Search Results (1,293)

Bacterial persistence, a non-heritable high-antibiotic-tolerance phenotype, is a key driver of recurrent clinical infections and antibiotic treatment failure. The pheromone-responsive pCF10 plasmid in Enterococcus faecalis (E. faecalis) mediates antibiotic resistance gene dissemination, but its role in bacterial persister formation remains unclear. This study systematically investigated the regulatory role of pheromone cCF10 in the persister phenotype of pCF10-carrying E. faecalis and its underlying molecular mechanisms. We confirmed that cCF10 enhanced persistence against levofloxacin in OG1RF (pCF10), with the persister frequency increasing from 0.291% to 16.466% upon treatment. Transcriptomic analysis revealed that cCF10 activated the (p)ppGpp-mediated stringent response and downregulated the expression of genes associated with energy-intensive pathways, including those involved in DNA repair, protein folding, and respiration. Concurrently, cCF10 enhanced the expression of genes related to biofilm formation and cell lysis resistance and downregulated components of its own sensing and uptake systems. These findings demonstrate that cCF10 induces transcriptional reprogramming associated with increased persister formation in E. faecalis carrying the pCF10 plasmid and identify potential targets within the stringent response and associated metabolic pathways for the development of anti-persister strategies. Full article

Hyperbranched poly(β-amino ester) (HPAE) is identified as a unique non-viral carrier capable of sustaining high-efficiency transfection under elevated plasmid concentrations, overcoming the aggregation and toxicity limitations of conventional lipid and PEI reagents. We demonstrate that transfection enhancement is driven by concentration-dependent synergism between membrane accumulation and endosomal escape. Guided by this mechanism, a half-volume transfection strategy was established to transiently elevate plasmid concentration without compromising cell viability, enabling superior lentivirus yield and purity. These findings define plasmid concentration as a previously overlooked regulatory axis in nanoparticle-mediated gene delivery and position HPAE as a high-performance platform for scalable therapeutic vector production. Full article

In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 10⁰ copies/μL for H. influenzae and L. monocytogenes, and 10¹ copies/μL for C. neoformans using recombinant plasmids, as well as 10⁻⁷ to 10⁻⁶ ng/μL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (κ = 0.861, p < 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted. Full article

Feline calicivirus (FCV) is a major pathogen that threatens feline health worldwide. Its global prevalence, extensive genetic variability, and limited cross-protection among strains present significant challenges for vaccine development. In this study, an infectious clone of the FCV-GDJM202201 strain was constructed using the eukaryotic expression plasmid pcDNA3.1 under the control of the cytomegalovirus (CMV) promoter. The rescued virus, rGDJM-A4822T, exhibited growth kinetics comparable to those of the parental strain in vitro. Subsequently, two recombinant viruses, rGDJM-VP1_JL and rGDJM-VP1_SH, were generated by replacing the VP1 gene in the GDJM202201 backbone with those from heterologous FCV strains. Notably, these recombinant viruses exhibited reduced viral titers compared to rGDJM-A4822T. Finally, neutralization assays revealed differential neutralizing antibody titers among the recombinant FCVs, with rGDJM-A4822T inducing higher neutralizing antibody titers and cross-neutralizing activity. Collectively, this study establishes an FCV infectious clone that can be used to rescue recombinant viruses carrying heterologous VP1 proteins and to evaluate neutralizing antibody responses. Full article

Lettuce (Lactuca sativa L.) is an important leafy vegetable crop, yet the efficiency and reliability of genome editing platforms in lettuce remain limited, particularly for precision base editing applications. In this study, we established an optimized PEG-mediated protoplast transformation system for lettuce through systematic evaluation of key parameters, including protoplast density, incubation time, plasmid size, and transformation method. Under optimized conditions, a maximum transient transformation efficiency of up to 81% was achieved. Using this optimized protoplast platform, we comparatively evaluated the performance of three single-base editing systems—adenosine base editor (ABE), glycosylase-based guanine base editor (gGBE), and rice alkylpurine DNA glycosylase-mediated A-to-K base editor (rAKBE)—targeting the LsALS gene, encoding acetolactate synthetase as a herbicide target with great value in weed control. Among the tested editors, ABE exhibited the highest A-to-G editing efficiency, reaching 9.3%. In contrast, gGBE and rAKBE showed lower editing efficiencies. Together, this study established a robust and reproducible protoplast-based platform for transient genome editing in lettuce and provides a practical framework for the rapid evaluation of base editing tools and target sites, firstly for gGBE and rAKBE evaluation in lettuce. The optimized system facilitates functional genomics studies and supports the development of precision breeding strategies in lettuce. Full article

Thirty 2-methyl-quinazolinone fussed hydroxamic acids (3-OH) and their 3-OEt and 3-OBn derivatives were evaluated for their affinity towards calf-thymus (CT) DNA using UV-vis absorption, viscosity and fluorescence spectroscopy. DNA photocleavage activity was assessed by incubating the compounds with plasmid DNA followed by UV-A and visible light irradiation, which enabled identification of the most potent derivatives active at concentrations of 100 nΜ and 10 μΜ, respectively. Mechanistic studies on the most active compounds indicated the formation of oxygen radical species and a decrease in efficiency under argon. Measurements of singlet oxygen release verified these findings. Molecular docking studies provided further insight into the interactions between the compounds and DNA. UV-A irradiation of the most potent DNA photocleavers in three cell lines, two malignant melanoma lines (A375 and COLO-800) and the immortalized keratinocyte line HaCaT, identified three derivatives that, at a concentration up to 10 μΜ, reduced cell viability by approximately 50%. Taken together, these results indicate that these 2-methylquinazolinone-based hydroxamic acid derivatives are promising candidates for the development of photodynamic therapy agents. Full article

Microplastics have emerged as a relatively new type of pollutant and have attracted significant global attention. This study focuses on toxicology of microplastics in ambient PM_2.5 and road dustfall in Beijing. It utilizes the Plasmid Scission Assay to toxicologically evaluate the oxidative damage capacity of microplastics as a component of PM_2.5. The Pollution Load Index (PLI) method, based on the mass concentration of microplastics in ambient air, was employed to assess the ecological risk of atmospheric dustfall microplastics in Beijing. The results showed that both standard microplastic samples and mixed samples of microplastics with ambient PM_2.5 exhibited a dose–response relationship in DNA damage rates. At the same dose, microplastic samples with smaller particle sizes have a higher DNA damage rate. Based on the PLI results, most road dustfall microplastics in Beijing exhibit significant spatial variation. Analysis of road dustfall along the east–west main road across Beijing’s urban area revealed that microplastic pollution levels are higher in the eastern zone than in the western zone. Comparisons of pollution levels across functional areas in Beijing showed that university areas > residential areas > industrial areas > commercial areas > agricultural areas. In vertically collected samples, higher elevations (PLI_13.6m = 3.54) exhibit greater pollution levels than lower (PLI_1.5m = 1), which warrants special attention. These findings highlight the complex relationship between atmospheric microplastic accumulation and their oxidative capacity, providing essential insights for the design of targeted emission reduction strategies. Full article

The COVID-19 pandemic highlighted both the transformative potential of mRNA vaccines and the structural challenges associated with their supply chains. Unlike traditional vaccine platforms, mRNA vaccines depend on highly specialized raw materials, including plasmid DNA (pDNA), nucleotides, enzymes, and lipid nanoparticles (LNP), that are produced by a limited number of global suppliers. These dependencies, combined with platform-specific manufacturing processes and stringent cold chain requirements, introduce vulnerabilities across production, distribution, and regulatory oversight. This narrative review examines the distinctive features of mRNA vaccine supply chains and identifies key challenges and opportunities across three interconnected domains: manufacturing systems, logistics and distribution, and regulatory governance. Drawing on literature published between January 2021 and March 2026, the review synthesizes evidence on supply chain bottlenecks revealed during the COVID-19 pandemic, including upstream raw-material dependencies, limitations in manufacturing scale-up, cold chain constraints, and regulatory fragmentation. Particular attention is given to the implications of these challenges for low- and middle-income countries, where infrastructure, technical capacity, and regulatory resources may limit participation in mRNA vaccine production and deployment. The review also highlights emerging strategies to strengthen supply chain resilience, including diversification of input suppliers, development of regional manufacturing hubs, improvements in vaccine thermostability, regulatory harmonization initiatives, and the use of digital technologies for supply chain management. By integrating insights from manufacturing, logistics, and regulatory perspectives, this study contributes to a better understanding of the structural characteristics shaping mRNA vaccine supply chains and identifies priority areas for strengthening global preparedness for future health emergencies. Full article

The moderate pathogenicity coupled with high host susceptibility of Eimeria maxima has precipitated substantial economic losses in the poultry industry. Addressing challenges such as emerging drug resistance underscores the imperative for innovative vaccine strategies. This study developed a novel DNA vaccine to solve this challenge by fusing E. maxima elongation factor-1α (EmEF1α) with chicken chemokine XCL1 (ChXCL1) in the pVAX1 vector. The recombinant plasmid, designated pVAX1-ChXCL1-EmEF1α, was successfully constructed and confirmed to express the ChXCL1-EmEF1α fusion protein in vitro. Immunization of chickens with this DNA vaccine elicited a robust and balanced immune response, characterized by significantly increased proportions of CD4⁺ (11.76%) and CD8⁺ (5.58%) T lymphocytes, elevated levels of Th1-associated cytokines (IFN-γ and IL-12), and strong antigen-specific IgG and IgA antibody responses. Following experimental challenge with E. maxima, vaccinated birds exhibited substantial protection: a 66.4% reduction in oocyst shedding, a 71.7% improvement in relative weight gain, marked attenuation of intestinal lesions, and an anticoccidial index (ACI) of 170. These findings demonstrate that the ChXCL1-EmEF1α DNA vaccine effectively enhances both cellular and humoral immunity. Collectively, this study validates ChXCL1 as a potent molecular adjuvant and establishes the “antigen–adjuvant” fusion DNA platform as a promising strategy for developing next-generation vaccines against avian coccidiosis. Full article

Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies/μL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions. Full article

Gut dysbiosis, defined as a disruption in the structure or function of the intestinal microbiota, is increasingly recognized as a key contributor to inflammatory, metabolic, and neuropsychiatric diseases. Conventional interventions such as broad-spectrum antibiotics, generic probiotics, and fecal microbiota transplantation (FMT) often show limited and inconsistent efficacy because they lack specificity, durability, and robust safety controls. In contrast, recent advances in DNA-based technologies are reshaping the therapeutic landscape by enabling targeted, programmable, and mechanistically informed modulation of the gut ecosystem. This review presents an integrated overview of three major domains driving this shift: CRISPR-based systems that selectively delete, silence, or reprogram microbial genes; synthetic biology-driven live therapeutics engineered to sense disease-associated cues and execute controlled responses; and metagenomics-informed strategies that tailor interventions to patient-specific microbial gene profiles and functional deficits. Additionally, we examine the continued evolution of FMT toward DNA-optimized workflows and defined microbial consortia that offer safer, more standardized alternatives to crude donor material. Across these domains, we discuss delivery platforms (including bacteriophages, conjugative plasmids, extracellular vesicles, and synthetic nanoparticles), and compare their efficiency, specificity, and scalability. We further highlight how DNA-guided interventions interface with host immunity—shaping Treg/Th17 balance, mucosal barrier function, and inflammatory signaling—while also analyzing ecological and evolutionary risks, biocontainment strategies, and regulatory classification gaps that will govern clinical translation. Together, these developments signal a transition from empirical microbiome manipulation to rational ecosystem engineering. DNA-guided therapies hold strong promise for precise and personalized management of gut-related diseases, but their success will depend on rigorous ecological risk assessment, long-term monitoring, and adaptive regulatory frameworks alongside continued technological innovation. Full article

Soybean (Glycine max) is relatively recalcitrant to genetic manipulations; hence, it is often interrogated with transient means such as virus-induced gene silencing (VIGS). We earlier modified cowpea severe mosaic virus (CPSMV) to develop a soybean-friendly VIGS system referred to as QUIN-FZ. Here we report additional calibrations of this system. We enhanced the intra-bacterial stability of plasmid QUIN, which contained a CPSMV RNA1 cDNA embedded with four introns, by adding a fifth intron, resulting in PENTIN. We separately upgraded the plasmid FZ, which contained a modified CPSMV RNA2 cDNA with a cloning site in the middle of the viral polyprotein, by creating another cloning site within the 3′ untranslated region, leading to ZY. We next used the new PENTIN-ZY system to investigate a putative soybean protein kinase designated QL18. Virus-mediated overexpression of two allelic, 147-amino-acid (aa) protein fragments, derived from two different QL18 orthologs, elicited drastically different responses in soybeans. While the fragment derived from soybean accession OX20-8 prevented the cognate virus from infecting top young leaves in at least 50% of inoculated seedlings, its allelic counterpart derived from soybean accession PI427105B elicited apical necrosis in 100% of soybean seedlings. By examining progeny viruses as well as viruses encoding chimeras of the two fragments, we identified more than a dozen mutations that abrogated these unique phenotypes. Our findings establish the PENTIN-ZY system as a versatile tool for overexpressing small proteins and protein fragments, accelerating their functional characterization. Full article

According to the World Health Organization, antibiotic research remains insufficient, emphasizing the urgent need for new active molecules, particularly against resistant bacteria. Based on known antibacterial scaffolds, new fluoroquinolone derivatives have been synthesized by our research group, including compound 7a, a difluoroboranyl-fluoroquinolone that previously demonstrated activity against sensitive strains. Methods: The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of compound 7a were determined against Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli. The selective development of ciprofloxacin-resistant S. aureus was induced by reseeding the isolate on seven consecutive days with an antibiotic concentration that was not capable of inhibiting its development. Pharmacokinetic and toxicological properties were predicted using SwissADME, Way2Drug, and molecular docking (AutoDock Vina). In vivo toxicity was evaluated in BALB/c mice through histopathological liver and kidney analysis and serum biochemical markers. The antibacterial efficacy of 7a (80 mg/kg/day) was assessed in a murine pneumonia model induced by ciprofloxacin-resistant S. aureus. DNA gyrase inhibition was confirmed through plasmid electrophoresis assays in E. coli DH5-α cells. Results: Compound 7a exhibited both MIC and MBC values of 0.25 μg/mL, while ciprofloxacin-resistant S. aureus strains did not exhibit a detectable MIC within the concentration range tested (up to 1024 μg/mL). In silico predictions revealed favorable ADME profiles, low toxicity, and strong interaction with DNA gyrase. In vivo, 7a showed no signs of hepatotoxicity or nephrotoxicity and effectively reduced pneumonic tissue to 1.99% in infected mice. Electrophoretic assays confirmed DNA gyrase inhibition consistent with the mechanism of fluoroquinolones. Conclusions: Compound 7a evidenced activity against ciprofloxacin-resistant S. aureus in vitro and reduced infection progression in vivo. It also displays favorable drug-like properties, low predicted toxicity, and DNA gyrase inhibition. Full article

Fecal microbiota are shaped by upstream digestive processes and reflect the outcome of host–microbe interactions, including the resistant microbial fraction that survives to be excreted. This is particularly crucial for assessing zoonotic risks and environmental contamination, as feces are the primary source of dissemination, which is considered an emerging One Health threat. Therefore, we conducted a pilot study to obtain the exploratory findings regarding the cattle GIT microbial composition, potential resistome, and their transmission drivers, such as plasmids, using metagenomic analysis from different districts in Khyber Pakhtunkhwa (KP) province, Pakistan. For this purpose, a total of 150 fecal samples (50 from each district) of healthy cattle were collected from various farms in Mardan (FC1), Peshawar (FC2), and Dera Ismail Khan (FC3) districts. Total DNA from each sample was extracted, pooled (FC1, FC2, and FC3), and sequenced via the Illumina platform. Bacteria were the highly abundant kingdom, while Pseudomonadota and Bacillota were dominant phyla in all samples. Caryophanon latum and Escherichia coli were highly abundant at the species level. A large resistome (40–49 genes), including critical genes, such as tet(X), bla_OXA-427, and plasmidomes (16–22), such as IncF, was detected in the samples. The prominence of certain commensal or opportunistic pathogens in the fecal microbiota may indicate the presence of sub-clinical gastrointestinal disruptions or disease that may affect cattle herds. The fecal resistome is extensive, identifying dairy cattle in these regions as important reservoirs for AMR genes capable of spreading via HGT. This pilot study establishes that the fecal microbiota of dairy cattle in this region are not merely a waste product but a complex ecosystem, rich in microbiota of One Health significance. Full article

Dogs, especially as pets but also an increasing number of stray dogs, share environments with humans, facilitating the transfer of antibiotic resistance genes (ARGs) between genetic compartments, with zoonotic and public health implications that must be addressed within One Health. In this cross-sectional comparative study, we explored the distribution of seven selected clinically relevant ARGs in both genomic DNA (gDNA) and plasmid DNA (pDNA), and the phenotypic resistance profile of the cultivable microbiota, between pet dogs (PeDs, n = 12) and free-roaming dogs (FRDs, n = 10) in Mexico. Tetracycline resistance genes (tetQ, tetW, and tetM) predominated in both compartments (40% to 100%), suggesting the presence of a core tetracycline-associated resistome. In contrast, plasmid-associated differences were group-specific: in pDNA cfxA was enriched in FRDs (90%) and tetK in PeDs (42%), whereas blaTEM-1 and ermC were absent in two dog populations. Cultivable bacteria from both groups exhibited phenotypic multidrug resistance, particularly by β-lactams, macrolides, lincosamides, and tetracyclines. FRDs also harbored pathogenic–zoonotic bacteria such as Yersinia enterocolitica, Campylobacter jejuni, and Enterococcus faecalis. Our findings indicated that FRDs and PeDs harbor substantial resistomes, with differences in plasmid-associated ARGs, revealing a transfer potential related to environmental exposure. Full article