Stable Isotope Kinetic Study of ApoM (Apolipoprotein M).

OBJECTIVE: ApoM (apolipoprotein M) binds primarily to high-density lipoprotein before to be exchanged with apoB (apolipoprotein B)-containing lipoproteins. Low-density lipoprotein (LDL) receptor-mediated clearance of apoB-containing particles could influence plasma apoM kinetics and decrease its antiatherogenic properties. In humans, we aimed to describe the interaction of apoM kinetics with other components of lipid metabolism to better define its potential benefit on atherosclerosis. APPROACH AND RESULTS: Fourteen male subjects received a primed infusion of 2H3-leucine for 14 hours, and analyses were performed by liquid chromatography-tandem mass spectrometry from the hourly plasma samples. Fractional catabolic rates and production rates within lipoproteins were calculated using compartmental models. ApoM was found not only in high-density lipoprotein (59%) and LDL (4%) but also in a non-lipoprotein-related compartment (37%). The apoM distribution was heterogeneous within LDL and non-lipoprotein-related compartments according to plasma triglycerides (r=0.86; P<0.001). The relationships between sphingosine-1-phosphate and apoM were confirmed in all compartments (r range, 0.55-0.89; P<0.05). ApoM fractional catabolic rates and production rates were 0.16±0.07 pool/d and 0.14±0.06 mg/kg per day in high-density lipoprotein and 0.56±0.10 pool/d and 0.03±0.01 mg/kg per day in LDL, respectively. Fractional catabolic rates of LDL-apoM and LDL-apoB100 were correlated (r=0.55; P=0.042). Significant correlations were found between triglycerides and production rates of LDL-apoM (r=0.73; P<0.004). CONCLUSIONS: In humans, LDL kinetics play a key role in apoM turnover. Plasma triglycerides act on both apoM and sphingosine-1-phosphate distributions between lipoproteins. These results confirmed that apoM could be bound to high-density lipoprotein after secretion and then quickly exchanged with a non-lipoprotein-related compartment and to LDL to be slowly catabolized.

Workflow4Metabolomics: a collaborative research infrastructure for computational metabolomics.

SUMMARY: The complex, rapidly evolving field of computational metabolomics calls for collaborative infrastructures where the large volume of new algorithms for data pre-processing, statistical analysis and annotation can be readily integrated whatever the language, evaluated on reference datasets and chained to build ad hoc workflows for users. We have developed Workflow4Metabolomics (W4M), the first fully open-source and collaborative online platform for computational metabolomics. W4M is a virtual research environment built upon the Galaxy web-based platform technology. It enables ergonomic integration, exchange and running of individual modules and workflows. Alternatively, the whole W4M framework and computational tools can be downloaded as a virtual machine for local installation. AVAILABILITY AND IMPLEMENTATION: http://workflow4metabolomics.org homepage enables users to open a private account and access the infrastructure. W4M is developed and maintained by the French Bioinformatics Institute (IFB) and the French Metabolomics and Fluxomics Infrastructure (MetaboHUB). CONTACT: contact@workflow4metabolomics.org.

Structure/function analysis of a type iii polyketide synthase in the brown alga Ectocarpus siliculosus reveals a biochemical pathway in phlorotannin monomer biosynthesis.

Brown algal phlorotannins are structural analogs of condensed tannins in terrestrial plants and, like plant phenols, they have numerous biological functions. Despite their importance in brown algae, phlorotannin biosynthetic pathways have been poorly characterized at the molecular level. We found that a predicted type III polyketide synthase in the genome of the brown alga Ectocarpus siliculosus, PKS1, catalyzes a major step in the biosynthetic pathway of phlorotannins (i.e., the synthesis of phloroglucinol monomers from malonyl-CoA). The crystal structure of PKS1 at 2.85-Å resolution provided a good quality electron density map showing a modified Cys residue, likely connected to a long chain acyl group. An additional pocket not found in other known type III PKSs contains a reaction product that might correspond to a phloroglucinol precursor. In vivo, we also found a positive correlation between the phloroglucinol content and the PKS III gene expression level in cells of a strain of Ectocarpus adapted to freshwater during its reacclimation to seawater. The evolution of the type III PKS gene family in Stramenopiles suggests a lateral gene transfer event from an actinobacterium.

Transcriptomic and metabolomic analysis of copper stress acclimation in Ectocarpus siliculosus highlights signaling and tolerance mechanisms in brown algae.

BACKGROUND: Brown algae are sessile macro-organisms of great ecological relevance in coastal ecosystems. They evolved independently from land plants and other multicellular lineages, and therefore hold several original ontogenic and metabolic features. Most brown algae grow along the coastal zone where they face frequent environmental changes, including exposure to toxic levels of heavy metals such as copper (Cu). RESULTS: We carried out large-scale transcriptomic and metabolomic analyses to decipher the short-term acclimation of the brown algal model E. siliculosus to Cu stress, and compared these data to results known for other abiotic stressors. This comparison demonstrates that Cu induces oxidative stress in E. siliculosus as illustrated by the transcriptomic overlap between Cu and H2O2 treatments. The common response to Cu and H2O2 consisted in the activation of the oxylipin and the repression of inositol signaling pathways, together with the regulation of genes coding for several transcription-associated proteins. Concomitantly, Cu stress specifically activated a set of genes coding for orthologs of ABC transporters, a P1B-type ATPase, ROS detoxification systems such as a vanadium-dependent bromoperoxidase, and induced an increase of free fatty acid contents. Finally we observed, as a common abiotic stress mechanism, the activation of autophagic processes on one hand and the repression of genes involved in nitrogen assimilation on the other hand. CONCLUSIONS: Comparisons with data from green plants indicate that some processes involved in Cu and oxidative stress response are conserved across these two distant lineages. At the same time the high number of yet uncharacterized brown alga-specific genes induced in response to copper stress underlines the potential to discover new components and molecular interactions unique to these organisms. Of particular interest for future research is the potential cross-talk between reactive oxygen species (ROS)-, myo-inositol-, and oxylipin signaling.

Mannitol metabolism in brown algae involves a new phosphatase family.

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase and a mannitol-1-phosphatase (M1Pase). Genome analysis of the brown algal model Ectocarpus siliculosus allowed identification of genes potentially involved in the mannitol cycle. Among these, two genes coding for haloacid dehalogenase (HAD)-like enzymes were suggested to correspond to M1Pase activity, and thus were named EsM1Pase1 and EsM1Pase2, respectively. To test this hypothesis, both genes were expressed in Escherichia coli. Recombinant EsM1Pase2 was shown to hydrolyse the phosphate group from mannitol-1-phosphate to produce mannitol but was not active on the hexose monophosphates tested. Gene expression analysis showed that transcription of both E. siliculosus genes was under the influence of the diurnal cycle. Sequence analysis and three-dimensional homology modelling indicated that EsM1Pases, and their orthologues in Prasinophytes, should be seen as founding members of a new family of phosphatase with original substrate specificity within the HAD superfamily of proteins. This is the first report describing the characterization of a gene encoding M1Pase activity in photosynthetic organisms.

Towards deciphering dynamic changes and evolutionary mechanisms involved in the adaptation to low salinities in Ectocarpus (brown algae).

Colonizations of freshwater by marine species are rare events, and little information is known about the underlying mechanisms. Brown algae are an independent lineage of photosynthetic and multicellular organisms from which few species inhabit freshwater. As a marine alga that is also found in freshwater, Ectocarpus is of particular interest for studying the transition between these habitats. To gain insights into mechanisms of the transition, we examined salinity tolerance and adaptations to low salinities in a freshwater strain of Ectocarpus on physiological and molecular levels. We show that this isolate belongs to a widely distributed and highly stress-resistant clade, and differed from the genome-sequenced marine strain in its tolerance of low salinities. It also exhibited profound, but reversible, morphological, physiological, and transcriptomic changes when transferred to seawater. Although gene expression profiles were similar in both strains under identical conditions, metabolite and ion profiles differed strongly, the freshwater strain exhibiting e.g. higher cellular contents of amino acids and nitrate, higher contents of n-3 fatty acids, and lower intracellular mannitol and sodium concentrations. Moreover, several stress markers were noted in the freshwater isolate in seawater. This finding suggests that, while high stress tolerance and plasticity may be prerequisites for the colonization of freshwater, genomic alterations have occurred that produced permanent changes in the metabolite profiles to stabilize the transition.

Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors.

KEY MESSAGE: Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses. Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.

Mass spectrometry-based metabolomics to elucidate functions in marine organisms and ecosystems.

Marine systems are very diverse and recognized as being sources of a wide range of biomolecules. This review provides an overview of metabolite profiling based on mass spectrometry (MS) approaches in marine organisms and their environments, focusing on recent advances in the field. We also point out some of the technical challenges that need to be overcome in order to increase applications of metabolomics in marine systems, including extraction of chemical compounds from different matrices and data management. Metabolites being important links between genotype and phenotype, we describe added value provided by integration of data from metabolite profiling with other layers of omics, as well as their importance for the development of systems biology approaches in marine systems to study several biological processes, and to analyze interactions between organisms within communities. The growing importance of MS-based metabolomics in chemical ecology studies in marine ecosystems is also illustrated.

Integrative analysis of metabolite and transcript abundance during the short-term response to saline and oxidative stress in the brown alga Ectocarpus siliculosus.

The model brown alga Ectocarpus siliculosus undergoes extensive transcriptomic changes in response to abiotic stress, many of them related to primary metabolism and particularly to amino acid biosynthesis and degradation. In this study we seek to improve our knowledge of the mechanisms underlying the stress tolerance of this alga, in particular with regard to compatible osmolytes, by examining the effects of these changes on metabolite concentrations. We performed extensive metabolic profiling (urea, amino acids, sugars, polyols, organic acids, fatty acids) of Ectocarpus samples subjected to short-term hyposaline, hypersaline and oxidative stress, and integrated the results with previously published transcriptomic data. The most pronounced changes in metabolite concentrations occurred under hypersaline stress: both mannitol and proline were accumulated, but their low final concentrations indicate that, in this stress condition, both compounds are not likely to significantly contribute to osmoregulation at the level of the entire cell. Urea and trehalose were not detected in any of our samples. We also observed a shift in fatty acid composition from n-3 to n-6 fatty acids under high salinities, and demonstrated the salt stress-induced accumulation of small amounts of γ-aminobutyric acid (GABA). GABA could be synthesized in E. siliculosus through a salt stress-induced putrescine-degradation pathway.

Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450.

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Release of volatile aldehydes by the brown algal kelp Laminaria digitata in response to both biotic and abiotic stress.

With a little kelp from my friends: In response to biotic and abiotic stress, the brown algal kelp Laminaria digitata releases volatile fatty acid aldehydes under laboratory conditions and in its natural environment (red). In response to 4-HHE treatment, L. digitata releases (13S)-HOTrE (green). These results support the hypothesis that these compounds may mediate kelp responses to stress.

Copper stress induces biosynthesis of octadecanoid and eicosanoid oxygenated derivatives in the brown algal kelp Laminaria digitata.

To better understand the toxicity and the orchestration of antioxidant defenses of marine brown algae in response to copper-induced stress, lipid peroxidation processes were investigated in the brown alga Laminaria digitata. The expression of genes involved in cell protection and anti-oxidant responses were monitored by semi-quantitative reverse transcriptase polymerase chain reaction and the lipid peroxidation products were further characterized by profiling oxylipin signatures using high-pressure liquid chromatography-mass spectrometry. Exposure to copper excess triggers lipoperoxide accumulation and upregulates the expression of stress related genes. It also increases the release of free polyunsaturated fatty acids, leading to an oxidative cascade through at least two distinct mechanisms. Incubations in presence of inhibitors of lipoxygenases and cycloxygenases showed that in addition to the reactive oxygen species-mediated processes, copper stress induces the synthesis of oxylipins through enzymatic mechanisms. Among complex oxylipins, cyclopentenones from C18 and C20 fatty acids such as 12-oxo-PDA and prostaglandins were detected for the first time in brown algae, as well as unique compounds such as the 18-hydroxy-17-oxo-eicosatetraenoic acid. These results suggest that lipid peroxidation participates in the toxic effects of copper and that lipid peroxidation derivatives may regulate protective mechanisms by employing plant-like octadecanoid signals but also eicosanoid oxylipins which are absent in vascular plants.

Determination of epoxyeicosatrienoic acids in human red blood cells and plasma by GC/MS in the NICI mode.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid involved in the regulation of vascular tone. Despite the importance of EETs in a variety of physiological effects, few methods have been developed to quantify them in human blood. This led us to develop a method by GC/MS with negative ion chemical ionization. As EETs are primarily located in phospholipids, red blood cells (RBCs) and plasma phospholipids were hydrolyzed with phospholipase A(2) after a solid phase extraction. Then, EETs were derivatized as pentafluorobenzyl esters, and [(2)H(8)]-arachidonic acid was used as internal standard for quantification. EETs were found to be at concentrations of 106+/-37ng mL(-1) in plasma and 33.4+/-8.5 ng/10(9) RBCs (mean+/-S.D.) in 10 healthy volunteers. Their amount in RBCs was 3-fold that in plasma; both parameters proved to be well correlated.

Consequences of blunting the mevalonate pathway in cancer identified by a pluri-omics approach.

We have previously shown that the combination of statins and taxanes was a powerful trigger of HGT-1 human gastric cancer cells' apoptosis1. Importantly, several genes involved in the "Central carbon metabolism pathway in cancer", as reported in the Kyoto Encyclopedia of Genes and Genomes, were either up- (ACLY, ERBB2, GCK, MYC, PGM, PKFB2, SLC1A5, SLC7A5, SLC16A3,) or down- (IDH, MDH1, OGDH, P53, PDK) regulated in response to the drug association. In the present study, we conducted non-targeted metabolomics and lipidomics analyses by complementary methods and cross-platform initiatives, namely mass spectrometry (GC-MS, LC-MS) and nuclear magnetic resonance (NMR), to analyze the changes resulting from these treatments. We identified several altered biochemical pathways involved in the anabolism and disposition of amino acids, sugars, and lipids. Using the Cytoscape environment with, as an input, the identified biochemical marker changes, we distinguished the functional links between pathways. Finally, looking at the overlap between metabolomics/lipidomics and transcriptome changes, we identified correlations between gene expression modifications and changes in metabolites/lipids. Among the metabolites commonly detected by all types of platforms, glutamine was the most induced (6-7-fold), pointing to an important metabolic adaptation of cancer cells. Taken together, our results demonstrated that combining robust biochemical and molecular approaches was efficient to identify both altered metabolic pathways and overlapping gene expression alterations in human gastric cancer cells engaging into apoptosis following blunting the cholesterol synthesis pathway.

Create, run, share, publish, and reference your LC-MS, FIA-MS, GC-MS, and NMR data analysis workflows with the Workflow4Metabolomics 3.0 Galaxy online infrastructure for metabolomics.

Metabolomics is a key approach in modern functional genomics and systems biology. Due to the complexity of metabolomics data, the variety of experimental designs, and the multiplicity of bioinformatics tools, providing experimenters with a simple and efficient resource to conduct comprehensive and rigorous analysis of their data is of utmost importance. In 2014, we launched the Workflow4Metabolomics (W4M; http://workflow4metabolomics.org) online infrastructure for metabolomics built on the Galaxy environment, which offers user-friendly features to build and run data analysis workflows including preprocessing, statistical analysis, and annotation steps. Here we present the new W4M 3.0 release, which contains twice as many tools as the first version, and provides two features which are, to our knowledge, unique among online resources. First, data from the four major metabolomics technologies (i.e., LC-MS, FIA-MS, GC-MS, and NMR) can be analyzed on a single platform. By using three studies in human physiology, alga evolution, and animal toxicology, we demonstrate how the 40 available tools can be easily combined to address biological issues. Second, the full analysis (including the workflow, the parameter values, the input data and output results) can be referenced with a permanent digital object identifier (DOI). Publication of data analyses is of major importance for robust and reproducible science. Furthermore, the publicly shared workflows are of high-value for e-learning and training. The Workflow4Metabolomics 3.0 e-infrastructure thus not only offers a unique online environment for analysis of data from the main metabolomics technologies, but it is also the first reference repository for metabolomics workflows.

Herbivore-induced chemical and molecular responses of the kelps Laminaria digitata and Lessonia spicata.

Kelps are founding species of temperate marine ecosystems, living in intertidal coastal areas where they are often challenged by generalist and specialist herbivores. As most sessile organisms, kelps develop defensive strategies to restrain grazing damage and preserve their own fitness during interactions with herbivores. To decipher some inducible defense and signaling mechanisms, we carried out metabolome and transcriptome analyses in two emblematic kelp species, Lessonia spicata from South Pacific coasts and Laminaria digitata from North Atlantic, when challenged with their main specialist herbivores. Mass spectrometry based metabolomics revealed large metabolic changes induced in these two brown algae following challenges with their own specialist herbivores. Targeted metabolic profiling of L. spicata further showed that free fatty acid (FFA) and amino acid (AA) metabolisms were particularly regulated under grazing. An early stress response was illustrated by the accumulation of Sulphur containing amino acids in the first twelve hours of herbivory pressure. At latter time periods (after 24 hours), we observed FFA liberation and eicosanoid oxylipins synthesis likely representing metabolites related to stress. Global transcriptomic analysis identified sets of candidate genes specifically induced by grazing in both kelps. qPCR analysis of the top candidate genes during a 48-hours time course validated the results. Most of these genes were particularly activated by herbivore challenge after 24 hours, suggesting that transcriptional reprogramming could be operated at this time period. We demonstrated the potential utility of these genes as molecular markers for herbivory by measuring their inductions in grazed individuals of field harvested L. digitata and L. spicata. By unravelling the regulation of some metabolites and genes following grazing pressure in two kelps representative of the two hemispheres, this work contributes to provide a set of herbivore-induced chemical and molecular responses in kelp species, showing similar inducible responses upon specialist herbivores in their respective ecosystems.

Determination of polyunsaturated fatty acid monoepoxides by high performance liquid chromatography-mass spectrometry.

Despite the implication of polyunsaturated fatty acid monoepoxides in a large panel of biological effects, few methods allowing their separation in a single run are available. We describe here a simple method based on reversed-phase ion-pair high-performance liquid chromatography (RP-HPLC) and developed to successfully separate the various monoepoxides of eicosatrienoic, arachidonic, eicosapentaenoic and docosahexaenoic acids. These compounds were easily identified by liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure chemical ionisation owing to the volatility of counter-ion species. Compared to established methods, this new protocol proved its ability to totally resolve, in a single run, all of the different regioisomeric epoxides. In the long run, this method will demonstrate its efficacy to give insights into the cytochrome P450-dependent metabolism of polyunsaturated fatty acids (PUFAs) and the generation of physiologically active epoxy-derivatives.

Microbiota Influences Morphology and Reproduction of the Brown Alga Ectocarpus sp.

Associated microbiota play crucial roles in health and disease of higher organisms. For macroalgae, some associated bacteria exert beneficial effects on nutrition, morphogenesis and growth. However, current knowledge on macroalgae-microbiota interactions is mostly based on studies on green and red seaweeds. In this study, we report that when cultured under axenic conditions, the filamentous brown algal model Ectocarpus sp. loses its branched morphology and grows with a small ball-like appearance. Nine strains of periphytic bacteria isolated from Ectocarpus sp. unialgal cultures were identified by 16S rRNA sequencing, and assessed for their effect on morphology, reproduction and the metabolites secreted by axenic Ectocarpus sp. Six of these isolates restored morphology and reproduction features of axenic Ectocarpus sp. Bacteria-algae co-culture supernatants, but not the supernatant of the corresponding bacterium growing alone, also recovered morphology and reproduction of the alga. Furthermore, colonization of axenic Ectocarpus sp. with a single bacterial isolate impacted significantly the metabolites released by the alga. These results show that the branched typical morphology and the individuals produced by Ectocarpus sp. are strongly dependent on the presence of bacteria, while the bacterial effect on the algal exometabolome profile reflects the impact of bacteria on the whole physiology of this alga.

Constitutive or Inducible Protective Mechanisms against UV-B Radiation in the Brown Alga Fucus vesiculosus? A Study of Gene Expression and Phlorotannin Content Responses.

A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds in brown algae.

Can we trust untargeted metabolomics? Results of the metabo-ring initiative, a large-scale, multi-instrument inter-laboratory study.

The metabo-ring initiative brought together five nuclear magnetic resonance instruments (NMR) and 11 different mass spectrometers with the objective of assessing the reliability of untargeted metabolomics approaches in obtaining comparable metabolomics profiles. This was estimated by measuring the proportion of common spectral information extracted from the different LCMS and NMR platforms. Biological samples obtained from 2 different conditions were analysed by the partners using their own in-house protocols. Test #1 examined urine samples from adult volunteers either spiked or not spiked with 32 metabolite standards. Test #2 involved a low biological contrast situation comparing the plasma of rats fed a diet either supplemented or not with vitamin D. The spectral information from each instrument was assembled into separate statistical blocks. Correlations between blocks (e.g., instruments) were examined (RV coefficients) along with the structure of the common spectral information (common components and specific weights analysis). In addition, in Test #1, an outlier individual was blindly introduced, and its identification by the various platforms was evaluated. Despite large differences in the number of spectral features produced after post-processing and the heterogeneity of the analytical conditions and the data treatment, the spectral information both within (NMR and LCMS) and across methods (NMR vs. LCMS) was highly convergent (from 64 to 91 % on average). No effect of the LCMS instrumentation (TOF, QTOF, LTQ-Orbitrap) was noted. The outlier individual was best detected and characterised by LCMS instruments. In conclusion, untargeted metabolomics analyses report consistent information within and across instruments of various technologies, even without prior standardisation.