RESUMO
Breast milk composition is influenced by maternal diet. This study aimed to evaluate if supplementation of maternal diet with a prebiotic fibre, through its potential effect on milk composition, can be a leverage to orientate the gut microbiota of infants in a way that would be beneficial for their health. Twelve sows received a diet supplemented with short chain fructo-oligosaccharides or maltodextrins during the last month of gestation and the lactation. Oligosaccharidic and lipidomic profiles of colostrum and mature milk (21 days), as well as faecal microbiota composition and metabolomic profile of 21 day-old piglets were evaluated. The total porcine milk oligosaccharide concentration tended to be lower in scFOS-supplemented sows, mainly due to the significant reduction of the neutral core oligosaccharides (in particular that of a tetrahexose). Maternal scFOS supplementation affected the concentration of 31 lipids (mainly long-chain triglycerides) in mature milk. Faecal short-chain fatty acid content and that of 16 bacterial metabolites were modified by scFOS supplementation. Interestingly, the integrative data analysis gave a novel insight into the relationships between (i) maternal milk lipids and PMOs and (ii) offspring faecal bacteria and metabolites. In conclusion, scFOS-enriched maternal diet affected the composition of mature milk, and this was associated with a change in the colonisation of the offspring intestinal microbiota.
Assuntos
Lactação , Leite , Animais , Suínos , Gravidez , Feminino , Humanos , Leite/metabolismo , Projetos Piloto , Suplementos Nutricionais/análise , Dieta/veterinária , Metaboloma , Oligossacarídeos/metabolismo , Lipídeos , Ração Animal/análiseRESUMO
BACKGROUND: In proteomics, the interpretation of mass spectra representing peptides carrying multiple complex modifications remains challenging, as it is difficult to strike a balance between reasonable execution time, a limited number of false positives, and a huge search space allowing any number of modifications without a priori. The scientific community needs new developments in this area to aid in the discovery of novel post-translational modifications that may play important roles in disease. RESULTS: To make progress on this issue, we implemented SpecGlobX (SpecGlob eXTended to eXperimental spectra), a standalone Java application that quickly determines the best spectral alignments of a (possibly very large) list of Peptide-to-Spectrum Matches (PSMs) provided by any open modification search method, or generated by the user. As input, SpecGlobX reads a file containing spectra in MGF or mzML format and a semicolon-delimited spreadsheet describing the PSMs. SpecGlobX returns the best alignment for each PSM as output, splitting the mass difference between the spectrum and the peptide into one or more shifts while considering the possibility of non-aligned masses (a phenomenon resulting from many situations including neutral losses). SpecGlobX is fast, able to align one million PSMs in about 1.5 min on a standard desktop. Firstly, we remind the foundations of the algorithm and detail how we adapted SpecGlob (the method we previously developed following the same aim, but limited to the interpretation of perfect simulated spectra) to the interpretation of imperfect experimental spectra. Then, we highlight the interest of SpecGlobX as a complementary tool downstream to three open modification search methods on a large simulated spectra dataset. Finally, we ran SpecGlobX on a proteome-wide dataset downloaded from PRIDE to demonstrate that SpecGlobX functions just as well on simulated and experimental spectra. We then carefully analyzed a limited set of interpretations. CONCLUSIONS: SpecGlobX is helpful as a decision support tool, providing keys to interpret peptides carrying complex modifications still poorly considered by current open modification search software. Better alignment of PSMs enhances confidence in the identification of spectra provided by open modification search methods and should improve the interpretation rate of spectra.
Assuntos
Peptídeos , Proteômica , Proteômica/métodos , Bases de Dados de Proteínas , Espectrometria de Massas/métodos , Software , AlgoritmosRESUMO
High consumption of plant sterols reduces the risk of cardiovascular diseases in humans and provides health benefits. Increasing the amount of plant sterols in the diet is therefore necessary to reach the recommended daily dietary intake. However, food supplementation with free plant sterols is challenging because of their low solubility in fats and water. The objectives of this study were to investigate the capacity of milk-sphingomyelin (milk-SM) and milk polar lipids to solubilise ß-sitosterol molecules in bilayer membranes organised as vesicles called sphingosomes. The thermal and structural properties of milk-SM containing bilayers composed of various amounts of ß-sitosterol were examined by differential scanning calorimetry (DSC) and temperature-controlled X-ray diffraction (XRD), the molecular interactions were studied using the Langmuir film technique, the morphologies of sphingosomes and ß-sitosterol crystals were observed by microscopy. We showed that the milk-SM bilayers devoid of ß-sitosterol exhibited a gel to fluid Lα phase transition for Tm = 34.5 °C and formed facetted spherical sphingosomes below Tm. The solubilisation of ß-sitosterol within milk-SM bilayers induced a liquid-ordered Lo phaseabove 25 %mol (1.7 %wt) ß-sitosterol and a softening of the membranes leading to the formation of elongated sphingosomes. Attractive molecular interactions revealed a condensing effect of ß-sitosterol on milk-SM Langmuir monolayers. Above 40 %mol (25.7 %wt) ß-sitosterol, partitioning occured with the formation of ß-sitosterol microcrystals in the aqueous phase. Similar results were obtained with the solubilization of ß-sitosterol within milk polar lipid vesicles. For the first time, this study highlighted the efficient solubilization of free ß-sitosterol within milk-SM based vesicles, which opens new market opportunities for the formulation of functional foods enriched in non-crystalline free plant sterols.
Assuntos
Leite , Fitosteróis , Humanos , Animais , Esfingomielinas , SitosteroidesRESUMO
Ovalbumin (OVA) is a food allergen whose allergenicity is modulated by heating. We aimed to establish a molecular connection between heat-induced structural modifications and the modulation of the IgE binding capacity of OVA. For this, we used model samples of heat-modified OVA with increasing complexity; glycated, aggregated, or glycated and aggregated. Using sera from egg-allergic individuals, we show that both aggregation and glycation strongly impacted IgE binding capacity, despite limited structural changes for glycated OVA. A molecular exploration at the amino acid level using high-resolution mass spectrometry revealed extensive cross-linking, mostly through disulfide and dehydroprotein bridges, and moderate but significant glycation. Structural modifications affected residues located within or at a few amino acids distance of known human linear IgE epitopes, such as C121, K123, S169, K190, K207, H332 and C368. We thus unveil key amino residues implicated in the changes in IgE binding of OVA induced by heating.
Assuntos
Hipersensibilidade Alimentar , Imunoglobulina E , Alérgenos/química , Alérgenos/genética , Calefação , Humanos , Imunoglobulina E/metabolismo , Espectrometria de Massas , Ovalbumina/químicaRESUMO
Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient's sera and with mouse monoclonal antibodies. Disruption of the C28-C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13-C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues (C13, C27, C28, C73 and K72), four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity.
Assuntos
Alérgenos , Triticum , Animais , Dissulfetos/química , Imunoglobulina E , Camundongos , Mutagênese Sítio-Dirigida , Proteínas de Plantas/metabolismo , Triticum/metabolismoRESUMO
SUMMARY: Oligator is software designed to assist scientists in their exploration of MS/MS experiments, especially for oligosaccharides bearing unreferenced chemical substitutions. Through a graphical interface, users have the total flexibility to build a candidate glycan structure and produce the corresponding theoretical MS/MS spectrum in accordance with the usual ion nomenclature. The structural information is saved using standard notations, in text format, which facilitates the capitalization and exchange of data as well as any other processing of the information. AVAILABILITY AND IMPLEMENTATION: Source code and user manual are freely available at https://github.com/vlollier/oligator. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Software , Espectrometria de Massas em Tandem , Oligossacarídeos , PolissacarídeosRESUMO
The authors wish to make the following corrections to this paper [...].
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The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.
Assuntos
Parede Celular/metabolismo , Grão Comestível/crescimento & desenvolvimento , Endosperma/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Sementes/metabolismo , Triticum/embriologia , Triticum/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Grão Comestível/citologia , Grão Comestível/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Polissacarídeos/metabolismoRESUMO
The remodeling of cell wall polysaccharides is controlled by cell wall proteins (CWPs) during the development of wheat grain. This work describes for the first time the cell wall proteomes of the endosperm and outer layers of the wheat developing grain, which have distinct physiological functions and technological uses. Altogether 636 nonredundant predicted CWPs are identified with 337 proteins in the endosperm and 594 proteins in the outer layers, among which 295 proteins are present in both tissues, suggesting both common and tissue specific remodeling activities. These proteins are distributed into eight functional classes. Approximatively a quarter of them were predicted to act on cell wall polysaccharides, with many glycosylhydrolases and also expansin, lyases, and carbohydrate esterases. Therefore, these results provide crucial data to go further in the understanding of relationship between tissue-specific morphogenesis and cell wall remodeling in cereals. Data are available via ProteomeXchange with identifier PXD010367.
Assuntos
Endosperma/metabolismo , Proteoma/análise , Triticum/metabolismo , Parede Celular/metabolismo , Grão Comestível/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Several proteomic database search engines that interpret LC-MS/MS data do not identify the same set of peptides. These disagreements occur even when the scores of the peptide-to-spectrum matches suggest good confidence in the interpretation. Our study shows that these disagreements observed for the interpretations of a given spectrum are almost exclusively due to the variation of what we call the "peptide space", i.e., the set of peptides that are actually compared to the experimental spectra. We discuss the potential difficulties of precisely defining the "peptide space." Indeed, although several parameters that are generally reported in publications can easily be set to the same values, many additional parameters-with much less straightforward user access-might impact the "peptide space" used by each program. Moreover, in a configuration where each search engine identifies the same candidates for each spectrum, the inference of the proteins may remain quite different depending on the false discovery rate selected.
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Interpretação Estatística de Dados , Proteômica/métodos , Ferramenta de Busca/normas , Espectrometria de Massas em Tandem/normas , Bases de Dados de Proteínas , Reações Falso-Positivas , Proteômica/normasRESUMO
Brachypodiumdistachyon is a suitable plant model for studying temperate cereal crops, such as wheat, barley or rice, and helpful in the study of the grain cell wall. Indeed, the most abundant hemicelluloses that are in the B. distachyon cell wall of grain are (1-3)(1-4)-ß-glucans and arabinoxylans, in a ratio similar to those of cereals such as barley or oat. Conversely, these cell walls contain few pectins and xyloglucans. Cell walls play an important role in grain physiology. The modifications of cell wall polysaccharides that occur during grain development and filling are key in the determination of the size and weight of the cereal grains. The mechanisms required for cell wall assembly and remodelling are poorly understood, especially in cereals. To provide a better understanding of these processes, we purified the cell wall at three developmental stages of the B. distachyon grain. The proteins were then extracted, and a quantitative and comparative LC-MS/MS analysis was performed to investigate the protein profile changes during grain development. Over 466 cell wall proteins (CWPs) were identified and classified according to their predicted functions. This work highlights the different proteome profiles that we could relate to the main phases of grain development and to the reorganization of cell wall polysaccharides that occurs during these different developmental stages. These results provide a good springboard to pursue functional validation to better understand the role of CWPs in the assembly and remodelling of the grain cell wall of cereals.
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Food allergy has become a major health issue in developed countries, therefore there is an urgent need to develop analytical methods able to detect and quantify with a good sensitivity and reliability some specific allergens in complex food matrices. In this paper, we present a targeted MS/MS approach to compare the relative abundance of the major recognized wheat allergens in the salt-soluble (albumin/globulin) fraction of wheat grains. Twelve allergens were quantified in seven wheat varieties, selected from three Triticum species: T. aestivum (bread wheat), T. durum (durum wheat), and T. monococcum. The allergens were monitored from one or two proteotypic peptides and their relative abundance was deduced from the intensity of one fragment measured in MS/MS. Whereas the abundance of some of the targeted allergens was quite stable across the genotypes, others like alpha-amylase inhibitors showed clear differences according to the wheat species, in accordance with the results of earlier functional studies. This study enriches the scarce knowledge available on allergens content in wheat genotypes, and brings new perspectives for food safety and plant breeding.
Assuntos
Alérgenos/imunologia , Espectrometria de Massas em Tandem/métodos , Triticum/imunologia , Alérgenos/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Análise de Componente PrincipalRESUMO
Cereal grain outer layers fulfil essential functions for the developing seed such as supplying energy and providing protection. In the food industry, the grain outer layers called 'the bran' is valuable since it is rich in dietary fibre and other beneficial nutriments. The outer layers comprise several tissues with a high content in cell wall material. The cell wall composition of the grain peripheral tissues was investigated with specific probes at a stage of active cell wall synthesis. Considerable wall diversity between cell types was revealed. To identify the cellular machinery involved in cell wall synthesis, a subcellular proteomic approach was used targeting the Golgi apparatus where most cell wall polysaccharides are synthesized. The tissues were dissected into outer pericarp and intermediate layers where 822 and 1304 proteins were identified respectively. Many carbohydrate-active enzymes were revealed: some in the two peripheral grain fractions, others only in one tissue. Several protein families specific to one fraction and with characterized homologs in other species might be related to the specific detection of a polysaccharide in a particular cell layer. This report provides new information on grain cell walls and its biosynthesis in the valuable outer tissues, which are poorly studied so far. A better understanding of the mechanisms controlling cell wall composition could help to improve several quality traits of cereal products (e.g. dietary fibre content, biomass conversion to biofuel).
Assuntos
Parede Celular/metabolismo , Fibras na Dieta/metabolismo , Triticum/metabolismo , Parede Celular/enzimologia , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Proteômica , Triticum/enzimologiaRESUMO
Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.
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Brachypodium/metabolismo , Parede Celular/metabolismo , Proteômica/métodos , Proteínas de Plantas/metabolismoRESUMO
IgE-binding epitopes are related to allergic symptoms by eliciting degranulation of special cells and release of molecules that trigger the hypersensitivity reaction. Little is known about what characterises allergen IgE-binding epitopes, although advances in analytical methods have led to the identification of a large number of them. To assess if a binary classification of allergen regions into epitopes or non-epitopes may accurately reflect biological reality, we computed the fraction of allergen amino acids that are involved in epitopes. A relationship between this fraction and the increasing number of literature references was modelled. Due to the wide variety of methods that are used in the literature, a peak in the number of matches between an allergen sequence and its epitopes confirms their validity. Accordingly, our graphical representation of positive assays along sequences provides an overview of epitope localisation, which should help to highlight major positions for IgE binding to allergens.
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Alérgenos/imunologia , Epitopos/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Alérgenos/química , Sequência de Aminoácidos , Mapeamento de Epitopos/métodos , Epitopos/química , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Methods that predict antibody epitopes could help to promote the development of diagnostic tools, vaccines or immunotherapies by affecting the epitope binding of antibodies during an immunological response to antigens. It is generally assumed that there is a direct relationship between antibody accessibility to antigens and accessible surface of proteins. Based on this assumption, prediction systems often includes solvent accessibility values calculated from the primary sequence of proteins or from their three dimensional structures as a predictive criterion. However, the current prediction systems seem weakly efficient in view of benchmark tests. We were interested in evaluating how amino acids that have been experimentally identified as epitopic elements could differ from the rest of the antigenic molecule at the level of surface exposure, hence we assessed the average accessibility of epitopes. The approach used here utilises published epitopes deduced from numerous identification techniques, including sequence scanning and structure visualisation after crystallography, and it involves many types of antigens from toxins to allergens. Our results show that epitopic residues are not distributed among any specific Relative Surface Accessibility and Protrusion Index values and that, in some cases, epitopes cover the entire antigenic sequence. These results led to the conclusion that the classification of known epitopes with respect to the experimental conditions used to identify them should be introduced before attempting to characterise epitopic areas in a generic way.
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Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , Proteínas/química , Proteínas/imunologia , Sequência de Aminoácidos , Aminoácidos/imunologia , Animais , Bases de Dados de Proteínas , Humanos , Dados de Sequência Molecular , Software , Propriedades de SuperfícieRESUMO
The tropical and subtropical parthenogenetic plant-parasitic nematodes Meloidogyne are polyphagous major agricultural pests. Implementing proper pest management approaches requires a good understanding of mechanisms, population structure, evolutionary patterns and species identification. A comparative analysis of the mitochondrial vs nuclear diversity was conducted on a selected set of Meloidogyne lines from various geographic origins. Mitochondrial co2-16S sequences and AFLP markers of total DNA were applied because of their ability to evidence discrete genetic variation between closely related isolates. Several distinct maternal lineages were present, now associated with different genetic backgrounds. Relative discordances were found when comparing mitochondrial and nuclear diversity patterns. These patterns are most likely related to crosses within one ancestral genetic pool, followed by the establishment of parthenogenesis. In this case, they mirror the genetic backgrounds of the original individuals. Another aspect could be that species emergence was recent or on process from this original genetic pool and that the relatively short time elapsed since then and before parthenogenesis settlement did not allow for lineage sorting. This could also be compatible with the hypothesis of hybrids between closely related species. This genetic pool would correspond to a species as defined by the species interbreeding concept, but also including the grey area of species boundaries. This complex process has implications on the way genotypic and phenotypic diversity should be addressed. The phenotype of parthenogenetic lines is at least for part determined by the ancestral amphimictic genetic background. A direct consequence is, therefore, in terms of risk management, the limited confidence one can have on the direct association of an agronomic threat to a simple typing or species delineation. Risk management strategies and tools must thus consider this complexity when designing quarantine implementation, resistance breeding programmes or molecular diagnostic.
Assuntos
Cruzamentos Genéticos , Variação Genética , Partenogênese/genética , Tylenchoidea/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Biodiversidade , Evolução Molecular , Genes Mitocondriais/genética , Especiação Genética , Partenogênese/fisiologia , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Infecções por Secernentea/parasitologia , Especificidade da Espécie , Clima Tropical , Tylenchoidea/classificaçãoRESUMO
Species delineation in parthenogenetic tropical species of Meloidogyne nematodes is particularly difficult although they are strictly apomictic. In fact, parthenogenesis in Meloidogyne nematodes is a recent phenomenon and the structure of the genetic diversity is mainly explained by crosses prior to the establishment of parthenogenesis. Under such hypothesis, increasing the size of a characterized sample by adding individuals should result in the decrease of the diversity structure. Twelve individuals from different geographical origins were added to the initial pool of 26 lines characterized in a previous study and an AFLP study was conducted on the whole set of 38 lines. As expected under the panmixy hypothesis, this resulted in a loss of genetic structure. This confirms thus that the genetic structure of tropical parthenogenetic Meloidogyne is due to crosses anterior to the establishment of apomixy.
Assuntos
Genes de Helmintos , Tylenchoidea/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Análise por Conglomerados , DNA de Helmintos/análise , DNA de Helmintos/genética , Evolução Molecular , Variação Genética , Partenogênese , Especificidade da EspécieRESUMO
M. chitwoodi and M. fallax populations are clustered and separated from the other species studied. The genetic diversity observed for M. incognita, M. arenaria, M. javanica, M. hapla, and M. mayaguensis correlates well with the previously validated species. Two main groups can be identified within the M. chitwoodi/M. fallax cluster, the first group comprises only M. chitwoodi populations whereas the second group is made of M. chitwoodi and M. fallax populations. Moreover, M. chitwoodi displays a higher genetic diversity than M. fallax and is characterised by the presence of several clusters.