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INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .
Assuntos
Metabolômica , Metadados , Curadoria de Dados/métodos , Espectrometria de Massas/métodos , Metaboloma , Metabolômica/métodosRESUMO
The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.
Assuntos
Apolipoproteínas/sangue , Apolipoproteínas/metabolismo , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Trypanosoma brucei gambiense/fisiologia , África , Animais , Animais Geneticamente Modificados , Apolipoproteína L1 , Apolipoproteínas/antagonistas & inibidores , Apolipoproteínas/toxicidade , Membrana Celular/química , Membrana Celular/metabolismo , Cisteína Proteases/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Hemólise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos , Lipoproteínas HDL/antagonistas & inibidores , Lipoproteínas HDL/química , Lipoproteínas HDL/toxicidade , Parasitos/patogenicidade , Parasitos/fisiologia , Estrutura Secundária de Proteína , Soro/química , Soro/parasitologia , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/patogenicidade , Tripanossomíase Africana/parasitologia , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
INTRODUCTION: In Northern Europe, maize early-sowing used to maximize yield may lead to moderate damages of seedlings due to chilling without visual phenotypes. Genetic studies and breeding for chilling tolerance remain necessary, and metabolic markers would be particularly useful in this context. OBJECTIVES: Using an untargeted metabolomic approach on a collection of maize hybrids, our aim was to identify metabolite signatures and/or metabolites associated with chilling responses at the vegetative stage, to search for metabolites differentiating groups of hybrids based on silage-earliness, and to search for marker-metabolites correlated with aerial biomass. METHODS: Thirty genetically-diverse maize dent inbred-lines (Zea mays) crossed to a flint inbred-line were sown in a field to assess metabolite profiles upon cold treatment induced by a modification of sowing date, and characterized with climatic measurements and phenotyping. RESULTS: NMR- and LC-MS-based metabolomic profiling revealed the biological variation of primary and specialized metabolites in young leaves of plants before flowering-stage. The effect of early-sowing on leaf composition was larger than that of genotype, and several metabolites were associated to sowing response. The metabolic distances between genotypes based on leaf compositional data were not related to the genotype admixture groups, and their variability was lower under early-sowing than normal-sowing. Several metabolites or metabolite-features were related to silage-earliness groups in the normal-sowing condition, some of which were confirmed the following year. Correlation networks involving metabolites and aerial biomass suggested marker-metabolites for breeding for chilling tolerance. CONCLUSION: After validation in other experiments and larger genotype panels, these marker-metabolites can contribute to breeding.
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Metabolômica , Melhoramento Vegetal , Zea mays/metabolismo , Biomarcadores/metabolismo , Cromatografia Líquida , Fenótipo , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles.
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Antígenos de Plantas/química , Bicamadas Lipídicas/química , Lipossomos/química , Proteínas de Plantas/química , Borracha/química , Alérgenos/química , Hevea/química , Látex/química , Espectroscopia de Ressonância Magnética , Proteínas Recombinantes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Ressonância de Plasmônio de SuperfícieRESUMO
Oleic acid vesicles represent good models of membrane protocells that could have existed in prebiotic times. Here, we report the formation, growth polymorphism, and dynamics of oleic acid spherical vesicles (1-10 µm), stable elongated vesicles (>50 µm length; 1-3 µm diameter), and chains of vesicles (pearl necklaces, >50 µm length; 1-3 µm diameter) in the presence of aminopropyl triethoxysilane and guanidine hydrochloride. These vesicles exhibit a remarkable behavior with temperature: spherical vesicles only are observed when keeping the sample at 4 °C for 2 h, and self-aggregated spherical vesicles occur upon freezing/unfreezing (-20/20 °C) samples. Rather homogeneous elongated vesicles are reformed upon heating samples at 80 °C. The phenomenon is reversible through cycles of freezing/heating or cooling/heating of the same sample. Deuterium NMR evidences a chain packing rigidity similar to that of phospholipid bilayers in cellular biomembranes. We expect these bilayered vesicles to be surrounded by a layer of aminosilane oligomers, offering a variant model for membrane protocells.
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Aminas/química , Grafite/química , Modelos Moleculares , Ácido Oleico/química , Silanos/química , Vesículas Transportadoras/química , Simulação de Dinâmica MolecularRESUMO
The dynamic in vivo profiling of lactate is of uppermost importance in both neuroenergetics and neuroprotection fields, considering its central suspected role as a metabolic and signaling molecule. For this purpose, we implemented proton magnetic resonance spectroscopy (1H-MRS) directly on brain microdialysate to monitor online the fluctuation of lactate contents during neuronal stimulation. Brain activation was obtained by right whisker stimulation of rats, which leads to the activation of the left barrel cortex area in which the microdialysis probe was implanted. The experimental protocol relies on the use of dedicated and sensitive home-made NMR microcoil able to perform lactate NMR profiling at submillimolar concentration. The MRS measurements of extracellular lactate concentration were performed inside a pre-clinical MRI scanner allowing simultaneous visualization of the correct location of the microprobe by MRI and detection of metabolites contained in the microdialysis by MRS. A 40% increase in lactate concentration was measured during whisker stimulation in the corresponding barrel cortex. This combination of microdialysis with online MRS/MRI provides a new approach to follow in vivo lactate fluctuations, and can be further implemented in physio-pathological conditions to get new insights on the role of lactate in brain metabolism and signaling.
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Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCgamma and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCgamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCgamma blocks nuclear envelope formation. In vivo, PLCgamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCgamma and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCgamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated.
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Membrana Nuclear/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipase C gama/metabolismo , Espermatozoides/citologia , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Lytechinus , Masculino , Dados de Sequência Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/química , Fosforilação , Espermatozoides/metabolismo , Strongylocentrotus purpuratus , Espectrometria de Massas em TandemRESUMO
In order to study metabolic processes in animal models of diseases and in patients, microdialysis probes have evolved as powerful tools that are minimally invasive. However, analyses of microdialysate, performed remotely, do not provide real-time monitoring of microdialysate composition. Microdialysate solutions can theoretically be analyzed online inside a preclicinal or clinical MRI scanner using MRS techniques. Due to low NMR sensitivity, acquisitions of real-time NMR spectra on very small solution volumes (µL) with low metabolite concentrations (mM range) represent a major issue. To address this challenge we introduce the approach of combining a microdialysis probe with a custom-built magnetic resonance microprobe that allows for online metabolic analysis (1H and 13C) with high sensitivity under continuous flow conditions. This system is mounted inside an MRI scanner and allows performing simultaneously MRI experiments and rapid MRS metabolic analysis of the microdialysate. The feasibility of this approach is demonstrated by analyzing extracellular brain cancer cells (glioma) in vitro and brain metabolites in an animal model in vivo. We expect that our approach is readily translatable into clinical settings and can be used for a better and precise understanding of diseases linked to metabolic dysfunction.
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Espectroscopia de Ressonância Magnética/métodos , Microdiálise/métodos , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Desenho de Equipamento , Feminino , Glioblastoma/metabolismo , Humanos , Imageamento por Ressonância Magnética/instrumentação , Espectroscopia de Ressonância Magnética/instrumentação , Microdiálise/instrumentação , Sistemas On-Line , Ratos , Ratos WistarRESUMO
The role of phosphoinositides has been thoroughly described in many signalling and membrane trafficking events but their function as modulators of membrane structure and dynamics in membrane fusion has not been investigated. We have reconstructed models that mimic the composition of nuclear envelope precursor membranes with naturally elevated amounts of phosphoinositides. These fusogenic membranes (membrane vesicle 1(MV1) and nuclear envelope remnants (NER) are critical for the assembly of the nuclear envelope. Phospholipids, cholesterol, and polyphosphoinositides, with polyunsaturated fatty acid chains that were identified in the natural nuclear membranes by lipid mass spectrometry, have been used to reconstruct complex model membranes mimicking nuclear envelope precursor membranes. Structural and dynamic events occurring in the membrane core and at the membrane surface were monitored by solid-state deuterium and phosphorus NMR. "MV1-like" (PCâ¶PIâ¶PIPâ¶PIP(2), 30â¶20â¶18â¶12, mol%) membranes that exhibited high levels of PtdIns, PtdInsP and PtdInsP(2) had an unusually fluid membrane core (up to 20% increase, compared to membranes with low amounts of phosphoinositides to mimic the endoplasmic reticulum). "NER-like" (PCâ¶CHâ¶PIâ¶PIPâ¶PIP(2), 28â¶42â¶16â¶7â¶7, mol%) membranes containing high amounts of both cholesterol and phosphoinositides exhibited liquid-ordered phase properties, but with markedly lower rigidity (10-15% decrease). Phosphoinositides are the first lipids reported to counterbalance the ordering effect of cholesterol. At the membrane surface, phosphoinositides control the orientation dynamics of other lipids in the model membranes, while remaining unchanged themselves. This is an important finding as it provides unprecedented mechanistic insight into the role of phosphoinositides in membrane dynamics. Biological implications of our findings and a model describing the roles of fusogenic membrane vesicles are proposed.
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Fusão de Membrana , Membranas Artificiais , Membrana Nuclear/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Colesterol/metabolismo , Fluidez de Membrana , FosforilaçãoRESUMO
The formation of the nuclear envelope (NE) typically occurs once during every mitotic cycle in somatic cells, and also around the sperm nucleus following fertilization. Much of our understanding of NE assembly has been derived from systems modeling the latter event in vitro. In these systems, demembranated sperm nuclei are combined with fertilized egg cytoplasmic extracts and an ATP-regenerating system and in a multistep process they form the functional double bilayer of the NE. Using a system that we developed from sea urchin gametes, we have demonstrated that NE assembly is regulated by membrane vesicles in a spatial and temporal fashion, emphasizing the roles of phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), diacylglycerols (DAG), and lipid-modifying enzymes in NE assembly.