Search Results (5,068)

Fruit-specific promoters (FSPs) are a class of regulatory DNA sequences that direct transgene expression exclusively in fruit tissues or during specific stages of fruit development. They are indispensable molecular tools in modern agricultural biotechnology, with broad applications in fruit quality improvement, nutritional enhancement, and shelf-life extension. By functioning as precise molecular switches that regulate fruit-specific gene expression, FSPs overcome the limitations of constitutive promoters and facilitate precision molecular breeding for fruit quality improvement. This review systematically summarizes recent advances in FSP research. First, based on their spatiotemporal expression patterns, FSPs are classified into four categories: immature fruit-specific, fruit ripening-specific, whole fruit development stage-specific, and dual-stage (immature fruit/ripening) promoters. Their origins, expression characteristics, and key cis-regulatory elements are comprehensively summarized. Second, the complex transcriptional regulatory network governing FSP activity is discussed from the perspectives of cis-regulatory elements, major transcription factor families (such as MADS-box and NAC proteins), and epigenetic regulation, including DNA methylation and histone modifications. Furthermore, recent advances in key methodologies, including promoter cloning, functional characterization, and CRISPR/Cas9-mediated precise editing of cis-regulatory elements, are reviewed, together with their applications in crop genetic improvement, plant molecular farming, and fundamental molecular biology research. Finally, this review highlights the major challenges limiting the application of FSPs, including the relatively weak transcriptional activity of natural promoters, insufficient tissue specificity, and limited cross-species applicability. Future perspectives are discussed, emphasizing the integration of artificial intelligence-assisted promoter design, high-throughput screening, and single-cell omics technologies to develop finely tunable synthetic promoters. These advances are expected to provide both a theoretical foundation and technical support for precision molecular breeding in fruit crops. Full article

Brucellosis remains among the most widespread global zoonoses, yet its genetic architecture—spanning host susceptibility and pathogen virulence—is increasingly being investigated. Advances in whole-genome sequencing, transposon mutagenesis, CRISPR screening, and machine learning-driven transcriptomics have accelerated marker discovery on both sides of the host–pathogen interface, although most signatures remain at discovery or early-validation stages. In hosts, polymorphisms in innate immune receptors (TLR2, TLR5, VDR), cytokine regulators (TNF, CTLA4), and novel candidates such as ASAP1 are linked to differential susceptibility in humans and livestock. In Brucella, functional genomics shows that virulence relies on interconnected regulatory circuits that control the type IV secretion system, lipopolysaccharide biosynthesis, and stress responses, with gene essentiality shifting according to the host immune context. Antimicrobial resistance may involve unconventional mechanisms, including metabolic and regulatory adaptation, that are not fully explained by classical resistance-gene paradigms and may implicate metabolic reprogramming and ribosomal modulation. This review synthesizes these converging lines of evidence, highlights key gaps—chiefly the scarcity of functional validation for host associations and limited multi-omics integration—and proposes a roadmap for translating genetic markers toward future diagnostic refinement and therapeutic target discovery, urging a shift from descriptive cataloging toward mechanistic and clinical validation. Full article

Cereal crops including wheat, rice, maize, barley, and sorghum collectively supply most global caloric and protein requirements, yet their productivity is increasingly constrained by combined abiotic stresses that co-occur under field conditions. Simultaneous drought, heat, salinity, and cold impose yield losses that consistently exceed those caused by individual stresses and elicit molecular responses that are qualitatively distinct from single-stress reactions and cannot be inferred from them. Despite this agronomic reality, the molecular mechanisms governing combined stress responses in cereals remain poorly resolved, and no integrated framework connecting the transcriptional, epigenetic, and genome-editing dimensions of combined stress tolerance has previously been articulated for this crop group. This review proposes a three-tier integrated framework for understanding and engineering combined abiotic stress tolerance in major cereals. The first tier encompasses transcription factor networks, including bZIP, WRKY, NAC, AP2/ERF, DREB, MYB, and HSF families, that translate combined stress signals into transcriptional reprogramming through ABA-dependent and ABA-independent pathways, hormonal crosstalk, and osmoprotectant and antioxidant defence systems. The second tier addresses the epigenetic regulatory layer, encompassing DNA methylation, histone modifications, and non-coding RNA pathways that gate TF binding site accessibility and encode stress memory in cereals. The third tier examines CRISPR-based tools, including multiplexed Cas9 editing and dCas9-based epigenome editing, that engineer validated targets from both tiers, while confronting polyploid off-target effects, growth penalties, and a laboratory-to-field validation gap. The three tiers are mechanistically coupled, with TF activity shaping epigenetic landscapes, epigenetic states gating TF access, and both providing precision engineering targets. Critical gaps include the absence of combined-stress epigenomic datasets, limited characterisation in barley and sorghum, and early-stage combined-stress-specific strategies. Full article

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that acts as a major signaling molecule across the nervous, cardiovascular, and immune systems. While GABA has historically been produced via chemical synthesis or plant extraction, microbial fermentation using lactic acid bacteria (LAB) provides a safe, sustainable, and food-grade alternative. This review details the recent progress of LAB-derived GABA, covering the workflow from strain selection to functional food applications. We discuss how modern screening methods combine high-throughput phenotypic testing with genomic mining of the gad operon to efficiently identify high-yielding strains. The biochemical mechanisms of the GABA shunt are also explained, alongside recent CRISPR-based metabolic engineering efforts designed to bypass natural yield limits. Furthermore, we address practical industrial challenges—such as the poor proteolytic ability of key producers like Levilactobacillus brevis—and evaluate viable solutions, including symbiotic co-cultures and optimized downstream purification steps. The review then summarizes the specific health benefits of dietary LAB-derived GABA, focusing on its ability to relieve anxiety via the microbiota-gut–brain axis, control blood pressure, and regulate immunity. Finally, we analyze the current regulatory and sensory hurdles, highlighting how integrating multi-omics data can help establish LAB-derived GABA as a reliable ingredient for functional foods and personalized nutrition. Full article

Adenylate cyclase 5 (Adcy5) generates cyclic adenosine monophosphate (cAMP) downstream of G protein-coupled receptor signaling, yet its role in vertebrate pigmentation remains incompletely understood. Here, we generated a CRISPR/Cas9-mediated adcy5 knockout line in Nile tilapia (Oreochromis niloticus) to investigate its function in chromatophore biology. Loss of adcy5 resulted in a pronounced disruption of body coloration, characterized by the absence of vertical black bars and a global reduction in pigmentation. Despite this, chromatophore number was largely unaffected, indicating that Adcy5 is not required for pigment cell specification but is essential for functional pigmentation. At the cellular level, pigment granules in melanophores and erythrophores failed to undergo dispersion and instead remained constitutively aggregated, revealing a primary defect in intracellular pigment granule transport. Consistently, adcy5 mutants exhibited reduced expression of key melanogenesis-associated genes, including mitfa, tyrb, tyrp1a, and tyrp1b, accompanied by decreased melanin content across multiple tissues. Pharmacological activation of cAMP signaling partially rescued the pigment dispersion defect, whereas stimulation of upstream α-MSH signaling produced only limited effects, placing Adcy5 upstream of intracellular cAMP production within the pigment regulatory cascade. Importantly, we further demonstrate that Adcy5 is required for erythrophore pigment granule dispersion, extending its functional role beyond melanophore biology. Together, these findings identify Adcy5 as a conserved regulator integrating cAMP-dependent pigment synthesis and granule transport across multiple chromatophore types in teleost fish. Full article

Soil salinization has become a major global abiotic threat restricting sustainable viticulture, especially in coastal and inland saline–alkali zones. Unlike cereal crops mainly suffering from sodium toxicity, grapevine (Vitis vinifera L.) is a typical chloride-sensitive woody perennial, subjected to superimposed damages of osmotic stress, ionic imbalance and secondary oxidative injury under saline conditions which severely suppress vegetative growth and degrade berry quality. This review systematically summarizes the multi-layered physiological adaptive mechanisms of grapevine against salt stress, including ion homeostasis maintained by salt overly sensitive (SOS), Na⁺/H⁺ exchanger (NHX) and chloride channel (CLC) transporter families, active accumulation of osmoprotectants, synergistic enzymatic and non-enzymatic antioxidant systems, and phytohormone crosstalk networks formed by endogenous phytohormones (abscisic acid, ABA; jasmonic acid, JA; salicylic acid, SA; brassinosteroid, BR) and small signaling molecules. We further elaborate comprehensive molecular regulatory cascades governing salt tolerance, covering core functional genes for ion transport, master transcription factor families WRKY, MYB, APETALA2/Ethylene Response Factor (AP2/ERF), NAC, basic helix–loop–helix (bHLH) and emerging epigenetic regulatory layers mediated by deoxyribonucleic acid (DNA) methylation, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). In addition, we integrate four categories of field mitigation strategies for saline vineyards: germplasm improvement via salt-tolerant rootstock grafting, rhizosphere soil basal amendment, exogenous biostimulant regulation, and precision agronomic optimization. Current experimental systems do not fully recapitulate complex field combined-stress conditions, as most studies rely on laboratory single-salt stress simulation. Meanwhile, multi-omics, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing and high-throughput phenotyping tools provide promising approaches to deepen our understanding of grape salt tolerance. This review constructs a comprehensive theoretical framework linking physiological responses, molecular regulatory networks and practical field technologies, offering systematic theoretical references and technical guidance for salt-tolerant germplasm innovation and environmentally sustainable viticulture on saline soils. Full article

Antimicrobial resistance (AMR) among pathogens involved in intra-abdominal infections (IAIs) represents a critical and escalating clinical challenge. The interconnected nature of antimicrobial resistance, spanning human medicine, veterinary practice, agricultural use and environmental reservoirs, has required coordinated international responses based on the ‘One Health’ principle. This study presents an update on efforts underway worldwide to develop new antibiotics, novel combined antimicrobial agents, and alternatives to classic therapies for IAIs. New antibiotics or compounds with antibacterial activity are currently in various stages of clinical trials, including several fluoroquinolones, beta-lactamase inhibitors, and polymyxin analogues. To reduce the risk of bacterial resistance, various additions to antimicrobial treatments are being explored, such as nanoparticles (NPs), antimicrobial peptides (AMPs), bacteriophages, the CRISPR/Cas system, and probiotics. Each modality offers distinct mechanisms that circumvent established resistance pathways, including multi-target membrane disruption, sequence-specific gene editing, and microbiome restoration. Current preclinical and clinical evidence is synthesized, and key translational barriers, including delivery challenges, safety concerns, regulatory complexity, and the need for IAI-specific pharmacokinetic data are critically examined. In conclusion, the convergence of novel antibiotic agents and non-traditional antimicrobial strategies reviewed herein provides the foundation for a new paradigm in the management of drug-resistant IAIs. The transition from a monotherapy-centric approach to an integrated, multi-modal treatment framework, guided by rapid diagnostics and informed by antimicrobial stewardship, will be essential to preserve therapeutic efficacy against AMR threats of the coming decades. Full article

The high-pathogenicity island (HPI) is a major virulence determinant in pathogenic Escherichia coli (E. coli), contributing to severe inflammation and tissue damage. Autophagy plays a critical role in clearing intracellular pathogens and modulating inflammation, but whether HPI manipulates this process remains unknown. Here, using a swine-pathogenic E. coli strain and its HPI-deficient mutant (Δirp2) generated by CRISPR/Cas9, we investigated the interplay between HPI and autophagy in RAW264.7 macrophages and a mouse intestinal infection model. We found that HPI⁺ infection induced autophagic activation, as evidenced by increased LC3 puncta (immunofluorescence), upregulated Beclin-1 and autophagy-related gene mRNA levels (qPCR), and downregulated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) expression at both mRNA (qPCR) and protein (immunohistochemistry) levels. In a mouse model, HPI⁺ infection upregulated intestinal Microfold (M) cell markers and secretory Immunoglobulin A (IgA), triggered robust production of pro-inflammatory cytokines, and induced more severe tissue pathology than the HPI-deficient mutant. Pharmacological activation of autophagy with rapamycin alleviated HPI-induced inflammation and injury, whereas inhibition of autophagy by 3-methyladenine (3-MA) or Beclin-1 silencing exacerbated damage. These findings suggest that HPI induces autophagy, but the endogenous autophagic response is insufficient to counteract HPI-induced pathology; pharmacological enhancement of autophagy partially alleviated this insufficiency and reduced tissue damage. Notably, Beclin-1 knockdown blunted HPI-induced upregulation of PI3K and autophagy-related genes, suggesting a role for Beclin-1 in the transcriptional regulation of these responses. In conclusion, HPI simultaneously exerts direct pro-inflammatory effects and induces Beclin-1-dependent autophagy. Enhancing this autophagic response pharmacologically, rather than relying on the endogenous level triggered by HPI alone, limits excessive tissue damage. Thus, boosting autophagy may represent a promising therapeutic strategy against HPI-bearing pathogenic E. coli infections. Full article

Pharyngeal infection with high-risk human papillomavirus (HPV), particularly HPV16, is biologically distinct from cervical infection because it occurs within the specialized lymphoepithelial environment of Waldeyer’s ring. This review evaluates nanoparticle materials for the prevention, detection, and treatment of pharyngeal HPV, with an emphasis on structure–property–function relationships, mucosal performance, and translational feasibility. Lipid nanoparticle platforms, polymeric nanoparticle platforms, inorganic systems, and hybrid platforms are compared with respect to composition, particle size distribution, surface charge, colloidal stability, biodegradability, payload compatibility, release behavior, and manufacturing complexity. Evidence suggests that lipid and polymeric systems are the most credible near-future candidates for mucosal vaccination and localized nucleic acid delivery because they offer the best balance between controllable fabrication, analytical tractability, and biologically plausible performance in mucus-exposed tissue. By contrast, the development of inorganic theranostics and CRISPR-enabled platforms remains at an earlier stage because repeated mucosal dosing, retention in lymphoid tissue, and combined product regulation impose substantial burdens. A translational roadmap is proposed in which material selection is guided by clinically relevant quality attributes, standardized saliva- and mucus-relevant assays, human tonsil organoid testing, and early attention to manufacturability, safety, and regulatory strategy. The field is promising, but direct pharyngeal HPV data remain limited; accordingly, there is an urgent need for comparative studies that connect nanoparticle architecture to measurable outcomes such as tonsillar deposition, epithelial uptake, immune activation, and local tolerability. Full article

Wheat (Triticum aestivum L.) is a major food crop that is severely affected by salt stress, resulting in significant yield losses. Glutamic acid decarboxylase (GAD) catalyzes the irreversible conversion of glutamic acid to γ-aminobutyric acid (GABA) and plays key roles in plant growth, development, and stress responses. However, the GAD gene family in hexaploid wheat and its role in salt tolerance remain poorly understood. In this study, the wheat GAD gene family was systematically identified. Genome-wide analysis revealed seven TaGAD genes with 19 gene copies. A Ka/Ks ratio < 1 indicates strong evolutionary conservation of this family. All TaGAD proteins contain a conserved Glu-decarb-GAD domain with similar motif composition and structural organization. Promoter analysis showed enrichment of stress-responsive cis-elements. Expression profiling demonstrated tissue-specific patterns, with several TaGAD genes significantly induced under salt stress. CRISPR/Cas9-mediated knockout of TaGAD1 led to markedly reduced salt tolerance, accompanied by decreased GABA content and GAD activity, reduced activities of antioxidant enzymes (SOD, POD, and CAT), and excessive accumulation of reactive oxygen species (ROS). These results demonstrate that TaGAD1 positively regulates salt tolerance in wheat through GABA-mediated ROS homeostasis. This study provides a systematic characterization of the wheat TaGAD gene family in the context of salt stress, laying a theoretical foundation for understanding GABA-mediated tolerance mechanisms and identifying TaGAD1 as a potential molecular breeding target for improving salt tolerance in wheat. Full article

Background: Live biotherapeutic products (LBPs) require robust genetic stability and effective biocontainment to support safe clinical translation and regulatory acceptance. Aim: This study presents a single-step chromosomal engineering strategy that integrates auxotrophy-mediated biocontainment with therapeutic gene insertion to support regulatory-oriented live biotherapeutic chassis design. Methods: A no-SCAR genome-editing approach combining CRISPR/Cas9 and λ-Red recombineering was used to generate an Escherichia coli MG1655 ΔilvC::hlyA strain by replacing ilvC with the hlyA gene encoding listeriolysin O. Chromosomal and episomal expression systems were compared for auxotrophy, growth, haemolytic activity, plasmid stability, and intracellular DNA delivery to RAW 264.7 macrophages. Results: Auxotrophy was successfully established and restored by branched-chain amino acid supplementation. Chromosomal integration preserved haemolytic activity and bacterial growth while improving long-term genetic stability and plasmid maintenance compared with episomal expression. Both systems supported intracellular DNA delivery, whereas the chromosomal construct showed improved host-cell preservation under higher bacterial challenge. Conclusions: This proof-of-concept study supports the feasibility of using a single-step chromosomal engineering strategy to combine intrinsic biocontainment with therapeutic-gene integration in an engineered bacterial chassis. Full article

Background: Providencia stuartii is an opportunistic pathogen associated with multidrug resistance. However, its occurrence, genomic characteristics, antimicrobial resistance profiles, and pathogenic potential in cattle-associated isolates remain poorly understood. Methods: A P. stuartii strain was isolated from a rectal swab of a diarrheic beef cattle individual during bacterial investigation on a commercial farm in Anhui Province, China. The strain was identified by Gram staining, biochemical tests, 16S rRNA sequencing, and whole-genome sequencing. Antimicrobial susceptibility was assessed using the Kirby–Bauer method, and resistance and virulence genes were detected by PCR and genomic analysis. Pathogenicity was evaluated in a murine infection model. Results: The isolate was confirmed as P. stuartii, with a 4.22 Mb genome and 41.28% GC content. It showed multidrug resistance, including resistance to eight antimicrobial agents. Genomic analysis revealed multiple resistance and virulence determinants, genomic islands, prophages, and CRISPR regions. Under high-dose intraperitoneal challenge conditions, the isolate caused dose-dependent mortality, systemic recovery from major organs, and histopathological lesions in mice. Conclusions: This study reports the genomic and antimicrobial resistance characteristics of a multidrug-resistant P. stuartii strain recovered from a rectal swab of diarrheic beef cattle in China. High-dose intraperitoneal inoculation demonstrated that the isolate possessed pathogenic potential in mice under experimental conditions. However, the murine model did not reproduce natural exposure in cattle, and the findings do not establish bovine tissue invasion, clinical pathogenicity, or a causal role in diarrhea. Further epidemiological investigations and cattle-relevant experimental studies are required to determine the clinical significance of this organism in bovine populations. Full article

During development, neurexin-2 (NRXN2) is a cell adhesion molecule localized to presynaptic terminals as well as axonal shafts and immature neurites, where it participates in the regulation of neuronal cell morphogenesis. Given its critical role in early neuronal development, NRXN2 is considered a susceptibility gene product for neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) and intellectual disability (ID). However, the intracellular signaling mechanisms linking NRXN2 deficiency to abnormal neuronal cell morphology remain unclear. Herein, we investigated the molecular basis of excessive cell morphogenesis induced by the knockdown of NRXN2 using the N1E-115 cell line, a model of neuronal morphogenesis characterized by neurite outgrowth. Silencing NRXN2 using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13 system resulted in a marked enhancement of process elongation. Mechanistically, we found that Girdin (also called GIV or CCDC88A), a non-receptor guanine nucleotide exchange factor for heterotrimeric G proteins, can mediate the excessive process length phenotype. Transfection of either the regulator of G protein signaling (RGS) domain of RGS3, a GTPase-activating protein for G proteins, or the G protein-binding domain of engulfment and cell motility 1 (ELMO1) rescued the excessive process formation. Similar results were obtained in primary cortical neurons. In addition, these interventions normalized downstream Rac1 activity in cells. Together, our findings elucidate Girdin signaling as a mediator of excessive neuronal process formation following NRXN2 knockdown, providing mechanistic insight into how the loss of function of NRXN2 leads to aberrant cell morphogenesis at least at the molecular and cellular levels. These results suggest that signaling through Girdin may contribute to the morphological abnormalities associated with NRXN2-related neurodevelopmental disorders. Full article

The symbiotic association between soybean and arbuscular mycorrhizal (AM) fungi enhances phosphorus acquisition, improving crop yield and quality. However, the molecular mechanisms underlying nutrient exchange in this symbiosis remain poorly understood. We performed transcriptomic profiling of Glycine max cv. Williams 82 roots across three time points during colonization by Rhizophagus irregularis BEG141, revealing distinct transcriptional reprogramming between mycorrhizal and non-mycorrhizal roots. Differentially expressed genes (DEGs) were significantly enriched in pathways related to fatty acid biosynthesis, carbon metabolism, and redox homeostasis. Time-course comparisons further identified DEGs associated with transcriptional regulation, biosynthetic processes, and nitrogen metabolism. Notably, five GDSL lipase genes (GmGELP6, GmGELP29, GmGELP140, GmGELP141, and GmGELP151) exhibited AM-associated expression patterns and were associated with AM-induced lipid-related transcriptional programs. CRISPR/Cas9-mediated disruption of these genes in soybean transgenic hairy roots altered arbuscule development and was associated with reduced expression of mycorrhiza-inducible fatty acid metabolism genes, suggesting that these genes are associated with normal AM colonization and arbuscule development. This study provides a transcriptomic resource and identifies AM-induced GmGELP candidate genes associated with normal soybean–AM symbiosis for future functional and mechanistic studies. Full article

The origin of life is commonly framed within the RNA World hypothesis, yet increasing evidence suggests that early evolution may have involved a broader diversity of hereditary systems than those preserved in modern biology. Alternative informational polymers, compositional inheritance, autocatalytic networks, and compartmentalized molecular communities have all been proposed as mechanisms that may have preceded modern genome-based heredity. Building upon these concepts, we propose that the fundamental unit of early evolution may have been neither the gene nor the organism, but persistent molecular networks capable of maintaining organizational continuity through time. We define Stable Molecular Networks (SMNs) as interacting systems of informational polymers, catalytic oligomers, metabolites, compartments, and environmental feedback processes that maintain continuity despite continual molecular turnover. We further propose that compartmentalized SMNs could form molecular ecosystems termed Precellular Polymer Consortia (PPCs), in which heredity emerges from network organization rather than genome replication alone. Within PPCs, selective interactions, molecular memory, ecological feedback, and distributed information exchange may generate lineage-like evolutionary processes that we term precellular speciation. To connect these concepts to extant biology, we examine archaeal and bacterial systems that preserve organizational principles potentially relevant to early evolution, including CRISPR-Cas adaptive memory systems, ancient RNA-based molecular machines such as the ribosome and RNase P, hydrothermal vent archaea, chemolithotrophic microorganisms, syntrophic consortia, and complex microbial communities. Although these systems are not direct descendants of precellular networks, they provide experimentally accessible examples of molecular memory, distributed information processing, protometabolism, ecological cooperation, and system-level organization. Finally, we propose PLURIBUS (Planetary Liquid Universal Polymer Identification By Ultrasensitive Sequencing) as a framework for discovering noncanonical informational polymers, novel RNA modifications, and alternative hereditary systems within Earth’s microbial biosphere, particularly among poorly characterized archaeal and extremophile communities. Knowledge gained from these environments may ultimately support the development of more general life-detection strategies for planetary exploration. Together, these perspectives suggest that extant microbial ecosystems provide valuable experimental windows into evolutionary processes that may have preceded modern cells, genomes, and the Last Universal Common Ancestor (LUCA). Full article