PubMed

NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery. Sci Rep. 2017 Feb 15;7:42324 Authors: Santana-Filho AP, Jacomasso T, Riter DS, Barison A, Iacomini M, Winnischofer SM, Sassaki GL Abstract Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of (1)H NMR data was able not only to differentiate the melanocyte cell line from the tumorigenic ones but also distinguish among the 3 tumorigenic cell lines. We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines. PMID: 28198377 [PubMed - in process]

Evaluating effects of L-carnitine on human bone-marrow-derived mesenchymal stem cells. Cell Tissue Res. 2017 Feb 14;: Authors: Fujisawa K, Takami T, Fukui Y, Quintanilha LF, Matsumoto T, Yamamoto N, Sakaida I Abstract Mesenchymal stem cells (MSCs) are multipotent cells showing potential for use in regenerative medicine. Culture techniques that are more stable and methods for the more efficient production of MSCs with therapeutic efficacy are needed. We evaluate the effects of growing bone marrow (Bm)-derived MSCs in the presence of L-carnitine, which is believed to promote lipid metabolism and to suppress apoptosis. The presence of L-carnitine decreased the degree of drug-induced apoptosis and suppressed adipogenic differentiation. Metabolomic analysis by means of the exhaustive investigation of metabolic products showed that, in addition to increased β-oxidation and the expression of all carnitine derivatives other than deoxycarnitine (an intermediate in carnitine synthesis), polysaturated and polyunsaturated acids were down-regulated. An integrated analysis incorporating both serial analysis of gene expression and metabolomics revealed increases in cell survival, suggesting the utility of carnitine. The addition of carnitine elevated the oxygen consumption rate by BmMSCs that had been cultured for only a few generations and those that had become senescent following repeated replication indicating that mitochondrial activation occurred. Our exhaustive analysis of the effects of various carnitine metabolites thus suggests that the addition of L-carnitine to BmMSCs during expansion enables efficient cell production. PMID: 28197778 [PubMed - as supplied by publisher]

Rats' urinary metabolomes reveal the potential roles of functional foods and exercise in obesity management. Food Funct. 2017 Feb 15;: Authors: Farag MA, Ammar NM, Kholeif TE, Metwally NS, El-Sheikh NM, Wessjohann LA, Abdel-Hamid AZ Abstract The complexity of the metabolic changes in obese individuals still presents a challenge for the understanding of obesity-related metabolic disruptions and for obesity management. In this study, a gas chromatography mass spectrometry (GC-MS) based metabolomics approach targeting urine metabolism has been applied to assess the potential roles of functional foods and exercise for obesity management in rats. Male albino rats diagnosed as obese via histopathology and biochemical assays were administered functional foods in common use for obesity management including pomegranate, grapefruit, and red cabbage juice extracts in parallel with swimming exercise. Urine samples were collected from these rats, and likewise from healthy control animals, for metabolite analysis using (GC-MS) coupled to multivariate data analysis. The results revealed a significant elevation in oxalate and phosphate levels in obese rat urine concurrent with lower lactate levels as compared to the control group. Furthermore, and to pinpoint the bioactive agents in the administered functional foods, ultra performance liquid chromatography (UPLC) coupled to high resolution time-of-flight mass spectrometry (TOF-MS) was employed for secondary metabolite profiling. The different phenolic classes found in the examined functional foods, viz. ellagitannins in pomegranate, flavanones in grapefruit and flavonols in red cabbage, are likely to mediate their anti-obesity effects. The results indicate that these functional foods and exercise were quite effective in reverting obesity-related metabolic disruptions back to normal status, as revealed by orthogonal partial least squares-discriminant analysis (OPLS-DA). PMID: 28197590 [PubMed - as supplied by publisher]

Prognostic impact of the expression of NCR1 and NCR3 NK cell receptors and PD-L1 on advanced non-small cell lung cancer. Oncoimmunology. 2017;6(1):e1163456 Authors: Fend L, Rusakiewicz S, Adam J, Bastien B, Caignard A, Messaoudene M, Iribarren C, Cremer I, Marabelle A, Borg C, Semeraro M, Barraud L, Limacher JM, Eggermont A, Kroemer G, Zitvogel L Abstract The putative contribution of natural killer (NK) cells to immunosurveillance in non-small cell lung cancer (NSCLC) has been an ongoing conundrum. Here, we used a readily standardizable quantitative real time polymerase chain reaction (qRT-PCR) to measure the expression of NK cell receptors in total peripheral blood mononuclear cells (PBMC) from healthy volunteers (HV), patients with gastrointestinal stromal tumors (GIST), neuroblastoma (NB), melanoma or NSCLC. We quantified NCR1 (which codes for NKp46) and NCR3 (which codes for NKp30), as well as that of three NCR3 splice variants (which give rise to immunostimulatory NKp30A and NKp30B, as well as to immunosuppressive NKp30C). NSCLC patients expressed lower levels of NCR1 than did HV. Remarkably, NCR3 was lower in NSCLC patients than in HV as well as in all other malignancies. Moreover, a discrete proportion of NSCLC patients exhibited a particular low ratio between NKp30B and NKp30C (ΔBC). In the overall cohort, low expression of NCR3 correlated with poor overall and progression-free survival (PFS). When patients were stratified according to the level of PD-L1 expression by NSCLC cells, within the PD-L1(high) category (>5% positive tumors), the sole parameter that affected prognosis was the expression of NCR1. However, in patients bearing tumors with negative PD-L1 expression on tumor or tumor-infiltrating stromal cells, the ΔBC(low) patients exhibited a dismal prognosis. Altogether, these results strongly suggest that NK cells mediate immunosurveillance against NSCLC and that measuring NK cell receptor expression by blood cells can yield useful biomarkers for patient stratification. PMID: 28197362 [PubMed - in process]

NKp30 isoforms and NKp30 ligands are predictive biomarkers of response to imatinib mesylate in metastatic GIST patients. Oncoimmunology. 2017;6(1):e1137418 Authors: Rusakiewicz S, Perier A, Semeraro M, Pitt JM, Pogge von Strandmann E, Reiners KS, Aspeslagh S, Pipéroglou C, Vély F, Ivagnes A, Jegou S, Halama N, Chaigneau L, Validire P, Christidis C, Perniceni T, Landi B, Berger A, Isambert N, Domont J, Bonvalot S, Terrier P, Adam J, Coindre JM, Emile JF, Poirier-Colame V, Chaba K, Rocha B, Caignard A, Toubert A, Enot D, Koch J, Marabelle A, Lambert M, Caillat-Zucman S, Leyvraz S, Auclair C, Vivier E, Eggermont A, Borg C, Blay JY, Le Cesne A, Mir O, Zitvogel L Abstract Despite effective targeted therapy acting on KIT and PDGFRA tyrosine kinases, gastrointestinal stromal tumors (GIST) escape treatment by acquiring mutations conveying resistance to imatinib mesylate (IM). Following the identification of NKp30-based immunosurveillance of GIST and the off-target effects of IM on NK cell functions, we investigated the predictive value of NKp30 isoforms and NKp30 soluble ligands in blood for the clinical response to IM. The relative expression and the proportions of NKp30 isoforms markedly impacted both event-free and overall survival, in two independent cohorts of metastatic GIST. Phenotypes based on disbalanced NKp30B/NKp30C ratio (ΔBC(low)) and low expression levels of NKp30A were identified in one third of patients with dismal prognosis across molecular subtypes. This ΔBC(low) blood phenotype was associated with a pro-inflammatory and immunosuppressive tumor microenvironment. In addition, detectable levels of the NKp30 ligand sB7-H6 predicted a worse prognosis in metastatic GIST. Soluble BAG6, an alternate ligand for NKp30 was associated with low NKp30 transcription and had additional predictive value in GIST patients with high NKp30 expression. Such GIST microenvironments could be rescued by therapy based on rIFN-α and anti-TRAIL mAb which reinstated innate immunity. PMID: 28197361 [PubMed - in process]

Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries. Front Plant Sci. 2017;8:53 Authors: Lecourieux F, Kappel C, Pieri P, Charon J, Pillet J, Hilbert G, Renaud C, Gomès E, Delrot S, Lecourieux D Abstract Reproductive development of grapevine and berry composition are both strongly influenced by temperature. To date, the molecular mechanisms involved in grapevine berries response to high temperatures are poorly understood. Unlike recent data that addressed the effects on berry development of elevated temperatures applied at the whole plant level, the present work particularly focuses on the fruit responses triggered by direct exposure to heat treatment (HT). In the context of climate change, this work focusing on temperature effect at the microclimate level is of particular interest as it can help to better understand the consequences of leaf removal (a common viticultural practice) on berry development. HT (+ 8°C) was locally applied to clusters from Cabernet Sauvignon fruiting cuttings at three different developmental stages (middle green, veraison and middle ripening). Samples were collected 1, 7, and 14 days after treatment and used for metabolic and transcriptomic analyses. The results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. When applied at the herbaceous stage, HT delayed the onset of veraison. Heating also strongly altered the berry concentration of amino acids and organic acids (e.g., phenylalanine, γ-aminobutyric acid and malate) and decreased the anthocyanin content at maturity. These physiological alterations could be partly explained by the deep remodeling of transcriptome in heated berries. More than 7000 genes were deregulated in at least one of the nine experimental conditions. The most affected processes belong to the categories "stress responses," "protein metabolism" and "secondary metabolism," highlighting the intrinsic capacity of grape berries to perceive HT and to build adaptive responses. Additionally, important changes in processes related to "transport," "hormone" and "cell wall" might contribute to the postponing of veraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HT-induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation. PMID: 28197155 [PubMed - in process]

Metabolomic and Metagenomic Analysis of Two Crude Oil Production Pipelines Experiencing Differential Rates of Corrosion. Front Microbiol. 2017;8:99 Authors: Bonifay V, Wawrik B, Sunner J, Snodgrass EC, Aydin E, Duncan KE, Callaghan AV, Oldham A, Liengen T, Beech I Abstract Corrosion processes in two North Sea oil production pipelines were studied by analyzing pig envelope samples via metagenomic and metabolomic techniques. Both production systems have similar physico-chemical properties and injection waters are treated with nitrate, but one pipeline experiences severe corrosion and the other does not. Early and late pigging material was collected to gain insight into the potential causes for differential corrosion rates. Metabolites were extracted and analyzed via ultra-high performance liquid chromatography/high-resolution mass spectrometry with electrospray ionization (ESI) in both positive and negative ion modes. Metabolites were analyzed by comparison with standards indicative of aerobic and anaerobic hydrocarbon metabolism and by comparison to predicted masses for KEGG metabolites. Microbial community structure was analyzed via 16S rRNA gene qPCR, sequencing of 16S PCR products, and MySeq Illumina shotgun sequencing of community DNA. Metagenomic data were used to reconstruct the full length 16S rRNA genes and genomes of dominant microorganisms. Sequence data were also interrogated via KEGG annotation and for the presence of genes related to terminal electron accepting (TEA) processes as well as aerobic and anaerobic hydrocarbon degradation. Significant and distinct differences were observed when comparing the 'high corrosion' (HC) and the 'low corrosion' (LC) pipeline systems, especially with respect to the TEA utilization potential. The HC samples were dominated by sulfate-reducing bacteria (SRB) and archaea known for their ability to utilize simple carbon substrates, whereas LC samples were dominated by pseudomonads with the genetic potential for denitrification and aerobic hydrocarbon degradation. The frequency of aerobic hydrocarbon degradation genes was low in the HC system, and anaerobic hydrocarbon degradation genes were not detected in either pipeline. This is in contrast with metabolite analysis, which demonstrated the presence of several succinic acids in HC samples that are diagnostic of anaerobic hydrocarbon metabolism. Identifiable aerobic metabolites were confined to the LC samples, consistent with the metagenomic data. Overall, these data suggest that corrosion management might benefit from a more refined understanding of microbial community resilience in the face of disturbances such as nitrate treatment or pigging, which frequently prove insufficient to alter community structure toward a stable, less-corrosive assemblage. PMID: 28197141 [PubMed - in process]

Metabolomics: A Primer. Trends Biochem Sci. 2017 Feb 11;: Authors: Liu X, Locasale JW Abstract Metabolomics generates a profile of small molecules that are derived from cellular metabolism and can directly reflect the outcome of complex networks of biochemical reactions, thus providing insights into multiple aspects of cellular physiology. Technological advances have enabled rapid and increasingly expansive data acquisition with samples as small as single cells; however, substantial challenges in the field remain. In this primer we provide an overview of metabolomics, especially mass spectrometry (MS)-based metabolomics, which uses liquid chromatography (LC) for separation, and discuss its utilities and limitations. We identify and discuss several areas at the frontier of metabolomics. Our goal is to give the reader a sense of what might be accomplished when conducting a metabolomics experiment, now and in the near future. PMID: 28196646 [PubMed - as supplied by publisher]

Sparse kernel canonical correlation analysis for discovery of nonlinear interactions in high-dimensional data. BMC Bioinformatics. 2017 Feb 14;18(1):108 Authors: Yoshida K, Yoshimoto J, Doya K Abstract BACKGROUND: Advance in high-throughput technologies in genomics, transcriptomics, and metabolomics has created demand for bioinformatics tools to integrate high-dimensional data from different sources. Canonical correlation analysis (CCA) is a statistical tool for finding linear associations between different types of information. Previous extensions of CCA used to capture nonlinear associations, such as kernel CCA, did not allow feature selection or capturing of multiple canonical components. Here we propose a novel method, two-stage kernel CCA (TSKCCA) to select appropriate kernels in the framework of multiple kernel learning. RESULTS: TSKCCA first selects relevant kernels based on the HSIC criterion in the multiple kernel learning framework. Weights are then derived by non-negative matrix decomposition with L1 regularization. Using artificial datasets and nutrigenomic datasets, we show that TSKCCA can extract multiple, nonlinear associations among high-dimensional data and multiplicative interactions among variables. CONCLUSIONS: TSKCCA can identify nonlinear associations among high-dimensional data more reliably than previous nonlinear CCA methods. PMID: 28196464 [PubMed - in process]

Related Articles Evaluation of different training systems on Annurca apple fruits revealed by agronomical, qualitative and NMR-based metabolomic approaches. Food Chem. 2017 May 01;222:18-27 Authors: D'Abrosca B, Scognamiglio M, Corrado L, Chiocchio I, Zampella L, Mastrobuoni F, Rega P, Scortichini M, Fiorentino A, Petriccione M Abstract Nine different training systems for "Annurca Rossa del Sud" apple fruits, including oblique palmette, free palmette, V-shaped, Tatura trellis, Bibaum®, modified Bibaum®, triple leader, slender spindle and Solaxe, were evaluated based on agronomic, qualitative and metabolomic traits. Fruits were analysed at harvest and after the reddening process. The slender spindle training system showed the highest cumulative efficiency yield compared to the others. Furthermore, an increase in the content of bioactive compounds in flesh and fruit peels was observed after the reddening process and was influenced by the different training systems. The metabolic variations in apple peel were measured and analysed. Changes in the metabolome highlight the influence of different training systems on apple quality. This multidisciplinary study expands our knowledge of the influence of training systems on a typical Italian apple cultivar. PMID: 28041554 [PubMed - indexed for MEDLINE]

Related Articles SSADH deficiency possibly associated with enzyme activity-reducing SNPs. Brain Dev. 2016 Oct;38(9):871-4 Authors: Akiyama T, Osaka H, Shimbo H, Kuhara T, Shibata T, Kobayashi K, Kurosawa K, Yoshinaga H Abstract BACKGROUND: Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare autosomal recessive disorder that affects the degradation of gamma-aminobutyric acid and leads to the accumulation of gamma-hydroxybutyric acid (GHB) in body fluids. Diagnosis of SSADH deficiency is challenging, since the neurological symptoms are non-specific. CASE: The patient is a nine-year-old Japanese boy who presented with developmental delay, autism, epilepsy, and episodic gait disturbance. Brain magnetic resonance imaging showed hyperintense lesions in the bilateral thalami, globus pallidi, substantia nigra, and dentate nuclei. Urine metabolome analysis revealed elevated GHB, which led to a biochemical diagnosis of SSADH deficiency. Genetic analysis of the ALDH5A1 gene revealed a novel missense mutation c.1586G>A inherited from his father. It also demonstrated three single nucleotide polymorphisms (SNPs) (c.106G>C, c.538C>T, and c.545C>T), all of which were inherited from his mother and are known to reduce SSADH enzyme activity. There were no duplications or deletions in other exons in the patient or his parents. No variants in the upstream, intronic, or downstream regions of the ALDH5A1 gene were found in the patient. Enzymatic assay revealed a marked reduction of SSADH enzyme activity (≈2% of the lower limit of the normal range). CONCLUSION: Although other mechanisms cannot be fully excluded, the clinical manifestation of SSADH deficiency in this patient may be attributed to the combined effect of the mutation and the three enzyme activity-reducing SNPs. Urine metabolome analysis effectively detected his elevated GHB and is thus considered to be a good screening method for this underdiagnosed and potentially manageable metabolic disorder. PMID: 27056292 [PubMed - indexed for MEDLINE]

Related Articles Newest Strategies in the Search for Bioactive Saponins from the Tropical Plant Biodiversity. Curr Drug Deliv. 2016;13(3):389-99 Authors: Lacaille-Dubois MA Abstract This review will focus on newest results leading to the discovery of new bioactive saponins by using a combination of successive advanced procedures in extraction, isolation, structure elucidation and bioassays. Microwave- and ultrasonic-assisted extractions, two recent advanced methods have been increasingly used in the last decade. Then, a multistep purification procedure was achieved by flash chromatography, vacuum liquid chromatography, low, medium- and high-pressure liquid chromatography on silica gel and reversed-phase silica gel RP-18 (VLC, LPLC, MPLC, HPLC). These successive chromatographic steps have been implemented in the author's laboratory in order to avoid the time-consuming traditional partitions between butanol and water, dialysis procedures or precipitations in diethyl/ether. The structural elucidation of complex saponins possessing from 5 to 8 sugar units is performed by a combination of extensive spectroscopic techniques including 1D- and 2D-NMR experiments (1H, 13C, DEPT, COSY, NOESY, TOCSY, HSQC, HMBC) and mass spectrometry (FAB-MS HRESIMS). The bioassays have been mainly carried out in the field of cancerology and inflammation, two closely related areas, and also in the field of immunology with recent literature results on Quillaja saponins in order to explore some structure/activity relationships. The more recent results of the author's laboratory will be presented with examples of saponins from the tropical plant biodiversity (Pittosporaceae, Polygalaceae, Mimosaceae, Sapindaceae, Apiaceae, Dioscoreaceae, and Asparagaceae). Furthermore, some new trends reported in the literature will be briefly reviewed concerning dereplication, and metabolomic approachs which are currently of considerable importance in the field of natural product discovery. PMID: 26521655 [PubMed - indexed for MEDLINE]

A Three-Minute Method for high-throughput quantitative metabolomics and quantitative tracing experiments of central carbon and nitrogen pathways. Rapid Commun Mass Spectrom. 2017 Feb 13;: Authors: Nemkov T, Hansen KC, D'Alessandro A Abstract RATIONALE: The implementation of mass spectrometry-based metabolomics is advancing many areas of biomedical research. The time associated with traditional chromatographic methods for resolving metabolites prior to mass analysis has limited the potential to perform large scale, highly powered metabolomics studies and clinical applications. METHODS: Here we describe a three-minute method for the rapid profiling of central metabolic pathways through UHPLC-MS, tracing experiments in vitro and in vivo, and targeted quantification of compounds of interest using spiked in heavy labeled standards. RESULTS: This method has shown to be linear, reproducible, selective, sensitive, and robust for the semi-targeted analysis of central carbon and nitrogen metabolism. Isotopically-labeled internal standards are used for absolute quantitation of steady-state metabolite levels and de novo synthesized metabolites in tracing studies. We further propose exploratory applications to biofluids, cell and tissue extracts derived from relevant biomedical/clinical samples. CONCLUSIONS: While limited to the analysis of central carbon and nitrogen metabolism, this method enables the analysis of hundreds of samples per day derived from diverse biological matrices. This approach makes it possible to analyze samples from large patient populations for translational research, personalized medicine, and clinical metabolomics applications. PMID: 28195377 [PubMed - as supplied by publisher]

Untargeted metabolomics of colonic digests reveals kynurenine pathway metabolites, dityrosine and 3-dehydroxycarnitine as red versus white meat discriminating metabolites. Sci Rep. 2017 Feb 14;7:42514 Authors: Rombouts C, Hemeryck LY, Van Hecke T, De Smet S, De Vos WH, Vanhaecke L Abstract Epidemiological research has demonstrated that the consumption of red meat is an important risk factor for the development of colorectal cancer (CRC), diabetes mellitus and cardiovascular diseases. However, there is no holistic insight in the (by-) products of meat digestion that may contribute to disease development. To address this hiatus, an untargeted mass spectrometry (MS)-based metabolomics approach was used to create red versus white meat associated metabolic fingerprints following in vitro colonic digestion using the fecal inocula of ten healthy volunteers. Twenty-two metabolites were unequivocally associated with simulated colonic digestion of red meat. Several of these metabolites could mechanistically be linked to red meat-associated pathways including N'-formylkynurenine, kynurenine and kynurenic acid (all involved in tryptophan metabolism), the oxidative stress marker dityrosine, and 3-dehydroxycarnitine. In conclusion, the used MS-based metabolomics platform proved to be a powerful platform for detection of specific metabolites that improve the understanding of the causal relationship between red meat consumption and associated diseases. PMID: 28195169 [PubMed - in process]

Mass Defect-Based N,N-Dimethyl Leucine Labels for Quantitative Proteomics and Amine Metabolomics of Pancreatic Cancer Cells. Anal Chem. 2017 Jan 17;89(2):1138-1146 Authors: Hao L, Johnson J, Lietz CB, Buchberger A, Frost D, Kao WJ, Li L Abstract Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS(1)-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells. PMID: 28194987 [PubMed - in process]

msPurity: Automated Evaluation of Precursor Ion Purity for Mass Spectrometry-Based Fragmentation in Metabolomics. Anal Chem. 2017 Feb 08;: Authors: Lawson TN, Weber RJ, Jones MR, Chetwynd AJ, Rodrı Guez-Blanco G, Di Guida R, Viant MR, Dunn WB Abstract Tandem mass spectrometry (MS/MS or MS(2)) is a widely used approach for structural annotation and identification of metabolites in complex biological samples. The importance of assessing the contribution of the precursor ion within an isolation window for MS(2) experiments has been previously detailed in proteomics, where precursor ion purity influences the quality and accuracy of matching to mass spectral libraries, but to date, there has been little attention to this data-processing technique in metabolomics. Here, we present msPurity, a vendor-independent R package for liquid chromatography (LC) and direct infusion (DI) MS(2) that calculates a simple metric to describe the contribution of the selected precursor. The precursor purity metric is calculated as "intensity of a selected precursor divided by the summed intensity of the isolation window". The metric is interpolated at the recorded point of MS(2) acquisition using bordering full-scan spectra. Isotopic peaks of the selected precursor can be removed, and low abundance peaks that are believed to have limited contribution to the resulting MS(2) spectra are removed. Additionally, the isolation efficiency of the mass spectrometer can be taken into account. The package was applied to Data Dependent Acquisition (DDA)-based MS(2) metabolomics data sets derived from three metabolomics data repositories. For the 10 LC-MS(2) DDA data sets with > ±1 Da isolation windows, the median precursor purity score ranged from 0.67 to 0.96 (scale = 0 to +1). The R package was also used to assess precursor purity of theoretical isolation windows from LC-MS data sets of differing sample types. The theoretical isolation windows being the same width used for an anticipated DDA experiment (±0.5 Da). The most complex sample had a median precursor purity score of 0.46 for the 64,498 XCMS determined features, in comparison to the less spectrally complex sample that had a purity score of 0.66 for 5071 XCMS features. It has been previously reported in proteomics that a purity score of <0.5 can produce unreliable spectra matching results. With this assumption, we show that for complex samples there will be a large number of metabolites where traditional DDA approaches will struggle to provide reliable annotations or accurate matches to mass spectral libraries. PMID: 28194963 [PubMed - as supplied by publisher]

Genetic and biochemical changes of the serotonergic system in migraine pathobiology. J Headache Pain. 2017 Dec;18(1):20 Authors: Gasparini CF, Smith RA, Griffiths LR Abstract Migraine is a brain disorder characterized by a piercing headache which affects one side of the head, located mainly at the temples and in the area around the eye. Migraine imparts substantial suffering to the family in addition to the sufferer, particularly as it affects three times more women than men and is most prevalent between the ages of 25 and 45, the years of child rearing. Migraine typically occurs in individuals with a genetic predisposition and is aggravated by specific environmental triggers. Attempts to study the biochemistry of migraine began as early as the 1960s and were primarily directed at serotonin metabolism after an increase of 5-hydroxyindoleacetic acid (5-HIAA), the main metabolite of serotonin was observed in urine of migraineurs. Genetic and biochemical studies have primarily focused on the neurotransmitter serotonin, considering receptor binding, transport and synthesis of serotonin and have investigated serotonergic mediators including enzymes, receptors as well as intermediary metabolites. These studies have been mainly assayed in blood, CSF and urine as the most accessible fluids. More recently PET imaging technology integrated with a metabolomics and a systems biology platform are being applied to study serotonergic biology. The general trend observed is that migraine patients have alterations of neurotransmitter metabolism detected in biological fluids with different biochemistry from controls, however the interpretation of the biological significance of these peripheral changes is unresolved. In this review we present the biology of the serotonergic system and metabolic routes for serotonin and discuss results of biochemical studies with regard to alterations in serotonin in brain, cerebrospinal fluid, saliva, platelets, plasma and urine of migraine patients. PMID: 28194570 [PubMed - in process]

Divergent Relationships between Fecal Microbiota and Metabolome following Distinct Antibiotic-Induced Disruptions. mSphere. 2017 Jan-Feb;2(1): Authors: Choo JM, Kanno T, Zain NM, Leong LE, Abell GC, Keeble JE, Bruce KD, Mason AJ, Rogers GB Abstract The intestinal microbiome plays an essential role in regulating many aspects of host physiology, and its disruption through antibiotic exposure has been implicated in the development of a range of serious pathologies. The complex metabolic relationships that exist between members of the intestinal microbiota and the potential redundancy in functional pathways mean that an integrative analysis of changes in both structure and function are needed to understand the impact of antibiotic exposure. We used a combination of next-generation sequencing and nuclear magnetic resonance (NMR) metabolomics to characterize the effects of two clinically important antibiotic treatments, ciprofloxacin and vancomycin-imipenem, on the intestinal microbiomes of female C57BL/6 mice. This assessment was performed longitudinally and encompassed both antibiotic challenge and subsequent microbiome reestablishment. Both antibiotic treatments significantly altered the microbiota and metabolite compositions of fecal pellets during challenge and recovery. Spearman's correlation analysis of microbiota and NMR data revealed that, while some metabolites could be correlated with individual operational taxonomic units (OTUs), frequently multiple OTUs were associated with a significant change in a given metabolite. Furthermore, one metabolite, arginine, can be associated with increases/decreases in different sets of OTUs under differing conditions. Taken together, these findings indicate that reliance on shifts in one data set alone will generate an incomplete picture of the functional effect of antibiotic intervention. A full mechanistic understanding will require knowledge of the baseline microbiota composition, combined with both a comparison and an integration of microbiota, metabolomics, and phenotypic data. IMPORTANCE Despite the fundamental importance of antibiotic therapies to human health, their functional impact on the intestinal microbiome and its subsequent ability to recover are poorly understood. Much research in this area has focused on changes in microbiota composition, despite the interdependency and overlapping functions of many members of the microbial community. These relationships make prediction of the functional impact of microbiota-level changes difficult, while analyses based on the metabolome alone provide relatively little insight into the taxon-level changes that underpin changes in metabolite levels. Here, we used combined microbiota and metabolome profiling to characterize changes associated with clinically important antibiotic combinations with distinct effects on the gut. Correlation analysis of changes in the metabolome and microbiota indicate that a combined approach will be essential for a mechanistic understanding of the functional impact of distinct antibiotic classes. PMID: 28194448 [PubMed - in process]

The impact of micronutrient status on health: correlation network analysis to understand the role of micronutrients in metabolic-inflammatory processes regulating homeostasis and phenotypic flexibility. Genes Nutr. 2017;12:5 Authors: van den Broek TJ, Kremer BH, Marcondes Rezende M, Hoevenaars FP, Weber P, Hoeller U, van Ommen B, Wopereis S Abstract BACKGROUND: Vitamins and carotenoids are key micronutrients facilitating the maintenance of health, as evidenced by the increased risk of disease with low intake. Optimal phenotypic flexibility, i.e., the ability to respond to a physiological challenge, is an essential indicator of health status. Therefore, health can be measured by applying a challenge test and monitoring the response of relevant phenotypic processes. In this study, we assessed the correlation of three fat-soluble vitamins, (i.e., vitamin A or retinol, vitamin D3, two homologues of vitamin E) and four carotenoids (i.e., α-carotene, β-carotene, β-cryptoxanthin, and lycopene), with characteristics of metabolic and inflammatory parameters at baseline and in response to a nutritional challenge test (NCT) in a group of 36 overweight and obese male subjects, using proteomics and metabolomics platforms. The phenotypic flexibility concept implies that health can be measured by the ability to adapt to a NCT, which may offer a more sensitive way to assess changes in health status of healthy subjects. RESULTS: Correlation analyses of results after overnight fasting revealed a rather evenly distributed network in a number of relatively strong correlations per micronutrient, with minor overlap between correlation profiles of each compound. Correlation analyses of challenge response profiles for metabolite and protein parameters with micronutrient status revealed a network that is more skewed towards α-carotene and γ-tocopherol suggesting a more prominent role for these micronutrients in the maintenance of phenotypic flexibility. Comparison of the networks revealed that there is merely overlap of two parameters (inositol and oleic acid (C18:1)) affirming that there is a specific biomarker response profile upon NCT. CONCLUSIONS: Our study shows that applying the challenge test concept is able to reveal previously unidentified correlations between specific micronutrients and health-related processes, with potential relevance for maintenance of health that were not observed by correlating homeostatic measurements. This approach will contribute to insights on the influence of micronutrients on health and help to create efficient micronutrient intervention programs. PMID: 28194237 [PubMed - in process]

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth. Neurosci Res. 2017 Feb 10;: Authors: Goschzik T, Cremer H, Gnanapragassam VS, Horstkorte R, Bork K, Diestel S Abstract The cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth. PMID: 28193531 [PubMed - as supplied by publisher]