Search Results (423)

H3 subtype avian influenza virus (AIV) is one of the most widely distributed and frequently detected low-pathogenicity avian influenza viruses (LPAIVs) in wild birds and poultry worldwide. Wild waterfowl are the natural reservoir of this virus. In recent years, driven by frequent genetic reassortment and continuous evolution, H3 subtype AIV has expanded its host range to include various mammalian species—dogs, cats, horses, seals, pigs (experimental infection), and humans. Among these, the H3N8 subtype shows the broadest cross-species transmissibility, having established sustained transmission lineages in horses and dogs, while also causing sporadic infections in seals and humans. Avian-origin H3N2 canine influenza virus has circulated stably in dog populations across Asia and North America for nearly two decades. Between 2022 and 2023, China reported three human cases of H3N8 subtype AIV infection, one of which was fatal (Guangdong, March 2023), indicating that this virus poses a potential zoonotic transmission risk. Novel triple-reassortant strains, including genotype G25 of H3N8 and H3N3, first detected in chicken flocks in late 2022, have continued to emerge. Their HA genes originate from H3 subtype viruses of wild bird or chicken origin, whereas their internal gene segments are predominantly acquired from H9N2 subtype AIV, resulting in enhanced mammalian adaptability. This review summarizes the global epidemiology of H3 subtype AIV (covering wild birds, poultry, and live poultry markets), its genetic evolution and reassortment patterns, and the molecular mechanisms underlying cross-species transmission. This review particularly highlights the regulatory roles of key amino acid mutations—such as PB2-D701N, M1-M192V, and HA-Q226L/G228S—in receptor binding preference, polymerase activity, and pathogenicity. This review also summarizes recent advances in detection techniques (multiplex RT-PCR, quantum dot-based immunochromatographic strips, RT-RAA, and CRISPR-Cas13a) and vaccine development (inactivated, adenovirus-vectored, and mRNA vaccines). Key prevention and control challenges—including covert circulation, surveillance gaps, and limited subtyping diagnostic capacity at primary laboratories—are also discussed. This review proposes that H3 subtype AIV should be recognized as a potential zoonotic threat warranting continued surveillance, although no evidence of sustained human-to-human transmission has been documented to date. Better understanding of cross-species mechanisms, stronger surveillance and early warning, and evidence-based control strategies are needed to inform future risk assessment. Full article

Osteoarthritis (OA) is one of the most prevalent degenerative joint diseases, and there is no approved pharmacological therapy that can reliably reverse cartilage degeneration or restore cartilage structure. Allogeneic mesenchymal stromal cells (MSCs) have shown therapeutic potential for OA, but inflammatory conditions can enhance HLA expression and increase allogeneic immune recognition risk. Here, we generated B2M and CIITA double-knockout induced pluripotent stem cells (dKO-iPSCs) using CRISPR/Cas9 and subsequently differentiated them into dKO-iMSCs. B2M/CIITA double-knockout did not affect iPSC pluripotency, karyotype, or teratoma formation. dKO-iMSCs retained characteristic MSC phenotypes, exhibited greater proliferative capacity than tissue-derived MSCs, and could be expanded to passage 18. Notably, HLA-ABC and HLA-DR expression remained undetectable in dKO-iMSCs even after IFN-γ stimulation. In co-culture assays with PHA-L-stimulated allogeneic PBMCs, dKO-iMSCs suppressed PBMC and T-cell proliferation as effectively as wild-type iMSCs, indicating preservation of their immunomodulatory activity. Intra-articular administration of dKO-iMSCs significantly reduced cartilage degeneration in the destabilization of the medial meniscus (DMM) mouse model of OA. The treatment showed greater therapeutic efficacy than wild-type iMSCs and tissue-derived MSCs and did not cause evident systemic organ toxicity. Single-cell RNA sequencing of BM-MSCs and dKO-iMSCs after in vitro IFN-γ stimulation revealed enhanced extracellular matrix-associated cell–cell communication and markedly reduced IL1B expression in dKO-iMSCs, indicating a less pro-inflammatory and more matrix-supportive cellular state under inflammatory conditions. Collectively, B2M/CIITA double-knockout enables sustained reduction in HLA expression while preserving the MSC phenotype and immunomodulatory activity, and enhances the therapeutic efficacy of iMSCs in the mouse OA model, providing a potential strategy for developing iPSC-derived MSCs with reduced HLA expression for allogeneic cell therapy. Full article

Diabetes mellitus affects nearly half a billion people worldwide and is primarily caused by the loss of insulin-producing cells, which are essential for maintaining normal blood glucose levels. In this study, we optimized a CRISPR activation (CRISPRa) system based on deactivated Cas9 (dCas9) to transfect HEK293 cells, which were subsequently differentiated into functional α-, β-, and δ-like cells as an engineering proof-of-concept. The CRISPRa system targeted multiple genes, including NGN3, NKX6.1 + MAFA, and PDX1, successfully inducing cell differentiation as confirmed by immunostaining. These cells were encapsulated in a 3D-printed GelMA disk, and the secretion of glucagon, insulin, and somatostatin was monitored for 30 days. Full article

Rice panicle architecture plays a critical role in determining grain yield; however, the regulatory networks influencing axillary meristem formation remain inadequately elucidated. A previous study identified LAX1 as a canonical bHLH transcription factor, detailing its direct targets and global transcriptomic alterations in lax1 mutants. In this study, an independent CRISPR/Cas9 LAX1 knockout mutant was generated, followed by transcriptome profiling of young panicles. The lax1-KO mutant displayed a reduction in secondary branches and grain number. Applying a more stringent threshold (|log₂ fold change (log₂FC)| ≥ 1), 518 high-confidence differentially expressed genes (DEGs) were identified, 131 of which overlapped with previously reported DEGs (direction concordance: 88.5%), supporting the core LAX1-regulated network. The analysis revealed member-specific PIN regulation (OsPIN1c/1d and OsPIN2 downregulated; OsPIN3t and OsPIN9 upregulated), expression changes in hormone-associated genes (OsPP2C09, OsPP2C49, OsRR3, OsRR9, OsGASR9) and OsTPP genes (OsTPP1, OsTPP4, OsTPP9), and altered expression of panicle regulators (IPA1, OsTB1, DEP1, DEP3, CUC, and MADS-box genes). Motif enrichment analysis further identified bHLH motifs as the most frequently represented, whereas CAMTA and FRS/FRF motifs exhibited the highest statistical significance in DEG promoters, suggesting a possible multi-layered transcriptional architecture. Collectively, these findings validate and refine the LAX1-regulated network, positioning LAX1 as a pivotal coordinator of rice panicle development. Full article

Essential oils are promising alternatives to conventional chemical pesticides due to their toxic, repellent or oviposition-deterrent effects against many kinds of pests. Sweet fennel essential oil is widely recognized for its pharmacological, antimicrobial, and acaricidal properties. Moreover, several studies have demonstrated its insecticidal and repellent activities against insects. In this study, we found that sweet fennel essential oil exhibited strong dose-dependent repellent activity in the dual-choice still-air assay and oviposition behavioral assays against Drosophila suzukii adults. Next, we confirmed that trans-anethole was the most abundant constituent in sweet fennel essential oil using GC-MS. In addition, the results of the behavioral assays indicated that trans-anethole displayed comparable repellency against D. suzukii. Furthermore, an Orco knockout homozygous strain (Orco^−/−) was generated using CRISPR–Cas9 genome editing, and the EAG responses of Orco^−/− flies to sweet fennel oil and trans-anethole were completely abolished. The repellency elicited by sweet fennel oil and trans-anethole were also dramatically reduced. Taken together, these laboratory results not only reveal that sweet fennel oil holds potential in pest management but also demonstrate that its repellency is mediated by the Orco pathway. Full article

Reduced expression of MAPK organiser 1 (MORG1) exerts renoprotective effects in MORG1^+/− mice in models of type 1 and type 2 diabetes. To investigate the regulation of MORG1 expression, we generated endogenous hMORG1-luciferase-tagged HEK293 cells carrying a firefly luciferase reporter inserted downstream of the human MORG1 gene using the CRISPR/Cas9 method. This study analysed the time-dependent effects of 20 mM D-glucose on MORG1-luciferase activity and examined whether 20 µM metformin influences MORG1 expression. HEK293 cells were genomically modified using CRISPR/Cas9 to insert the firefly luciferase gene downstream of MORG1. Stable clones were selected with puromycin. The influence of D-glucose concentration on MORG1 expression was analysed in a dose- and time-dependent manner. Luciferase assays assessed MORG1 transcriptional activity, while Western blotting determined protein expression. qRT-PCR was used to assess MORG1 mRNA levels. The pharmacological effect of metformin was evaluated in parental and tagged HEK293 cells and TKPTS cells, simultaneously exposed to 5.5 mM or 20 mM D-glucose in the presence or absence of 20 µM metformin. High glucose significantly increased luciferase activity and MORG1 protein expression compared with 5.5 mM glucose controls. Time course experiments demonstrated significant induction of MORG1 transcription, confirmed by corresponding increases in protein levels. Metformin treatment significantly reduced glucose-induced luciferase activity, MORG1 mRNA levels, and MORG1 protein expression at 24 h. Analyses of TKPTS cells show that 20 mM D-glucose elevated Morg1 mRNA levels, while this induction was attenuated in the presence of 20 µM metformin. High glucose induces time-dependent upregulation of MORG1 transcription and protein expression in MORG1-luciferase-tagged HEK293 cells. Metformin application attenuates, but does not fully prevent, glucose-induced MORG1 mRNA and protein expression. Full article

The spatial organization of the genome has emerged as a central regulator of gene expression and cellular function. Chromatin architecture is organized hierarchically across multiple spatial scales and involves chromatin loops, topologically associating domains (TADs), chromatin compartments, and specialized nuclear environments that collectively shape regulatory interactions within the nucleus. Disruption of these structures contributes to a wide range of diseases, including developmental disorders, cancer, and laminopathies, stimulating growing interest in technologies capable of programmable manipulation of genome topology. The emergence of CRISPR/dCas-based technologies has transformed the field from descriptive 3D genomics to programmable genome engineering. Catalytically inactive Cas proteins fused to architectural or epigenetic effectors enable targeted manipulation of chromatin loops, loop extrusion, subnuclear positioning, and local chromatin states without altering the underlying DNA sequence. In this review, we summarize current CRISPR/dCas-based approaches for engineering three-dimensional genome architecture, discuss their mechanistic basis and applications, and highlight emerging therapeutic opportunities and major technical challenges in the field. Full article

Genetic transformation is a core technology for wheat molecular breeding. Transformation efficiency and genotype compatibility determine the pace of targeted genetic improvement. Mature embryos are readily accessible but are associated with low transformation efficiency and strong genotype dependence. Using mature wheat embryos as explants, we screened eight wheat varieties representing three ecological types to develop genotype-adapted tissue culture systems. Treatments A5 (4 mg/L 2,4-D + 2 mg/L Dicamba), A7 (4 mg/L 2,4-D + 6 mg/L Dicamba), and A8 (4 mg/L 2,4-D + 8 mg/L Dicamba) were optimal for callus induction in winter, semi-winter, and spring wheat, respectively. WF3 (a 1/2 MS-based medium) was broadly applicable to callus differentiation, whereas IBA produced the highest rooting efficiency in regenerated plantlets. The optimal transformation conditions comprised ultrasound treatment (2.5 min) followed by vacuum infiltration (5 min), supplementation of the infection solution with PEG₆₀₀₀ (250 mg/L) and Tween20 (0.01%), and an OD₆₀₀ of 0.6–0.8. Transformation efficiencies using mature embryos of the semi-winter wheat varieties YX288 and ZM578 were 2.5% and 1.8%, respectively. Using this transformation platform, the dwarfing genes Rht12 in ZM578 and Rht8 in YX288 were targeted by CRISPR/Cas9-mediated editing. Multiple single-base substitutions at the target sites were detected in ZM578 rht12 mutants, which exhibited significantly reduced height, increased tiller number, and a compact plant architecture. However, no successfully edited rht8 lines were obtained in YX288. These findings indicate that this study established a reliable mature-embryo transformation system for wheat and successfully validated gene editing in the semi-winter variety ZM578. The system provides a technical platform for the functional analysis of key genes and molecular breeding in bread wheat. Full article

Transmissible gastroenteritis (TGE) and pseudorabies (PR) remain important viral diseases causing massive economic losses and posing a continuous burden on the global swine industry. The continuous emergence of variant strains of transmissible gastroenteritis virus (TGEV) and pseudorabies virus (PRV) has gradually weakened the protective efficacy of traditional vaccines, highlighting the urgent need for next-generation preventive vaccine candidates. Here, we constructed a recombinant pseudorabies virus named rPRV-AD expressing the major neutralizing epitopes A and D of the TGEV spike protein via homologous recombination combined with CRISPR/Cas9-gene editing technology and then evaluated its biological characteristics in vitro and immunogenicity in piglets. The results showed that this recombinant virus exhibited similar replication kinetics and biological properties to the parental strain. Immunization of 2-week-old piglets with rPRV-AD caused no obvious adverse effects and induced specific antibody and neutralizing antibody responses against both TGEV and PRV. Following virulent TGEV challenge, compared with the DMEM control group, rPRV-AD immunization alleviated clinical signs of piglets and significantly reduced viral load in intestine and feces, although its protective efficacy was lower than that of the commercial TGEV vaccine. Moreover, rPRV-AD provided effective clinical protection against challenge with the virulent PRV NY strain. In summary, these findings suggest that rPRV-AD represents a vaccine candidate that provides partial protection and warrants further optimization, and yet shows short-term protective efficacy against both PRV and TGEV in pigs. Full article

Adseverin is a Ca²⁺-dependent, actin severing protein that promotes fusion of osteoclast precursors in osteoclast formation. Currently it is not understood how actin severing activity is regulated in osteoclastogenesis. Mass spectrometry of adseverin immunoprecipitates from osteoclasts showed that adseverin associated with Serpin D1. Purified Serpin D1 interacted with adseverin in vitro in a Ca²⁺-dependent manner. Ca²⁺ increased actin severing by ~2-fold. Cells that differentiated into osteoclasts expressed an intracellular, truncated Serpind1 isoform that lacked exons 1 and 2, and part of exon 3. This truncated Serpin D1 isoform was distinct from the secreted, full-length Serpin D1 protein that inhibits thrombin activity. The expression of truncated Serpin D1 in RAW264.7 cells appeared to be further increased following TNF-α treatment during RANKL-induced osteoclastogenesis. CRISPR/Cas9-mediated partial deletion of exon 3 of Serpind1, which resulted in Serpind1 knockout, increased the abundance of subcortical actin filaments. This treatment also altered the spatial distribution of adseverin, inhibited the expression of osteoclast-specific genes and reduced the formation of multinucleated osteoclasts by >90% in vitro. We conclude that a truncated, intracellular variant of Serpin D1 interacts with adseverin to promote actin severing and osteoclast formation. Full article

Rice (Oryza sativa L.) is a staple food crop worldwide, and improving disease resistance is a core target in rice breeding. In this study, we employed CRISPR/Cas9 genome editing to modify the coding sequence (CDS) of two susceptibility genes, Bsr-d1 and Pi21, in the elite maintainer line Gengxiang B to enhance its blast resistance. We generated Bsr-d1/Pi21 double homozygous mutants via Agrobacterium-mediated genetic transformation. Quantitative RT-PCR revealed significantly suppressed transcript accumulation of both target genes in the edited lines compared with the wild type Gengxiang B. Upon inoculation with Magnaporthe oryzae, multiple defense-related marker genes were markedly upregulated in the double mutants. Phenotypic assays demonstrated significantly reduced disease severity for both leaf and panicle blast in the edited lines compared with the wild type. Importantly, no statistically detectable differences were found between the double mutants and wild-type plants for key agronomic or grain quality traits. Collectively, these results demonstrate that CRISPR/Cas9-mediated editing of susceptibility loci generates genetically stable blast-resistant rice germplasm without compromising agronomic traits or grain quality, providing valuable genetic resources for future rice varietal improvement. Full article

Water deficit severely limits rice productivity. The elite Dian (D1)-type hybrid japonica rice ‘Dianheyou 615 (ZH1)’ exhibits exceptional drought adaptation in high-altitude rainfed uplands of the Yungui Plateau, yet the underlying molecular mechanisms remain unknown. We compared phenotypic and transcriptomic responses of ZH1 and six other japonica cultivars under well-watered and water-deficit conditions. Water-deficit stress significantly impaired agronomic traits across all cultivars; however, ZH1 uniquely maintained relatively stable flag leaf morphology and seed-setting rate, and displayed distinctive stomatal traits, in stark contrast to its parental lines and other cultivars. Transcriptomic profiling at the jointing-to-booting stage defined a core drought response module of 174 conserved genes across all cultivars. Critically, by intersecting 1097 ZH1-specific genes with drought-responsive elements, we pinpointed 15 core, cultivar-specific regulatory genes. These candidates are enriched in functions related to cuticle formation, carbohydrate metabolism, and stress signaling; among them, a DREB transcription factor (LOC4347618) is a prime candidate. qRT-PCR validated their expression. Using CRISPR/Cas9-mediated gene editing, we generated homozygous knockout mutants for LOC4333842, LOC4347618, and LOC4328441. Under 20% PEG-6000-simulated drought stress, all three mutant lines showed significantly increased drought susceptibility relative to wild-type controls, confirming the positive regulatory roles of these genes in drought stress tolerance in japonica rice. These results establish these three genes as promising targets for molecular breeding aimed at enhancing drought resistance in rice. Full article

DNA methylation has long been regarded as a stable, maintenance-based epigenetic marker. However, this classical binary model struggles to fully explain the dynamic and situational dependence of methylation regulation at the multi-biological level. This review defines DNA methylation as a programmable information layer that systematically integrates the latest advances in three interrelated dimensions of molecular coding, disease status indication, and epigenomic engineering. At the molecular level, this paper describes how the chemical diversity of cytosine modification, the writing–erasing enzyme network, and the three-dimensional structure of chromatin jointly construct a methylated polymorphic coding system and evaluates the performance of emerging sequencing technologies in DNA integrity, reading length, modification resolution, and analytical complexity through a multidimensional scoring framework. At the cellular and clinical levels, this paper comprehensively demonstrates methylation as a quantifiable indicator of cell identity, biological aging and disease status, covering circulating free DNA biomarkers and spatial heterogeneity analysis. Critically, this paper evaluates how the clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing platform achieves causal inference and promotes the transformation of methylation from related biomarkers to functional therapeutic targets. At the same time, persistent challenges such as off-target specificity, in vivo delivery, and spatiotemporal regulation encountered in epigenetic gene editing are discussed. This review reveals the paradigm shift of DNA methylation from passive observation markers to actively engineered regulatory parameters, which has direct therapeutic application prospects. Full article

Acinetobacter baumannii is a critical WHO priority pathogen due to its multidrug resistance and high mortality in carbapenem-resistant infections. Resistance is predominantly mediated by class D carbapenemase genes bla_OXA-23 and bla_OXA-40, which spread rapidly via horizontal gene transfer in healthcare settings. To address the lack of a rapid assay capable of detecting both blaOXA-23 and blaOXA-40 in a single analytical workflow, we developed a multiplex two-step RPA–CRISPR/Cas12a assay. Since infections caused by strains harboring either gene require identical therapeutic management, their co-detection in a single reaction is clinically justified. Although simultaneous use of two crRNAs within a single CRISPR/Cas12a reaction is often considered technically challenging due to potential inter-crRNA competition, here it advantageously enables dual-target coverage without compromising sensitivity. The assay demonstrated high specificity with no cross-reactivity against a panel of clinically relevant bacterial species, including closely related Acinetobacter spp. Evaluation using genomic DNA extracted from 63 cultured clinical A. baumannii isolates revealed bla_OXA-23 in 19 isolates (30.2%), bla_OXA-40 in 28 (44.4%), and co-carriage of both genes in 9 (14.3%), with at least one resistance gene detected in 60.3% of isolates. The complete workflow was accomplished within 45 min without specialized equipment, offering a rapid, sensitive, and cost-effective solution for point-of-care molecular surveillance of carbapenem-resistant A. baumannii in clinical and resource-limited settings. Full article

The designation of O RhD-negative blood as a “universal donor” misrepresents the complexity of red blood cell (RBC) compatibility. With 47 recognized blood group systems encompassing 366 antigens, non-ABO antigen exposure drives alloimmunization in 20–50% of chronically transfused patients. A narrative review of PubMed, Scopus, and EMBASE was conducted for studies published between 2016 and 2025, with selective inclusion of foundational pre-2016 works. Enzymatic approaches using Flavonifractor plautii CAZymes and Akkermansia muciniphila exoglycosidase cocktails enable near-complete ABO antigen removal, though group A conversion remains incomplete. CRISPR-Cas9 multiplex editing of immortalized erythroblasts has achieved simultaneous deletion of ABO, Rh, Kell, Duffy, and MNS antigens. Induced pluripotent stem cell platforms enable scalable manufacture; however, enucleation efficiency (40–70%), yield (4.6 × 10³ RBCs/iPSC), and fetal hemoglobin prevalence remain translational barriers. Convergence of enzymatic and gene-editing technologies on GMP-compatible iPSC platforms represents the most credible pathway to truly universal RBCs, although current evidence remains largely confined to immortalized cell lines and early-stage iPSC proof-of-concept studies rather than clinically validated products. Regulatory frameworks, scalability, and rigorous off-target profiling define the key challenges ahead. Full article