected into LMH-tetR24 cells, and recombinant FAdV4-CF1K-CtG and FAdV4-CF1K-CtHA viruses were successfully rescued, amplified, and purified. When infected with FAdV4-CF1K-CtG at various multiplicities of infection (MOI), the progeny virus yield from LMH-tetR24 cells was 4–10 times higher than that from LMH-C3532 cells. For FAdV4-CF1K-CtHA, the yield difference between the two cell lines was even more pronounced, reaching 3–4 orders of magnitude. Overexpression of HA in LMH-C3532 cells negatively affected FAdV4-CF1K-CtHA replication, resulting in smaller and fewer plaques. In conclusion, by separately integrating tetR into packaging cells and TetO into the adenoviral plasmid, we established a system that can be routinely used to package FAdV-4 vectors. Notably, this system facilitates the propagation of FAdV-4 vectors carrying toxic transgenes. Full article

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is an adult-onset inflammatory disorder caused by acquired mutations in UBA1, the gene encoding the primary ubiquitin-activating enzyme. The recognition of VEXAS has transformed the current understanding of autoinflammatory disease by demonstrating that somatic alterations arising within hematopoietic stem cells can precipitate severe, multisystem inflammation in later life. While pathogenic UBA1 variants are essential to disease pathogenesis, many affected individuals also harbor additional somatic mutations associated with clonal hematopoiesis, most commonly involving DNMT3A and TET2. These concurrent mutations may contribute to clonal architecture; however, their independent impact on inflammatory phenotype and hematologic manifestations remains incompletely defined. Emerging evidence suggests that co-occurring clonal hematopoiesis mutations may be independently associated with poorer overall survival, though their causal role remains unestablished. This review examines the evolving genetic framework of VEXAS syndrome, emphasizing UBA1 as the obligate driver mutation while reviewing current evidence regarding non-Met41 UBA1 variants and co-occurring somatic mutations. Full article

Background: Clonal hematopoiesis of indeterminate potential (CHIP) can lead to adverse outcomes and may begin early in life. This study aimed to investigate the association between early-life events and CHIP. Methods: In total, 456,658 participants from U.K. Biobank without baseline hematologic malignancies were enrolled. Exposures included 17 early-life events, including reproductive, childhood adversity, and pre-adulthood development factors. CHIP was derived from whole-exome sequencing for mutations in 74 driver genes. Logistic regressions were used to estimate associations between early-life events and the presence of any CHIP or gene-specific CHIP mutations. Results: Overall, 17,513 (3.8%) individuals with any CHIP were identified, among which the most common subtype was DNMT3A (2.4%), followed by TET2 (0.6%) and ASXL1 (0.4%). Compared with participants without sexual abuse in childhood, those who experienced such abuse were positively associated with CHIP (OR 1.35, 95% CI 1.02–1.80), especially among ASXL1, JAK2, and TP53 mutations. Long-term/recurrent antibiotic use as a child or teenager was positively associated with CHIP (OR 1.11, 95% CI 1.02–1.21), especially among DNMT3A, ASXL1, and EP300 mutations. Sex-specific differences were observed, including sexual abuse associated with ASXL1-CHIP in males and JAK2/TP53-CHIP in females and long-term/recurrent antibiotic use associated with DNMT3A/EP300-CHIP in males and ASXL1-CHIP in females. Furthermore, we identified circulating proteomic biomarkers shared by six pairs of early-life factors and gene-specific CHIP mutations, including B2M for sexual abuse and JAK2-CHIP. Conclusions: Early-life factors, especially sexual abuse and long-term/recurrent antibiotic use, were positively associated with the presence of CHIP, particularly among specific gene mutations, offering potential targets for susceptibility and pathogenesis exploration. Full article

Cronobacter spp. are opportunistic foodborne pathogens that can cause neonatal meningitis, necrotizing enterocolitis, and sepsis. This study conducted a systematic contamination survey and whole-genome epidemiological analysis of 562 low-water-activity food samples in Hunan Province of China. The results showed an overall Cronobacter spp. detection rate of 41.99% (236/562), with spices exhibiting the highest contamination rate (60.06%), and with high-level contamination samples (>110 MPN/g) concentrated in this category. The 236 isolates comprised 6 species, 120 sequence types, and 39 clonal complexes, with C. sakazakii being the most frequently isolated species (64.83%) and high-risk clones ST4, ST1, ST148, and ST64 prevailing. Multiple virulence genes (TraJ, fur, rcsAB, rpoS) and antimicrobial resistance genes (qnrS1, blaTEM-1, blaCTX-M-55, blaLAP-2, aac(3)-IId, aadA2, tet(A), floR, mcr-9.1, sul2) were detected. Core genome multilocus sequence typing (cgMLST) identified two clustering patterns: Cluster C, whose genetic clustering was consistent with transmission associated with potential common upstream raw materials across different brands and provinces, and Cluster G, whose clustering suggested potential persistent colonization in the production environment across multiple batches of the same brand. This study elucidates the contamination characteristics of Cronobacter spp. in low-water-activity foods from Hunan Province and provides a basis for WGS-based active surveillance and supply chain traceability. Full article

This study aimed to investigate the molecular basis of high-level tetracycline resistance to tetracycline antibiotics in Aeromonas hydrophila isolated from fish farming. Comparative genomic analysis of the tetracycline-sensitive strain NJ-35 and the tetracycline-resistant strain AH823 revealed that the tetracycline repressor gene tetR in AH823 had undergone base mutations, resulting in premature translational termination. The tetR gene in NJ-35 was inhibited using a plasmid-based antisense RNA strategy, and the knockdown efficiency was confirmed by RT-qPCR. The resulting tetR antisense RNA-expressing strain, AHtetR-as, exhibited significantly increased resistance to tetracycline antibiotics (minocycline, tetracycline, and doxycycline), but did not affect biofilm formation or hemolysis. Moreover, tetR knockdown in NJ-35 was associated with increased efflux activity and reduced intracellular doxycycline accumulation. Transcriptomic analysis revealed that genes encoding the 30S ribosomal subunit proteins showed a differential expression pattern, with rpsO upregulated and rpsD and rpsP downregulated. These findings suggest that tetR contributes to tetracycline resistance in A. hydrophila and is associated with broad transcriptional changes related to cellular transport and ribosomal function. Full article

Central chondrosarcoma is the second most common primary malignant bone tumour, and grade progression markedly worsens prognosis. The contributions of lipid metabolic reprogramming and epigenetic co-dysregulation to grade progression remain poorly characterised. We integrated a bulk RNA-seq discovery cohort of 53 graded central chondrosarcomas (GSE299759) with a single-cell analysis of eight chondrosarcomas (GSE184118). Because the atypical cartilaginous tumour (ACT) and dedifferentiated groups each comprised only three samples, the Grade 3 versus Grade 2 contrast was pre-specified as the primary comparison. Curated panels of 44 lipid metabolism genes and 50 epigenetic regulators were assessed by differential expression and a correlation-based connectivity ranking, evaluated by permutation testing. In the primary Grade 3 versus Grade 2 comparison, SQLE, ACACA, and FASN were upregulated (FDR < 0.05), indicating a grade-associated increase in de novo lipogenesis. In the exploratory Grade 3 versus ACT comparison, additional lipid genes (HMGCR, LDLRAP1) and the epigenetic regulators EHMT2 and SIRT2 showed altered expression, although the small ACT group limits these estimates. A connectivity ranking highlighted FASN, KMT2C, TET2, SETD5, and KDM5B; permutation testing confirmed this co-expression structure was non-random (p < 0.0001). Single-cell analysis showed FASN, SETD5, and KDM5B are expressed predominantly in malignant cells, whereas KMT2C and TET2 are not, indicating cell-type heterogeneity. De novo lipogenesis upregulation is the most consistent lipid alteration in high-grade central chondrosarcoma, nominating SQLE, ACACA, and FASN as candidates for experimental investigation. Full article

Enterococci are among the most frequently isolated environmental bacteria that cause mastitis in cows. This study aimed to determine the prevalence of virulence genes, as well as phenotypic and genotypic antibiotic resistance, among eighty enterococcal isolates from cases of bovine mastitis in Polish herds. The presence of virulence and antibiotic resistance genes was determined by PCR. E. faecalis isolates were found to carry more virulence genes than E. faecium isolates, including the efaA_fs (100%), ace (98.1%), gelE (86.5%), asa1 (63.5%), esp (57.7%) and cylA (17.3%) genes. The efaA_fm gene was the only virulence gene detected in E. faecium isolates. This study revealed that E. faecalis showed a higher virulence gene burden. The ermB gene was present in 90.9% of the Enterococcus spp. that were phenotypically resistant to erythromycin. Almost all tetracycline-resistant Enterococcus isolates carried the tet(M) gene (94.3%), either alone or in combination with the tet(L) and tet(O) genes. Three isolates harboured vanC genes and were susceptible to vancomycin (MIC = 4 μg/mL). The results confirm the high level of antimicrobial resistance of enterococci isolated from cows with mastitis and indicate the genes that may be responsible for this resistance. Full article

Antibiotic resistance genes (ARGs) are increasingly recognized as a concern in drinking water, yet the factors influencing their persistence from raw to finished water in drinking water treatment plants remain poorly understood. This study investigated the occurrence, removal, and potential factors associated with the persistence of ARGs (sul1, sul2, and tetG) in a full-scale rapid sand filtration drinking water treatment system with intermediate and post-chlorination. ARGs were detected in raw water at a median total concentration of 10⁶ copies/L and remained detectable in finished water at 10⁴ copies/L. Relative ARG abundance increased after treatment despite substantial absolute reductions (2.1–3.6 log). Intermediate chlorination achieved the greatest ARG log reduction value (0.53–2.4 log), likely due to higher chlorine dose and lower pH favoring HOCl formation. By contrast, post-chlorination at higher pH provided limited additional removal, possibly due to predominance of less reactive OCl⁻ and survival of chlorine-tolerant bacteria. Multivariate analyses showed a shift from particle-bound ARGs in raw water to dissolved organic matter (DOM) and fine-particle-associated fractions along the treatment train. These findings suggest that reducing fine particles and DOM, together with optimized disinfection, may help lower ARG-associated risk in finished water. Full article

Acinetobacter is one of the most relevant pathogenic and nosocomial bacteria in human medicine. However, in veterinary medicine, particularly in Spain, there are very few studies on the impact and frequency of infections due to this genus. The main objective of this study was to characterise Acinetobacter isolates recovered at the Complutense Veterinary Teaching Hospital of the Complutense University of Madrid (HCV-UCM), with special emphasis on detecting antimicrobial resistance. A total of 23 isolates obtained from different animal species and samples over a 25-year period were included in the study, based on their identification as Acinetobacter by VITEK-2. Identification was made by using MALDI-TOF, VITEK-2, whole-genome sequencing (WGS) and a chromogenic medium. WGS confirmed that 13 of the 23 isolates belonged to Acinetobacter spp. Antimicrobial susceptibility was interpreted according to CLSI guidelines using the Kirby–Bauer disk diffusion and broth microdilution method. The proportion of clinical isolates identified as Acinetobacter spp. at the HCV-UCM was 0.3%. Of these, 15.4% (2/13 isolates) were classified as multidrug-resistant. Two isolates with the highest MIC for tigecycline carried the tet(X) gene, and two isolates harboured mutations in both gyrA and parC QRDR regions. The results of this study suggest that, in this hospital, antimicrobial resistance among Acinetobacter isolates may not yet be widespread. Full article

DNA methylation, the covalent addition of methyl groups to cytosine (5mC) or adenine (6mA) in DNA, is a fundamental mechanism of epigenetic inheritance conserved from bacteria to humans. Fungi provide a uniquely informative window into the evolutionary logic of methylation systems. Spanning more than 1 billion years of diversification, the kingdom encompasses species that have lost cytosine methylation entirely, lineages that use 5mC to silence transposons and drive the irreversible genome-defense process known as repeat-induced point mutation (RIP), and early-diverging lineages, in which 6mA has emerged as a prominent chromatin mark. The methyltransferases underlying these strategies (DIM-2, RID, DNMT1-RFD, DNMT5, and the MT-A70 complex) and the recently characterized demethylases Dmt1 and CcTet are structurally and mechanistically distinct from their mammalian counterparts. Here we review the mechanisms, targets, and biological functions of fungal DNA methyltransferases and demethylases, incorporating cryo-EM structural insights into DIM-2 and DNMT5 catalysis, analyses of DNMT gene loss as a continuous evolutionary process, the antiviral role of DIM-2 in vegetative hyphae, and the emerging model of 6mA as a heritable regulatory mark in early-diverging lineages. By integrating these advances, this review offers the updated and comprehensive account of DNA methylation across fungi. Full article

Background/Objectives: Chromosomal microarray analysis (CMA) is an essential tool in modern cytogenetics for detecting copy number alterations and copy-neutral loss of heterozygosity (CN-LOH). As optical genome mapping (OGM) emerges as a potential replacement for traditional cytogenetic methods, the extent to which CMA remains necessary in routine diagnostic workflows remains to be elucidated. Methods: We retrospectively reviewed 53 primary neoplastic cases, selected from a larger cohort of 327 hematologic malignancy specimens, in which CMA identified one or more CN-LOH events. Event size, genomic content, and correlation with next-generation sequencing (NGS) findings were assessed. A separate cohort of newly diagnosed B-cell acute lymphoblastic leukemia (B-ALL) was analyzed to evaluate disease-specific CN-LOH frequency. Results: Nearly half of CN-LOH events detected were <25 Mb, below the current detection threshold of OGM inferred from published benchmarks and validated workflows. Many encompassed clinically relevant genes, including FLT3, JAK2, TET2, TP53, and RUNX1. Additionally, two-thirds of cases harbored pathogenic or likely pathogenic variants by NGS within the corresponding CN-LOH regions, further underscoring the clinical value of detecting these copy-neutral events. In contrast, CN-LOH was uncommon in B-ALL, and most alterations identified by CMA would be detectable by OGM. Many of these patients also harbored complex structural rearrangements that required multiple conventional assays for full characterization; these could be resolved by OGM in a single analysis. Conclusions: Our findings indicate that although OGM excels at resolving complex structural variants, CMA remains essential for detecting copy-neutral events. Until OGM achieves improved sensitivity for CN-LOH, an integrated approach utilizing conventional cytogenetics, CMA, NGS, and OGM provides the most reliable framework for comprehensive genomic assessment across cancer types. Full article

The emergence of plasmid-mediated tigecycline resistance in food-producing animals poses a significant threat to global public health by compromising a last-resort antimicrobial. This study investigated the prevalence, antimicrobial susceptibility profiles, and molecular genetic characteristics of tigecycline-resistant Escherichia coli (E. coli) isolated from broiler cecal samples in the Jinan region of Shandong Province, China, between 2020 and 2024. Antimicrobial susceptibility testing showed that high-level resistance to colistin (87.5%, 14/16), florfenicol (81.3%, 13/15), and enrofloxacin (75.0%, 12/16) was observed. Notably, a single isolate was resistant to meropenem (6.3%, 1/16). Whole-genome sequencing and subsequent in silico analysis demonstrated that all 16 tigecycline-resistant isolates harbored the tigecycline resistance gene tet(X4), the primary determinant of resistance. Molecular typing identified a diverse population structure, with sequence type 224 (ST224) being the dominant clone, accounting for 37.5% (6/16) of the isolates. The genetic milieu of resistance was complex, characterized by the co-existence of tet(X4) with multiple other clinically important resistance genes, including the mobile colistin resistance gene mcr-1 (87.5%, 14/16) and various extended-spectrum β-lactamase genes such as blaCTX-M variants and, critically, blaNDM-5. Furthermore, an array of virulence factor genes was identified, with a particularly high prevalence of the toxin gene astA (68.8%, 11/16) and the bacteriocin gene cma (50.0%, 8/16), indicating pathogenic potential. This convergence of resistance and virulence in a foodborne pathogen highlights an urgent need for continuous One Health surveillance to mitigate the risks posed by these potentially untreatable “superbugs” to both animal and human health. Full article

Pulmonary arterial hypertension (PAH) is a progressive, fatal disease of the pulmonary vasculature characterized by obliterative remodeling of small pulmonary arteries, leading to sustained elevation of pulmonary vascular resistance, right ventricular failure, and premature death. The diagnostic gold standard remains right heart catheterization, requiring a mean pulmonary artery pressure greater than 20 mmHg at rest, a pulmonary arterial wedge pressure of 15 mmHg or below, and a pulmonary vascular resistance exceeding 2 Wood units. PAH is an autosomal dominant disorder with markedly incomplete penetrance of approximately 20–30%, indicating that germline mutations alone are insufficient to cause disease. Disease manifestation requires additional “second hits”, including chronic hypoxia, systemic inflammation, hemodynamic stress, hormonal influences, and common genetic modifiers such as single-nucleotide polymorphisms (SNPs). This genetic and environmental complexity underpins the broad clinical heterogeneity observed across PAH subtypes, which include idiopathic PAH, heritable PAH, and disease associated with connective tissue disorders, HIV infection, portal hypertension, congenital heart disease, schistosomiasis, and drug or toxin exposure. This review provides a comprehensive and critical appraisal of the molecular-genetic architecture of PAH. Thirty genes have now been implicated in disease pathogenesis, spanning seven functional categories: receptors of the TGF-β/BMP signaling family (BMPR2, ACVRL1, ENG, BMPR1B); circulating BMP ligands (GDF2, BMP10); transcription factors (TBX4, SOX17, KLF4, FOXF1, SMAD1, SMAD4, SMAD9); membrane and polyamine transporters (ATP13A3, AQP1); potassium channel regulators (KCNA5, KCNK3, ABCC8); metabolic and mitochondrial genes (EIF2AK4, NFU1, GGCX); signaling receptors and structural proteins (NOTCH3, KDR, CAV1, PLEKHH2); vasoactive and extracellular matrix regulators (KLK1, CBLN2, CD248); and epigenetic regulators (TET2, TOPBP1). Among these, BMPR2 is the dominant contributor, accounting for 53–86% of heritable PAH and 14–35% of idiopathic cases. The remaining genes each account for fewer than 5% of cases individually, collectively reflecting a broad landscape of rare and ultra-rare genetic contributions. For each gene, we critically evaluate the strength of genetic evidence, pathogenic mechanisms, degree of mechanistic resolution, and clinical relevance. We further discuss the contribution of emerging technologies, including whole-genome sequencing, single-cell and spatial transcriptomics, multi-omics integration, iPSC-derived vascular models, and artificial intelligence, to expanding the PAH genetic architecture beyond single-gene discovery. A key theme across this landscape is convergence: despite mechanistic diversity at the gene level, most PAH-associated variants ultimately impair endothelial quiescence, promote smooth muscle proliferation, and drive apoptosis resistance through disruption of BMP signaling amplitude, transcriptional stability, ion channel homeostasis, metabolic integrity, or epigenetic regulation. This convergence supports both a unified therapeutic rationale and a precision medicine framework for genotype-stratified intervention in PAH. Full article

Poultry is the main reservoir of Campylobacter spp. and most human cases result from consuming undercooked poultry or handling raw meat. In 2022, a total of 55 samples, including neck skin, cecal contents, and processing waters, were collected at two poultry slaughterhouses in Italy and analysed according to ISO 10272-2:2017 at the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata laboratories. Overall, 51/55 (92.72%) samples tested positive for Campylobacter. Among the isolates, 64.71% were identified as C. coli, and 35.29% as C. jejuni. Phenotypic and genotypic analysis were performed to assess antimicrobial resistance and virulence characteristics. All C. jejuni isolates and 72.72% of C. coli showed resistance to fluoroquinolones. Resistances to tetracycline and carbapenem were observed in 60.78% and 45.09% of isolates, respectively. Genomic analysis confirmed the presence of the tet(O) gene, conferring tetracycline resistance. In addition, OXA-450 and OXA-466 genes, conferring beta-lactam resistance, were detected in 78.43% and 3.92% of isolates. Virulence-associated genes were detected. Specifically, the ciaB gene was found in 50/51 (98.04%) of isolates, whereas jlpA, cdtA, cdtB, and ctdC genes were exclusively identified in C. jejuni strains. The high prevalence of pathogenic and antimicrobial-resistant Campylobacter strains highlights the need for strengthened control measures along the poultry production chain. Full article

Background: Despite significant progress in management of acute coronary syndromes (ACSs), they continue to be a major cause of death worldwide due to residual inflammatory risk (RIR). Aim: This study reviews existing clinical evidence for anti-inflammatory therapies in coronary heart disease (CHD) and assesses precision medicine in classifying patients from clinical, immunological, and genetic perspectives. Results: Large clinical trials confirm the inflammatory hypothesis of atherosclerosis. Therapies targeted at the specific NLRP3 inflammasome/interleukin-1β (IL-1β)/interleukin-6 (IL-6) pathway reduce major adverse cardiovascular events (MACEs), while broad immunosuppression fails. This highlights the need for molecular specificity. Precision cardiology aims to identify high-risk inflammatory phenotypes through clonal hematopoiesis of indeterminate potential (CHIP). Mutations in genes such as TET2 and ASXL1 lead to macrophage hyperreactivity and increased plaque vulnerability. Available data suggest that the effectiveness of immunomodulatory treatment strongly depends on timing. Starting therapy early with SGLT2 inhibitors (SGLT2is) or agents that target temporarily activated receptors like P2Y11 seems to be essential for managing harmful inflammation while supporting myocardial repair. Conclusions: Precision cardiology aims to integrate targeted anti-inflammatory therapies with established clinical markers, while future pathways may incorporate advanced immunophenotyping and genetic risk assessment as they undergo clinical validation. Full article