Search Results (1,136)

Skeletal muscle development is a critical economic trait in livestock, governed by myogenic satellite cell regulation. Integrins mediate mechanical anchorage to the ECM and enable ECM–intracellular signaling. CIB2, as an EF-hand-domain protein involved in mechanotransduction, shows significant developmental regulation in goat muscle. Although the role of CIB2 in skeletal muscle growth is poorly characterized, we observed pronounced developmental upregulation of IB2 in postnatal goat muscle. CIB2 expression increased >20-fold by postnatal day 90 (P90) compared to P1, sustaining elevation through P180 (p < 0.05). Functional investigations indicated that siRNA-mediated knockdown of CIB2 could inhibit myoblast proliferation by inducing S-phase arrest (p < 0.05) and downregulating the expression of CDK4/Cyclin D/E. Simultaneously, CIB2 interference treatment was found to decrease the proliferative activity of goat myogenic satellite cells, yet it significantly promoted differentiation by upregulating the expression of MyoD/MyoG/MyHC (p < 0.01). Mechanistically, CTCF was identified as a transcriptional repressor binding to an intragenic region of the CIB2 gene locus (ChIP enrichment: 2.3-fold, p < 0.05). Knockdown of CTCF induced upregulation of CIB2 (p < 0.05). RNA-seq analysis established CIB2 as a calcium signaling hub: its interference activated IL-17/TNF and complement cascades, while overexpression suppressed focal adhesion/ECM–receptor interactions and enriched neuroendocrine pathways. Collectively, this study identifies the CTCF-CIB2–integrin α7β1–PI3K/AKT axis as a novel molecular mechanism that regulates the balance of myogenic fate in goats. These findings offer promising targets for genomic selection and precision breeding strategies aimed at enhancing muscle productivity in ruminants. Full article

SnRK kinases, central regulators of plant stress response, remain uncharacterized in Taxus—an ancient gymnosperm valued for paclitaxel production. This study aimed to identify the Taxus SnRK family and elucidate its functional roles. Specifically, we identified SnRK genes through genomic analysis and assessed tissue-specific expression via transcriptomics, while regulatory networks were deciphered using WGCNA. To overcome experimental constraints, a PEG-mediated protoplast transient expression system was developed using calli, followed by dual-luciferase assays. Consequently, 19 SnRK genes (2 SnRK1, 4 SnRK2, 13 SnRK3) were identified, with tissue-specific expression revealing TaSnRK1.2 upregulation under methyl jasmonate (MeJA) and in stress-resilient tissues (bark/root). Subsequently, WGCNA uncovered a bark/root-specific module containing TaSnRK1.2 with predicted TF interactions (TaGRAS/TaERF). Critically, homologous dual-luciferase assays demonstrated TaSnRK1.2 activates TaGRAS and TaERF promoters (4.34-fold and 3.11-fold induction, respectively). This study establishes the Taxus SnRK family and identifies TaSnRK1.2 as a hub integrating stress signals (e.g., MeJA) to modulate downstream TF networks, while the novel protoplast system enables future functional studies in this medicinal plant. Full article

The recent discovery of Orthonairovirus songlingense (SGLV) and Norwavirus beijiense (BJNV) in China has raised significant concern due to their potential to cause severe human disease. However, little is known about the structural features and function of their nucleoproteins, which play a key role in the viral life cycle. By combining small-angle X-ray scattering (SAXS) data and AlphaFold 3 simulations, we reconstructed the BJNV and SGLV nucleoprotein structures for the first time. The SGLV and BJNV nucleoproteins have structures that are broadly similar to those of Orthonairovirus haemorrhagiae (CCHFV) nucleoproteins despite low sequence similarity. Based on structural analysis, several residues located in the positively charged region of BJNV and SGLV nucleoproteins have been indicated to be important for viral RNA binding. A positively charged RNA-binding crevice runs along the interior of the SGLV and BJNV ribonucleoprotein complex (RNP), shielding the viral RNA. Despite the high structural similarity between SGLV and BJNV nucleoprotein monomers, their RNPs adopt distinct conformations. These findings provide important insights into the molecular mechanisms of viral genome packaging and replication in these emerging pathogens. Also, our work demonstrates that experimental SAXS data can validate and improve predicted AlphaFold 3 structures to reflect their solution structure and also provides the first low-resolution structures of the BJNV and SGLV nucleoproteins for the future development of POC tests, vaccines, and antiviral drugs. Full article

Background/Objectives: Listeria monocytogenes is a foodborne pathogen of major public health concern due to its ability to invade host cells and cause severe illness. This study aimed to develop and validate a multi-tiered screening pipeline to identify Bacillus strains with probiotic potential against L. monocytogenes. Methods: A total of 26 Bacillus isolates were screened for antimicrobial activity, gastrointestinal resilience, and host cell adhesion. A fluorescence-based infection assay using mCherry-expressing HCT 116 cells was used to assess cytoprotection against L. monocytogenes NCTC 7973. Eight strains significantly improved host cell viability and were validated by quantification of intracellular CFU. Two top candidates were tested in a murine model of listeriosis. The genome of the lead strain was sequenced to evaluate safety and biosynthetic potential. Results: B. subtilis CECT 8266 completely inhibited intracellular replication of L. monocytogenes in HCT 116 cells, reducing bacterial recovery to undetectable levels. In vivo, it decreased splenic bacterial burden by approximately 6-fold. Genomic analysis revealed eight bacteriocin biosynthetic clusters and silent antibiotic resistance genes within predicted genomic islands, as determined by CARD and Alien Hunter analysis. The strain also demonstrated bile and acid tolerance, as well as strong adhesion to epithelial cells. Conclusions: The proposed pipeline enables efficient identification of probiotic Bacillus strains with intracellular protective activity. B. subtilis CECT 8266 is a promising candidate for translational applications in food safety or health due to its efficacy, resilience, and safety profile. Full article

Various SARS-CoV-2 remdesivir resistance-associated substitutions (RAS) have been reported, but a comprehensive comparison of their resistance levels is lacking. We identified novel RAS and performed head-to-head comparisons with known RAS in Vero E6 cells. A remdesivir escape polyclonal virus exhibited a 3.6-fold increase in remdesivir EC₅₀ and mutations throughout the genome, including substitutions in nsp12 (E796D) and nsp14 (A255S). However, in reverse-genetics infectious assays, viruses harboring both these substitutions exhibited only a slight decrease in remdesivir susceptibility (1.3-fold increase in EC₅₀). The nsp12-E796D substitution did not impair viral fitness (Vero E6 cells or Syrian hamsters) and was reported in a remdesivir-treated COVID-19 patient. In replication assays, a subgenomic replicon containing nsp12-E796D+nsp14-A255S led to a 16.1-fold increase in replication under remdesivir treatment. A comparison with known RAS showed that S759A, located in the active site of nsp12, conferred the highest remdesivir resistance (106.1-fold increase in replication). Nsp12-RAS V166A/L, V792I, E796D or C799F, all adjacent to the active site, caused intermediate resistance (2.0- to 11.5-fold), whereas N198S, D484Y, or E802D, located farther from the active site, showed no resistance (≤2.0-fold). In conclusion, our classification system, correlating replication under remdesivir treatment with RAS location in nsp12, shows that most nsp12-RAS cause moderate resistance. Full article

Tellurite (TeO₃²⁻) is a highly soluble and toxic oxyanion that inhibits the growth of Escherichia coli at concentrations as low as ~1 µg/mL. This toxicity has been primarily attributed to the generation of reactive oxygen species (ROS) during its intracellular reduction by thiol-containing molecules and NAD(P)H-dependent enzymes. However, under anaerobic conditions, E. coli exhibits significantly increased tellurite tolerance—up to 100-fold in minimal media—suggesting the involvement of additional, ROS-independent mechanisms. In this study, we combined chemical-genomic screening, untargeted metabolomics, and targeted biochemical assays to investigate the effects of tellurite under both aerobic and anaerobic conditions. Our findings reveal that tellurite perturbs amino acid and nucleotide metabolism, leading to intracellular imbalances that impair protein synthesis. Additionally, tellurite induces notable changes in membrane lipid composition, particularly in phosphatidylethanolamine derivatives, which may influence biophysical properties of the membrane, such as fluidity or curvature. This membrane remodeling could contribute to the increased resistance observed under anaerobic conditions, although direct evidence of altered membrane fluidity remains to be established. Overall, these results demonstrate that tellurite toxicity extends beyond oxidative stress, impacting central metabolic pathways and membrane-associated functions regardless of oxygen availability. Full article

Background/Objectives: Ewing sarcoma (ES), a highly aggressive bone and soft tissue cancer occurring in children and young adults, is defined by the ETS fusion oncoprotein EWS::FLI1. Although event-free survival rates remain high in ES patients with localized disease, those with metastatic or relapsed disease face poor long-term survival odds. Topoisomerase 1 (TOP1) inhibitors are commonly used therapeutics in ES relapse regimens. Methods: In this work, we used a genome-wide CRISPR knockout library screen to identify the deletion of the TOP1 gene as a mechanism for resistance to topoisomerase 1 inhibitors. Using isogenic cell line models, we performed a high-throughput small-molecule screen to discover a small molecule, GNF-7, which had an IC50 that was 10-fold lower in TOP1-deficient cells when compared to the wild-type cells. Results: The characterization of GNF-7 demonstrated the molecule was highly active in the inhibition of CSK, p38α, EphA2, Lyn, and ZAK and specifically downregulated genes induced by the EWS::FLI1 fusion oncoprotein. Conclusions: Together, these results suggest that GNF-7 or small molecules with a similar kinase profile could be effective treatments for ES patients in combination with TOP1 inhibitors or for those patients who have developed resistance to TOP1 inhibitors. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article

Mitochondria are critical for cellular energy, and while large deletions in their genome (mtDNA) are linked to primary mitochondrial diseases, their significance in cancer is less understood. Given cancer’s metabolic nature, investigating mtDNA deletions in tumors at various stages could provide insights into disease origins and treatment responses. In this study, we analyzed 148 bone marrow samples from 129 pediatric patients with B-cell (B-ALL) and T-cell (T-ALL) acute lymphoblastic leukemia at diagnosis, remission, and relapse using long-range PCR, next-generation sequencing, and the Splice-Break2 pipeline. Both T-ALL and B-ALL exhibited significantly more mtDNA deletions than did the controls, with T-ALL showing a ~100-fold increase and B-ALL a ~15-fold increase. The T-ALL samples also exhibited larger deletions (median size > 2000 bp) and greater heterogeneity, suggesting increased mitochondrial instability. Clustering analysis revealed distinct deletion profiles between ALL subtypes and across disease stages. Notably, large clonal deletions were detected in some B-ALL remission samples, including one affecting up to 88% of mtDNA molecules, which points toward treatment-driven selection or toxicity. A multivariate analysis confirmed that disease type, timepoint, and WHO subtype significantly influenced mtDNA deletion metrics, while age and gender did not. These findings suggest that mtDNA deletion profiling could serve as a biomarker for pediatric ALL and may indicate mitochondrial toxicity contributing to late effects in survivors. Full article

Grass carp is an economically important cultured species in China. Triploid embryo production is widely applied in aquaculture to achieve reproductive sterility, improve somatic growth, and reduce ecological risks associated with uncontrolled breeding. In this study, a simple cold shock method for inducing triploid grass carp was developed. The triploid induction rate of 71.73 ± 5.00% was achieved by applying a cold treatment at 4 °C for 12 min, starting 2 min after artificial fertilization. Flow cytometry and karyotype analysis revealed that triploid individuals exhibited a 1.5-fold increase in DNA content compared to diploid counterparts, with a chromosomal composition of 3n = 72 (33m + 36sm + 3st). Additionally, embryonic transcriptome analysis demonstrated that, in the cold shock-induced embryos, genes associated with abnormal mesoderm and dorsal–ventral axis formation, zygotic genome activation (ZGA), and anti-apoptosis were downregulated, whereas pro-apoptotic genes were upregulated, which may contribute to the higher abnormal mortality observed during embryonic development. Overall, this study demonstrates optimized conditions for inducing triploidy in grass carp via cold shock and provides insights into the transcriptomic changes that take place in cold shock-induced embryos, which could inform future grass carp genetic breeding programs. Full article

Rouxiella badensis DSM 100043^T had been previously proven to produce a novel glucoselipid biosurfactant which has a very low critical micelle concentration (CMC) as well as very good stability against a wide range of pH, temperature, and salinity. In this study, we performed a function-based library screening from a R. badensis DSM 100043^T genome library to identify responsible genes for biosynthesis of this glucoselipid. The identified open reading frames (ORFs) were cloned into several constructs in Escherichia coli for gene permutation analysis and the individual products were analyzed using high-performance thin-layer chromatography (HPTLC). Products of interest from positive expression strains were purified and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) for further structure elucidation. Function-based screening of 5400 clones led to the identification of an operon containing three ORFs encoding acetyltransferase GlcA (ORF1), acyltransferase GlcB (ORF2), and phosphatase/HAD GlcC (ORF3). E. coli pCAT2, with all three ORFs, resulted in the production of identical R. badensis DSM 100043^T glucosedilipid with Glu-C_10:0-C_12:1 as the main congener. ORF2-deletion strain E. coli pAFP1 primarily produced glucosemonolipids, with Glu-C_10:0,3OH and Glu-C_12:0 as the major congeners, predominantly esterified at the C-2 position of the glucose moiety. Furthermore, fed-batch bioreactor cultivation of E. coli pCAT2 using glucose as the carbon source yielded a maximum glucosedilipid titer of 2.34 g/L after 25 h of fermentation, which is 55-fold higher than that produced by batch cultivation of R. badensis DSM 100043^T in the previous study. Full article

The poll gland, a specialized tissue of male Bactrian camels, undergoes seasonal enlargement and marked metabolic activation during the rutting season. However, the metabolic mechanisms of the poll gland and its role in rutting activities and inducing estrus are still not fully understood. This study aimed to investigate the contribution of fatty acid metabolic pathways, specifically those mediated by carnitine palmitoyltransferase 1A (CPT1A), in poll gland activity during the breeding season; poll gland tissue, neck mane, and urine samples were systematically collected from healthy male Bactrian camels stratified into breeding and non-breeding season groups for integrated proteomic, metabolomic, and biochemical assays. Histological and immunohistochemical analyses revealed reduced adipocytes but elevated ATP production in rutting camels, suggesting increased mitochondrial activity and enhanced oxidative phosphorylation. Proteomic analyses identified 119 differentially expressed proteins (DEPs) linked to fatty acid metabolism, with CPT1A, a key regulator of mitochondrial fatty acid oxidation, emerging as a central hub. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further confirmed enrichment in fatty acid biosynthesis, degradation, and PPAR/AMPK signaling. The metabolomic analysis identified 14 metabolites, including acetylcarnitine and glycine, that were closely correlated with CPT1A expression, suggesting their potential involvement in regulating fatty acid metabolism during the breeding season. Quantitative expression analyses revealed that CPT1A in glandular acini was significantly upregulated in the breeding group compared to the non-breeding group across all assays: qPCR (2.53-fold, p < 0.05), Western blot (3.5-fold, p < 0.05), and immunohistochemistry (1.5-fold, p < 0.05). This demonstrated that CPT1A-mediated fatty acid metabolism plays a pivotal role in energy provision for reproductive activities. The results suggested that CPT1A-mediated fatty acid oxidation sustains poll gland function and reproductive behaviors in male Bactrian camels. This study provided a theoretical basis for understanding the role of CPT1A-mediated fatty acid oxidation in maintaining poll gland function and supporting reproductive activities in male Bactrian camels. Full article

The TGACG motif-binding factor (TGA) family is an important group of basic region/leucine zipper (bZIP) transcription factors in plants, playing crucial roles in plant development and stress responses. This study conducted a comprehensive genome-wide analysis of the TGA transcription factor (TF) family in common wheat (Triticum aestivum L.). A total of 48 wheat TGAs were identified and classified into four subgroups. Collinearity analysis of the TGAs between wheat and other species identified multiple duplicated gene pairs and highlighted the presence of highly conserved TGAs in wheat. Whole-genome and segmental duplications were identified as the primary drivers of TaTGA expansion. Expression pattern analysis indicated that TaTGAs are involved in plant development and responses to abiotic stresses, including drought, heat, and cold treatment. Among these, TaTGA16-2D was significantly upregulated under both drought and heat stresses, showing more than a five-fold increase in expression. Subcellular localization confirmed its nucleus localization. Functional validation through ectopic expression in Arabidopsis demonstrated that transgenic lines overexpressing TaTGA16-2D exhibited significantly improved stress tolerance. Under heat stress, the survival rates of transgenic lines exceeded 34%, compared to less than 18% in wild-type plants. Overall, this study provides valuable insights into the evolution and functional roles of TaTGAs and identifies TaTGA16-2D as a promising candidate to enhance abiotic stress tolerance in wheat via molecular breeding. Full article

Arterial aneurysms are vascular conditions associated with life-threatening consequences in patients, such as dissection and rupture. Understanding their genetic basis is an evolving field, driven by the robust reporting of genetic variants associated with aneurysms in patients. In this study, we present clinical and genetic data from nine unrelated subjects with arterial aneurysms who were identified to harbor rare variants in the TNXB gene, mainly affecting fibronectin type III (FNIII) domains. The cohort included three female and six male subjects with a mean age of 53.5 years (SD = 14.4). The most frequently affected vascular territory was the thoracic ascending aorta (n = 7). A range of pathogenic impacts was predicted via multiple in silico tools that analyze evolutionary conservation and biochemical properties. Computational protein structure modeling with AlphaFold 3 predicted domain-specific alterations across multiple FNIII regions for four unique missense variants and one in-frame deletion, and premature protein truncation resulting from two frameshift variants. To our knowledge, this study is one of the first and largest to associate TNXB variants with arterial aneurysmal disease. Our findings demonstrate the potential of computational genomics and structural modeling to advance the understanding of extracellular matrix gene alterations in aneurysm pathogenesis. Full article

Background/Objectives: Ceftiofur hydrochloride (CEF) is a third-generation cephalosporin widely used in cattle to treat various disease. The recommended dosage was 1.1 to 2.2 mg/kg BW for 3 to 5 consecutive days by intramuscular or subcutaneous injection. Incomplete treatment, overuse, or misuse, often observed in clinical practice, are major contributors to resistance development. This study aims to explore how different concentrations, durations, and dosing frequencies affect susceptibility and bactericidal efficacy of Staphylococcus aureus to optimize CEF dosage regimens. Methods: First, CEF was intermittently administered at 1/2 × minimum inhibitory concentration (MIC), 2 × MIC, 6 × MIC, and 100 × MIC for 30 cycles. Second, CEF was continuously administered for 48, 72, 96, 120, 144, and 168 h. Bacterial susceptibility, regrowth, survival rate, and the emergence of persisters or tolerant phenotypes were assessed. Genetic mutations were identified by whole-genome resequencing. Membrane permeability, integrity, and efflux pump activity were analyzed to elucidate the mechanism of CEF. Results: After 30 cycles, the MIC increased eight-fold in the 2 × MIC group. No significant MIC increase was found in other groups, but a progression from susceptibility to persistence and then to tolerance was observed in the 100 × MIC intermittent group. The survival rate increased both in the 2 × MIC and 100 × MIC groups. With continuous exposure to ≥6 × MIC over 120 h, strains were completely eradicated without MIC increase. Resistance-associated single-nucleotide polymorphism (SNP) mutations were detected only in strains of the 2 × MIC and 100 × MIC intermittent groups. CEF altered the membrane hydrophobicity, damaging membrane integrity after 30 cycles. Conclusions: These findings suggest that high-dose, prolonged exposure is more effective for eliminating Staphylococcus aureus and avoiding resistance, whereas intermittent dosing may promote persistence, tolerance, and resistance evolution. Full article