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N6-Methyladenosine (m6A), one of the most abundant internal modification of eukaryotic mRNAs, participates in the post-transcriptional control of gene expression through recruitment of specific m6A readers. In Saccharomyces cerevisiae, the m6A methyltransferase Ime4 is expressed only during meiosis and its deletion impairs this process. To elucidate how m6A control gene expression, we investigated the function of the budding yeast m6A reader Pho92. We show that Pho92 is an early meiotic factor that promotes timely meiotic progression. High-throughput RNA sequencing and mapping of Pho92-binding sites following UV-crosslinking reveal that Pho92 is recruited to specific mRNAs in an m6A-dependent manner during the meiotic prophase, preceding their down-regulation. Strikingly, point mutations altering m6A sites in mRNAs targeted by Pho92 are sufficient to delay their down-regulation and, in one case, to slow down meiotic progression. Altogether, our results indicate that Pho92 facilitate the meiotic progression by accelerating the down-regulation of timely-regulated mRNAs during meiotic recombination.mRNAs molecules carry information contained in genes to direct the formation of proteins. In specific circumstances, the cellular machinery modifies some mRNAs through the formation of m6A residues. To understand the function of these m6A marks, the authors used the yeast Saccharomyces cerevisiae in which their formation only occurs during meiosis that leads to spore formation. Characterization of the Pho92 protein that specifically recognizes m6A residues revealed its importance for meiosis. m6A sites bound by Pho92 were identified and shown to be biologically functional. Unexpectedly, Pho92 was found to regulate an early step of meiosis by controlling DNA recombination. Overall, this study provides important clues on the role of m6A residues in mRNAs.

SUMMARYN6-methyladenosine (m6A), the most abundant internal modification of eukaryotic mRNAs, participates in the post-transcriptional control of gene expression. In Saccharomyces cerevisiae, m6A is only found during meiosis. Although the deletion of the m6A- methyltransferase Ime4 impairs this process, the molecular impact of m6A on gene expression remains ill defined. Here we investigated the function of the budding yeast m6A reader Pho92. We found that Pho92 is specifically expressed during meiosis and impacts meiotic progression. We used high-throughput RNA sequencing and mapping of Pho92-binding sites following UV-crosslinking to show that Pho92 is recruited to specific mRNAs in an m6A-dependent manner during the meiotic prophase, preceding their down-regulation. Strikingly, point mutations altering m6A sites in mRNAs targeted by Pho92 are sufficient to delay their down-regulation and, in one case, to impact meiotic progression. Altogether, our results indicate that Pho92 facilitate the meiotic progression by accelerating the down-regulation of timely-regulated mRNAs during meiotic recombination.

In human society, there is a demand for sustainable solutions for water preservation and efficient treatment systems. An important water reservoir is road runoff defined as rainwater leaching from roads, loading micropollutants and infiltrating the soil. We aimed to study this poorly understood feature using large-scale metabolomic analysis coupled with analysis of soil physico-chemical properties and molecular chemical similarity enrichment. A total of 2406 micropollutants were assayed to understand their distribution and assess the trapping abilities of a road runoff infiltration system composed of a sedimentation pond and an infiltration pond. Here, we confirm the essential role of the infiltration pond in preventing environmental contamination and propose a model correlating micropollutant abundance and the soil physico-chemical properties. We demonstrate that sand in infiltration ponds is a key player, helping retain 86% of the micropollutant abundance and propose a model that could be easily applied for road runoff management.

Jasmonate signaling for adaptative or developmental responses generally relies on an increased synthesis of the bioactive hormone jasmonoyl-isoleucine (JA-Ile), triggered by environmental or internal cues. JA-Ile is embedded in a complex metabolic network whose upstream and downstream components strongly contribute to hormone homeostasis and activity. We previously showed that JAO2, an isoform of four Arabidopsis JASMONIC ACID OXIDASES, diverts the precursor jasmonic acid (JA) to its hydroxylated form HO-JA to attenuate JA-Ile formation and signaling. Consequently, JAO2-deficient lines have elevated defenses and display improved tolerance to biotic stress. Here we further explored the organization and regulatory functions of the JAO pathway. Suppression of JAO2 enhances the basal expression of nearly 400 JA-regulated genes in unstimulated leaves, many of which being related to biotic and abiotic stress responses. Consistently, non-targeted metabolomic analysis revealed the constitutive accumulation of several classes of defensive compounds in jao2-1 mutant, including indole glucosinolates and breakdown products. The most differential compounds were agmatine phenolamides, but their genetic suppression did not alleviate the strong resistance of jao2-1 to Botrytis infection. Furthermore, jao2 alleles and a triple jao mutant exhibit elevated survival capacity upon severe drought stress. This latter phenotype occurs without recruiting stronger abscisic acid responses, but relies on enhanced JA-Ile signaling directing a distinct survival pathway with MYB47 transcription factor as a candidate mediator. Our findings reveal the selected spectrum of JA responses controlled by the JAO2 regulatory node and highlight the potential of modulating basal JA turnover to pre-activate mild transcriptional programs for multiple stress resilience.

Nitrate, one of the main nitrogen (N) sources for crops, acts as a nutrient and key signaling molecule coordinating gene expression, metabolism and various growth processes throughout the plant life cycle. It is widely accepted that nitrate-triggered developmental programs cooperate with hormone synthesis and transport, to finely adapt plant architecture to N availability. Here, we report that nitrate, acting through its signaling pathway, promotes growth in Arabidopsis and wheat, in part by modulating the accumulation of gibberellin (GA)-regulated DELLA growth repressors. We show that nitrate reduces the abundance of DELLAs by increasing GA contents through activation of GA metabolism gene expression. Consistently, the growth restraint conferred by nitrate deficiency is partially rescued in global-DELLA mutant that lacks all DELLAs. At the cellular level, we show that nitrate enhances both cell proliferation and elongation in a DELLA-dependent and -independent manner, respectively. Our findings establish a connection between nitrate and GA signaling pathways that allow plants to adapt their growth to nitrate availability.

The RNA exosome is a key 3’−5’ exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans., Cytosolic RNA degradation by the RNA exosome requires the Ski complex. Here the authors show that the proteins RST1 and RIPR assist the RNA exosome and the Ski complex in RNA degradation, thereby preventing the production of secondary siRNAs from endogenous mRNAs.

Increased metabolism is one of the main causes for evolution of herbicide resistance in weeds, a major challenge for sustainable food production. The molecular drivers of this evolution are poorly understood. We tested here the hypothesis that a suitable context for the emergence of herbicide resistance could be provided by plant enzymes with high innate promiscuity with regard to their natural substrates. A selection of yeast-expressed plant cytochrome P450 enzymes with well documented narrow to broad promiscuity when metabolizing natural substrates was tested for herbicide metabolism competence. The positive candidate was assayed for capacity to confer herbicide tolerance in Arabidopsis thaliana. Our data demonstrate that Arabidopsis thaliana CYP706A3, with the most promiscuous activity on monoterpenes and sesquiterpenes for flower defence, can also oxidize plant microtubule assembly inhibitors, dinitroanilines. Ectopic overexpression of CYP706A3 confers dinitroaniline resistance. We show, in addition, that the capacity to metabolize dinitroanilines is shared by other members of the CYP706 family from plants as diverse as eucalyptus and cedar. Supported by three-dimensional (3D) modelling of CYP706A3, the properties of enzyme active site and substrate access channel are discussed together with the shared physicochemical properties of the natural and exogenous substrates to explain herbicide metabolism.

To investigate mechanisms by which hibernators avoid atherogenic hyperlipidemia during hibernation, we assessed lipoprotein and cholesterol metabolisms of free-ranging Scandinavian brown bears (Ursus arctos). In winter- and summer-captured bears, we measured lipoprotein sizes and sub-classes, triglyceride-related plasma-enzyme activities, and muscle lipid composition along with plasma-levels of antioxidant capacities and inflammatory markers. Although hibernating bears increased nearly all lipid levels, a 36%-higher cholesteryl-ester transfer-protein activity allowed to stabilize lipid composition of high-density lipoproteins (HDL). Levels of inflammatory metabolites, i.e., 7-ketocholesterol and 11ß-prostaglandin F2α, declined in winter and correlated inversely with cardioprotective HDL2b-proportions and HDL-sizes that increased during hibernation. Lower muscle-cholesterol concentrations and lecithin-cholesterol acyltransferase activity in winter suggest that hibernating bears tightly controlled peripheral-cholesterol synthesis and/or release. Finally, greater plasma-antioxidant capacities prevented excessive lipid-specific oxidative damages in plasma and muscles of hibernating bears. Hence, the brown bear manages large lipid fluxes during hibernation, without developing adverse atherogenic effects that occur in humans and non-hibernators.

International audience; Sulfur metabolism in fuel-biodesulfurizing bacteria and the underlying physiological adaptations are not understood, which has impeded the development of a commercially viable bioprocess for fuel desulfurization. To fill these knowledge gaps, we performed comparative proteomics and untargeted metabolomics in cultures of the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 grown on either inorganic sulfate or the diesel-borne organosulfur compound dibenzothiophene as a sole sulfur source. Dibenzothiophene significantly altered the biosynthesis of many sulfur metabolism proteins and metabolites in a growth phase-dependent manner, which enabled us to reconstruct the first experimental model for sulfur metabolism in a fuel-biodesulfurizing bacterium. All key pathways related to assimilatory sulfur metabolism were represented in the sulfur proteome, including uptake of the sulfur sources, sulfur acquisition, and assimilatory sulfate reduction, in addition to biosynthesis of key sulfur-containing metabolites such as S-adenosylmethionine, coenzyme A, biotin, thiamin, molybdenum cofactor, mycothiol, and ergothioneine (low-molecular weight thiols). Fifty-two proteins exhibited significantly different abundance during at least one growth phase. Sixteen proteins were uniquely detected and 47 proteins were significantly more abundant in the dibenzothiophene culture during at least one growth phase. The sulfate-free dibenzothiophene-containing culture reacted to sulfate starvation by restricting sulfur assimilation, enforcing sulfur-sparing, and maintaining redox homeostasis. Biodesulfurization triggered alternative pathways for sulfur assimilation different from those operating in the inorganic sulfate culture. Sulfur metabolism reprogramming and metabolic switches in the dibenzothiophene culture were manifested in limiting sulfite reduction and biosynthesis of cysteine, while boosting the production of methionine via the cobalamin-independent pathway, as well as the biosynthesis of the redox buffers mycothiol and ergothioneine. The omics data underscore the key role of sulfur metabolism in shaping the biodesulfurization phenotype and highlight potential targets for improving the biodesulfurization catalytic activity via metabolic engineering. IMPORTANCE For many decades, research on biodesulfurization of fossil fuels was conducted amid a large gap in knowledge of sulfur metabolism and its regulation in fuel-biodesulfurizing bacteria, which has impeded the development of a commercially viable bioprocess. In addition, lack of understanding of biodesulfurization-associated metabolic and physiological adaptations prohibited the development of efficient biodesulfurizers. Our integrated omics-based findings reveal the assimilatory sulfur metabolism in the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 and show how sulfur metabolism and oxidative stress response were remodeled and orchestrated to shape the biodesulfurization phenotype. Our findings not only explain the frequently encountered low catalytic activity of native fuel-biodesulfurizing bacteria but also uncover unprecedented potential targets in sulfur metabolism that could be exploited via metabolic engineering to boost the biodesulfurization catalytic activity, a prerequisite for commercial application.

Wastewater is one of the major sources of micropollutant release into the environment. In order to reduce the impact of wastewater, wastewater treatment plants (WWTP) have been set up, in the instance of vertical flow constructed wetlands (VFCWs). Besides, micropollutants could represent a vast diversity of compounds and compound's choice could bias studies focused on their fate. To overcome this bias, non-targeted screening approaches can be performed. Therefore, the diffusion of micropollutants from raw wastewater in the VFCW compartments (wastewater, plants and sludge) as well as their fate have been investigated using this non-target approach with liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) and gas chromatography (GC) coupled to mass spectrometry. To help the operators in their sludge management, this study will be focused on the following question: Is there a specific distribution of micropollutants according to sludge layers? To eliminate the background contamination found both inside the CW and in the surrounding environment, a control coring was performed in bank. A specific distribution could be observed in the top (191 compounds) and bottom layers (38 compounds). However, a distribution over the whole depth for xenobiotics was observed. Micropollutants classes and the main microbial productivity were preferably found in the top layer. The micropollutants fate could however not be restricted to the sludge compartment. Therefore, the specific micropollutants distribution was analyzed in the outputs of the system in their interactions with wastewater (effluent, sludge, and reed rhizomes) to understand their fate. In our study, the results highlighted a consistent part of compounds found in at least two or three of these compartments, with a similar trend in each compartment. These results underline the interactions between the compartments and the global issues of micropollutants distribution as well as its wide spreading in the whole CW ecosystem.

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii., SCOPUS: ar.j, info:eu-repo/semantics/published

Fragile X syndrome (FXS), the leading cause of familial intellectual disability, is an uncured disease caused by the absence or loss of function of the FMRP protein. FMRP is an RNA binding protein that controls the translation of specific proteins in neurons. A main target of FMRP in neurons is diacylglycerol kinase kappa (DGKk) and the loss of FMRP leads to a loss of DGK activity causing a diacylglycerol excess in the brain. Excessive diacylglycerol signaling could be a significant contributor to the pathomechanism of FXS. Here we tested the contribution of DAG-signaling inFmr1-KO mouse model of FXS and we show that pioglitazone, a widely prescribed drug for type 2 diabetes, has ability to correct excessive DAG signaling in the brain and rescue behavioral alterations of theFmr1-KO mouse. This study highlights the role of lipid signaling homeostasis in FXS and provides arguments to support the testing of pioglitazone for treatment of FXS.

Jasmonate synthesis and signalling are essential for plant defense upregulation upon herbivore or microbial attacks. Stress-induced accumulation of jasmonoyl-isoleucine (JA-Ile), the bioactive hormonal form triggering transcriptional changes, is dynamic and transient because of the existence of potent removal mechanisms. Two JA-Ile turnover pathways operate in Arabidopsis, consisting in cytochrome P450 (CYP94)-mediated oxidation and deconjugation by the amidohydrolases IAR3/ILL6. Understanding their impacts was previously blurred by gene redundancy and compensation mechanisms. Here we address the consequences of blocking these pathways on jasmonate homeostasis and defenses in double-2ah, triple-3cyp mutants, and a quintuple-5ko line deficient in all known JA-Ile-degrading activities. These lines reacted differently to either mechanical wounding/insect attack or fungal infection. Both pathways contributed additively to JA-Ile removal upon wounding, but their impairement had opposite impacts on insect larvae feeding. By contrast, only the ah pathway was essential for JA-Ile turnover upon infection by Botrytis, yet only 3cyp was more fungus-resistant. Despite building-up extreme JA-Ile levels, 5ko displayed near-wild-type resistance in both bioassays. Molecular analysis indicated that restrained JA-Ile catabolism resulted in enhanced defense/resistance only when genes encoding negative regulators were not simultaneously overstimulated. This occurred in discrete stress- and pathway-specific combinations, providing a framework for future defense-enhancing strategies.

International audience; Plants are able to sense a rise in temperature of several degrees, and appropriately adapt their metabolic and growth processes. To this end, plants produce various signalling molecules that act throughout the plant body. Here, we report that root-derived GA$_{12}$, a precursor of the bioactive gibberellins, mediates thermo-responsive shoot growth in $Arabidopsis$. Our data suggest that root-to-shoot translocation of GA$_{12}$ enables a flexible growth response to ambient temperature changes.

Jasmonates (JAs) orchestrate immune responses upon wound/herbivore injury or infection by necrotrophic pathogens. Elucidation of catabolic routes has revealed new complexity in jasmonate metabolism. Two integrated pathways attenuate signaling by turning over the active hormone jasmonoyl-isoleucine (JA-Ile) through ω-oxidation or deconjugation, and define an indirect route forming the derivative 12OH-JA. Here, we provide evidence for a second 12OH-JA formation pathway by direct jasmonic acid (JA) oxidation. Three jasmonic acid oxidases (JAOs) of the 2-oxoglutarate dioxygenase family catalyze specific oxidation of JA to 12OH-JA, and their genes are induced by wounding or infection by the fungus Botrytis cinerea. JAO2 exhibits the highest basal expression, and its deficiency in jao2 mutants strongly enhanced antifungal resistance. The resistance phenotype resulted from constitutive expression of antimicrobial markers rather than from their higher induction in infected jao2 plants and could be reversed by ectopic expression of any of the three JAOs in jao2. Elevated defense in jao2 was dependent on the activity of JASMONATE RESPONSE 1 (JAR1) and CORONATINE-INSENSITIVE 1 (COI1) but was not correlated with enhanced JA-Ile accumulation. Instead, jao2 mutant lines displayed altered accumulation of several JA species in healthy and challenged plants, suggesting elevated metabolic flux through JA-Ile. Collectively, these data identify the missing enzymes hydroxylating JA and uncover an important metabolic diversion mechanism for repressing basal JA defense responses.

Background: Pueraria candollei var. mirifica , a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results: A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers.A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidatesinvolved in these biosyntheticpathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica . To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor ( At MYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins ledto the production of the isoflavone genistein. Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for unraveling biosynthetic pathways in plants.

Background Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. Conclusions Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

Plants are able to sense a rise in temperature of several degrees, and appropriately adapt their metabolic and growth processes. To this end, plants produce various signalling molecules that act throughout the plant body. Here, we report that root-derived GA

Barley (Hordeum vulgare) is the fourth crop cultivated in the world for human consumption and animal feed, making it important to breed healthy and productive plants. Among the threats for barley are lodging, diseases, and pathogens. To avoid lodging, dwarf and semi-dwarf mutants have been selected through breeding processes. Most of these mutants are affected on hormonal biosynthesis or signalling. Here, we present the metabolic characterization of a brassinosteroid insensitive semi-dwarf mutant, BW312. The hormone profile was determined through a targeted metabolomics analysis by UHPLC-triple quadrupole-MS/MS, showing an induction of gibberellic acid and jasmonic acid in the semi-dwarf mutant. A non-targeted metabolomics analysis by UHPLC-QTOF-MS/MS revealed a differential metabolic profile, with 16 and 9 metabolites showing higher intensities in the mutant and wild-type plants respectively. Among these metabolites, azelaic acid was identified. Gibberellic acid, jasmonic acid, and azelaic acid are involved in pathogen resistance, showing that this semi-dwarf line has an enhanced basal pathogen resistance in absence of pathogens, and therefore is of interest in breeding programs to fight against lodging, but also probably to increase pathogen resistance.

The metabolome, final downstream product of the genome and exogenous sources, is characterized by a large number of small molecules exhibiting a huge diversity of chemical structures and abundances, requiring complementary analytical platforms to reach its extensive coverage. The metabolome is dynamic, reflecting the continuous fluxes of metabolic and signalling pathways. Metabolomics is the detection and quantitation of metabolites in biological samples. Furthermore, recently emerged technologies have enabled us to determine spatiotemporal distribution of biological molecules present in tissues. In a two-part article, we present a description of usual metabolomics technologies, workflows and strategies leading to the implementation of new clinical biomarkers. In this first part, after introducing metabolomics concepts, we review analytical techniques used in this setting.