Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, lacks the genes for de novo riboflavin biosynthesis and depends on host-derived riboflavin to generate flavin cofactors FMN and FAD. Riboflavin salvage by Bb has demonstrated importance to infection and metabolic homeostasis, though the critical roles of flavin cofactors to central metabolism are not well defined. Here, we demonstrate that riboflavin availability directly controls glycolytic flux through regulation of intracellular redox balance. Disruption of riboflavin uptake by deletion of gene bb0318 encoding the putative ATPase component of the riboflavin transporter resulted in impaired glycolysis as indicated by reduced intracellular pyruvate and a perturbed NADH/NAD+ ratio driven by faulty NAD+ regeneration. We identified coenzyme A disulfide reductase (CoADR) as a key flavin-dependent mediator of glycolytic flux. A Δcdr mutant mimicked the glycolytic dysregulation of the Δbb0318 mutant, but was not rescued by exogenous riboflavin, placing BbCoADR downstream of riboflavin salvage. Biochemical analyses of recombinant BbCoADR demonstrated FAD-dependent NADH oxidase activity. Targeted mutagenesis of predicted FAD-binding residues impaired cofactor binding and NADH oxidase activity, further confirming the dependence of BbCoADR on FAD. Together, this work reveals a direct link between riboflavin acquisition and core metabolic processes driven by BbCoADR-mediated flavin-dependent redox metabolism.

Effect of pH on the formation mechanism of riboflavin mediated photocrosslinking hydrogel of soybean protein nanofibrils.

Current pharyngitis treatment relies heavily on antibiotics and glucocorticoids, leading to increasing bacterial resistance and significant adverse drug reactions, while the traditional efficacy of Fructus Chebulae is to reduce swelling and relieve sore throat, yet its key components and mechanism against pharyngitis need systematic research. Therefore, this study aimed to integrate MALDI-MSI spatial metabolomics and network pharmacology to systematically analyze Fructus Chebulae's therapeutic mechanism against acute pharyngitis, identifying ellagic acid as the core component, along with key targets and spatial metabolic pathways. Using a 'disease-component-metabolic pathways' strategy, network pharmacology was combined with in vivo validation in an acute pharyngitis animal model induced by 5% ammonia oropharyngeal spray, followed by spatial metabolomics analysis. The results showed that network pharmacology identified ellagic acid and SRC protein as the core ingredient and key target. Ellagic acid spraying dose-dependently repaired pharyngeal damage, significantly reduced TNF-α and oxidative stress, and inhibited SRC protein expression. Furthermore, MALDI-MSI visualized metabolites, screening 376 differentially expressed metabolites and pinpointing 49 biomarkers. Specifically, ellagic acid inhibited inflammation, scavenged radicals, and promoted regeneration by regulating amino acid biosynthesis, riboflavin, and lipid metabolism. In conclusion, this study multidimensionally analyzed Fructus Chebulae's traditional efficacy using network pharmacology, confirming the anti-pharyngitis efficacy of ellagic acid, and for the first time via MALDI-MSI illustrated its core spatial metabolic pathways regulating inflammation, oxidative stress, and tissue repair.

Microbial artificial photosynthesis offers a promising strategy for light-driven biomanufacturing, yet its efficiency remains limited by the non-selective conversion of photogenerated electrons into metabolically usable reducing power, causing energy dissipation and weak coupling between light capture and metabolic reactions. Here, we report a rational strategy using riboflavin (RF), a membrane-permeable and biocompatible flavin photosensitizer, to selectively channel photonic energy into intracellular NADPH regeneration. Quantum chemical calculations and spectroscopic analyses reveal that light-excited RF exhibits a specific binding affinity and favorable electron transfer trend toward NADP+. In vivo, RF activation markedly elevated intracellular NADPH levels and enhanced the synthesis of NADPH-dependent metabolites through NADPH reductase-associated pathways. Transcriptomic and inhibition analyses linked RF-mediated NADPH regeneration to NADP+/NADPH redox enzymes rather than glucose-6-phosphate dehydrogenase-mediated flux, while NADH-related redox genes remained largely unaffected, demonstrating the selectivity of this reductive route. Cross-species and multi-product validations consistently reproduced these results, underscoring the generality of this mechanism across distinct NADPH-dependent microbial chassis. This work establishes a mechanistically defined and broadly applicable framework for directing photogenerated electrons into specific cellular reducing equivalents, paving the way for efficient artificial photosynthetic and bioelectrochemical platforms.

Ticks (Ixodida) are the second most important vectors of infectious diseases in vertebrates, after mosquitoes. Beyond vector roles, they maintain mutualistic associations with bacteria, including endosymbionts that provide essential B vitamins lacking in their blood-based diet. The most extensively studied endosymbionts belong to the genera Coxiella, Midichloria, and Francisella. The genus Francisella encompasses endosymbionts (FE), pathogens (FP), opportunistic pathogens (FO) and free-living environmental strains (FL), making it a powerful system for evolutionary and comparative genomic analyses. In this study, total DNA from six adult female ticks of the genera Hyalomma and Amblyomma was sequenced to generate new FE genomes. Seven deeply sequenced public metagenomes were also assembled, yielding 71 Francisella and three Allofrancisella strains. This dataset supported phylogenomic reconstruction and comparison of genomic features, including vitamin biosynthesis and virulence pathways, with a focus on transitions to tick endosymbiosis. A densely sampled MLST phylogeny was constructed to explore biogeographic patterns. Our results show that, except for FE, no ecological trait is monophyletic, supporting an origin of Francisella diversity from free-living ancestors. Biogeography suggests Palearctic and Afrotropical FE strains are derived and may involve horizontal transfers. Francisella comparative genomics reveals two contrasting profiles: environmental generalists and host-restricted specialists. These findings reinforce the role of tick FEs as nutritional mutualists, retaining key pathways such as riboflavin, shikimate, and biotin biosynthesis. In contrast, virulence is not ancestrally conserved but an innovation in pathogenic lineages, largely degraded in tick FEs. These results advance understanding of endosymbiont evolution and provide genomic insights with potential for disease control.

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by vascular injury and progressive fibrosis. Although microvascular injury is an inciting event and pericytes are recognized as a major source of myofibroblasts, the precise phenotypic heterogeneity of pericytes in the SSc microenvironment and the genetic mechanisms driving their pathological transition remain elusive.MethodsThis study systematically explored the cellular and genetic basis of pericyte dysfunction by integrating single‐cell RNA sequencing (scRNA‐seq) data from SSc patients with genome‐wide association study (GWAS) data using bidirectional Mendelian randomization (MR) analysis. Pseudotime trajectory analysis was used to reconstruct developmental lineages, while cell–cell communication and metabolic pathway analyses were conducted to uncover underlying mechanisms. Multiomics validation was performed using external bulk RNA‐seq datasets.ResultsSingle‐cell analysis revealed significant heterogeneity in pericyte subpopulations, specifically identifying a marked expansion of progenitor‐like pericytes, which were positioned at the root of the differentiation trajectory toward fibrotic phenotypes. Bidirectional MR analysis identified RAC1 as a significant causal risk factor for SSc (OR = 2.0756, p = 0.0046). Mechanistically, RAC1‐positive pericytes exhibited enhanced proinflammatory crosstalk with macrophages via the MIF‐(CD74 + CD44) signaling axis. Furthermore, these activated pericytes displayed distinct metabolic reprogramming, characterized by the upregulation of riboflavin and thiamine metabolism to support their bioenergetic demands. Transcriptomic validation further confirmed the aberrant overexpression of RAC1 in SSc tissues.ConclusionThis study establishes a mechanistic link between RAC1‐mediated activation of progenitor‐like pericytes and SSc pathogenesis. RAC1 acts as a causal driver promoting pathological pericyte transition and orchestrates a proinflammatory microenvironment through metabolic reprogramming and immune recruitment, offering a novel therapeutic target for SSc.

Antibiotic treatment failure is frequently driven by bacterial insusceptibility, yet understanding of the full repertoire of genes contributing to this in Gram-negative bacteria remains incomplete. We used TraDIS-Xpress, a high-density transposon mutagenesis approach, to systematically define the genetic and physiological determinants of Escherichia coli K-12 susceptibility to 13 antibiotics spanning all major drug classes and multiple concentrations. Beyond known resistance determinants, our data reveal that the E. coli core genome plays a central role in modulating susceptibility. Disruption of metabolic, energy-generating, growth-associated, and amino acid biosynthetic pathways commonly enhanced survival under antibiotic stress, indicating that reduced metabolic activity and growth modulation broadly promote survival during exposure to bactericidal drugs. In contrast, pathways involved in DNA replication, peptidoglycan and lipopolysaccharide synthesis, aminoacyl-tRNA biosynthesis, riboflavin metabolism, and terpenoid backbone biosynthesis consistently aided survival. Experimental validation using defined mutants confirmed that disruption of selected metabolic and respiratory pathways confers survival benefits across multiple antibiotic classes. Together, this work provides a genome-scale framework for understanding antibiotic susceptibility in E. coli, identifies conserved physiological pathways underpinning survival, and offers new opportunities for strategies aimed at reversing insusceptibility and improving antibiotic efficacy.

A multi-omics investigation of pulmonary and hepatic toxicity induced by polyamide and polyacrylonitrile microplastics via ingestion and inhalation exposures.

Single-cell and metabolomic profiling reveals multi-lineage hepatic perturbations and metabolic reprogramming in pregnant mice exposed to dimethomorph

ObjectiveThis research explored the effect of grape exosome–like nanovesicles (GELNs) on reversing prediabetic conditions in mice and investigated the underlying mechanisms via metabolomic and gut microbiota analyses.MethodsTwenty‐four C57BL/6J mice were divided into four groups: normal control, prediabetic model, GELN intervention, and nutrient intervention. Prediabetic models were induced in all but the control group, and then the intervention groups were treated for 8 weeks.ResultsGELNs significantly improved fasting blood glucose, 2‐h postprandial glucose, insulin levels, and total cholesterol. Metabolomic analysis found enriched differential metabolites, highlighting tryptophan and riboflavin metabolism. 16S rDNA sequencing showed no difference in α‐diversity but differences in β‐diversity. At the phylum level, the abundance of Firmicutes and Desulfobacterota was higher in the GELN group (Group W), while Bacteroidota and Proteobacteria were more abundant in the model group (Group T). At the genus level, Akkermansia was significantly reduced in the GELN group (Group W), indicating a notable shift in gut microbiota composition.ConclusionGELNs may reverse prediabetes in mice by improving metabolic health and gut homeostasis, offering new insights for prediabetes prevention and treatment.

This in vitro study evaluated the effect of collagen crosslinking agents (grape seed extract, riboflavin, and chitosan nanoparticles with UVA light) on hybrid layer thickness, resin tag length, and shear bond strength (SBS) of a fifth-generation adhesive resin to caries-affected dentin of primary molars. Seventy-five carious human primary molars were selected, exhibiting lesions that reached the middle third of the dentin, according to the International Caries Detection and Assessment System (ICDAS) criteria 5. Infected dentin was removed. The crowns were shortened in height up to CAD, and etching was performed, followed by division of the samples into five groups (n = 15 each). Group 1: No collagen crosslinking (CCL) agent; Group 2:6.5% Grape Seed Extract (GSE); Group 3:0.1% riboflavin (RF)-UVA; Group 4:0.2% chitosan nanoparticles (CHNPs)-UVA; and Group 5: chlorhexidine (CHX). Composite restorations were built using a fifth-generation etch-and-rinse (ER) adhesive, followed by artificial aging. Scanning electron microscopy (SEM) was used to determine the resin tag length (RTL) and hybrid layer (HL) thickness. The SBS and failure modes were assessed using a universal testing machine and a stereomicroscope. One-way ANOVA and post-hoc Tukey tests were used to evaluate the means across all tested groups (p < 0.05). Group 4-CHNPs-UVA demonstrated the highest RTL (20.03 ± 3.54μm) and thickest HL (5.65 ± 0.42μm). Group 1 (No CCL agent) displayed the shortest RTL (7.11 ± 1.05μm) and thinnest HL (1.23 ± 0.11μm). The CHNPs-UVA pretreated samples showed maximum bond strength (10.31 ± 0.23MPa). Group 1 (No CCL agent) tested samples presented minimum bond integrity (6.18 ± 0.33MPa). Within the limitations of this in vitro study, CHNPs-UVA appears promising as a biomodification strategy for CAD in primary molars and warrants further long-term in vivo investigations before clinical recommendations can be made.

Riboflavin is an essential water-soluble vitamin that serves as a precursor for the biosynthesis of the flavin cofactors FMN and FAD, which play pivotal roles in numerous redox and energy metabolism reactions. With the growing global demand for sustainable vitamin production, microbial fermentation has become an attractive alternative to chemical synthesis due to its environmental and economic advantages. Among microbial hosts, Bacillus subtilis has emerged as a leading cell factory for riboflavin production owing to its GRAS status, well-characterized genetics, and efficient protein secretion system. This review provides a comprehensive overview of recent advances in metabolic engineering strategies to enhance riboflavin biosynthesis in B. subtilis. Key topics include strengthening biosynthetic and precursor pathways, relieving feedback inhibition, balancing metabolic flux and cell growth, employing adaptive laboratory evolution, and utilizing omics-guided optimization and 13C metabolic flux analysis. Moreover, the integration of synthetic biology tools such as riboswitch engineering, regulatory element design, and high-throughput screening has significantly accelerated strain improvement. Despite remarkable progress, challenges remain in achieving precise regulatory control, optimizing multi-gene expression, and enhancing genome integration efficiency. Future research combining multi-omics data, synthetic regulatory design, and machine learning-driven predictive modeling is expected to further advance the development of intelligent B. subtilis cell factories. However, the practical implementation of these systems remains constrained by the metabolic burden of overproduction and the lack of universal regulatory models that can predict strain performance across varying industrial scales.

Riboflavin (vitamin B₂; RF) and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are indispensable cofactors for redox reactions in plants. While higher plants possess a conserved pathway for de novo riboflavin biosynthesis, how flavins are transported and spatially distributed between tissues remains unresolved. In particular, the molecular identity of plasma membrane transporters involved in flavin transport in plants remains largely unknown. Here, we identify Arabidopsis purine permease 5 (AtPUP5) as a plasma membrane-localized protein associated with riboflavin transport and its distribution in plants. Using a riboflavin-auxotrophic yeast mutant, we show that AtPUP5 enhances intracellular accumulation of RF and, to a lesser extent, FMN, whereas FAD accumulation is inefficient under the conditions tested. In planta, AtPUP5 overexpression increases RF accumulation following external application. In contrast, loss-of-function mutants do not display defects in bulk RF uptake at the whole-plant level, indicating that AtPUP5 is not essential for global riboflavin acquisition. Notably, AtPUP5 deficiency results in RF overaccumulation in reproductive organs, including inflorescences, siliques, and seeds, irrespective of external RF supply. This organ-specific phenotype is fully suppressed by genetic complementation and coincides spatially with strong AtPUP5 promoter activity in reproductive tissues. These findings suggest that AtPUP5 contributes to the regulation of localized riboflavin distribution in reproductive tissues. Together, our study provides a molecular framework for plasma membrane-mediated regulation of riboflavin distribution in plants and highlights spatial regulation as an important component of flavin homeostasis.

The biosynthesis of flavin adenine dinucleotide (FAD), an essential redox coenzyme, is limited by inefficient substrate utilization and low catalytic activity. To address this, an "Uptake-Catalysis" system was constructed in Bacillus subtilis for FAD synthesis from riboflavin (RF). First, the Streptomyces davawensis transporter (RibM) was coexpressed with native FAD synthase (BsFADs) at a 2:1 ratio, which increased RF consumption to 51%. Subsequently, the isothermal compressibility perturbation engineering (ICPE) strategy was adopted, generating the optimum mutant BsFADsP61W+R202Y, which increased catalytic efficiency (kcat/Km) toward RF and FMN by 83% and 73%, respectively. Mechanistic analysis revealed that this improvement was due to a 39% shortening of RF substrate channel. Finally, FAD titer peaked at 563 mg/L with a conversion rate (cr-FAD) of 92% in shaking-flask fermentation optimization. This work achieved a 225-fold increase in FAD production, validating the effectiveness of the uptake-catalysis synergistic strategy, thereby establishing an efficient and scalable synthesis paradigm.

Gut microbiota alterations have been linked to childhood eating disorders, but the functional and metabolic changes in non-organic anorexia (NOA) remain poorly understood. This study aimed to characterize the gut microbial composition, function, and metabolic profiles in children with NOA using an integrated multi-omics approach. A case-control study was conducted involving 88 children aged 1-5years (48 NOA, 40 healthy controls). Gut microbiota composition was assessed via 16S rRNA gene sequencing of all fecal samples. Subsequently, the five most representative samples from each group were selected for deep shotgun metagenomic sequencing and liquid chromatography-mass spectrometry (LC-MS) based non-targeted metabolomics. NOA children showed significantly higher microbial richness and diversity (Chao1, Shannon; P < 0.001). The NOA group had elevated Firmicutes, Bacteroidota, Bacteroides, Faecalibacterium, Subdoligranulum, and Roseburia, but reduced Actobacteriota, Bifidobacterium, and Enterococcus. Metagenomics revealed downregulated riboflavin metabolism and upregulated fat digestion/absorption pathways in NOA (P < 0.05). Metabolomics identified 26 differential fecal metabolites, including decreased L-carnitine derivatives and elevated tyramine glucuronide involved in bile secretion. These metabolites were significantly correlated with altered bacterial genera. Our integrated multi-omics analysis demonstrates that NOA in children is associated with a specific gut ecosystem characterized by altered microbiota structure, perturbed microbial metabolic functions (particularly riboflavin metabolism), and corresponding host-microbiota co-metabolic disturbances. These findings provide novel evidence for the disrupted "microbiota-metabolite" axis in NOA, offering new mechanistic insights.

Plant growth-promoting rhizobacteria (PGPR) enhance plant growth and development through diverse mechanisms, including phytohormone production, nutrient acquisition, and stress mitigation. This study describes the isolation and characterization of two bacterial strains, DT1 and S10, from the rhizospheres of Diplotaxis tenuifolia and Cynodon dactylon, respectively that exhibit multiple plant growth‑promoting traits, including phosphate and zinc solubilization, nitrogen metabolism and the production of indole acetic acid (IAA) and siderophores. Using whole genome sequencing and taxonomic analyses, these two strains were identified as Acinetobacter calcoaceticus (DT1) and Citrobacter braakii (S10). Functional genomic annotation revealed numerous genes associated with key plant growth-promoting traits, including those involved in indole-3-acetic acid (IAA) (trpABCDE, ipdC), cytokinin (miaABE), and riboflavin biosynthesis, which were further supported by targeted metabolomic analyses. In addition, genes associated with nitrogen metabolism, including nitrate/nitrite reduction (nirB, narGHI), as well as phosphate solubilization (gcd, phoARP, pstABCS, pqqEFG) were identified and supported by phenotypic assays. Interestingly, biosynthetic gene clusters for the secondary metabolites enterobactin, bacillibactin, and staphyloferrin B, known to contribute to plant growth promotion, were identified in both genomes. Both strains also harbored genes potentially involved in stress-related metabolic processes. Furthermore, non-targeted metabolomic analysis revealed that DT1 and S10 produced a range of intracellular and extracellular metabolites associated with plant growth promotion and stress resilience, including cadaverine, biotin, arginine, and GABA. Collectively, these findings position DT1 and S10 as promising bioinoculant candidates, offering an integrative genomic and metabolic foundation for their application in next-generation sustainable agricultural strategies.

A comprehensive genetic landscape of antibiotic sensitivity in Staphylococcus aureus is lacking. Using genome-scale CRISPR-interference libraries, we systematically quantified global gene fitness across 10 antibiotics and uncovered hundreds of significant antibiotic-gene interactions. Essential genes dominated these interactions, a finding not revealed by transposon-based studies. CRISPR interference repression of transcriptional and translational processes desensitized bacteria to multiple antibiotics. In contrast, repression of cell wall synthesis/cell division (CC), DNA replication/DNA recombination (DD), coenzyme A biosynthesis, and riboflavin metabolism strongly sensitized bacteria to antibiotics. Network and genetic analyses further revealed synergistic genetic interactions (GIs) within these bioprocesses, including an extensive CC-DD subnetwork. Only a subset of CC-DD synergies was dependent on the cell division inhibitor SosA. Informed by these GIs, we identified multiple drug-drug combinations with potent synergistic activity against multidrug-resistant S. aureus. Our detailed profiling of drug-gene, gene-gene, and drug-drug interactions reveals important functional relationships among essential genes and defines a vulnerability landscape to guide drug target discovery and effective combination therapies.

Antioxidants are essential for mitigating oxidative stress and preserving cellular homeostasis, underscoring the need for sustainable, analytically robust detection platforms. Here, we propose an environmentally friendly and effective method for producing fluorescent carbon dots (CDs) from clove (Syzygium aromaticum) under hydrothermal conditions utilizing a deep eutectic solvent (choline chloride and urea). This ecologically friendly method eliminates the need for harmful reagents and produces very uniform CDs with an average particle size of ∼2.1 nm and a fluorescence quantum yield of 22.5%. Detailed structural and compositional investigations employing HR-TEM, FT-IR, and XPS reveal effective carbonization as well as the presence of several surface functional groups that contribute to the excellent optical characteristics. The as-prepared CDs show excitation-dependent fluorescence emission, outstanding photostability, and remarkable stability throughout a wide pH, ionic strength, and irradiation time range. With low detection limits of 0.25 µM and 0.14 µM, respectively, the CDs are extremely sensitive and selective fluorescent nanoprobes for quercetin (QT) and riboflavin (RF). The improved sensing performance is ascribed to the effective interactions between the analytes and the surface functionalities of the CDs. Overall, this study demonstrates how deep eutectic solvent engineering and biomass-derived precursors may work together to create functional nanomaterials in a sustainable manner. The proposed CDs have considerable potential for use in antioxidant sensing as well as in extensive bioanalytical and environmental monitoring systems.

Exploration of MAIT cell-activating ligands and their potential as vaccine adjuvants for infectious diseases.

To evaluate the protective effects of Hydrocotyle asiatica extract (HAE), aloe polysaccharide (AP), and Ginkgo biloba extract (GBE) against ovalbumin (OVA)-induced damage in canine intestinal epithelial cells and to elucidate the underlying mechanisms using a multiomics approach. The study was conducted from January 1 through September 30, 2025. An in vitro model of OVA-induced injury in canine intestinal epithelial cells was established. The effects of the 3 extracts were assessed through integrated metabolomic and transcriptomic analyses. All 3 extracts (HAE, 320 µg/mL; AP, 1 µg/mL; GBE, 1 µg/mL) significantly mitigated OVA-induced damage: they restored antioxidant activity (eg, superoxide dismutase increased 105% to 137%), suppressed inflammation (eg, tumor necrosis factor-α reduced by up to 25%), and enhanced barrier protein expression. Multiomics analysis specifically linked HAE to the restoration of metabolic pathways (eg, riboflavin metabolism, oxidative phosphorylation) and the upregulation of genes involved in epithelial adhesion and junction assembly. HAE, AP, and GBE exert protective effects against OVA-induced intestinal epithelial cell injury by mitigating oxidative stress, inflammation, and barrier dysfunction. Hydrocotyle asiatica extract specifically modulates key metabolic and transcriptional pathways involved in intestinal integrity. These findings provide valuable insights into the mechanisms by which HAE preserves intestinal barrier function and underscore its potential application in enhancing intestinal barrier integrity, which may be relevant for managing food allergies and intestinal inflammatory disorders in clinical settings.