Exogenous Metabolites Research Articles

Metabolomic Profiling Identifies Exogenous and Microbiota-Derived Metabolites as Markers of Methotrexate Efficacy in Juvenile Idiopathic Arthritis.

Variability in methotrexate (MTX) efficacy represents a barrier to early and effective disease control in the treatment of juvenile idiopathic arthritis (JIA). This work seeks to understand the impact of MTX on the plasma metabolome and to identify metabolic biomarkers of MTX efficacy in a prospective cohort of children with JIA. Plasma samples from a cohort of children with JIA (n = 30) collected prior to the initiation of MTX and after 3 months of therapy were analyzed using a semi-targeted global metabolomic platform detecting 673 metabolites across a diversity of biochemical classes. Disease activity was measured using the 71-joint count juvenile arthritis disease activity score (JADAS-71) and clinical response to MTX was based on achievement of ACR Pedi 70 response. Metabolomic analysis identified 50 metabolites from diverse biochemical classes that were altered following the initiation of MTX (p < 0.05) with 15 metabolites reaching a false-discovery rate adjusted p-value (q-value) of less than 0.05. Enrichment analysis identified a class-wide reduction in unsaturated triglycerides following initiation of MTX (q = 0.0009). Twelve of the identified metabolites were significantly associated with disease activity by JADAS-71. Reductions in three metabolites were found to be associated with clinical response by ACR Pedi 70 response criteria and represented several microbiota and exogenously derived metabolites including: dehydrocholic acid, biotin, and 4-picoline. These findings support diverse metabolic changes following initiation of MTX in children with JIA and identify metabolites associated with microbial metabolism and exogenous sources associated with MTX efficacy.

Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth

BackgroundPyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1–4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth.MethodsTo isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice.ResultsIn this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice.ConclusionsWe found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo.

Deciphering metabolomic alterations in seminal plasma of crossbred (Bos taurus X Bos indicus) bulls through comparative deep metabolomic analysis.

The incidence of sub-fertility is higher in crossbred bulls compared to zebu bulls. In the present study, we analysed the metabolomic profile of seminal plasma from crossbred and zebu bulls and uncovered differentially expressed metabolites between these two breeds. Using a high-throughput LC-MS/MS-based approach, we identified 990 and 1,002 metabolites in crossbred and zebu bull seminal plasma respectively. After excluding the exogenous metabolites, we found that 50 and 68 putative metabolites were unique to crossbred and zebu bull seminal plasma, respectively, whilst 87 metabolites were common to both. After data normalisation, 63 metabolites were found to be dysregulated between crossbred and zebu bull seminal plasma. Observed pathways included Linoleic acid metabolism (observed metabolite was phosphatidylcholine) in crossbred bull seminal plasma whereas inositol phosphate metabolism (observed metabolites were phosphatidylinositol-3,4,5-trisphosphate/inositol 1,3,4,5,6-pentakisphosphate/myo-inositol hexakisphosphate) was observed in zebu bull seminal plasma. Abundance of Tetradecanoyl-CoA was significantly higher, whilst abundance of Taurine was significantly lower in crossbred bull seminal plasma. In conclusion, the present study established the seminal plasma metabolomic profile in crossbred and zebu bulls and suggest that increased lipid peroxidation coupled with low concentrations of antioxidants in seminal plasma might be associated with high incidence of sub-fertility in crossbred bulls.

Metabolomics profiling of human exhaled breath condensate by SPME/GC × GC-ToFMS: Exploratory study on the use of face masks at the level of lipid peroxidation volatile markers

Metabolomics encompasses the acquisition of large-scale datasets from a single sample to identify and/or elucidate physiological or pathological mechanisms, or even to assess environmental exposure, being sample preparation and instrumental analysis two fundamental steps in the construction of a metabolomics workflow. The study of the human exhaled breath condensate (EBC) has gained tremendous interest recently owing to the advances in analytical techniques. Thus, this study aims to develop a methodology for EBC metabolites profiling based on headspace solid phase microextraction (SPME) combined with comprehensive two-dimensional gas chromatography (GC × GC). Metabolite’s extraction efficiency was improved by the optimization of four SPME parameters using a real EBC matrix: fibre coating, sample pH and volume, and extraction temperature. EBC analysis was performed according to the optimized parameters, using aliquots of 500 μL of EBC, at its physiological pH, with the DVB/CAR/PDMS fibre coating, at 40.0 ± 0.1 °C, being able to detect analytes at pg/mL, with acceptable reproducibility. EBC volatile composition was provided, with around 400 chemical features detected, in which 130 were putatively identified as belonging to hydrocarbons, aldehydes, alcohols, ketones, esters, monoterpenic and sesquiterpenic compounds, ethers, norisoprenoids and furans, revealing therefore its high complexity. This research represents the most detailed study on the volatile composition of EBC and allows the assessment of a broad range of endogenous and exogenous metabolites, with further relevance on clinical or environmental exposure studies. To test the applicability of this methodology, an exploratory study was carried out to assess the impact of the use of surgical facemasks on young researcher’s lipid peroxidation volatile markers during a normal working day in a laboratory. In young healthy adults (age ≤ 32), the use of surgical facemasks seems not significantly affect the levels of the volatile markers of lipid peroxidation (aliphatic alkanes and aldehydes), neither the O2 saturation level - SpO2. This methodology shows promise as a convenient way to follow specific physiological or pathological conditions based on EBC metabolome and may be paired as part of an aggregate analytical structure.

Tooth biomarkers to characterize the temporal dynamics of the fetal and early-life exposome.

Background:Teeth have unique histology that make this biomatrix a time-capsule for retrospective exposure analysis of fetal and early life. However, most analytic methods require pulverizing the whole tooth, which eliminates exposure timing information. Further, the range of chemicals and endogenous exposures that can be measured in teeth has yet to be fully characterized.Methods:We performed untargeted metabolomics on micro-dissected layers from naturally shed deciduous teeth. Using four liquid-chromatography high-resolution mass spectrometry analytical modes, we profiled small molecules (<1000 Da) from prenatal and postnatal tooth fractions. In addition, we employed linear regression on the tooth fraction pairs from 31 children to identify metabolites that discriminate between prenatal and postnatal exposures.Results:Of over 10,000 features measured in teeth dentin, 390 unique compounds were annotated from 62 chemical classes. The class with the largest number of compounds was carboxylic acids and their derivatives (36%). Of the annotated exogenous metabolites (phthalates, parabens, perfluoroalkyl compounds, and cotinine) and endogenous metabolites (fatty acids, steroids, carnitines, amino acids, and others), 91 are linked to 256 health conditions through published literature. Differential analysis revealed 267 metabolites significantly different between the prenatal and the postnatal tooth fractions (adj. p-value < 0.05, Bonferroni correction), and 21 metabolites exclusive to the prenatal fraction.Conclusions:The prenatal and early postnatal exposome revealed from dental biomarkers represents a broad range of endogenous and exogenous metabolites for a comprehensive characterization in environmental health research. Most importantly, this technology provides a direct window into fetal exposures that is not possible by maternal biomarkers. Indeed, we identified several metabolites exclusively in the prenatal fraction, suggesting unique fetal exposures that are markedly different to postnatal exposures. Expansion of databases that include tooth matrix metabolites will strengthen biological interpretation and shed light on exposures during gestation and early life that may be causally linked with later health conditions.

Development of specific and selective bactericide by introducing exogenous metabolite of pathogenic bacteria

The widespread and repeated use of broad-spectrum bactericides has led to an increase in resistance. Developing novel broad-spectrum bactericides cannot solve the resistance problem, and may even aggravate it. The design of specific and selective bactericides has become urgent. A specific bactericidal design strategy was proposed by introducing exogenous metabolites in this study. This strategy was used to optimize two known antibacterial agents, luteolin (M) and Isoprothiolane (D), against Xoo. Based on the prodrug principles, target compound MB and DB were synthesized by combing M or D with exogenous metabolites, respectively. Bactericidal activity test results demonstrated that while the antibacterial ability of target compounds was significantly improved, their selectivity was also well enhanced by the introducing of exogenous metabolites. Comparing with the original compound, the antibacterial activity of target compound was significantly increased 92.0% and 74.5%, respectively. The optimized target compounds were more easily absorbed, and the drug application concentrations were much lower than those of the original agents, which would greatly reduce environmental pollution and relieve resistance risk. Our proposed strategy is of great significance for exploring the specific and selective bactericides against other pathogens.

Could metabolomics drive the fate of COVID-19 pandemic? A narrative review on lights and shadows.

Human Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection activates a complex interaction host/virus, leading to the reprogramming of the host metabolism aimed at the energy supply for viral replication. Alterations of the host metabolic homeostasis strongly influence the immune response to SARS-CoV-2, forming the basis of a wide range of outcomes, from the asymptomatic infection to the onset of COVID-19 and up tolife-threatening acute respiratory distress syndrome, vascular dysfunction, multiple organ failure, and death. Deciphering the molecular mechanisms associated with the individual susceptibility to SARS-CoV-2 infection calls for a system biology approach; this strategy can address multiple goals, including which patients will respond effectively to the therapeutic treatment. The power of metabolomics liesin the ability to recognize endogenous and exogenous metabolites within a biological sample, measuring their concentration, and identifying perturbations of biochemical pathways associated with qualitative and quantitative metabolic changes. Over the last year, a limited number of metabolomics- and lipidomics-based clinical studies in COVID-19 patients have been published and are discussed in this review. Remarkable alterations in the lipid and amino acid metabolism depict the molecular phenotype ofsubjects infected by SARS-CoV-2; notably, structural and functionaldata on the lipids-virus interaction may open new perspectives on targeted therapeutic interventions. Several limitations affect most metabolomics-based studies, slowing the routine application of metabolomics. However, moving metabolomics from bench to bedside cannot imply the mere determination of a given metabolite panel; rather, slotting metabolomics into clinical practice requires the conversion of metabolic patient-specific data into actionable clinical applications.

Impaired anaplerosis is a major contributor to glycolysis inhibitor toxicity in glioma

BackgroundReprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates and must be replenished (anaplerosis), mainly from pyruvate and glutamine. We recently described a novel enolase inhibitor, HEX, and its pro-drug POMHEX. Since glycolysis inhibition would deprive the cell of a key source of pyruvate, we hypothesized that enolase inhibitors might inhibit anaplerosis and synergize with other inhibitors of anaplerosis, such as the glutaminase inhibitor, CB-839.MethodsWe analyzed polar metabolites in sensitive (ENO1-deleted) and resistant (ENO1-WT) glioma cells treated with enolase and glutaminase inhibitors. We investigated whether sensitivity to enolase inhibitors could be attenuated by exogenous anaplerotic metabolites. We also determined the synergy between enolase inhibitors and the glutaminase inhibitor CB-839 in glioma cells in vitro and in vivo in both intracranial and subcutaneous tumor models.ResultsMetabolomic profiling of ENO1-deleted glioma cells treated with the enolase inhibitor revealed a profound decrease in the TCA cycle metabolites with the toxicity reversible upon exogenous supplementation of supraphysiological levels of anaplerotic substrates, including pyruvate. ENO1-deleted cells also exhibited selective sensitivity to the glutaminase inhibitor CB-839, in a manner rescuable by supplementation of anaplerotic substrates or plasma-like media PlasmaxTM. In vitro, the interaction of these two drugs yielded a strong synergistic interaction but the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood-brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors.ConclusionTogether, these data suggest that at least for ENO1-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of in vitro culture and suggest that cell culture media nutrient composition more faithful to the in vivo environment will more accurately predict in vivo efficacy of metabolism targeting drugs.

Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states.

Metabolism negotiates cell-endogenous requirements of energy, nutrients and building blocks with the immediate environment to enable various processes, including growth and differentiation. While there is an increasing number of examples of crosstalk between metabolism and chromatin, few involve uptake of exogenous metabolites. Solute carriers (SLCs) represent the largest group of transporters in the human genome and are responsible for the transport of a wide variety of substrates, including nutrients and metabolites. We aimed to investigate the possible involvement of SLC-mediated solutes uptake and cellular metabolism in regulating cellular epigenetic states. Here, we perform a CRISPR-Cas9 transporter-focused genetic screen and a metabolic compound library screen for the regulation of BRD4-dependent chromatin states in human myeloid leukaemia cells. Intersection of the two orthogonal approaches reveal that loss of transporters involved with purine transport or inhibition of de novo purine synthesis lead to dysfunction of BRD4-dependent transcriptional regulation. Through mechanistic characterization of the metabolic circuitry, we elucidate the convergence of SLC-mediated purine uptake and de novo purine synthesis on BRD4-chromatin occupancy. Moreover, adenine-related metabolite supplementation effectively restores BRD4 functionality on purine impairment. Our study highlights the specific role of purine/adenine metabolism in modulating BRD4-dependent epigenetic states.

Exogenous microbiota-derived metabolite trimethylamine N-oxide treatment alters social behaviors: Involvement of hippocampal metabolic adaptation

Increasing evidence indicates that gut microbiota and its metabolites can influence the brain function and the related behaviors. Trimethylamine N-oxide (TMAO), an indirect metabolite of gut microbiota, has been linked to aging, cognitive impairment, and many brain disorders. However, the potential effects of TMAO on social behaviors remain elusive. The present study investigated the effects of early life systemic TMAO exposure and intra-hippocampal TMAO infusion during adulthood on social behaviors in mice. We also analyzed the effects of intra-hippocampus infusion of TMAO during adulthood on levels of metabolites. The results showed that both systemic TMAO exposure in the post-weaning period and intra-hippocampal TMAO infusion during adulthood decreased social rank and reduced sexual preference in adult mice. Data from LC-MS metabolomics analysis showed that intra-hippocampal TMAO infusion induced a total 207 differential metabolites, which belongs to several metabolic or signaling pathways, especially FoxO signaling pathway and retrograde endocannabinoid signaling pathway. These data suggest that TMAO may affect social behaviors by regulating metabolites in the hippocampus, which may provide a new insight into the role of gut microbiota in regulating social behaviors.

Analysis of Anti-Doping Rule Violations That Have Impacted Medal Results at the Summer Olympic Games 1968-2012.

IntroductionSince 2004, the International Olympic Committee (IOC) store all samples collected at summer Olympic Games (OG) for retrospective re-analysis with more advanced analytical techniques to catch doping athletes.MethodsAll announced Anti-Doping Rule Violations (ADRVs) from IOC re-tests of the 2004, 2008 and 2012 OG (via IOC, International Federations and Athletics Integrity Unit public data) and other ADRVs confirmed to impact OG results from 1968 to 2012 (via the list of Doping Irregularities on olympedia.org) were collated to investigate how many medals have been impacted by ADRVs, when the ADRV was identified relative to the OG in question and its cause.ResultsOne hundred and thirty-four medals were impacted by ADRVs but only 26% of these ADRVs were identified at the time of the OG. Most ADRVs impacting medal results (74%) were identified retrospectively, either from events prior to the OG (17%) or via IOC re-tests of samples from 2004, 2008 and 2012 (57%). ADRVs impacting medal results from these re-tests took a mean of 6.8 ± 2.0 years to be announced relative to the end of the OG in which the medal was originally won. Exogenous Anabolic Androgenic Steroid metabolites were present in 90% of all athlete (n = 142) samples from IOC re-tests with dehydrochloromethyltestosterone and stanozolol accounting for 79% of detected substances. Athletics (n = 64) and weightlifting (n = 62) were the most affected sports.ConclusionThis analysis shows the frequency of targeted pre-OG Out-of-Competition testing should increase. We advocate for long-term sample storage to continue and additionally incorporate novel and potentially complementary technologies/sample matrices.

Plasma metabolomics of autism spectrum disorder and influence of shared components in proband families.

Prevalence of autism spectrum disorder (ASD) has been increasing in the United States in the past decades. The exact mechanisms remain enigmatic, and diagnosis of the disease still relies primarily on assessment of behavior. We first used a case–control design (75 idiopathic cases and 29 controls, enrolled at Boston Children’s Hospital from 2007-2012) to identify plasma biomarkers of ASD through a metabolome-wide association study approach. Then we leveraged a family-based design (31 families) to investigate the influence of shared genetic and environmental components on the autism-associated features. Using untargeted high-resolution mass spectrometry metabolomics platforms, we detected 19 184 features. Of these, 191 were associated with ASD (false discovery rate < 0.05). We putatively annotated 30 features that had an odds ratio (OR) between <0.01 and 5.84. An identified endogenous metabolite, O-phosphotyrosine, was associated with an extremely low autism odds (OR 0.17; 95% confidence interval 0.06-0.39). We also found that glutathione metabolism was associated with ASD (P = 0.048). Correlations of the significant features between proband and parents were low (median = 0.09). Of the 30 annotated features, the median correlations within families (proband–parents) were −0.15 and 0.24 for the endogenous and exogenous metabolites, respectively. We hypothesize that, without feature identification, family-based correlation analysis of autism-associated features can be an alternative way to assist the prioritization of potentially diagnostic features. A panel of ASD diagnostic metabolic markers with high specificity could be derived upon further studies.

Untargeted analysis of first trimester serum to reveal biomarkers of pregnancy complications: a case\u2013control discovery phase study

Understanding of causal biology and predictive biomarkers are lacking for hypertensive disorders of pregnancy (HDP) and preterm birth (PTB). First-trimester serum specimens from 51 cases of HDP, including 18 cases of pre-eclampsia (PE) and 33 cases of gestational hypertension (GH); 53 cases of PTB; and 109 controls were obtained from the Global Alliance to Prevent Prematurity and Stillbirth repository. Metabotyping was conducted using liquid chromatography high resolution mass spectroscopy and nuclear magnetic resonance spectroscopy. Multivariable logistic regression was used to identify signals that differed between groups after controlling for confounders. Signals important to predicting HDP and PTB were matched to an in-house physical standards library and public databases. Pathway analysis was conducted using GeneGo MetaCore. Over 400 signals for endogenous and exogenous metabolites that differentiated cases and controls were identified or annotated, and models that included these signals produced substantial improvements in predictive power beyond models that only included known risk factors. Perturbations of the aminoacyl-tRNA biosynthesis, l-threonine, and renal secretion of organic electrolytes pathways were associated with both HDP and PTB, while pathways related to cholesterol transport and metabolism were associated with HDP. This untargeted metabolomics analysis identified signals and common pathways associated with pregnancy complications.

Integrated metabonomics and serum pharmacochemistry strategy for ascertaining the material basis of Huangqi Jianzhong Tang against chronic atrophic gastritis

Huangqi Jianzhong Tang (HQJZ) is a classical traditional Chinese prescription for treating chronic atrophic gastritis (CAG). However, its material basis is still unclear. Thus, we constructed a novel strategy to illustrate its pharmaco basis coupled with metabolomics and serum pharmacochemistry. Plasma metabolic profile including control group, model group, HQJZ treated control group, and HQJZ treated model group were firstly detect by Ultra-high performance liquid chromatography coupled with quadrupole-Exactive mass spectrometry. Eight endogenous metabolites were screened as the pharmaco markers of HQJZ, which were significantly ameliorated by HQJZ in model group but no significant change in control group. A total of 11 prototypical compounds and 13 metabolites of HQJZ were screening and identified in HQJZ intervention groups. Further pearson correlative analysis were applied to discover the effective compounds for HQJZ, which were significantly correlated with those pharmaco markers. At last, 12 prototypical compounds excepting isoliquiritigenin and 7 metabolites were considered as potential pharmacodynamic basis of HQJZ, which could hit more metabolite targets. The results revealed the links between serum exogenous and endogenous metabolites involved into the protection of HQJZ, which showed a powerful tool to obtain the material basis of TCM formula.

Exogenous metabolites spray, which identified from metabolomics analysis and transcriptomic analysis, can improve salt tolerance of Chinese cabbages (Brassica rapa L.ssp pekinensis)*

ABSTRACT Soil salinization is one of the causes of the decline of the available cultivated land. By improving the salt tolerance of vegetables, the abandoned salinized land can be effectively utilized, saving the arable land for major crops. We investigated the salt tolerance of hundreds of Chinese cabbage cultivars, by which the two most tolerant and sensitive cultivars were selected and studied by metabolome and transcriptome analyses. The results showed that, under salt stress, metabolites’ response was not closely correlated with gene expressions in the tested Chinese cabbages. However, some KEGG pathways were significantly regulated at both metabolic and transcriptional levels. Furthermore, we identified several critical metabolites in the co-regulated pathways, including acetyl-CoA, pyruvic acid, ATP, nicotinic acid and Coenzyme Q10, which could alleviate the salt stress level in the tested Chinese cabbage. Thus, our findings provide candidate agents which can be used to improve salt tolerance in Chinese cabbage.

Enhancing Salt Tolerance of Plants: From Metabolic Reprogramming to Exogenous Chemical Treatments and Molecular Approaches.

Plants grow on soils that not only provide support for root anchorage but also act as a reservoir of water and nutrients important for plant growth and development. However, environmental factors, such as high salinity, hinder the uptake of nutrients and water from the soil and reduce the quality and productivity of plants. Under high salinity, plants attempt to maintain cellular homeostasis through the production of numerous stress-associated endogenous metabolites that can help mitigate the stress. Both primary and secondary metabolites can significantly contribute to survival and the maintenance of growth and development of plants on saline soils. Existing studies have suggested that seed/plant-priming with exogenous metabolites is a promising approach to increase crop tolerance to salt stress without manipulation of the genome. Recent advancements have also been made in genetic engineering of various metabolic genes involved in regulation of plant responses and protection of the cells during salinity, which have therefore resulted in many more basic and applied studies in both model and crop plants. In this review, we discuss the recent findings of metabolic reprogramming, exogenous treatments with metabolites and genetic engineering of metabolic genes for the improvement of plant salt tolerance.

Comparison of emerging dried capillary blood collection devices for untargeted metabolomics in epidemiological studies

Background: Capillary blood collection is minimally invasive, easy to perform, and when dried, is more stable at higher temperatures than liquid blood, making dried capillary blood microsamplers a promising alternative to venous blood for epidemiological research, particularly for vulnerable populations. Newly developed microsampler technologies are making headway as replacements to venous blood sampling in nonclinical pharmaceutical research, suggesting their potential for use in epidemiological research. However, their compatibility with untargeted metabolomics assays has not been fully evaluated.Methods: Using commercially purchased whole blood, we performed untargeted metabolomics profiling of 5-mm Guthrie card punches (~8-10 µL of whole blood), Volumetric Absorptive Microsampling (VAMS®) tips (10 µL of whole blood), HemaXisTM whole spots (10 µL of whole blood), and a Noviplex™ plasma discs (2.74 µL of plasma). Samples were analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) for measurement of polar compounds (ZHP) and semi- and non-polar compounds (RPN). Molecular features with a fold-change &gt; 3, compared to equivalent matrix blank were retained for analysis.Results: Endogenous and exogenous metabolites, environmental chemicals, and contaminants were measured in all four samplers. 4690 and 1605 peaks were measured in all four methods for ZHP and RPN analysis, respectively. An additional 3669 and 1848 peaks were shared by VAMS, Guthrie and HemaXis, but not found in Noviplex for ZPH and RPN analysis, respectively. Coefficients of variation calculated in replicate samples indicated higher reproducibility in VAMS and Guthrie card punches, than from HemaXisTM and Noviplex™. Furthermore, Noviplex™ extracts contained large contamination peaks not found in other devices.Conclusions: While we found that all microsamplers were amenable to untargeted metabolomics, differences were observed in the total number of metabolites measured, reproducibility of features, and background contamination levels. VAMS and Guthrie cards are the most promising devices for further untargeted metabolomics development for use in epidemiological research.

Combating Antibiotic Tolerance Through Activating Bacterial Metabolism.

The emergence of antibiotic tolerance enables genetically susceptible bacteria to withstand the killing by clinically relevant antibiotics. As is reported, an increasing body of evidence sheds light on the critical and underappreciated role of antibiotic tolerance in the disease burden of bacterial infections. Considering this tense situation, new therapeutic strategies are urgently required for combating antibiotic tolerance. Herein, we provide an insightful illustration to distinguish between antibiotic resistance and tolerance, and highlight its clinical significance and complexities of drug-tolerant bacteria. Then, we discuss the close relationship between antibiotic tolerance and bacterial metabolism. As such, a bacterial metabolism-based approach was proposed to counter antibiotic tolerance. These exogenous metabolites including amino acids, tricarboxylic acid cycle (TCA cycle) metabolites, and nucleotides effectively activate bacterial metabolism and convert the tolerant cells to sensitive cells, and eventually restore antibiotic efficacy. A better understanding of molecular mechanisms of antibiotic tolerance particularly in vivo would substantially drive the development of novel strategies targeting bacterial metabolism.

Short-Chain Fatty Acids Promote Mycobacterium avium subsp. hominissuis Growth in Nutrient-Limited Environments and Influence Susceptibility to Antibiotics.

Mycobacterium avium subsp. hominissuis (MAH) is a common intracellular pathogen that infects immunocompromised individuals and patients with pre-existing chronic lung diseases, such as cystic fibrosis, who develop chronic and persistent pulmonary infections. The metabolic remodeling of MAH in response to host environmental stresses or within biofilms formed in bronchial airways plays an important role in development of the persistence phenotype contributing to the pathogen’s tolerance to antibiotic treatment. Recent studies suggest a direct relationship between bacterial metabolic state and antimicrobial susceptibility, and improved antibiotic efficacy has been associated with the enhanced metabolism in bacteria. In the current study, we tested approximately 200 exogenous carbon source-dependent metabolites and identified short-chain fatty acid (SCFA) substrates (propionic, butyric and caproic acids) that MAH can utilize in different physiological states. Selected SCFA enhanced MAH metabolic activity in planktonic and sessile states as well as in the static and established biofilms during nutrient-limited condition. The increased bacterial growth was observed in all conditions except in established biofilms. We also evaluated the influence of SCFA on MAH susceptibility to clinically used antibiotics in established biofilms and during infection of macrophages and found significant reduction in viable bacterial counts in vitro and in cultured macrophages, suggesting improved antibiotic effectiveness against persistent forms of MAH.

Metabolome of canine and human saliva: a non-targeted metabolomics study

IntroductionSaliva metabolites are suggested to reflect the health status of an individual in humans. The same could be true with the dog (Canis lupus familiaris), an important animal model of human disease, but its saliva metabolome is unknown. As a non-invasive sample, canine saliva could offer a new alternative material for research to reveal molecular mechanisms of different (patho)physiological stages, and for veterinary medicine to monitor dogs’ health trajectories.ObjectivesTo investigate and characterize the metabolite composition of dog and human saliva in a non-targeted manner.MethodsStimulated saliva was collected from 13 privately-owned dogs and from 14 human individuals. We used a non-targeted ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS) method to measure metabolite profiles from saliva samples.ResultsWe identified and classified a total of 211 endogenous and exogenous salivary metabolites. The compounds included amino acids, amino acid derivatives, biogenic amines, nucleic acid subunits, lipids, organic acids, small peptides as well as other metabolites, like metabolic waste molecules and other chemicals. Our results reveal a distinct metabolite profile of dog and human saliva as 25 lipid compounds were identified only in canine saliva and eight dipeptides only in human saliva. In addition, we observed large variation in ion abundance within and between the identified saliva metabolites in dog and human.ConclusionThe results suggest that non-targeted metabolomics approach utilizing UHPLC-qTOF-MS can detect a wide range of small compounds in dog and human saliva with partially overlapping metabolite composition. The identified metabolites indicate that canine saliva is potentially a versatile material for the discovery of biomarkers for dog welfare. However, this profile is not complete, and dog saliva needs to be investigated in the future with other analytical platforms to characterize the whole canine saliva metabolome. Furthermore, the detailed comparison of human and dog saliva composition needs to be conducted with harmonized study design.