PeakForest: a multi-platform digital infrastructure for interoperable metabolite spectral data and metadata management.

INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .

Untargeted plasma metabolomic profiles associated with overall diet in women from the SU.VI.MAX cohort.

PURPOSE: Dietary intakes are reflected in plasma by the presence of hundreds of exogenous metabolites and variations in endogenous metabolites. The exploration of diet-related plasma metabolic profiles could help to better understand the impact of overall diet on health. Our aim was to identify metabolomic signatures reflecting overall diet in women from the French general population. METHODS: This cross-sectional study included 160 women in the SU.VI.MAX cohort with detailed dietary data (≥ 10 24-h dietary records) selected according to their level of adherence to the French dietary recommendations, represented by the validated score mPNNS-GS; 80 women from the 10th decile of the score were matched with 80 women from the 1st decile. Plasma metabolomic profiles were acquired using untargeted UPLC-QToF mass spectrometry analysis. The associations between metabolomic profiles and the mPNNG-GS, its components and Principal Component Analyses-derived dietary patterns were investigated using multivariable conditional logistic regression models and partial correlations. RESULTS: Adherence to the dietary recommendations was positively associated with 3-indolepropionic acid and pipecolic acid (also positively associated with fruit and vegetable intake and a healthy diet)-2 metabolites linked to microbiota and inversely associated with lysophosphatidylcholine (LysoPC(17:1)), acylcarnitine C9:1 (also inversely associated with a healthy diet), acylcarnitine C11:1 and 2-deoxy-D-glucose. Increased plasma levels of piperine and Dihydro4mercapto-3(2H) furanone were observed in women who consumed a Western diet and a healthy diet, respectively. Ethyl-ß-D-glucopyranoside was positively associated with alcohol intake. Plasma levels of LysoPC(17:1), cholic acid, phenylalanine-phenylalanine and phenylalanine and carnitine C9:1 decreased with the consumption of vegetable added fat, sweetened food, milk and dairy products and fruit and vegetable intakes, respectively. CONCLUSION: This study highlighted several metabolites from both host and microbial metabolism reflecting the long-term impact of the overall diet. TRIAL REGISTRATION: SU.VI.MAX, clinicaltrials.gov NCT00272428. Registered 3 January 2006, https://clinicaltrials.gov/show/NCT00272428.

Intracerebral synthesis of glutamine from hyperpolarized glutamate.

PURPOSE: Changes in glutamate (Glu) levels occur in a number of neurodegenerative diseases. We proposed the use of 13 C spectroscopy and the highly amplified signal generated by hyperpolarization to achieve spatial and temporal resolutions adequate for in vivo studies of Glu metabolism in the healthy rat brain. Thus, we investigated uptake of hyperpolarized [1-13C ]Glu after a temporary blood-brain barrier (BBB) disruption protocol and its conversion to glutamine (Gln) in the brain. METHODS: [1-13 C]Glu was hyperpolarized using the dynamic nuclear polarization process. A temporary BBB disruption using mannitol allowed hyperpolarized [1-13 C]Glu to reach the brain. Then, hyperpolarized [1-13 C]Glu brain metabolism was observed in vivo by MR spectroscopy experiments at 3T. Products synthesized from [1-13 C]Glu were assigned via liquid chromatography-mass spectrometry. RESULTS: Hyperpolarized [1-13 C]Glu reached 20% ± 2.3% polarization after 90 min. After validation of the BBB disruption protocol, hyperpolarized [1-13 C]Glu (175.4 ppm) was detected inside the rat brain, and the formation of [1-13 C]Gln at 174.9 ppm was also observed. CONCLUSION: The Gln synthesis from hyperpolarized [1-13 C]Glu can be monitored in vivo in the healthy rat brain after opening the BBB. Magn Reson Med 78:1296-1305, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Metabolomic study of the response to cold shock in a strain of Pseudomonas syringae isolated from cloud water.

INTRODUCTION: Active microorganisms have been recently discovered in clouds, thus demonstrating the capacity of microorganisms to exist in harsh environments, including exposure to UV and oxidants, osmotic and cold shocks, etc. It is important to understand how microorganisms respond to and survive such stresses at the metabolic level. OBJECTIVES: The objective of this work is to assess metabolome modulation in a strain of Pseudomonas syringae isolated from cloud water and facing temperature downshift from 17 to 5 °C by identifying key molecules and pathways of the response/adaptation to cold shock. METHODS: Bacterial extracts from suspensions of cells grown at 17 °C and further incubated in microcosms at 5 and 17 °C to mimic cloud conditions were analysed by combining LC-MS and NMR; the results were evaluated in comparison to similar suspensions kept at constant temperature. The differences in the metabolome profiles were deciphered using multivariate statistics (PLS-DA). RESULTS: Key cold shock biomarkers were observed, including cryoprotectants (trehalose, glucose, glycerol, carnitine, glutamate), antioxidants (glutathione and carnitine) and their precursors, alkaloids (bellendine and slaframine) and metabolites involved in energy metabolism (ATP, carbohydrates). Furthermore, new short peptides (nine dipeptides and a tetrapeptide) were found that have no known function. CONCLUSIONS: This study shows that in response to cold temperatures, Pseudomonas syringae PDD-32b-74 demonstrates numerous metabolism modifications to counteract the impacts of low temperatures.

Untargeted metabolomic insights into plastisphere communities in European rivers.

Every year, rivers introduce a staggering amount of hundred kilotons of plastic into the Oceans. This plastic is inhabited by microorganisms known as the plastisphere, which can be transferred between different ecosystems through the transport of microplastics. Here, we simulated the microbial colonization of polyethylene-based plastic pellets that are classically used to manufacture large-scale plastic products. The pellets were immersed for 1 month in four to five sampling stations along the river-to-sea continuum of nine of the major European rivers. This study presents the first untargeted metabolomics analysis of the plastisphere, by using ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The plastisphere metabolomes were similar in the Rhine and Rhone rivers, while being different from the Tiber and Loire rivers, which showed greater similarity to the Thames and Seine rivers. Interestingly, we found a clear distinction between plastisphere metabolomes from freshwater and marine water in most of the river-to-sea continuum, thus suggesting a complete segregation in plastisphere metabolites that is not consistent with a major transfer of microorganisms between the two contrasted ecosystems. Putative annotations of 189 discriminating metabolites suggested that lipid metabolism was significantly modulated. These results enlightened the relevance of using environmental metabolomic as complementary analysis to the current OMICs analysis.

Validation of a LC-MS/MS method for the quantitative analysis of four antibiotics in pig tissues and plasma to assess the risk of transfer of residues to edible matrices after exposure to cross-contaminated feed.

Cross-contamination between medicated and non-medicated feed can occur during production, processing, transport or storage of animal feed. This may lead to the presence of low concentrations of antibiotics in supposedly drug-free feed for food production animals, which potentially could also harm consumers due to residues. In addition, consumption of sub-therapeutic concentrations of antibiotics may increase the risk of emergence of resistant bacteria. In this study, LC-MS/MS methods were developed to quantify four antibiotics (sulfadimethoxine, oxytetracycline, trimethoprim and amoxicillin) in several pig matrices, i.e. plasma, muscle, liver, kidneys and faeces. All methods were validated using the accuracy profile, except for amoxicillin in faeces, for which extraction could not be optimised for low concentrations. These methods were then applied as part of an animal study during which several pigs received contaminated feed at a concentration corresponding to 2% of therapeutic dose, in order to evaluate the risk of the presence of residues in animal faeces and tissues. The results showed that sulfadimethoxine is well absorbed and accumulates in the muscle, kidneys and liver, where concentrations were higher than the maximum residue limits (MRLs) authorised in EU legislation. Conversely, oxytetracycline was mostly found in faeces as its oral absorption is very low. Trimethoprim concentrations were slightly higher than the tolerated MRL in the kidneys, but they were below this level in the other tissues. Finally, amoxicillin concentrations remained below the lower limit of quantification of the methods in all matrices.

A liquid chromatography coupled to tandem mass spectrometry method for the quantification of spiramycin and its active metabolite neospiramycin in milk of major and minor species: Validation using the accuracy profile.

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of residues of spiramycin, a macrolide antibiotic, and its active metabolite neospiramycin in cow's milk as well as in minor species 'milk, goat and ewe. Spiramycin-d3 was used as internal standard for quantification of both analytes. This analytical method was validated using a global accuracy profile as a graphical decision tool built according to the trueness and the precision of the method. A unique and optimal linear model with logarithm transformation (with a determination coefficient of 0.9991) allowed the measurement of both analytes in the milks of the three animal species, in a wide range from 0.2 to 10 times the Maximal Residue Limit (MRL) (40-2000 µg.kg-1). The limits of detection and quantification were 13 µg.kg-1 and 40 µg.kg-1, respectively. The accuracy profile was established to get 80% of future measurements in routine assays that will fall within the acceptance limits. Trueness of the method, expressed as relative bias, was comprised between -1.6% and 5.7% over the whole range of concentrations. The mean relative standard deviation for repeatability and intermediate precision were comprised between 1.1% and 2.7%; 2.5 and 4.2%, respectively, in all levels of concentration for the three milks. Moreover, a two-order polynomial function was used to model the relative expanded uncertainty with a determination coefficient of 0.834. This function aimed to determine the uncertainty of the future quantifications within the validated dosing range. Overall, the global accuracy profile highlighted the reliability of the method for the routine assays of spiramycin and neospiramycin even in milk from minor species (goat, ewe) by using the most accessible milk (often from cow), while guaranteeing a very high proportion of samples within the fixed acceptance limits. The applicability of this method was tested during a depletion study of spiramycin and neospiramycin in the milk of cow, goat and ewe. The developed analytical method will be useful to assess the distribution profile of the antibiotic and its metabolite in milk of minor species where few studies are available.

Study of in vitro interaction between Fusarium verticillioides and Streptomyces sp. using metabolomics.

The Streptomyces sp. strain AV05 isolated from an organic amendment was found to impact both growth and fumonisin production of Fusarium verticillioides during in vitro direct confrontation. In order to investigate the interactions between the Streptomyces sp. strain AV05 and F. verticillioides, a metabolomic approach was used. The study of the endometabolomes of the microorganisms was carried out in two different conditions: the microorganisms were cultivated alone or in confrontation. The aim of this study was to examine the modifications of the endometabolome of F. verticillioides in confrontation with the Streptomyces strain. The metabolites involved in these modifications were identified using 2D NMR. Many metabolites were found to be overproduced in confrontation assays with the Streptomyces strain, notably 16 proteinogenic amino acids, inosine, and uridine. This suggested that fungal metabolic pathways such as protein synthesis have been affected due to interaction. Thus, metabolomic studies, as well as proteomics or transcriptomics, are useful for deciphering the mechanisms of interactions between biological control agents and mycotoxigenic fungi. This comprehension is one of the key elements of the improvement of the selection and use of antagonistic agents.

Diet-Related Metabolomic Signature of Long-Term Breast Cancer Risk Using Penalized Regression: An Exploratory Study in the SU.VI.MAX Cohort.

BACKGROUND: Diet has been recognized as a modifiable risk factor for breast cancer. Highlighting predictive diet-related biomarkers would be of great public health relevance to identify at-risk subjects. The aim of this exploratory study was to select diet-related metabolites discriminating women at higher risk of breast cancer using untargeted metabolomics. METHODS: Baseline plasma samples of 200 incident breast cancer cases and matched controls, from a nested case-control study within the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) cohort, were analyzed by untargeted LC-MS. Diet-related metabolites were identified by partial correlation with dietary exposures, and best predictors of breast cancer risk were then selected by Elastic Net penalized regression. The selection stability was assessed using bootstrap resampling. RESULTS: 595 ions were selected as candidate diet-related metabolites. Fourteen of them were selected by Elastic Net regression as breast cancer risk discriminant ions. A lower level of piperine (a compound from pepper) and higher levels of acetyltributylcitrate (an alternative plasticizer to phthalates), pregnene-triol sulfate (a steroid sulfate), and 2-amino-4-cyano butanoic acid (a metabolite linked to microbiota metabolism) were observed in plasma from women who subsequently developed breast cancer. This metabolomic signature was related to several dietary exposures such as a "Western" dietary pattern and higher alcohol and coffee intakes. CONCLUSIONS: Our study suggested a diet-related plasma metabolic signature involving exogenous, steroid metabolites, and microbiota-related compounds associated with long-term breast cancer risk that should be confirmed in large-scale independent studies. IMPACT: These results could help to identify healthy women at higher risk of breast cancer and improve the understanding of nutrition and health relationship.

Metabolic modulations of Pseudomonas graminis in response to H2O2 in cloud water.

In cloud water, microorganisms are exposed to very strong stresses especially related to the presence of reactive oxygen species including H2O2 and radicals, which are the driving force of cloud chemistry. In order to understand how the bacterium Pseudomonas graminis isolated from cloud water respond to this oxidative stress, it was incubated in microcosms containing a synthetic solution of cloud water in the presence or in the absence of H2O2. P. graminis metabolome was examined by LC-MS and NMR after 50 min and after 24 hours of incubation. After 50 min, the cells were metabolizing H2O2 while this compound was still present in the medium, and it was completely biodegraded after 24 hours. Cells exposed to H2O2 had a distinct metabolome as compared to unexposed cells, revealing modulations of certain metabolic pathways in response to oxidative stress. These data indicated that the regulations observed mainly involved carbohydrate, glutathione, energy, lipid, peptides and amino-acids metabolisms. When cells had detoxified H2O2 from the medium, their metabolome was not distinguishable anymore from unexposed cells, highlighting the capacity of resilience of this bacterium. This work illustrates the interactions existing between the cloud microbial metabolome and cloud chemistry.

Plasma Metabolomic Signatures Associated with Long-term Breast Cancer Risk in the SU.VI.MAX Prospective Cohort.

BACKGROUND: Breast cancer is a major cause of death in occidental women. The role of metabolism in breast cancer etiology remains unclear. Metabolomics may help to elucidate novel biological pathways and identify new biomarkers to predict breast cancer long before symptoms appear. The aim of this study was to investigate whether untargeted metabolomic signatures from blood draws of healthy women could contribute to better understand and predict the long-term risk of developing breast cancer. METHODS: A nested case-control study was conducted within the SU.VI.MAX prospective cohort (13 years of follow-up) to analyze baseline plasma samples of 211 incident breast cancer cases and 211 matched controls by LC/MS. Multivariable conditional logistic regression models were computed. RESULTS: A total of 3,565 ions were detected and 1,221 were retained for statistical analysis. A total of 73 ions were associated with breast cancer risk (P < 0.01; FDR ≤ 0.2). Notably, we observed that a lower plasma level of O-succinyl-homoserine (OR = 0.70, 95%CI = [0.55-0.89]) and higher plasma levels of valine/norvaline [1.45 (1.15-1.83)], glutamine/isoglutamine [1.33 (1.07-1.66)], 5-aminovaleric acid [1.46 (1.14-1.87)], phenylalanine [1.43 (1.14-1.78)], tryptophan [1.40 (1.10-1.79)], γ-glutamyl-threonine [1.39 (1.09-1.77)], ATBC [1.41 (1.10-1.79)], and pregnene-triol sulfate [1.38 (1.08-1.77)] were associated with an increased risk of developing breast cancer during follow-up.Conclusion: Several prediagnostic plasmatic metabolites were associated with long-term breast cancer risk and suggested a role of microbiota metabolism and environmental exposure. IMPACT: After confirmation in other independent cohort studies, these results could help to identify healthy women at higher risk of developing breast cancer in the subsequent decade and to propose a better understanding of the complex mechanisms involved in its etiology.

Effectiveness of Interventions to Modulate the Rumen Microbiota Composition and Function in Pre-ruminant and Ruminant Lambs.

Modulating the assembly of the ruminal microbiota might have practical implications in production. We tested how an early-life dietary intervention in lambs influences the diversity and function of the ruminal microbiota during and after the intervention. Microbiota resilience during a repeated dietary intervention was also tested. The treatment, aiming to mitigate enteric methane emissions, combined garlic essential oil and linseed oil. Fifty-six lambs and their dams were allocated to two groups and treatment (T1) or placebo (C1) was drenched from birth until 10 weeks of life. Lambs were weaned at 8 weeks. From 16 to 20 weeks, lambs in each group were divided in two subgroups that received (T1-T2 and C1-T2) or not (T1-C2 and C1-C2) the same treatment. Measurements were done at 8, 14, and 20 weeks. Average daily gain was similar between groups. Methane production was reduced by treatment at 8 and 20 weeks but at 14 weeks it was similar between C1 and T1. Interestingly, early-life treated lambs displayed a numerical increase (P = 0.12) in methane emissions at 20 weeks compared with non-treated lambs. Concentration of VFA was not affected by the intervention at 8 or 14 weeks but a lower concentration was observed in T2 lambs compared with C2 at week 20. Metataxonomics (rRNA gene) revealed differences in archaeal communities between groups of lambs when treatment was applied (weeks 8 and 20); whereas, in accord with methane emissions, these differences disappeared when treatment was discontinued (week 14). Protozoal community structure was not affected by treatment. In contrast, bacterial community structure differed between treated and non-treated lambs during and after the intervention. Rumen and urine LC-MS and NMR metabolomics at week 20 separated C2 from T2 lambs and correlation analysis highlighted interactions between microbes and metabolites, notably that of methylated compounds and Methanomassiliicocceae methanogens. This study demonstrates that a long-term early-life intervention induced modifications in the composition of the rumen bacterial community that persisted after the intervention ceased with little or no effect on archaeal and protozoal communities. However, there was no persistency of the early-life intervention on methanogenesis indicating resilience for this function.