Integrative Molecular Phenotyping
INTEGRATIVE MOLECULAR
PHENOTYPING
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY

PubMed

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PubMed
NCBI: db=pubmed; Term=metabolomics
Updated: 2 hours 52 min ago

Effect of Melittin on Metabolomic Profile and Cytokine Production in PMA-Differentiated THP-1 Cells.

Wed, 17/10/2018 - 12:58
Related Articles Effect of Melittin on Metabolomic Profile and Cytokine Production in PMA-Differentiated THP-1 Cells. Vaccines (Basel). 2018 Oct 13;6(4): Authors: Alqarni AM, Ferro VA, Parkinson JA, Dufton MJ, Watson DG Abstract Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1β, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addition of melittin enhanced the release of inflammatory cytokines induced by LPS. Effective chromatographic separation of metabolites was obtained by liquid chromatography-mass spectrometry (LC-MS) using a ZIC-pHILIC column and an ACE C4 column. The levels of 108 polar and non-polar metabolites were significantly changed (p ˂ 0.05) following cell activation by the combination of LPS and melittin when compared to untreated control cells. Overall, the findings of this study suggested that melittin might have a potential application as a vaccine adjuvant. PMID: 30322119 [PubMed]

Metabonomics delineates allergic reactions induced by Shuang-huang-lian injection in rats using ultra performance liquid chromatography-mass spectrometry.

Wed, 17/10/2018 - 12:58
Related Articles Metabonomics delineates allergic reactions induced by Shuang-huang-lian injection in rats using ultra performance liquid chromatography-mass spectrometry. Chin J Nat Med. 2018 Aug;16(8):628-640 Authors: Gu YY, Shi L, Zhang DD, Huang X, Chen DZ Abstract Shuang-huang-lian Injection (SHLI) is the first successfully developed drug from traditional Chinese medicine (TCM) powder for injection, since its use for the treatment of acute respiratory tract infection, pneumonia, influenza, etc. At the same time, its allergic reactions have also emerged, which limits clinical applications. However, few scholars pay attention to the mechanism of allergic reactions. In this present study, metabonomics technology was used to explore the changes in endogenous metabolites in urine of the rat model of SHLI induced allergic reaction; we and analyzed the metabolites, metabolic pathway, and the mechanism which were closely related to the allergic reactions. The levels of serum histamine and tryptase were examined and changes in histomorphology were also observed. Based on the UPLC-Q-TOF/MS metabonomics, we carried out the pattern recognition analysis, selected potential biomarkers associated with allergic reactions, and explored the pathological mechanism for SHLI induced allergic reaction, which laid the foundation for the safety research of SHLI. Our results showed that SHLI increased the levels of serum histamine and tryptase in rats with allergic reaction; we determined 15 biomarkers in rat allergic reaction model induced by SHLI and found multiple metabolic pathways involved, such as metabolism of linolenic acid, phenylalanine, amino acid, 2-oxo acid, and purine and other metabolic pathways. PMID: 30197129 [PubMed - indexed for MEDLINE]

Individual susceptibility to arsenic-induced diseases: the role of host genetics, nutritional status, and the gut microbiome.

Wed, 17/10/2018 - 12:58
Related Articles Individual susceptibility to arsenic-induced diseases: the role of host genetics, nutritional status, and the gut microbiome. Mamm Genome. 2018 02;29(1-2):63-79 Authors: Chi L, Gao B, Tu P, Liu CW, Xue J, Lai Y, Ru H, Lu K Abstract Arsenic (As) contamination in water or food is a global issue affecting hundreds of millions of people. Although As is classified as a group 1 carcinogen and is associated with multiple diseases, the individual susceptibility to As-related diseases is highly variable, such that a proportion of people exposed to As have higher risks of developing related disorders. Many factors have been found to be associated with As susceptibility. One of the main sources of the variability found in As susceptibility is the variation in the host genome, namely, polymorphisms of many genes involved in As transportation, biotransformation, oxidative stress response, and DNA repair affect the susceptibility of an individual to As toxicity and then influence the disease outcomes. In addition, lifestyles and many nutritional factors, such as folate, vitamin C, and fruit, have been found to be associated with individual susceptibility to As-related diseases. Recently, the interactions between As exposure and the gut microbiome have been of particular concern. As exposure has been shown to perturb gut microbiome composition, and the gut microbiota has been shown to also influence As metabolism, which raises the question of whether the highly diverse gut microbiota contributes to As susceptibility. Here, we review the literature and summarize the factors, such as host genetics and nutritional status, that influence As susceptibility, and we also present potential mechanisms of how the gut microbiome may influence As metabolism and its toxic effects on the host to induce variations in As susceptibility. Challenges and future directions are also discussed to emphasize the importance of characterizing the specific role of these factors in interindividual susceptibility to As-related diseases. PMID: 29429126 [PubMed - indexed for MEDLINE]

metabolomics; +17 new citations

Tue, 16/10/2018 - 15:37
17 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2018/10/16PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

metabolomics; +17 new citations

Tue, 16/10/2018 - 12:31
17 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2018/10/16PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Integrated analysis of proteomics-delineated and metabolomics-delineated hepatic metabolic responses to (-)-hydroxycitric acid in chick embryos.

Mon, 15/10/2018 - 12:11
Integrated analysis of proteomics-delineated and metabolomics-delineated hepatic metabolic responses to (-)-hydroxycitric acid in chick embryos. J Cell Biochem. 2018 Oct 14;: Authors: Peng M, He Q, Li S, Li L, Ma H Abstract (-)-Hydroxycitric acid [(-)-HCA] is widely used as a nutritional supplement to control body weight and fat accumulation in animals and humans, whereas the underlying biochemical mechanism is unclear. Broiler chicken was used as a model for studies of obesity due to its natural hyperglycemia and being insulin resistant. The current study aimed to obtain a systematic view of serum metabolites and hepatic proteins and well understand the mechanism of hepatic metabolic response to (-)-HCA treatment in chick embryos. The results showed that 22, 90, and 82 of differentially expressed proteins were identified at E14d, E19d, and H1d in chick embryos treated with (-)-HCA, respectively. Meanwhile, 5, 83, and 88 of serum metabolites significantly changed at E14d, E19d, and H1d in chick embryos after (-)-HCA treatment. Bioinformatics analysis showed that the key proteins and metabolites, which were significantly altered in chick embryos treated with (-)-HCA, were mainly involved in the citrate cycle, glycolysis/gluconeogenesis, fatty acid metabolism, and pyruvate metabolism. Our data indicated that (-)-HCA treatment might promote fat metabolism via regulating the key protein expression levels and metabolite contents in the citrate cycle, glycolysis/gluconeogenesis, and oxidative phosphorylation during chicken embryonic development. These results will deepen our understanding of the mechanism of fat reduction by (-)-HCA and provide substantial information for (-)-HCA as a nutritional supplement to control body weight gain and curb obesity-related diseases. PMID: 30317645 [PubMed - as supplied by publisher]

Study on urine metabolic profile of Aβ25-35-induced Alzheimer's disease by using UHPLC-Q-TOF-MS.

Mon, 15/10/2018 - 12:11
Study on urine metabolic profile of Aβ25-35-induced Alzheimer's disease by using UHPLC-Q-TOF-MS. Neuroscience. 2018 Oct 11;: Authors: Liu Y, Wei M, Yue K, Hu M, Li S, Men L, Pi Z, Liu Z, Liu Z Abstract Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with no effective method for its treatment so far. The pathogenesis of AD has been reported, but the endogenous metabolic profile and disease-related biomarkers are still not clear. To better understand AD, an AD model induced by injecting β-amyloid 25-35 (Aβ 25-35) solution into bilateral hippocampus was developed on Sprague-Dawley rats. After 8 weeks of modeling, the impairment of spatial learning and memory ability in AD rats were assessed by Morris water maze task. Hematoxylin and eosin staining and immunohistochemistry were used to investigate the pathological changes of hippocampus. The neurotransmitter concentrations in the hippocampus were measured by using UHPLC-TQ-MS. Urinary metabolomics based on UHPLC-Q-TOF-MS was established to delineate the alterations of endogenous metabolites in AD rats. The results showed that compared with healthy control rats, AD rats were suffered from cognitive dysfunction, hippocampus damage, Aβ formation and tau phosphorylation at 8 weeks after surgery, suggesting that the Aβ25-35-induced AD model was successfully established. In addition, the levels of γ-aminobutyric acid, acetylcholine, glycine, norepinephrine, serotonin, taurine and dopamine decreased and glutamate and aspartic acid increased in hippocampal tissue of AD rats. 45 altered metabolites mainly involved in 8 metabolic pathways were identified as the endogenous biomarkers of AD. According to the analysis of the biological significance of metabolic profiles, the pathogenesis of AD was mainly due to gut microbiome dysbiosis, inhibition of energy metabolism, oxidative stress injury and loss of neuronal protective substances. PMID: 30316910 [PubMed - as supplied by publisher]

Comparison of sequential derivatization with concurrent methods for GC/MS-based metabolomics.

Mon, 15/10/2018 - 12:11
Comparison of sequential derivatization with concurrent methods for GC/MS-based metabolomics. J Biosci Bioeng. 2018 Oct 10;: Authors: Miyagawa H, Bamba T Abstract The gas chromatography/mass spectrometry (GC/MS)-based metabolomics requires a two-step derivatization procedure consisting of oximation and silylation. However, due to the incomplete derivatization and degeneration of the metabolites, good repeatability is difficult to obtain during the batch derivatization, as the time between completing the derivatization process and GC analysis differs from sample to sample. In this research, we successfully obtained good repeatability for the peak areas of 52 selected metabolites by sequential derivatization and interval injection, in which the oximation and silylation times were maintained at constant values. In addition, the derivatization times and amount of reagents employed were varied to confirm that the optimal derivatization conditions differed for the various metabolites. In conventional batch derivatization, six metabolites, viz. glutamine, glutamic acid, histidine, alanine, asparagine, and tryptophan, exhibited fluctuations in their peak areas. Indeed, we found that for all six metabolites these differences originated from the silylation process, while the variations for glutamine and glutamic acid were related to the oximation process. PMID: 30316697 [PubMed - as supplied by publisher]

High-throughput metabolomics for discovering metabolic biomarkers from intestinal tumorigenesis in APC min/+ mice based on liquid chromatography/mass spectrometry.

Sun, 14/10/2018 - 14:50
Related Articles High-throughput metabolomics for discovering metabolic biomarkers from intestinal tumorigenesis in APC min/+ mice based on liquid chromatography/mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Oct 02;1100-1101:131-139 Authors: Guo XD, Liu L, Xiao HY Abstract As a major public health concern, colon cancer is one of the most common cancer types, which is also the second cause of cancer death in developed countries and the third most common cancer in other parts of the world. It was reported that patients diagnosed at early stage have a chance to obtain 5-year survival rates at least compared to patients with late stage. Facing the multistep process in intestinal tumorigenesis, there is an urgent need to develop more effective early detection strategies for ameliorating the patient clinical outcome. Metabolomics open up a novel avenue of seeking valuable potential biomarkers for assessing disease severity and prognosticating course by dynamic snapshot of small molecule metabolites. The study aims to provide deeper insights into the discovery, identification and functional pathways analysis of differentially expressed metabolites in intestinal tumorigenesis in APC min/+ mice used by the serum metabolomics, and bring about useful information for further effective prevention and treatment of the disease. 17 marker metabolites and related metabolism pathway were identified using non-targeted metabolomics based on liquid chromatography/mass spectrometry (LC/MS) associated with multivariate statistical analysis. The ingenuity pathway analysis platform involved multiple-pathways was applied to metabolic network analysis for further understanding the relationship between functional metabolic pathways and disease. PMID: 30316137 [PubMed - as supplied by publisher]

Urinary metabonomic study of patients with acute coronary syndrome using UPLC-QTOF/MS.

Sun, 14/10/2018 - 14:50
Related Articles Urinary metabonomic study of patients with acute coronary syndrome using UPLC-QTOF/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Oct 07;1100-1101:122-130 Authors: Wang Y, Sun W, Zheng J, Xu C, Wang X, Li T, Tang Y, Li Z Abstract This urinary metabonomic study aimed to identify the potential metabolic biomarkers in acute coronary syndrome (ACS) patients. Ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) was used to analyze the urine samples from ACS patients and healthy controls. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were applied to characterizing the endogenous metabolites and potential biomarker, respectively. Among twenty biomarkers that functioned in nine metabolic pathways, nine biomarkers were found up-regulated significantly, including of isobutyryl‑l‑carnitine, 3‑methylglutarylcarnitine, cinnavalininate, l‑tryptophan, 3‑methyldioxyindole, palmitic acid, N4‑acetylaminobutanal, 3‑sulfino‑l‑alanine and S‑adenosyl‑l‑homocysteine. The other eleven biomarkers were showed down-regulated, including of l‑lactic acid, trigonelline, nicotinuric acid, l‑alanine, d‑alanyl‑d‑alanine, creatine, N4‑acetylaminobutanoate, glutathionyl spermidine, 5‑methoxytryptamine, kynurenic acid and xanthurenic acid. This study also implied that fatty acid metabolism, fatty acid β‑oxidation metabolism, amino acid metabolism and TCA cycle played important roles in ACS. Therefore, urinary metabolomics may improve the diagnosis efficacy of ACS and make it more accurate and comprehensive for ACS diagnosis. PMID: 30316136 [PubMed - as supplied by publisher]

Targeted metabolomics to understand the association between arsenic metabolism and diabetes-related outcomes: Preliminary evidence from the Strong Heart Family Study.

Sun, 14/10/2018 - 14:50
Related Articles Targeted metabolomics to understand the association between arsenic metabolism and diabetes-related outcomes: Preliminary evidence from the Strong Heart Family Study. Environ Res. 2018 Sep 27;168:146-157 Authors: Spratlen MJ, Grau-Perez M, Umans JG, Yracheta J, Best LG, Francesconi K, Goessler W, Bottiglieri T, Gamble MV, Cole SA, Zhao J, Navas-Acien A Abstract BACKGROUND: Inorganic arsenic exposure is ubiquitous and both exposure and inter-individual differences in its metabolism have been associated with cardiometabolic risk. A more efficient arsenic metabolism profile (lower MMA%, higher DMA%) has been associated with reduced risk for arsenic-related health outcomes. This profile, however, has also been associated with increased risk for diabetes-related outcomes. OBJECTIVES: The mechanism behind these conflicting associations is unclear; we hypothesized the one-carbon metabolism (OCM) pathway may play a role. METHODS: We evaluated the influence of OCM on the relationship between arsenic metabolism and diabetes-related outcomes (HOMA2-IR, waist circumference, fasting plasma glucose) using metabolomic data from an OCM-specific and P180 metabolite panel measured in plasma, arsenic metabolism measured in urine, and HOMA2-IR and FPG measured in fasting plasma. Samples were drawn from baseline visits (2001-2003) in 59 participants from the Strong Heart Family Study, a family-based cohort study of American Indians aged ≥14 years from Arizona, Oklahoma, and North/South Dakota. RESULTS: In unadjusted analyses, a 5% increase in DMA% was associated with higher HOMA2-IR (geometric mean ratio (GMR)= 1.13 (95% CI: 1.03, 1.25)) and waist circumference (mean difference=3.66 (0.95, 6.38). MMA% was significantly associated with lower HOMA2-IR and waist circumference. After adjustment for OCM-related metabolites (SAM, SAH, cysteine, glutamate, lysophosphatidylcholine 18.2, and three phosphatidlycholines), associations were attenuated and no longer significant. CONCLUSIONS: These preliminary results indicate that the association of lower MMA% and higher DMA% with diabetes-related outcomes may be influenced by OCM status, either through confounding, reverse causality, or mediation. PMID: 30316100 [PubMed - as supplied by publisher]

From Plants to Pharmacy Shelf: Focus on Toxicology.

Sun, 14/10/2018 - 14:50
Related Articles From Plants to Pharmacy Shelf: Focus on Toxicology. Food Chem Toxicol. 2018 Oct 10;: Authors: Georgiev MI, Sieniawska E PMID: 30315823 [PubMed - as supplied by publisher]

Urinary and Plasma Metabolomics Identify the Distinct Metabolic Profile of Disease State in Chronic Mouse Model of Multiple Sclerosis.

Sun, 14/10/2018 - 14:50
Related Articles Urinary and Plasma Metabolomics Identify the Distinct Metabolic Profile of Disease State in Chronic Mouse Model of Multiple Sclerosis. J Neuroimmune Pharmacol. 2018 Oct 12;: Authors: Singh J, Cerghet M, Poisson LM, Datta I, Labuzek K, Suhail H, Rattan R, Giri S Abstract Identification of non-invasive biomarkers of disease progression in multiple sclerosis (MS) is critically needed for monitoring the disease progression and for effective therapeutic interventions. Urine is an attractive source for non-invasive biomarkers because it is easily obtained in the clinic. In search of a urine metabolite signature of progression in chronic experimental autoimmune encephalomyelitis (EAE), we profiled urine at the chronic stage of the disease (day 45 post immunization) by global untargeted metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found 105 metabolites (P < 0.05) significantly altered at the chronic stage, indicating a robust alteration in the urine metabolite profile during disease. Assessment of altered metabolites against the Kyoto Encyclopedia of Genes and Genomes revealed distinct non-overlapping metabolic pathways and revealed phenylalanine-tyrosine and associated metabolism being the most impacted. Combined with previously performed plasma profiling, eight common metabolites were significantly altered in both of the biofluids. Metaboanalyst analysis of these common metabolites revealed that phenylalanine metabolism and Valine, leucine, and isoleucine biosynthetic pathways are central metabolic pathways in both bio-fluids and could be analyzed further, either for the discovery of therapeutics or biomarker development. Overall, our study suggests that urine and plasma metabolomics may contribute to the identification of a distinct metabolic fingerprint of EAE disease discriminating from the healthy control which may aid in the development of an objective non-invasive monitoring method for progressive autoimmune diseases like MS. Graphical Abstract Untargeted urinary metabolomics of a chronic mouse model of multiple sclerosis identified Phenylalanine, tyrosine & tryptophan metabolism as the significantly altered metabolic pathway. Eight common metabolites were identified when we combined urinary and plasma metabolic signature, which revealed a perturbation of Phenylalanine metabolism and valine, leucine & isoleucine metabolic pathways, involved in CNS dysfunction during diseases. The identified eight metabolic signature of urine and plasma may be of clinical relevance as potential biomarkers and guide towards the identification of specific metabolic pathways as novel drug targets. PMID: 30315511 [PubMed - as supplied by publisher]

Measuring Rates of ATP Synthesis.

Sun, 14/10/2018 - 14:50
Related Articles Measuring Rates of ATP Synthesis. Methods Mol Biol. 2019;1862:97-107 Authors: Bird MJ, Radenkovic S, Vermeersch P, Cassiman D Abstract Here, we offer you a high-throughput assay to measure the ATP synthesis capacity in cells or isolated mitochondria. More specifically, the assay is linked to the mitochondrial' electron transport chain components of your interest being either through complex I (with or without a linkage to pyruvate dehydrogenase activity), through complex II, or through the electron transport flavoprotein and complex I (β-oxidation of fatty acids). PMID: 30315462 [PubMed - in process]

Measuring In Vivo Tissue Metabolism Using 13C Glucose Infusions in Mice.

Sun, 14/10/2018 - 14:50
Related Articles Measuring In Vivo Tissue Metabolism Using 13C Glucose Infusions in Mice. Methods Mol Biol. 2019;1862:67-82 Authors: Broekaert D, Fendt SM Abstract Metabolic alterations are a hallmark of cancer. While determining metabolic changes in vitro has delivered valuable insight into the metabolism of cancer cells, it emerges that determining the in vivo metabolism adds an additional layer of information. Here, we therefore describe how to measure the in vivo metabolism of cancer tissue using 13C glucose infusions in mice. PMID: 30315460 [PubMed - in process]

13C Tracer Analysis and Metabolomics in 3D Cultured Cancer Cells.

Sun, 14/10/2018 - 14:50
Related Articles 13C Tracer Analysis and Metabolomics in 3D Cultured Cancer Cells. Methods Mol Biol. 2019;1862:53-66 Authors: van Gorsel M, Elia I, Fendt SM Abstract Metabolomics and 13C tracer analysis are state-of-the-art techniques that allow determining the concentration of metabolites and the activity of metabolic pathways, respectively. Three dimensional (3D) cultures of cancer cells constitute an enriched in vitro environment that can be used to assay anchorage-independent growth, spheroid formation, and extracellular matrix production by (cancer) cells. Here, we describe how to perform metabolomics and 13C tracer analysis in 3D cultures of cancer cells. PMID: 30315459 [PubMed - in process]

Measuring the Nutrient Metabolism of Adherent Cells in Culture.

Sun, 14/10/2018 - 14:50
Related Articles Measuring the Nutrient Metabolism of Adherent Cells in Culture. Methods Mol Biol. 2019;1862:37-52 Authors: Ogrodzinski MP, Teoh ST, Yu L, Broadwater D, Ensink E, Lunt SY Abstract Metabolite extraction from cells cultured in vitro enables the comprehensive measurement of intracellular metabolites. These extracts can be analyzed using techniques such as liquid chromatography-mass spectrometry (LC-MS). This chapter describes in detail a method for metabolite extraction from cultured adherent mammalian cells to collect both polar and nonpolar intracellular metabolites. This chapter also describes experimental design considerations for performing stable isotope labeling experiments, and the use of chemical derivatization to increase the number of compounds that can be detected using one chromatography method. PMID: 30315458 [PubMed - in process]

A Protocol to Compare Methods for Untargeted Metabolomics.

Sun, 14/10/2018 - 14:50
Related Articles A Protocol to Compare Methods for Untargeted Metabolomics. Methods Mol Biol. 2019;1862:1-15 Authors: Wang L, Naser FJ, Spalding JL, Patti GJ Abstract There are thousands of published methods for profiling metabolites with liquid chromatography/mass spectrometry (LC/MS). While many have been evaluated and optimized for a small number of select metabolites, very few have been assessed on the basis of global metabolite coverage. Thus, when performing untargeted metabolomics, researchers often question which combination of extraction techniques, chromatographic separations, and mass spectrometers is best for global profiling. Method comparisons are complicated because thousands of LC/MS signals (so-called features) in a typical untargeted metabolomic experiment cannot be readily identified with current resources. It is therefore challenging to distinguish methods that increase signal number due to improved metabolite coverage from methods that increase signal number due to contamination and artifacts. Here, we present the credentialing protocol to remove the latter from untargeted metabolomic datasets without having to identify metabolite structures. This protocol can be used to compare or optimize methods pertaining to any step of the untargeted metabolomic workflow (e.g., extraction, chromatography, mass spectrometer, informatic software, etc.). PMID: 30315456 [PubMed - in process]

De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever.

Sun, 14/10/2018 - 14:50
Related Articles De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever. J Biol Chem. 2018 Oct 12;: Authors: Bitew MA, Khoo CA, Neha N, De Souza DP, Tull D, Wawegama NK, Newton HJ, Sansom FM Abstract Coxiella burnetii is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of C. burnetii pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that nadB, encoding a putative L-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the wildtype, confirming the involvement of NadB in de novo NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of Escherichia coli NadB, and expression of wildtype and R275L GST-NadB fusion proteins in E. coli JM109, we found that purified recombinant wildtype GST-NadB has L-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to wildtype levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii. This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections. PMID: 30315113 [PubMed - as supplied by publisher]

Assessment of flux through oleoresin biosynthesis in epithelial cells of loblolly pine resin ducts.

Sat, 13/10/2018 - 14:24
Related Articles Assessment of flux through oleoresin biosynthesis in epithelial cells of loblolly pine resin ducts. J Exp Bot. 2018 Oct 11;: Authors: Turner GW, Parrish AN, Zager JJ, Fischedick JT, Lange BM Abstract The shoot system of pines contains abundant resin ducts, which harbor oleoresins that play important roles in constitutive and inducible defenses. In a pilot study, we assessed the chemical diversity of oleoresins obtained from mature tissues of loblolly pine trees (Pinus taeda L.). Building on these data sets, we designed experiments to assess oleoresin biosynthesis in needles of 2-year-old saplings. Comparative transcriptome analyses of single cell types indicated that genes involved in the biosynthesis of oleoresins are significantly enriched in isolated epithelial cells of resin ducts, compared with those expressed in mesophyll cells. Simulations using newly developed genome-scale models of epithelial and mesophyll cells, which incorporate our data on oleoresin yield and composition as well as gene expression patterns, predicted that heterotrophic metabolism in epithelial cells involves enhanced levels of oxidative phosphorylation and fermentation (providing redox and energy equivalents). Furthermore, flux was predicted to be more evenly distributed across the metabolic network of mesophyll cells, which, in contrast to epithelial cells, do not synthesize high levels of specialized metabolites. Our findings provide novel insights into the remarkable specialization of metabolism in epithelial cells. PMID: 30312429 [PubMed - as supplied by publisher]

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