Plasma metabolomics reveals the shared and distinct metabolic disturbances associated with cardiovascular events in coronary artery disease.

Risk prediction for subsequent cardiovascular events remains an unmet clinical issue in patients with coronary artery disease. We aimed to investigate prognostic metabolic biomarkers by considering both shared and distinct metabolic disturbance associated with the composite and individual cardiovascular events. Here, we conducted an untargeted metabolomics analysis for 333 incident cardiovascular events and 333 matched controls. The cardiovascular events were designated as cardiovascular death, myocardial infarction/stroke and heart failure. A total of 23 shared differential metabolites were associated with the composite of cardiovascular events. The majority were middle and long chain acylcarnitines. Distinct metabolic patterns for individual events were revealed, and glycerophospholipids alteration was specific to heart failure. Notably, the addition of metabolites to clinical markers significantly improved heart failure risk prediction. This study highlights the potential significance of plasma metabolites on tailed risk assessment of cardiovascular events, and strengthens the understanding of the heterogenic mechanisms across different events.

Lysophosphatidylcholine binds α-synuclein and prevents its pathological aggregation.

Accumulation of aggregated α-synuclein (α-syn) in Lewy bodies is the pathological hallmark of Parkinson's disease (PD). Genetic mutations in lipid metabolism are causative for a subset of patients with Parkinsonism. The role of α-syn's lipid interactions in its function and aggregation is recognized, yet the specific lipids involved and how lipid metabolism issues trigger α-syn aggregation and neurodegeneration remain unclear. Here, we found that α-syn shows a preference for binding to lysophospholipids (LPLs), particularly targeting lysophosphatidylcholine (LPC) without relying on electrostatic interactions. LPC is capable of maintaining α-syn in a compact conformation, significantly reducing its propensity to aggregate both in vitro and within cellular environments. Conversely, a reduction in the production of cellular LPLs is associated with an increase in α-syn accumulation. Our work underscores the critical role of LPLs in preserving the natural conformation of α-syn to inhibit improper aggregation, and establishes a potential connection between lipid metabolic dysfunction and α-syn aggregation in PD.

Yersinia infection induces glucose depletion and AMPK-dependent inhibition of pyroptosis in mice.

Nutritional status and pyroptosis are important for host defence against infections. However, the molecular link that integrates nutrient sensing into pyroptosis during microbial infection is unclear. Here, using metabolic profiling, we found that Yersinia pseudotuberculosis infection results in a significant decrease in intracellular glucose levels in macrophages. This leads to activation of the glucose and energy sensor AMPK, which phosphorylates the essential kinase RIPK1 at S321 during caspase-8-mediated pyroptosis. This phosphorylation inhibits RIPK1 activation and thereby restrains pyroptosis. Boosting the AMPK-RIPK1 cascade by glucose deprivation, AMPK agonists, or RIPK1-S321E knockin suppresses pyroptosis, leading to increased susceptibility to Y. pseudotuberculosis infection in mice. Ablation of AMPK in macrophages or glucose supplementation in mice is protective against infection. Thus, we reveal a molecular link between glucose sensing and pyroptosis, and unveil a mechanism by which Y. pseudotuberculosis reduces glucose levels to impact host AMPK activation and limit host pyroptosis to facilitate infection.

Dysregulations of amino acid metabolism and lipid metabolism in urine of children and adolescents with major depressive disorder: a case-control study.

RATIONALE: The mechanisms underlying major depressive disorder (MDD) in children and adolescents are unclear. Metabolomics has been utilized to capture metabolic signatures of various psychiatric disorders; however, urinary metabolic profile of MDD in children and adolescents has not been studied. OBJECTIVES: We analyzed urinary metabolites in children and adolescents with MDD to identify potential biomarkers and metabolic signatures. METHODS: Here, liquid chromatography-mass spectrometry was used to profile metabolites in urine samples from 192 subjects, comprising 80 individuals with antidepressant-naïve MDD (AN-MDD), 37 with antidepressant-treated MDD (AT-MDD) and 75 healthy controls (HC). We performed orthogonal partial least squares discriminant analysis to identify differential metabolites and employed logistic regression and receiver operating characteristic analysis to establish a diagnostic panel. RESULTS: In total, 143 and 71 differential metabolites were identified in AN-MDD and AT-MDD, respectively. These were primarily linked to lipid metabolism, molecular transport, and small molecule biochemistry. AN-MDD additionally exhibited dysregulated amino acid metabolism. Compared to HC, a diagnostic panel of seven metabolites displayed area under the receiver operating characteristic curves of 0.792 for AN-MDD, 0.828 for AT-MDD, and 0.799 for all MDD. Furthermore, the urinary metabolic profiles of children and adolescents with MDD significantly differed from those of adult MDD. CONCLUSIONS: Our research suggests dysregulated amino acid metabolism and lipid metabolism in the urine of children and adolescents with MDD, similar to results in plasma metabolomics studies. This contributes to the comprehension of mechanisms underlying children and adolescents with MDD.

Identifying plasma metabolic characteristics of major depressive disorder, bipolar disorder, and schizophrenia in adolescents.

Major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SCZ) are classified as major mental disorders and together account for the second-highest global disease burden, and half of these patients experience symptom onset in adolescence. Several studies have reported both similar and unique features regarding the risk factors and clinical symptoms of these three disorders. However, it is still unclear whether these disorders have similar or unique metabolic characteristics in adolescents. We conducted a metabolomics analysis of plasma samples from adolescent healthy controls (HCs) and patients with MDD, BD, and SCZ. We identified differentially expressed metabolites between patients and HCs. Based on the differentially expressed metabolites, correlation analysis, metabolic pathway analysis, and potential diagnostic biomarker identification were conducted for disorders and HCs. Our results showed significant changes in plasma metabolism between patients with these mental disorders and HCs; the most distinct changes were observed in SCZ patients. Moreover, the metabolic differences in BD patients shared features with those in both MDD and SCZ, although the BD metabolic profile was closer to that of MDD than to SCZ. Additionally, we identified the metabolites responsible for the similar and unique metabolic characteristics in multiple metabolic pathways. The similar significant differences among the three disorders were found in fatty acid, steroid-hormone, purine, nicotinate, glutamate, tryptophan, arginine, and proline metabolism. Interestingly, we found unique characteristics of significantly altered glycolysis, glycerophospholipid, and sphingolipid metabolism in SCZ; lysine, cysteine, and methionine metabolism in MDD and BD; and phenylalanine, tyrosine, and aspartate metabolism in SCZ and BD. Finally, we identified five panels of potential diagnostic biomarkers for MDD-HC, BD-HC, SCZ-HC, MDD-SCZ, and BD-SCZ comparisons. Our findings suggest that metabolic characteristics in plasma vary across psychiatric disorders and that critical metabolites provide new clues regarding molecular mechanisms in these three psychiatric disorders.

Shaping immune landscape of colorectal cancer by cholesterol metabolites.

Cancer immunotherapies have achieved unprecedented success in clinic, but they remain largely ineffective in some major types of cancer, such as colorectal cancer with microsatellite stability (MSS CRC). It is therefore important to study tumor microenvironment of resistant cancers for developing new intervention strategies. In this study, we identify a metabolic cue that determines the unique immune landscape of MSS CRC. Through secretion of distal cholesterol precursors, which directly activate RORγt, MSS CRC cells can polarize T cells toward Th17 cells that have well-characterized pro-tumor functions in colorectal cancer. Analysis of large human cancer cohorts revealed an asynchronous pattern of the cholesterol biosynthesis in MSS CRC, which is responsible for the abnormal accumulation of distal cholesterol precursors. Inhibiting the cholesterol biosynthesis enzyme Cyp51, by pharmacological or genetic interventions, reduced the levels of intratumoral distal cholesterol precursors and suppressed tumor progression through a Th17-modulation mechanism in preclinical MSS CRC models. Our study therefore reveals a novel mechanism of cancer-immune interaction and an intervention strategy for the difficult-to-treat MSS CRC.

Intestinal epithelial dopamine receptor signaling drives sex-specific disease exacerbation in a mouse model of multiple sclerosis.

Although the gut microbiota can influence central nervous system (CNS) autoimmune diseases, the contribution of the intestinal epithelium to CNS autoimmunity is less clear. Here, we showed that intestinal epithelial dopamine D2 receptors (IEC DRD2) promoted sex-specific disease progression in an animal model of multiple sclerosis. Female mice lacking Drd2 selectively in intestinal epithelial cells showed a blunted inflammatory response in the CNS and reduced disease progression. In contrast, overexpression or activation of IEC DRD2 by phenylethylamine administration exacerbated disease severity. This was accompanied by altered lysozyme expression and gut microbiota composition, including reduced abundance of Lactobacillus species. Furthermore, treatment with N2-acetyl-L-lysine, a metabolite derived from Lactobacillus, suppressed microglial activation and neurodegeneration. Taken together, our study indicates that IEC DRD2 hyperactivity impacts gut microbial abundances and increases susceptibility to CNS autoimmune diseases in a female-biased manner, opening up future avenues for sex-specific interventions of CNS autoimmune diseases.

Neutral lysophosphatidylcholine mediates α-synuclein-induced synaptic vesicle clustering.

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.

AllCCS2: Curation of Ion Mobility Collision Cross-Section Atlas for Small Molecules Using Comprehensive Molecular Representations.

The development of ion mobility-mass spectrometry (IM-MS) has revolutionized the analysis of small molecules, such as metabolomics, lipidomics, and exposome studies. The curation of comprehensive reference collision cross-section (CCS) databases plays a pivotal role in the successful application of IM-MS for small-molecule analysis. In this study, we presented AllCCS2, an enhanced version of AllCCS, designed for the universal prediction of the ion mobility CCS values of small molecules. AllCCS2 incorporated newly available experimental CCS data, including 10,384 records and 7713 unified values, as training data. By leveraging a neural network trained on diverse molecular representations encompassing mass spectrometry features, molecular descriptors, and graph features extracted using a graph convolutional network, AllCCS2 achieved exceptional prediction accuracy. AllCCS2 achieved median relative error (MedRE) values of 0.31, 0.72, and 1.64% in the training, validation, and testing sets, respectively, surpassing existing CCS prediction tools in terms of accuracy and coverage. Furthermore, AllCCS2 exhibited excellent compatibility with different instrument platforms (DTIMS, TWIMS, and TIMS). The prediction uncertainties in AllCCS2 from the training data and the prediction model were comprehensively investigated by using representative structure similarity and model prediction variation. Notably, small molecules with high structural similarities to the training set and lower model prediction variation exhibited improved accuracy and lower relative errors. In summary, AllCCS2 serves as a valuable resource to support applications of IM-MS technologies. The AllCCS2 database and tools are freely accessible at http://allccs.zhulab.cn/.

Exhaustion-associated cholesterol deficiency dampens the cytotoxic arm of antitumor immunity.

The concept of targeting cholesterol metabolism to treat cancer has been widely tested in clinics, but the benefits are modest, calling for a complete understanding of cholesterol metabolism in intratumoral cells. We analyze the cholesterol atlas in the tumor microenvironment and find that intratumoral T cells have cholesterol deficiency, while immunosuppressive myeloid cells and tumor cells display cholesterol abundance. Low cholesterol levels inhibit T cell proliferation and cause autophagy-mediated apoptosis, particularly for cytotoxic T cells. In the tumor microenvironment, oxysterols mediate reciprocal alterations in the LXR and SREBP2 pathways to cause cholesterol deficiency of T cells, subsequently leading to aberrant metabolic and signaling pathways that drive T cell exhaustion/dysfunction. LXRß depletion in chimeric antigen receptor T (CAR-T) cells leads to improved antitumor function against solid tumors. Since T cell cholesterol metabolism and oxysterols are generally linked to other diseases, the new mechanism and cholesterol-normalization strategy might have potential applications elsewhere.

Encoding LC-MS-Based Untargeted Metabolomics Data into Images toward AI-Based Clinical Diagnosis.

Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics provides comprehensive and quantitative profiling of metabolites in clinical investigations. The use of whole metabolome profiles is a promising strategy for disease diagnosis but technically challenging. Here, we developed an approach, namely MetImage, to encode LC-MS-based untargeted metabolomics data into multi-channel digital images. Then, the images that represent the comprehensive metabolome profiles can be employed for developing deep learning-based AI models toward clinical diagnosis. In this work, we demonstrated the application of MetImage for clinical screening of esophageal squamous cell carcinoma (ESCC) in a clinical cohort with 1104 participants. A convolutional neuronal network-based AI model was trained to distinguish ESCC screening positive and negative subjects using their serum metabolomics data. Superior performances such as sensitivity (85%), specificity (92%), and area under curve (0.95) were validated in an independent testing cohort (N = 442). Importantly, we demonstrated that our AI-based ESCC screening model is not a "black box". The encoded images reserved the characteristics of mass spectra from the raw LC-MS data; therefore, metabolite identifications in key image features were readily achieved. Altogether, MetImage is a unique approach that encodes raw LC-MS-based untargeted metabolomics data into images and facilitates the utilization of whole metabolome profiles for AI-based clinical applications with improved interpretability.

A mass spectrum-oriented computational method for ion mobility-resolved untargeted metabolomics.

Ion mobility (IM) adds a new dimension to liquid chromatography-mass spectrometry-based untargeted metabolomics which significantly enhances coverage, sensitivity, and resolving power for analyzing the metabolome, particularly metabolite isomers. However, the high dimensionality of IM-resolved metabolomics data presents a great challenge to data processing, restricting its widespread applications. Here, we develop a mass spectrum-oriented bottom-up assembly algorithm for IM-resolved metabolomics that utilizes mass spectra to assemble four-dimensional peaks in a reverse order of multidimensional separation. We further develop the end-to-end computational framework Met4DX for peak detection, quantification and identification of metabolites in IM-resolved metabolomics. Benchmarking and validation of Met4DX demonstrates superior performance compared to existing tools with regard to coverage, sensitivity, peak fidelity and quantification precision. Importantly, Met4DX successfully detects and differentiates co-eluted metabolite isomers with small differences in the chromatographic and IM dimensions. Together, Met4DX advances metabolite discovery in biological organisms by deciphering the complex 4D metabolomics data.

ONE-seq: epitranscriptome and gene-specific profiling of NAD-capped RNA.

The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.

Serum metabolic traits reveal therapeutic toxicities and responses of neoadjuvant chemoradiotherapy in patients with rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) has become the standard treatment for patients with locally advanced rectal cancer (LARC). Therapeutic efficacy of nCRT is significantly affected by treatment-induced diarrhea and hematologic toxicities. Metabolic alternations in cancer therapy are key determinants to therapeutic toxicities and responses, but exploration in large-scale clinical studies remains limited. Here, we analyze 743 serum samples from 165 LARC patients recruited in a phase III clinical study using untargeted metabolomics and identify responsive metabolic traits over the course of nCRT. Pre-therapeutic serum metabolites successfully predict the chances of diarrhea and hematologic toxicities during nCRT. Particularly, levels of acyl carnitines are linked to sex disparity in nCRT-induced diarrhea. Finally, we show that differences in phenylalanine metabolism and essential amino acid metabolism may underlie distinct therapeutic responses of nCRT. This study illustrates the metabolic dynamics over the course of nCRT and provides potential to guide personalized nCRT treatment using responsive metabolic traits.

Metabolite annotation from knowns to unknowns through knowledge-guided multi-layer metabolic networking.

Liquid chromatography - mass spectrometry (LC-MS) based untargeted metabolomics allows to measure both known and unknown metabolites in the metabolome. However, unknown metabolite annotation is a major challenge in untargeted metabolomics. Here, we develop an approach, namely, knowledge-guided multi-layer network (KGMN), to enable global metabolite annotation from knowns to unknowns in untargeted metabolomics. The KGMN approach integrates three-layer networks, including knowledge-based metabolic reaction network, knowledge-guided MS/MS similarity network, and global peak correlation network. To demonstrate the principle, we apply KGMN in an in vitro enzymatic reaction system and different biological samples, with ~100-300 putative unknowns annotated in each data set. Among them, >80% unknown metabolites are corroborated with in silico MS/MS tools. Finally, we validate 5 metabolites that are absent in common MS/MS libraries through repository mining and synthesis of chemical standards. Together, the KGMN approach enables efficient unknown annotations, and substantially advances the discovery of recurrent unknown metabolites for common biological samples from model organisms, towards deciphering dark matter in untargeted metabolomics.

Four-Dimensional Untargeted Profiling of N-Acylethanolamine Lipids in the Mouse Brain Using Ion Mobility-Mass Spectrometry.

N-Acylethanolamines (NAE) are a class of essential signaling lipids that are involved in a variety of physiological processes, such as energy homeostasis, anti-inflammatory responses, and neurological functions. NAE lipids are functionally different yet structurally similar and often have low concentrations in biological systems. Therefore, the comprehensive analysis of NAE lipids in complex biological matrices is very challenging. In this work, we developed an ion mobility-mass spectrometry (IM-MS) based four-dimensional (4D) untargeted technology for comprehensive analysis of NAE lipids. First, we employed the picolinyl derivatization to significantly improve ionization sensitivity of NAE lipids by 2-9-fold. Next, we developed a two-step quantitative structure-retention relationship (QSRR) strategy and used the AllCCS software to curate a 4D library for 170 NAE lipids with information on m/z, retention time, collision cross-section, and MS/MS spectra. Then, we developed a 4D untargeted technology empowered by the 4D library to support unambiguous identifications of NAE lipids. Using this technology, we readily identified a total of 68 NAE lipids across different biological samples. Finally, we used the 4D untargeted technology to comprehensively quantify 47 NAE lipids in 10 functional regions in the mouse brain and revealed a broad spectrum of the age-associated changes in NAE lipids across brain regions. We envision that the comprehensive analysis of NAE lipids will strengthen our understanding of their functions in regulating distinct physiological activities.

Global stable-isotope tracing metabolomics reveals system-wide metabolic alternations in aging Drosophila.

System-wide metabolic homeostasis is crucial for maintaining physiological functions of living organisms. Stable-isotope tracing metabolomics allows to unravel metabolic activity quantitatively by measuring the isotopically labeled metabolites, but has been largely restricted by coverage. Delineating system-wide metabolic homeostasis at the whole-organism level remains challenging. Here, we develop a global isotope tracing metabolomics technology to measure labeled metabolites with a metabolome-wide coverage. Using Drosophila as an aging model organism, we probe the in vivo tracing kinetics with quantitative information on labeling patterns, extents and rates on a metabolome-wide scale. We curate a system-wide metabolic network to characterize metabolic homeostasis and disclose a system-wide loss of metabolic coordinations that impacts both intra- and inter-tissue metabolic homeostasis significantly during Drosophila aging. Importantly, we reveal an unappreciated metabolic diversion from glycolysis to serine metabolism and purine metabolism as Drosophila aging. The developed technology facilitates a system-level understanding of metabolic regulation in living organisms.

Trapped ion mobility spectrometry-mass spectrometry improves the coverage and accuracy of four-dimensional untargeted lipidomics.

Lipids play vital roles in many physiological and pathological processes in living organisms. Due to the high structural diversity and the numerous isomers and isobars of lipids, high-coverage and high-accuracy lipidomic analysis of complex biological samples remain the bottleneck to investigate lipid metabolism. Here, we developed the trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) based four-dimensional untargeted lipidomics to support accurate lipid identification and quantification in biological samples. We first demonstrated that the TIMS based multi-dimensional separation improved the differentiations of isomeric and isobaric lipids, and increased the purity of precursor ion isolation and the quality of MS/MS spectra. Hyphenation of TIMS and PASEF technologies significantly improved the coverages of MS/MS spectra. These technological advantages jointly improved the coverage and accuracy of lipid identification in untargeted lipidomics. We further demonstrated that the CCS values of lipids acquired using TIMS were highly consistent with those from drift tube ion mobility spectrometry (DTIMS). Lipid identification and quantification results of NIST human plasma samples were also verified with inter-laboratory reports. Finally, we applied the TIMS-MS based untargeted lipidomics to characterize the spatial distributions of 1393 distinctive lipids in the mouse brain, and demonstrated that diverse lipid distributions and compositions among brain regions contributed to different functions of brain regions. Altogether, TIMS-MS based four-dimensional untargeted lipidomics significantly improved the coverage and accuracy of untargeted metabolomics, thereby facilitating a system-level understanding of lipid metabolism in biological organisms.

metID: an R package for automatable compound annotation for LC-MS-based data.

SUMMARY: Accurate and efficient compound annotation is a long-standing challenge for LC-MS-based data (e.g. untargeted metabolomics and exposomics). Substantial efforts have been devoted to overcoming this obstacle, whereas current tools are limited by the sources of spectral information used (in-house and public databases) and are not automated and streamlined. Therefore, we developed metID, an R package that combines information from all major databases for comprehensive and streamlined compound annotation. metID is a flexible, simple and powerful tool that can be installed on all platforms, allowing the compound annotation process to be fully automatic and reproducible. A detailed tutorial and a case study are provided in Supplementary Materials. AVAILABILITY AND IMPLEMENTATION: https://jaspershen.github.io/metID. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.