Artificial sweeteners are widely used sugar substitutes, yet their effects on bacterial physiology and antimicrobial resistance dissemination remain poorly understood. This study evaluated the impact of the sucralose-based artificial sweetener Zerocal on bacterial growth, biofilm formation, and plasmid-mediated gene transfer in gut-associated opportunistic bacteria including extended-spectrum β-lactamase producing Escherichia coli, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. Bacterial growth was monitored spectrophotometrically, biofilm formation was quantified using a crystal violet assay, and conjugative plasmid transfer was assessed using donor-recipient co-culture experiments. Exposure to Zerocal at 0.5 mM and 1 mM did not significantly alter planktonic growth of any tested strain during 24 h incubation. In contrast, Zerocal markedly enhanced biofilm formation in a strain-dependent manner. K. pneumoniae exhibited the greatest increase, with biofilm biomass rising by approximately 260% at 0.5 mM and ~ 200% at 1 mM relative to controls. ESBL E. coli and E. faecalis showed increases of approximately 222% and ~ 150%, respectively, whereas no change was observed in E. coli. Zerocal also significantly enhanced conjugative plasmid transfer from K. pneumoniae to E. coli, increasing the number of transconjugants by 61.7% at 12 h compared with untreated controls. Co-exposure with 0.1 mM zinc sulfate reduced Zerocal-associated biofilm formation and partially attenuated conjugation. Together, these results suggest that sucralose-containing sweeteners may promote bacterial traits associated with persistence and antimicrobial resistance dissemination. These findings raise the possibility that widely consumed artificial sweeteners may unintentionally influence bacterial traits associated with persistence and antimicrobial resistance dissemination within the gut environment.

The CRISPR/Cas system has emerged as a transformative tool for nucleic acid detection, offering significant potential for point-of-care testing (POCT). However, translating CRISPR/Cas-based assays into practical POCT devices critically depends on the development of portable, sensitive, and user-friendly signal readout modalities. This review systematically compares four major readout modalities: fluorescence, electrochemical, colorimetric, and distance‑based readout, analyzing their mechanisms, analytical performance, and practical limitations. Key challenges, including sample preparation, amplification-free detection, multiplexing, and commercialization barriers, are critically assessed. Finally, future perspectives are proposed: integrating microfluidics with smartphone‑based readout, leveraging artificial intelligence and the Internet of Things for automated signal interpretation and cloud connectivity, and establishing regulatory pathways for clinical translation. This review aims to provide actionable insights for researchers developing next‑generation CRISPR diagnostics and to accelerate the transition from laboratory prototypes to deployable POCT devices.

Antimicrobial resistance (AMR) and biofilm-associated infections caused by multidrug-resistant pathogens, particularly vancomycin-resistant Staphylococcus aureus (VRSA) and Pseudomonas aeruginosa (ceftazidime-resistant), pose serious healthcare challenges. This study aimed to develop PEGylated niosomes co-loaded with nisin (Nis) and biosynthesized zinc oxide nanoparticles (Nio-Nis/ZnO@PEG) to enhance antibacterial and anti-biofilm efficacy. The formulation was prepared using thin-film hydration followed by PEGylation and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and fourier transform infrared spectroscopy (FTIR). Nio-Nis/ZnO@PEG exhibited high encapsulation efficiency, sustained-release kinetics, and good physicochemical stability. Antibacterial activity and anti-biofilm effects were evaluated using standard microbiological assays. The PEGylated formulation showed significantly lower minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) values, larger inhibition zones, and sustained bactericidal activity against VRSA and ceftazidime-resistant P. aeruginosa compared to free agents or non-PEGylated niosomes (P < 0.001). Anti-biofilm assays demonstrated > 80% biomass reduction and the lowest minimum biofilm eradication concentration (MBEC) values across clinical and reference strains. The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed marked downregulation of all target genes. Cytotoxicity assays using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay confirmed minimal adverse effects on mammalian cells. Overall, PEGylated niosomal co-delivery of Nis and ZnO nanoparticles provides a potent, broad-spectrum, and biocompatible strategy to combat multidrug-resistant bacteria and disrupt biofilms, supporting its potential for further in vivo evaluation.

Effective immunomodulation of natural killer (NK) cells holds promise for enhancing innate immune responses in cancer and infectious diseases; however, free immunostimulants often induce only transient activation. In this study, we developed peptidoglycan (PG)-loaded poly(lactic-co-glycolic acid)-polyvinyl alcohol nanoparticles (PGN) as a biocompatible platform for sustained immune signalling. PGN fabricated via nanoprecipitation exhibited a spherical morphology with an average diameter of 150 nm, a zeta potential of - 15 mV, and approximately 18% loading efficiency. 50% encapsulation efficiency was achieved, with release studies confirming controlled PG delivery over 48 h. Functional assays in NK-92 cells demonstrated that while both free PG and PGN upregulated key effector molecules, including perforin, granzyme B, and interferon-γ (IFN-γ), PGN treatment uniquely maintained significantly elevated IFN-γ expression at transcriptional level even after an PAMP withdrawal and a subsequent rest period, indicating prolonged immune stimulation compared to the transient effects of free PG. Mechanistically, PGN sustained NOD2 receptor gene expression, suggesting prolonged modulation of pattern recognition receptor signalling and delayed signal attenuation relative to bare PG. Notably, this sustained post-stimulation transcriptional response exhibited features conceptually consistent with innate immune memory-like or trained immunity-associated phenomena previously described in NK cells and monocytes. These findings highlight the potential of PGN-mediated delivery to extend the temporal window of NK cell activation and support its application as a nanocarrier strategy for next-generation NK cell-based immunotherapies.

Aspergillus flavus is a globally significant threat to peanut (Arachis hypogaea L.) production, causing yellow mold disease and contaminating crops with carcinogenic aflatoxins. This review synthesizes current knowledge on the origins, pathogenesis, diagnosis, and sustainable management of this disease, with a specific focus on pre-harvest interventions. The disease cycle originates from soil-borne inoculum and spreads via wind and insects. As a saprophytic opportunist, A. flavus deploys hydrolytic enzymes, with host lipids governing sporulation and aflatoxin biosynthesis. Pathogenicity is orchestrated by an intricate signalling network: GPCRs sense host oxylipins, activating cAMP/PKA and three MAPK cascades (Fus3, HOG, CWI), while the TOR pathway functions as an independent nutrient sensor; these converge on the velvet complex (VeA, VelB, LaeA) to regulate development and virulence. Diagnosis integrates visual scales, culture, chemical (TLC, HPLC-MS/MS), immunological (ELISA), and molecular tools (PCR, qPCR, LAMP). Management strategies have advanced through breeding partially resistant cultivars (leveraging defense-related genes, microRNAs, and CRISPR-based tools) and biological control using non-aflatoxigenic A. flavus strains, beneficial bacteria, microbial cell-free supernatants, and plant metabolites. However, we critically evaluate concerns that non-aflatoxigenic biocontrol strains may retain plant-pathogenic potential independent of aflatoxin production. Because complete genetic resistance remains elusive, we advocate integrated disease management combining host resistance, biocontrol, cultural practices, and climate-adaptive surveillance. Future priorities include elucidating the ecological role of sexual recombination, validating field-deployable diagnostics, implementing digital phenotyping, and developing predictive models that incorporate climate change scenarios to safeguard peanut production and global food safety.

Limitations in current wound management strategies of infections with antibiotic resistance Pseudomonas aeruginosa, have prompted the search for alternative therapeutic approaches. Employing the probiotic-derived products was the approach of this investigation by using probiotic cell-free supernatants (CFSs) through controlling infections and mitigating the cytotoxic effects of pathogenic metabolites on fibroblast survival. Four probiotic CFSs were analyzed using antimicrobial broth microdilution assays, GC-MS analysis, antioxidant activity assays, and hemolysis assays. The effects of probiotic CFSs, both alone and in combination with P. aeruginosa CFS, were investigated using cellular and molecular assays. Lactobacillus paracasei CASEI431 exhibited antimicrobial activity against Staphylococcus aureus ATCC 25,923, and P. aeruginosa ATCC 27,853, with a minimum inhibitory concentration of 6.25 mg/mL. However, Bifidobacterium animalis CFS showed the strongest toxicity to fibroblast cells, while L. paracasei CFS had the most positive effect on fibroblast proliferation. Following treatment of cells with the pathogenic CFS, viability of HFFs was significantly reduced. Although co-treatment of cells with L. paracasei and pathogenic CFSs did not significantly increase cell viability by MTT assay, it caused a significant upregulation of the anti-apoptotic gene BCL2 by approximately 3-fold and 36-fold after 24 and 72 h, respectively comparing pathogenic CFS treatment alone. Overall, this study suggests probiotic derived products as a complementary therapy accelerating chronic wounds healing through infection control. However, further research including in vivo studies, comprehensive metabolite profiling, and elucidation of the underlying signaling pathways are necessary to validate and expand upon these findings.

The gastrointestinal tract operates as a highly integrated, multi-layer signaling ecosystem in which microbial communities, metabolite flux, epithelial receptors, immune circuits, and neuroendocrine pathways form a coordinated regulatory network rather than isolated biological compartments. The Microbiome-Metabolite Signaling Network (MMSN) framework conceptualizes gastrointestinal homeostasis and disease as emergent properties of dynamic cross-layer interactions. This review explores the framework principally on inflammatory bowel disease and irritable bowel syndrome as representative gastrointestinal disorders, drawing on other conditions only for illustrative contrast. Within this architecture, microbiota-derived metabolites including short-chain fatty acids, bile acid derivatives, and tryptophan catabolites serve as biochemical intermediaries that relay ecological signals to host receptor systems such as GPR41/43, FXR, TGR5, the aryl hydrocarbon receptor, and innate immune sensors. These receptor-mediated inputs converge on intracellular signaling hubs, including NF-κB, STAT3, inflammasomes, and neuroimmune mediators, which act as high-centrality nodes governing epithelial integrity, cytokine gradients, metabolic coordination, and visceral sensitivity. Signaling hubs are mechanistic convergence nodes that integrate diverse upstream perturbations into coordinated inflammatory or regulatory outputs. In contrast, network fragility denotes the loss of redundancy, modularity, and buffering capacity, predisposing the system to nonlinear amplification and pathological attractor states. Gastrointestinal disorders are therefore more accurately interpreted as manifestations of network rewiring characterized by hub centralization, metabolite imbalance, and strengthened inter-module coupling rather than simple microbial compositional shifts. This review explains the clinical heterogeneity, fluctuating disease trajectories, and variable therapeutic responsiveness. A network-based translational strategy emphasizes hub stabilization, metabolite recalibration, and restoration of distributed connectivity, shifting precision therapeutics toward topology-informed intervention. Integration of microbiology, immunology, neuroscience, systems biology, and computational medicine establishes a pathway toward predictive, mechanistically grounded gastrointestinal network care.

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight, poses a persistent threat to rice production, with virulent pathotypes overcoming major resistance (R) genes. To investigate virulence diversity among Indian isolates, we performed complete genome sequencing of four Indian Xoo strains (IX-123, IX-188, IX-249, and IX-423) using a hybrid Illumina-Oxford Nanopore approach to characterize their transcription activator-like effector (TALE) repertoires. Each strain harbored 17-19 tal genes, revealing extensive TALome variation. Effector binding element (EBE) predictions identified multiple TALEs targeting promoters of clade-III OsSWEET genes. Comparative analysis showed several Indian TALEs shared EBE similarities with known virulence effectors such as PthXo1 and AvrXa7, suggesting conserved targeting mechanisms. Quantitative RT-PCR based expression profiling confirmed strain-specific induction of five clade III SWEET genes in the susceptible cultivar IR24 upon infection. Collectively, this study uncovers the complexity of Indian Xoo TALomes and the central role of SWEET genes as universal susceptibility hubs, providing genomic insights for developing SWEET-promoter-edited rice lines with durable, region-specific resistance to bacterial blight.

This study aimed to identify and characterize indigenous yeast strains suitable for the fermentation of Fragaria × ananassa Duchesne (strawberry) wine. A total of 58 yeast isolates were obtained from spontaneously fermented strawberry juice. Preliminary screening based on colony morphology on Wallerstein Laboratory (WL) nutrient agar yielded 15 candidate strains. Subsequent evaluation of CO2 production using Durham tube fermentation assays further narrowed the selection to 12 high-performing isolates. Lysine utilization testing indicated that strain Z-36 belonged to Saccharomyces cerevisiae, whereas the remaining 11 isolates were classified as non-Saccharomyces yeasts. Comprehensive physiological tolerance profiling revealed distinct functional characteristics among the selected strains: strain Z-36 exhibited robust tolerance to multiple stress conditions, including cold stress at 4 °C, ethanol concentrations up to 15% (v/v), sucrose levels of 400 g/L, SO2 concentrations of 360 mg/L, and low pH, rendering it especially well-suited for demanding fermentation environments; strain Z-1 demonstrated superior thermotolerance at 40 °C; strain Z-50 displayed exceptional resistance to high glucose concentration (400 g/L), strong resistance to common microbial inhibitors, and notably high ester biosynthetic capacity; and both strains Z-25 and Z-36 achieved outstanding ethanol yields. Molecular identification, based on sequencing of the D1/D2 domain of the large subunit rRNA gene, classified the 12 strains as follows: five Pichia fermentans, four Pichia kudriavzevii, two Hanseniaspora spp., and one S. cerevisiae. Integrating morphological, biochemical, physiological, and molecular data, strains Z-36 (S. cerevisiae) and Z-50 (Hanseniaspora sp.) were selected as optimal candidates for strawberry wine fermentation. However, we emphasize that these conclusions are drawn from in vitro screening assays; validation through actual strawberry wine fermentations with chemical and sensory analyses is required to confirm their technological suitability. This work provides a scientifically grounded foundation for the targeted development of starter cultures and informs future studies on strain optimization and mixed-fermentation strategies.

While small colony variants (SCVs) of Staphylococcus aureus have been extensively documented, little is known about the properties of menadione-dependent SCVs from cow raw milk and how heme affects their ability to form biofilms. In this study, gentamicin-induced SCVs were generated from mastitis-derived Staphylococcus aureus, and the underlying mechanisms were investigated through nutrient dependency assays combined with genomic analysis. A comparative assessment of biological characteristics-including physiological and biochemical properties, antibiotic susceptibility, biofilm formation, and hemolytic activity-was conducted between the SCV and wild-type strains. Additionally, the effects of exogenous heme and menadione on biofilm formation were evaluated. The results revealed a frameshift deletion mutation due to a 95-nucleotide deletion in the menE gene. This mutation altered the corresponding amino acid sequence, correlating with the menadione-dependent SCV phenotype. Contrary to the typical SCV phenotype, the isolated SCV strain not only presented classic traits, such as slow growth and increased resistance to aminoglycoside antibiotics, but also exhibited atypical characteristics, including significantly decreased biofilm formation capacity and increased hemolytic activity. Exogenous supplementation with menadione or heme markedly promoted biofilm formation in the SCV strain. These results suggest that the reduced biofilm-forming ability of the SCV strain may stem from compromised metabolic activity. Furthermore, the SCV strain may be capable of acquiring exogenous heme via unknown pathways, a hypothesis that warrants further investigation, thereby restoring its biofilm formation capacity. In conclusion, this atypical SCV exhibits reduced biofilm formation and increased hemolytic activity, and heme partially restores biofilm via increased EPS and surface hydrophobicity. These findings are derived from a single SCV-parent pair; further isolates are needed to determine generalizability.

The method used to preserve samples prior to DNA extraction is crucial for the accurate characterization of microbial diversity. This study evaluated the effects of different storage conditions on microbial DNA preservation in Coffea arabica fruit samples. Coffee cherries were harvested directly from plants, placed in plastic tubes, and stored at 4 °C before being subjected to four treatments: lyophilization, cryopreservation at - 80 °C, and refrigeration at 4 °C. Samples were stored for 3 and 13 days. Microbial communities were characterized by next-generation sequencing. Lyophilization retained more than 80% of the ASVs shared between days 3 and 13 of storage, whereas refrigeration at 4 °C retained less than 50%. These findings demonstrate that preservation method significantly affects the integrity of bacterial and fungal microbiomes in C. arabica beans. The absence of a time-zero control, chemical preservative comparisons, and the study's limited scope (single variety, location, and short storage period) warrant cautious interpretation of the findings. Although lyophilization was the best-performing physical preservation strategy evaluated here, broader validation across coffee cultivars, environments, and storage durations is still required before it can be considered a standard preservation protocol for preserving microbial materials derived from coffee samples.

The increasing incidence of antifungal resistance in Candida species underscores the urgent need for new agents that effectively inhibit fungal growth and virulence while exhibiting minimal host toxicity. In this study, DBE-7 was identified as a potent antifungal compound with strong activity against Candida albicans. DBE-7 inhibited fungal growth at low micromolar concentrations (MIC = 3.37 µM) and exhibited fungicidal rather than fungistatic activity. The compound markedly impaired key virulence attributes, including hyphal morphogenesis and biofilm formation. Quantitative image analysis demonstrated a concentration-dependent reduction in hyphal elongation and filamentous cell formation, with inhibitory effects comparable to those of fluconazole. DBE-7 also significantly reduced biofilm biomass, indicating its ability to disrupt biofilm architecture. DBE-7 effectively eradicated preformed mature biofilms in a concentration-dependent manner, indicating its ability not only to prevent biofilm development but also to disrupt established biofilm. Kill kinetics revealed rapid and sustained fungicidal activity in a time- and concentration-dependent manner. DBE-7 also displayed low cytotoxicity toward mammalian cell lines, maintaining high cell viability within its antifungal concentration range. Molecular docking revealed a strong binding affinity of DBE-7 toward lanosterol 14-α-demethylase (LDM), while the marked reduction in cellular ergosterol levels in DBE-7-treated C. albicans further supports disruption of the ergosterol biosynthetic pathway, suggesting potential targeting of LDM. Collectively, these findings highlight DBE-7 as a promising antifungal candidate that effectively targets C. albicans growth and virulence while exhibiting a favorable safety profile, supporting its further preclinical development.

Conversion of unstructured biomedical literature into structured knowledge for identifying cross-domain associations between biological entities remains a challenging task. SigMine is an automated pipeline constructed to mine biomedical literature to identify significantly associated biological entities. SigMine performs biomedical entity recognition from PMC articles using the EuropePMC Annotation API. Advanced entity recognition was performed using Python scripting, NCBI E-Utilities, and an n-gram algorithm followed by extensive data cleaning and mapping against standard databases. Statistical evaluation identified significantly co-occurring entities. The entire workflow was automated through a modular framework developed in Python v3.13 with a Tkinter-based Graphical User Interface. SigMine enhances usability while retaining the flexibility to use new dictionaries for annotation. SigMine was used to construct a literature-derived potential human Opportunistic Pathogens Database (OPathDb), housing 5,626 potential opportunistic pathogens significantly co-occurring with 1440 diseases and 7121 genes mined from 25,000 PMC articles. Additional annotation of 598 significantly co-occurring metabolites and 30 affected tissues is available for 3204 and 227 pathogens, respectively. OpathDb has a user-friendly query interface searchable by organism, disease, tissue, gene, protein and metabolite available at https://www.opathdb.cbsblab-nsut.in . Organism-entity associations can be visualized as weighted networks, with color-coded nodes and significance-scaled edges. Significant associations of opportunistic pathogens like Akkermansia mucinifila with colorectal cancer and Segatella copri with glucose intolerance can be identified through OpathDb. Through this database, the SigMine framework demonstrates conversion of unstructured text in vast and heterogenous corpora into standardized and well-organized information. Statistically inferred associations in OPathDb are potential candidates for clinical and experimental validation.

Xylitol is a sugar alcohol widely used in the food industry due to its lower caloric value compared to sucrose. Industrial xylitol production relies primarily on chemical hydrogenation processes that require high energy input. Although biotechnological routes represent a more sustainable alternative, their industrial implementation is often limited by enzyme stability and cofactor costs. This study presents the design, expression, and biochemical analysis of recombinant xylose reductase from Neurospora crassa (rNcXR). The optimized gene was cloned into a salt-inducible T7-based expression system and expressed in Escherichia coli (E. coli) BL21-SI. The design incorporated an OmpT signal peptide to encourage secretion into the periplasm, and a 6×His tag at the C-terminus to simplify purification. The recombinant enzyme exhibited a specific activity of 20.87 U/mg, significantly exceeding values previously reported for both native and recombinant xylose reductases. Kinetic characterization revealed that the engineered rNcXR maintains a high substrate affinity while achieving a remarkably enhanced catalytic turnover compared to traditional fungal biocatalysts. Furthermore, experimental validation confirmed an optimized cofactor-binding pocket, supporting the structural enhancements predicted by molecular docking. The rNcXR displayed robust thermodynamic stability at an alkaline pH and maintained high catalytic efficiency at elevated temperatures up to 55 °C. These results demonstrate that the engineered rNcXR represents a resilient and high-performance biocatalyst with strong potential for continuous enzymatic xylitol production in industrial biorefineries.