The majority of metabolic pathways rely on the production of activated electrophilic intermediates, e.g., coenzyme-A esters, and their chemical structures and abundances are central to understanding enzyme function and biochemical mechanisms. However, most electrophilic metabolites are lost in traditional metabolomic analysis and thus remain poorly characterized. Here we introduce a biochemical probe, O-(trimethylammoniobutyl)-hydroxylamine (TAMOHA), that enables comprehensive profiling of electrophilic species such as coenzyme-A esters, ketones, and aldehydes. TAMOHA incorporates a highly nucleophilic hydroxyl amine that reacts quickly with electrophilic species upon tissue lysis, trapping them as stable derivatives that feature a tetraalkylammonium moiety whose constitutive charge and characteristic MS2 fragmentation fingerprint enable their highly sensitive detection. Using TAMOHA to survey the electrophilomes of E. coli, C. elegans, and mouse revealed several thousand electrophilic metabolites, most of which have not been characterized. We then demonstrate trapping of electrophilic metabolites with TAMOHA in the context of specific biochemical pathways, confirming previously proposed functions of two fatty acid metabolism enzymes, detecting formaldehyde production in mice, and providing new insights into the biosynthesis of ascaroside pheromones in C. elegans. We anticipate that use of TAMOHA for the profiling of electrophilic species will help clarify enzyme function and uncover previously elusive biochemical mechanisms in a wide range of biological systems.

Bifidobacteria dominate the gut microbiota of breast-fed infants, and are strongly associated with human health, including immune regulation, colonization resistance, and protection against inflammation. However, bifidobacteria persist at high abundance after weaning in only a subset of individuals, and the factors that regulate intestinal persistence of bifidobacteria are poorly understood. Using gnotobiotic mouse models, we identified a common dietary fiber, raffinose, as a critical determinant of bifidobacterial persistence during microbial transitions associated with weaning. Bifidobacterial persistence depends on an intact raffinose utilization operon and is associated with disease resistance and restrained inflammation in adult mice. Specific dietary fiber recommendations commencing at weaning are a potential strategy to maintain bifidobacteria persistence beyond infancy, with potential long-term benefits for host resilience and reduced risk of inflammatory disease.

Nematodes communicate via diverse sex pheromones, including long-range volatile signals, short-range chemical cues, and contact-dependent molecules. While the ascaroside family of small molecules that mediate short-range attraction is well characterized, the identities and roles of volatile sex pheromones (VSPs) that act over longer ranges remain unknown. Using GC–MS analysis of crude VSP extracts, we identified cyclohexyl acetate (CA) as a candidate mimic, sharing retention time and mass spectral features with natural VSPs. Behavioral assays demonstrated that CA acts as a concentration-dependent, male-specific attractant in Caenorhabditis. Pre-exposure to VSPs induced cross-adaptation to CA, suggesting shared sensory processing. Surprisingly, genetic and calcium imaging analyses revealed that CA perception is mediated primarily by AWCon (str-2-expressing) neurons and involves VSP chemoreceptor srd-1-independent pathways, which are distinct from the neural pathways involved in natural VSP perception. These data indicate that CA is unlikely to be a major VSP constituent; rather, it is a structural analog that elicits male-specific attraction via a parallel sensory circuit. The endogenous source of CA in C. remanei remains unresolved; our data do not establish whether females produce CA. Its structural and behavioral mimicry provides new insights into the complexity of chemosensory signaling and the potential for interspecies chemical eavesdropping in nematode ecology.

Recent studies demonstrated that the predacious fungi Arthrobotrys oligospora emits a mixture of volatile chemical cues that function to attract nematode prey. The strong attraction elicited by one of the mixture components, methyl 3-methyl-2-butenoate (MMB), was highly female- and hermaphrodite-specific within several Caenorhabditis species, including C. remanei and C. elegans, suggesting that MMB might function as a mimic of an endogenous, male-produced, volatile sex pheromone (VSP) within these species. Here, we report evidence that MMB is produced by C. remanei males at levels that are attractive to C. remanei females and C. elegans hermaphrodites. Notably, MMB production was not detected for C. elegans males; a finding which correlates with behavioral assays for which worm-conditioned media (WCM) prepared from C. remanei, but not from C. elegans adult males is strongly attractive to both C. remanei females and C. elegans hermaphrodites. Our findings establish MMB as the first chemically identified VSP in nematodes and show that A. oligospora exploits a dual strategy of chemical deception-mimicry and eavesdropping-to enhance prey capture.

Plant monoterpene indole alkaloids (MIAs) exhibit important pharmacological activities, yet understanding of their biosyntheses remains incomplete. Since protein-protein interactions (PPIs) represent a conserved regulatory mechanism in MIA-producing plants, we developed a large-scale, yeast-based screening pipeline to profile PPIs of a key enzyme, strictosidine β-D-glucosidase (SGD) from Mitragyna speciosa (kratom). This screen identified six novel medium-chain dehydrogenases/reductases (MDRs) as high-confidence interaction partners of SGD. Biochemical characterization revealed that all six MsMDRs produce an MIA we named charlamine by acting directly on the reactive strictosidine aglycone intermediate, preventing its spontaneous rearrangement and establishing a functional rationale for SGD-MDR interaction. One MsMDR additionally catalyzed the reduction of vallesiachotamine, derived from the spontaneous rearrangement of strictosidine aglycone, to another previously unreported MIA, vallesiachotaminol. Parallel transcriptomics and genomics analyses uncovered a biosynthetic gene cluster containing a dihydrocorynantheine aldehyde esterase, functioning downstream of MsMDRs. Collectively, these findings demonstrate the utility of interactomics-driven plant pathway discovery.

Gene family expansions are critical for functional diversification, yet the contributions of paralogs to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family-enzymes that transfer acyl groups to hydroxylated substrates-remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones-airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate the synthesis and secretion of nonvolatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.

Abstract Mass spectrometry (MS) has revealed millions of small organic molecules across organisms, yet most remain uncharacterized, limiting progress in biology and medicine. Despite computational advances, MS workflows rely heavily on expert input and reference libraries that cover only a fraction of known chemical space. Here, we introduce AIMe (AI Molecule Explorer), a multi-agent neuro-symbolic AI framework that transforms the interpretation of unknown spectra into an omics-scale exploration across the known structural space, providing chemically interpretable annotations. At its core, AIMe combines chemical reasoning with structure- informed learning to predict MS 2 spectra by modeling fragmentation as a sequence of actions, outperforming existing methods. AIMe dynamically constructs fragmentation pathways by assigning likelihoods to individual fragmentation actions, linking spectral peaks to explicit fragment molecular formulas and structures. At scale, AIMe predicted MS 2 spectra for over 100 million small organic molecules in PubChem and organized them into MS 2 KOSMOS, a substructure-informed community resource comprising over 800 million predicted spectra that expands the searchable small-molecule universe by roughly three orders of magnitude relative to experimental libraries. Analogous to sequence homology-based searches in genomics and proteomics, AIMe maps unknown spectra to molecular neighborhoods in MS 2 KOSMOS. Exact- formula indexing enables ranked retrieval of candidates and related structures, with peak-level structural and fragmentation-pathway annotations. Applied to mouse microbiota-dependent metabolites, AIMe enabled putative annotation of knowns and guided structure elucidation of unknowns, revealing previously unreported types of microbiota-dependent polyamines that also occur in humans. At repository scale, AIMe enabled putative annotation of roughly a third of 7 million spectral clusters representing most of the unknowns in the GNPS database. By extending MS 2 annotation beyond curated-library matching to interpretable search across the known small-molecule universe, AIMe accelerates discovery and large-scale exploration of small molecules across biomedicine, agriculture, and ecology.

Plants sense a diverse array of small molecules and macromolecules derived from their natural environment, including diverse microbe-associated molecular patterns (MAMPs) that can trigger defense responses. Several MAMPs have been shown to prime plants for enhanced defense, providing extended protection against pathogens with minimal fitness costs. However, the extent to which conserved small molecule signatures of other phyla contribute to priming of plant defenses is unclear. Here, we demonstrate that exposure of seeds or plants to the ascaroside ascr#18, a pheromone secreted by plant-parasitic and free-living soil nematodes, primes immune genes for enhanced expression upon pathogen challenge, thereby increasing resistance to diverse microbial pathogens. We further show that ascr#18-induced priming is associated with the formation of open chromatin in the regulatory regions of defense genes. Defense priming and disease protection by ascr#18 is compromised in Arabidopsis mutants defective in the receptor of ascr#18, the leucine-rich repeat receptor-like kinase NILR1. Defense priming by ascr#18 is retained under field conditions, demonstrating potential of ascaroside treatment as a crop protection strategy to reduce pesticide usage. Our findings provide insight into the molecular basis of defense priming by ascr#18 and demonstrate how evolutionarily conserved small molecule signatures of plant-associated macrobiota modulate immunity.

Protein posttranslational modifications (PTMs) play a central role for regulating protein function and cellular processes, with many PTMs arising from reactions with electrophilic metabolites. Here we extend the known landscape of PTMs with the identification of “lysine C 3 -iminylation,” the conjugation of protein lysine residues with propionaldehyde. To stabilize iminylation for mass spectrometric analyses and distinguish it from other isomeric PTMs, we developed a fixation and stable-isotope labeling approach based on parallel reduction of proteome with sodium borohydride and borodeuteride. Analyses of protein hydrolysates confirmed the presence of C 3 -iminylation in Caenorhabditis elegans and mouse. Additionally, proteomics results demonstrated specificity of this PTM in vitro and in vivo and revealed C 3 -iminylation in proteins related to critical metabolic pathways. Importantly, collective evidence from isotope tracing as well as genetic, dietary, and pharmacological manipulation experiments uncovered that C 3 -iminylation originates from cytochrome P450 (CYP)-mediated oxidation of omega-3 fatty acids. Correspondingly, C 3 -iminylation levels were elevated in C. elegans daf-2 ( e1370 ) mutants, an aging model, in which CYP activity is generally increased. These findings not only expand our understanding of the biochemical diversity of PTMs but also underscore the complex interplay between lipid metabolism and protein modifications, enabling further exploration of their biological and clinical implications.

Metabolites derived from the intestinal microbiota, including bile acids (BA), extensively modulate vertebrate physiology, including development1, metabolism2, 3-4, immune responses5, 6-7 and cognitive function8. However, to what extent host responses balance the physiological effects of microbiota-derived metabolites remains unclear9,10. Here, using untargeted metabolomics of mouse tissues, we identified a family of BA-methylcysteamine (BA-MCY) conjugates that are abundant in the intestine and dependent on vanin 1 (VNN1), a pantetheinase highly expressed in intestinal tissues. This host-dependent MCY conjugation inverts BA function in the hepatobiliary system. Whereas microbiota-derived free BAs function as agonists of the farnesoid X receptor (FXR) and negatively regulate BA production, BA-MCYs act as potent antagonists of FXR and promote expression of BA biosynthesis genes in vivo. Supplementation with stable-isotope-labelled BA-MCY increased BA production in an FXR-dependent manner, and BA-MCY supplementation in a mouse model of hypercholesteraemia decreased lipid accumulation in the liver, consistent with BA-MCYs acting as intestinal FXR antagonists. The levels of BA-MCY were reduced in microbiota-deficient mice and restored by transplantation of human faecal microbiota. Dietary intervention with inulin fibre further increased levels of both free BAs and BA-MCY levels, indicating that BA-MCY production by the host is regulated by levels of microbiota-derived free BAs. We further show that diverse BA-MCYs are also present in human serum. Together, our results indicate that BA-MCY conjugation by the host balances host-dependent and microbiota-dependent metabolic pathways that regulate FXR-dependent physiology.

Neural circuits that consolidate sensory cues are essential for neurological functioning. Neural circuits that perform sensory integration can vary greatly because the sensory processing regions of the brain employ various neural motifs. Here, we investigate a neural circuit that mediates the response to conflicting stimuli in Caenorhabditis elegans We concurrently expose animals to an aversive dispersal pheromone, osas#9, and an attractive bacterial extract. While worms usually avoid osas#9 alone, they suppress this avoidance behavior in the presence of a bacterial extract. Loss-of-function mutants and cell-specific rescues reveal that signaling from the ADF and NSM neurons is essential for bacterial extract-induced osas#9 avoidance attenuation. The inhibitory serotonin receptor, MOD-1, which is widely expressed on interneurons and motor neurons, is required for this sensory integration, suggesting that serotonin acts in an inhibitory manner. By performing calcium imaging on the ADF neurons in synaptic signaling (unc-13) and peptidergic (unc-31) signaling mutant backgrounds, we show that the ADF neurons require input from other neurons to respond to food extracts. We reveal a cue integration neural circuit in which serotonergic signaling and sensory neurons silence an aversive neural signal.

Physiological stress in non-neural tissues drives aversive learning for sensory cues associated with stress. However, the identities of signals derived from non-neural tissues and the mechanisms by which these signals mediate aversive learning remain elusive. Here, we show that intercellular sphingolipid signaling contributes to aversive learning under mitochondrial stress in C. elegans. We found that stress-induced aversive learning requires sphingosine kinase, SPHK-1, the enzyme that produces sphingosine-1-phosphate (S1P). Genetic and biochemical studies revealed an intercellular signaling pathway in which intestinal or hypodermal SPHK-1 signals through the neuronal G protein-coupled receptor, SPHR-1, and modulates responses of the octopaminergic RIC neuron to promote aversive learning. We further show that SPHK-1-mediated sphingolipid signaling is required for learned aversion of Chryseobacterium indologenes, a bacterial pathogen found in the natural habitats of C. elegans, which causes mitochondrial stress. Taken together, our work reveals a sphingolipid signaling pathway that communicates from intestinal or hypodermal tissues to neurons to promote aversive learning in response to mitochondrial stress and pathogen infection.

Intricate coupling between metabolism and protein post-translational modifications (PTMs) has emerged as a fundamental aspect of cellular regulation. Recent studies demonstrate that protein modifications can originate from diverse metabolites, and that their regulation is closely tied to the cellular metabolic state. Here we explore recently uncovered PTMs, including the concept of ‘modification of a modification’, as well as associated feedback and feedforward regulatory mechanisms, in which modified proteins impact not only related metabolic pathways but also other signaling cascades affecting physiology and diseases. The recently uncovered role of nucleus-localized metabolic enzymes for histone modifications additionally highlights the importance of cell-compartment-specific metabolic states. We further comment on the utility of untargeted metabolomics and proteomics for previously unrecognized PTMs and associated metabolic patterns. Together, these advances have uncovered a dynamic interplay between metabolism and PTMs, offering new perspectives for understanding metabolic regulation and developing targeted therapeutic strategies. This Perspective highlights how metabolic states regulate diverse protein modifications that affect physiology. In addition, the roles of subcellular localization of metabolic enzymes and the importance of untargeted omics approaches are discussed.

Widespread anthelmintic resistance has complicated the management of parasitic nematodes. Resistance to the benzimidazole (BZ) drug class is nearly ubiquitous in many species and is associated with mutations in beta-tubulin genes. However, mutations in beta-tubulin alone do not fully explain all BZ resistance. We performed a genome-wide association study using a genetically diverse panel of Caenorhabditis elegans strains to identify loci that contribute to resistance to the BZ drug thiabendazole (TBZ). We identified a quantitative trait locus (QTL) on chromosome V independent of all beta-tubulin genes and overlapping with two promising candidate genes, the cytochrome P450 gene cyp-35D1 and the nuclear hormone receptor nhr-176. Both genes were previously demonstrated to play a role in TBZ metabolism. NHR-176 binds TBZ and induces the expression of CYP-35D1, which metabolizes TBZ. We generated single gene deletions of cyp-35D1 and nhr-176 and found that both genes play a role in TBZ response. A predicted high-impact lysine-to-glutamate substitution at position 267 (K267E) in CYP-35D1 was identified in a sensitive strain, and reciprocal allele replacement strains in different genetic backgrounds were used to show that the lysine allele conferred increased TBZ resistance. Using competitive fitness assays, we found that neither allele was deleterious, but the lysine allele was selected in the presence of TBZ. Additionally, we found that the lysine allele significantly increased the rate of TBZ metabolism compared to the glutamate allele. Moreover, yeast expression assays showed that the lysine version of CYP-35D1 had twice the enzymatic activity of the glutamate allele. To connect our results to parasitic nematodes, we analyzed four Haemonchus contortus cytochrome P450 orthologs but did not find variation at the 267 position in fenbendazole-resistant populations. Overall, we confirmed that variation in this cytochrome P450 gene is the first locus independent of beta-tubulin to play a role in BZ resistance.

Sexually-dimorphic neural circuits play a critical role in shaping sex-specific animal behaviors. Maps of the structural dimorphisms in these circuits have been explored by analyzing "synaptic connectomes", electron micrograph reconstructions of synaptic connectivity. Nevertheless, recent studies in the model organism C. elegans have shown little to no correlation between the synaptic connectome and dynamic neural activity. Therefore, the extent of sexual dimorphism in functional neural activity remains unknown. To determine the extent of functional sexual-dimorphisms in C. elegans we compared activity, neuron-by-neuron, across all neurons in the heads of both sexes. To sample a broad view of responses to different sensory modalities, we tested a diverse panel of ethologically-relevant olfactory, gustatory, and chemical stimuli, representing both attractive and aversive cues. We found that nearly every sensory neuron responded dimorphically to at least one cue and monomorphically to other cues, indicating that sexually-dimorphic circuits are pervasive and stimulus dependent. This dimorphic and monomorphic activity was present to a lesser extent in downstream interneurons and even less so in motoneurons, implicating sensory neurons as the primary source and location of sexually-dimorphic activity. Comparing the functional activity we measured to the published synaptic connectomes of both sexes revealed that sexual dimorphism in functional connectivity was distinct from and complementary to sexual dimorphism in synaptic connectivity. Our results provide a first-of-its-kind comparison of whole-brain dynamics between sexes at the level of single neurons, serving as an extensive resource for further investigations of functional sex differences.

Toward characterization of protein-metabolite interactomes, we recently introduced PROMIS, a co-fractionation-based mass spectrometry approach. However, the challenge lies in distinguishing true interactors from coincidental co-elution when a metabolite co-fractionates with numerous proteins. To address this, we integrated two chromatographic techniques-size exclusion and ion exchange-to enhance the mapping of protein-metabolite interactions (PMIs) in Escherichia coli. This integration aims to refine the PMI network by considering size and charge characteristics, resulting in 994 interactions involving 51 metabolites and 465 proteins. The PMI network is enriched for known and predicted interactions, providing validation. Furthermore, analyzing protein targets for different metabolites revealed functional insights, such as a connection between proteinogenic dipeptides and fatty acid biosynthesis. Notably, we uncovered an inhibitory interaction between the riboflavin degradation product lumichrome and orotate phosphoribosyltransferase, a key enzyme in de novo pyrimidine synthesis affecting biofilm formation. In summary, our integrated chromatographic approach significantly advances PMI mapping.

Lactate has emerged as a key metabolite involved in multiple physiological processes, including memory formation, immune response regulation, and muscle biogenesis. However, its role in aging and cellular protection remains unclear. Here, we show that lactate promotes longevity in C. elegans through a mechanism that requires early-life intervention, indicating a hormetic priming effect. This pro-longevity action depends on its metabolic conversion via LDH-1 and NADH, which drives redox-dependent metabolic reprogramming. Multi-omics approaches revealed that lactate induces early-stage metabolic adaptations, with a strong modulation of lipid metabolism, followed by late-life transcriptional remodeling. These shifts are characterized by enhanced stress response pathways and suppression of energy-associated metabolic processes. Our genetic screening identified sir-2.1/SIRT1 and rict-1/RICTOR as essential for lactate-mediated lifespan extension. Our findings establish lactate as a pro-longevity metabolite that couples redox signaling with lipid remodeling and nutrient-sensing pathways. This work advances our understanding of lactate's dual role as a metabolic intermediary and geroprotector signaling molecule, offering insights into therapeutic strategies for age-related metabolic disorders.

To prevent their detection, bacteria inhibit plant enzymes with a small molecule.

Animals must flexibly respond to environmental stimuli to survive, and optimal responses critically depend on the organism's current needs. Many organisms have evolved both cell-intrinsic and intertissue signaling pathways that integrate metabolic status. However, how this information is encoded in molecular signals is currently not well understood. Here we show that the nematode C. elegans employs lipidated neurohormones that combine the neurotransmitter octopamine and fat metabolism-derived building blocks to relay information about lipid metabolic status and drive inhibition of aversive olfactory responses during food removal. Using targeted metabolomics, we show that lipidated neurohormone synthesis requires the carboxylesterase CEST-2.1, which links octopamine-glucosides with endogenous methyl-branched or diet-derived cyclopropane fatty acids that act as agonists of the nuclear receptor and master regulator of fat metabolism, NHR-49/PPARα. Loss of cest-2.1, loss of bacterial cyclopropane fatty acid production, or loss of endogenous biosynthesis of the methyl-branched fatty acid substrates of CEST-2.1 mimics the behavioral responses of animals lacking octopamine, indicating that regulation of neurotransmitter-dependent behavior is linked to the coordination of fat metabolism via NHR-49/PPARα. Biosynthesis and subsequent neuromodulation via lipidated neurohormone relies on an intertissue trafficking pathway in which octopamine is shuttled first into the intestine where it is chemically modified, which is likely followed by neuronal import and intracellular hydrolysis to finally release free octopamine. We propose that esterase-dependent synthesis and subsequent hydrolysis of lipidated neurohormones represents a chemical encoding mechanism by which animals integrate information from neurotransmitter signaling and lipid homeostasis to direct appropriate behaviors.