Metabolic Evaluation of Lupin-Enriched Yogurt by Nuclear Magnetic Resonance Metabolomics.

Untargeted nuclear magnetic resonance (NMR) metabolomics was used to evaluate compositional changes during yogurt fermentation upon lupin enrichment compared to traditional conditions. Lupin significantly changed the sample metabolic profile and its time course dynamics, seemingly delaying microbial action. The levels of organic and amino acids were significantly altered, along with those of some sugars, nucleotides, and choline compounds. Lupin seemed to favor acetate and formate synthesis, compared to that of citrate and fumarate; a higher formate levels may suggest increased levels of Streptococcus thermophilus action, compared toLactobacillus bulgaricus. Lupin-yogurt was poorer in hippurate, lactose (and hence lactate), galactose, glucose-1-phosphate, and galactose-1-phosphate, containing higher orotate levels (possibly related to increased uridine derivatives), among other differences. Trigonelline was confirmed as a lupin marker, possibly together with glutamate and histidine. Other metabolite trajectories remained unchanged upon lupin addition, unveiling unaffected underlying processes. These results demonstrate the usefulness of untargeted NMR metabolomics to understand/develop new foodstuffs and their production processes, highlighting the identity of a variety of bioactive metabolites with importance for human health.

Impact of Sea Warming and 17-α-Ethinylestradiol Exposure on the Lipid Metabolism of Ruditapes philippinarum Clams.

This paper reports on an NMR metabolomics study of lipophilic extracts of Ruditapes philippinarum clams exposed to the hormonal contaminant 17-α-ethinylestradiol (EE2), at 17 °C and 21 °C. The results reveal that exposure at 17 °C triggers a weak response at low EE2 concentrations, suggestive of a slight increase in membrane rigidity, followed by lipid metabolic stability at higher EE2 concentrations. On the other hand, at 21 °C, lipid metabolism begins to respond at 125 ng/L EE2, with antioxidant docosahexaenoic acid (DHA) helping to tackle high-oxidative-stress conditions, in tandem with enhanced storage of triglycerides. Exposure to 625 ng/L EE2 (highest concentration) enhances phosphatidylcholine (PtdCho) and polyunsaturated fatty acid (PUFA) levels, their direct intercorrelation suggesting PUFA incorporation in new membrane phospholipids. This should lead to increased membrane fluidity, probably aided by a decrease in cholesterol. PUFA levels, considered a measure of membrane fluidity, were strongly (and positively) correlated to intracellular glycine levels, thus identifying glycine as the main osmolyte entering the cells under high stress. Membrane fluidity also seems to elicit the loss of taurine. This work contributes to the understanding of the mechanisms of response of R. philippinarum clams to EE2 in tandem with warming while unveiling novel potential markers of stress mitigation, namely high levels of PtdCho, PUFAs (or PtdCho/glycerophosphocholine and PtdCho/acetylcholine ratios) and linoleic acid and low PUFA/glycine ratios.

The influence of temperature rise on the metabolic response of Ruditapes philippinarum clams to 17-α-ethinylestradiol.

Untargeted Nuclear Magnetic Resonance metabolomics was employed to study the effects of warming conditions (17-21 °C) and exposure to 17-α-ethinylestradiol (EE2) on the polar metabolome of Ruditapes philippinarum clams, to identify metabolic markers for monitoring/prediction of deviant environmental conditions. Warming alone triggered changes in alanine/aspartate/glutamate, aromatic amino acids, taurine/hypotaurine and homarine/trigonelline pathways, as well as in energy metabolism, suggesting osmoregulatory adaptations and glycolytic/tricarboxylic acid (TCA) cycle activation, possibly accompanied to some extent by gluconeogenesis to preserve glycogen reserves. At 17 °C, the lowest EE2 concentration (5 ng/L) specifically engaged branched-chain and aromatic amino acids to activate the glycolysis/TCA cycle. Notably, a partial metabolic recovery was observed at 25 ng/L, whereas higher EE2 concentrations (125 and 625 ng/L) again induced significant metabolic disturbances. These included enhanced glycogen biosynthesis and increased lipid reserves, sustained by low-level glutathione-based antioxidative mechanisms that seemed active. At 21 °C, response to EE2 was notably weak at low/intermediate concentrations, becoming particularly significant at the highest EE2 concentration (625 ng/L), suggesting higher protection capacity of Ruditapes philippinarum clams under warming conditions. At 625 ng/L, disturbances in alanine/aspartate/glutamate and taurine/hypotaurine metabolisms were observed, with no evidence of enhanced carbohydrate/protein catabolism. This low energy function profile was accompanied by marked antioxidative mechanisms and choline compounds modulation for cell membrane protection/repair. These results help monitor clams´ response to temperature rise and EE2 exposure, paving the way for future effective guidance and prediction of environmental damaging effects.

Different approaches for estimation of the expanded uncertainty of an analytical method developed for determining pharmaceutical active compounds in wastewater using solid-phase extraction and a liquid chromatography coupled with tandem mass spectrometry method.

Although the evaluation of the uncertainty of an analytical method is a mandatory step in the method's validation, its applicability to the monitoring of trace compounds in complex samples is not simple, nor is it part of the routine of most laboratories, namely those dedicated to research. This manuscript focuses on the full validation of an analytical procedure for determining trace concentrations of twenty-four pharmaceutical active compounds (PhACs) in wastewaters using solid-phase extraction (SPE) and ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method optimization was performed on different wastewater matrices, namely influents and final effluents from two distinct wastewater treatment plants (WWTPs). Matrix effects and extraction efficiency (absolute recovery) of the developed method were determined. Validation was performed to obtain the method's linearity/working range, precision, trueness, method detection limits (MDLs) and method quantification limits (MQLs). The expanded uncertainty of the data obtained was estimated according to the requirements of international procedures dedicated to the expression of uncertainty. Different approaches for the estimation of uncertainty were applied. The validated method was used in the analysis of target PhACs in wastewater samples collected at two WWTPs. The obtained results facilitated the introduction of a validated method for routine measurement of PhACs in wastewater samples and allowed method accreditation by the competent national authority.

Chitosan Film as a Replacement for Conventional Sulphur Dioxide Treatment of White Wines: A 1H NMR Metabolomic Study.

Chitosan-genipin (Ch-Ge) films have been proposed for the replacement of sulfur dioxide (SO2) in white wines preservation to circumvent the adverse health consequences caused by SO2 intake. To assess the effects of different-sized Ch-Ge films (25 and 100 cm2) on wine composition compared to SO2-treated and untreated wines, nuclear magnetic resonance metabolomics was applied. Relative to SO2, 100 cm2 films induced significant changes in the levels of organic acids, sugars, amino acids, 5-hydroxymethylfurfural, among other compounds, while 25 cm2 films appeared to induce only small variations. The observed metabolite variations were proposed to arise from the mitigation of fermentative processes, electrostatic interactions between acids and the positively charged films and the promotion of Maillard and Strecker reactions. Qualitative sensory analysis showed that wines maintained overall appropriate sensory characteristics, with 100 cm2 film treated wines showing slightly higher attributes. Based on these results, the possibility of using Ch-Ge films as a replacement for SO2 treatment is discussed.

N6-methyladenosine in poly(A) tails stabilize VSG transcripts.

RNA modifications are important regulators of gene expression1. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally2. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown3. Here we found that m6A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m6A is located in the poly(A) tail of the actively expressed VSG transcripts. m6A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m6A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m6A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation.

African trypanosomes expressing multiple VSGs are rapidly eliminated by the host immune system.

Trypanosoma brucei parasites successfully evade the host immune system by periodically switching the dense coat of variant surface glycoprotein (VSG) at the cell surface. Each parasite expresses VSGs in a monoallelic fashion that is tightly regulated. The consequences of exposing multiple VSGs during an infection, in terms of antibody response and disease severity, remain unknown. In this study, we overexpressed a high-mobility group box protein, TDP1, which was sufficient to open the chromatin of silent VSG expression sites, to disrupt VSG monoallelic expression, and to generate viable and healthy parasites with a mixed VSG coat. Mice infected with these parasites mounted a multi-VSG antibody response, which rapidly reduced parasitemia. Consequently, we observed prolonged survival in which nearly 90% of the mice survived a 30-d period of infection with undetectable parasitemia. Immunodeficient RAG2 knock-out mice were unable to control infection with TDP1-overexpressing parasites, showing that the adaptive immune response is critical to reducing disease severity. This study shows that simultaneous exposure of multiple VSGs is highly detrimental to the parasite, even at the very early stages of infection, suggesting that drugs that disrupt VSG monoallelic expression could be used to treat trypanosomiasis.

Protein O-Fucosyltransferase 2 Is Not Essential for Plasmodium berghei Development.

Thrombospondin type I repeat (TSR) domains are commonly O-fucosylated by protein O-fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite Plasmodium falciparum expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are O-fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant P. falciparum and Plasmodium berghei (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, P. berghei PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of O-fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology.

Mass spectrometry-based plant metabolomics: Metabolite responses to abiotic stress.

Metabolomics is one omics approach that can be used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include analysis of a wide range of chemical species with diverse physical properties, from ionic inorganic compounds to biochemically derived hydrophilic carbohydrates, organic and amino acids, and a range of hydrophobic lipid-related compounds. This complexitiy brings huge challenges to the analytical technologies employed in current plant metabolomics programs, and powerful analytical tools are required for the separation and characterization of this extremely high compound diversity present in biological sample matrices. The use of mass spectrometry (MS)-based analytical platforms to profile stress-responsive metabolites that allow some plants to adapt to adverse environmental conditions is fundamental in current plant biotechnology research programs for the understanding and development of stress-tolerant plants. In this review, we describe recent applications of metabolomics and emphasize its increasing application to study plant responses to environmental (stress-) factors, including drought, salt, low oxygen caused by waterlogging or flooding of the soil, temperature, light and oxidative stress (or a combination of them). Advances in understanding the global changes occurring in plant metabolism under specific abiotic stress conditions are fundamental to enhance plant fitness and increase stress tolerance. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:620-649, 2016.

Sugar activation and glycosylation in Plasmodium.

Glycoconjugates are important mediators of host-pathogen interactions and are usually very abundant in the surface of many protozoan parasites. However, in the particular case of Plasmodium species, previous works show that glycosylphosphatidylinositol anchor modifications, and to an unknown extent, a severely truncated N-glycosylation are the only glycosylation processes taking place in the parasite. Nevertheless, a detailed analysis of the parasite genome and the recent identification of the sugar nucleotide precursors biosynthesized by Plasmodium falciparum support a picture in which several overlooked, albeit not very prominent glycosylations may be occurring during the parasite life cycle. In this work, the authors review recent developments in the characterization of the biosynthesis of glycosylation precursors in the parasite, focusing on the outline of the possible fates of these precursors.

Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei.

Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known ß1-4-galactosyltransferase or ß1-2- or ß1-6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian ß1-3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:α3-D-mannoside ß1-2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical "pseudohybrid" glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man3GlcNAc2, but not to triantennary Man5GlcNAc2, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the ß3-glycosyltransferase family to catalyze ß1-2 linkages.

Evaluation of beer deterioration by gas chromatography-mass spectrometry/multivariate analysis: a rapid tool for assessing beer composition.

Beer stability is a major concern for the brewing industry, as beer characteristics may be subject to significant changes during storage. This paper describes a novel non-targeted methodology for monitoring the chemical changes occurring in a lager beer exposed to accelerated aging (induced by thermal treatment: 18 days at 45 °C), using gas chromatography-mass spectrometry in tandem with multivariate analysis (GC-MS/MVA). Optimization of the chromatographic run was performed, achieving a threefold reduction of the chromatographic time. Although losing optimum resolution, rapid GC runs showed similar chromatographic profiles and semi-quantitative ability to characterize volatile compounds. To evaluate the variations on the global volatile signature (chromatographic profile and m/z pattern of fragmentation in each scan) of beer during thermal deterioration, a non-supervised multivariate analysis method, Principal Component Analysis (PCA), was applied to the GC-MS data. This methodology allowed not only the rapid identification of the degree of deterioration affecting beer, but also the identification of specific compounds of relevance to the thermal deterioration process of beer, both well established markers such as 5-hydroxymethylfufural (5-HMF), furfural and diethyl succinate, as well as other compounds, to our knowledge, newly correlated to beer aging.

Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases.

Asparagine-linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single-subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Deltastt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N-glycosylation showed that TbSTT3A selectively transfers biantennary Man(5)GlcNAc(2) to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man(9)GlcNAc(2) to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single-subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N-glycosylation could have therapeutic potential against trypanosomiasis.

Bioactivity studies and chemical profile of the antidiabetic plant Genista tenera.

AIM OF THE STUDY: Genista tenera is a plant endemic to the island of Madeira and is used in folk medicine to control diabetes. In the present work we evaluate the antihyperglycaemic activity of its n-butanol extract and determine its chromatographic profile. In addition, this extract, the ethyl acetate and diethyl ether plant extracts were studied in order to assess the plant antioxidant and acetylcholinesterase inhibitory activities, as well as its cyto- and genotoxicities. MATERIALS AND METHODS: HPLC-DAD-ESI-MS was used to analyze the flavonoid profile of the n-butanol extract. The antihyperglycaemic activity of this extract was performed over streptozotocin induced diabetic Wistar rats (200 mg/kg, bw/day), for 15 days. Antioxidant activity (DPPH assay) and acetylcholinesterase inhibitory effect (Ellman method) were also performed. Acute cytotoxicity and genotoxicity were assessed by proliferative index quantification and the short-term chromosomal aberration technique, after exposure of lymphocytes to the extracts. RESULTS AND CONCLUSIONS: The n-butanol extract, where 21 monoglycosyl and 12 diglycosyl flavonoids were detected, significantly lowered blood glucose levels, bringing them to normal values after 15 days of treatment. The best radical scavenging activity was observed for the ethyl acetate extract (48.7% at 139.1 microg/mL), which was also the most effective one at the minimal concentration tested. The highest acetylcholinesterase inhibitory activity (77.0% at 70.0 microg/mL) was also obtained with the ethyl acetate extract. In vitro toxicity studies showed no evidence for acute cytotoxicity or genotoxicity. This is the first report on antidiabetic activity of genus Genista.

Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase has unusual substrate specificity and protects the parasite from stress.

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37 degrees C, it is essential for parasite growth and survival at 40 degrees C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.

NopM and NopD are rhizobial nodulation outer proteins: identification using LC-MALDI and LC-ESI with a monolithic capillary column.

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLC-MS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD.

Capillary electrophoresis-mass spectrometry characterisation of secondary metabolites from the antihyperglycaemic plant Genista tenera.

Genista tenera is endemic to the Portuguese island of Madeira, where an infusion of the aerial parts of the plant is used in folk medicine as an antidiabetic agent. Consequently the medicinal properties of the secondary metabolites of this plant have been the subject of an ongoing study. A recently reported LC-MS method using a 100 min separation allowed identification of five flavonoid components in an extract of the aerial parts of this plant. In order to obtain additional information on the range and complexity of the plant's secondary metabolite components a CE-MS method has been developed and applied for the analysis of an extract of G. tenera. Twenty-six different components are distinguished in an analysis time of only 10 min. Results demonstrate that CE-MS/MS rapidly generates data complementary to those obtainable by LC-MS/MS and is particularly suited to the analysis of plant metabolites where concentration is not limiting.

Structural analysis of the capsular polysaccharide from Sinorhizobium fredii HWG35.

We have determined the structure of a capsular polysaccharide from Sinorhizobium fredii HWG35. This polysaccharide was isolated following the standard protocols applied for lipopolysaccharide isolation. On the basis of monosaccharide analysis, methylation analysis, mass spectrometric analysis, one-dimensional (1)H and (13)C NMR, and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the following disaccharide repeating unit: -->6)-2,4-di-O-methyl-alpha-d-Galp-(1-->4)-beta-d-GlcpA-(1-->. Strain HWG35 produces a capsular polysaccharide that does not show the structural motif (sugar-Kdx) observed in those S. fredii strains that, while effective with Asiatic soybean cultivars, are unable to form nitrogen-fixing nodules with American soybean cultivars. Instead, the structure of the capsular polysaccharide of S. fredii HWG35 is in line with those produced by strains HH303 (rhamnose and galacturonic acid) and B33 (4-O-methylglucose-3-O-methylglucuronic acid), two S. fredii strains that form nitrogen-fixing nodules with both groups of soybean cultivars. Hence, in these three strains that effectively nodulate American soybean cultivars, the repeating unit of the capsular polysaccharide is composed of two hexoses, one neutral (methylgalactose, rhamnose, or methylglucose) and the other acidic (glucuronic, galacturonic, or methylglucuronic acid).