Search Results (5,917)

Background/Objectives: Rapid, equipment-free molecular detection of bacterial pathogens at the point of care (POC) remains a critical challenge. Staphylococcus aureus is a leading cause of severe infections, necessitating simple and sensitive diagnostic tools. Methods: We developed an integrated assay combining helicase-dependent amplification (HDA) and rolling circle amplification (RCA) in a sequential ‘one-pot’ format. Asymmetric HDA generates short, single-stranded amplicons from S. aureus DNA, enabling specific padlock probe ligation and subsequent exponential RCA. For equipment-free visual detection, biotin-labeled nucleotides are incorporated during RCA, and products are captured on a silica membrane and detected using a streptavidin-HRP conjugate with 3,3′,5,5′-tetramethylbenzidine substrate, producing an unambiguous blue color. Results: The assay detected as few as 10¹ genome copies of S. aureus per reaction. Evaluation against a panel of nine non-target respiratory pathogens and human genomic DNA demonstrated 100% specificity, with no cross-reactivity. The entire procedure is performed isothermally at 65 °C in a single tube with a total assay time of approximately 90 min. Conclusions: This ‘one-pot’ HDA-RCA colorimetric assay combines high sensitivity and specificity for S. aureus in a user-friendly, almost equipment-free format. Its simplicity and robust visual readout make it a promising tool for POC diagnostics in resource-limited settings, enabling rapid clinical decisions without specialized instrumentation. 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

Cultivated lettuce (Lactuca sativa L.) is a major leafy crop and an emerging model for functional genomics within the Asteraceae family, supported by high-quality reference genomes and efficient transformation systems. Although CRISPR/Cas technology offers powerful opportunities for crop improvement, editing efficiency depends on optimized construct architecture and reliable guide RNA (gRNA) validation. However, a rapid platform for evaluating CRISPR reagents in lettuce is still lacking. Here, we developed an efficient hairyroot-based system to accelerate CRISPR/Cas genome editing optimization in L. sativa. Four Agrobacterium rhizogenes strains were compared for hairy root induction in two cultivars, ‘Saladin’ and ‘Osiride’, identifying strain ATCC15834 as the most effective based on transformation frequency and root production. Using this platform, we evaluated multiple CRISPR construct configurations, including alternative promoters for nuclease and gRNA expression. A plant-derived promoter combined with At-pU6-26 variant significantly improved editing efficiency. As a proof of concept, we targeted LsHB2, the putative ortholog of Arabidopsis thaliana ATHB2, a key regulator of the shade avoidance response using SpCas9, SaCas9, and LbCas12a nucleases. The system enabled rapid genotyping and quantitative indel profiling. Overall, this workflow provides a robust framework for efficient guide selection and construct optimization in lettuce genome editing. Full article

Artificial intelligence (AI) is increasingly used to support tuberculosis (TB) screening and diagnosis, particularly through computer-aided detection (CAD) applied to chest radiography (CXR). However, the programmatic value of AI depends not only on diagnostic accuracy but also on implementation context, threshold calibration, and integration into diagnostic pathways. We conducted a narrative, state-of-the-art review of AI applications across the TB diagnosis pathway. Evidence was synthesized from World Health Organization policy documents, independent validation initiatives, and peer-reviewed studies published between 2010 and 2026, with a structured selection process aligned with PRISMA principles. CAD for CXR is the most mature AI application and is recommended by WHO for TB screening and triage among individuals aged ≥15 years in specific contexts. Across studies, CAD-CXR demonstrates sensitivity comparable to human readers, although performance varies by product, population, and imaging conditions, necessitating local threshold calibration. Evidence from implementation studies suggests improvements in screening efficiency and potential cost-effectiveness in high-burden settings. Other AI modalities, including computed tomography (CT)-based imaging analysis, point-of-care ultrasound interpretation, cough or stethoscope sound analysis, clinical risk models, and genomic resistance prediction show promising but heterogeneous results, with most requiring further independent validation and prospective evaluation. AI has the potential to strengthen TB screening and diagnostic pathways, but its impact depends on integration into health systems and evaluated using patient- and program-level outcomes rather than accuracy alone. A differentiated approach is needed, with responsible scale-up of policy-endorsed tools alongside rigorous evaluation of emerging technologies to support effective and equitable TB care. Full article

Lactiplantibacillus plantarum LP28 (LP28), isolated from traditional Taiwanese dried tofu, has been demonstrated to have substantial probiotic potential because it increases the production of short-chain fatty acids (SCFAs) and strengthens anti-inflammatory responses. In this study, the safety of LP28 was assessed using both in vitro and in vivo approaches, including whole-genome sequence analysis, the Ames bacterial reverse mutation assay, a chromosomal aberration test, a rodent peripheral blood micronucleus test, a 28-day subacute oral toxicity assay, and an assessment of hemolytic activity. In vitro phenotypic evaluation revealed that LP28 exhibited no hemolytic activity and was susceptible to all the tested antibiotics except kanamycin. In vivo assessments revealed no significant alterations in reticulocyte counts or micronuclei incidence in ICR mice, and SD rats exhibited no subacute toxicity at an oral LP28 dosage of 2000 mg/kg body weight/day for 28 days. Moreover, a whole-genome sequence analysis of LP28 revealed the absence of antimicrobial resistance genes, harmful virulence factors, and genes associated with biogenic amine synthesis. Additionally, the presence of genes involved in stress responses (e.g., acid, bile salt, heat, osmotic, and oxidative stresses) and adhesion-related genes was confirmed. Furthermore, LP28 contains six genes (plnA, plnE, plnF, plnJ, plnK, and plnN) that encode bacteriocin precursor peptides, suggesting the potential for enhanced probiotic effects through the production of antimicrobial plantaricins. These findings highlight the potential of LP28 as a safe and effective probiotic for human consumption. Full article

Genetically modified (GM) plants have been commercially grown for 30 years, and their acceptance depends on a thorough risk assessment. Environmental Risk Assessment (ERA) evaluates potential impacts of releasing GM plants into the environment, whether through cultivation or import for food, feed, and processing. A key component is assessing potential gene flow to crop wild relatives or non-GM crops. For gene flow to significantly affect the environment, transferred genes must provide a selective advantage. Since most GM plants are engineered for herbicide tolerance, insect resistance, or stacked traits, evaluating such advantages is relatively straightforward. New genomic techniques (NGTs) can generate plants with a wider range of traits, including tolerance to biotic and abiotic stress. Although still considered GM in the EU, their genomic changes can complicate detection, identification, and ERA, especially when such traits may offer advantages under stress conditions. This scoping review focuses on gene flow in two crops: oilseed rape (canola) (Brassica napus L.) and potato (Solanum tuberosum L.). In canola, transgene movement can increase weediness, fitness, herbicide resistance, or genetic diversity in feral or related populations. Gene flow in potato is less studied, with concerns centered on contamination risks in the Andean diversity center. Limited data exist for NGT plants, though many are expected to resemble conventionally bred varieties, suggesting comparable environmental impacts. Full article

The genus Lasiodiplodia includes numerous plant-pathogenic species whose delimitation is complicated by overlapping morphological traits and limited resolution of common genetic markers. Lasiodiplodia brasiliensis was described as a species closely related to L. theobromae; however, its taxonomic status remains controversial. In this study, we re-evaluated the species boundaries between L. theobromae and L. brasiliensis using an integrative approach that combined multilocus and genome-scale phylogenetic analyses with morphological comparisons. Multilocus phylogenetic analyses based on ITS, tef1-α, tub2, and rpb2 revealed an unresolved relationship between the two taxa. The L. theobromae clade had low bootstrap support, whereas the ancestral node connecting both species had high support. In contrast, genome-scale phylogenetic analysis using hundreds of single-copy orthologous genes strongly supported a single monophyletic clade encompassing isolates assigned to both L. theobromae and L. brasiliensis. Morphological analyses further revealed that conidial dimensions and other diagnostic characteristics largely overlapped between the two taxa, rendering them unreliable criteria for species separation. Considering the combined molecular and morphological evidence, our results support treating L. brasiliensis as a synonym of L. theobromae. Clarifying species boundaries within this group helps stabilize the taxonomy of Lasiodiplodia and provides a reliable foundation for accurate pathogen identification and disease management. Full article

Foodborne pathogens such as Klebsiella aerogenes pose a threat to food safety, highlighting the need for rapid, reliable detection methods amid rising contamination risks in production chains. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated to detect the histidine decarboxylase (HDC) gene of K. aerogenes. The assay was optimized for specificity and sensitivity, tested on pure bacterial genomic DNA and artificially contaminated food matrices (vegetables and meats), and evaluated against real-time PCR (qPCR). To evaluate performance under different DNA quality conditions and simulate laboratory versus on-site workflows, two extraction approaches were compared: a standard laboratory protocol yielding high-purity DNA and a crude extraction method producing low-purity DNA, mimicking the presence of inhibitors commonly encountered in routine analysis and enabling practical on-site detection where commercial kits are not feasible. The developed LAMP assay achieved maximum specificity with no cross-reactivity to related species, limits of detection of 240 fg/reaction for pure bacterial DNA and 0.4 pg/µL in K. aerogenes artificially contaminated food samples, and a reaction time under 30 min—outperforming real-time PCR in speed and robustness. This cost-effective method provides a scalable tool for near-real-time monitoring of K. aerogenes in food production, enhancing safety and enabling early outbreak detection. Full article

Synthetic dyes, such as methylene blue (MB), constitute a major category of environmental pollutants due to their toxicity, persistence, and resistance to standard treatment methods. In this study, Bacillus cereus BC WW Saida was isolated from the heavily polluted Saida dumpsite in Lebanon and evaluated for its MB degradation efficiency. The isolate was identified through whole-genome sequencing, which revealed the presence of key enzymatic systems involved in azo dye degradation. Under optimized conditions, the strain achieved 82% decolorization, as determined by optical density measurements using a microplate reader. The process was further examined using High-Performance Liquid Chromatography (HPLC), which revealed a significant reduction in the original dye peak and the emergence of new intermediate products. These findings suggest the strong biodegradation capability of B. cereus BC WW Saida isolated from contaminated environments and highlight its potential application in the eco-friendly treatment of azo dye-contaminated wastewater. Full article

Objectives: Platinum resistance remains a critical bottleneck in ovarian cancer management, yet reliable pre-treatment predictive tools are lacking. Existing markers like the platinum-free interval are retrospective, while genomic profiling is often cost-prohibitive. This study aimed to develop an accessible, machine learning-based dynamic weighted fusion (DWF) model using routine laboratory data to provide bidirectional risk stratification, particularly to reliably rule out platinum resistance before treatment initiation. Methods: In this retrospective study (2019–2023), seventy baseline clinical features were collected to differentiate platinum-resistant from platinum-sensitive ovarian cancer patients. We developed a DWF framework that dynamically integrates the top-performing classifiers from a library of 168 algorithms (combining 14 feature selection and 12 machine learning methods). Class imbalance was addressed via oversampling, and model efficacy was evaluated using area under the curve (AUC), accuracy, sensitivity, and specificity. Results: The DWF model achieved a robust AUC of 0.760 (95% CI: 0.683–0.837), outperforming all individual base classifiers. Subgroup analysis demonstrated highly consistent overall discrimination across initial treatment strategies (AUC of 0.755 for primary debulking surgery and 0.761 for neoadjuvant chemotherapy). Feature interpretation highlighted that resistance is driven by synergistic dysregulation of systemic inflammation and hypercoagulability, rather than single biomarkers. Conclusions: The proposed DWF model effectively leverages low-cost, standardized clinical data to serve as a robust bidirectional stratification tool. Its exceptional ability to rule out resistance provides clinicians with the evidence-based confidence to proceed with standard therapies, while its high-risk alerts identify candidates for early therapeutic adjustments and enhanced surveillance in ovarian cancer care. Full article

Background: Ambient air pollution, particularly particulate matter (PM), has been linked to metabolic disorders, including diabetes. We evaluated associations between long-term exposure to coarse particulate matter (PM₁₀) and fine particulate matter (PM_2.5) and glycated hemoglobin (HbA1c) levels in a Korean population and assessed whether specific subgroups exhibited heightened susceptibility. Methods: We analyzed 6940 participants without diabetes from the Korean Genome and Epidemiology Study (KoGES) Ansan-Ansung cohort. Participants contributed 35,395 observations across a mean follow-up of 5.1 visits (2005–2017). Linear mixed models estimated associations between PM exposure and HbA1c while adjusting for covariates, including body mass index (BMI), time, and region. Subgroup analyses stratified by sex, age, BMI, region, education, smoking status, drinking status, and exercise. Results: Higher long-term PM₁₀ exposure was associated with elevated HbA1c (β = 0.0347 per interquartile range [IQR] increase of 9.48 μg/m³; 95% CI: 0.0220, 0.0473; p < 0.001). PM_2.5 showed a comparable positive association (β = 0.0166 per IQR of 8.67 μg/m³; 95% CI: 0.0010, 0.0321; p = 0.037). Associations were stronger among older adults (≥60 years: β = 0.0789 vs. <60 years: β = 0.0210; p-interaction < 0.001), rural Ansung residents (β = 0.0963 vs. Ansan: β = 0.0398; p-interaction < 0.001), participants with lower educational attainment (≤middle school: β = 0.0637; p-interaction < 0.001), and never smokers (β = 0.0455; p-interaction = 0.035). Conclusions: Among nondiabetic Korean adults, long-term PM₁₀ exposure was associated with higher HbA1c; PM_2.5 demonstrated a similar positive association. Associations were more pronounced among older adults, rural residents, individuals with lower educational attainment, and never smokers. These findings support ambient air pollution as an environmental contributor to subclinical glycemic changes and underscore the need for targeted public health strategies for vulnerable populations. Full article

In the post-antibiotic era, the Bacillus-based direct-fed beneficial microorganisms are emerging as a cornerstone for sustainable animal farming. This study aimed to screen and evaluate Bacillus strains with probiotic potential for use as feed additives. A total of 394 Bacillus strains were initially screened based on their extracellular enzyme production (cellulase, protease, and amylase) and antibacterial activities against Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Two strains, Bacillus velezensis FJAT-10508 and FJAT-13563, were selected and subsequently subjected to in vitro probiotic characterization, safety assessment, and whole-genome analysis. The results demonstrated that both strains exhibited α-hemolysis, acceptable antibiotic susceptibility profiles, absence of invasion and cytotoxicity effect on the Caco-2 cells, and no mobile virulence or antibiotic resistance genes, indicating their safety as probiotic candidates. High endospore-forming efficiencies (72.4–90.8%), strong auto-aggregation (74–85%) and co-aggregation abilities (52–82%) were observed. In addition, both strains showed considerable tolerance to simulated gastrointestinal conditions, with vegetative cell and endospore survival rates of 28.33–38.33% and 85–89.67% at pH 2.0, and 38.33–43.33% and 90.33–96.33% in 0.3% bile salts, respectively. Overall, B. velezensis FJAT-10508 and FJAT-13563 demonstrated robust in vitro probiotic properties, supporting their potential application as reliable Bacillus-based feed additives. Full article

The genus Listeria comprises diverse bacteria with significant public health relevance, particularly Listeria monocytogenes. A comparative genomic analysis of ten representative Listeria species was conducted using 33 high-quality genome assemblies to investigate core and accessory genome dynamics and identify candidate diagnostic loci. Pangenome reconstruction was performed using the Roary Integer Linear Programming Bacterial Annotation Pipeline (RIBAP) to classify core, soft-core, and accessory genes, while average nucleotide identity (ANI) analysis assessed genomic relatedness across thresholds of 60–95%. Functional annotation of core and species-specific genes was conducted using Genome Annotation and Information Analysis (GAIA). Core genes were highly conserved and associated with essential cellular functions, whereas the accessory genome contributed to species-level diversification and ecological adaptation. Candidate molecular markers were derived from accessory genes and evaluated based on presence/absence across genomes, retaining loci present in ≥80% of target strains and absent in non-target strains. Experimental validation of selected primers was performed using two L. monocytogenes reference strains (ATCC 19117 and ATCC BAA-679) with conventional PCR and gel electrophoresis to confirm expected amplicon sizes and specificity. These findings establish a genome-informed, specificity-driven framework for marker development and highlight the accessory genome as a valuable source of diagnostic loci, supporting accurate detection, epidemiological surveillance, and food safety monitoring. Full article

Members of the genus Brucella are bacterial pathogens of global importance, and their increasing detection in marine mammals has raised concerns for wildlife conservation and public health. In this study, we evaluated the biological and pathogenic characteristics of two Brucella sp. sequence type 27 (ST27) isolates obtained from a dwarf sperm whale (Kogia sima). We compared them with terrestrial and marine Brucella reference strains. We assessed resistance to polymyxin B and human serum complement, intracellular infection dynamics in HeLa epithelial cells, persistence in a murine model, and associated hematological and histopathological changes, and analyzed lipopolysaccharide (LPS) profiles. The Kogia isolates exhibited resistance to polymyxin B and serum complement, comparable to that of B. abortus 2308W and marine mammal Brucella strains. In HeLa cells, the isolates displayed distinct, strain-specific intracellular infection dynamics. In the murine model, both isolates persisted in the spleen and induced granulomatous lesions. However, splenic bacterial loads and histopathological scores were generally lower than those observed with B. abortus 2308W, which exhibited the highest virulence among the strains evaluated. Hematological alterations associated with Kogia isolates were also less pronounced than those induced by B. abortus 2308W, indicating an intermediate and strain-dependent virulence phenotype without evidence of enhanced virulence relative to the terrestrial reference strain. Western blot analyses showed that Brucella sp. ST27 isolates were not recognized by anti-B. abortus or anti-O-antigen monoclonal antibodies, while exhibiting a distinct recognition pattern with anti-B. canis serum, indicating differences in surface antigen composition. Comparative whole-genome analysis identified a limited number of isolate-specific variants affecting coding and intergenic regions. Collectively, these findings highlight phenotypic and genetic features of Brucella sp. ST27 from Kogia sima, which distinguishes it from other marine and terrestrial Brucella strains and supports further investigation into its biological behavior and potential public health relevance. Full article

Background: Kleefstra syndrome (KS) is a rare neurodevelopmental disorder caused by haploinsufficiency of EHMT1; it is characterized by global developmental delay, intellectual disability, hypotonia, distinctive facial features, and variable congenital anomalies. Autistic features, behavioral abnormalities and severe speech impairment are frequently reported. However, molecularly confirmed cases of KS from Africa remain extremely limited, largely due to restricted access to genomic diagnostic infrastructures. Methods: We describe a 15-month-old patient from Rwanda presenting with neonatal hypotonia, global developmental delay, short stature, and characteristic dysmorphic facial features. Comprehensive clinical evaluation was performed, followed by trio-based exome sequencing to identify the underlying genetic cause of this neurodevelopmental disorder. Results: Exome sequencing identified a de novo heterozygous frameshift variant in EHMT1 (NM_024757.5: c.2871dup; p. Phe958Leufs*219), confirming the diagnosis of KS. Conclusions: This report presents the first molecularly confirmed case of KS in Rwanda. It highlights additional clinical features like bilateral 5th toe clinodactyly, short stature and absence of obesity in KS. There is a need to further delineate the study of EHMT1 and investigate the natural history of KS across different populations for optimal patient management and to reduce diagnostic odyssey. The diagnostic utility of exome sequencing for neurodevelopmental disorders needs to be strengthened, with strong emphasis on expanding genomic medicine to help diagnose rare diseases in resource-limited settings. Full article