PubMed

Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose-phosphate pathways in embryonic heart. J Biol Chem. 2018 Mar 14;: Authors: Peoples JNR, Maxmillian T, Le Q, Nadtochiy SM, Brookes PS, Porter GA, Davidson VL, Ebert SN Abstract Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart.  Here, we show that embryos lacking adrenergic stimulation have approximately 10-fold less cardiac ATP compared to littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation or degradation were affected in adrenergic-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational.  We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using liquid chromatography/mass spectrometry (LC/MS). Our results showed glucose-1-phosphate and glucose-6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentose-phosphate pathways were significantly lower compared to controls.  Further, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80% and 40%, respectively) lower in adrenergic-deficient hearts.  Addition of pyruvate and to a lesser extent, ribose, led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival. PMID: 29540484 [PubMed - as supplied by publisher]

Related Articles Cellular and Subcellular Level Localization of Maize Lipids and Metabolites Using High-Spatial Resolution MALDI Mass Spectrometry Imaging. Methods Mol Biol. 2018;1676:217-231 Authors: Dueñas ME, Feenstra AD, Korte AR, Hinners P, Lee YJ Abstract Recent technological advances have pushed the achievable spatial resolution for mass spectrometry imaging (MSI) to cellular and subcellular levels. Direct visualization of maize tissues by this tool has provided key insights into the localization of metabolites and lipids. This chapter outlines methodology for sample preparation, data acquisition, and data analysis of maize tissue sections using high-spatial resolution matrix-assisted laser desorption ionization (MALDI)-MSI, as well as the incorporation of a multi-resolution optical system, which allows for simple inter-conversion between different resolution setups (5, 10, and 50 μm imaging). PMID: 28986913 [PubMed - indexed for MEDLINE]

Related Articles Coupled brain and urine spectroscopy - in vivo metabolomic characterization of HMG-CoA lyase deficiency in 5 patients. Mol Genet Metab. 2017 Jun;121(2):111-118 Authors: Roland D, Jissendi-Tchofo P, Briand G, Vamecq J, Fontaine M, Ultré V, Acquaviva-Bourdain C, Mention K, Dobbelaere D Abstract BACKGROUND: 3-Hydroxy-3-Methylglutaryl-Coenzyme A (HMG-CoA) lyase deficiency is a rare inborn error of leucine metabolism and ketogenesis. Despite recurrent hypoglycemia and metabolic decompensations, most patients have a good clinical and neurological outcome contrasting with abnormal brain magnetic resonance imaging (MRI) signals and consistent abnormal brain proton magnetic resonance spectroscopy (1H-MRS) metabolite peaks. Identifying these metabolites could provide surrogate markers of the disease and improve understanding of MRI-clinical discrepancy and follow-up of affected patients. METHODS: Urine samples, brain MRI and 1H-MRS in 5 patients with HMG-CoA lyase deficiency (4 boys and 1 girl aged from 25days to 10years) were, for each patient, obtained on the same day. Brain and urine spectroscopy were performed at the same pH by studying urine at pH 7.4. Due to pH-induced modifications in chemical shifts and because reference 1H NMR spectra are obtained at pH 2.5, spectroscopy of normal urine added with the suspected metabolite was further performed at this pH to validate the correct identification of compounds. RESULTS: Mild to extended abnormal white matter MRI signals were observed in all cases. Brain spectroscopy abnormal peaks at 0.8-1.1ppm, 1.2-1.4ppm and 2.4ppm were also detected by urine spectroscopy at pH 7.4. Taking into account pH-induced changes in chemical shifts, brain abnormal peaks in patients were formally identified to be those of 3-hydroxyisovaleric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxy-3-methylglutaric acids. CONCLUSION: 3-Methylglutaric, 3-hydroxyisovaleric and 3-hydroxy-3-methylglutaric acids identified on urine 1H-NMR spectra of 5 patients with HMG-CoA lyase deficiency are responsible for the cerebral spectroscopy signature seen in these patients, validating their local involvement in brain and putative contribution to brain neuropathology. PMID: 28396157 [PubMed - indexed for MEDLINE]

Diet, Genetics, and the Gut Microbiome Drive Dynamic Changes in Plasma Metabolites. Cell Rep. 2018 Mar 13;22(11):3072-3086 Authors: Fujisaka S, Avila-Pacheco J, Soto M, Kostic A, Dreyfuss JM, Pan H, Ussar S, Altindis E, Li N, Bry L, Clish CB, Kahn CR Abstract Diet, genetics, and the gut microbiome are determinants of metabolic status, in part through production of metabolites by the gut microbiota. To understand the mechanisms linking these factors, we performed LC-MS-based metabolomic analysis of cecal contents and plasma from C57BL/6J, 129S1/SvImJ, and 129S6/SvEvTac mice on chow or a high-fat diet (HFD) and HFD-treated with vancomycin or metronidazole. Prediction of the functional metagenome of gut bacteria by PICRUSt analysis of 16S sequences revealed dramatic differences in microbial metabolism. Cecal and plasma metabolites showed multifold differences reflecting the combined and integrated effects of diet, antibiotics, host background, and the gut microbiome. Eighteen plasma metabolites correlated positively or negatively with host insulin resistance across strains and diets. Over 1,000 still-unidentified metabolite peaks were also highly regulated by diet, antibiotics, and genetic background. Thus, diet, host genetics, and the gut microbiota interact to create distinct responses in plasma metabolites, which can contribute to regulation of metabolism and insulin resistance. PMID: 29539432 [PubMed - in process]

Metabolomic profiling of macrophages determines the discrete metabolomic signature and metabolomic interactome triggered by polarising immune stimuli. PLoS One. 2018;13(3):e0194126 Authors: Rattigan KM, Pountain AW, Regnault C, Achcar F, Vincent IM, Goodyear CS, Barrett MP Abstract Priming and activating immune stimuli have profound effects on macrophages, however, studies generally evaluate stimuli in isolation rather than in combination. In this study we have investigated the effects of pro-inflammatory and anti-inflammatory stimuli either alone or in combination on macrophage metabolism. These stimuli include host factors such as IFNγ and ovalbumin-immunoglobulin immune complexes, or pathogen factors such as LPS. Untargeted LC-MS based metabolomics provided an in-depth profile of the macrophage metabolome, and revealed specific changes in metabolite abundance upon either individual stimuli or combined stimuli. Here, by factoring in an interaction term in the linear model, we define the metabolome interactome. This approach allowed us to determine whether stimuli interact in a synergistic or antagonistic manner. In conclusion this study demonstrates a robust approach to interrogate immune-metabolism, especially systems that model host-pathogen interactions. PMID: 29538444 [PubMed - in process]

Metabolomics: A High-Throughput Platform for Metabolite Profile Exploration. Methods Mol Biol. 2018;1754:265-292 Authors: Cheng J, Lan W, Zheng G, Gao X Abstract Metabolomics aims to quantitatively measure small-molecule metabolites in biological samples, such as bodily fluids (e.g., urine, blood, and saliva), tissues, and breathe exhalation, which reflects metabolic responses of a living system to pathophysiological stimuli or genetic modification. In the past decade, metabolomics has made notable progresses in providing useful systematic insights into the underlying mechanisms and offering potential biomarkers of many diseases. Metabolomics is a complementary manner of genomics and transcriptomics, and bridges the gap between genotype and phenotype, which reflects the functional output of a biological system interplaying with environmental factors. Recently, the technology of metabolomics study has been developed quickly. This review will discuss the whole pipeline of metabolomics study, including experimental design, sample collection and preparation, sample detection and data analysis, as well as mechanism interpretation, which can help understand metabolic effects and metabolite function for living organism in system level. PMID: 29536449 [PubMed - in process]

Promises and pitfalls of untargeted metabolomics. J Inherit Metab Dis. 2018 Mar 13;: Authors: Gertsman I, Barshop BA Abstract Metabolomics is one of the newer omics fields, and has enabled researchers to complement genomic and protein level analysis of disease with both semi-quantitative and quantitative metabolite levels, which are the chemical mediators that constitute a given phenotype. Over more than a decade, methodologies have advanced for both targeted (quantification of specific analytes) as well as untargeted metabolomics (biomarker discovery and global metabolite profiling). Untargeted metabolomics is especially useful when there is no a priori metabolic hypothesis. Liquid chromatography coupled to mass spectrometry (LC-MS) has been the preferred choice for untargeted metabolomics, given the versatility in metabolite coverage and sensitivity of these instruments. Resolving and profiling many hundreds to thousands of metabolites with varying chemical properties in a biological sample presents unique challenges, or pitfalls. In this review, we address the various obstacles and corrective measures available in four major aspects associated with an untargeted metabolomics experiment: (1) experimental design, (2) pre-analytical (sample collection and preparation), (3) analytical (chromatography and detection), and (4) post-analytical (data processing). PMID: 29536203 [PubMed - as supplied by publisher]

Multiomics tools for the diagnosis and treatment of rare neurological disease. J Inherit Metab Dis. 2018 Mar 13;: Authors: Crowther LM, Poms M, Plecko B Abstract Conventional workup of rare neurological disease is frequently hampered by diagnostic delay or lack of diagnosis. While biomarkers have been established for many neurometabolic disorders, improved methods are required for diagnosis of previously unidentified or underreported causes of rare neurological disease. This would result in a higher diagnostic yield and increased patient numbers required for interventional studies. Recent studies using next-generation sequencing and metabolomics have led to identification of novel disease-causing genes and biomarkers. This combined approach can assist in overcoming challenges associated with analyzing and interpreting the large amount of data obtained from each technique. In particular, metabolomics can support the pathogenicity of sequence variants in genes encoding enzymes or transporters involved in metabolic pathways. Moreover, metabolomics can show the broader perturbation caused by inborn errors of metabolism and identify a metabolic fingerprint of metabolic disorders. As such, using "omics" has great potential to meet the current needs for improved diagnosis and elucidation of rare neurological disease. PMID: 29536202 [PubMed - as supplied by publisher]

Physiological and Metabolic Responses Triggered by Omeprazole Improve Tomato Plant Tolerance to NaCl Stress. Front Plant Sci. 2018;9:249 Authors: Rouphael Y, Raimondi G, Lucini L, Carillo P, Kyriacou MC, Colla G, Cirillo V, Pannico A, El-Nakhel C, De Pascale S Abstract Interest in the role of small bioactive molecules (< 500 Da) in plants is on the rise, compelled by plant scientists' attempt to unravel their mode of action implicated in stimulating growth and enhancing tolerance to environmental stressors. The current study aimed at elucidating the morphological, physiological and metabolomic changes occurring in greenhouse tomato (cv. Seny) treated with omeprazole (OMP), a benzimidazole inhibitor of animal proton pumps. The OMP was applied at three rates (0, 10, or 100 μM) as substrate drench for tomato plants grown under nonsaline (control) or saline conditions sustained by nutrient solutions of 1 or 75 mM NaCl, respectively. Increasing NaCl concentration from 1 to 75 mM decreased the tomato shoot dry weight by 49% in the 0 μM OMP treatment, whereas the reduction was not significant at 10 or 100 μM of OMP. Treatment of salinized (75 mM NaCl) tomato plants with 10 and especially 100 μM OMP decreased Na+ and Cl- while it increased Ca2+ concentration in the leaves. However, OMP was not strictly involved in ion homeostasis since the K+ to Na+ ratio did not increase under combined salinity and OMP treatment. OMP increased root dry weight, root morphological characteristics (total length and surface), transpiration, and net photosynthetic rate independently of salinity. Metabolic profiling of leaves through UHPLC liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry facilitated identification of the reprogramming of a wide range of metabolites in response to OMP treatment. Hormonal changes involved an increase in ABA, decrease in auxins and cytokinin, and a tendency for GA down accumulation. Cutin biosynthesis, alteration of membrane lipids and heightened radical scavenging ability related to the accumulation of phenolics and carotenoids were observed. Several other stress-related compounds, such as polyamine conjugates, alkaloids and sesquiterpene lactones, were altered in response to OMP. Although a specific and well-defined mechanism could not be posited, the metabolic processes involved in OMP action suggest that this small bioactive molecule might have a hormone-like activity that ultimately elicits an improved tolerance to NaCl salinity stress. PMID: 29535755 [PubMed]

Genome-Wide Association Study on Immunoglobulin G Glycosylation Patterns. Front Immunol. 2018;9:277 Authors: Wahl A, van den Akker E, Klaric L, Štambuk J, Benedetti E, Plomp R, Razdorov G, Trbojević-Akmačić I, Deelen J, van Heemst D, Slagboom PE, Vučković F, Grallert H, Krumsiek J, Strauch K, Peters A, Meitinger T, Hayward C, Wuhrer M, Beekman M, Lauc G, Gieger C Abstract Immunoglobulin G (IgG), a glycoprotein secreted by plasma B-cells, plays a major role in the human adaptive immune response and are associated with a wide range of diseases. Glycosylation of the Fc binding region of IgGs, responsible for the antibody's effector function, is essential for prompting a proper immune response. This study focuses on the general genetic impact on IgG glycosylation as well as corresponding subclass specificities. To identify genetic loci involved in IgG glycosylation, we performed a genome-wide association study (GWAS) on liquid chromatography electrospray mass spectrometry (LC-ESI-MS)-measured IgG glycopeptides of 1,823 individuals in the Cooperative Health Research in the Augsburg Region (KORA F4) study cohort. In addition, we performed GWAS on subclass-specific ratios of IgG glycans to gain power in identifying genetic factors underlying single enzymatic steps in the glycosylation pathways. We replicated our findings in 1,836 individuals from the Leiden Longevity Study (LLS). We were able to show subclass-specific genetic influences on single IgG glycan structures. The replicated results indicate that, in addition to genes encoding for glycosyltransferases (i.e., ST6GAL1, B4GALT1, FUT8, and MGAT3), other genetic loci have strong influences on the IgG glycosylation patterns. A novel locus on chromosome 1, harboring RUNX3, which encodes for a transcription factor of the runt domain-containing family, is associated with decreased galactosylation. Interestingly, members of the RUNX family are cross-regulated, and RUNX3 is involved in both IgA class switching and B-cell maturation as well as T-cell differentiation and apoptosis. Besides the involvement of glycosyltransferases in IgG glycosylation, we suggest that, due to the impact of variants within RUNX3, potentially mechanisms involved in B-cell activation and T-cell differentiation during the immune response as well as cell migration and invasion involve IgG glycosylation. PMID: 29535710 [PubMed]

Quality Variation of Goji (Fruits of Lycium spp.) in China: A Comparative Morphological and Metabolomic Analysis. Front Pharmacol. 2018;9:151 Authors: Yao R, Heinrich M, Zou Y, Reich E, Zhang X, Chen Y, Weckerle CS Abstract Goji (fruits of Lycium barbarum L. and L. chinense Mill.) has been used in China as food and medicine for millennia, and globally has been consumed increasingly as a healthy food. Ningxia, with a semi-arid climate, always had the reputation of producing best goji quality (daodi area). Recently, the increasing market demand pushed the cultivation into new regions with different climates. We therefore ask: How does goji quality differ among production areas of various climatic regions? Historical records are used to trace the spread of goji production in China over time. Quality measurements of 51 samples were correlated with the four main production areas in China: monsoon (Hebei), semi-arid (Ningxia, Gansu, and Inner Mongolia), plateau (Qinghai) and arid regions (Xinjiang). We include morphological characteristics, sugar and polysaccharide content, antioxidant activity, and metabolomic profiling to compare goji among climatic regions. Goji cultivation probably began in the East (Hebei) of China around 100 CE and later shifted westward to the semi-arid regions. Goji from monsoon, plateau and arid regions differ according to its fruit morphology, whereas semi-arid goji cannot be separated from the other regions. L. chinense fruits, which are exclusively cultivated in Hebei (monsoon), are significantly lighter, smaller and brighter in color, while the heaviest and largest fruits (L. barbarum) stem from the plateau. The metabolomic profiling separates the two species but not the regions of cultivation. Lycium chinense and samples from the semi-arid regions have significantly (p < 0.01) lower sugar contents and L. chinense shows the highest antioxidant activity. Our results do not justify superiority of a specific production area over other areas. Instead it will be essential to distinguish goji from different regions based on the specific morphological and chemical traits with the aim to understand what its intended uses are. PMID: 29535631 [PubMed]

Comparative metabolism of tripolide and triptonide using metabolomics. Food Chem Toxicol. 2018 Mar 10;: Authors: Hu DD, Chen XL, Xiao XR, Wang YK, Liu F, Zhao Q, Li X, Yang XW, Li F PMID: 29534979 [PubMed - as supplied by publisher]

Laboratory evolution reveals regulatory and metabolic trade-offs of glycerol utilization in Saccharomyces cerevisiae. Metab Eng. 2018 Mar 10;: Authors: Strucko T, Zirngibl K, Pereira F, Kafkia E, Mohamed ET, Rettel M, Stein F, Feist AM, Jouhten P, Patil KR, Forster J Abstract Most microbial species, including model eukaryote Saccharomyces cerevisiae, possess genetic capability to utilize many alternative nutrient sources. Yet, it remains an open question whether these manifest into assimilatory phenotypes. Despite possessing all necessary pathways, S. cerevisiae grows poorly or not at all when glycerol is the sole carbon source. Here we discover, through multiple evolved lineages, genetic determinants underlying glycerol catabolism and the associated fitness trade-offs. Most evolved lineages adapted through mutations in the HOG pathway, but showed hampered osmotolerance. In the other lineages, we find that only three mutations cause the improved phenotype. One of these contributes counter-intuitively by decoupling the TCA cycle from oxidative phosphorylation, and thereby hampers ethanol utilization. Transcriptomics, proteomics and metabolomics analysis of the re-engineered strains affirmed the causality of the three mutations at molecular level. Introduction of these mutations resulted in improved glycerol utilization also in industrial strains. Our findings not only have a direct relevance for improving glycerol-based bioprocesses, but also illustrate how a metabolic pathway can remain unexploited due to fitness trade-offs in other, ecologically important, traits. PMID: 29534903 [PubMed - as supplied by publisher]

Nanoparticle-Assisted Metabolomics. Metabolites. 2018 Mar 13;8(1): Authors: Zhang B, Xie M, Bruschweiler-Li L, Brüschweiler R Abstract Understanding and harnessing the interactions between nanoparticles and biological molecules is at the forefront of applications of nanotechnology to modern biology. Metabolomics has emerged as a prominent player in systems biology as a complement to genomics, transcriptomics and proteomics. Its focus is the systematic study of metabolite identities and concentration changes in living systems. Despite significant progress over the recent past, important challenges in metabolomics remain, such as the deconvolution of the spectra of complex mixtures with strong overlaps, the sensitive detection of metabolites at low abundance, unambiguous identification of known metabolites, structure determination of unknown metabolites and standardized sample preparation for quantitative comparisons. Recent research has demonstrated that some of these challenges can be substantially alleviated with the help of nanoscience. Nanoparticles in particular have found applications in various areas of bioanalytical chemistry and metabolomics. Their chemical surface properties and increased surface-to-volume ratio endows them with a broad range of binding affinities to biomacromolecules and metabolites. The specific interactions of nanoparticles with metabolites or biomacromolecules help, for example, simplify metabolomics spectra, improve the ionization efficiency for mass spectrometry or reveal relationships between spectral signals that belong to the same molecule. Lessons learned from nanoparticle-assisted metabolomics may also benefit other emerging areas, such as nanotoxicity and nanopharmaceutics. PMID: 29533993 [PubMed]

Quantitation of the anticancer drug abiraterone and its metabolite Δ(4)-abiraterone in human plasma using high-resolution mass spectrometry. J Pharm Biomed Anal. 2018 Mar 06;154:66-74 Authors: Bhatnagar A, McKay MJ, Crumbaker M, Ahire K, Karuso P, Gurney H, Molloy MP Abstract Abiraterone acetate is administered as a prodrug to patients with metastatic, castration-resistant prostate cancer (mCRPC) and is readily metabolized into the potent 17a-hydroxylase/17,20-lyase (CYP17) enzyme inhibitor and androgen receptor inhibitor abiraterone and Δ(4)-abiraterone (D4A), respectively. To investigate pharmacokinetic variability in abiraterone acetate metabolism we developed highly sensitive liquid chromatography/mass spectrometry (LC/MS) assays for the simultaneous quantitation of abiraterone and D4A in human plasma using high-resolution mass spectrometry (HRMS) on an Orbitrap mass spectrometer. This study demonstrates the quantitative performance of HRMS and compares the conventional Parallel Reaction Monitoring (PRM) mode of quantitation with the unconventional Full scan MS mode conducted at high resolution (>70,000 resolution). The use of HRMS for quantitation of abiraterone and D4A yielded assays that were linear over a broad concentration range (0.074-509.6 ng/mL for abiraterone; 0.075-59.93 ng/mL for D4A) in both Full scan MS and PRM modes. The assay precision for abiraterone and D4A was below 5% in PRM mode and 7% in Full scan MS mode. Accuracies fell within 98-107% for abiraterone and 104-112% for D4A in PRM mode, and 96-116% for abiraterone and 96-105% for D4A in Full scan MS mode, each meeting the acceptance criteria of FDA approved guidelines for bioanalytical methods The PRM analysis of abiraterone and D4A provided high specificity and reduced background interference, however the Full scan MS detection at a resolution of 70,000 was advantageous in that it required minimal optimization, was simple to implement, yielded comparable quantitative characteristics to PRM and the data is useful for re-analysis. Use of the assays were demonstrated for quantitation of these metabolites in steady state trough level plasma of seventeen (17) patients with mCRPC, demonstrating the inter-patient variability of up to 10-fold concentration. PMID: 29533860 [PubMed - as supplied by publisher]

Morphology of glandular trichomes of Japanese catnip (Schizonepeta tenuifolia Briquet) and developmental dynamics of their secretory activity. Phytochemistry. 2018 Mar 10;150:23-30 Authors: Liu C, Srividya N, Parrish AN, Yue W, Shan M, Wu Q, Lange BM Abstract Schizonepeta tenuifolia Briquet, commonly known as Japanese catnip, is used for the treatment of colds, headaches, fevers, and skin rashes in traditional Asian medicine (China, Japan and Korea). The volatile oil and its constituents have various demonstrated biological activities, but there is currently limited information regarding the site of biosynthesis. Light microscopy and scanning electron microscopy indicated the presence of three distinct glandular trichome types which, based on their morphological features, are referred to as peltate, capitate and digitiform glandular trichomes. Laser scanning microscopy and 3D reconstruction demonstrated that terpenoid-producing peltate glandular trichomes contain a disk of twelve secretory cells. The oil of peltate glandular trichomes, collected by laser microdissection or using custom-made micropipettes, was demonstrated to contain (-)-pulegone, (+)-menthone and (+)-limonene as major constituents. Digitiform and capitate glandular trichomes did not contain appreciable levels of terpenoid volatiles. The yield of distilled oil from spikes was significantly (44%) higher than that from leaves, while the composition of oils was very similar. Oils collected directly from leaf peltate glandular trichomes over the course of a growing season contained primarily (-)-pulegone (>80% at 32 days after germination) in young plants, while (+)-menthone began to accumulate later (>75% at 80 days after germination), at the expense of (-)-pulegone (the levels of (+)-limonene remained fairly stable at 3-5%). The current study establishes the morphological and chemical characteristics of glandular trichome types of S. tenuifolia, and also provides the basis for unraveling the biosynthesis of essential oil in this popular medicinal plant. PMID: 29533838 [PubMed - as supplied by publisher]

Interrogation of Benzomalvin Biosynthesis using FAC-MS: Discovery of a Benzodiazepine Synthase Activity. Biochemistry. 2018 Mar 13;: Authors: Clevenger KD, Ye R, Bok JW, Thomas PM, Islam MN, Miley GP, Robey M, Chen C, Yang K, Swyers M, Wu E, Gao P, Wu C, Keller NP, Kelleher NL Abstract The benzodiazepine benzomalvin A/D is a fungal-derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene non-ribosomal peptide synthetase cluster. Here, we utilize <u>f</u>ungal <u>a</u>rtificial <u>c</u>hromosomes with <u>m</u>etabolomic <u>s</u>coring (FAC-MS) to carry out molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncover evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities. PMID: 29533658 [PubMed - as supplied by publisher]

Probing the application range and selectivity of a differential mobility spectrometry-mass spectrometry platform for metabolomics. Anal Bioanal Chem. 2018 Mar 12;: Authors: Wernisch S, Afshinnia F, Rajendiran T, Pennathur S Abstract Metabolomics applications of differential mobility spectrometry (DMS)-mass spectrometry (MS) have largely concentrated on targeted assays and the removal of isobaric or chemical interferences from the signals of a small number of analytes. In the work reported here, we systematically investigated the application range of a DMS-MS method for metabolomics using more than 800 authentic metabolite standards as the test set. The coverage achieved with the DMS-MS platform was comparable to that achieved with chromatographic methods. High orthogonality was observed between hydrophilic interaction liquid chromatography and the 2-propanol-mediated DMS separation, and previously observed similarities were confirmed for the DMS platform and reversed-phase liquid chromatography. We describe the chemical selectivity observed for selected subsets of the metabolite test set, such as lipids, amino acids, nucleotides, and organic acids. Furthermore, we rationalize the behavior and separation of isomeric aromatic acids, bile acids, and other metabolites. Graphical abstract Differential mobility spectrometry-mass spectrometry (DMS-MS) facilitates rapid separation of metabolites of similar mass-to-charge ratio by distributing them across the compensation voltage range on the basis of their different molecular structures. PMID: 29532192 [PubMed - as supplied by publisher]

Metabolic Heterogeneity Evidenced by MRS among Patient-Derived Glioblastoma Multiforme Stem-Like Cells Accounts for Cell Clustering and Different Responses to Drugs. Stem Cells Int. 2018;2018:3292704 Authors: Grande S, Palma A, Ricci-Vitiani L, Luciani AM, Buccarelli M, Biffoni M, Molinari A, Calcabrini A, D'Amore E, Guidoni L, Pallini R, Viti V, Rosi A Abstract Clustering of patient-derived glioma stem-like cells (GSCs) through unsupervised analysis of metabolites detected by magnetic resonance spectroscopy (MRS) evidenced three subgroups, namely clusters 1a and 1b, with high intergroup similarity and neural fingerprints, and cluster 2, with a metabolism typical of commercial tumor lines. In addition, subclones generated by the same GSC line showed different metabolic phenotypes. Aerobic glycolysis prevailed in cluster 2 cells as demonstrated by higher lactate production compared to cluster 1 cells. Oligomycin, a mitochondrial ATPase inhibitor, induced high lactate extrusion only in cluster 1 cells, where it produced neutral lipid accumulation detected as mobile lipid signals by MRS and lipid droplets by confocal microscopy. These results indicate a relevant role of mitochondrial fatty acid oxidation for energy production in GSCs. On the other hand, further metabolic differences, likely accounting for different therapy responsiveness observed after etomoxir treatment, suggest that caution must be used in considering patient treatment with mitochondria FAO blockers. Metabolomics and metabolic profiling may contribute to discover new diagnostic or prognostic biomarkers to be used for personalized therapies. PMID: 29531533 [PubMed]

CoA synthase regulates mitotic fidelity via CBP-mediated acetylation. Nat Commun. 2018 Mar 12;9(1):1039 Authors: Lin CC, Kitagawa M, Tang X, Hou MH, Wu J, Qu DC, Srinivas V, Liu X, Thompson JW, Mathey-Prevot B, Yao TP, Lee SH, Chi JT Abstract The temporal activation of kinases and timely ubiquitin-mediated degradation is central to faithful mitosis. Here we present evidence that acetylation controlled by Coenzyme A synthase (COASY) and acetyltransferase CBP constitutes a novel mechanism that ensures faithful mitosis. We found that COASY knockdown triggers prolonged mitosis and multinucleation. Acetylome analysis reveals that COASY inactivation leads to hyper-acetylation of proteins associated with mitosis, including CBP and an Aurora A kinase activator, TPX2. During early mitosis, a transient CBP-mediated TPX2 acetylation is associated with TPX2 accumulation and Aurora A activation. The recruitment of COASY inhibits CBP-mediated TPX2 acetylation, promoting TPX2 degradation for mitotic exit. Consistently, we detected a stage-specific COASY-CBP-TPX2 association during mitosis. Remarkably, pharmacological and genetic inactivation of CBP effectively rescued the mitotic defects caused by COASY knockdown. Together, our findings uncover a novel mitotic regulation wherein COASY and CBP coordinate an acetylation network to enforce productive mitosis. PMID: 29531224 [PubMed - in process]