Alan K. Jarmusch

Extensive characterization of xenobiotic metabolites remains a major challenge in mass spectrometry-based exposomics, as untargeted high-resolution mass spectrometry (HRMS) can detect extensive chemical diversity but often lacks sufficient structural annotation and prioritization strategies for unknown metabolites. Here, we developed a built-for-purpose HRMS-based workflow combining untargeted metabolomics, in silico metabolite prediction, structural annotation, and toxicity prioritization to characterize xenobiotic biotransformation products using Disperse Orange 61 (DO61) as a case study. Sprague–Dawley rats were exposed to DO61 via oral gavage, and liver, plasma, and urine samples were analyzed by ultra-high performance liquid chromatography (UHPLC) coupled to HRMS in positive and negative ionization modes. Statistically significant features were prioritized and screened against a BioTransformer-generated suspect library containing predicted Phase I, Phase II, reductive, and microbial transformations. HRMS-based annotation, supported by bromine isotope pattern analysis and MS/MS fragmentation, identified putative DO61 metabolites, while SIRIUS-derived molecular formula predictions provided additional confidence through fragmentation-based structural evaluation. The workflow experimentally confirmed reductive azo cleavage as an early DO61 transformation, producing two aromatic amine products, and revealed extensive downstream metabolism of the parent compound and both cleavage products. Computational toxicity prediction further prioritized confirmed and putatively annotated metabolites with potential mutagenic relevance for a future evaluation. This workflow demonstrates how integrating untargeted HRMS with computational approaches can facilitate metabolite discovery, improve structural annotation confidence, and prioritize metabolites for toxicological evaluation, providing a hypothesis discovery strategy for characterizing xenobiotic metabolism and supporting exposure and hazard assessment within the field of exposomics research.

Glaucoma is a group of eye diseases that affects 4 million people in the US and is one of the leading causes of vision loss due to damage to the eye’s optic nerve (ON) which is composed of axons from retinal ganglion cells (RGCs) that transmit visual information to the brain. Injury to the ON often triggers RGC death and subsequent loss of visual function. Despite its increasing prevalence worldwide, effective therapies for glaucoma remain elusive. Notably, the thirteen-lined ground squirrel (TLGS) exhibits intrinsic neuroprotection during hibernation; however, reproducing this protective state pharmacologically has proven challenging. To elucidate the metabolic mechanisms underlying this resilience, we conducted untargeted metabolomic analyses on TLGS retinas at 6 hours, 3 days, and 7 days following ON crush. Retinas from awake and hibernating animals were compared to identify temporal and state-dependent metabolic signatures. Distinct metabolomic profiles were observed in hibernating animals relative to their awake counterparts. Pathway analyses revealed coordinated regulation of amino acid, lipid, and purine metabolism that likely contributes to hibernation-induced resilience. Furthermore, our findings indicate that hibernating TLGS retinas increase exosome biogenesis, prompting in vitro validation using TLGS-derived exosomes, which demonstrated robust neuroprotective and anti-inflammatory effects. Proteomic and transcriptomic characterization of exosomal cargo identified conserved miRNAs, mRNAs, and proteins implicated in redox balance, cytoskeletal stabilization, and stress-response regulation. Collectively, these data support the hypothesis that metabolic reprogramming and exosome-mediated intercellular signaling underlie hibernation-associated neuroprotection. Modulating these pathways may provide a blueprint for novel therapeutic strategies to mitigate neurodegeneration and promote recovery following optic nerve injury. Graphical Illustration depicting state-dependent metabolic responses to optic nerve crush (ONC) injury in Thirteen-lined Ground Squirrels (TLGS). In Awake animals, injury triggers enhanced ATP production through the TCA cycle, leading to excessive reactive oxygen species (ROS) generation and subsequent retinal ganglion cell (RGC) death. In contrast, Hibernating animals shift toward lipid metabolism and utilize ATP for the biosynthesis of ceramides and sphingolipids, promoting membrane integrity and exosomal signaling. Additionally, a range of metabolites associated with hibernation-linked neuroprotection are elevated, contributing to enhanced RGC survival.

Introduction: Juvenile dermatomyositis (JDM) is a rare autoimmune inflammatory myopathy characterized by muscle weakness and distinctive skin rashes. Despite advancements in the clinical understanding of JDM, metabolic disturbances underlying the disease remain poorly understood. Objectives: This study aimed to investigate serum metabolite differences in JDM compared to age- and sex-matched unaffected siblings (US) and unrelated healthy controls (HC), and to identify metabolite abundance differences associated with disease severity. Methods: Serum samples from JDM (n = 16) and adult dermatomyositis (DM; n = 15) patients and corresponding US and HC underwent untargeted metabolomics profiling. Multivariate, univariate, and correlation analyses were employed to identify metabolites differentiating groups and correlating with Physician Global Damage (PGD) scores. Results: JDM patients exhibited modest but discernible alterations in serum metabolites compared to controls, many of which also correlated with PGD. Several bioactive lipids and pyroglutamic acid were upregulated in JDM and positively correlated with PGD. Changes in xanthine, methionine and N-acetylneuraminic acid also indicated increased oxidative stress and inflammation. Markers of increased energy demand and muscle damage, including acylcarnitines, creatine, 4-guanidinobutyric acid, glutamine, and phenylacetylglutamine, were differential and correlated with PGD in some cases. A metabolite abundance gradient from JDM to US to HC groups suggests that siblings help account for genetic and environmental influences on the metabolome. DM patients did not show significant serum changes compared to US. Conclusion: Untargeted metabolomics revealed distinct serum metabolite alterations in JDM, providing insights into disease-related metabolic perturbations. These findings enhance understanding of JDM pathophysiology and inform future large-scale, targeted studies.

Nicotinamide adenine dinucleotide (NAD) is synthesized through both amidated salvage and deamidated pathways. Although NAD-producing enzymes are often overexpressed in cancer cells to meet the high metabolic demands of rapid proliferation and are considered oncogenic, we report that physiological levels of nicotinic acid phosphoribosyl transferase (NAPRT), the first enzyme in the Preiss-Handler arm of the deamidated pathways, suppress tumorigenesis. We show that NAPRT is enriched in gut epithelial cells, where it sustains the NAD pool for an efficient response to stress-induced acute NAD depletion. Consequently, NAPRT deficiency impairs the activity of poly-(ADP-ribose) polymerases and DNA repair, sensitizes mice to chemical-induced colitis and tumorigenesis, as well as to age-associated spontaneous tumor development. Moreover, low NAPRT expression correlates with poor prognosis in several human cancer types. Thus, homeostatic levels of deamidated NAD biosynthesis contribute to tumor suppression, and boosting this pathway may offer a strategy for cancer prevention.

Microbial metabolites play a critical role in regulating ecosystems, including the human body and its microbiota. However, understanding the physiologically relevant role of these molecules, especially through liquid chromatography tandem mass spectrometry (LC-MS/MS)-based untargeted metabolomics, poses significant challenges and often requires manual parsing of a large amount of literature, databases, and webpages. To address this gap, we established the Collaborative Microbial Metabolite Center knowledgebase (CMMC-KB), a platform that fosters collaborative efforts within the scientific community to curate knowledge about microbial metabolites. The CMMC-KB aims to collect comprehensive information about microbial molecules originating from microbial biosynthesis, drug metabolism, exposure-related molecules, food, host-derived molecules, and, whenever available, their known activities. Molecules from other sources, including host-produced, dietary, and pharmaceutical compounds, are also included. By enabling direct integration of this knowledgebase with downstream analytical tools, including molecular networking, we can deepen insights into microbiota and their metabolites, ultimately advancing our understanding of microbial ecosystems.

Per- and polyfluoroalkyl substances (PFASs) are a class of manmade chemicals linked to numerous health outcomes including cancers and increased cholesterol. Consequently, legacy PFASs such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) have been voluntarily phased out of production within the United States since the early 2000s. However, as replacements, emerging PFASs have been introduced over the last 25 years. The presence and accumulation of these emerging PFASs remains largely unexplored due to the complexity of their identification and quantification. Here, we analyzed a unique set of serum samples (n = 156) collected from 2003 to 2021 consisting of three groups: an autoimmune proband, a healthy sibling, and an unrelated control. Quantitative analyses revealed no discernible link between PFAS exposure and autoimmune disease diagnosis; however, nontargeted analysis revealed concerning temporal trends in emerging replacement PFASs the significance of which is unknown. We detected a concerning increase in the chlorinated PFOS-replacement 9Cl-PF3ONS and polyfluoroalkyl phosphate species including 6:2 DiPAP and perfluoroalkyl phosphinic acids (PFPis) in human serum across the US since 2003. In addition, we documented the presence of a novel PFAS chloroperfluorononylphosphonic acid (Cl-PFNPA) in human serum. These analyses provide an assessment of how PFAS exposure has changed since 2003 while exploring potential linkages to autoimmune diseases.

Despite extensive efforts, extracting medication exposure information from clinical records remains challenging. To complement this approach, here we show the Global Natural Product Social Molecular Networking (GNPS) Drug Library, a tandem mass spectrometry (MS/MS) based resource designed for drug screening with untargeted metabolomics. This resource integrates MS/MS references of drugs and their metabolites/analogs with standardized vocabularies on their exposure sources, pharmacologic classes, therapeutic indications, and mechanisms of action. It enables direct analysis of drug exposure and metabolism from untargeted metabolomics data, supporting flexible summarization at multiple ontology levels to align with different research goals. We demonstrate its application by stratifying participants in a human immunodeficiency virus (HIV) cohort based on detected drug exposures. We uncover drug-associated alterations in microbiota-derived N -acyl lipids that are not captured when stratifying by self-reported medication use. Overall, GNPS Drug Library provides a scalable resource for empirical drug screening in clinical, nutritional, environmental, and other research disciplines, facilitating insights into the ecological and health consequences of drug exposures. While not intended for immediate clinical decision-making, it supports data-driven exploration of drug exposures where traditional records are limited or unreliable.

The microbiome plays an important role in immune responses and inflammation in HIV-1 infection. Hence, a deeper understanding of the changes in the microbiome, its function and metabolites, and their interactions prior to HIV-1 infection is potentially important for HIV-1 prevention strategies. Using stool, oral washes, and plasma biospecimens obtained from men who have sex with men (MSM) and who were without HIV-1, we found several differences in microbial ecologies, gene functions, and metabolites between MSM who became HIV-1 infected (Pre-HIV) within six months and those who remained HIV-1 uninfected (Non-HIV). The Pre-HIV group had an enrichment of enzymes involved in purine metabolism, lower amino acid metabolism, and higher oxidative stress before the infection compared to the Non-HIV group. We also introduced a novel and broadly applicable taxon-specific measure of DISruption in COrrelations (DISCO) with other features, such as microbial taxa and metabolites in a given group (e.g., Pre-HIV group) relative to a reference group (e.g., Non-HIV group). Using DISCO, we identified several gut and oral species with disrupted correlations prior to HIV-1 infection. Application of DISCO to external datasets revealed that Prevotella spp . are consistently disrupted in their correlations across multiple cohorts prior to or following HIV-1 infection.

Metformin (Met) and liraglutide (Lira) are preferred diabetes therapies that may improve glycemia by modulating the gut microbiome, but the mechanisms and pathways are unknown and few data exist in youth-onset type 2 diabetes (Y-T2D). In a 3-month parallel clinical trial in African American Y-T2D randomized to Met (n = 14) or Met+Lira (n = 11), we compared gut microbial composition and metabolomic profiles and determined the relationship of changes in microbial abundance with glycemia and plasma metabolites. After 3 months, Met was associated with greater relative abundance of Eubacterium and Eubacterium rectale and lower Bacteroides ovates (p < 0.05). Met+Lira was associated with greater Bacteroides fragilis and lower Streptococcus thermophilus (p < 0.05). Met group had increased (>1.5-fold) plasma cholic secondary bile acids (sulfochenodeoxycholic acid, nutriacholic acid, alpha-muricholic acid, and C24 dihydroxy bile acid; p ≤ 0.002). The change in nutriacholic acid correlated with lower fasting glucose (r = -0.7, p < 0.05). Shifts in microbiota taxa were not associated with plasma short-chain fatty acids (SCFA), hemoglobin A1c or glucose. Short-term Met and Met+Lira in Y-T2D were related to distinct shifts toward bile acid and SCFA-producing gut microbiota taxa, and secondary bile acid metabolites correlated with improved glycemia, suggesting bile acid pathways may be important modulators of glycemia in youth on metformin.Clinical trials.gov identifier: NCT02960659.

Rationale: Reproducible analytical instrumentation system performance is critical for mass spectrometry, particularly metabolomics, aptly named system suitability testing. We identified a need based on literature reports that stated only 2% of papers performed system suitability testing. Methods: We report MassQLab, built upon open‐source, vendor‐agnostic software called the mass spectrometry query language (MassQL). MassQL, implemented in MassQLab, provides freedom for researchers to choose their analyte/s, mass spectrometry system (including liquid chromatography—mass spectrometry), and metrics of performance. Results: In this report, we describe the use of MassQLab, demonstrate the construction of the required MassQL query, common metrics of performance (i.e., extracted ion chromatograms), uncommon metrics (i.e., MS/MS product ion spectra), and discuss insights gained about performance—including issues requiring correction prior to sample analysis. Conclusions: MassQLab is a flexible solution for system suitability testing for mass spectrometry‐based analytical measurements. Deficits in analytical performance, while unavoidable and rare, were noted prior to data collection and corrected. The open‐source and adaptable nature of MassQLab will empower researchers and lead to improved implementation of system suitability testing.