Metabolomics

Background & Aims: Per- and polyfluoroalkyl substances (PFAS) are persistent endocrine-disrupting chemicals associated with metabolic dysfunction, including metabolic dysfunction-associated steatotic liver disease (MASLD). While PFAS perturb lipid and bile acid (BA) metabolism in a sex-specific manner, the underlying mechanisms remain unclear. We tested whether steroid hormones mediate PFAS-associated metabolic alterations. Methods: In 104 patients with biopsy-characterized MASLD, we performed sex-stratified analyses applied liquid chromatography coupled to mass spectrometry (LC-MS) for chemical analysis, integrating circulating steroids, PFAS exposure, hepatic lipidomics and BA profiles. Results: Steroid hormones were associated with MASLD severity in a sexually-dimorphic manner. Dihydrotestosterone showed consistent inverse associations with steatosis, fibrosis, necroinflammation and insulin resistance, particularly in females. PFAS exposure was associated with altered steroid profiles, predominantly indicating suppressed steroidogenesis in females. These PFAS-associated hormonal changes were linked to downstream alterations in hepatic lipids and BAs. Mediation analysis supported indirect effects of PFAS on metabolic pathways via steroids, including testosterone/epi-testosterone-mediated effects on ether phospholipids and estradiol-mediated effects on lithocholic acid. Females exhibited stronger PFAS-steroid-BA associations, whereas males showed weaker, lipid-centric effects. Conclusions: PFAS exposure is associated with sex-specific disruption of steroid hormone pathways that may link environmental exposure to lipid and BA dysregulation in MASLD. These findings identify steroid hormones as potential key mediators of PFAS-associated metabolic dysfunction and highlight sex as a critical determinant in environmental liver disease.

Background and aimsCirculating complement is associated with occurrence of alcohol-associated hepatitis (AH) and is a potential biomarker to distinguish AH from alcohol cirrhosis (AC). Complement contributes to kidney injury, a condition often occurring in patients with alcohol-associated liver disease (ALD). However, little is known regarding complement in cross talk between liver and kidney in ALD. Here we tested the hypothesis that urinary complement would provide potential biomarkers for ALD and insights into mechanisms of liver-kidney crosstalk in the pathogenesis of ALD. MethodsPlasma and urine were collected at admission from patients with sAH, healthy controls (HC), and heavy drinkers without liver disease (HD) (from the multicenter Alcohol Hepatitis Network) and with AC (from the Northern Ohio Alcohol Center). Urine was subjected to unbiased proteomics analysis and plasma complement assessed by multiplex/ELISA assays. 30- and 90-day mortality was tracked in patients with sAH. ResultsAll three complement activation pathways were perturbed in plasma and urine of patients with sAH and AC compared to HC and HD. Components of the lectin and classical pathways in urine were associated with 30- and 90-day mortality in patients with sAH. When 4 complement proteins were combined, they distinguished sAH from AC (AUC 0.78), equivalent to that of MELD (AUC 0.65). There was no correlation between complement in plasma and urine, suggesting an independent impact of sAH on complement in kidney and liver. ConclusionThe urinary proteome revealed complement protein signatures associated with sAH and AC, providing valuable insights into the potential for complement biomarkers and the mechanisms of liver-kidney crosstalk in ALD.

IntroductionRetinoids are bioactive vitamin A derivatives that regulate cellular differentiation and gene expression, yet their reliable quantification remains challenging due to low abundance, structural isomerism, and sensitivity to ionization conditions while handling. ObjectivesIn this study, we performed a systematic optimization of liquid chromatography-mass spectrometry (LC-MS)-based detection of retinoids across tissues and biofluids. MethodsChromatographic separation, adduct formation, ionization parameters, fragmentation behavior, and extraction procedures were evaluated in an integrated workflow. ResultsChromatographic conditions influenced not only retention time but also the ionic species detected, affecting precursor selection for MS{superscript 2} analysis. Retinoids exhibited compound-dependent responses to electrospray ionization and collision energy, requiring tailored acquisition parameters. Extraction experiments demonstrated differential recovery among retinoid classes and revealed matrix-dependent behavior, indicating that protocols used for tissues cannot be directly transferred to low-abundance biofluids. Using optimized conditions, retinoids were detected in mouse cerebrospinal fluid (CSF) at concentrations approaching the analytical detection limit, where MS{superscript 2} confirmation was necessary for reliable identification. ConclusionTogether, our results provide a framework for reproducible retinoid profiling across biological matrices and enables comparative studies of retinoid biology in low-volume and low-abundance biofluids.

Pseudomonas putida strain KT2440 is a crucial model organism for synthetic biology and bioengineering applications, yet there currently exists no comprehensive metabolomics database comparable to those available for other model organisms. This gap hinders the use of untargeted metabolomics for exploratory analyses in this system. We developed the P. putida metabolome reference database (PPMDB v1) to address this limitation by consolidating metabolite information from multiple sources and expanding coverage through computational predictions. The database was constructed by curating metabolites from BioCyc, BiGG, and other literature sources, then computationally expanding this collection using BioTransformer environmental transformation predictions to generate additional predicted metabolites. We enhanced the databases utility for molecular annotation in metabolomics studies by incorporating analytical properties including collision cross-sections, tandem mass spectra, and gas-phase infrared spectra. These analytical properties were gathered from existing measurement data or predicted using computational tools. We further augmented the database through inclusion of reaction information and pathway annotations, facilitating biological interpretation of metabolomics data. This publicly available resource fills a critical gap in P. putida research infrastructure, supporting metabolite annotation and biological interpretation in untargeted metabolomics studies and enabling in-depth exploratory analyses of this important synthetic biology platform at the molecular level. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713193v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c8828forg.highwire.dtl.DTLVardef@1f3a5c5org.highwire.dtl.DTLVardef@1084535org.highwire.dtl.DTLVardef@1f7ca4a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Identifying metabolites and metabolic reactions specific to a cellular state, such as inflammatory state in immune cells, is of great interest, as it can provide important biomarkers and point to compounds and reactions of specific biological functions. However, many cell state-specific metabolites remain in the unannotated part of metabolome. Here we identified a series of sulfur-containing metabolites that are actively produced in macrophages upon classical activation, but not in resting state or alternative activation state. Isotopic tracing, in vitro assays and genetic perturbations further revealed that they are formed from reactions between free cysteine and several important intermediates in glycolysis and TCA cycle. Upon classical activation, macrophages specifically upregulate the import of cystine via Slc7a11, supporting the production of these adducts. Their production dynamically responds to changes in central metabolism, environmental nutrient levels, and is regulated by nitric oxide. Finally, we confirmed these newly identified compounds also present in human samples, and most of them are significantly elevated in inflammatory granuloma annulare lesions. This work elucidated a previously uncharted part of metabolic network that is associated with inflammation and metabolic stress condition, which has important implications and set foundation for many future discoveries.

Background & Aims: People with HIV (PWH) who achieve hepatitis C virus (HCV) cure may retain persistent metabolic alterations, particularly those with advanced fibrosis or cirrhosis. This study aimed to characterize plasma metabolomic and lipidomic profiles associated with cirrhosis in PWH at one and five years post-HCV therapy. Methods: Two cross-sectional studies evaluated PWH one (n=48) and five (n=30) years post-HCV therapy. Cirrhosis was defined as a liver stiffness measurement (LSM)[≥]12.5 kPa. Metabolomics and lipidomics were performed using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS), respectively. Data were analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA) and generalized linear models (GLM), adjusting for relevant covariates. Results: At one and five years, 32 (66.7%) and 10 (33.3%) participants, respectively, had cirrhosis. OPLS-DA identified 235 and 229 metabolites with variable importance in projection (VIP)scores >1. At one year, cirrhosis was associated with elevated levels of glycerophospholipids, sphingomyelins, and amino acids, and lower levels of triglycerides. At five years, cirrhotic PWH exhibited higher levels of glycerophospholipids and acyl-carnitines, together with lower levels of triglycerides and amino acids. Conclusions: PWH with cirrhosis post-HCV cure exhibits a persistently altered metabolic profile stable for five years, suggesting ongoing liver disease progression. These findings underscore the need for continued long-term monitoring of this population.

Background: Adolescence is a critical period of neurodevelopment with the emergence of chronic medical conditions and increasing exposure to long-term medications. P-tau217 is a sensitive blood-based biomarker of neuropathology in older adults, yet its developmental behavior and susceptibility to common clinical factors in youth are unclear. Here we tested whether p-tau217 varies with age, comorbidity, or medication use during adolescence; and whether collection method (venous vs Tasso+ capillary) yields comparable concentrations. Methods: In an adolescent cohort, plasma p-tau217 was measured by Simoa-X. Paired venous and Tasso+ capillary samples were also analyzed from adult volunteers for methodological comparison Results: In adolescents (n=41; mean age 16{+/-}2.6 years), p-tau217 did not correlate with age or BMI z-score and did not differ by psychiatric, cardiometabolic, or gastrointestinal comorbidity, nor by corresponding medication use. In contrast, p-tau217 concentrations were >10-fold higher in Tasso+ capillary plasma than venous plasma, a discordance replicated in paired adult samples. Conclusion: Plasma p-tau217 appears physiologically stable across common clinical variables in adolescence, but highly sensitive to biospecimen collection method. Venous and Tasso+ capillary plasma should not be directly compared or pooled until methodological differences are resolved. These data provide a developmental baseline and critical methodological caution for pediatric neuroscience and decentralized biomarker studies.

Metabolic syndrome (MetS), characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension, affects a substantial proportion of the global population and increases the risk for cardiovascular disease, diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD). Despite its prevalence, there are currently no effective pharmacological therapies targeting MetS, highlighting the need to identify novel etiological mechanisms, particularly within the gastrointestinal (GI) tract. Using a mouse model of MetS and healthy lean controls, we assessed the colonic microenvironment through metabolomic, transcriptomic, and microbiome analyses. Colonic organoids were cultured to further explore epithelial alterations. Additionally, human MetS fecal metabolomics data were cross-compared with the mouse model to validate translational relevance. MetS mice exhibited upregulation of colonic anabolic pathways, including glycolysis, the pentose phosphate pathway, and the tryptophan/kynurenine pathway, without evidence of intestinal inflammation. Microbiome analysis revealed an increased abundance of the genus Lactobacillus in MS NASH mice. Colonic organoids from MetS mice showed altered goblet cell differentiation. Comparative analysis with human MetS fecal metabolomics demonstrated similar dysregulated pathways, underscoring the translational relevance of these findings. Our study reveals significant metabolic and microbial alterations in the colon of MS NASH mice, implicating a dysfunctional GI tract as a potential etiological factor in MetS. These findings highlight specific metabolic pathways and microbial signatures that could serve as future therapeutic targets for MetS. NEW & NOTEWORTHYThis study identifies the colon as a metabolically active tissue affected in metabolic syndrome. Despite the absence of intestinal inflammation, MS NASH mice displayed altered colonic metabolism and microbiota composition, with conserved metabolite changes matching those seen in humans with metabolic syndrome. These findings highlight colonic metabolic dysfunction as a potential driver of gut dysbiosis and disease progression in metabolic syndrome and MASLD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/716131v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1b7c685org.highwire.dtl.DTLVardef@4a832aorg.highwire.dtl.DTLVardef@1e95c66org.highwire.dtl.DTLVardef@1b14209_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe composition and function of the gut microbiome have been shown to contribute to both health and disease. One of the most powerful modulators of microbial composition and function is diet. Materials & MethodsUsing the E{micro}-TCL1 murine model of B-cell chronic lymphocytic leukemia (CLL), we assigned male and female mice to a high-fat, high-carbohydrate Western diet (HF) or standard chow (CH) diet. ResultsMice consuming a HF diet had significantly shorter survival than those consuming a CH diet, irrespective of sex, with female mice exhibiting particularly poor outcomes. We also observed a significant increase in splenic involvement by CLL in the HF diet-fed mice at time of sacrifice. Mice receiving the HF diet demonstrated immediate and profound effects on the gut microbiome, marked by reduced alpha diversity and significantly different community composition as measured by beta diversity. Notably, there was a sustained increase in Akkermansia muciniphila and Bacteroidetes thetaiotaomicron in HF diet-fed mice, coupled with a corresponding increase in microbiome functional pathways related to arginine and histidine biosynthesis, chitin degradation, and nucleotide biosynthesis. DiscussionCollectively our data provides evidence of the profound and sustained impact of a high-fat Western diet upon the gut microbiome community and CLL pathogenesis in the E{micro}-TCL1 murine model of CLL.

BackgroundElevated postprandial hypertriglyceridemia (PP-HTG) is a significant risk factor for development of cardiovascular diseases, however, the mechanisms underlying its exaggerated rise remains poorly understood. MicroRNAs (miRs) are known to be implicated in the regulation of lipid metabolism, thus identifying them as potential key players. We presently investigated whether miRs may control postprandial triglyceride (PP-TG) response. MethodsPostprandial changes in circulating miR expression as a function of the degree of postprandial TG response were evaluated in non-dyslipidemic healthy subjects (n=32). The impact of miR-100-5p on hepatic gene expression was evaluated in differentiated Caco2 and HepG2 cells by analysis of hepatic transcriptome (RNAseq), western blot and ELISA. In vivo studies were conducted in C57BL/6J mice overexpressing mimic miR-100-5p. ResultsPostprandial variation in circ-miR-100-5p levels inversely correlate with PP-TG response. Cir-miR-100-5p was preferentially associated with TGRL particles of intestinal origin in subjects exhibited a low PP TG response. Differential analysis of transcriptome from HepG2 cells transfected by either mimic miR-100-5p or scrambled mimic miR as control allowed us to identify PCSK9 as a down-regulated gene. Overexpression of miR-100-5p in HepG2 cells significantly decreased PCSK9 mRNA levels by 52% (p<0.0001), cellular protein content by 28 % (p<0.0001) as well as PCSK9 secretion by 39% (p<0.0001). In vivo systemic delivery of mimic miR-100-5p induced a two-fold reduction (p<0.0001) on PP-TG in mice, such effect being abolished by blocking the circulating form of PCSK9 with alirocumab. Finally, we revealed a significant inverse relationship between circulating miR-100-5p expression levels and both PCSK9 levels and the magnitude of postprandial hypertriglyceridemia. ConclusionTaken together, our observations reveal that miR-100-5p regulates postprandial hypertriglyceridemia by targeting PCSK9, thus enhancing hepatic triglyceride-rich lipoproteins (TGRL) uptake. Our findings allow us to propose circ-miR-100-5p as a potential biomarker for early identification of subjects at high cardiovascular risk, prior to appearance of classical clinical features of metabolic disorders. Postprandial clinical study, HDL-PP (NCT03109067) Lay summaryThis study examined whether miRs may control postprandial triglyceride response Key findingsOur data reveal that miR-100-5p regulates postprandial hypertriglyceridemia by targeting PCSK9 Our observations allow us to propose miR-100-5p as a potential biomarker for early identification of subjects at high cardiovascular risk

Structured AbstractO_ST_ABSINTRODUCTIONC_ST_ABSAdults with Down syndrome (DS) have a higher risk of developing Alzheimers disease (AD). As gut microbiota (GM) alterations have been reported in AD, we investigated their association with cognitive decline and plasma AD biomarkers in DS. METHODSFecal and plasma samples were collected from 58 adults with DS (21-75 years) and 30 euploid controls (CTRL; 25-83 years). GM was profiled using 16S rRNA sequencing. Major Neurocognitive Disorder (NcD) was diagnosed according to DSM-5 criteria. Plasma levels of p-Tau181, NfL, and GFAP were measured using the Simoa platform. RESULTSCompared with CTRL, DS showed significant changes in UBA1819 and Intestinibacter genera, previously reported to be associated with mild cognitive impairment. Furthermore, DS with NcD were characterized by a reduced abundance of Roseburia genus, which was also negatively associated with plasma levels of AD biomarkers. CONCLUSIONAdults with DS display AD-associated changes in GM partially resembling those previously reported in euploid AD patients

ObjectiveTo establish a fetal lamb model of complex gastroschisis and characterize the impact on the intestines over time. Summary Background DataGastroschisis is a congenital abdominal wall defect and in its complex form is associated with serious morbidity. Robust large-animal models may help understanding are lacking. MethodsAt gestational day 75, gastroschisis was induced by creating a 1-cm abdominal wall defect reinforced by a silicone ring. Fetuses were assessed either at term or at mid-gestation (13-21 days post-induction). The primary outcome was complex gastroschisis occurrence, defined by bowel stenosis, atresia, volvulus, perforation or necrosis; otherwise classified as simple. At mid-gestation, occurrence was compared between early (13-16 days) and late (17-21 days) intervals. Secondary outcomes included prenatal ultrasound findings, in vivo bowel motility and morphology, ex-vivo bowel contractility, amniotic fluid composition, and histology across complex, simple, and normal groups. ResultsGastroschisis was induced in 32 fetuses. At term (n=14), all survivors (7/14; 50%) had complex gastroschisis, with impaired bowel motility, altered enteric neural contractile responses and smooth muscle remodeling. At mid-gestation (n=18), complex gastroschisis occurred more frequently in the late than in the early group (71% vs. 11%; p=0.035). Mid-gestation gastroschisis fetuses showed greater intra-abdominal bowel dilatation on ultrasound and higher amniotic fluid digestive enzyme levels compared with non-operated littermates, with the greatest dilation observed in complex gastroschisis. ConclusionsThis model consistently reproduces complex gastroschisis in term survivors. After induction, complex gastroschisis occurrence increases with disease duration and is accompanied by structural and functional bowel changes.

Age-related macular degeneration is a common ocular disease that causes vision loss in the elderly, with a complex set of risk factors and proposed mechanisms of pathogenesis. A powerful method for investigating changes in disease is metabolomics, by which small molecules can be identified and quantified simultaneously. We report here the metabolic analysis of human RPE-choroid tissue in aging and macular degeneration (AMD), as well as comparisons of human macular and extramacular RPE-choroid and neural retina. Levels of 215 metabolites were determined in young donors, AMD donors (early/intermediate, geographic atrophy, and neovascularization) and age-matched controls. The largest number of metabolite differences were observed between young and healthy aged controls, as opposed to between aged controls and any stage of AMD. Two notable metabolites found to be increased in aging choroids are trimethylamine N-oxide and uric acid, both of which were significant after Bonferroni correction. A mouse endothelial cell line treated with a high concentration of uric acid exhibited reduced migration in a wound closure assay. This study provides initial insights into the metabolome of human choroids in varying states of age and macular degeneration, as well as functional implications of these changes in the aging choroid.

Background & AimsMetabolic disorders such as dyslipidemia, metabolic dysfunction-associated steatotic liver disease (MASLD), and diabetes are promoted by chronic pro-inflammatory and pro-oxidative states. Paraoxonase 1 (PON1), a liver-derived HDL-associated enzyme, plays an important antioxidant role by hydrolyzing oxidized lipids and protecting against oxidative stress- induced damage. Genetic variation in PON1, particularly in promoter and coding regions, modulates enzyme expression and activity, thereby influencing susceptibility to metabolic and cardiovascular diseases. This study investigated the genetic determinants of serum paraoxonase (PONase) activity and their relationship with dysmetabolic phenotypes. MethodsA genome-wide association study was conducted in 922 Portuguese individuals from the PREVADIAB2 cohort. Genetic variants and haplotypes related to PONase activity were analyzed, and associations with dysglycemia and liver fibrosis were evaluated in individuals aged over 55 years. ResultsWe identified two key PON1 variants as determinants of PONase activity: rs2057681 (in strong linkage disequilibrium with the non-synonymous Q192R variant) and rs854572 (located in the promoter region). Analysis of rs854572-rs2057681 haplotypes revealed that specific combinations differentially modulate PONase activity and confer risk or protection for dysglycemia and liver fibrosis, depending on the rs2057681 genotype context. Notably, although PONase activity was strongly associated with PON1 variants, it did not directly correlate with dysmetabolic phenotypes, suggesting that genetic context and haplotype structure, rather than enzyme activity alone, shape disease susceptibility. ConclusionsThese findings highlight the complex genetic architecture of PON1 and its role in metabolic disease risk, supporting the use of PON1 genetic information to uncover predisposition to dysmetabolic conditions. Our results provide insights into the interplay between PON1 genetics, enzyme function, and dysmetabolism, with implications for risk stratification in metabolic liver disease. Lay SummaryPON1 is a liver-derived gene that encodes an enzyme involved in protection against oxidative stress, a key contributor to metabolic liver disease and diabetes. In this study, we found that specific combinations of PON1 genetic variants are associated with abnormalities in blood glucose regulation and with markers of liver fibrosis. These associations were dependent on genetic configuration rather than enzyme activity alone, suggesting that PON1 genetic information may help identify individuals at higher risk of metabolic liver disease.

Harbour porpoises (Phocoena phocoena) in the North and Baltic Seas are increasingly impacted by anthropogenic pressures, including underwater noise, fisheries and pollution. These pressures correlate with declining population health, particularly affecting the respiratory system. Growing pathological lesions, partly resulting from high prevalence of parasitic infestations and subsequent diseases, can impair tissue function and oxygen supply to distant end-organs. In this study, we applied an integrative MultiOmics approach (proteomics, metabolomics, lipidomics) to analyse the lungs and muscles of 12 wild harbour porpoises with compromised respiratory health. Our aim was to identify dysregulated biological pathways across omics layers to advance insights into adaptive physiological responses and to define disease-associated molecular signatures that could assist health assessments. Our analysis revealed pronounced immune system and antioxidative responses in the lungs and muscles, indicated by enhanced immunoglobulins, plasmalogens and glutathione-related proteins. In the lungs, high cardiolipin levels and reduced collagen suggest impaired tissue structure and function, while tissue maintenance processes were elevated in the muscle. Both tissues exhibited metabolic alterations suggestive of energetic imbalance, including increased purine metabolism in the lung and decreased lipid metabolism in the muscle. Several dysregulated molecules were shared across tissues, pointing to pathophysiological effects. The proposed disease-associated molecular signatures included the protein SLC25A4, the metabolite O-phosphoethanolamine and the lipid TG O-16:0_16:0_20:4 for the lung, and the protein SPEG, the metabolite pipecolic acid, and the lipid BMP 18:1_22:6 in the muscle. Our findings elucidate the complexity of molecular mechanisms linking anthropogenic and environmental stressors with vulnerability and resilience in a marine sentinel species. Furthermore, this study highlights the potential of integrative omics to define disease-related marker panels, thereby supporting ongoing and future health monitoring and conservation efforts.

ObjectiveA catheter-free, non-radiolabeled method that permits in vivo measurement of tissue-specific glucose uptake does not exist. To address this gap, we sought to develop and validate a new, higher throughput mass spectrometry (MS)-based method that combines an injection of insulin with a non-radiolabeled glucose tracer, 2-fluoro-2-deoxyglucose (2FDG), to determine insulin-stimulated tissue-specific glucose clearance in conscious, unrestrained mice. MethodsInjections of saline or insulin with 2FDG were coupled with LC-Q Exactive Hybrid Quadrupole-Orbitrap (LC) MS-based measures of plasma 2FDG and tissue (2-fluoro-2-deoxyglucose-6-phosphate) 2FDGP to determine glucose clearance in mice under several different conditions. ResultsThe newly developed method was first applied to a dose response experiment in mice. Next, the ability of this method to quantify changes in glucose clearance in response to an insulin stimulus was assessed, and glucose clearance was compared between chow and high fat fed mice. Results from these studies showed that insulin-stimulated skeletal muscle and heart glucose clearance can be estimated following a bolus injection of tracer, and these fluxes are blunted in diet-induced obese mice. The broad applicability of this approach was then demonstrated by assessing glucose clearance in a mouse model with anticipated changes in insulin-stimulated skeletal muscle glucose metabolism. ConclusionsThe results validated a new LC-MS method to quantify insulin-stimulated tissue-specific glucose clearance in vivo without the use of catheters or radiolabeled tracers. The method offers great potential because it is designed for application to pre-clinical studies seeking high throughput tests and/or assays that can be coupled with discovery technologies such as genomics, proteomics and metabolomics. HIGHLIGHTSO_LIIn vivo glucose clearance can be estimated by a new non-radiolabeled method. C_LIO_LIThe plasma tracer to tracee ratio is required to determine tissue tracer phosphorylation. C_LIO_LIMeasures of plasma glucose and tracer kinetics are critical for data interpretation. C_LIO_LIThe new method can be combined with omics technologies such as metabolomics. C_LI

Understanding pathogen metabolism is critical for identifying key functions for drug targeting, establishing effective in vitro experimental systems, etc., particularly for metabolically unique organisms such as Leptospira. Pathogenic Leptospira are thought to infect humans from environmental sources; however, direct isolation from environmental samples remains technically challenging and is not yet well established. Here, we report that a ubiquitous environmental bacterium, Massilia sp., produces metabolites that promote the growth of Leptospira interrogans, encountered through an incidental contamination event, and identified in this study. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis showed demonstrated that cultivating of Massilia sp. in R2A medium resulted in the accumulation of metabolites, including branched-chain amino acid (BCAA) intermediates, compared to fresh medium. By combining genome-scale metabolic modeling with experimental validation using cell-free culture supernatant supplementation assays, we demonstrate that BCAA intermediates, particularly 2-ketoisocaproic acid (4-methyl-2-oxopentanoate; 4MOP), a leucine biosynthetic intermediate produced by Massilia sp., enhance Leptospira growth. To investigate the metabolic role of 4MOP, we incorporated transcriptomic data into a genome-scale metabolic network model to generate condition-specific models. Resulted flux distributions indicated that Leptospira catabolized imported 4MOP to produce acetyl-CoA. Our results reveal a previously unrecognized metabolic interaction where metabolites produced by environmental bacteria support the growth of pathogenic Leptospira, offering mechanistic insight into its metabolic requirement. These findings have implications to understand the environmental persistence of Leptospira through its metabolic dependencies on coexisting microbes, and they also help develop better strategies for this pathogen. ImportancePathogenic Leptospira persist in environmental reservoirs, yet the mechanisms supporting their growth remain poorly defined. Here, we find that metabolites produced by common environmental bacteria, Massilia sp., can promote Leptospira growth, suggesting a previously unrecognized metabolic dependency on coexisting microbes. Importantly, this study indicates that combining genome-scale metabolic modeling with experimental validation provides a useful framework for identifying metabolic interactions that are otherwise difficult to resolve using conventional culture-based approaches. Current strategy may facilitate the systematic identification of growth-supporting metabolites and provide a basis for improving selective cultivation for uncultured or difficult to culture organisms. Determination of growth promoting metabolites advances our understanding of pathogen persistence in natural environments and offers a generalized framework to study metabolically dependent microorganisms.

Background and Aims: Alterations in immunoglobulin G (IgG) N-glycosylation are implicated in inflammatory bowel disease (IBD); however, the robustness of IgG glycan signatures across IBD cohorts with diverse demographics and geographic origins remains underexplored. We aimed to determine whether compositional data analysis (CoDA) and machine learning (ML) can identify IBD-related IgG N-glycan signatures and whether these signatures capture disease-associated acceleration of biological aging. Methods: We analyzed the IgG glycome profiles of 1,367 plasma samples collected from healthy controls (HC), symptomatic controls (SC), and people with newly diagnosed Crohn's (CD), and ulcerative colitis (UC) across four cohorts (UK, Italy, United States, and Netherlands). IgG glycosylation was analyzed by ultra-high-performance liquid chromatography, yielding 24 total-area-normalized glycan peaks (GPs). Analyses were performed using cross-sectional data obtained at baseline. CoDA-powered association analyses were used to identify disease-related effects on GPs while controlling for demographic covariates. ML models were trained and evaluated to assess generalizability to unseen cohorts and demographic subgroups, with a focus on discrimination and reliability. Results: Across all cohorts, people with IBD demonstrated accelerated biological aging as quantified by the GlycanAge index. This was accompanied by consistent reductions in IgG galactosylation, with effects partially modulated by age. Classification models trained on glycomics and demographics achieved robust discrimination (AUROC~0.80) between non-IBD (HC+SC) and IBD across cohorts. Conclusion: These findings reveal accelerated biological aging in people with IBD and support the translational potential of IgG glycans as biomarkers and a novel route toward clinically interpretable personalized risk estimates.

BackgroundChronic gastroduodenal symptoms are challenging to diagnose and treat. Body surface gastric mapping provides non-invasive biomarkers of gastric function, but the requirement of a standard meal for postprandial assessment can be difficult for severely symptomatic patients. AimsTo assess the impact of reduced meal sizes and fasting on body surface gastric mapping metrics to determine clinical interpretability under non-standard nutritional loads. MethodsHealthy controls (n=60) underwent a 4.5-hour Gastric Alimetry test. Three age, sex, and BMI-matched groups (n=20 each) were compared: Standard Meal (482 kCal), Nutrient bar + Water (250 kcal), and Fasted (no meal). Principal Gastric Frequency, Gastric Alimetry Rhythm Index, BMI-Adjusted Amplitude, and fed:fasted Amplitude Ratio were analyzed against normative intervals. ResultsMeal status significantly affected amplitude-based metrics; the Standard Meal group exhibited higher BMI-Adjusted Amplitude (p<0.001) and fed:fasted Amplitude Ratio (p=0.001) than Fasted and Bar + Water groups. Frequency and rhythm-based metrics were resilient; Principal Gastric Frequency (p=0.245) and Gastric Alimetry Rhythm Index (p=0.336) showed no significant differences across conditions. While amplitude deviations were common in the Fasted group (20% fell below the normative range), Gastric Alimetry Rhythm Index and Principal Gastric Frequency remained within normal reference ranges for 95% of participants across all conditions. ConclusionsWhile consuming <50% of the standard meal significantly reduces gastric amplitude, gastric rhythm remains stable. Principal Gastric Frequency and Gastric Alimetry Rhythm Index function as reliable biomarkers of gastric myoelectrical function regardless of nutritional state.

Background and AimFecal microbiota transplantation (FMT) in Alcohol-related liver disease (ALD) has shown therapeutic potential, with variable efficacy and unclear mechanism. Because dietary protein influences gut microbiota composition, we hypothesized that donor dietary preconditioning could enhance FMT efficacy. We therefore examined in a murine ALD model if high-protein donor diet improves FMT outcome. MethodsALD was induced in C57BL/6N mice using a Lieber-DeCarli ethanol diet combined with thioacetamide administration for 12 weeks. FMT was performed using stool from diet-modulated donors, and recovery was assessed on day7 post-FMT. Multi-omics analysis using 16s rRNA and mass spectroscopy was performed for Gut microbiota composition, plasma- and stool-metabolome, and hepatic proteomes. Multi-omics outcomes were validated in ALD animal and Huh7 hepatocytes. ResultsBoth protein-based FMTs improved ALD recovery; Veg-FMT demonstrated superior efficacy, significantly reducing hepatic injury (AST 1.2-fold, p=0.002; bilirubin 1.2-fold, p=0.03; steatosis 1.7-fold,p=0.01) and restoring gut barrier integrity (occludin 1.5-fold,p=0.04; mucin 2 2.2-fold, p=002; and plasma endotoxin 1.7-fold, p=0.02). A significant 2-fold increase was observed in Lachnospiraceae NK4A136, Coriobacteriaceae UCG-002, and short-chain fatty acids, particularly caproic acid. Functional validation confirmed that caproic acid promoted hepatic fatty acid {beta}-oxidation through PPAR-dependent mechanisms, reducing triglyceride accumulation and lipogenesis in both cellular and animal models. ConclusionDonor preconditioning with a plant-protein enriched diet enhances FMT efficacy in ALD by gut microbiota modulation with increased metabolites like caproic acid. These findings highlight a microbiota-metabolite-host axis through which diet-modulated FMT improves hepatic lipid metabolism and injury, and identifies a pathway via which FMT imparts its effect. SignificanceThis study identifies a mechanistic basis for improving fecal microbiota transplantation (FMT) efficacy in alcohol-related liver disease (ALD) by demonstrating that dietary preconditioning of donor microbiota improves therapeutic outcomes. We show that plant protein-modulated donor microbiota supplements abstinence-associated recovery through increased production of the microbial metabolite caproic acid, which promotes hepatic fatty acid {beta}-oxidation via PPAR signaling. These findings highlight donor dietary conditioning and microbiota-derived metabolites, rather than microbial composition alone, as important determinants of FMT efficacy. The results suggest that microbial metabolites such as caproic acid may represent potential therapeutic targets or biomarkers to enhance and standardize microbiota-based interventions in ALD. Although the current work is based on a murine model, the identified microbiota-metabolite-host metabolic axis provides a framework for future translational studies aimed at optimizing FMT strategies in liver disease.