Gut Microbes

Background and AimsThe rising incidence of Crohns disease (CD) in Westernized countries has been linked to changes in diet and increased consumption of food additives, yet the mechanisms by which these factors fuel intestinal inflammation remain unclear. Adherent-invasive Escherichia coli (AIEC), a pathobiont involved in CD pathogenesis, lacks a clear genetic hallmark but exhibits intestinal colonization and virulence traits, raising questions about the evolutionary forces promoting its emergence among select individuals. Here, we investigated how chronic exposure to two common dietary emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (P80), along with host inflammation, drives AIEC genomic evolution and pathogenic potential. MethodsWild-type and Il10-deficient mice were monocolonized with AIEC and chronically exposed to CMC, P80, or water. Bacterial isolates were collected and analyzed for genomic diversification, mutations, and phenotype both in vitro and in vivo. ResultsEmulsifiers accelerated AIEC genomic diversification and selected for mutations linked to increased motility, invasion, and pro-inflammatory activity. Moreover, dietary emulsifier-evolved strains displayed a marked fitness advantage in vivo, outcompeting their counterparts in murine hosts, with the greatest advantage observed when evolution occurred under inflammatory conditions. Notably, evolutionary pathways and phenotypic outcomes were shaped by both emulsifier and the hosts inflammatory status, highlighting synergy between diet and host genetics in fostering pro-inflammatory pathobionts. ConclusionThese findings provide an evolutionary framework connecting modern dietary habits to the emergence of pathogenic AIEC strains, and underscore the importance of dietary interventions in individuals at risk for inflammatory bowel disease.

Diet is an important ecological modulator of the oral microbiome, yet population-level evidence on a broader spectrum of food components remains limited. This cross-sectional study investigated associations among dietary intake, oral rinse microbiome, and oral disease conditions in a nationally representative sample of United States adults from the National Health and Nutrition Examination Survey. A total of 3,254 participants with oral rinse microbiome sequencing data were included, with oral conditions classified as oral health, caries-only, periodontitis-only, or co-existing disease. Dietary intake was assessed using 24-hour dietary recalls and summarized as dietary indices and energy-adjusted food components. Associations between diet and the oral microbiome were evaluated using community-level analyses, regression models, mediation analyses, and unsupervised clustering, while accounting for oral conditions. This study found that dietary intake, as a combined variable set, explained 3.6% of the variance in oral rinse microbial community structure; this was comparable to oral disease status or smoking and larger than sociodemographic factors. Healthier dietary profiles, including higher health-associated dietary index scores and greater vegetable and fruit intake, were associated with taxa commonly linked to oral health (e.g., Neisseria, Cardiobacterium and Lautropia). In contrast, added sugars, alcoholic drinks, cured meat, potatoes, dairy products, and higher dietary inflammatory index scores showed opposite association patterns. Mediation analyses suggested that coordinated microbial groups may partly link dietary exposures with oral disease outcomes, particularly for vegetables and added sugars. Additionally, three population-level dietary patterns were identified, among which the plant-rich pattern was associated with more favorable oral health and microbial profiles enriched in nitrate-reducing commensals, including Neisseria and Haemophilus. Overall, dietary intake was associated with oral microbiota composition and oral health conditions, supporting ecological influences of dietary components beyond sugar on oral bacteria and dental diseases. Longitudinal studies are needed to clarify the direction and causality of these relationships.

Gut microbial biotransformation of poorly absorbable polyphenols into bioactive, bioavailable metabolites is increasingly recognized as a key mechanism underlying their health benefits of polyphenols. Microbial ellagic acid (EA)-to-urolithin conversion represents a typical example, but the environmental factors that facilitate such metabolism remain underexplored. We discovered that urolithin production by a gut commensal bacterium, Gordonibacter urolithinfaciens (G. uro), is metabolically repressed by arginine. To overcome such limitations, we developed PhenolBoost Medium (PBM) that induces a metabolic shift by suppressing the arginine deiminase pathway while activating pyruvate metabolism and hydrogen production in G. uro, thereby driving urolithin dehydroxylation. Transcriptomic profiling and 13C-isotopic tracing analysis revealed that pyruvate metabolism in PBM upregulates hydrogenase expression, facilitating the dehydroxylation of EA. PBM also promoted the complete conversion of EA to urolithin A in G. uro-Enterocloster bolteae co-culture, and other polyphenol biotransformations. In addition, co-culturing G. uro with hydrogen-producing Bacteroides species significantly increased urolithin production. Furthermore, an arginine-limited, pyruvate-enriched dietary regimen proved effective in vivo, resulting in significantly higher urolithin production and bioavailability in a mouse model. Our findings reveal the critical role of hydrogen in facilitating polyphenol dehydroxylation, and offer a viable nutritional strategy for boosting microbial production of beneficial metabolites from polyphenols.

Emerging infectious diseases and antimicrobial resistance (AMR) have surfaced as two major public health threats over the past two decades. Consequently, integrative surveillance systems capable of detecting both emerging pathogens and resistance-carrying bacteria are crucial. With advances in next-generation sequencing, simultaneous detection of pathogens and AMR is increasingly feasible. In this study, we used short-read metatranscriptomics complemented by total 16S rRNA metagenomic long-read sequencing to analyze paired oral and plasma samples from a cohort of febrile individuals at two locations in Senegal. Oral microbiomes differed in community composition between locations, and reduced diversity and richness were significantly associated with high fever. We identified at least one known pathogen in 15.33 % (23/150) of samples, with Borrelia crocidurae as the most frequently detected pathogen. We detected both pathogenic and non-pathogenic viruses in oral (10/72) and plasma (09/78) samples. Finally, we observed a high frequency of genes associated with resistance and virulence: 10% of samples expressed at least one AMR gene (ARG), and 24% expressed virulence factor genes. Resistance to widely used beta-lactam antibiotics was the most prevalent. Our findings provide critical data on oral and plasma microbiomes in the context of acute febrile illness in Senegal while expanding understanding of circulating ARGs.

The human gut microbiome plays a critical role in host health, yet its functional organization in disease remains poorly understood. Most studies focus on taxonomic composition or pathway abundance, which fail to capture higher-order interactions governing system-level behavior. Here, we investigated microbiome functional organization in inflammatory bowel disease (IBD), including Crohns disease (CD), ulcerative colitis (UC), and healthy controls (HC), using a network-based framework across 60 metagenomic samples. Functional pathway profiles were used to construct correlation-based interaction networks, followed by analysis of network topology, functional redundancy, keystone pathway architecture, and system robustness. Disease-associated networks (CD and UC) exhibited reduced global connectivity, increased modular fragmentation, and centralization of keystone pathways, indicating a shift from distributed organization to more fragmented and fragile network structures compared to healthy controls. Notably, machine learning models demonstrated that network-derived features achieved higher classification performance (accuracy up to 0.824) compared to redundancy-based measures. These findings reveal that microbiome dysfunction in IBD is driven by large-scale reorganization of functional interaction networks rather than loss of functional capacity. This study highlights the importance of network-level analysis in understanding microbiome-associated disease and provides a systems-level framework for future research.

Recent advances in high-throughput sequencing and novel culture techniques have revolutionized our understanding of the human microbiota. However, most studies primarily focused on bacterial communities, often overlooking the fungal component. Building upon our previous metagenomic analysis of the Inuit oropharyngeal microbiome 1, this study used culturomics to provide a more comprehensive view of both bacterial and fungal communities. We analyzed oropharyngeal swabs from the Qanuilirpitaa? 2017 Inuit Health Survey 2, demonstrating the complementarity of metagenomic and culturomic approaches. Our findings highlight the importance of culturomics in revealing low-abundance microorganisms, particularly fungi, which are often underrepresented in metagenomics data. Moreover, we designed an approach to isolate previously uncultivated species. We described two Pauljensenia sp., and provided insights into the phylogenetic relationship between Schaalia and Pauljensenia genera. This study underscores the necessity of a holistic approach to microbiome research, combining multiple techniques to fully elucidate microbial diversity in unique populations like the Inuit.

Gingival inflammation is associated with dysbiotic oral biofilms characterized by reduced nitrate-reducing capacity and diminished nitric oxide (NO) bioavailability. While dietary nitrate has been shown to influence oral microbial activity, the effects of sustained, localized nitrate delivery on oral biofilm ecology and gingival inflammation remain incompletely defined. In this randomized, double-blind, placebo-controlled trial, 30 adults with gingival bleeding were assigned to receive localized prebiotic nitrate (~0.989 mmol per dose) or placebo for 21 days. The primary outcome was mean bleeding on probing (mBOP). Secondary outcomes included modified Gingival Index (mGI), Quigley-Hein plaque index (QHPI), salivary nitrite (as a proxy for NO bioavailability), oral pH, and microbiome composition assessed by 16S rRNA gene sequencing. Prebiotic nitrate supplementation formulation delivered in a slow-release chewing gum significantly reduced mBOP (25.7% to 15.3%; p = 0.0002) compared to placebo chewing gum. Salivary nitrite levels and oral pH increased, indicating enhanced nitrate metabolism. Microbiome analysis demonstrated enrichment of nitrate-reducing taxa, including Rothia mucilaginosa and Neisseria spp., and a relative reduction in inflammation-associated genera such as Prevotella and Porphyromonas. Localized prebiotic nitrate formula delivered in a functional chewing gum was associated with reduced gingival inflammation and shifts in oral microbiome composition consistent with enhanced nitrate-reducing capacity critical in nitric oxide formation. These findings support a role for biofilm-directed nutritional modulation as a non-antimicrobial approach for managing gingival inflammation and improving nitric oxide bioavailability.

Intestinal epithelial cells are central players in mucosal barrier integrity and host-microbe interactions. Genetic studies have revealed that epithelial dysfunction is a key contributor to the pathogenesis of inflammatory bowel disease. Non-SMC condensin II complex subunit D3 (NCAPD3) is essential for chromatin organization and stability. NCAPD3 also promotes antimicrobial defense and autophagy responses in vitro. NCAPD3 expression is decreased in intestinal epithelial cells from patients with ulcerative colitis; however, it is not known whether loss of NCAPD3 expression drives intestinal barrier dysfunction or is a result of disease-associated inflammation. To investigate this relationship in vivo, a tissue-specific approach was required, as global constitutive knockout of NCAPD3 is embryonic lethal. Therefore, a transgenic mouse line with doxycycline-inducible expression of a short hairpin RNA targeting NCAPD3 restricted to villin-expressing cells was generated (NCAPD3KD mice) to enable the study of NCAPD3 function in the intestinal epithelium. Treatment of NCAPD3KD mice with 9-tert-butyl doxycycline resulted in [~]75% reduction of NCAPD3 protein in EpCAM intestinal cells. Short-term epithelial NCAPD3 knockdown did not induce spontaneous colitis but was associated with increased serum amyloid A and a trend towards increased intestinal permeability. Upon dextran sodium sulfate or Salmonella enterica serovar Typhimurium {Delta}AroA challenge, NCAPD3KD mice exhibited exacerbated weight loss, higher disease activity, increased histopathological damage, abnormal colonic cytokines and chemokines, and significantly increased intestinal permeability. These results indicate that NCAPD3 expression in the intestinal epithelium is required for optimal barrier maintenance and antimicrobial defense under chemical or microbial stress. These findings support prior in vitro observations and solidify NCAPD3 as a regulator of intestinal epithelial barrier function and mucosal host defense. Author SummaryNCAPD3 is a multifunctional protein with established roles in chromatin organization, genome stability, mitochondrial function, and antimicrobial defense. Dysregulated NCAPD3 is implicated in human diseases, such as inflammatory bowel disease (IBD) and microcephaly; however, due to its essential role in cellular division, determination of whether NCAPD3 loss drives these pathologies in vivo has been lacking. Using a new transgenic mouse model that selectively reduces NCAPD3 expression in intestinal epithelial cells, our study establishes NCAPD3 as an epithelial regulator of the mammalian intestine that enhances epithelial barrier resilience and antimicrobial defense during stress. Although dispensable for short-term basal homeostasis, NCAPD3 function becomes critical during epithelial injury and enteric infection. Reduced NCAPD3 expression may therefore lower the threshold for inflammatory disease by weakening barrier integrity, amplifying inflammatory cascades, and impairing antimicrobial defenses. These findings position NCAPD3 as a potential modulator of IBD susceptibility and highlight chromatin organization as an important, previously underappreciated layer of intestinal epithelial regulation.

Extreme environments impose severe physicochemical stresses that drive microorganisms to evolve specialized survival strategies. Microbial secondary metabolites determined by biosynthetic gene clusters (BGCs) are recognized as important mediators of microbial adaptation to environmental stress. However, their ecological roles, particularly habitat-dependent preferences across different environments, remain poorly understood. Although extreme environments provide opportunities to mine microbiomes for unique adaptations, such research is hampered by a lack of systematic overview of its genomic diversity, BGC diversity, and the relationships between them. Here, we constructed a standardized extremophilic genomic catalogue (SEGC) from 1,462 metagenomic samples spanning seven representative extreme habitats. The catalogue comprised 54,661 metagenome-assembled genomes representing 21,805 species, 66.1% of which were previously uncharacterized. With this catalogue, we identified 162,855 BGCs distributed across 81.5% of MAGs. Gene cluster family analysis showed the strong habitat dependence largely explained by species-level habitat specificity. Terpene biosynthetic pathways illustrated habitat-linked adaptive strategies, with hopan-22-ol biosynthesis enriched in acid mine, deep sea and hydrothermal plume environments, while retinal-based phototrophy predominated in cryosphere and saline-alkaline habitats. Metatranscriptomic analyses supported in situ activity of these pathways. In conclusion, we presented a global atlas of biosynthetic potential across extreme-environment microbiota and revealed habitat-dependent patterns of secondary metabolism linked to microbial survival.

ObjectivesAntimicrobial resistance (AMR) in Gram-negative pathogens is driven by complex and coordinated molecular mechanisms that remain incompletely characterized. This study integrated phenotypic, genomic, and quantitative proteomic analyses to characterize multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria circulating in an intensive care unit (ICU) in Northeastern Brazil. MethodsA total of 259 Gram-negative isolates collected between 2019 and 2021 underwent species identification, antimicrobial susceptibility testing, and targeted qPCR for resistance genes. Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa representing susceptible, MDR, and XDR phenotypes were selected for whole-genome sequencing and label-free quantitative proteomics. Differential protein abundance was assessed using Limma with |log2FC| > 1 and p < 0.05. ResultsK. pneumoniae (47%), A. baumannii (24%), and P. aeruginosa (21%) predominated. Carbapenem resistance reached 44%, 93%, and 61%, respectively, and MDR/XDR phenotypes occurred in >30% of isolates. Genomic analyses revealed dense resistomes with coexisting {beta}-lactamases (blaKPC, blaNDM, blaCTX-M, OXA) and widespread efflux systems. Proteomic profiling demonstrated phenotype-associated differences in outer membrane proteins, transport systems, regulatory proteins, and metabolic pathways. XDR isolates showed additional enrichment of envelope remodeling proteins, stress response mechanisms, and proteostasis-associated factors. ConclusionsMDR and XDR Gram-negative ICU pathogens exhibit coordinated resistance architecture characterized by accumulation of resistance genes and adaptive proteomic remodeling. Integrated multi-omics approaches provide mechanistic insight into antimicrobial resistance and support improved surveillance and therapeutic strategies. What is known?O_LIAntimicrobial resistance is a priority and a serious problem in global health, resulting in high rates of morbidity and mortality. C_LIO_LIKlebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are on the World Health Organizations (WHO) priority list as major causes of morbidity and mortality worldwide. C_LIO_LIClassical characterization of susceptibility and resistance phenotypes does not capture the complexity of antimicrobial resistance and hampers effective control measures and actions to minimize the evolutionary dynamics of resistance in these bacteria. C_LI What is new?O_LIThe study characterizes the phenotypic pattern of antimicrobial susceptibility, the presence and sequencing of the resistome and virulome, and analyzes the label-free quantitative proteome of susceptible, MDR, and XDR phenotypes in strains of K. pneumoniae, A. baumannii, and P. aeruginosa circulating in hospital ICUs in Brazil. C_LIO_LIMDR and XDR gram-negative phenotypes are associated with a dense resistome, with widespread dissemination of beta-lactamase genes (bla_KPC, bla_NDM, bla_CTX-M, and OXA) and RND-type (MEXs) and acrAB-tolC efflux pumps, without changes in virulence genes. C_LIO_LIProteomic analysis demonstrated increased production of beta-lactamases, components of efflux pump systems, outer membrane protein synthesis, protection for oxidative stress mechanisms, proteins for iron acquisition, and systemic regulators. XDR strains additionally showed enhanced remodeling of the cell envelope, activation of proteostasis, and metabolic adaptation. C_LI

Fusobacterium nucleatum is an anaerobic bacterium strongly associated with the development and progression of colorectal cancer (CRC). Its pathogenic mechanisms involve the LuxS/AI-2 quorum sensing (QS) system, which regulates biofilm formation, virulence factor expression, and host immune evasion. Targeting LuxS represents a promising anti-virulence strategy that could disrupt bacterial communication without inducing selective pressure for antibiotic resistance. In this study, we employed a computational drug repurposing pipeline to identify FDA-approved drugs capable of inhibiting the LuxS enzyme in F. nucleatum. We performed structure-based virtual screening of 9,466 compounds from DrugBank using AutoDock Vina against the AlphaFold-predicted LuxS structure (UniProt: A0A133NIU3). From 1,082 initial hits (binding energy [&le;] - 7.0 kcal/mol), we applied ADMET filtering and composite scoring to select the top 5 candidates. Molecular dynamics simulations (10 ns each) using OpenMM with the AMBER14 force field confirmed the stability of all five protein-ligand complexes (RMSD < 2.0 [A]). The most promising candidates include Tubocurarine ({Delta}G = -16.97 kcal/mol, RMSD = 1.87 [A]), Docetaxel ({Delta}G = -13.22 kcal/mol, RMSD = 1.81 [A]), Metyrosine ({Delta}G = -13.78 kcal/mol, RMSD = 1.97 [A]), and Ergometrine ({Delta}G = -13.22 kcal/mol, RMSD = 1.92 [A]). These results constitute an exploratory computational basis that requires subsequent experimental validation through in vitro and in vivo assays, and provide candidates for testing as anti-quorum sensing agents against F. nucleatum, with potential implications for CRC prevention and treatment.

O_LIWD40 is a highly conserved protein domain in eukaryotes, playing a critical role in various cellular process. C_LIO_LIWe conducted genome-wide functional analysis of WD40 genes in Fusarium graminearum--a phytopathogenic fungus that causes severe yield loss and mycotoxin contamination in major cereal crops. C_LIO_LIComprehensive phenome analysis of 119 WD40 gene deletion mutants across 22 distinct phenotypic traits revealed phenotypic divergence within the phenome, establishing a strong correlation between virulence and sexual reproduction. Notably, 21 "core WD40 genes" were identified, offering valuable insights into divergent biological processes. C_LIO_LIPilot interactome studies of Fgwd101 and Fgwd133 provided further insights into their potential pathobiological functions. Our investigation contributes to broadening our knowledge of the biological mechanisms underlying fungal pathogenesis and may assist in the identification of targets for antifungal agents. C_LI

Diet is a major determinant of gut microbiome structure and function, yet the role of dietary electrolytes--particularly sodium--remains poorly defined. Here, we identify dietary sodium availability as a key regulator of gut microbial fermentation and host energy harvest. Using a controlled sodium-sufficient versus sodium-deprived dietary intervention in rats, we integrated shotgun metagenomic sequencing, functional pathway analysis, targeted short-chain fatty acid (SCFA) quantification, and host physiological phenotyping. Sodium deprivation induced a coordinated restructuring of the gut microbiome, characterized by depletion of classical saccharolytic Firmicutes, including multiple Lactobacillus species, and enrichment of stress-tolerant, metabolically flexible taxa. Functional profiling revealed a shift away from growth-associated metabolic programs toward stress-adaptive and nutrient-scavenging pathways. Consistent with these changes, fecal concentrations of key SCFAs--including acetate, butyrate, hexanoate, and valerate--were significantly reduced, indicating impaired microbial fermentative capacity. These microbiome-level alterations translated into measurable host phenotypes, including reduced cecal mass and attenuated weight gain, consistent with decreased microbial energy harvest. Together, these findings establish a functional link between luminal sodium availability, microbial metabolic efficiency, and host energy balance, extending the framework of diet-microbiome interactions beyond macronutrients to include dietary electrolytes. This work identifies sodium as a previously underappreciated ecological constraint shaping gut microbial metabolism and suggests that modulation of dietary sodium intake may influence host metabolic outcomes through microbiome-mediated mechanisms.

Background: Acute necrotizing encephalopathy (ANE) is a rare and severe neurologic complication of viral infection in children, thought to result from a hyperacute cytokine storm causing blood-brain barrier disruption and central nervous system injury. Despite characteristic clinical and radiologic features, ANE remains poorly understood at the molecular level, with no validated biomarkers or targeted therapies. We aimed to determine whether genetic predisposition to ANE due to RANBP2 variants is associated with a distinct immunologic signature. Methods: We conducted a prospective biological study of familial ANE (ANE1, NCT06731790). We included 23 heterozygous carriers of the RANBP2 c.1754C>T (p.Thr585Met) variant from 10 families, and 28 noncarriers (median age, 40 years [range, 4-72]). Soluble immune mediators, transcriptomic analyses, multiparameter flow cytometry, and cellular imaging were analysed in peripheral blood mononuclear cells (PBMCs) and monocytes. Baseline and resiquimod stimulated immune responses were analysed within the same statistical model, with genetic status as the primary predictor. Findings: The RANBP2 Thr585Met mutation was associated with a dysregulated inflammatory phenotype characterized by reduced basal mediator production and exaggerated TNF- responses following stimulation (estimated difference, +2,098 pg/mL; 95% CI, 1,121 to 3,076; P=0.0001). Transcriptomic and flow cytometry analyses showed broad reprogramming of myeloid cells with enrichment of CXCR3-high CD14-high subsets. Expansion of these populations was associated with increased long-term disease burden. The RANBP2 variant was the only independent factor associated this inflammatory phenotype. Interpretation: RANBP2-associated ANE is characterised by a distinct immunological signature that can inform disease stratification and support the development of targeted immunotherapeutic approaches.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease and strongly linked to obesity and insulin resistance. We previously reported that the common nuclear envelope variant rs6461378 (g.842031C>T; SUN1 H118Y) associated with MASLD and related traits including insulin resistance. To gain insight into how wild-type (WT) and H118Y SUN1 might differentially impact insulin signaling, we performed affinity purification-mass spectrometry (AP-MS) in human liver-derived cells stably expressing WT or H118Y SUN1. Unbiased AP-MS revealed a novel SUN1-CUL3 interaction, with comparative analysis showing that WT SUN1 interacted robustly with CUL3, while CUL3 interaction was markedly diminished with H118Y SUN1. Cells in which SUN1 was silenced via siRNA, or in which H118Y SUN1 was ectopically expressed, showed increased CUL3 neddylation, which is required for cullin RING ligase (CRL)-mediated ubiquitination of insulin receptor substrate (IRS) proteins. Inhibition of neddylation restored IRS-1 levels and insulin signaling in H118Y SUN1-expressing cells. Together, our findings provide a potential mechanism of H118Y SUN1-driven insulin resistance and a viable therapeutic approach for its reversal.

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported a protective role for IFN-{gamma} signaling in host defense against Ot infection; however, the underlying mechanisms remain obscure. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-{gamma} signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2-/- and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2-/- mice developed eschar-like lesions similar to those observed in Ifngr1-/- mice, indicating a critical role for the IFN-{gamma}/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2-/- mice displayed impaired bacterial clearance during the recovery phase (day 42), with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-{gamma}-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Author summaryScrub typhus is a potentially severe infectious disease caused by the bacterium Orientia tsutsugamushi (Ot), which is transmitted to humans through the bite of infected mites. Despite its global impact and expanding geographic distribution, the immune mechanisms that protect against this infection remain incompletely understood. In this study, we examined the role of inducible nitric oxide synthase (iNOS), an immune effector molecule that helps the host control infection. Using mouse models, we found that iNOS plays dual and stage-specific roles during Ot infection. Mice lacking iNOS developed dysregulated immune homeostasis during acute infection and exhibited skin lesions resembling the eschars observed in some patients with scrub typhus. In addition, these mice showed delayed bacterial clearance, prolonged inflammation, and increased tissue damage during the recovery phase. Our findings indicate that iNOS contributes not only to host antimicrobial defense but also to the control of excessive inflammation following infection. These results provide new insight into host defense mechanisms in scrub typhus and may help inform future therapeutic or preventive strategies.

Obesity affects one in three adults and is complicated by adipose inflammation, lipotoxicity and cell death. We previously identified RIPK1 as a genetic determinant of human obesity risk and adipose inflammation. Because RIPK1 is the apical kinase in the necroptosis pathway upstream of RIPK3 and the executioner protein MLKL, and emerging evidence links MLKL to lipid metabolism, MLKL has surfaced as a potential metabolic regulator. However, conflicting findings in Mlkl knockout mice fed a high fat diet have left its therapeutic relevance unresolved. MLKL has not been previously targeted through therapeutic knockdown in vivo in the context of diet-induced obesity. Here, we evaluated two independent MLKL antisense oligonucleotides (ASOs) in high fat diet (HFD)-fed C57BL/6J mice. In a 24-week progression model, MLKL ASO markedly reduced body weight, fat mass and hepatic steatosis compared with controls, while preserving lean mass. MLKL knockdown also lowered the respiratory exchange ratio, indicating a shift toward increased fat oxidation. In the intervention model, once obesity was established after 12 weeks of HFD feeding, both MLKL ASOs, and similarly, two independent RIPK1 ASOs, reversed weight gain and improved systemic glucose control. In vitro, MLKL-CRISPR/Cas9 knockout blocked 3T3-L1 adipogenesis, indicating a requirement for MLKL during adipocyte differentiation. However, in mature adipocytes, MLKL siRNA reduced palmitic acid-induced lipid accumulation, increased isoprenaline-stimulated lipolysis, and prevented TNF-mediated suppression of insulin-mediated AKT signalling and glucose uptake. Collectively, these findings demonstrate that partial MLKL suppression reprograms whole-body energy metabolism, enhances insulin sensitivity and limits diet-induced adiposity. MLKL, therefore, represents a promising and mechanistically novel therapeutic target for obesity and insulin resistance.

Bacteria use diverse mechanisms to interact with each other and with eukaryotic hosts, thereby shaping microbiome composition and influencing host health. One of these mechanisms is the production of outer membrane vesicles (OMVs), nanoscale structures that bud off from bacterial cells into the extracellular space. OMVs can deliver bioactive cargoes, including enzymes, RNA and DNA, enabling functions such as cell-to-cell communication, nutrient acquisition and immunomodulation. However, the role of OMVs in beneficial host-associated microbiomes remains unclear. Here, we investigated OMV production in the gut bacteria of the western honey bee (Apis mellifera), which forms a highly conserved and stable microbial community. Using electron microscopy, fluorescence labelling, and nanoparticle tracking analysis, we detected OMV production in every gram-negative species of the normal bee microbiota that we investigated. Vesicles were observed in gut contents of wild and laboratory-inoculated bees, but absent in bees lacking a microbiota. OMVs contained nucleic acids, with more RNA than DNA. Bacterial strains varied in OMV properties, including abundance, size, and zeta potential. These findings indicate that OMVs are likely significant mediators of interbacterial and host-microbe interactions in the bee gut.

Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder characterized by persistent hyperglycemia, insulin resistance, and chronic low-grade inflammation. Despite the widespread use of established therapies such as metformin, long-term glycemic control remains suboptimal, and disease progression is often not adequately prevented. This highlights the need for novel therapeutic strategies that address both metabolic dysfunction and the underlying immunometabolic components of the disease. In this study, GLX10 (GLXM100) was evaluated as a novel immune modulator in a high-fat diet (HFD) and low-dose streptozotocin (STZ)-induced rat model of T2DM over a 91-day period. Glycemic outcomes were assessed using terminal random blood glucose and oral glucose tolerance testing (OGTT), with glucose exposure quantified by area under the curve (AUC 0-120). Complementary in vitro investigations were performed in hepatic and macrophage cell models to assess cytocompatibility, nitric oxide production, and modulation of pro-inflammatory cytokines, including IL-6 and TNF-. GLX10 treatment resulted in a significant reduction in random blood glucose levels and a marked improvement in glucose tolerance compared to diabetic control animals. Importantly, GLX10 demonstrated greater improvement in OGTT AUC compared to metformin under the same experimental conditions, indicating enhanced dynamic glucose regulation. In vitro, GLX10 maintained viability in normal hepatic cells while significantly suppressing nitric oxide production and inflammatory cytokine outputs in macrophages, supporting a favorable safety and immune profile. Collectively, these findings demonstrate that GLX10 exerts robust antidiabetic activity through a dual mechanism involving metabolic regulation and suppression of inflammatory signaling. The integration of in vivo efficacy with supportive in vitro safety and mechanistic data provides a strong preclinical foundation and supports the further development of GLX10 as a promising therapeutic candidate for T2DM.

Aim: To comprehensively characterize the salivary proteome in periodontitis using Orbitrap Astral data-independent acquisition mass spectrometry (DIA-MS), identify an atlas of differentially expressed proteins (DEPs), and develop a machine learning-derived multi-protein biomarker panel for non-invasive diagnosis of stage III/IV periodontitis. Materials and Methods: Unstimulated saliva samples from 199 participants (periodontal health/gingivitis, n=120; stage III/IV periodontitis, n=79) were analyzed by Orbitrap Astral DIA-MS. DEPs were identified, and pathway enrichment analysis was performed. A two-tier machine learning pipeline, integrating pathway-based feature selection with cross-validated evaluation, was applied to identify the optimal diagnostic panel. Results: Orbitrap Astral DIA-MS quantified 5,597 salivary proteins and 1,966 DEPs (|log2FC|>0.5, FDR<0.05). Pathway analysis identified 14 periodontitis-relevant KEGG pathways, including Th17 cell differentiation, IL-17 signaling, neutrophil extracellular trap formation, and complement and coagulation cascades. A four-protein panel (TEC, RAC1, MAPK14, KRT17) achieved an area under the curve (AUC) of 0.985 plus-or-minus sign 0.010, with 83% sensitivity and 100% specificity. The panel was corroborated using public datasets. Conclusions: To our knowledge, this study represents the first application of Orbitrap Astral DIA mass spectrometry in periodontitis research, establishing a disease-specific DEPs atlas and a salivary biomarker panel with high diagnostic accuracy for stage III/IV periodontitis, providing a foundation for future external validation studies.