Global Metabolomics Research Articles

Liquid Crystal Monomer: A Potential PPARγ Antagonist.

Liquid crystal monomers (LCMs) are a large family of artificial ingredients that have been widely used in global liquid crystal display (LCD) industries. As a major constituent in LCDs as well as the end products of e-waste dismantling, LCMs are of growing research interest with regard to their environmental occurrences and biochemical consequences. Many studies have analyzed LCMs in multiple environmental matrices, yet limited research has investigated the toxic effects upon exposure to them. In this study, we combined in silico simulation and in vitro assay validation along with omics integration analysis to achieve a comprehensive toxicity elucidation as well as a systematic mechanism interpretation of LCMs for the first time. Briefly, the high-throughput virtual screen and reporter gene assay revealed that peroxisome proliferator-activated receptor gamma (PPARγ) was significantly antagonized by certain LCMs. Besides, LCMs induced global metabolome and transcriptome dysregulation in HK2 cells. Notably, fatty acid β-oxidation was conspicuously dysregulated, which might be mediated through multiple pathways (IL-17, TNF, and NF-kB), whereas the activation of AMPK and ligand-dependent PPARγ antagonism may play particularly important parts. This study illustrated LCMs as a potential PPARγ antagonist and explored their toxicological mode of action on the trans-omics level, which provided an insightful overview in future chemical risk assessment.

Microbiome Dysbiosis Shows Strong Association of Gut-Derived Altered Metabolomic Profile in Gulf War Chronic Multisymptom Illness Symptom Persistence Following Western Diet Feeding and Development of Obesity.

The pathophysiology of Gulf War Illness (GWI) remains elusive even after three decades. The persistence of multiple complex symptoms along with metabolic disorders such as obesity worsens the health of present Gulf War (GW) Veterans often by the interactions of the host gut microbiome and inflammatory mediators. In this study, we hypothesized that the administration of a Western diet might alter the host metabolomic profile, which is likely associated with the altered bacterial species. Using a five-month symptom persistence GWI model in mice and whole-genome sequencing, we characterized the species-level dysbiosis and global metabolomics, along with heterogenous co-occurrence network analysis, to study the bacteriome-metabolomic association. Microbial analysis at the species level showed a significant alteration of beneficial bacterial species. The beta diversity of the global metabolomic profile showed distinct clustering due to the Western diet, along with the alteration of metabolites associated with lipid, amino acid, nucleotide, vitamin, and xenobiotic metabolism pathways. Network analysis showed novel associations of gut bacterial species with metabolites and biochemical pathways that could be used as biomarkers or therapeutic targets to ameliorate symptom persistence in GW Veterans.

Global Metabolomic Profiling of Host Red Blood Cells Infected with Babesia divergens Reveals Novel Antiparasitic Target Pathways.

Babesia divergens is an apicomplexan parasite that infects human red blood cells (RBCs), initiating cycles of invasion, replication, and egress, resulting in extensive metabolic modification of the host cells. Babesia is an auxotroph for most of the nutrients required to sustain these cycles. There are currently limited studies on the biochemical pathways that support these critical processes, necessitating the high-resolution global metabolomics approach described here to uncover the metabolic interactions between parasite and host RBC. Our results reveal an extensive parasite-mediated modulation of RBC metabolite levels of all classes, including lipids, amino acids, carbohydrates, and nucleotides, with numerous metabolic species varying in proportion to the level of infection. Many of these molecules are scavenged from the host RBCs. This is in accord with the needs of a rapidly proliferating parasite with limited biosynthetic capabilities. Probing these pathways in depth, we used growth inhibition assays to quantitate parasite susceptibility to drugs targeting these pathways and stimulated emission depletion (STED) microscopy to obtain high-resolution images of drug-treated parasites to correlate changes in morphology with specific metabolic blocks in order to validate the data generated by the untargeted metabolomics platform. Thus, interruption of cholesterol scavenging from the host cell led to premature parasite egress, while chemical targeting of the hydrolysis of acyl glycerides led to the buildup of malformed parasites that could not successfully egress. This is the first report detailing the global metabolomic profile of the B. divergens-infected RBC. Besides deciphering diverse aspects of the host-parasite relationship, our results can be exploited by others to uncover further drug targets in the host-parasite biochemical network. IMPORTANCE Human babesiosis is caused by apicomplexan parasites of the Babesia genus and is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations. Through its continuous cycles of invasion, proliferation, and egress, B. divergens radically changes the metabolic environment of the host red blood cell, allowing us opportunities to study potential chemical vulnerabilities that can be targeted by drugs. This is the first global metabolomic profiling of Babesia-infected human red blood cells, and our analysis revealed perturbation in all biomolecular classes at levels proportional to the level of infection. In particular, lipids and energy flux pathways in the host cell were altered by infection. We validated the changes in key metabolic pathways by performing inhibition assays accompanied by high-resolution microscopy. Overall, this global metabolomics analysis of Babesia-infected red blood cells has helped to uncover novel aspects of parasite biology and identified potential biochemical pathways that can be targeted for chemotherapeutic intervention.

Global Metabolome of Palmer Amaranth (Amaranthus palmeri) Populations Highlights the Specificity and Inducibility of Phytochemical Responses to Abiotic Stress.

Commonalities in adaptive responses to abiotic stressors could contribute to the development of cross-resistance in weeds. The degree to which herbicide-induced changes in weeds parallel those induced by other abiotic stress remains unknown. We investigated the specificity of metabolic perturbations induced by glyphosate and drought across three glyphosate-resistant (GR) and two glyphosate-susceptible (GS) biotypes of Palmer amaranth (Amaranthus palmeri) using global metabolomics approaches. Compared to GS-biotypes, in the absence of stress, the GR-biotypes had a higher abundance of primary metabolites, including sugars, nonaromatic amino acids, and organic acids. However, despite having a higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy number that could upregulate the phenylpropanoid metabolism, the nonstressed GR-biotypes were less abundant in specialized (secondary) metabolites. Under glyphosate stress, 80% of metabolites, including shikimate, that accumulated in GS-biotypes also increased in the GR-biotypes. However, glyphosate triggered the preferential accumulation of glycosides of dihydroxylated and methoxylated flavanols with higher antioxidant potential, and ferulic acid derivatives, specifically in GR-biotypes. The disruption of the shikimate pathway and the accumulation of phenylpropanoids upon glyphosate exposure suggest that the stress response of GR-biotypes could be partly induced. This differential response was less evident in other phytochemical classes and under drought, highlighting that the phytochemical responses are stress-specific rather than biotype-specific.

Abstract WP241: Phenylalanine, Apob/apoa-1 Ratio, Ketone Bodies, Fatty Acids, And Microrna 9-3p Patterns Are Altered In Acute Ischemic Stroke

Background: Stroke is a significant health issue in the United States and identifying biomarkers for prevention and functional recovery of acute ischemic stroke (AIS) remains the highest priority. This study aims to identify metabolite and microRNA (miRNA) signatures that may be associated with AIS pathophysiology by performing longitudinal and case-control studies. Methods: We evaluated 260 patients during acute (<72 hours, T1), and chronic (3-5 months, T2) phases of stroke with 160 controls by performing global metabolomics using high-throughput nuclear magnetic resonance spectroscopy. In a subset, we also examined differential expression patterns of blood and serum exosome miRNAs between the T1 and T2 using RNA sequencing. Results: Our study identified 19 metabolites to be significantly perturbed during the T1 using the orthogonal partial least square discrimination analysis and multivariate mixed regression. Phenylalanine showed a 15.9-fold increase in T1 and remained high at the T2 stage. The Apolipoprotein B/Apolipoprotein A-1 ratio and ketone bodies were markedly increased while several essential fatty acids and amino acids were decreased in the T1. Our data also identified 3 exosomal miRNAs upregulated in the T1 stage. The miR-9-3p showed a 14.7-fold increase in T1 (Bonferroni p= 9.44x10 -6 ) which is strongly supported by the literature to trigger brain injury and cell death via the caspase pathway. Increased expression of miR-4508 correlated positively with phenylalanine both in T1 (r = 0.88, p = 0.001) and T2 (r = 0.71, p = 0.02). Conclusion: This study elucidates significant alterations in amino acids, fatty acids, and apolipoprotein patterns between the acute and chronic stages of stroke compared to controls. Our results on exosomal miRNAs and pathway analysis using metabolites identified four major pathways; apoptosis, excitotoxicity, oxidative stress, and stroke-mediated inflammation to play a critical role in the AIS leading to neuronal cell death. Further validation in independent datasets is warranted, and if confirmed some of these may become clinically meaningful biomarkers to predict AIS in humans.

Ultra-High Performance Liquid Chromatography-Mass Spectrometry-based Serum Metabolomics for Early Diagnosis of Refractory Tumor-induced Osteomalacia: A Case-control Study.

Nearly 20% patients with tumor-induced osteomalacia (TIO) experienced recurrence or nonrecovery after surgery. Serum fibroblast growth factor 23 and phosphate concentrations are not sufficient for prognosis in such cases. Despite its importance for understanding of prognosis and underlying pathogenesis, the alteration of systemic metabolism in refractory TIO remains unclear. We aimed to find the metabolomic characteristics of refractory TIO and establish a novel predictive model for early discriminating refractory TIO based on their serum metabolomics. Cross-section study for comparison of metabolomic profile between TIO and normal control and longitudinal study for identifying prognostic model. Based on liquid chromatography-tandem mass spectrometry, we analyzed the global metabolomes of preoperative sera from 86 samples (32 TIO recovery patients, 11 nonremission patients, and 43 matched controls). Statistical analyses, pathway enrichment, and receiver operating characteristic analysis were performed to identified and evaluate potential markers. Sparse partial least squares discriminant analysis indicated a clear separation of metabolomic profiles between healthy controls (HC) and TIO patients. The serum metabolites altered in different prognostic groups. L-pipecolic acid, 2-dodecylbenzenesulfonic acid, and 2-deoxygalactopyranose were the top 3 metabolites that were significantly perturbed. A combination of L-pipecolic acid and 2-dodecylbenzenesulfonic acid demonstrated a high-performance panel for TIO prognosis evaluated by random forest algorithm (area under the curve = 0.921, 95% CI, 0.787-0.995). We investigate the global metabolomes of refractory TIO and identify potential prognostic biomarkers preliminarily. A high sensitivity and specificity panel were identified as promising discriminating predictors, which need to be verified in more patients. This work may demonstrate novel insights into TIO prognosis and pathogenesis.

Maternal Exercise Protects Male Offspring from Maternal Diet Programmed Non-Alcoholic Fatty Liver Disease Progression.

Maternal obesity programs risk for development of non-alcoholic fatty liver disease (NAFLD) in offspring. Maternal exercise is a potential intervention to prevent developmentally programmed phenotypes. We hypothesized that maternal exercise would protect from progression of NAFLD in offspring previously exposed to maternal obesogenic diet. Female mice were fed chow (CON) or HFFC and bred with lean males. A subset had an exercise wheel introduced 4 weeks after starting diet to allow for voluntary exercise. The offspring were weaned to HFFC diet for 7 weeks to induce NAFLD. Serum, adipose, and liver tissue were collected for metabolic, histologic, and gene expression analyses. Cecal contents were collected for 16S sequencing. Global metabolomics was performed on liver. Female mice fed HFFC diet had increased body weight prior to adding an exercise wheel. Female mice fed HFFC diet had an increase in exercise distance relative to CON during the pre-conception period. Exercise distance was similar between groups during pregnancy and lactation. CON-Active and HFFC-Active offspring exhibited decreased inflammation compared to offspring from sedentary dams. Fibrosis increased in offspring from HFFC-sedentary dams compared to CON-sedentary. Offspring from exercised HFFC dams exhibited less fibrosis than offspring from sedentary HFFC dams. While maternal diet significantly affected the microbiome of offspring, the effect of maternal exercise was minimal. Metabolomics analysis revealed shifts in multiple metabolites including several involved in bile acid, one-carbon, histidine, and acylcarnitine metabolism. This study provides pre-clinical evidence that maternal exercise is a potential approach to prevent developmentally programmed liver disease progression in offspring.

Sensitive quantification of short-chain fatty acids combined with global metabolomics in microbiome cultures.

The microbiome has been identified to have a key role for the physiology of their human host. One of the major impacts is the clearance of bacterial pathogens. We have now developed a chemoselective probe methodology for the absolute quantification of short-chain fatty acids at low nM concentrations, with high reproducibility and spiked isotope labelled internal standards. Immobilization to magnetic beads allows for separation from the matrix and the tagged metabolites upon bioorthogonal cleavage can be analyzed via UHPLC-MS. The major advantage of our sensitive method is the simple combination with global metabolomics analysis as only a small sample volume is required. We have applied this chemical metabolomics strategy for targeted SCFA analysis combined with global metabolomics on gut microbiome co-cultures with Salmonella and investigated the effect of antibiotic treatment.

Investigation of cystine as differential diagnostic biomarker between astrocytomas and oligodendrogliomas based on global- and targeted analysis using liquid chromatography/tandem mass spectrometric analysis

Astrocytoma and oligodendroglioma are primary brain tumors classified as gliomas. Because there is difference in the prognostic significance of the extent of resection between IDH-mutant astrocytoma and oligodendroglioma, intraoperative differential diagnosis between them provides important information for optimal extent of resection. Although the characteristics of genetic mutations and chromosomal aberrations in both tumors have been reported, there is no molecular diagnostic methods that is able to be used quickly and simply for differentiate the two tumors. Therefore, we aimed to search for biomarker candidates for differentiating them with metabolome analysis using liquid chromatography/tandem mass spectrometry and develop a molecular diagnostic method based on quantitative analysis. We searched for peaks that differed in two types of gliomas using global metabolomics. Next, we identified five biomarker candidates as hypoxanthine, inosine, cystine, proline and uric acid, respectively. Next, we developed an LC/MS/MS analytical method for five biomarker candidates and quantified them in brain tumors. Cystine had significantly lower amounts in astrocytomas than in oligodendrogliomas. We developed two prediction models for differentiation of the two gliomas and validated them using the separated two dataset. The logistic regression model with only cystine provided the best prediction performance. It was suggested that mass spectrometric analysis of cystine in surgery might be useful for differentiating astrocytoma and oligodendroglioma with 91.7% positive prediction value and 80.0% specificity whereas negative predictive value and sensitivity was lesser than 70%, so that further exploration for unknown metabolite is mandatory.

Comprehensive investigation of pathway enrichment methods for functional interpretation of LC-MS global metabolomics data.

Global or untargeted metabolomics is widely used to comprehensively investigate metabolic profiles under various pathophysiological conditions such as inflammations, infections, responses to exposures or interactions with microbial communities. However, biological interpretation of global metabolomics data remains a daunting task. Recent years have seen growing applications of pathway enrichment analysis based on putative annotations of liquid chromatography coupled with mass spectrometry (LC-MS) peaks for functional interpretation of LC-MS-based global metabolomics data. However, due to intricate peak-metabolite and metabolite-pathway relationships, considerable variations are observed among results obtained using different approaches. There is an urgent need to benchmark these approaches to inform the best practices. We have conducted a benchmark study of common peak annotation approaches and pathway enrichment methods in current metabolomics studies. Representative approaches, including three peak annotation methods and four enrichment methods, were selected and benchmarked under different scenarios. Based on the results, we have provided a set of recommendations regarding peak annotation, ranking metrics and feature selection. The overall better performance was obtained for the mummichog approach. We have observed that a ~30% annotation rate is sufficient to achieve high recall (~90% based on mummichog), and using semi-annotated data improves functional interpretation. Based on the current platforms and enrichment methods, we further propose an identifiability index to indicate the possibility of a pathway being reliably identified. Finally, we evaluated all methods using 11 COVID-19 and 8 inflammatory bowel diseases (IBD) global metabolomics datasets.

New insights into the antibacterial mode of action of quercetin against uropathogen Serratia marcescens in-vivo and in-vitro

In the course of a quest for therapeutic agents inhibiting uropathogens, the rise and universal blowout of antibiotic-resistant organisms is a wide problem. To overcome this matter, exploration of alternative antimicrobials is necessary. The antimicrobial potential of quercetin has been widely described against some pathogenic microorganisms, but to the best of our knowledge, no report exists against the pathogenicity of uropathogenic Serratia marcescens. Hence, the present study focused on the antibacterial mechanism of action of quercetin, a flavonoid against the uropathogen Serratia marcescens. Quercetin was evaluated for its anti-QS activity, and the attained outcomes showed that quercetin inhibited QS-mediated virulence factors such as biofilm formation, exopolysaccharides, swarming motility and prodigiosin in Serratia marcescens. The proposed mechanism of action of quercetin greatly influences cell metabolism and extracellular polysaccharide synthesis and damages the cell membrane, as revealed through global metabolome profiling. In vivo experiments revealed that treatment with quercetin prolonged the life expectancy of infected Caenorhabditis elegans and reduced the colonization of Serratia marcescens. Hence, the current study reveals the use of quercetin as a probable substitute for traditional antibiotics in the treatment of uropathogen infections driven by biofilms.

Global Metabolomics Discovers Two Novel Biomarkers in Pyridoxine-Dependent Epilepsy Caused by ALDH7A1 Deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive developmental and epileptic encephalopathy caused by pathogenic variants in the ALDH7A1 gene (PDE-ALDH7A1), which mainly has its onset in neonates and infants. Early diagnosis and treatment are crucial to prevent severe neurological sequelae or death. Sensitive, specific, and stable biomarkers for diagnostic evaluations and follow-up examinations are essential to optimize outcomes. However, most of the known biomarkers for PDE lack these criteria. Additionally, there is little discussion regarding the interdependence of biomarkers in the PDE-ALDH7A1 metabolite profile. Therefore, the aim of this study was to understand the underlying mechanisms in PDE-ALDH7A1 and to discover new biomarkers in the plasma of patients using global metabolomics. Plasma samples from 9 patients with genetically confirmed PDE-ALDH7A1 and 22 carefully selected control individuals were analyzed by ultra high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Two novel and reliable pyridoxine-independent diagnostic markers, 6-hydroxy-2-aminocaproic acid (HACA) and an isomer of C9H11NO4, were identified. Furthermore, a possible reaction mechanism is proposed for HACA. This study demonstrates the capability of global metabolomics in disease screening to detect established and novel biomarkers.

Serum Phospholipids Are Potential Therapeutic Targets of Aqueous Extracts of Roselle (Hibiscus sabdariffa) against Obesity and Insulin Resistance

Roselle (Hibiscus sabdariffa) is rich in phenolic compounds with antiobesogenic and antidiabetic effects. In this study, the effects of aqueous extracts of two varieties of Hibiscus sabdariffa, Alma blanca (white-yellow color) and Cuarenteña (purple color), were evaluated for the prevention of obesity and insulin resistance in rats fed a high-fat and high-fructose diet (HFFD), identifying targeted molecules through global metabolomics. After sixteen weeks, both roselle aqueous extracts prevented body weight gain, and white roselle extract ameliorated insulin resistance and decreased serum free fatty acid levels. Moreover, white roselle extract decreased 18:0 and 20:4 lysophosphatidylethanolamines and purple roselle extract increased 16:0 and 20:4 lysophosphatidylinositol compared to HFFD-fed rats. These results demonstrate that roselle's beneficial health effects are variety-dependent. Interestingly, the white roselle extract showed a greater beneficial effect, probably due to its high contents of organic and phenolic acids, though its consumption is not as popular as that of the red/purple varieties.

The mechanism of Renshen-Fuzi herb pair for treating heart failure-Integrating a cardiovascular pharmacological assessment with serum metabolomics.

Background: Renshen-Fuzi herb pair (RS-FZ) is often used in the clinical treatment of heart failure (HF) and has a remarkable therapeutic effect. However, the mechanism of RS-FZ for treating HF remains unclear. In our study, we explored the mechanism of RS-FZ for treating HF. Methods: Evaluation of RS-FZ efficacy by cardiovascular pharmacology. Moreover, Global metabolomics profiling of the serum was detected by UPLC-QTOF/MS. Multivariate statistics analyzed the specific serum metabolites and corresponding metabolic pathways. Combining serum metabolomics with network pharmacology, animal experiments screened and validated the critical targets of RS-FZ intervention in HF. Results: RS-FZ significantly ameliorated myocardial fibrosis, enhanced cardiac function, and reduced the serum HF marker (brain natriuretic peptide) level in rats with HF. Through topological analysis of the "Metabolite-Target-Component" interaction network, we found that 79 compounds of RS-FZ directly regulated the downstream specific serum metabolites by acting on four critical target proteins (CYP2D6, EPHX2, MAOB, and ENPP2). The immunohistochemistry results showed that RS-FZ observably improved the expression of CYP2D6 and ENPP2 proteins while decreasing the expression of EPHX2 and MAOB proteins dramatically. Conclusion: The integrated cardiovascular pharmacological assessment with serum metabolomics revealed that RS-FZ plays a crucial role in the treatment of HF by intervening in CYP2D6, EPHX2, MAOB, and ENPP2 target proteins. It provides a theoretical basis for RS-FZ for treating HF.

Abstract B020: Biomarkers of IDO1 inhibition in patients with locally advanced rectal cancer treated with radiation and chemotherapy

Abstract Background: To date, immunotherapy has not advanced the initial care of locally advanced rectal cancer (LARC) where a combination of radiation and chemotherapy remains the standard of care. In evaluation of other targets, we identified that the immune-oncology target indoleamine 2,3 dioxygenase-1 (IDO1) is universally overexpressed in rectal cancers after radiation therapy (RT) regardless of MSI status. IDO1 initiates tryptophan metabolism along the kynurenine pathway (KP) and activation of the KP exerts immune-suppressive effects in the tumor microenvironment. Our preclinical data identified that epacadostat, a potent inhibitor of IDO1, reduced CRC growth in vitro and in vivo. Furthermore, we found that epacadostat augmented the anti-tumor effects of RT by directly increasing apoptosis in neoplastic cells as well as by relieving RT induced immune-suppression. Based on these findings, we initiated a Phase I study in patients with LARC where epacadostat was used in combination with short-course RT (5 Gy x 5) and chemotherapy. This study is ongoing with the primary endpoint of determining the recommended phase II dose (RP2D) of epacadostat in this population. The study is a dose escalating design including 300, 400, and 600 mg BID. Aim: To present preliminary results from the corollary studies on sequentially acquired biospecimens. Methods: We collected tumor tissue and blood pre-treatment, post-radiation/pre-chemo, sequentially throughout chemo, and at treatment completion or surgery. Analyses include global metabolomics, focused evaluation of tryptophan pathway metabolites, single cell RNAseq, flow cytometry, ctDNA and tissue analysis for IDO1 and other immune checkpoint targets. We also established tumor-derived organoids/tumoroids and have established a “tumor-on-a-chip” model to conduct ex vivo tumor modeling. Results: Preliminary studies confirm an elevation of kynurenine to tryptophan ratio after SCRT which is reversed by epacadostat. Changes in the global metabolomics profiles and immune profiles shift from baseline through the stages of SCRT and chemotherapy revealing consistent patterns. Changes in ctDNA were assessed using the CAPP-Seq based AVENIO platform providing a personalized oncogenomic analysis and demonstrated of rapid clearance from post-SCRT which persisted throughout chemotherapy. Studies with tumoroids and the analysis of scRNAseq data are ongoing and will be presented. Conclusions: Preliminary analysis of biospecimens from this actively accruing study of epacadostat plus RT and chemotherapy reveals distinct changes in metabolomic, immunologic, and genomic profiling across the stages of treatment. These results have the potential to inform a precision design for the Phase II study and will also inform the most promising disease and treatment related biomarkers. Citation Format: Matthew A. Ciorba, Katrina Pedersen, Lauren Henke, Aadel Chaudhuri, Matthew Mutch, Hyun Kim, Haeseong Park. Biomarkers of IDO1 inhibition in patients with locally advanced rectal cancer treated with radiation and chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B020.

Immunometabolic profiling of cervicovaginal lavages identifies key signatures associated with adenomyosis.

Adenomyosis is a burdensome gynecologic condition that is associated with pelvic pain, dysmenorrhea, and abnormal uterine bleeding, leading to a negative impact on quality of life; and yet is often left undiagnosed. We recruited 108 women undergoing hysterectomy for benign gynecologic conditions and collected non-invasive cervicovaginal lavage samples for immunometabolic profiling. Patients were grouped according to adenomyosis status. We investigated the levels of 72 soluble immune proteins and >900 metabolites using multiplex immunoassays and an untargeted global metabolomics platform. There were statistically significant alterations in the levels of several immune proteins and a large quantity of metabolites, particularly cytokines related to type II immunity and amino acids, respectively. Enrichment analysis revealed that pyrimidine metabolism, carnitine synthesis, and histidine/histamine metabolism were significantly upregulated pathways in adenomyosis. This study demonstrates utility of non-invasive sampling combined with immunometabolic profiling for adenomyosis detection and a greater pathophysiological understanding of this enigmatic condition.

Combined transcriptome and metabolome analysis revealed pathways involved in improved salt tolerance of Gossypium hirsutum L. seedlings in response to exogenous melatonin application

BackgroundSalinization is major abiotic stress limiting cotton production. Melatonin (MT) has been implicated in salt stress tolerance in multiple crops including upland cotton. Here, we explored the transcriptomic and metabolomic response of a salt-tolerant self-bred high-yielding cotton line SDS-01, which was exogenously sprayed with four MT concentrations (50, 100, 200, and 500 μM).ResultsHere we found that MT improves plant biomass and growth under salt stress. The combined transcriptome sequencing and metabolome profiling approach revealed that photosynthetic efficiency is improved by increasing the expressions of chlorophyll metabolism and antenna proteins in MT-treated seedlings. Additionally, linoleic acid and flavonoid biosynthesis were improved after MT treatment. The Na+/K+ homeostasis-related genes were increasingly expressed in salt-stressed seedlings treated with MT as compared to the ones experiencing only salt stress. Melatonin treatment activated a cascade of plant-hormone signal transduction and reactive oxygen scavenging genes to alleviate the detrimental effects of salt stress. The global metabolome profile revealed an increased accumulation of flavonoids, organic acids, amino acids and derivatives, saccharides, and phenolic acids in MT-treated seedlings. Interestingly, N, N′-Diferuloylputrescine a known antioxidative compound was highly accumulated after MT treatment.ConclusionCollectively, our study concludes that MT is a salt stress regulator in upland cotton and alleviates salt-stress effects by modulating the expressions of photosynthesis (and related pathways), flavonoid, ROS scavenging, hormone signaling, linoleic acid metabolism, and ion homeostasis-related genes.

Mixture and individual effects of benzene, toluene, and formaldehyde in zebrafish (Danio rerio) development: Metabolomics, epigenetics, and behavioral approaches

In this study, we aimed to investigate the potential hazards of volatile organic compounds (VOCs) on the development of zebrafish. To this end, zebrafish embryos were exposed in two different windows, either alone or in a mixture with VOCs (benzene, toluene, and formaldehyde) [EW1: 4 ± 2 h post-fertilization (hpf) to 24 hpf and EW2: 24 ± 2 hpf to 48 hpf]. Alterations in global DNA methylation and related gene expression, behavioral responses, and stress-related gene expression were observed. In addition to these endpoints, non-targeted NMR-based global metabolomics followed by pathway analysis showed significant changes in the metabolism of various amino acids during VOC exposure. Regardless of the analyzed endpoints, toluene was the most toxic chemical when exposed individually and possibly played the most pivotal role in the mixture treatment conditions. In conclusion, our data show that exposure to VOCs at embryonic developmental stages causes physiological perturbations and adverse outcomes at later life stages.

Biomarkers of oxidative stress in urine and plasma of operators at six Singapore printing centers and their association with several metrics of printer-emitted nanoparticle exposures

Inhalation of nanoparticles emitted from toner-based printing equipment (TPE), such as laser printers and photocopiers, also known as PEPs, has been associated with systemic inflammation, hypertension, cardiovascular disease, respiratory disorders, and genotoxicity. Global serum metabolomics analysis in 19 healthy TPE operators found 52 dysregulated biomolecules involved in upregulation of inflammation, immune, and antioxidant responses and downregulation of cellular energetics and cell proliferation. Here, we build on the metabolomics study by investigating the association of a panel of nine urinary OS biomarkers reflecting DNA/RNA damage (8OHdG, 8OHG, and 5OHMeU), protein/amino acid oxidation (o-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine), and lipid oxidation (8-isoprostane, 4-hydroxy nonenal, and malondialdehyde [MDA]), as well as plasma total MDA and total protein carbonyl (TPC), with several nanoparticle exposure metrics in the same 19 healthy TPE operators. Plasma total MDA, urinary 5OHMeU, 3-chlorotyrosine, and 3-nitrotyrosine were positively, whereas o-tyrosine inversely and statistically significantly associated with PEPs exposure in multivariate models, after adjusting for age and urinary creatinine. Urinary 8OHdG, 8OHG, 5OHMeU, and total MDA in urine and plasma had group mean values higher than expected in healthy controls without PEPs exposure and comparable to those of workers experiencing low to moderate levels of oxidative stress (OS). The highest exposure group had OS biomarker values, most notably 8OHdG, 8OHG, and total MDA, that compared to workers exposed to welding fumes and titanium dioxide. Particle number concentration was the most sensitive and robust exposure metric. A combination of nanoparticle number concentration and OS potential of fresh aerosols is recommended for larger scale future studies.

MLL (KMT2A)-rearranged Acute Lymphoblastic Leukemias Are Addicted to S-Adenosyl Methionine (SAM): Implications for Therapy

MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is an aggressive subset associated with poor survival and relapse representing 5% of childhood ALL cases and 75% of infant leukemias. Though current intensive chemotherapy regimens have improved survival in childhood ALL overall, they fail to cure many of these high-risk patient subgroups and inflict significant long-term side effects, demonstrating the urgent need for novel therapies. While exploring the effect of different amino acid depletion strategies, we observed rapid induction of apoptosis upon methionine restriction (MR) in MLLr cells. The histone methyltransferase function of the MLL fusion protein complex requires increased amounts of the methionine metabolite s-adenosylmethionine (SAM) to maintain its hypermethylated state, creating a selective sensitivity of MLLr ALL for perturbations in methionine availability. To explore how MR impinges on MLLr cells' metabolic state we performed global metabolomics on SEM cells and observed rapid effects on metabolism, with 416 metabolites out of the 650 measured differing significantly upon 12 hours of MR. We performed in parallel mRNA sequencing on SEM cells to understand the effect of MR on gene expression and identified 1351 differentially expressed genes. Integrated transcriptomics and metabolomics analysis revealed several key pathways interlinked with SAM, including the folate cycle, polyamine synthesis, arginine/redox metabolism, nucleotide synthesis, and the creatine/urea cycle, with several SAM-dependent enzymes and key byproducts in these pathways being significantly reduced upon MR. Moreover, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells, confirming the critical role of SAM in this leukemia subset. To better understand the effect of methionine and SAM restriction on epigenetic modifications, we looked at histone methylation in a panel of MLLr and non-MLLr B-ALL cell lines and observed a global suppression of methylation upon MR specifically in the pediatric MLLr cells. Additionally, gene expression analysis revealed downregulation of key MLL fusion target genes associated with proliferation and maintenance of the oncogenic phenotype in response to MR, including ZMYND8, ERG, BCL2, PROM1, HMX3, HOXA10, HMGA2, and PBX3. Several of these gene targets are known to be transcriptionally upregulated in MLLr leukemia via aberrant H3K79 methylation. Consistent with this notion, knockdown of the H3K79 demethylase, KDM2B, displayed a more MR-resistant phenotype than its wildtype counterparts. Taken together, MR has a direct effect on global histone methylation in MLLr ALL cells, which in turn disturbs the MLL gene signature critical for their survival. Lastly, immunocompromised mice were engrafted with MLLr SEM cells and treated with a 95% MR diet while leukemia progression was monitored by flow cytometry. The MR diet was well tolerated with minimal weight loss, methionine levels in plasma were roughly 50% reduced compared to controls, and that alone was enough to significantly delay leukemic growth (p = 0.015). While several compounds targeting MAT2A, the enzyme that produces SAM from methionine, are currently in Phase 1 clinical trials, the availability of an FDA approved methionine-free formula for infants allows for rapid translation to the clinic. As a follow-up, we also performed a drug screen using 214 compounds to identify drugs that interact with MR, showing that vinca-alkaloids, including vincristine, strongly synergize. Validation experiments demonstrate that MR triggers a stress induced G2 arrest, priming cells for mitotic spindle destruction with vincristine. In vivo experiments are currently underway using patient-derived xenografts exploring synergistic potential of vincristine with targeting methionine metabolism. In summary, we found that MLLr leukemic cells are selectively vulnerable to perturbations of the methionine cycle due to their increased need for SAM to maintain hypermethylation and aberrant gene activation. Limiting methionine and/or SAM availability provides an attractive adjuvant therapy potentiating the effects of current or novel (targeted) therapies for MLLr leukemia.