[U-13C]-Glu SIRM study of A549 cells with QDA treatment
LC-MS based metabolomic analysis of 55 milk samples from 3 individual panda mothers during early lactation period.
Phytoplankton are significant producers of dissolved organic matter (DOM) in marine ecosystems but the identity and dynamics of this DOM remain poorly constrained. Knowledge on the identity and dynamics of DOM are crucial for understanding the molecular-level reactions at the base of the global carbon cycle. Here we apply emerging analytical and computational tools from metabolomics to investigate the composition of DOM produced by the centric diatom Thalassiosira pseudonana. We assessed both intracellular metabolites within T. pseudonana (the endo-metabolome) and extracellular metabolites released by T. pseudonana (the exo-metabolome). The intracellular metabolites had a more variable composition than the extracellular metabolites. We putatively identified novel compounds not previously associated with T. pseudonana as well as compounds that have previously been identified within T. pseudonana’s metabolic capacity (e.g. dimethylsulfoniopropionate and degradation products of chitin). The resulting information will provide the basis for future experiments to assess the impact of T. pseudonana on the composition of dissolved organic matter in marine environments.
Dissolved organic matter produced by Thalassiosira pseudonana
Weight comparision of obese and lean patients.
IWMS Study 1:Weight comparison of obese and lean patients
Secondary metabolites are frequently involved in mechanisms of the plant tolerance to environmental stresses. Determining metabolic biomarkers for drought tolerance may be a useful implementation in increasing and stabilizing crop plant productivity. The most accurate and fast tool for this purpose is the ultra-high performance liquid chromatography (UHPLC). It enabled a rapid discrimination between samples from plants grown in different water limitation regimes. Multivariate statistical analysis was applied to handle and interpret the complexity of the generated data. UHPLC raw data was mathematically pre-processed to reduce the effects of instrumental factors. The multivariate analysis of variance was used to find the significant changes in the secondary metabolites composition induced by the drought stress. Tests were performed on 9 varieties of spring barley (Hordeum vulgare L.) originating from Europe, North America and Syria. Drought stress was applied at different developmental stages. The UHPLC results suggest that the changes in metabolite profiles are a stage-specific trait and alter in the life cycle. In addition, there is variability in metabolites selected as significant for the reaction to drought depending on sampling time and variety. In parallel, an ion trap mass spectrometer with an electrospray ion source coupled to a high performance liquid chromatography (HPLC) and UV detector was used for qualitative analysis of phenolic compounds and identification of the potential biomarkers.
Profiles of phenolic secondary metabolites in plants of barley (Hordeum vulgare L.)
Hemorrhagic shock with injury results in alterations of the metabolic state of an organism, which contribute to organ dysfunction and death. Previous investigations have explored the effects of carbohydrate prefeed in murine models but few in clinically relevant large animal models. We performed carbohydrate prefeed in pigs undergoing simulated polytrauma and hemorrhagic shock with resuscitation to determine if carbohydrate prefeeding if the metabolic response to shock is dependent on fed state. Sixty-four Yorkshire pigs were divided into two experimental groups: fasted and prefed in additon to two Control groups. Experimental animals were subjected to a standardized hemorrhagic shock protocol, including pulmonary contusion and liver crush injury. To determine molecular alterations in response to trauma as a result of prefeeding, liver and muscle biopsies in addition to serum and urine samples were obtained at set timepoints throughout the procedure. The samples were prepared and analyzed by NMR spectroscopy.
Identification of Biomarkers using Metabolomics in Porcine Hemorrhagic Shock
In this study broncho-alveolar lavage fluid (BALF) from mice infected with influenza A virus, PR8 (200 PFU and 2x10^5 PFU), X31 (2x10^5 PFU) was analyzed for lipidomic host response in comparison with the mock infected animals. Time Points: 3, 5, 7, 9, 11, 13, 19 days post infection, done in 8-10 replicates.)
Lipid Mediators of inflammation in BALF 3-19 days after infection with Influenza
This West Coast Metabolomics Center pilot and feasibility project granted to Peter Kaiser (UC Irvine), aims to achieve understanding of a unique metabolic dependence of cancer cells to explore development of novel unconventional therapeutic strategies that exploit dependence of cancer cells on methyl-donor abundance. The past few years have highlighted the role of altered metabolism in cancer. While mechanistic insight into changed metabolism in cancer is very limited, the importance of the metabolic pathway surrounding homocysteine and methionine for cancer cell proliferation has been known for over 30 years. These findings, generally summarized as methionine-dependence or methionine stress sensitivity, describe the phenomenon that most cancer cells cannot proliferate in growth medium where the amino acid methionine is replaced with its direct metabolic precursor homocysteine. Importantly, non-tumorigenic cells are unaffected by replacing methionine with homocysteine in the growth medium. For the past years we have been studying methionine dependence of breast and prostate cancer and demonstrated that methionine-dependence is caused by insufficient flux through this pathway to sustain synthesis of the downstream metabolite and the principal methyl-donor S-adenosylmethionine (SAM). We have isolated rare cell clones from MDA-MB468 breast cancer cells (referred to as MB468RES) that are no longer methionine dependent and proliferate in homocysteine medium. Interestingly, MB468RES have lost their ability for anchorage independent growth, a hallmark of cancer. The MB468 and MB468RES cell line pair confirms other observations showing that methionine dependence is tightly linked to tumorigenicity. Importantly, this cell line pair is an ideal model to identify metabolite signatures linked to cancer cell methionine dependence. We propose to characterize the metabolic changes triggered by the shift from normal growth medium to homocysteine medium in MB468 breast cancer cells and the methionine stress insensitive MB468RES derivatives. In addition we have developed cancer cell lines with inducible shRNAs targeting methionine adenosyltransferase (MAT), the enzyme catalyzing synthesis of SAM from methionine and ATP. Inducible knockdown of MAT allows us to specifically reduce SAM synthesis. Our previous results suggest that SAM limitation is the critical trigger for cancer cell methionine dependence. Thus metabolite profiling using the MAT knockdown system will provide an independent dataset that together with metabolite profiles from the MB468 and MB468RES cell line pair will define critical metabolic profiles related to cancer cell methionine dependence. In the current investigation, untargeted analysis of primary metabolites and complex lipids, coupled with quantitative analysis of methionine pathway intermediates (folate and respective derivatives, s-adenosylmethoinine, s-adenosylhomocysteine, choline, betaine) and metabolic flux will be conducted on MB468, MB468RES and MB468shRNA following the switch from methionine containing media to homocysteine containing media over the course of 0, 2, 4, 8, 12, 24 and 48 hours. The primary objectives were to 1) characterize the metabolic response to methionine stress and SAM limitation and 2) correlate the metabolic signatures with cancer cell proliferation arrest and death.
Metabolite changes associated with methionine stress sensitivity of cancer (CSH QTOF MS analysis)
Indole-3-acetaldoxime (IAOx) represents an early intermediate of the biosynthesis of a variety of indolic secondary metabolites including the phytoanticipin indol-3-ylmethyl glucosinolate and the phytoalexin camalexin (3-thiazol-2′-yl-indole). Arabidopsis thaliana cyp79B2 cyp79B3 double knockout plants are completely impaired in the conversion of tryptophan to indole-3-acetaldoxime and do not accumulate IAOx-derived metabolites any longer. Consequently, comparative analysis of wild-type and cyp79B2 cyp79B3 plant lines has the potential to explore the complete range of IAOx-derived indolic secondary metabolites.
Comparative LC/MS-based profiling of silver nitrate-treated Arabidopsis thaliana leaves of wild-type and cyp79B2 cyp79B3 double knockout plants
Metabolite profiling of herbivory-induced changes in Nicotiana attenuata was used to construct a correlation-weighted network to visualize intra- and inter-compound spectra relationships from individual mass features, and to annotate 12 major network hubs, including 4 O-acyl sugars and 6 17-hydroxygeranyllinalool diterpene glycosides, metabolites with recently uncovered anti-herbivore functions.
Metabolomics profile of herbivory-induced Nicotiana attenuata plants
In the EstroSTAT sub-study, a total of 18 samples from 23 week old, female C57BL/6 mice; comprised of 6 urine samples, 6 serum samples and 6 liver tissue samples were analyzed. Three mice/matrix were given STAT penicillin and 3 mice/matrix were non-treated Controls. The mice were housed with conventional bedding and fed a Low phyto-estrogen diet.
Metabolomics Involved in Early Life Antibiotic Exposures(EstroSTAT-Liver )
An integrative genomics approach for deciphering the complex interactions between ascorbate metabolism and fruit growth and composition in tomato.
Isolated skelelal muscle mitochondria incubated in the presence of fatty acid oxidation precursors, palmitic acid and palmitoyl carnitine
Skeletal Muscle Treatment (palmitate/palmitoyl carnitine)
We showed earlier that nutritional stress like starvation or high fat diet resulted in phenotypic changes in the lipidomes of hepatocyte lipid droplets (LDs), representative for the pathophysiological status of the mouse model. Here we extend our former study by adding genetic stress due to knock-out (KO) of adipocyte triglyceride lipase (ATGL), the rate limiting enzyme in LD lipolysis. An intervention trial for 6 weeks with male wild-type (WT) and ATGL-KO mice was carried out; both genotypes were fed lab chow or were exposed to short-time starvation. Isolated LDs were analyzed by LC-MS/MS. Triacylglycerol, diacylglycerol and phosphatidylcholine lipidomes, in that order, provided best phenotypic signatures characteristic for respective stresses applied to the animals. This was evidenced at lipid species level by principal component analysis, calculation of average values for chain-lengths and numbers of double bonds, and by visualization in heat maps. Structural backgrounds for analyses and metabolic relationships were elaborated at lipid molecular species level. Relating our lipidomic data to non-alcoholic fatty liver diseases of nutritional and genetic etiologies with or without accompanying insulin resistance, phenotypic distinction in hepatocyte LDs dependent on insulin status emerged. Taken together, lipidomes of hepatocyte lipid droplets are sensitive responders to nutritional and genetic stress.
The impact of genetic stress by ATGL deficiency on the lipidome of lipid droplets from murine hepatocytes
Recently, multicellular spheroids were isolated from a well-established epithelial ovarian cancer cell line, OVCAR-3, and were propagated in vitro. These spheroid derived cells displayed numerous hallmarks of cancer stem cells, which are chemo and radioresistant cells thought to be a significant cause of cancer recurrence and resultant mortality. Gene set enrichment analysis of expression data from the OVCAR-3 cells and the spheroid-derived putative cancer stem cells identified several metabolic pathways enriched in differentially expressed genes. Before this, there had been little previous knowledge or investigation of systems-scale metabolic differences between cancer cells and cancer stem cells, and no knowledge of such differences in ovarian cancer stem cells. To determine if there were substantial metabolic changes corresponding with these transcriptional differences, we used two-dimensional gas chromatography coupled to mass spectrometry to measure the metabolite profiles of the two cell lines. These two cell lines exhibited significant metabolic differences in both intracellular and extracellular metabolite measurements. Principal components analysis, an unsupervised dimensional reduction technique, showed complete separation between the two cell types based on their metabolite profiles. Pathway analysis of intracellular metabolomics data revealed close overlap with metabolic pathways identified from gene expression data, with four out of six pathways found enriched in gene-level analysis also enriched in metabolite-level analysis. Some of those pathways contained multiple metabolites that were individually statistically significantly different between the two cell lines, with one of the most broadly and consistently different pathways, arginine and proline metabolism, suggesting an interesting hypothesis about cancerous and stem-like metabolic phenotypes in this pair of cell lines. Overall, we demonstrate for the first time that metabolism in an ovarian cancer stem cell line is distinct from that of more differentiated isogenic cancer cells, supporting the potential importance of metabolism in the differences between cancer cells and cancer stem cells.
OVCAR-3 spheroid-derived cells display distinct metabolic profiles
SIRM Analysis of MYC inducible P493 cells under normoxia and hypoxia in [U-13C] labeled Glucose medium
SIRM Analysis of human P493 cells under hypoxia in [U-13C] labeled Glucose medium
Absolute quantification of metabolites in tomato fruit extracts by fast 2D NMR
Vitis vinifera cultivar Corvina clone 48 berries were harvested from different vineyards, each located in one of the three most important wine production macro-areas of the Verona region: Bardolino, Valpolicella and Soave, on the basis of the site geographical coordinates. For each of the selected vineyards, specific environmental conditions (altitude and type of soil) and farming and agricultural practices used (training system, rows facing direction, planting layout, vineyard age and rootstock type) were recorded. Vineyards were selected in order to maximize differences in locations and in microenvironmental and farming conditions. Berries were harvested at three different developmental stages: véraison, mid-ripening and harvest; each sample was collected in three biological replicates, to cover the whole vineyard variability. The same sampling procedure had been repeated over three consecutive vintages (2006, 2007 and 2008).
The plasticity of the grapevine berry transcriptome
Carotenoids are important secondary metabolites, which have been recognized as an essential component of the human diet because of their valuable beneficial health effects. With this rationale, there is a continuous aim to define the distribution of these compounds in plants, better understand their metabolism and try to increase their concentration levels in fruits and vegetables. This study aimed at deepening the knowledge on the regulatory role of phytohormones in carotenoid metabolism. More specifically, it was envisaged to reveal the phytohormones involved in the metabolism of ?-carotene, ?-carotene, lycopene, lutein and zeaxanthin. For this purpose, the phytohormone profiles of 50 tomato fruits were defined by using high-resolution Orbitrap mass spectrometry and evaluated with respect to the associated carotenoid concentrations. Data mining was performed by differential expression and orthogonal partial least squares analyses. This metabolomic approach was able to reveal 5 phytohormonal metabolites, which were assigned strong influence (Variable Importance in Projection scores ? 0.89) towards carotenoid metabolism. These metabolites were identified as cis-zeatin-O-glucoside, 1-acetylindole-3-carboxaldehyde, 2-oxindole-3-acetic acid, cis-12-oxo-phytodienoic acid and cucurbic acid. Furthermore, the significance of the individual phytohormones towards carotenoid metabolism was investigated by regression analysis (P-values ? 0.05, R2 varying between 0.259 and 0.760). It was concluded that these phytohormones all have significant contributing value in the regulation of carotenoid metabolism, thereby exhibiting both down- and up-regulating influences. As a result, this knowledge encloses the potential for improving tomato fruit nutritional quality by targeted control of agronomic conditions, exogenous use of plant bioregulators, or genetic engineering.
A metabolomics approach to unravel the regulating role of phytohormones towards carotenoid metabolism in tomato fruit. (B-carotene metabolism)
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative pathogen that uses complex mechanisms to invade and proliferate within mammalian host cells. To investigate possible contributions of metabolic processes to virulence in S. Typhimurium grown under conditions known to induce expression of virulence genes, we used a metabolomics-driven systems biology approach coupled with genome scale modeling. First, we identified distinct metabolite profiles associated with bacteria grown in either rich or virulence-inducing media and report the most comprehensive coverage of the S. Typhimurium metabolome to date. Second, we applied an omics-informed genome scale modeling analysis of the functional consequences of adaptive alterations in S. Typhimurium metabolism during growth under our conditions. Modeling efforts highlighted a decreased cellular capability to both produce and utilize intracellular amino acids during stationary phase culture in virulence conditions, despite significant abundance increases for these molecules as observed by our metabolomics measurements. Furthermore, analyses of omics data in the context of the metabolic model indicated rewiring of the metabolic network to support pathways associated with virulence. For example, cellular concentrations of polyamines were perturbed, as well as the predicted capacity for secretion and uptake.
Salmonella Modulates Metabolism during Growth under Conditions that Induce Expression of Virulence Genes
The hydrophilic metabolome of heterotrophic Arabidopsis thaliana cells grown in suspension, a cellular model of plant sink tissues. Water-soluble metabolites were extracted using four protocols: perchloric acid, boiling ethanol, methanol and methanol/chloroform. Only representative data of arabidopsis metabolome obtained with methanol/chloroform/water extraction is loaded in MeRy-B.
Arabidopsis thaliana hydrophilic metabolome
Compare highly motile MDA-MB-231 breast cancer cells with less motile, yet highly proliferative gamma p38 knockdowns
MDA-MB-231 cells and p38 gamma knockdown
The aim of the present study was to investigate, under commercial culture conditions, the effect of the harvest time during the production season and that of the type of nutrient solution handling on some determinants of the organoleptic and nutritional quality of tomato fruit through analysis of its major biochemical components using metabolic profiling.
Greenhouse Tomato - Hortis - Metabolomics 2009
The legume genus Lotus includes glycophytic forage crops and other species adapted to extreme environments, such as saline soils. Understanding salt tolerance mechanisms will contribute to the discovery of new traits which may enhance the breeding efforts towards improved performance of legumes in marginal agricultural environments. Here, we used a combination of ionomic and gas chromatography-mass spectrometry (GC-MS)-based metabolite profilings of complete shoots (pooling leaves, petioles and stems) to compare the extremophile Lotus creticus, adapted to highly saline coastal regions, and two cultivated glycophytic grassland forage species, Lotus corniculatus and Lotus tenuis. L. creticus exhibited better survival after exposure to long-term lethal salinity and was more efficient at excluding Cl- from the shoots than the glycophytes. In contrast, Na+ levels were higher in the extremophile under both control and salt stress, a trait often observed in halophytes. Ionomics demonstrated a differential rearrangement of shoot nutrient levels in the extremophile upon salt exposure. Metabolite profiling showed that responses to NaCl in L. creticus shoots were globally similar to those of the glycophytes, providing little evidence for metabolic pre-adaptation to salinity. This study is the first comparing salt acclimation responses between extremophile and non-extremophile legumes, and challenges the generalization of the metabolic salt pre-adaptation hypothesis.
Metabolomic responses to long-term salt stress in related Lotus species (B)
This experiment tests the consequence of a mutation at the FatB gene (At1g08510) in the wound-response of Arabidopsis. The FatB mutant allele (fatb KD J. Ohlrogge (Plant Cell 2003, Vol 15, 1020-1033)) was obtained from Dr. Katayonn Dehesh, University of California, Davis, Davis, CA. This allele is in the Ws background.The standardized growth conditions are as follows: 1. Seeds (between 14 and 16) are sown on media in 100 x 100 x 15mm square Falcon Petri Dishes (Fisher Scientific, catalogue #08-757-11A). Seeds were arranged on the plates in a single horizontal line at the 1-cm mark from the top of the plate.2. Each plate contains between 20 and 25-ml of sterile MS media containing 0.1% (w/v) sucrose.3. Prior to sowing, seeds were sterilized by treating for 1 minute at room temperature with a 300-l solution of 50% (v/v) ethanol, this solution was removed and replaced with a 300-l solution consisting of 1% (v/v) Tween 20 (Fischer BioReagents, catalogue #BP33750), and 50% (v/v) bleach solution (Clorox), and incubated at room temperature for 10-minutes. The seeds were then washed with three changes of 0.3-ml of sterile water.
Glycolysis/TCA/Nucleotide analysis (especially interested in alpha-ketoglutarate) and NAD+ and related metabolite analysis for parp1 ko and parp1 wild type lung tissue after saline or bleomycin treatment
Metabolic analysis of Normal Mouse Lung Fiboblasts with/without TGFbeta treatment
Perinatal DDT causes dysfunctional lipid metabolism underlying metabolic syndrome
Two groups of mixtures with 5 replicates each were each made using 20 synthetic metabolites. Some metabolites were at equal concentrations in both groups, and 10 metabolites differed between groups with half higher in group A, and half higher in group B.
NMR analysis of Synthetic Mixture Analysis
Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally. Thus, improving gene annotation remains a major challenge in plant science. Because metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation of plant metabolism, it provides clues to the function of genes of interest. In this regard, we chose 50 Arabidopsis mutants including a set of characterized and uncharacterized mutants, which resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry (GC-MS). To make the dataset available as an efficient public functional genomics tool for hypothesis generation, we developed the MeKO database, which allows evaluation of whether a mutation affects metabolism during normal growth. This database contains images of mutants, statistical data analyses, and data on differential metabolite accumulation. Non-processed data, including chromatograms, mass spectra, and experimental meta-data, follow the guidelines of the Metabolomics Standards Initiative (MSI) and are freely downloadable. Proof-of-concept analysis suggests that the MeKO database is highly useful for gene annotation and for generation of hypotheses for genes of interest. MeKO is publicly available at http://prime.psc.riken.jp/meko/.
Metabolomic Characterization of Knock-Out Mutants in Arabidopsis - Development of a Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO)
Scarring alopecia consists of a collection of disorders characterized by destruction of hair follicles, replacement with fibrous scar tissue, and irreversible hair loss. Alopecia affects men and women worldwide and can be a significant source of psychological stress and depression for affected individuals. The purpose of this study was to explore metabolic profiles in scalp tissue samples from normal control subjects (n=6) and in matched samples obtained from affected (n=12) and unaffected (n=12) areas of the scalp in patients with lymphocytic Frontal Fibrosing Alopecia (FFA). Frontal fibrosing alopecia results from destruction of hair follicles by an inflammatory lymphocytic infiltrate that is localized around the upper portion of the hair follicle.
Metabolomics Analysis of Frontal Fibrosing Alopecia
Here we present a metabolic profiling strategy employing direct infusion Orbitrap mass spectrometry (MS) and gas chromatography-mass spectrometry (GC/MS) for the monitoring of soybean's (Glycine max L.) global metabolism regulation in response to Rhizoctonia solani infection in a time-course. Key elements in the approach are the construction of a comprehensive metabolite library for soybean, which accelerates the steps of metabolite identification and biological interpretation of results, and bioinformatics tools for the visualization and analysis of its metabolome. The study of metabolic networks revealed that infection results in the mobilization of carbohydrates, disturbance of the amino acid pool, and activation of isoflavonoid, α-linolenate and phenylpropanoid biosynthetic pathways of the plant. Components of these pathways include phytoalexins, coumarins, flavonoids, signaling molecules, and hormones, many of which exhibit antioxidant properties and bioactivity helping the plant to counterattack the pathogen's invasion. Unraveling the biochemical mechanism operating during soybean-Rhizoctonia interaction in addition to its significance towards the understanding of the plant's metabolism regulation under biotic stress, provides valuable insights with potential for applications in biotechnology, crop breeding, and agrochemical and food industries.
A Metabolic Profiling Strategy for the Dissection of Plant Defense against Fungal Pathogens
Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.
Lipidomic analysis of lipid droplets from murine hepatocytes reveals distinct signatures for nutritional stress.
A metabolomics approach using 1H-NMR and GC-EI-TOF-MS profiling was employed to characterise three F1 hybrid cultivars (Cézanne, Escrito and Hugo) of Charentais melon (Cucumis melo L.). STREP META-PHOR
Charentais Melon Fruit 2008 - Anal Chem 2009
In the TranSTAT sub-study, a total of 18 samples from 8 week old, female Swiss webster mice; comprised of 6 serum samples, 6 cecal content samples and 6 liver tissue samples were analyzed. Three mice/matrix were inoculated with cecal contents from STAT mice and 3 mice/matrix were inoculated with cecal contents from Control mice. The mice were housed with conventional bedding and fed a high fat diet.
Metabolomics Involved in Early Life Antibiotic Exposures(TranSTAT-Liver)
Meal test of bariatric surgery patients before and after weight loss
NMR analysis of sugars and organic acids in strawberry fruits.
The cell expansion phase of tomato fruit development was explored at the level of transcriptome and metabolome to identify regulatory genes involved in the control of developmental and metabolic processes that may impact fruit quality. Attention was focussed on the two major expanding tissues in tomato fruit, mesocarp and locular tissue.
The complex nature of the mechanisms behind cardiovascular diseases prevents the detection of latent early risk conditions. Network representations are ideally suited to investigate the complex interconnections between the individual components of a biological system underlying complex diseases. Here we investigate the patterns of correlations of an array of 29 metabolites identified and quantified in the plasma of 864 healthy blood donors and use a systems biology approach to define metabolite probabilistic networks specific for low and high latent cardiovascular risk. We adapted methods based on the likelihood of correlation and methods from information theory and combined them with resampling techniques. Our results show that plasma metabolite networks can be defined that associate with latent cardiovascular disease risk. The analysis of the networks supports our previous finding of a possible association between cardiovascular risk and impaired mitochondrial activity and highlights post-translational modifications (glycosilation and oxidation) of lipoproteins as a possible target-mechanism for early detection of latent cardiovascular risk.
Probabilistic networks of blood metabolites in healthy subjects as indicators of latent cardiovascular risk
New metabolic profiling technologies provide data on a wider range of metabolites than traditional targeted approaches. Metabolomic technologies currently facilitate acquisition of multivariate metabolic data using diverse, mostly hyphenated, chromatographic detection systems, such as GC-MS or liquid chromatography coupled to mass spectrometry, Fourier-transformed infrared spectroscopy or NMR-based methods. Analysis of the resulting data can be performed through a combination of non-supervised and supervised statistical methods, such as independent component analysis and analysis of variance, respectively. These methods reduce the complex data sets to information, which is relevant for the discovery of metabolic markers or for hypothesis-driven, pathway-based analysis. Plant responses to salinity involve changes in the activity of genes and proteins, which invariably lead to changes in plant metabolism. Here, we highlight a selection of recent publications in the salt stress field, and use gas chromatography time-of-flight mass spectrometry profiles of polar fractions from the plant models, Arabidopsis thaliana, Lotus japonicus and Oryza sativa to demonstrate the power of metabolite profiling. We present evidence for conserved and divergent metabolic responses among these three species and conclude that a change in the balance between amino acids and organic acids may be a conserved metabolic response of plants to salt stress.
Mining for metabolic responses to long-term salt stress: a case study on Arabidopsis thaliana Col-0 (C)
Storage conditions and duration have a considerable influence on wine quality. Optimum temperature and humidity conditions may improve wine quality through ageing, while incorrect or excessively long storage leads to negative results. In order to evaluate the global effects of storage on red wine composition, 20 Sangiovese wines were stored in two different conditions (cellar or house) for a period of 2 years and analysed every 6 months. Untargeted LC-MS analysis showed various putative markers for the type and length of conservation (i.e. pigments, flavanols, pantothenic acid etc.), while targeted LC-MS confirmed and expanded these results within specific metabolic groups. The results of multivariate analysis showed that wines stored in the cellar changed little even after 2 years of storage, while wines stored in typical domestic conditions (house) developed approximately four times faster, reaching after just 6 months a composition similar to wines stored in the cellar for 2 years. The formation of several monosulfonated flavanols during the domestic ageing provided the first evidence in wine of a reaction between wine tannins - both catechins and proanthocyanidins - and the exogenous antioxidant bisulfite. Moreover, ageing under domestic conditions appeared to induce an accelerated decrease of wine pigments, while specifically promoting the formation of the pinotin A-like pigments.
The influence of storage on the “chemical age” of red wines
One mg of RESV dissolved in ethanol was applied directly (Et) or mixed with hydrophilic ointment (HO), macrogol (Ma) or CMC gel (CMC), and swabbed on mouse dorsal skin. After 4 h mice were sacrificed, metabolites were extracted from tissues and analyzed by LC-MS. Peak areas of metabolites were normalized using peak areas of spiked internal standards (HEPES and PIPES).
Absorption efficiency of RESV through mouse skin using 3 bases in different tissues
In this study broncho-alveolar lavage fluid (BALF) from mice infected with influenza A virus, PR8 (200 PFU) or X31 (1e5 PFU) was analyzed for lipidomic host response in comparison with the mock infected animals. Time Points: 3, 5, 6, 7, 9, 10 and 13 days post infection.)
Lipid Mediators of inflammation in BALF 3-13 days after infection with Influenza
Phénotypage haut débit du fruit de tomate en vue de lintégration avec des données Omics. Caractérisation de fruits de tomate à 2 stades de développement (expansion et fruits orange rouge) issus de 12 génotypes contrastés (8 lignées et 4 hybrides F1 qui en dérivent).
In the VG STAT sub-study, a total of 18 samples from 7 week old, male C57BL/6 mice; comprised of 9 cecal content samples and 9 liver tissue samples were analyzed. Three mice/matrix were given penicillin VK, 3 were given penicillin G, and 3 mice/matrix were non-treated Controls. The mice were housed with conventional bedding and fed a normal diet.
Metabolomics Involved in Early Life Antibiotic Exposures(VGSTAT-Cecal)
To determine the basis of running capacity and health differences in outbread male N/NIH rats selected for high capacity (HCR) and low capacity (LCR) running (a surrogate for VO2max) (see:Science. 2005 Jan 21;307(5708):418-20). Plasma collected at 12 months of age in generation 28 rats after ad lib feeding or 40% caloric restriction beginning at week 8 of age. All animals fasted 4 hours prior to collection between 5-8 pm.
Analyzis of the (h)GSH metabolism in galls, evaluation of the importance of these tripeptides in the Root-knot nematode Meloidogine incognita development cycle in Medicago truncatula and examination of the metabolism modifications occurring in galls under (h)GSH deficiency.
Metabolomic profiling of melon fruit quality as affected by genotype and environment
Melon France META-PHOR (Metabolomics 2013)
Phytoplankton are known to release organic compounds that fuel secondary production by heterotrophic bacteria. Here we show that an abundant marine cyanobacterium, Synechococcus elongatus, contributes a variety of nitrogen-rich and sulfur-containing compounds to dissolved organic matter. A combination of targeted and untargeted metabolomics and genomic tools was used to characterize the intracellular and extracellular metabolites of S. elongatus. Aromatic compounds such as 4-hydroxybenzoic acid and phenylalanine, as well as nucleosides (e.g., thymidine, 5’-methylthioadenosine, xanthosine), the organosulfur compound 3-mercaptopropionate, and the plant auxin indole 3-acetic acid, were detected in the extracellular metabolites at multiple time points during the growth of S. elongatus. Further, the amino acid kynurenine was found to accumulate in the media even though it was not included in the predicted metabolome of S. elongatus. This suggests that not all metabolites produced by an organism can be predicted from its genome sequence. Some metabolites may be products of non-enzymatic reactions and are likely excreted into the environment as waste. The compounds described herein provide excellent targets for quantitative analysis in field settings to assess the source and lability of dissolved organic matter in situ.
Release of Ecologically Relevant Metabolites by the Cyanobacterium, Synechococcus elongatus CCMP 1631
Metabolomics analysis of wild-type S. enterica, single RidA knock-out and triple Rid (ridA Rid2 Rid7) knock-out S.enterica cells
Metabolomics analysis of multiple metabolic functions in the YjgF/YER057c/UK114 (Rid) protein family GC-MS (Part 1)
In this study, we established a profiling method for wax esters and lipophilic compounds in Euglena using gas chromatography-mass spectrometry (GC-MS). Using this method, we compared accumulation profiles of wax esters and lipophilic compounds between a wild type Euglena Z strain and a bleached SM-ZK strain.
Wax ester and lipophilic compound profiling of Euglena gracilis by gas chromatography-mass spectrometry: toward understanding of anoxia wax ester fermentation
Spring wheat (Triticum aestivum L.) was grown in a free-air carbon dioxide (CO2) enrichment (FACE) field experiment. Grain and biomass yield and its components were determined at maturity and the grain metabolome was analysed by gas chromatography-mass spectrometry (GC-MS) for elevated and ambient CO2 (537 versus 409 μl/l) concentration for samples grown in two successive years (2005, 2006).
Combining peak- and chromatogram-based retention time alignment algorithms for multiple chromatography-mass spectrometry datasets
Criteria for the diagnosis of amnestic MCI included: (i) memory complaint documented by the patient and collateral source; (ii) impairment in 1 or more of the 4 cognitive domains (memory, executive functioning/attention, visuospatial, or language); (iii) essentially normal functional activities of daily living; and (iv) absence of dementia. In general, the amnestic MCI determination is made when the memory measures fall 1.0â1.5 SD below the means for age and education appropriate individuals in our community; however, rigid cutoffs on psychometric scores were not used to establish the diagnosis of amnestic MCI which was made on clinical grounds. The diagnosis of dementia was made using DSM-IV criteria, and the diagnosis of AD was made using established criteria. Subjects were considered to be CN if they performed within the normative range and did not meet criteria for MCI or dementia.
Identification of altered metabolic pathways in Alzheimer's disease, mild cognitive impairment and cognitively normals using Metabolomics (plasma)
The purpose of this study was to examine the metabolic profiles of mayfly (Centroptilum triangulifer) larvae subjected to thermal challenge. This species is unusual in terms of its ease of culture, and its suitability as a laboratory test organism. Our purpose here was to examine how an environmentally realistic thermal challenge affects the physiology of this organism. In this study, we obtained several types of insect species and we were able to show that GC-MS Metabolomics could be used to distinguish among the different types of larvae.
Metabolomics Analysis of Thermally Challenged Mayfly Larvae (GCMS analysis)
The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) in collaboration with the National Institute of Standards (NIST) recently produced a human plasma standard reference material (SRM 1950) for metabolite analysis. The SRM was prepared by obtaining plasma samples from 100 individuals between 40 and 50 years of age, whose ethnicity was representative of the US population and that included an equal number of men and women. The intent of the NIDDK/NIST project was to provide a reference material that would be publically available to researchers and that could be used by the clinical chemistry community to identify plasma metabolites for diagnostic purposes. Signature metabolites could then be further probed for their usefulness as disease biomarkers. The LIPID MAPS Consortium has undertaken the task to analyze this SRM by systematically identifying and quantifying the lipid molecular species in the six main categories of mammalian lipids. The quantitative levels of over 500 different lipids present in this reference human plasma sample are presented here.
Lipidomics studies on NIDDK / NIST human plasma samples
Noxa regulation of malate aspartate shuttle
Changes in cellular lipid metabolism are a common feature in most solid tumors, which occur already in early stages of the tumor progression. However, it remains unclear if the tumor-specific lipid changes can be detected at the level of systemic lipid metabolism. The objective of this study was to perform comprehensive analysis of lipids in breast cancer patient serum samples. Lipidomic profiling using an established analytical platform was performed in two cohorts of breast cancer patients receiving neoadjuvant chemotherapy. The analyses were performed for 142 patients before and after neoadjuvant chemotherapy, and the results before chemotherapy were validated in an independent cohort of 194 patients. The analyses revealed that in general the tumor characteristics are not reflected in the serum samples. However, there was an association of specific triacylglycerols (TGs) in patients' response to chemotherapy. These TGs containing mainly oleic acid (C18:1) were found in lower levels in those patients showing pathologic complete response before receiving chemotherapy. Some of these TGs were also associated with estrogen receptor status and overall or disease-free survival of the patients. The results suggest that the altered serum levels of oleic acid in breast cancer patients are associated with their response to chemotherapy.
Monounsaturated fatty acids in serum triacylglycerols are associated with response to neoadjuvant chemotherapy in breast cancer patients
Endocannabinoids belong to a diverse family of endogenous lipid bioregulators acting as physiological ligands of cannabinoid receptor type 1 and cannabinoid receptor type 2 in the central and peripheral nervous system. They are also present in nmol L−1 concentrations in human blood plasma; however, their association with possible molecular carriers remains poorly characterized. Here we report on the quantification of 46 endogenous molecular species from five major classes of endocannabinoids and endocannabinoid-related compounds in three lipoprotein fractions of human blood plasma: (VLDL), LDL, HDL, and in the plasma lipoproteinfree fraction. Although sizable quantities of endocannabinoid related molecules are associated with lipoproteins, we identified the lipoprotein-free fraction as a major carrier of endocannabinoids in blood circulation with the exception of 2-acylglycerols, which are markedly abundant in VLDL.
Quantitative profiling of endocannabinoids in lipoproteins by LC–MS/MS
Urease pre-treatment of urine has been utilized since the early 1960s to remove high levels of urea from samples prior to further processing and analysis by gas chromatography–mass spectrometry (GC–MS). Aside from the obvious depletion or elimination of urea, the effect, if any, of urease pre-treatment on the urinary metabolome has not been studied in detail. Here, we report the results of three separate but related experiments that were designed to assess possible indirect effects of urease pre-treatment on the urinary metabolome as measured by GC–MS. In total, 235 GC–MS analyses were performed and over 106 identified and 200 unidentified metabolites were quantified across the three experiments. The results showed that data from urease pre-treated samples (1) had the same or lower coefficients of variance among reproducibly detected metabolites, (2) more accurately reflected quantitative differences and the expected ratios among different urine volumes, and (3) increased the number of metabolite identifications. Overall, we observed no negative consequences of urease pre-treatment. In contrast, urease pre-treatment enhanced the ability to distinguish between volume-based and biological sample types compared to no treatment. Taken together, these results show that urease pre-treatment of urine offers multiple beneficial effects that outweigh any artifacts that may be introduced to the data in urinary metabolomics analyses.
A statistical analysis of the effects of urease pre-treatment on the measurement of the urinary metabolome by gas chromatography–mass spectrometry
The study investigated the interaction between omental adipocytes and OvCa cells, as a follow up to preliminary data indicating this leads to reprograming of metabolic (especially lipid) profiles in both adipocytes and OvCa cells as ovarian cancer cells (OvCa) readily metastasize to the omental fat pad in the abdomen and stimulate the release of fatty acids. In order to mimic the interaction between OvCa and omental adipocytes during metastasis, a coculture system was used that employed OvCa cells and primary human adipocytes isolated from omentum. Human primary adipocytes were isolated from omental explants from patients undergoing surgery for benign conditions. After surgical removal, omental tissue was digested with collagenase I, and primary cultures of adipocytes were established, characterized, and incorporated into the co-culture. The primary adipocytes were isolated and co-cultured with the OvCa cell line Skov3ip1. In this current submission, the the samples will be collected at 4, 18 and 24 hour time points post co-culture to determine the time dependent effect on lipid mediators, including oxylipins and ceramides. The study results included in this DRCC submission were the 18 hour time point data for oxylipins and ceramides from targeted metabolomic analysis of lipid mediators performed by the Newman lab.
A study of changes in lipid metabolism of ovarian cancer cells co-cultured with adipocytes: UPLC-QTRAP MS analysis
Characterization of various melon cultivars via a new methodology for qualitative analysis of aroma related volatile organic compounds and quantification of amino acids.
Single-cell level measurements are necessary to characterize the intrinsic biological variability in a population of cells. In a study of the metabolic response of a clonal Saccharomyces cerevisiae population challenged by the glycolytic inhibitor 2-deoxy-D-glucose (2-DG), this variability is shown here, by comparing measurements at the population and the single-cell level. A novel microarray platform and MALDI mass spectrometric read-out allow us to follow 24 parameters simultaneously, in up to 100 single-cell measurements. Correlation plots for metabolites from the glycolytic pathway, the pentose phosphate pathway, and the observed ATP/ADP ratio as a measure of cellular energy charge give new insight into biochemical and metabolic pathways, which would not be accessible from population-level metabolomics data. Single-cell level measurements are necessary to characterize the intrinsic biological variability in a population of cells. In this study we demonstrate that with the Microarrays for Mass Spectrometry (MAMS) platform we are able to observe this variability. We monitor environmentally-(2-deoxy-d-glucose) and genetically-(∆PFK2) perturbed Saccharomyces cerevisiae cells both at the population and near single-cell levels. Correlation plots between metabolites from the glycolytic pathway, as well as with the observed ATP/ADP ratio as a measure of cellular energy charge give new biological insight that is not accessible from population-level metabolomic data.
Single-cell and bulk level metabolomic study of the glycolysis inhibition by 2-deoxy-d-glucose in Saccharomyces cerevisiae
This West Coast Metabolomics Center pilot and feasibility project granted to Graeme Bell (University of Chicago) and collaborator Manami Hara uses a multi-omics approach to investigate the metabolome (primary metabolites), lipidome (complex lipids) and signaling lipids (oxylipins) in the plasma of Non-obese Diabetic (NOD) mice which progressed to Type 1 Diabetes Mellitus (T1D) and those that did not. NOD Mice (n=71) were assessed as diabetic or non-diabetic based on their fasting (4hr) blood glucose levels at sacrifice, which defined 30 hyperglycemic (glucose = 250 mg/dL) and 41 normoglycemic animals. The primary objective of this study was to identify candidate biomarkers associated with beta-cell destruction/survival and T1D progression.
Combined Metabolomics and Lipidomics of Type 1 Diabetes (QTOF)
In the NOD sub-study, a total of 18 samples from 6 week old, male NOD/ShiLtj mice; comprised of 6 serum samples, 6 cecal content samples and 6 liver tissue samples were analyzed. Three mice/matrix were exposed to PAT and 3 mice/matrix were non-exposed Controls. The mice were housed with SPF (Helicobacter neg/MNV neg) bedding and fed a normal diet.
Metabolomics Involved in Early Life Antibiotic Exposures(NOD-Liver)
Metabolomic profiles in short or long day, before and after bolting, single leaf analysis, Arabidopsis thaliana
Metabolomic profiles in short day, before bolting, single leaf analysis, Arabidopsis thaliana
Steel Ball/ Glass bead + Chloroform/ Isopropanol treatment
The aim of this work was to determine changes in metabolite fingerprints of grape berry skins of Merlot noir, Carbernet franc or Cabernet Sauvignon cultivars harvested in 2002, 2003 and 2004 from five geographical locations in Bordeaux (south-west of France) to better understand the factors influencing the grape berry composition.
Grape berry - vintage terroir Bordeaux - ACA 2006
Tissue extracts from metastatic melanoma mouse spleen and controls were compared via NMR based metabolomic analysis
1H NMR Metabolomics Study of Metastatic Melanoma in C57BL/6J Mouse Spleen
Direct-infusion mass spectrometry (DIMS) metabolomics is an important approach for characterising molecular responses of organisms to disease, drugs and the environment. Increasingly large-scale metabolomics studies are being conducted, necessitating improvements in both bioanalytical and computational workflows to maintain data quality. This dataset represents a systematic evaluation of the reproducibility of a multi-batch DIMS metabolomics study of cardiac tissue extracts. It comprises of twenty biological samples (cow vs. sheep) that were analysed repeatedly, in 8 batches across 7 days, together with a concurrent set of quality control (QC) samples. Data are presented from each step of the workflow and are available in MetaboLights. The strength of the dataset is that intra- and inter-batch variation can be corrected using QC spectra and the quality of this correction assessed independently using the repeatedly-measured biological samples. Originally designed to test the efficacy of a batch-correction algorithm, it will enable others to evaluate novel data processing algorithms. Furthermore, this dataset serves as a benchmark for DIMS metabolomics, derived using best-practice workflows and rigorous quality assessment.
Direct infusion mass spectrometry metabolomics dataset: a benchmark for data processing and quality control
Cancer metabolism is emerging as an important focus area in cancer research. However, the in vitro cell culture conditions under which much cellular metabolism research is performed differ drastically from in vivo tumor conditions, which are characterized by variations in the levels of oxygen, nutrients like glucose, and other molecules like chemotherapeutics. Moreover, it is important to know how the diverse cell types in a tumor, including cancer stem cells that are believed to be a major cause of cancer recurrence, respond to these variations. Here, in vitro environmental perturbations designed to mimic different aspects of the in vivo environment were used to characterize how an ovarian cancer cell line and its derived, isogenic cancer stem cells metabolically respond to environmental cues. Mass spectrometry was used to profile metabolite levels in response to in vitro environmental perturbations. Docetaxel, the chemotherapeutic used for this experiment, caused significant metabolic changes in amino acid and carbohydrate metabolism in ovarian cancer cells, but had virtually no metabolic effect on isogenic ovarian cancer stem cells. Glucose deprivation, hypoxia, and the combination thereof altered ovarian cancer cell and cancer stem cell metabolism to varying extents for the two cell types. Hypoxia had a much larger effect on ovarian cancer cell metabolism, while glucose deprivation had a greater effect on ovarian cancer stem cell metabolism. Core metabolites and pathways affected by these perturbations were identified, along with pathways that were unique to cell types or perturbations. The metabolic responses of an ovarian cancer cell line and its derived isogenic cancer stem cells differ greatly under most conditions, suggesting that these two cell types may behave quite differently in an in vivo tumor microenvironment. While cancer metabolism and cancer stem cells are each promising potential therapeutic targets, such varied behaviors in vivo would need to be considered in the design and early testing of such treatments.
Distinct metabolic responses of an ovarian cancer stem cell line using gas chromatography mass spectrometry
In this study, the metabolic difference resulting in H_2 production yield between the C. reinhardtii wild type stem cc406 (wt) and the high H_2-producing strain Stm6Glc4 (mut) at two different time points was compared before (t1) and during (t2) the H_2 production phase.
Evaluation of algorithms for multiple alignment of GCxGC-MS data
The potential for commensal microorganisms indigenous to a host (the microbiome or microbiota) to alter infection outcome by influencing host-pathogen interplay is largely unknown. We used a multi-omics systems approach, incorporating proteomics, metabolomics, glycomics, and metagenomics, to explore the molecular interplay between the murine host, the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), and commensal gut microorganisms during intestinal infection with S. Typhimurium. We find proteomic evidence that S. Typhimurium thrives within the infected 129/SvJ mouse gut without antibiotic pre-treatment, inducing inflammation and disrupting the intestinal microbiome (e.g., suppressing Bacteroidetes and Firmicutes while promoting growth of Salmonella and Enterococcus). Alteration of the host microbiome population structure was highly correlated with gut environmental changes, including the accumulation of metabolites normally consumed by commensal microbiota. Finally, the less characterized phase of S. Typhimuriums lifecycle was investigated, and both proteomic and glycomic evidence suggests S. Typhimurium may take advantage of increased fucose moieties to metabolize fucose while growing in the gut. The application of multiple omics measurements to Salmonella-induced intestinal inflammation provides insights into complex molecular strategies employed during pathogenesis between host, pathogen, and the microbiome.
A Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection
A metabolomics approach to unravel the regulating role of phytohormones towards carotenoid metabolism in tomato fruit. (Lutein Metabolism)
Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth.
Growth control of the eukaryote cell: a systems biology study in yeast
This West Coast Metabolomics Center pilot and feasibility project was granted to Roel Ophoff (UC Los Angeles ) and Steve Horwath (UC Los Angeles ) . Major depressive disorder (MDD) is one of the most debilitating disorders in the United States with a 12-month prevalence of 6.7% in the adult population. The disorder affects millions of Americans daily and is a major health concern with enormous economic cost the society at large. Criteria for MDD diagnosis and treatment are based on various signs and symptoms not always fitting into strict diagnostic categories such as DSM-IV. Despite various known risk factors (such as family history, age, and gender), biological markers supporting diagnosis or prediction of MDD are unavailable. We have collected cerebrospinal fluid (CSF) and peripheral blood of more than 600 subjects from the general population. For each of the participants we also obtained biometric information as well as behavioral trait measures. One of the measures is the Beck Depression Inventory (BDI), a well-established questionnaire for measuring severity of depression. Based on the BDI, roughly 5% of participants suffer from severe depressive symptoms while most of these subjects are not under treatment or receiving any medication for depression In the current investigation, untargeted analysis of primary metabolites was conducted on age and gender matched human cerebrospinal fluid (CSF) and plasma from subjects suffering with MDD ( n=50) and control subjects (n=50). Subjects were diagnosed as having MDD based on the Beck Depression Inventory. The primary objectives of this study were to 1) identify metabolites which discriminate between subjects with and without depression symptoms in the CSF and plasma and 2) how changes correlate between CSF and plasma.
Biomarkers for Depression in Human Plasma in a Population Sample
Human subjects were given high PUFA diet for 21 days then converted to high carbohydrate diet for another 21 days. Plasma samples were drawn during the feeding process on 7 visits.
Metabolomics, a new branch of chemical biology, provides compositional and quantitative information about the state of organism or cell at the levels of metabolite constituents. We here report non-targeted metabolome of human blood that is made up of plasma and red blood cells (RBCs), using liquid-chromatography and mass spectrometry (LC-MS). Previously, metabolome of a microbe, the fission yeast Schizosaccharomyces pombe, was reported. Two sets of metabolome results are highly similar in their compositions: among 133 compounds identified in human blood, 101 (75%) are also present in S. pombe, and among 57 compounds enriched in RBC, 45 (78%) are also present in S. pombe. Most abundant metabolites are ATP, glutathione and glutamine. Many of other relatively abundant metabolites identified are also implicated in energy, anti-oxidant and amino acid metabolism. We show fourteen newly-identified blood compounds; citramalate, GDP-glucose, trimethyl-histidine, trimethyl-phenylalanine, trimethyl-tryptophan, trimethyl-tyrosine, UDP-acetyl-glucosamine, UDP-glucuronate, dimethyl-lysine, glutamate methyl ester, N-acetyl-(iso)leucine, N-acetyl-glutamate, N2-acetyl-lysine, and N6-acetyl-lysine (the first eight are enriched in RBC). Ten of them are also detected in S. pombe, and ten of them are methylated or acetylated, amino acids. Trimethylated or acetylated free amino acids are also abundant in white blood cell. Their physiologic role may be investigated in the future by yeast genetics.
Unexpected similarities between the Schizosaccharomyces and human blood metabolomes, and novel human metabolites (Blood fraction)
Steroids play a broad and vital role in regulation of gene expression, secondary sexual characteristics, maturation, reproduction, and neurological functions; but an imbalance in steroid metabolism is also linked to development and progression of many diseases including autism. Prenatal stress of different nature has been demonstrated to affect both the mother and the offspring. Adverse nutritional conditions during gestation can impair the maternal hypothalamic-pituitary-adrenal axis (HPA) and expose the fetus to high levels of glucocorticoids (GC). Evenwhen GC are required for normal brain development; an increased exposure of the fetus to GC as a consequence of prenatal stress can affect fetal hypothalamic-pituitary-gonad axis (HPG) development, impair neurogenesis, and have a long term impact on the offsprings mental health. Decreased zinc availability can occur during pregnancy as a consequence of different conditions (nutritional deficiency, infections, diabetes, alcohol consumption, and exposure to certain toxicants). Importantly, several of these gestational conditions have been linked to autism. In fact, alterations in maternal zinc homeostasis upon exposure to select environmental stressors (e.g. the phthalate plasticizer bis-2-ethylhexyl phthalate (DEHP)) that have become increasingly common since the industrial revolution may underlie the recent rise in the incidence of autism.Alterations in maternal zinc homeostasis could expose the fetus to high GC concentrations secondary to a high maternal GC production and/or to a decreased capacity of the placenta to metabolize GC to inactive metabolites. The overall goal of this proposal is to investigate if alterations in zinc homeostasis during gestation triggered by either a marginal zinc nutrition or exposure to an environmental pollutant (the phthalate plasticizer bis-2-ethylhexyl phthalate (DEHP)) can impair maternal and fetal endocrine signaling leading to impaired fetal brain development.
Disruption of Zinc homeostasis can impair maternal glucocorticoid metabolism: consequences on the developing fetus
LC-MS untargeted analysis of leaves from two fungus-resistant grape varieties (PIWI) grape varieties (Regent and Phoenix) collected from two countries (Italy and Germany).
MetaDB a Data Processing Workflow in Untargeted MS-Based Metabolomics Experiments
Deciphering the metabolome is essential for a better understanding of the cellular metabolism as a system. Typical metabolomics data show a few but significant correlations among metabolite levels when data sampling is repeated across individuals grown under strictly controlled conditions. Although several studies have assessed topologies in metabolomic correlation networks, it remains unclear whether highly connected metabolites in these networks have specific functions in known tissue- and/or genotype-dependent biochemical pathways. In our study of metabolite profiles we subjected root tissues to gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) and used published information on the aerial parts of 3 Arabidopsis genotypes, Col-0 wild-type, methionine over-accumulation 1 (mto1), and transparent testa4 (tt4) to compare systematically the metabolomic correlations in samples of roots and aerial parts. We then applied graph clustering to the constructed correlation networks to extract densely connected metabolites and evaluated the clusters by biochemical-pathway enrichment analysis. We found that the number of significant correlations varied by tissue and genotype and that the obtained clusters were significantly enriched for metabolites included in biochemical pathways. We demonstrate that the graph-clustering approach identifies tissue- and/or genotype-dependent metabolomic clusters related to the biochemical pathway. Metabolomic correlations complement information about changes in mean metabolite levels and may help to elucidate the organization of metabolically functional modules.
Metabolomic correlation-network modules in Arabidopsis based on a graph-clustering approach
We obtained plasma samples from 7 c-peptide negative type 1 diabetic individuals (T1D) and 7 non-diabetic controls (Con) that were matched for age (T1D = 31.1±2.9 yrs, Con = 30.2±3.4 yrs), body mass (T1D = 80.2±4.7kg, Con = 81.9±7.4 kg) and BMI (T1D = 26.5±1.2 kg/m2, Con = 25.2±1.3 kg/m2). Type 1 diabetic people were studied while treated with insulin and also after 8 hours of insulin deprivation
Quantitative Metabolomics by 1H-NMR and LC-MS/MS Confirms Altered Metabolic Pathways in Diabetes
In light of global reef decline new methods to accurately, cheaply, and quickly evaluate coral metabolic states are needed to assess reef health. Metabolomic profiling can describe the response of individuals to disturbance (i.e., shifts in environmental conditions) across biological models and is a powerful approach for characterizing and comparing coral metabolism. For the first time, we assess the utility of a proton-nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomics approach in characterizing coral metabolite profiles by 1) investigating technical, intra-, and inter-sample variation, 2) evaluating the ability to recover targeted metabolite spikes, and 3) assessing the potential for this method to differentiate among coral species. Our results indicate 1H-NMR profiling of Porites compressa corals is highly reproducible and exhibits low levels of variability within and among colonies. The spiking experiments validate the sensitivity of our methods and showcase the capacity of orthogonal partial least squares discriminate analysis (OPLS-DA) to distinguish between profiles spiked with varying metabolite concentrations (0 mM, 0.1 mM, and 10 mM). Finally, 1H-NMR metabolomics coupled with OPLS-DA, revealed species-specific patterns in metabolite profiles among four reef-building corals (Pocillopora damicornis, Porites lobata, Montipora aequituberculata, and Seriatopora hystrix). Collectively, these data indicate that 1H-NMR metabolomic techniques can profile reef-building coral metabolomes and have the potential to provide an integrated picture of the coral phenotype in response to environmental change.
Application of 1H-NMR Metabolomic Profiling for Reef-Building Corals.
In an effort to identify novel biomarkers capable of predicting the development of acute kidney injury (AKI) after cardiac surgery with cardiopulmonary bypass (CPB) use, urine specimens were collected before and at 4 and 24 hours after surgery from 106 patients and analyzed by means of nuclear magnetic resonance (NMR) spectroscopy and machine learning methods.
Analysis of human urine reveals metabolic changes related to the development of acute kidney injury following cardiac surgery
In the DuraSTAT sub-study, a total of 18 samples from 8 week old, female C57BL/6 mice; comprised of 6 urine samples, 6 cecal content samples and 6 liver tissue samples were analyzed. Three mice/matrix were given STAT penicillin and 3 mice/matrix were non-treated Controls. The mice were housed with conventional bedding and fed a high fat diet.
Metabolomics Involved in Early Life Antibiotic Exposures(DuraSTAT-Urine)
Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo), gives rise to devastating crop losses in rice. Disease resistant rice cultivars are the most economical way to combat the disease. The TP309 cultivar is susceptible to infection by Xoo strain PXO99. A transgenic variety, TP309_Xa21, expresses the pattern recognition receptor Xa21, and is resistant. PXO99?raxST, a strain lacking the raxST gene, is able to overcome Xa21-mediated immunity. We used a single extraction solvent to demonstrate comprehensive metabolomics and transcriptomics profiling under sample limited conditions, and analyze the molecular responses of two rice lines challenged with either PXO99 or PXO99?raxST. LCTOF raw data file filtering resulted in better within group reproducibility of replicate samples for statistical analyses. Accurate mass match compound identification with molecular formula generation (MFG) ranking of 355 masses was achieved with the METLIN database. GCTOF analysis yielded an additional 441 compounds after BinBase database processing, of which 154 were structurally identified by retention index/MS library matching. Multivariate statistics revealed that the susceptible and resistant genotypes possess distinct profiles. Although few mRNA and metabolite differences were detected in PXO99 challenged TP309 compared to mock, many differential changes occurred in the Xa21-mediated response to PXO99 and PXO99?raxST. Acetophenone, xanthophylls, fatty acids, alkaloids, glutathione, carbohydrate and lipid biosynthetic pathways were affected. Significant transcriptional induction of several pathogenesis related genes in Xa21 challenged strains, as well as differential changes to GAD, PAL, ICL1 and Glutathione-S-transferase transcripts indicated limited correlation with metabolite changes under single time point global profiling conditions.
Thirty participants were randomized to 3 groups of 10 each with each participant studied twice. Study groups comprised (1) low and high insulin, (2) low insulin with and without EAAs, and (3) high insulin with and without EAAs.
High Insulin Combined With Essential Amino Acids Stimulates Skeletal Muscle Mitochondrial Protein Synthesis While Decreasing Insulin Sensitivity in Healthy Humans
type profiles of different tissues of tomato: pericarp (35 and 45 DPA) epiderm (45 DPA) seed
Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host-pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.
Decreased abundance of type III secretion systeminducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae
In present study, a 1H NMR based untargeted metabolomics approach was used to evaluate the subacute effects of oral gavage adminstration of HBCD (10mg/kg, 50mg/kg) on mice urine metabolome. Multivariate statistical analysis by PLS-DA showed that the metabolites in exposure groups were clearly distinct from those of control group. The distinguishing metabolites citric acid and 2-ketoglutarate were significantly up-regulated, displaying the disturbance of energy metabolism after HBCD treatment. While some metabolites including acetic acid, malonic acid and 3-hydroxybutyrate were reduced in low or high dosed groups，indicating the impaired lipid metabolism. Other influenced metabolites consisted of formate, trimethylamine and several amino acids. Furthermore, a targeted UPLC-MS/MS method was performed for metabolic profiling of twenty amino acids. Quantitation results identified alanine, lysine and phenylalanine as significantly disturbed amino acids. Overall, these results suggested that subchronic exposure to HBCD caused a disturbance of mice metabolism, especially in TCA cycle, lipid metabolism and homeostasis of amino acids, and the analysis of metabolic profiles can provide more comprehensive information for risk assessment of environment pollutants.
1H NMR and LC–MS/MS based urine metabolomic investigation of the subacute effects of HBCD in mice
We use metabolite profiles of the model plant Arabidopsis thaliana measured on an UPLC-ESI/QqTOF-MS as a pilot study to estimate variance levels which then can be used to obtain several statistical parameters (e.g. minimum detectable effect, number of required replicates and error probabilities) which influence the design of a subsequent study.
Assessment of Biological and Technical Variation
Seventeen northern elephant seal pups constituting four different cohorts at Año Nuevo State Reserve were studied at two post-weaning periods: early (1-2 wk post weaning; n=5) and late (6-7 weeks post weaning; n=12). Study #1: Prior to each infusion protocol, plasma U/kg). Following the infusion, blood samples were collected at 10,To determine the effects of prolonged fasting on peripheral insulin activity and function, samples were collected. Ten fasting seal pups (n=5 early, n=5 late) were infused (i.v.) with a mass-specific dose of insulin (0.065 U/kg). Following the infusion, blood samples were collected at 10, 30, 60, 90, and 120 minutes. Study #2: Seven, late-fasted seal pups were administered either a low (LDG; 10 pmol/kg; n=3) or high (HDG; 100 pmol/kg; n=4) dose of GLP-1 immediately following a glucose bolus (0.5 g/kg) (i.v.) infused within 2 mins. the infusions, blood samples were collected at 10, 30, 60, 90, 120, and 150 minutes.
Metabolomic and lipidomic profiles in response to exogenous insulin and GLP-1 infusions during prolonged fasting (LCMS)
The Uppsala Longitudinal Study of Adult Men (ULSAM) study is an ongoing, longitudinal, epidemiologic study started in 1970 and based on men born between 1920 and 1924 in Uppsala, Sweden which were invited to participate at age 50 (N=2,841) [PMID: 9484991]; 81.7% (N=2,322) participated. Individuals were reinvestigated at the ages of 60, 70, 77, 82 and 88 years. Information collected includes a medical questionnaire, blood pressure and anthropometric measurements, glucose tolerance test and 24-hour ambulatory blood pressure. Metbaolomics has been performed on plasma samples from age 70. In the final analysis we took samples from 1,138 individuals.
In this study broncho-alveolar lavage fluid (BALF) from mice infected with influenza A virus, PR8 (280 PFU) was analyzed for lipidomic host response in comparison with the mock infected animals. Time Points: 6, 9, 11, 13 and 19 days post infection, done in 8-10 replicates.)
Lipid Mediators of inflammation in BALF 6-19 days after infection with Influenza
Women with polycystic ovary syndrome (PCOS) have metabolic disturbances that closely resemble the metabolic syndrome, suggesting an increased risk for type 2 diabetes and possibly for cardiovascular disease. With the aim of determining the underlying mechanism by which PCOS is associated with these long-term health risks, here we use NMR and MS-based metabolomics to compare the lipoprotein profile and the serum metabolome of young and non-obese PCOS patients with age- and weight-matched control women. We demonstrate that elevated levels of methionine sulfoxide in PCOS serum are strongly correlated with the degree of oxidation of methionine residues in apolipoprotein-A1. As a result, there is an impaired maturation of HDL reflected in a decline of large HDL particles in women with PCOS. Remarkably, these metabolic disturbances were partially restored after 18 months of treatment with a novel combination of insulin sensitizers plus an anti-androgen (pioglitazone, metformin and flutamide). We postulate that hyperandrogenism in PCOS is the root cause of the oxidative damage in lipoproteins. Dyslipidemic features and hyperinsulinemia precede any other metabolic complication associated with the increased risk for diabetes and cardiovascular disease, including impaired glucose tolerance and insulin resistance. Taken together, these metabolite changes might also constitute early biomarkers of metabolic syndrome.
Long-term health risks in PCOS occur prematurely with serum markers of oxidative stress impacting HDL maturation through oxidation of methionine residues in apolipoprotein A1
Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the delta-9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. Gas chromatography mass spectrometry (GC-MS) was used to define the metabolome of all the possible knock-outs for the delta 9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence delta 9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. The propagation of metabolic changes across the network of metabolism demonstrates that modification of the delta 9 desaturases places C.elegans into a catabolic state compared with wildtype controls.
A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans
Combined Metabolomics and Lipidomics of Type 1 Diabetes (GCMS)
Metabolomics Involved in Early Life Antibiotic Exposures(EstroSTAT-Serum)
Isolated skelelal muscle mitochondria incubated in the presence of fatty acid oxidation precursor and inhibitors
Skeletal Muscle Treatment (palmitate and inhibitors)
Experiments were conducted using 13-wk-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME). Mice were housed individually with free access to water and chow (TD.10112; Harlan Laboratories, Indianapolis, IN), with a 12:12-h light-dark cycle and temperature and humidity control. Mice were acclimated for 1 wk prior to the beginning of the experiment. The protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Following a 6-h fast, mice were given intraperitoneal injections of STZ (125 mg/kg; in sodium acetate buffer, pH = 4.5) (67). Injections were repeated on the following day. Control animals received intraperitoneal injection of vehicle. Only mice that displayed blood glucose ?300 mg/dl and an increase in blood ketones (both values by Precision Xtra glucometer; Abbott Laboratories, Abbott Park, IL), hyperphagia, and polyuria and were positive for urine glucose presence via dipstick (Uristix, Bayer, Pittsburgh, PA) on day 7 after the first STZ dose were included in the experiment. Animals that were positive for STZ diabetes received LinBit subcutaneous insulin implant (LinShin Canada, Toronto, ON, Canada) (79) under pentobarbital sodium anesthesia (Nebutal, 40 mg/kg of body wt) according to the manufacturer's protocol. Each animal received two subcutaneous implants (total dose: 0.2 U/24 h for >30 days, 10 U/kg for 20-g mice). Insulin treatment was continued for 3 wk. Control animals (C; n = 13) received blank implants. Diabetic control was confirmed by biweekly measurements of blood and urinary glucose. In some cases, when urine glucose was present and blood glucose was >288 mg/dl, the animal received a third implant. The insulin treatment was continued until initially lower plasma glucose content in diabetic animals reached control values. Three weeks following implantation, diabetic mice were divided randomly into diabetic-treated (D + I; n = 13) and diabetic-deprived (D ? I; n = 13) groups. Insulin implants were removed from the D ? I group under pentobarbital anesthesia, which led to the return of the diabetic phenotype within 24 h. Animals from the D + I group continued on insulin treatment (Fig. 1). At the age of 18 wk, animals from all groups were analyzed for body composition by an Echo-MRI Body Composition Analyzer (EchoMRI, Houston, TX) and euthanized by decapitation 5 wk after the initial STZ or vehicle dose. Figure 1 depicts the timeline of the experiment and blood glucose profiles for each experimental group. Additional animals were used for estimation of skeletal muscle insulin sensitivity by acute insulin stimulation. The mice were divided into the C (n = 6), D ? I (n = 7), and D + I (n = 7) groups and followed appropriate experimental treatment, except for acute insulin stimulation 10 min prior to euthanization by pentobarbital overdose. Figure 1 of the attached PDF of the article summarizes the study design
Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice
Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by progressive ectopic mineralization of the skin, eyes and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding ABCC6 (MRP6), an ATP-dependent efflux transporter that is mainly present in the liver. Abcc6-/- mice have been instrumental in the demonstration that PXE is a metabolic disease caused by the absence of an unknown factor in the circulation, the presence of which depends on ABCC6 in the liver. Why absence of this factor results in PXE remained a mystery. We now show that medium from HEK293 cells overexpressing either human or rat ABCC6 potently inhibits mineralization in vitro, whereas medium of HEK293 control cells does not. Untargeted metabolomics revealed that cells expressing ABCC6 excrete large amounts of nucleoside triphosphates, even though ABCC6 itself did not transport nucleoside triphosphates. Extracellularly, ectonucleotidases hydrolyzed the excreted nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), a strong inhibitor of mineralization that plays a pivotal role in several mineralization disorders similar to PXE. The in vivo relevance of our data was demonstrated in Abcc6-/- mice, which had plasma PPi levels that were less than 40% of those found in wild-type mice. This study provides the first insight into how ABCC6 affects PXE. Our data indicate that the factor that normally prevents PXE is PPi, which is provided to the circulation in the form of nucleoside triphosphates via an as yet unidentified, but ABCC6-dependent mechanism.
LC-MS metabolic profiling of mouse plasma and cell culture medium in relation to ABCC6 expression
Metabolites are not only the catalytic products of enzymatic reactions but also the active regulators or the ultimate phenotype of metabolic homeostasis in highly complex cellular processes. The modes of regulation at the metabolome level can be revealed by metabolic networks. We investigated the metabolic network between wild-type and 2 mutant (methionine-over accumulation 1 [mto1] and transparent testa4 [tt4]) plants regarding the alteration of metabolite accumulation in Arabidopsis thaliana. In the GC-TOF/MS analysis, we acquired quantitative information regarding over 170 metabolites, which has been analyzed by a novel score (ZMC, z-score of metabolite correlation) describing a characteristic metabolite in terms of correlation. Although the 2 mutants revealed no apparent morphological abnormalities, the overall correlation values in mto1 were much lower than those of the wild-type and tt4 plants, indicating the loss of overall network stability due to the uncontrolled accumulation of methionine. In the tt4 mutant, a new correlation between malate and sinapate was observed although the levels of malate, sinapate, and sinapoylmalate remain unchanged, suggesting an adaptive reconfiguration of the network. Gene-expression correlations presumably responsible for these metabolic networks were determined using the metabolite correlations as clues. Two Arabidopsis mutants, mto1 and tt4, exhibited the following changes in entire metabolome networks: the overall loss of metabolic stability (mto1) or the generation of a metabolic network of a backup pathway for the lost physiological functions (tt4). The expansion of metabolite correlation to gene-expression correlation provides detailed insights into the systemic understanding of the plant cellular process regarding metabolome and transcriptome.
Unbiased characterization of genotype-dependent metabolic regulations by metabolomic approach in Arabidopsis thaliana
A metabolomics approach to unravel the regulating role of phytohormones towards carotenoid metabolism in tomato fruit. (Lycopene metabolism)
Although hepatocellular carcinoma (HCC) has been subjected to continuous investigation and its symptoms are well-known, early stage diagnosis of this disease remains difficult and the survival rate after diagnosis is typically very low (3−5%). Early and accurate detection of metabolic changes in the sera of patients with liver cirrhosis can help improve the prognosis of HCC and lead to a better understanding of its mechanism at the molecular level, thus providing patients with in-time treatment of the disease. In this study, we compared metabolite levels in sera of 40 HCC patients and 49 cirrhosis patients from Egypt by using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from cirrhotic controls are selected by statistical methods. Putative metabolite identifications for these ions are obtained through mass-based database search. The identities of some of the putative identifications are verified by comparing their MS/MS fragmentation patterns and retention times with those from authentic compounds. Finally, the serum samples are reanalyzed for quantitation of selected metabolites as candidate biomarkers of HCC. This quantitation was performed using isotope dilution by selected reaction monitoring (SRM) on a triple quadrupole linear ion trap (QqQLIT) coupled to UPLC. Statistical analysis of the UPLC-QTOF data identified 274 monoisotopic ion masses with statistically significant differences in ion intensities between HCC cases and cirrhotic controls. Putative identifications were obtained for 158 ions by mass based search against databases. We verified the identities of selected putative identifications including glycholic acid (GCA), glycodeoxycholic acid (GDCA), 3β, 6β-dihydroxy-5β-cholan-24-oic acid, oleoyl carnitine, and Phe-Phe. SRM-based quantitation confirmed significant differences between HCC and cirrhotic controls in metabolite levels of bile acid metabolites, long chain carnitines and small peptide. Our study provides useful insight into appropriate experimental design and computational methods for serum biomarker discovery using LC−MS/MS based metabolomics. This study has led to the identification of candidate biomarkers with significant changes in metabolite levels between HCC cases and cirrhotic controls. This is the first MS-based metabolic biomarker discovery study on Egyptian subjects that led to the identification of candidate metabolites that discriminate early stage HCC from patients with liver cirrhosis.
LC−MS Based Serum Metabolomics for Identification of Hepatocellular Carcinoma Biomarkers in Egyptian Cohort
Mining for robust transcriptional and metabolic responses to long-term salt stress: a case study on the model legume Lotus japonicus. Translational genomics, the use of model species to generate knowledge about biological processes and the functions of genes, offers great promise to biotechnologists. Few studies have sought robust responses of model plants to environmental stresses, such as salinity, by altering the stress dosage or by repeating experiments in consecutive years and/or different seasons.We mined our published and unpublished data on legume salt acclimation for robust system features at the ionomic, transcriptomic and metabolomic levels. We analysed data from the model legume Lotus japonicus, obtained through six independent, longterm, non-lethal salt stress experiments which were carried out over two consecutive years. Best possible controlled greenhouse conditions were applied and two main questions asked: how reproducible are results obtained from physiologically meaningful salinity experiments, and what degree of bias may be expected if conclusions are drawn from less well-repeated sampling?A surprisingly large fraction of the transcriptional and metabolic responses to salt stress were not reproducible between experiments. A core set of robust changes was found that was shared between experiments. Many of these robust responses were qualitatively and quantitatively conserved between different accessions of the same species, indicating that the robust responses may be a sound starting point for translational genomics.
Mining for metabolic responses to long-term salt stress: a case study on the model legume Lotus japonicus (A)
This West Coast Metabolomics Center pilot and feasibility project was granted to Johanna Lampe (Fred Hutchinson Cancer Research Center at Univ. of Washington, Seattle). In the current investigation, unbiased profiling of the metabolome and lipidome of adipose tissue samples (visceral(VAT) and subcutaenous (SAT)) and serum of 50 CRC patients, including stages I-IV, from the Fred Hutchinson Cancer Center (Seattle,WA) and the German Cancer Research Center (Heidelberg, Germany) was conducted. The lipidome and metabolome of adipose tissue (VAT/SAT) and serum were analyzed using established UPLC-QTOFMS analysis and GC-TOFMS analyses, respectively. The primary objectives of this project were to 1) compare the metabolome and lipidome of matched VAT and SAT adipose tissue of n=50 Colorectal Cancer Cell (CRC) patients, 2) characterize the associations between the lipidome and metabolome in adipose tissue (VAT/SAT) and serum of n=50 CRC patients and 3) test the associations between the lipidome/metabolome of VAT and serum with the tumor stage of CRC patients.
Metabolic Profiling of Visceral and Subcutaneous Adipose Tissue from Colorectal Cancer Patients: GC-TOF MS analyis of serum samples
Pilot experiment looking for the existence of certain molecules in pancreatic cancer cells
Intestinal Samples II pre/post transplantation
Labeling of cells was carried out in triplicate with each unstimulated sample containing 20e6 cells and stimulated cells containing 12.8e6 cells. Cells were deprived of glucose or glutamine for 1 h then resuspended in 10mM [13C1,2] D-glucose labeled medium. At this time cells were also activated with 1 ug/ml anti-CD3 and anti-CD28. Cells were labeled (and activated) for 12 h when 1 ml of spent medium was collected and cells were resuspended in 200 ul ice cold methanol. All samples were immediately stored in -80°C.
The PIVUS study started in 2001 with the primary aim to investigate the predicive power of different measurements of endothelial function and arterial compliance in a random sample of 1000 subjects aged 70 living in the community of Uppsala.In March 2006 a reinvesigation of the cohort at the age of 75 was started. The major measurements performed at age 70 were also repeated at age 75.In the spring of 2011 we started the 80-year reinvestigation of the cohort. This round will continue until the summer of 2014 and we expect that 500-600 of the original sample will attend. Here we make publically available all the metabolomics analysis on serum samples from the investigation at age 70.
Prospective Investigation of the Vasculature in Uppsala Seniors - Metabolomics
In colorectal cancer, p53 is commonly inactivated, associated with chemo-resistance, and marks the transition from non-invasive to invasive disease. Cancers, including colorectal cancer, are thought to be diseases of aberrant stem cell populations, as stem cells are able to self-renew, making them long-lived enough to acquire mutations necessary to manifest the disease. We have shown that extracts from sweet sorghum stalk components eliminate colon cancer stem cells (CCSC) in a partial p53-dependent fashion. However, the underlying mechanisms are unknown. In the present study, CCSC were transfected with short hairpin-RNA against p53 (CCSC p53 shRNA) and treated with sweet sorghum phenolics extracted from different plant components (dermal layer, leaf, seed head and whole plant). While all components demonstrated anti-proliferative and pro-apoptotic effects in CCSC, phenolics extracted from the dermal layer and seed head were more potent in eliminating CCSC by elevating caspases 3/7 activity, PARP cleavage, and DNA fragmentation in a p53-dependent and p53-independent fashion, respectively. Further investigations revealed that the anti-proliferative and pro-apoptotic effects were associated with decreases in ?-catenin protein levels, and ?-catenin targets cyclin D1, cMyc, and survivin. These results suggest that the anti-proliferative and pro-apoptotic effects of sweet sorghum extracts against human colon cancer stem cells are via suppression of Wnt/?-catenin pro-survival signaling in a p53-dependent (dermal layer) and partial p53-independent (seed head) fashion. LCMS used to identify phenolic compounds associated with extract activity
Extracts from sweet sorghum (Sorghum bicolor), a feedstock for biorefining, suppressed proliferation and elevated apoptosis in human colon cancer stem cells via suppression of Wnt/?-catenin signaling pathway
Metabolites of cold hardy versus cold susceptible flies were compared using N less than R-based metabolomics. We used 8 replicates per line (2 hardy lines, two susceptible lines), and sampled each line at three time points (before, during and after cold), giving rise to 96 samples total.
Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster
Several xylem-associated regulatory genes have been identified that control processes associated with wood formation in poplar. Prominent among these are the NAC domain transcription factors (NACs). We evaluated the putative involvement of Populus NAC154, a co-ortholog of the Arabidopsis gene SND2, as a regulator of secondary biosynthetic processes in stem internode tissues by interrogating methanol:water extracts from control and transgenic trees. Comprehensive untargeted metabolite profiling was accomplished with a liquid chromatography-mass spectrometry platform that utilized two different chromatographic supports (HILIC and reversed phase) and both positive and negative ionization modes. Evaluation of current and previous year tissues provided datasets for assessing the effects of NAC154 overexpression in wood maturation processes. Phenolic glycoside levels as well as those of oligolignols, sucrose and arginine were modulated with phenotypic and chemotypic traits exhibiting similar trends. Specifically, increased levels of arginine in the NAC154 overexpressing tissues supports a role for the transcription factor in senescence/dormancy-associated processes.
Effect of NAC154 transcription factor overexpression on the field grown poplar stem wood metabolome
Variations in tissue development and spatial composition of melon (Cucumis melo L.), a ripe fleshy fruit. To gain insight into the mechanisms involved in these changes we identified key metabolites for rational food quality design.
Charentais Melon Fruit 2009-Spatial 2- METAPHOR NP
Global approach to characterize changes in metabolic profiles in mesocarp tissues of palm date (Deglet Noor) fruit during fruit development.
Metabolomics Involved in Early Life Antibiotic Exposures(TranSTAT-Serum)
Characterization of changes in leaf metabolic profiles at two ontogenis stages (mature and juvenile) from tomato plants in response to a long-term cadmium exposure (90 days).
Tomato Leaf and long-term cadmium stress - 2010
There is a lack of comprehensive studies documenting the impact of sample collection conditions on metabolic composition of human urine. To address this issue, two experiments were performed at a three-month interval, in which midstream urine samples from healthy individuals were collected, pooled, divided into several aliquots and kept under specific conditions (room temperature, 4°C, with or without preservative) up to 72 h before storage at -80°C. Samples were analyzed by high-performance liquid chromatography coupled to high-resolution mass spectrometry and bacterial contamination was monitored by turbidimetry. Multivariate analyses showed that urinary metabolic fingerprints were affected by the presence of preservatives and also by storage at room temperature from 24 to 72 hours, whereas no change was observed for urine samples stored at 4°C over a 72-hour period. Investigations were then focused on 280 metabolites previously identified in urine: 19 of them were impacted by the kind of sample collection protocol in both experiments, including 12 metabolites affected by bacterial contamination and 7 exhibiting poor chemical stability. Finally, our results emphasize that the use of preservative prevents bacterial overgrowth, but does not avoid metabolite instability in solution, whereas storage at 4°C inhibits bacterial overgrowth at least over a 72-hour period and slows the chemical degradation process. Consequently, and for further LC/MS analyses, human urine samples should be maintained at 4°C if their collection is performed over 24 hours.
Impact of collection conditions on the metabolite content of human urine samples as analyzed by liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectroscopy
The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. To investigate potential systemic effects of the mutation, 1H NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.
Impacts of the Callipyge mutation on ovine plasma metabolites
SIRM Analysis of MYC inducible P493 cells under normoxia and hypoxia in [U-13C/15N] labeled Glutamine medium
SIRM Analysis of human P493 cells under hypoxia in [U-13C/15N] labeled Glutamine medium (Both positive and ion mode FTMS)
Global approach to characterize changes in metabolic profiles in two interdependent tissues Seed and Flesh from the same tomato fruits during tomato fruit development.
Cells were treated with 0, 20 or 200 μM RESV for 4 h. After washing with PBS, cells were lysed, and metabolites were extracted and analyzed by LC-MS. Peak areas metabolites were normalized by peak areas of spiked internal standards (HEPES and PIPES). 3 samples for each condition (except for HaCaT 200 μM, 2 samples).
Resveratrol metabolism in HepG2 (human hepatocytes), HaCaT (human keratinocytes), and C2C12 (mouse myoblasts)
Metabolic Profiling of Visceral and Subcutaneous Adipose Tissue from Colorectal Cancer Patients: UHPLC-QTOF MS analyis of subcutaneous and visceral adipose tissue samples
To provide useful data for development and validation of the PyMS Mass Spectrometry software, a test dataset was run at Metabolomics Australia. A biologically complex mix of a biological background material (foetal calf serum), spiked with 2-fold increasing amounts of a mix of metabolite standards. In addition, a single sample consisting of a simple mix of 45 metabolites representing a variety of chemical classes (sufars, organic acids, amino acids, sugar phosphates), was run through a standard Metabolomics Australia GC-MS analysis. The resulting data is a valuable tool in testing GC-MS data analysis software.
Development and validation of the PyMS Mass Spectrometry software
Metabolomics Involved in Early Life Antibiotic Exposures(VGSTAT-Liver)
Metabolism and water status of galls induced by root-knot nematode in Medicago truncatula. 2 infestation stages 21 and 35 days.
Medicago Roots and Galls development 2013
Metabolomic profiling of Israeli melon fruit quality as affected by genotype and environment : Field versus greenhouse culture.
Melon Israel META-PHOR (Metabolomics 2013)
The aim of the study is to investigate the stability of isoprenoids in grape extracts using HPLC-DAD. It is shown that the addition of triethylamine is beneficial in that carotenoid, chlorophyll and tocopherol compounds are much more stable and in this way can be kept up to at least thirty days without any sign of degradation.
High-throughput carotenoid profiling using multivariate curve resolution (Stability of isoprenoids in grape extract)
1H high resolution magic angle spinning (HR-MAS) NMR spectroscopy was applied in combination with multivariate statistical analyses to study the metabolic response of whole cells to the treatment with a hexacationic ruthenium metallaprism [1]6+ as potential anticancer drug. Human ovarian cancer cells (A2780), the corresponding cisplatin resistant cells (A2780cisR), and human embryonic kidney cells (HEK-293) were each incubated for 24 h and 72 h with [1]6+ and compared to untreated cells. Different responses were obtained depending on the cell type and incubation time. Most pronounced changes were found for lipids, choline containing compounds, glutamate and glutathione, nucleotide sugars, lactate, and some amino acids. Possible contributions of these metabolites to physiologic processes are discussed and lead to the conclusion that different cell death pathways and temporal stages thereof occur with A2780 cells most likely following necrotic cell death, while HEK-293 cells and A2780cisR rather follow apoptotic cell death.
Cell metabolic response to treatment with hexacationic Ru-complex
Metabolomics analysis of multiple metabolic functions in the YjgF/YER057c/UK114 (Rid) protein family GCMS (part 2)
Caloric restriction increases life-span of a number of different organisms including mammalian species such as dogs and rats. The underlying mechanisms are yet unknown, but appear to be related to changes of the metabolism. In order to shed more light on the metabolic response of caloric restriction we here present the first NMR-spectroscopy based study of how the blood metabolite profiles of rats are influenced by graded levels of caloric restriction. The study involved three groups of obese rats exposed for five days to 0%, 20%, and 40% caloric restriction respectively. Blood serum samples from each individual were analyzed by 1H NMR and the resulting spectra were subjected to multivariate analysis by unsupervised (PCA) and supervised (OPLS-DA) methods. The analyzes revealed that a response to caloric restriction is clearly observable already at 20% caloric restriction. The metabolites that distinguish the metabolic profiles at 20% caloric restriction deviated to some extent from those at 40% caloric restriction. The metabolic profile changes induced by caloric restriction were most clearly observed as an increased level of 3-hydroxybutyrate, and decreased levels of lipids and pyruvate. These metabolic responses are in agreement with a switch in metabolism from anabolic pathways towards fatty acid catabolism and gluconeogenesis.
NMR-based Blood Metabolic Profiles of Rats Exposed to Different Levels of Short Term Caloric Restriction
This West Coast Metabolomics Center pilot and feasibility project was granted to Ernst Lengyel (University of Chicago). The biology of ovarian cancer (OvCa) is clearly distinct from that of most epithelial tumors, in that hematogenous metastases are rare, and ovarian tumors remain confined to the peritoneal cavity. The omentum, a large pad of fat tissue (20x13x3cm) covering the bowel, is the most common site of OvCa metastasis. It consists primarily of adipocytes, which become the principal microenvironment for the OvCa cells. The underlying hypothesis for this application is that, in the presence of adipocytes, the metabolism of OvCa cells is reprogramed and shifts towards lipid utilization, which provides energy that facilitates tumor growth and metastasis. Preliminary results suggest that primary human omental adipocytes secrete cytokines which promote the metastasis of OvCa cells to the omentum and their subsequent invasion. Once metastasis has occurred, OvCa cells induce lipolysis in omental adipocytes, and use the energy derived from these lipids to proliferate. To study the metabolic changes in the tumor microenvironment we have established a 3D organotypic culture of the human omentum using primary human cells established from patient tissue. Metabolic studies will be performed on adipocytes and OvCa cells individually, on conditioned media and on adipocytes and OvCa cells co-cultured in our 3D model, with the goal of arriving at a comprehensive analysis of primary metabolites and lipids in the tumor microenvironment. In the current investigation, untargeted analysis of primary metabolites and complex lipids were conducted on adipocytes and OvCa cells individually, on conditioned media and on adipocytes and OvCa cells co-cultured in our 3D model. Analysis of oxylipins was conducted on conditioned media. To gain better understanding of the dynamic regulation of metabolic pathways we will also perform metabolic flux analysis using labeled cells (13C-glucose, 13C-glutamine) in the 3D culture model. The primary objective of this study is to gain insight into the dynamic interactions between OvCa cells and human adipocytes with the anticipation of elucidating targets of therapeutic intervention.
A study of changes in lipid metabolism of ovarian cancer cells co-cultured with adipocytes: UHPLC-QTOF MS analysis
Criteria for the diagnosis of amnestic MCI included: (i) memory complaint documented by the patient and collateral source; (ii) impairment in 1 or more of the 4 cognitive domains (memory, executive functioning/attention, visuospatial, or language); (iii) essentially normal functional activities of daily living; and (iv) absence of dementia. In general, the amnestic MCI determination is made when the memory measures fall 1.0⿿1.5 SD below the means for age and education appropriate individuals in our community; however, rigid cutoffs on psychometric scores were not used to establish the diagnosis of amnestic MCI which was made on clinical grounds. The diagnosis of dementia was made using DSM-IV criteria, and the diagnosis of AD was made using established criteria. Subjects were considered to be CN if they performed within the normative range and did not meet criteria for MCI or dementia.
Identification of altered metabolic pathways in Alzheimer's disease, mild cognitive impairment and cognitively normals using Metabolomics (CSF)
Metabolomics Involved in Early Life Antibiotic Exposures(DuraSTAT-Cecal)
Lipidomics studies on macrophages - RAW 264.7 cells treated with Kdo2-Lipid A and compactin. Experiments were conducted with RAW264.7 cells fed 10% fetal calf serum. 8-timepoint study: Measurements were taken at 0, 0.5,1,2,4,8, 12, and 24hrs for: (i) compactin, (ii) Kdo2-Lipid A, (iii) compactin + Kdo2-Lipid A. and (iv) control
Timecourse on RAW 264.7 cells treated with Kdo2-Lipid A and compactin
Jurkat parental cells (JP6) or cells stably expressing the pCDNA Noxa vector (N5) cells were cultured to confluence and counted. 20E6 cells in triplicate were resuspended in glucose free or glutamine free medium for 2 hours. Cells were then pelleted and resuspended in 13C-1,2 Glucose (10mM) or 13C-U-glutamine (4mM) for 24 hours. Cells were pelleted and spent medium was collected. Cell pellets were washed 1X with ice cold PBS and cell pellets were resuspended in 400ul -20 methanol. Cells and media were snap frozen in liquid nitrogen and stored at -80.
The TwinGene project, conducted between 2004 and 2008, is a population-based Swedish study of twins born between 1911 and 1958. The study participants have previously participated in a telephone interview called Screening Across the Lifespan Twin Study, conducted between 1998 and 2002. To be included in TwinGene, both twins within a pair had to be alive. The zygosity of the twins was based on self-reported childhood resemblance, or by using DNA markers (for 18% of the total sample). In total, 12591 individuals participated by donating blood to the study, and by answering questionnaires about life style and health. For the purpose of metabolomic profiling, we designed a case-cohort of incident coronary heart disease events and a matched sub-cohort (controls) stratified on age and sex. In the final analysis we included serum samples from 1,670 individuals. Here we make publically available all the metabolomics analysis on serum samples.
Large-scale metabolomics in the TwinGene study
In this study, a combination of targeted and untargeted metabolomics/lipidomics approach was used in conjunction with statistical analysis for identification of a bio-signature of pre-clinical Alzheimer’s disease. Alzheimer’s disease causes a progressive dementia that currently affects over 35 million individuals worldwide and is expected to affect 115 million by 2050. There are no cures or disease-modifying therapies, and this may be due to our inability to detect the disease before it has progressed to produce evident memory loss and functional decline. Herein, we describe our lipidomic approach to detecting preclinical Alzheimer’s disease in a group of cognitively normal older adults. We discovered and validated a set of ten lipids from peripheral blood that predicted phenoconversion to either amnestic mild cognitive impairment or Alzheimer’s disease within a 2–3 year timeframe with over 90% accuracy.
Plasma Lipidomics for the Identification of Anticedent Memory Impairment
Combustible cigarette smoking has major impact on human health. In order to reduce smoking-related health risk, the tobacco industry is developing modified risk tobacco products (MRTP). To assess the potential of such a prototype (pMRTP) to reduce health risk, we used C57BL/6 mice, model of chronic obstructive pulmonary disease (COPD), in a systems toxicology approach to investigate in a large, controlled study, classical toxicology end points in parallel to transcriptomics, lipidomics, and proteomics profiles in mice exposed to conventional cigarette smoke (3R4F) or a pMRTP aerosol for up to 7 months. We also included a cessation group and a switching-to-pMRTP group (after 2 months of 3R4F exposure), in addition to the control (fresh air exposed) group, to understand the potential benefit of switching to pMRTP for smokers who cannot or do not want to quit smoking. The present manuscript describes in detail the study design, setup, implementation, as well as generation, processing and quality control analysis of the toxicology and omics dataset accessible in public repositories for further studies.
Comprehensive systems biology analysis of a seven-month cigarette smoke inhalation study in C57BL/6 mice
This experiment is investigating the metabolome of men, healthy women, and women with Polycystic Ovary Syndrome (PCOS) with and without Obstructive Sleep Apnea (OSA).
Twenty replicates of each genetic line (cold hardy and cold tolerant) were collected with 40 flies per replicate.
The rapidly growing family of coregulators of nuclear receptors includes coactivators that promote transcription and corepressors that harbor the opposing function. In recent years, coregulators have begun to emerge as important regulators of metabolic homeostasis, including the p160 Steroid Receptor Coactivator (SRC) family. Members of the SRC family have been ascribed important roles in control of gluconeogenesis in liver and fatty acid oxidation in skeletal muscle. To provide a deeper and more granular understanding of the metabolic impact of SRC family members, we have performed targeted metabolomics analysis of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice with global knockout of SRC-1, SRC-2, or SRC-3. We measured amino acids, acyl carnitines, and organic acids in five tissues with key metabolic functions (liver, heart, skeletal muscle, brain, plasma) isolated from SRC-1, -2, or -3 knockout mice and their wild-type littermates in the fed and fasted conditions, thereby unveiling unique metabolic functions of each SRC coactivator. Overall, we observed entire groups or subgroups of metabolites belonging to discrete metabolic pathways that were influenced in different tissues and/or feeding states due to ablation of individual SRC isoforms. Surprisingly, we identified very few metabolites that changed universally across the three SRC knockout models. These findings demonstrate that coactivator function has very limited redundancy even within the homologous SRC coactivator family. Furthermore, this work also demonstrates the use of metabolomics as a means for identifying novel metabolic regulatory functions of transcriptional coregulators.
Tissue- and Pathway-Specific Metabolomic Profiles of the Steroid Receptor Coactivator (SRC) family
13C-Glu and 13C/15N-Gln SIRM study of A549 cells
Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with uni- and multivariate statistics was applied to examine the urinary metabolic changes in Human type 2 diabetes mellitus patients compared to the control group. The human population were un medicated diabetic patients who have good daily dietary control over their blood glucose concentrations by following the guidelines on diet issued by the American Diabetes Association. Note: This is part of a larger study, please refer to the original paper below.
NMR based metabolomics of Human Type 2 Diabetes urine samples
Metabolomics Involved in Early Life Antibiotic Exposures(NOD-Serum)
This experiment tests the effect of individual mutations on the metabolome of Arabidopsis. The standardized growth conditions are as follows: 1. Seeds (between 14 and 16) are sown on media in 100 x 100 x 15mm square Falcon Petri Dishes (Fisher Scientific, catalogue #08-757-11A). Seeds were arranged on the plates in a single horizontal line at the 1-cm mark from the top of the plate. 2. Each plate contains between 20 and 25-ml of sterile MS media containing 0.1% (w/v) sucrose. 3. Prior to sowing, seeds were sterilized by treating for 1-minute at room temperature with a 300-l solution of 50% (v/v) ethanol, this solution was removed and replaced with a 300-l solution consisting of 1% (v/v) Tween 20 (Fischer BioReagents, catalogue #BP33750), and 50% (v/v) bleach solution (Clorox), and incubated at room temperature for 10-minutes. The seeds were then washed with three changes of 0.3-ml of sterile water. 4. Upon sowing with seeds, the plates were wrapped with Micropore tape (3M Health Care, catalogue #1530-0), and then stored horizontally for 4-days at 4 °C, with illumination of 1 mol/m2. 5. On the 5th day, plates were moved to the growth room, and held in a vertical position in Plexi-glass holders for 17-days this growth room condition is labeled in Table I. 6. On 18th day Petri plates were opened and the aerial portions of these plants were harvested immediately upon plate opening. 7. Upon harvesting, plant material was quenched by immersion in liquid nitrogen and stored at 70 °C.
Mining for metabolic responses to long-term salt stress: a case study on the model legume Lotus japonicus (C)
Mining for metabolic responses to long-term salt stress: a case study on Arabidopsis thaliana Col-0 (A)
Metabolomics analysis was performed on DU145 prostate cancer cells and PNT1A non-tumorigenic prostate cells after treatment with selenomethionine and Se-methylselenocysteine using 800 MHz Bruker NMR spectrometer on 18 cell samples.
Monitoring In Vitro Response of Selenium-Treated Prostate Cells by 1H NMR Spectroscopy
SIRM Analysis of human P493 cells under hypoxia in [U-13C/15N] labeled Glutamine medium (Positive ion mode FTMS)
The development and the validation of innovative approaches for biomarker selection are of paramount importance in many -omics technologies. Unfortunately, the actual testing of new methods on real data is difficult, because in real data sets, one can never be sure about the “true” biomarkers. In this paper, we present a publicly available metabolomic ultra performance liquid chromatography–mass spectrometry spike-in data set for apples. The data set consists of 10 control samples and three spiked sets of the same size (G1,G2,G3), where naturally occurring compounds are added in different concentrations. In this sense, the data set can serve as a test bed to assess the performance of new algorithms (eg. biomarker selection, peak picking, ...) and compare them with previously published results. The dataset includes the raw CDF files for all the injections. The raw data are organized in three assays: positive ion mode, negative ion mode, and injections of the standard mixes. The spiked compounds are: epicatechin (CHEBI:90), catechin (CHEBI:15600), cyanidin-3-galactoside (CHEBI:27475), quercetin-3-galactoside (CHEBI:67486), quercetin-3-glucoside (CHEBI:28299), quercetin-3-rhamnoside (CHEBI:17558), trans-resveratrol (CHEBI:45713), phloridzin (CHEBI:8113), and quercetin (CHEBI:16243). All the compounds except resveratrol and cyanidin-3-galactoside are known to be present in the apple extracts. Spiking concentrations have been defined, with the ones measured in a pooled apple extract taken as a reference. A detailed description of the spiking concentration in the three groups is included in the spiking.pdf file.
A benchmark spike-in data set for biomarker identification in metabolomics
Mining for metabolic responses to long-term salt stress: a case study on Arabidopsis thaliana Col-0 (B)
A metabolomics approach to unravel the regulating role of phytohormones towards carotenoid metabolism in tomato fruit. (A-carotene metabolism)
A collection of biologically-relevant plant metabolite standards. The standards are fully semantically annotated with peak information, ChEBI identifiers, and chemical ontology. The standards were acquired on on a high-resolution LC-MS/MS Orbitrap instrument.
Metabolite Standards for the development and validation of MassCascade
Macrophages are central players in immune response, manifesting divergent phenotypes to control inflammation and innate immunity through release of cytokines and other signaling factors. Recently, the focus on metabolism has been reemphasized as critical signaling and regulatory pathways of human pathophysiology, ranging from cancer to aging, often converge on metabolic responses. Here, we used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features that are critical for macrophage activation. A genome-scale metabolic network for the RAW 264.7 cell line was constructed to determine metabolic modulators of activation. Metabolites well-known to be associated with immunoactivation (glucose and arginine) and immunosuppression (tryptophan and vitamin D3) were among the most critical effectors. Intracellular metabolic mechanisms were assessed, identifying a suppressive role for de-novo nucleotide synthesis. Finally, underlying metabolic mechanisms of macrophage activation were identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. This study demonstrates that the role of metabolism in regulating activation may be greater than previously anticipated and elucidates underlying connections between activation and metabolic effectors. This submission corresponds to the metabolomics data from this study.
Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation
Metabolic profiling of transgenic tomato fruits highly accumulating polyamines by the expression of spermidine synthase gene.
Characterizing the metabolic changes pertaining to hepatocellular carcinoma (HCC) in patients with liver cirrhosis is believed to contribute towards early detection, treatment, and understanding of the molecular mechanisms of HCC. we compare metabolite levels in sera of 78 HCC cases with 184 cirrhotic controls by using ultra performance liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). Several candidate metabolic biomarkers for early detection of HCC cases in high risk population of cirrhotic patients are identified using mass spectrum.
Utilization of Metabolomics to Identify Serum Biomarkers for Hepatocellular Carcinoma in Patients with Liver Cirrhosis
Biomarkers for Depression in Human Cerebrospinal Fluid in a Population Sample
BALF comparison of healthy controls vs patients with Acute Lung Injury.
[U-13C]-Glu SIRM study of H1299 cells with QDA treatment
Blood was separated by low speed (120 g) centrifugation for 15 min. Resulting supernatant (plasma fraction) and pellet (RBC fraction) were collected (each 0.2 ml). Blood was donated four times within 24 hours bysingle person, samples were processed separetely. By measuring metabolites we could determine their distribution between plasma and RBC fractions.
Unexpected similarities between the Schizosaccharomyces and human blood metabolomes, and novel human metabolites (Blood plasma and RBC fractions)
We examined the effect of several commonly-used methods of anesthesia and euthanasia for collection of skeletal muscle, liver, heart, adipose and serum of C57BL/6J mice. The data revealed tissue-specific impacts of different anesthesia and euthanasia strategies. Based on these findings, we present a more optimal collection strategy mammalian tissues and recommend that rodent tissues intended for metabolomics studies be collected under anesthesia rather than post-euthanasia.
Impact of anesthesia and euthanasia on metabolomics of mammalian tissues: studies in a C57BL/6J mouse model
Effet de la teneur en CO2 et de la photopériode sur les métabolites extraits d'Arabidopsis
Schizosaccharomyces pombe metabolome at the onset of nitrogen starvation (1 hour time course)
Metabolomic Analysis of Fission Yeast at the Onset of Nitrogen Starvation
Global Metabolomics of C. elegans using an augmented reference IROA design
Here we present a metabolic profiling strategy employing direct infusion Orbitrap mass spectrometry (MS) and gas chromatography-mass spectrometry (GC/MS) for the monitoring of soybean's (Glycine max L.) global metabolism regulation in response to Rhizoctonia solani infection in a time-course. Key elements in the approach are the construction of a comprehensive metabolite library for soybean, which accelerates the steps of metabolite identification and biological interpretation of results, and bioinformatics tools for the visualization and analysis of its metabolome. The study of metabolic networks revealed that infection results in the mobilization of carbohydrates, disturbance of the amino acid pool, and activation of isoflavonoid, α-linolenate and phenylpropanoid biosynthetic pathways. Components of these pathways include phytoalexins, coumarins, flavonoids, signaling molecules, and hormones, many of which exhibit antioxidant properties and bioactivity helping the plant to counterattack the pathogen's invasion. Unraveling the biochemical mechanism operating during soybean-Rhizoctonia interaction in addition to its significance towards the understanding of the plant's metabolism regulation under biotic stress, provides valuable insights with potential for applications in biotechnology, crop breeding, and agrochemical and food industries.
A Large-scale Metabolic Profiling Strategy for the Dissection of Plant Defense against Fungal Pathogens
The identification and the present wide acceptance of cardiovascular risk factors such as age, sex, hypertension, hyperlipidemia, smoking, obesity, diabetes, and physical inactivity have led to dramatic reductions in cardiovascular morbidity and mortality. However, novel risk predictors present opportunities to identify more patients at risk and to more accurately define the biochemical signature of that risk. In this paper, we present a comprehensive metabonomic analysis of 864 plasma samples from healthy volunteers, through Nuclear Magnetic Resonance (NMR) and multivariate statistical analysis (regression and classification). We have found that subjects that are classified as at high or at low risk using the common clinical markers can also be discriminated using NMR metabonomics. This discrimination is not only due to common markers (such as total cholesterol, triglycerides, LDL, HDL), but also to (p < 0.05 after Bonferroni correction) other metabolites (e.g., 3-hydroxybutyrate, α-ketoglutarate, threonine, dimethylglycine) previously not associated with cardiovascular diseases.
The cardiovascular risk of healthy individuals studied by NMR metabonomics of plasma samples.
Urease pre-treatment of urine has been utilized since the early 1960s to remove high levels of urea from samples prior to further processing and analysis by gas chromatographymass spectrometry (GCMS). Aside from the obvious depletion or elimination of urea, the effect, if any, of urease pre-treatment on the urinary metabolome has not been studied in detail. Here, we report the results of three separate but related experiments that were designed to assess possible indirect effects of urease pre-treatment on the urinary metabolome as measured by GCMS. In total, 235 GCMS analyses were performed and over 106 identified and 200 unidentified metabolites were quantified across the three experiments. The results showed that data from urease pre-treated samples (1) had the same or lower coefficients of variance among reproducibly detected metabolites, (2) more accurately reflected quantitative differences and the expected ratios among different urine volumes, and (3) increased the number of metabolite identifications. Overall, we observed no negative consequences of urease pre-treatment. In contrast, urease pre-treatment enhanced the ability to distinguish between volume-based and biological sample types compared to no treatment. Taken together, these results show that urease pre-treatment of urine offers multiple beneficial effects that outweigh any artifacts that may be introduced to the data in urinary metabolomics analyses.
A statistical analysis of the effects of urease pre-treatment on the measurement of the urinary metabolome by gas chromatographymass spectrometry
To test the quantification performance of MetaboQuant and other NMR quantification tools, a set of eight metabolites were added to replicates of a urine sample in varying concentrations. The metabolites were alanine, creatinine, histidine, betaine, acetate, phosphocreatine, β-hydroxybutyrate (BHBA) and lactate. These were added in concentrations of 6000, 3000, 1500, 750, 375, 188, 94, and 47 micromol/L in a Latin Square design. 2D 1H-13C-HSQC spectra were recorded on a 600 MHz Bruker Avance III spectrometer employing a triple-resonance cryoprobe and z-gradients.
Urine Latin Square Spike-In for Testing MetaboQuant
Metabolomic profiles in short day, after bolting, single leaf analysis, Arabidopsis thaliana
Metabolomic responses to long-term salt stress in related Lotus species (A)
Metabolomics Involved in Early Life Antibiotic Exposures(TranSTAT-Cecal)
Metabolomics analysis was performed on 112 serum samples that were collected in India at baseline (before Cholera vaccination) and 28 days after Cholera vaccination under the Population Genetics Project (National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) contract HHSN2004000676C). The 112 samples were selected based on titer results which indicated subjects who were either high responders or low responders to the vaccination. 950MHz 1H NMR spectra were acquired for the 112 serum samples.
Metabolomics Analysis of Population Genetics (PopGen) for Response to Cholera Vaccination)
Metabolomic profiles in long day, after bolting, single leaf analysis, Arabidopsis thaliana
The potential for commensal microorganisms indigenous to a host (the microbiome or microbiota) to alter infection outcome by influencing host-pathogen interplay is largely unknown. We used a multi-omics systems approach, incorporating proteomics, metabolomics, glycomics, and metagenomics, to explore the molecular interplay between the murine host, the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), and commensal gut microorganisms during intestinal infection with S. Typhimurium. We find proteomic evidence that S. Typhimurium thrives within the infected 129/SvJ mouse gut without antibiotic pre-treatment, inducing inflammation and disrupting the intestinal microbiome (e.g., suppressing Bacteroidetes and Firmicutes while promoting growth of Salmonella and Enterococcus). Alteration of the host microbiome population structure was highly correlated with gut environmental changes, including the accumulation of metabolites normally consumed by commensal microbiota. Finally, the less characterized phase of S. Typhimurium's lifecycle was investigated, and both proteomic and glycomic evidence suggests S. Typhimurium may take advantage of increased fucose moieties to metabolize fucose while growing in the gut. The application of multiple omics measurements to Salmonella-induced intestinal inflammation provides insights into complex molecular strategies employed during pathogenesis between host, pathogen, and the microbiome.
Microbial autotroph-heterotroph interactions influence biogeochemical cycles on a global scale, but the diversity and complexity of natural systems and their intractability to in situ manipulation make it challenging to elucidate the principles governing these interactions. The study of assembling phototrophic biofilm communities provides a robust means to identify such interactions and evaluate their contributions to the recruitment and maintenance of phylogenetic and functional diversity overtime. To examine primary succession in phototrophic communities, we isolated two unicyanobacterial consortia from the microbial mat in HotLake, Washington, characterizing the membership and metabolic function of each consortium. We then analyzed the spatial structures and quantified the community compositions of their assembling biofilms. The consortia retained the same suite of heterotrophic species, identified as abundant members of the mat and assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes. Autotroph growth rates dominated early in assembly, yielding to increasing heterotroph growth rates late in succession. The two consortia exhibited similar assembly patterns, with increasing relative abundances of members from Bacteroidetes and Alphaproteobacteria concurrent with decreasing relative abundances of those from Gamma proteobacteria. Despite these similarities at higher taxonomic levels, the relative abundances of individual heterotrophic species were substantially different in the developing consortial biofilms. This suggests that, although similar niches are created by the cyanobacterial metabolisms, the resulting webs of autotroph-heterotroph and heterotroph-heterotroph interactions are specific to each primary producer. The relative simplicity and tractability of the Hot Lake unicyanobacterial consortia make them useful model systems for deciphering interspecies interactions and assembly principles relevant to natural microbial communities.
Phototrophic biofilm assembly in microbial-mat-derived unicyanobacterial consortia: model systems for the study of autotroph-heterotroph interactions
Metabolic variations occur during normal pregnancy to provide the growing fetus with a supply of nutrients required for its development and to ensure the health of the woman during gestation. Mass spectrometry-based metabolomics was employed to study the metabolic phenotype variations in the maternal plasma that are induced by pregnancy in each of its three trimesters. Nontargeted metabolomics analysis showed that pregnancy significantly altered the profile of metabolites in maternal plasma. The levels of six metabolites were found to change significantly throughout pregnancy, with related metabolic pathway variations observed in biopterin metabolism, phospholipid metabolism, amino acid derivatives, and fatty acid oxidation. In particular, there was a pronounced elevation of dihydrobiopterin (BH2), a compound produced in the synthesis of dopa, dopamine, norepinephrine, and epinephrine, in the second trimester, whereas it was markedly decreased in the third trimester. The turnover of BH2 and tryptophan catabolites indicated that the fluctuations of neurotransmitters throughout pregnancy might reveal the metabolic adaption in the maternal body for the growth of the fetus. Furthermore, 11 lipid classes and 41 carnitine species were also determined and this showed variations in the presence of long-chain acylcarnitines and lysophospholipids in later pregnancy, suggesting changes of acylcarnitines and lysophospholipids to meet the energy demands in pregnant women.
Pregnancy-Induced Metabolic Phenotype Variations in Maternal Plasma
RESV (1 mg in 200 ul saline) was orally administered to hairless mice or 1 mg RESV dissolved in ethanol was applied on the dorsal skin of hairless mice. After 4 h mice were sacrificed, metabolites were extracted from the tissues and analyzed by the LC-MS. Peak areas of metabolites were normalized by the peak areas of spiked internal standards (HEPES and PIPES).
Distribution of RESV and its metabolite peaks in mouse tissues after oral and skin administration
In this study broncho-alveolar lavage fluid (BALF) from mice infected with influenza A virus, PR8 (280 PFU) was analyzed for lipidomic host response in comparison with the mock infected animals. Time Points: 3, 6, 9 and 11 days post infection.
Lipid Mediators of inflammation in BALF 3-11 days after infection with Influenza
In this study, the metabolic response to activation and inhibition of Y2R was studied in the human neuroblastoma cell line SK-N-BE2 was studied using high-resolution nuclear magnetic resonance spectroscopy (NMR). Three treatments were evaluated: (1) activation of Y2R with NPY, (2) blocking of Y2R with BIIE, and (3) treatment of cells with both NPY and BIIE.
NMR based metabonomics study of Y2 Receptor activation by neuropeptide Y in SK-N-BE2, a human neuroblastoma cell line
Most analytical methods in metabolomics are based on one of two strategies. The first strategy is aimed at specifically analysing a limited number of known metabolites or compound classes. Alternatively, an unbiased approach can be used for profiling as many features as possible in a given metabolome without prior knowledge of the identity of these features. Using high-resolution mass spectrometry with instruments capable of measuring m/z ratios with sufficiently low mass measurement uncertainties and simultaneous high scan speeds, it is possible to combine these two strategies, allowing unbiased profiling of biological samples and targeted analysis of specific compounds at the same time without compromises. Such high mass accuracy and mass resolving power reduces the number of candidate metabolites occupying the same retention time and m/z ratio space to a minimum. In this study, we demonstrate how targeted analysis of phospholipids as well as unbiased profiling is achievable using a benchtop orbitrap instrument after high-speed reversed-phase chromatography. The ability to apply both strategies in one experiment is an important step forward in comprehensive analysis of the metabolome.
High-resolution extracted ion chromatography, a new tool for metabolomics and lipidomics using a second-generation orbitrap mass spectrometer
Metabolite changes associated with methionine stress sensitivity of cancer (GC TOF MS analysis)
Metabolomics Involved in Early Life Antibiotic Exposures(EstroSTAT-Urine)
Determination of NPM-ALK regulated global metabolic environment
1H nuclear magnetic resonance metabolic profiles confirmed the impact of the bt2-H2328 mutation on these pathways and revealed that the bt2-H2328 mutation did not only affect the endosperm, but also the embryo at the metabolic level. These data suggest that, in the bt2-H2328 endosperms, regulatory networks are activated that redirect excess carbon into alternative biosynthetic pathways (amino acid synthesis) or into other tissues (embryo).
In this study, GC/LC-MS-based metabolomic analyses in conjuction with multivariate statistics were applied to investigate the effect of exogenous MeJA on the matabolome of Centella asiatica cells. C. asiatica is an important medicinal plant with a wide range of bioactivities associated with its secondary metabolites. Concentration-dependent changes in the metabolite profiles, indicative of the cellular response to MeJA, were observed. Annotated metabolites included a range of sesquiterpenoids that illustrates the stimulatory effect of MeJA on terpenoid pathways. In contrast, flavanoids were mostly negatively correlated with the treatment. The presence of the five bioactive cyclic sesquiterpenoids in MeJA-elicited cells and other annotated metabolites (abscisic acid, fatty acids, phytosterols and metabolites of shikimate-phenylpropanoid pathways) indicates that the changes in the metabolome are associated with a defensive function in response to elicitation by MeJA, rather than just the amplication of existing terpene pathways. These results provided a detailed and comprehensive picture of metabolic changes occurring in C. asiatica cells in response to MeJA elicitation and contribute to the understanding of flexible and controllable aspects of metabolic manipulation
Metabolomics-derived insights into the manipulation of terpenoid sysnthesis in Centella asiatica cells by methyl-jasmonate
Phosphorus limitation is pervasive in the oligotrophic surface ocean and marine microorganisms use different strategies to survive, and thrive, at these low nutrient levels. Eukaryotic algae such as diatoms are extremely sensitive to phosphorus limitation and recent transcriptomics work has suggested that multiple cellular processes are affected under these growth conditions. Metabolomics is the systematic study of intra- and extracellular metabolites, i.e. the end products of microbial metabolism. As such, metabolomics complements genomics and transcriptomics through the identification and quantification of metabolic intermediates that reflect cellular physiology. Here, we applied intracellular metabolomics to examine the differential response of the model diatom Thalassiosira pseudonana to phosphate-replete and phosphate-limited growth conditions. We focused on metabolites from the purine and pyrimidine biochemical pathways, due to their role in phosphorus cycling associated with nucleic acid synthesis. Under phosphate-replete conditions, T. pseudonana stored nucleotides with phosphate moieties such as adenosine 5'-monophosphate (AMP) and, to a lesser extent, inosine 5'-monophosphate (IMP). In contrast, under phosphate-limited conditions, T. pseudonana had higher concentrations of adenine, inosine, and adenosine all of which lack phosphate moieties. Furthermore, based on previously published transcriptomics data, T. pseudonana differentially regulates select genes that can alter these nucleic acid precursors through the gain or loss of the phosphate moiety. Thus, our analysis of the metabolomics and transcriptomics data converged upon the sensitivity of the purine biochemical pathway to phosphorus availability
Tps6: Thalassiosira pseudonana varies accumulation of purine metabolites as a function of phosphate availability
The parental P. acidipropionici and its genome-shuffled mutant were compared via metabolomics to find the key metabolic nodes influencing PA production.
Comparative metabolomics analysis of the key metabolic nodes in propionic acid synthesis in Propionibacterium acidipropionici
Fusarium graminearum and related species commonly infest grains causing the devastating plant disease Fusarium head blight (FHB) and the formation of trichothecene mycotoxins. The most relevant toxin is deoxynivalenol (DON), which acts as a virulence factor of the pathogen. FHB is difficult to control and resistance to this disease is a polygenic trait, mainly mediated by the quantitative trait loci (QTL) Fhb1 and Qfhs.ifa-5A. In this study we established a targeted GC-MS based metabolomics workflow comprising a standardized experimental setup for growth, treatment and sampling of wheat ears and subsequent GC-MS analysis followed by data processing and evaluation of QC measures using tailored statistical and bioinformatics tools. This workflow was applied to wheat samples of six genotypes with varying levels of Fusarium resistance, treated with either DON or water, and harvested 0, 12, 24, 48 and 96 hours after treatment. The results suggest that the primary carbohydrate metabolism and transport, the citric acid cycle and the primary nitrogen metabolism of wheat are clearly affected by DON treatment. Most importantly significantly elevated levels of amino acids and derived amines were observed. In particular, the concentrations of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan increased. No clear QTL specific difference in the response could be observed except a generally faster increase in shikimate pathway intermediates in genotypes containing Fhb1. The overall workflow proved to be feasible and facilitated to obtain a more comprehensive picture on the effect of DON on the central metabolism of wheat.
GC-MS based targeted metabolic profiling identifies changes in the wheat metabolome following deoxynivalenol treatment
A study of changes in lipid metabolism of ovarian cancer cells co-cultured with adipocytes: GC-TOF MS analysis
This study deals with the annotation of the human adult urinary metabolome and metabolite identification from electrospray-mass spectrometry (ESI-MS)based metabolomics datasets. Metabolite identification was achieved using two complementary approaches: (i) formal identification by matching chromatographic retention times, mass spectra and also product ion spectra (if required) of metabolites to be characterized in biological datasets to those of reference compounds, and, (ii) putative identification from biological data thanks to MS/MS experiments for metabolites not available in our chemical library. By these means, 384 metabolites corresponding to 1484 annotated features (659 in negative ion mode and 825 in positive ion mode) were characterized in human urine samples. Of these metabolites, 191 and 67 were formally and putatively identified, respectively.
Annotation of the human adult urinary metabolome and metabolite identification using ultra high performance liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer
Non-diabetic controls whose metabolites were compared to T1D patients with and without insulin. Seven C-peptidenegative T1D subjects were studied on two occasions: one during insulin treatment and the other following withdrawal of insulin for 8 h and compared with matched healthy ND participants
Plasma metabolomics: Comparison of non-diabetic controls with T1D patients
mouse embryonic fibroblasts, embryonic stem cells, and induced pluripotent stem cells were compared via metabolomic analysis
Metabolomic analysis of mouse embryonic fibroblasts, embryonic stem cells, and induced pluripotent stem cells
Cells were deprived of glucose or glutamine for 1 h then resuspended in 10mM [13C1,2] D-glucose labeled or 4 mM [13C5]-U-glutamine labeled medium, respectively. At this time cells were also activated with 5 ug/ml anti-CD3 and anti-CD28. Cells were labeled (and activated) for 12 h when samples were spun down, and cells were resuspended in 200 ul ice cold methanol
Biochemical diversity in three commercially-relevant waxy rice cultivars important to food production in the Lao Peoples Democratic Republic (PDR). Following cultivation with different nitrogen fertiliser regimes, multiple metabolomic data sets, including minerals, were produced and analysed using multivariate statistical methods to reveal the degree of similarity between the genotypes and to identify discriminatory compounds supported by multiple technology platforms.
Mining for metabolic responses to long-term salt stress: a case study on the model legume Lotus japonicus (B)
Targeted metabolomic analysis of bile acids was performed on 15 mouse liver samples collected from mice euthanized at 9 months following consumption of a high fat diet w/o perinatal DDT exposure. Funded by the National Institute of Health (R00 ES019919, R03 DK082724, U24 DK092993, U24 DK097154, T32 ES007059, and P60 DK020541), the American Diabetes Association, and USDA-ARS intramural Project 5306-51530-019-00D. Samples were analyzed by UPLC-MS/MS using a Waters Acquity UPLC and detected on an API 4000 QTrap (AB Sciex, Framingham, MA, USA) by multiple reaction monitoring (MRM) after negative mode electrospray ionization.
Dysfunctional lipid metabolism underlies the effect of perinatal DDT exposure on the development of metabolic syndrome
Lung cancer remains the most common cause of cancer deaths world-wide. Non-invasive diagnostic and prognostic biomarkers that would identify the need for additional diagnostic testing and guide treatment decisions are still lacking. Methods: A first-of-its-kind effort was undertaken to identify noninvasive biomarkers using metabolic profiling of urine samples from 469 lung cancer patients and 536 population controls. Putative biomarkers were technically validated by quantitation at two different time points. Further validation was performed in an independent sample set comprising 158 samples. Results: Four urinary biomarkers for early detection of non-small cell lung cancer (NSCLC) were identified, including N-acetylneuraminic acid, cortisol sulfate, an unidentified metabolite referred to as 561+, and a novel molecule identified as creatine ribose. Intraclass correlation coefficients between 0.82 and 0.99 indicate high measurement reproducibility. Elevated levels in lung cancer patients were confirmed in an independent validation set (P<0.04). The four diagnostic biomarkers are also associated with survival (P<0.02), adjusted for gender, race histology, smoking status, interview year and urine collection time. Importantly, creatine ribose and metabolite 561+ are associated with prognosis in early stage I and II lung cancers (P=0.03 and 0.0006, respectively). Furthermore, creatine ribose and N-acetylneuraminic acid are significantly more abundant in the tumor than in adjacent normal lung tissue (P<0.03). Conclusions: Four robust urinary biomarkers were identified in association with lung cancer diagnosis and prognosis, two of which are linked to tumor metabolism. The results of this research provide basis for future clinical applications related to lung cancer diagnosis, prognosis and therapeutic outcome.
Diagnostic and prognostic utility of urinary metabolites in lung cancer patients
Global approach to characterize changes in metabolic profiles in mesocarp tissues of oil palm fruit during fruit development.
Metabolomics Involved in Early Life Antibiotic Exposures(NOD-Cecal)
Compare metabolomic, phenotypic and genetic diversity across various groups of Africans.
Screening of transgenic tomato plants using a metabolome fingerprinting approach by 1H-NMR ; deutered methanol extract of red ripe fruit
The aqueous extract of yerba mate, a South American tea beverage made from Ilex paraguariensis leaves, has demonstrated bactericidal and inhibitory activity against bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). The gas chromatography-mass spectrometry (GC-MS) analysis of two unique fractions of yerba mate aqueous extract revealed 8 identifiable small molecules in those fractions with antimicrobial activity. For a more comprehensive analysis, a data analysis pipeline was assembled to prioritize compounds for antimicrobial testing against both MRSA and methicillin-sensitive S. aureus using forty-two unique fractions of the tea extract that were generated in duplicate, assayed for activity, and analyzed with GC-MS. As validation of our automated analysis, we checked our predicted active compounds for activity in literature references and with used authentic standards to test for antimicrobial activity. 3,4-dihydroxybenzaldehyde showed the most antibacterial activity against MRSA at low concentrations in our bioassays. In addition, quinic acid and quercetin were identified using random forests analysis and 5-hydroxy pipecolic acid was identified using linear discriminant analysis. We additionally also generated a ranked list of unidentified compounds that may contribute to the antimicrobial activity of yerba mate against MRSA. Here we utilized GC-MS data to implement an automated analysis that resulted in a ranked list of compounds that likely contribute to the antimicrobial activity of aqueous yerba mate extract against MRSA.
Solvent fractions of an aqueous extract of yerba mate (Ilex paraguariensis)
The amount of inorganic carbon represents one of the main environmental factors determining productivity of photoautotrophic organisms. Using the model cyanobacterium Synechocystis sp. PCC 6803, we performed a first metabolome study with cyanobacterial cells shifted from high CO2 (5% in air) into conditions of low CO2 (LC; ambient air with 0.035% CO2). Using gas chromatography-mass spectrometry, 74 metabolites were reproducibly identified under different growth conditions. Shifting wild-type cells into LC conditions resulted in a global metabolic reprogramming and involved increases of, for example, 2-oxoglutarate (2OG) and phosphoenolpyruvate, and reductions of, for example, sucrose and fructose-1,6-bisphosphate. A decrease in Calvin-Benson cycle activity and increased usage of associated carbon cycling routes, including photorespiratory metabolism, was indicated by synergistic accumulation of the fumarate, malate, and 2-phosphoglycolate pools and a transient increase of 3-phosphoglycerate. The unexpected accumulation of 2OG with a concomitant decrease of glutamine pointed toward reduced nitrogen availability when cells are confronted with LC. Despite the increase in 2OG and low amino acid pools, we found a complete dephosphorylation of the PII regulatory protein at LC characteristic for nitrogen-replete conditions. Moreover, mutants with defined blocks in the photorespiratory metabolism leading to the accumulation of glycolate and glycine, respectively, exhibited features of LC-treated wild-type cells such as the changed 2OG to glutamine ratio and PII phosphorylation state already under high CO2 conditions. Thus, metabolome profiling demonstrated that acclimation to LC involves coordinated changes of carbon and interacting nitrogen metabolism. We hypothesize that Synechocystis has a temporal lag of acclimating carbon versus nitrogen metabolism with carbon leading.
Inorganic Carbon Limitation in Cells of the Wild Type and Photorespiratory Mutants of the Cyanobacterium Synechocystis sp. Strain PCC 6803
Metabolomic responses to long-term salt stress in related Lotus species (C)
Use extra plates seeded for workshop to investigate biological variation with no tracer and no treatment
Study of biological variation in PC9 cell culture
Here, metabolome analysis using LC-QTOF-MS was conducted to figure out the metabolome alternations in Arabidopsis overaccumulating and lacking flavonoids.
Metabolome profiling using LC-MS in Arabidopsis overaccumulating and lacking flavonoids
Corynebacterium glutamicum strain ATCC 21831 is a producer of L-arginine that was created by random mutagenesis. It is resistant to the arginine structural analogue canavanine. In order to identify potential bottlenecks in the biosynthetic pathway that leads to this industrially important amino acid, relative metabolite abundances of biosynthetic intermediates were determined in comparison to the type strain ATCC 13032. An extract of U13C-labeled biomass was used as internal standard, to correct for different ionization efficiencies. Metabolites were idientified using the ALLocator web platform.
Comparative LC/MS-based profiling of L-arginine producing, canavanine resistant Corynebacterium glutamicum strain ATCC 21831 and type strain ATCC 13032
A metabolomics approach to unravel the regulating role of phytohormones towards carotenoid metabolism in tomato fruit. (Zeaxanthin metabolism)
The purpose of this study was to highlight metabolic differences between different cultivars of tomato throughout development and ripening.
Metabolic differences in ripening of wild and mutant cultivars of Solanum lycopersicum
Metabolic Profiling of Visceral and Subcutaneous Adipose Tissue from Colorectal Cancer Patients: UHPLC-QTOF MS analyis of serum samples
Metabolomic profiles long day and before bolting, single leaf analysis, Arabidopsis thaliana
Metabolic Profiling of Visceral and Subcutaneous Adipose Tissue from Colorectal Cancer Patients: GC-TOF MS analyis of subcutaneous and visceral adipose tissue samples
Heatshock response of C. elegans using IROA (I)
We demonstrate the global metabolic analysis ofCaenorhabditis elegansstress responses using a mass-spectrometry-based technique called isotopic ratio outlier analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95 and 5%13C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis.To illustrate the utility of IROA for global metabolomics, we exposed wild-type (N2) worms to a heat shock (30 min heat shock at 33 C), which causes significant, widespread changes in metabolism. We collected and analyzed material from the exometabolome (all material that worms release in the supernatant) and the endometabolome (homogenized total extracts from the worm bodies).
Determine purity and quality of IROA labelled glucose
In this study, metabolomic analysis with multivariate statistics was performed to examine the serum metabolic changes in human participants, which are divided into three groups (So-Yang, So-Eum and Tae-Eum) according to Sasang Constitution Medicine (SCM), Korean Traditional Oriental Medicine, that has bean an important tool in diagnosis and treatment of patients.
UPLC-Q/TOF-MS based metabolomics of Human Serum
Selection of the optimal injection solvent composition for the analysis of cassava extracts with LC-DAD.
Metabolite profiling in LC-DAD using multivariate curve resolution: the alsace package in R
Microbial alteration and remineralization of organic matter in the ocean play essential roles in the carbon cycle. The impact of individual compounds as either growth substrates or infochemicals is still poorly constrained in models, particularly for organisms with relatively versatile genomes. Here, we use metabolomics techniques to characterize the response of a heterotrophic marine bacterium, Ruegeria pomeroyi DSS-3, to growth on the abundant algal metabolite dimethylsulfoniopropionate (DMSP). DMSP is a valuable source of reduced carbon and sulfur as well as a chemo-attractant. It is present at higher concentrations during phytoplankton blooms. When cultivated on DMSP, R. pomeroyi synthesized a quorum sensing molecule, N-(3-oxotetradecanoyl)-L-homoserine lactone, which was not synthesized at the same levels during growth on propionate, another carbon substrate. More broadly, we observed differential production of intra- and extracellular metabolites with implications for nitrogen assimilation and microbial cross-feeding. This multi-faceted response suggests that R. pomeroyi uses a two-component regulatory system dependent on the presence of DMSP and high cell densities to activate a suite of metabolic changes that may shape how it accesses nutrients and impacts the surrounding microbial consortium.
Evidence for quorum sensing and differential metabolite production by Ruegeria pomeroyi in response to DMSP
Metabolomics analysis of multiple metabolic functions in the YjgF/YER057c/UK114 (Rid) protein family LCMS
Comparison of caloric restriction vs medications that prolong life
This application requests funding to access state-of-the-art metabolomics and lipidomic platforms at the NIH West Coast Metabolomics Center to analyze plasma samples from recent insulin and glucagon-like peptide-1 (GLP-1) infusion experiments performed in prolong-fasted elephant seals. This suite of studies was designed to better assess the mechanisms contributing to the onset of an insulin resistantlike condition induced by prolonged food deprivation/starvation in mammals. Because elephant seals have evolved robust physiological mechanisms that have allowed them to naturally tolerate such protracted bouts of fasting, they provide an ideal model to address our central hypothesis that increased lipid utilization late in the fast contributes to insulin resistance in elephant seals. Insulin resistance is a common consequence of fasting in mammals and, while the mechanisms by which it manifests are still unclear, a metabolic shift favoring increased mobilization and utilization of lipids during prolonged food deprivation may be a principal causative factor. Insulin resistance has a negative connotation due to its association with obesity and diabetes among humans, but it has been suggested to be an adaptive response to food deprivation.
Metabolomic & lipidomic profiles in response to exogenous insulin & GLP-1 infusions during prolonged fasting (GCMS)
Metabolomics Involved in Early Life Antibiotic Exposures(DuraSTAT-Liver)
In this study, seventeen white wines including Chardonnays, Viogniers, Pinot gris, Rieslings and Sauvignon blancs (which were part of a M.S. study in the Viticulture & Enology Department on white wine mouthfeel properties), were analyzed by GC-TOF. Additionally, chemical data obtained will be mined with the sensory data collected to further investigate the chemical basis for mouthfeel properties in wine.