Search Results (707)

Based on PCR with complementary primer pairs and Pfu DNA polymerase, QuickChange site-directed mutagenesis has been widely employed, but its efficiency varies from mutation to mutation. An alternative strategy relies on partially overlapping primer pairs with 3′-overhangs, and this strategy has led to the recent development of P3a and P3b site-directed mutagenesis, in which the use of SuperFi II and Q5 polymerases raises the mutagenesis efficiency to ~100%. It is unclear whether these two DNA polymerases also improve the QuickChange method. Herein, we have evaluated this possibility by engineering 46 mutations on seven expression plasmids, two of which possess extremely GC-rich sequences. As Pfu DNA polymerase is a slow enzyme, its replacement with SuperFi II and Q5 polymerases reduced PCR length. Moreover, the average efficiency for each of the seven plasmids ranged from 48% to 69%, thereby outperforming the original QuickChange method. However, this efficiency is still lower than that from the P3a and P3b methods, supporting the superiority of primer pairs with 3′-overhangs. Analysis of the incorrect plasmids from the improved QuickChange method revealed frequent insertions at primer sites. The insertions were derived from primers and varied from mutation to mutation, with certain sites much more prone to such insertions. In comparison, these insertions occurred at a much lower frequency with the P3a and P3b methods, suggesting that primer pairs with 3′-overhangs enhance mutagenesis efficiency by reducing the likelihood to introduce insertions at primer sites. Thus, this study improves the QuickChange mutagenesis method, supports the superiority of the P3a and P3b methods, and uncovers a novel molecular mechanism by which the efficiency of PCR-based mutagenesis with completely overlapping primer pairs is negatively affected. Full article

Background: Coffin–Siris syndrome 12 (CSS12) is a recently described neurodevelopmental disorder caused by heterozygous pathogenic variants in BICRA, a gene encoding a core subunit of the non-canonical BAF (ncBAF) chromatin-remodeling complex. The condition is characterized by developmental delay, hypotonia, hypertrichosis, and joint laxity. However, long-term data remain limited, and systemic manifestations are incompletely defined. Case Description: We report a 22-year-old male with a de novo BICRA frameshift variant, c.2479_2480delinsA (p.Ala827Thrfs*15), previously included in the original cohort reported by Barish et al. Longitudinal follow-up revealed an expanded phenotype extending beyond neurodevelopmental features. Early findings included global developmental delay, growth hormone deficiency, short stature, and joint hypermobility. In adolescence and adulthood, he developed severe intestinal dysmotility requiring total colectomy, recurrent spontaneous pneumothoraces from bilateral apical bullous disease, and portal-vein thrombosis, representing visceral and vascular complications not previously emphasized in BICRA-related disorders. The identified BICRA variant truncates the coiled-coil domain critical for BRD9/BRD4 interaction, consistent with a loss-of-function mechanism. The patient’s systemic features suggest that BICRA haploinsufficiency affects not only neurodevelopmental pathways but also smooth-muscle and connective-tissue integrity. Conclusions: This case expands the phenotypic spectrum of BICRA-related CSS12, demonstrating that visceral and vascular involvement can occur alongside neurodevelopmental and connective-tissue features. Recognition of these broader manifestations underscores the need for lifelong multidisciplinary surveillance and contributes to understanding the diverse biological roles of the ncBAF complex in human development. Full article

Bovine Respiratory Disease (BRD) represents one of the largest causes of economic loss and animal morbidity in the global cattle industry, second only to neonatal diarrhea. Its etiology is complex, originating from a multifactorial combination of host susceptibility, environmental stressors, viral infections, and secondary bacterial pathogens. Although viruses are often the initial cause of disease, suppressing the host’s respiratory defense mechanisms, most of the severe pneumonic damage and clinical signs can be attributed to bacterial infections. This review provides an overview of the primary bacterial agents identified within the BRD complex, including Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. We discuss their role as commensals that then become opportunistic pathogens, and further how they interact in a synergistic relationship with a primary viral insult, leading to the resulting pathogenesis and the development of pneumonia. This manuscript discusses in further detail some of the challenges in BRD management, such as the limitations of current diagnostic methodologies, overreliance on antimicrobial therapy, and the growing concern of antimicrobial resistance. Lastly, the need for integrated approaches in management, better husbandry and biosecurity, coupled with the development of novel therapeutic alternatives, is underlined as a means of assuring a sustainable control of this serious syndrome. Full article

Radiotherapy is an effective treatment for cancer; however, radioresistant cancer cells result in recurrence. Therefore, elucidating the mechanisms of radioresistance is urgently needed. Super-enhancers (SEs) are clusters of enhancers occupied by a high density of master transcription factors, mediators, and bromodomain protein BRD4. Recently, we reported that ΔNp63, an oncogenic transcription factor, promotes radioresistance in human head and neck squamous cell carcinoma (HNSCC) cells. As ΔNp63 establishes SEs in HNSCC cells, SEs may be involved in radioresistance. Here, we investigated the role of the SE component BRD4 in the radiation responses of HNSCC cells using a BRD4 degrader ARV-771 or BRD4 knockdown. First, Western blotting confirmed that ARV-771 decreased BRD4 protein expression. ARV-771 treatment resulted in reduced cell proliferation and enhanced apoptosis in irradiated HNSCC cells. Moreover, colony formation assays revealed that both ARV-771 and BRD4 knockdown enhanced the radiosensitivity of HNSCC cells, suggesting BRD4 contributes to the radioresistance of HNSCC cells. Furthermore, fluorescence immunostaining revealed distinct localization patterns of γH2AX, a marker of DNA double-strand breaks, compared with BRD4 and ΔNp63 in irradiated cells. Notably, ARV-771 and BRD4 knockdown decreased ΔNp63 and BRD4 protein expression, whereas ΔNp63 knockdown had minimal impact on BRD4 expression. Taken together, these findings suggest that BRD4-dependent maintenance of ΔNp63 expression may contribute, at least in part, to the regulation of radioresistance in HNSCC cells. Full article

The interplay between chromatin structure and phase-separating proteins is an emerging topic in cell biology with implications for understanding disease states. Here, we investigate the functional relationship between bromodomain protein 4 (BRD4) and chromatin architecture. By combining molecular dynamics simulations with live-cell imaging, we demonstrate that BRD4, when mutated at specific N-terminus sites, significantly impacts the organization and dynamics of chromatin nanodomains, known as nucleosome clutches. Our findings reveal that a constitutively phosphorylated mutant of BRD4 condenses nucleosome clutches, while treatment with (+)-JQ1 increases the diffusion dynamics of single nucleosomes and decondenses nucleosome clutches. Simultaneously, we demonstrate that BRD4 mutations can alter localization of BRD4 to chromatin as well as modify single nucleosome dynamics. These results suggest that both chromatin binding and phase separation of BRD4 could co-regulate the nanoscale chromatin architecture and the chromatin microenvironment. Our observations shed light on the nuanced regulation of chromatin structure by BRD4, offering insights into its role in maintaining the nuclear architecture and transcriptional activity. Full article

Background/Objectives: Polycystic ovary syndrome (PCOS) is a common endocrine–metabolic disorder in which skeletal muscle insulin resistance contributes substantially to cardiometabolic risk. Pioglitazone improves insulin sensitivity in women with PCOS, yet the underlying transcriptional changes and their potential as treatment-response biomarkers remain incompletely defined. We aimed to reanalyse skeletal muscle gene expression from pioglitazone-treated PCOS patients using modern machine learning and network approaches to identify candidate biomarkers and regulatory hubs that may support precision therapy. Methods: Public microarray data (GSE8157) from skeletal muscle of obese women with PCOS and healthy controls were reprocessed. Differentially expressed genes (DEGs) were identified and submitted to Ingenuity Pathway Analysis to infer canonical pathways, upstream regulators, and disease functions. Four supervised machine learning algorithms (logistic regression, random forest, support vector machines, and gradient boosting) were trained using multi-step feature selection and 3-fold stratified cross-validation to provide superior Exploratory Gene Analysis. Gene co-expression networks were constructed from the most informative genes to characterize network topology and hub genes. A simulated multi-omics framework combined selected transcripts with representative clinical variables to explore the potential of integrated signatures. Results: We identified 1459 DEGs in PCOS skeletal muscle following pioglitazone, highlighting immune and fibrotic signalling, interferon and epigenetic regulators (including IFNB1 and DNMT3A), and pathways linked to mitochondrial function and extracellular matrix remodelling. Within this dataset, all four machine learning models showed excellent cross-validated discrimination between PCOS and controls, based on a compact gene panel. Random forest feature importance scoring and network centrality consistently prioritized ITK, WT1, BRD1-linked loci and several long non-coding RNAs as key nodes in the co-expression network. Simulated integration of these transcripts with clinical features further stabilized discovery performance, supporting the feasibility of multi-omics biomarker signatures. Conclusions: Reanalysis of skeletal muscle transcriptomes from pioglitazone-treated women with PCOS using integrative machine learning and network methods revealed a focused set of candidate genes and regulatory hubs that robustly separate PCOS from controls in this dataset. These findings generate testable hypotheses about the immunometabolism and epigenetic mechanisms of pioglitazone action and nominate ITK, WT1, BRD1-associated loci and related network genes as promising biomarkers for future validation in larger, independent PCOS cohorts. Full article

This review summarizes a growing collection of data on adult neurogenesis in various vertebrate species, with a focus on teleost fish and mammals. Teleost fish serve as exceptional models for studying the dynamics of the cell cycle and the functions of adult neural stem progenitor cells (aNSPCs) throughout the central nervous system (CNS). New information about the characteristics of cells in various areas of the telencephalon of non-model objects—juvenile masu salmon Oncorhynchus masou and chum salmon Oncorhynchus keta—during postembryonic ontogenesis and after traumatic injury expands the current understanding of the issue. The expression of molecular markers of adult-type glial precursors in the model zebrafish and non-model objects, juveniles O. masou and O. keta, was presented. Immunohistochemical (IHC) verification of BrdU and PCNA made it possible to identify a population of rapidly and slowly proliferating cells in the pallium of intact O. masou and after traumatic brain injury (TBI). In salmonids, unlike in mammals, progenitor cells are able to differentiate into neurons after injury. The expression of vimentin and GFAP in the aNSCPs has functional specificity. A comparative analysis of the expression of Pax transcription factors in various vertebrates and juveniles O. masou is presented. Pax genes maintain cells in an undifferentiated state and ensure the spatiotemporal formation of mature cell types in changing developing neurogenic niches. The functions of glutamine synthetase (GS) and H₂S in the brains of vertebrates and juvenile chum salmon under intact conditions and after TBI are characterized. In fish, unlike mammals, as a result of TBI, neuronal conduction is restored in the injury area, whereas in mammals the regenerative process is complicated by neuroinflammation and culminates in the formation of a glial scar. Full article

Background/Objectives: Astrocytic redox-inflammatory signaling has been implicated in Parkinson’s disease (PD) pathology and may constrain hippocampal neurogenesis. We previously identified an astrocytic NOX4–MPO–OPN axis associated with impaired neurogenic capacity. Here, we tested whether a saffron-derived antioxidant (SDA; Crocus sativus extract) and Passiflora incarnata L. extract (PI) modulate this pathway in an MPTP-induced PD mouse model. Methods: Male C57BL/6J mice were randomized to Sham, MPTP, and treatment groups (n = 9/group for behavior; n = 4–5/group for histology/immunoblotting). SDA or PI (50 mg/kg/day, oral, 5 weeks) was administered, with resveratrol as a positive control. Behavioral, histological, and molecular analyses were performed by investigators blinded to group allocation where feasible. Results: SDA and PI were associated with reduced NOX4/MPO/OPN signals, mainly in GFAP-positive astrocytes, along with recovery of neurogenesis markers (Ki67, DCX, BrdU/NeuN) and synaptic markers (PSD95, synaptophysin), and improved motor performance. Mitochondrial and oxidative injury markers (TIM23, TOM20, OXPHOS subunits; 4-HNE) and apoptotic markers (Bax, cleaved caspase-3, Bcl-2) also shifted toward Sham levels. Given previous reports of Passiflora extracts’ sedative effects, we note that metabolic measures (body weight, food intake, and water intake) were similar across groups; however, specific tests for sedation or arousal were not conducted. Conclusions: These findings offer preclinical evidence that SDA and PI modulate redox-inflammatory and mitochondrial stress signatures and are associated with neurogenic, synaptic, and behavioral improvements in an acute MPTP model. Further validation in chronic/genetic PD models and pharmacokinetic/brain exposure studies will be necessary to confirm their translational potential. Full article

Synovial sarcoma (SySa) is a malignant soft tissue tumor that is characterized by an SS18::SSX fusion protein, which integrates into BAF chromatin remodeling complexes and alters global gene transcription. Despite its uniform genetic driver, SySa displays striking histomorphological and phenotypic heterogeneity, including spindle cell, glandular and poorly differentiated patterns. Prognosis is variable, with around 50% of patients developing metastases. Limited response to chemotherapy highlights the need for a better understanding of the underlying molecular mechanisms to guide alternative therapeutic strategies. Given the pivotal function of BAF complexes in SySa and their recently described impact on cellular differentiation processes, this study aims to investigate the role of SS18::SSX and specific BAF subunits in SySa differentiation. Nanostring analysis revealed that silencing of SS18::SSX and the GBAF subunit BRD9 modulates the cellular differentiation pathways. SS18::SSX and BRD9 were found to regulate epithelial–mesenchymal-transition (EMT)-associated factors of Snail and Slug on different levels, with SS18::SSX repressing E-Cadherin expression. Published single-cell RNA sequencing data were analyzed to validate our finding that BRD9 contributes to SySa EMT regulation. Our study provides novel insights into the multilayered regulation of key EMT players by SS18::SSX and BRD9 in SySa, thereby defining tumor phenotype and (potentially) prognosis. 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

Uterine Fibroids (UFs) are the most common benign tumors in women of reproductive age, affecting ~77% of women overall and are clinically manifest in ~25% by age 50. Bromodomain and extra-terminal domain (BET) proteins play key roles in epigenetic transcriptional regulation, influencing many biological processes, such as proliferation, differentiation, and DNA damage response. Although BET dysregulation contributes to various diseases, their specific role in the pathogenesis of UFs remains largely unexplored. The present study aimed to determine the expression pattern of BET proteins in UFs and matched myometrium and further assess the impact of BET inhibitors on UF phenotype and epigenetic changes. Our studies demonstrated that the levels of Bromodomain-containing protein (BRD)2 and detection rate of BRD4 were significantly altered in UFs compared to matched myometrium, suggesting that aberrant BET protein expression may contribute to the pathogenesis of UFs. To investigate the biological effects of BET proteins, two small-molecule inhibitors, JQ1 and I-BET762, were used to assess their impact on UF cell behavior and transcriptomic profiles. Targeted inhibition of BET proteins markedly reduced UF cell viability compared with myometrial cells and induced cell cycle arrest. Unbiased transcriptomic profiling coupled with bioinformatic analysis revealed that BET inhibition altered multiple biological pathways, including G2M checkpoint, E2F targets, mitotic spindle, mTORC1 signaling, TNF-α signaling via NF-κB, and inflammatory response, as well as reprogrammed the UF cell epigenome. Notably, BET inhibition decreased the expression of several genes encoding extracellular matrix (ECM) proteins, a hallmark of UFs. Collectively, these results support that BET proteins play a pivotal role in regulating key signaling pathways and cellular processes in UFs. Targeting BET proteins may therefore represent a promising non-hormonal therapeutic strategy for UF treatment. Full article

Background: Maize (Zea mays L.) is a vital global crop, and yield improvement through dwarfing breeding—inspired by the Green Revolution—holds promise for addressing food security challenges. Despite the identification of over 60 dwarf genes in maize, their genetic diversity remains limited. Brassinosteroids (BRs) are key phytohormones that regulate plant height, and mutations in BR-related genes often result in dwarf phenotypes. Methods: The zmbrd1 mutant was generated via EMS mutagenesis in the B73 background. Phenotypic traits (plant height, root length) and histological features (e.g., mesocotyl cell length) were compared between mutant and wild-type plants. Transcriptome sequencing of leaves and root tips identified differentially expressed genes (DEGs), followed by GO and KEGG enrichment analyses. Key hormone-related genes were validated by means of qRT-PCR. Results: The zmbrd1 mutant exhibited severe dwarfism and reduced root length, primarily due to inhibited longitudinal cell elongation in internodes. Transcriptome analysis revealed 1652 DEGs in leaves and 1450 DEGs in roots. Enriched pathways included BR biosynthesis, plant hormone signal transduction, and glutathione metabolism. In leaves, upregulated genes were linked to hormone signaling and chloroplast function, while downregulated genes involved oxidoreductase activity and stress response. In roots, DEGs were enriched in ethylene signaling, MAPK pathways, and plant–pathogen interaction, suggesting impaired defense responses. qRT-PCR confirmed dysregulation of hormone-related genes: GA biosynthesis genes were downregulated, whereas auxin-related genes were upregulated in leaves but downregulated in roots. Conclusions: The dwarf phenotype of zmbrd1 stems from disrupted BR biosynthesis, leading to hormonal imbalance (particularly in GA and auxin pathways), oxidative stress, and suppressed cell elongation. Our results suggest that ZmBRD1 plays a key role in integrating aboveground and underground growth likely through modulating hormone crosstalk. This study elucidates BR-mediated height regulation and provides genetic resources for maize breeding. Full article

Pradofloxacin is a third-generation dual enzyme targeting bactericidal veterinary fluoroquinolone, recently approved for use in cattle for bovine respiratory disease, which is active against Gram-positive/negative, atypical and anaerobic bacteria. We compared in vitro killing by pradofloxacin to that by ceftiofur, danofloxacin, enrofloxacin, florfenicol, marbofloxacin, tildipirosin, tilmicosin and tulathromycin against bovine isolates of Mannheimia haemolytica and Pasteurella multocida over a range of bacterial densities (10⁶–10⁹ cfu/mL). Drug concentrations used in the kill assays included the minimum inhibitory and mutant prevention drug concentrations and maximum serum and maximum tissue drug concentrations. Regardless of bacteria density tested and drug concentration used, pradofloxacin consistently killed as many or more (but not fewer) bacterial cells than any other drug tested against M. haemolytica strains. At the 10⁸–10⁹ cfu/mL densities, pradofloxacin killed 99–99.9%, 100% and 100% of bacterial cells at the MPC, maximum serum and maximum tissue drug concentrations, respectively, following 24 h of drug exposure. Indeed, pradofloxacin killed 99.9–99.99% of cells following 30–60 min of exposure to the maximum serum concentration. Similar trends were seen with killing of P. multocida strains by pradofloxacin. Against high-density bacterial populations, pradofloxacin was rapidly bactericidal and consistently killed more cells than the other agents tested. This manuscript represents the most comprehensive comparative in vitro kill study completed to date. Full article

Bovine respiratory disease (BRD) is a multifactorial syndrome and a leading cause of morbidity and economic loss in global cattle production. Next-generation sequencing (NGS) platforms, including Illumina and Oxford Nanopore Technologies (ONT), have enabled high-resolution profiling of the bovine respiratory microbiome and virome, revealing novel viral contributors such as bovine rhinitis A virus (BRAV) and influenza D virus (IDV). Transcriptomic approaches, including RNA sequencing (RNA-Seq) and microRNA (miRNA) profiling, provide insights into host immune responses and identify potential biomarkers for disease prediction. Traditional diagnostic methods—culture, ELISA, and immunohistochemistry—are increasingly complemented by PCR-based and metagenomic techniques, improving sensitivity and specificity. Despite technological progress, gaps remain in virome characterization, miRNA function, and the integration of multi-omics data. Standardized protocols and longitudinal studies are needed to validate microbial signatures and support field-deployable diagnostics. Advances in bioinformatics, particularly network-based integrative pipelines, are becoming essential for harmonizing multi-omics datasets and revealing complex host–pathogen interactions. The objective of this comprehensive review was to synthesize current understanding of the bovine transcriptomic response to BRD as well as the respiratory microbiome and virome, emphasizing how advanced sequencing technologies have transformed microbial profiling and molecular diagnostics in BRD. Full article

Macrolides are crucial for the management and treatment of bovine respiratory disease (BRD). However, antimicrobial resistance (AMR) threatens the efficacy of these and other antimicrobials. We developed real-time recombinase polymerase amplification (RPA) assays targeting three clinically relevant macrolide antimicrobial resistance genes (ARGs)—msrE-mphE and erm42—in ≤30 min using extracted DNA. A set of 199 deep nasopharyngeal swabs (DNPS) collected from feedlot calves near the time of arrival were selected based on bacterial culture (BC) results for Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni and antimicrobial susceptibility testing (AST) for tulathromycin, tilmicosin, tildipirosin, or gamithromycin. Samples were also tested for the same targets using RPA and polymerase chain reaction (PCR). In samples that were culture-positive for one or more macrolide-resistant BRD-associated bacteria (n = 101), msrE-mphE and/or erm42 were detected in 95% of cases using RPA. The remaining 98 samples were either culture-negative, or the recovered bacteria were macrolide-susceptible: 43% of these were RPA-positive for at least one macrolide ARG. Together with BC-AST and PCR, Bayesian latent class modelling estimated the clinical sensitivity of RPA for macrolide ARGs to be 95% and specificity to be 58%, with moderate agreement between RPA and BC-AST (κ = 0.52) or PCR (κ = 0.55). The estimated sensitivity of the RPA multiplex assay for the targeted macrolide ARGs was very good, although estimated specificity was limited. However, Sanger sequencing confirmed RPA detection of msrE-mphE in BC-AST/PCR-negative samples (n = 23), reflecting the presence of this locus in non-target bacteria, as well as potential ARG variants among BRD bacteria. These findings support the potential of RPA for rapid ARG detection from extracted DNA. Continued assay optimization and evaluation for detection of respiratory bacteria and ARGs will further enhance its diagnostic utility. Full article