Quantitative Analytical and Computational Workflow for Large-Scale Targeted Plasma Metabolomics.

Quantifying metabolites from various biological samples is necessary for the clinical and biomedical translation of metabolomics research. One of the ongoing challenges in biomedical metabolomics studies is the large-scale quantification of targeted metabolites, mainly due to the complexity of biological sample matrices. Furthermore, in LC-MS analysis, the response of compounds is influenced by their physicochemical properties, chromatographic conditions, eluent composition, sample preparation, type of MS ionization source, and analyzer used. To facilitate large-scale metabolite quantification, we evaluated the relative response factor (RRF) approach combined with an integrated analytical and computational workflow. This approach considers a compound's individual response in LC-MS analysis relative to that of a non-endogenous reference compound to correct matrix effects. We created a quantitative LC-MS library using the Skyline/Panorama web platform for data processing and public sharing of data. In this study, we developed and validated a metabolomics method for over 280 standard metabolites and quantified over 90 metabolites. The RRF quantification was validated and compared with conventional external calibration approaches as well as literature reports. The Skyline software environment was adapted for processing such metabolomics data, and the results are shared as a "quantitative chromatogram library" with the Panorama web application. This new workflow was found to be suitable for large-scale quantification of metabolites in human plasma samples. In conclusion, we report a novel quantitative chromatogram library with a targeted data analysis workflow for biomedical metabolomic applications.

Comparative Genomics of Members of the Genus Defluviicoccus With Insights Into Their Ecophysiological Importance.

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca. Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published (Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca. Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanusT , the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus.

Influence of Extraction Solvent on Nontargeted Metabolomics Analysis of Enrichment Reactor Cultures Performing Enhanced Biological Phosphorus Removal (EBPR).

Metabolome profiling is becoming more commonly used in the study of complex microbial communities and microbiomes; however, to date, little information is available concerning appropriate extraction procedures. We studied the influence of different extraction solvent mixtures on untargeted metabolomics analysis of two continuous culture enrichment communities performing enhanced biological phosphate removal (EBPR), with each enrichment targeting distinct populations of polyphosphate-accumulating organisms (PAOs). We employed one non-polar solvent and up to four polar solvents for extracting metabolites from biomass. In one of the reactor microbial communities, we surveyed both intracellular and extracellular metabolites using the same set of solvents. All samples were analysed using ultra-performance liquid chromatography mass spectrometry (UPLC-MS). UPLC-MS data obtained from polar and non-polar solvents were analysed separately and evaluated using extent of repeatability, overall extraction capacity and the extent of differential abundance between physiological states. Despite both reactors demonstrating the same bioprocess phenotype, the most appropriate extraction method was biomass specific, with methanol: water (50:50 v/v) and methanol: chloroform: water (40:40:20 v/v) being chosen as the most appropriate for each of the two different bioreactors, respectively. Our approach provides new data on the influence of solvent choice on the untargeted surveys of the metabolome of PAO enriched EBPR communities and suggests that metabolome extraction methods need to be carefully tailored to the specific complex microbial community under study.

Metabolic Traits of Candidatus Accumulibacter clade IIF Strain SCELSE-1 Using Amino Acids As Carbon Sources for Enhanced Biological Phosphorus Removal.

Despite recent evidence from full-scale plants suggesting that Candidatus Accumulibacter may be capable of using amino acids, this metabolic trait has never been confirmed in a bioreactor experiment. Here we show that an enriched culture of Ca. Accumulibacter clade IIF strain SCELSE-1 could metabolize 11 of 20 α-amino acids, with aspartate, glutamate, asparagine, and glutamine resulting in the highest phosphorus removal. The anaerobic uptake of aspartate and glutamate was achieved through a glutamate/aspartate-proton symporter fully powered by the proton motive force (PMF). Under anaerobic conditions aspartate was deaminized and routed into core carbon metabolic pathways to form polyhydroxyalkanoates (PHA). The lack of genes encoding NADH dependent isocitrate dehydrogenase in the Ca. Accumulibacter genome resulted in a kinetic barrier for glutamate to be channelled to the TCA cycle. Glutamate was stored as glutamate polymer. When amino acids (aspartate or glutamate) and acetate were supplied together, Ca. Accumulibacter took up both carbon sources simultaneously, with the uptake rate of each carbon source largely preserved. Overall energy savings (up to 17%) were achieved under mixed carbon scenarios, due to the ability of Ca. Accumulibacter to rearrange its anaerobic carbon metabolism based on the reducing power, PMF and ATP balance.

Applying generalized additive models to unravel dynamic changes in anthocyanin biosynthesis in methyl jasmonate elicited grapevine (Vitis vinifera cv. Gamay) cell cultures.

Plant cell cultures represent important model systems to understand metabolism and its modulation by regulatory factors. Even in controlled conditions, cell metabolism is highly dynamic and can be fully characterized only by time course experiments. Here, we show that statistical analysis of this type of data gains power if it moves to approaches able to compare the 'trends' of the different metabolites. In particular, we show how generalized additive models can be used to model the time-dependent profile of anthocyanin synthesis in grapevine cell suspension cultures (Vitis vinifera cv. Gamay), following treatment with 100 µm methyl jasmonate. The sampling was performed daily for 20 days of culturing following elicitation at day 5. All samples were analyzed by UPLC-MS/MS for the identification and quantification of fifteen anthocyanin compounds. The models confirmed the separation in the anthocyanin biosynthetic pathway between delphinidin-based and cyanidin-based compounds, showing that methyl jasmonate modulates the anthocyanin concentration profiles. Our results clearly indicate that the combination of high-throughput metabolomics and state of the art statistical modeling is a powerful approach to study plant metabolism. This approach is expected to gain popularity due to the growing availability of low-cost high-throughput 'omic' assays.

Effects of different encapsulation agents and drying process on stability of betalains extract.

Red beet plants are rich in betalains that can be used as food natural colorants. Betalains were extracted from red beet and encapsulated with different carrier agents and freeze or spray dried. Effect of different encapsulating agents as maltodextrin, guar gum, gum Arabic, pectin and xanthan gum with different concentration (as encapsulating agents) were studied on the betalain stability. Encapsulated betalains with xanthan gum with maltodextrin showed about 65 % more recovery than the control. Encapsulation showed a higher recovery of betalains during freeze drying by 1.3 times than during spray drying. Spray dried samples has L* (lightness) higher than the freeze dried samples. The variations of maltodextrin with xanthan and guar gum freeze dried have highest chroma value of 21. The stabilization of pure betalain pigments may boost the use of these colouring molecules in the food industry and promote their application.

Investigation of Phenolic Acids in Suspension Cultures of Vitis vinifera Stimulated with Indanoyl-Isoleucine, N-Linolenoyl-L-Glutamine, Malonyl Coenzyme A and Insect Saliva.

Vitis vinifera c.v. Muscat de Frontignan (grape) contains various high valuable bioactive phenolic compounds with pharmaceutical properties and industrial interest which are not fully exploited. The focus of this investigation consists in testing the effects of various biological elicitors on a non-morphogenic callus suspension culture of V. vinifera. The investigated elicitors: Indanoyl-isoleucine (IN), N-linolenoyl-L-glutamine (LG), insect saliva (IS) and malonyl coenzyme A (MCoA) were aimed at mimicking the influence of environmental pathogens on plants in their natural habitats and at provoking exogenous induction of the phenylpropanoid pathway. The elicitors' indanoyl-isoleucine (IN), N-linolenoyl-L-glutamine (LG) and insect saliva (IS), as well as malonyl coenzyme A (MCoA), were independently inoculated to stimulate the synthesis of phenylpropanoids. All of the enhancers positively increased the concentration of phenolic compounds in grape cells. The highest concentration of phenolic acids was detected after 2 h for MCoA, after 48 h for IN and after 24 h for LG and IS respectively. At the maximum production time, treated grape cells had a 3.5-fold (MCoA), 1.6-fold (IN) and 1.5-fold (IS) higher phenolic acid content compared to the corresponding control samples. The HPLC results of grape cells showed two major resveratrol derivatives: 3-O-Glucosyl-resveratrol and 4-(3,5-dihydroxyphenyl)-phenol. Their influences of the different elicitors, time of harvest and biomass concentration (p < 0.0001) were statistically significant on the synthesis of phenolic compounds. The induction with MCoA was found to demonstrate the highest statistical effect corresponding to the strongest stress response within the phenylpropanoid pathway in grape cells.