Relations structure-allergénicité dans les protéines de blé

in "INRA en lumière - 5 ans de collaborations avec SOLEIL" [Rapport de recherche

Grãos de sorgo imaturos como promoção da bioeconomia e da nutrição humana

International audienceObjetivo: Caracterizar o perfil de compostos fenólicos e atividade antioxidante de diferentes genótipos de sorgo ao longo da maturação do grão usando abordagens metabolômicas para valorização da cultura destinada à alimentação humana deste cereal no Brasil. Metodologia: Amostras de sorgo (Sorghum bicolor L., caudatum) foram cultivadas em 2017 (Mauguio, França) e fornecidas pela unidade “Melhoramento genético e adaptação de plantas tropicais e mediterrâneas” (CIRAD, INRA, Montpellier). Cinco estágios de maturação de grãos (S1-S4 e maduro) do genótipo IS15752 foram colhidos em 7, 21, 29, 37 e 40 dias após a antese, respectivamente, e liofilizados até 12% de umidade (Figura 1). Os grãos inteiros foram pesados, moídos criogenicamente usando moinho de bolas e analisados por colorimetria (a*, b*, L*). Os compostos livres foram extraídos em solução etanólica 80% e os compostos ligados a partir dos pellets após hidrólise alcalina seguida por hidrólise ácida. A quantificação do teor de compostos redutores (TCR) totais nos extratos foi feito em microplacas (FlexStation III, Molecular Devices) com modificações (Bobo-Garcia et al., 2015) e os resultados foram expressos pela média das triplicatas em mg de equivalentes de ácido gálico (EAG) por grama de grão (bs). A atividade antioxidante pela inibição do Radical DPPH foi adaptado para microplacas, de acordo com Sompong et al. (2011) e os dados foram expressos pela média das triplicatas em mg de equivalentes de Trolox (TE) por grama de grão (bs). Para a determinação do perfil dos compostos fenólicos por UPLC-MSE foi utilizado o sistema UPLC Acquity acoplado ao Xevo G2-S Q-ToF (Waters) com fonte de ionização por eletrospray (ESI) e analisadores de massas tipo quadrupolo e tempo de voo (QToF). Resultados: A evolução do peso seco médio do grão ao longo do desenvolvimento permite a compreensão das fases de maturação do grão. O TCR decaiu nos extratos livres, enquanto nos extratos ligados houve uma maior variação que não acompanhou a maturação do grão. Os grãos mais imaturos (S1) apresentaram TCR significativamente maior nos extratos livres, enquanto os estágios mais maduros (S3-maduro) apresentaram maior teor de compostos ligados, provavelmente devido a mudanças oxidativas ou enzimáticas sofridas pelos compostos na matriz do grão de sorgo. Os resultados da capacidade antioxidante seguiram o mesmo perfil do TCR, apresentando forte correlação positiva. Na análise metabolômica, foram tentativamente identificados 45 compostos fenólicos livres e 68 ligados. O extrato ligado corresponde a 70% da abundância total de ions e estão presentes principalmente em S1, S2 e maduro; enquanto os fenólicos livres solúveis foram maiores nos estágios imaturos, diminuindo ao longo da maturação dos grãos. Conclusão: Este trabalho parece ser o primeiro a revelar as diferenças nas composições fenólicas e capacidade antioxidante durante o desenvolvimento de grãos de sorgo e permite que seja utilizado como alimento funcional voltado à promoção da saúde pública

Transcriptomic analysis of developing sorghum grains to detect genes related to cell wall biosynthesis and remodelling

Abstract Objective Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important grain produced in the world. Interest for cultivating sorghum is increasing all over the world in the context of climate change, due to its low input and water requirements. Like other cultivated cereals, sorghum has significant nutritional value thanks to its protein, carbohydrate and dietary fiber content, these latter mainly consisting of cell wall polysaccharides. This work describes for the first time a transcriptomic analysis dedicated to identify the genes involved in the biosynthesis and remodelling of cell walls both in the endosperm and outer layers of sorghum grain during its development. Further analysis of these transcriptomic data will improve our understanding of cell wall assembly, which is a key component of grain quality. Data description This research delineates the steps of our analysis, starting with the cultivation conditions and the grain harvest at different stages of development, followed by the laser microdissection applied to separate the endosperm from the outer layers. It also describes the procedures implemented to generate RNA libraries and to obtain a normalized and filtered table of transcript counts, and finally determine the number of putative cell wall-related genes already listed in literature

Évolution du protéome de la graine de sorgho au cours de sa germination

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Mechanisms regulating the peripheral utilisation of glucose: involvement of AMPK

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IgE-binding epitopes on wheat LTP1 for patients with food allergy to wheat and experimentally sensitized mice

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Changes in metabolomic profile and antioxidant activity during sorghum grain growth

International audienceThe comprehensive phenolic profile, antioxidant activity and color of sorghum (Sorghum bicolor L., caudatum) grains were investigated in five different stages of growth (from cellular division to maturity). Grains were harvested (2018, Montpellier-France), freeze-dried and cryogenically ground. Colorimetric parameters (L*, a*, b*) were determined and free (FPC) and bound (BPC) phenolic compounds were extracted from wholegrain flours. Antioxidant capacity was determined by DPPH and Folin-Ciocalteu methods in microplates. Phenolic profile was analyzed by UPLC-ESI-QTOF-MSE in negative mode. Data were processed with Progenesis QI using a customized database (PubChem and Phenol explorer) applying: mass error (80%) and reproducibility (3/3). Sorghum grain flours became significantly darker and redness throughout maturation. FPC and BPC ranged from 207.19 to 1052.77 and 268.71 to 624.75 mg GAE/100g (db), respectively; and antioxidant activity between 10.84 to 108.68 and 29.92 to 67.50 mg TE/100g (db). The most immature stage (S1) had the highest FPC content and antioxidant activity in free extracts, while the highest values of BPC were found in mature grains (S4, mature). S2 represents the grain filling stage and showed the same values in both extracts. Globally, 69 FPC and 120 BPC were tentatively identified with 18 compounds present in both extracts. The first stages (S1-S3) showed the highest number and abundance of phenolic compounds. Bound phenolics were the most abundant (72%), mainly in S2, while free soluble phenolics were higher in immature stages, decreasing along grain maturation. PCA allowed a distinguished phenolic profile in each stage, where S1, S2 and mature presented more than 10 unique PC. This work seems to be the first to reveal the phenolic profiling based on metabolomics during sorghum grain growth. These data can be hereafter correlated with proteomic analysis, in order to better understand the digestion problematic involving tannins and kafirins in this cereal matrix

Cytidine deaminase deficiency in tumor cells is associated with sensitivity to a naphthol derivative and a decrease in oncometabolite levels

International audienceIdentifying new molecular targets for novel anticancer treatments is a major challenge in clinical cancer research. We have shown that cytidine deaminase (CDA) expression is downregulated in about 60% of cancer cells and tissues. In this study, we aimed to develop a new anticancer treatment specifically inhibiting the growth of CDA-deficient tumor cells. High-throughput screening of a chemical library led to the identification of a naphthol derivative, X55, targeting CDA-deficient tumor cells preferentially, without affecting the growth of non-tumoral cells regardless of CDA expression status. Metabolomic profiling revealed that CDA-deficient HeLa cells differed markedly from control HeLa cells. X55 treatment had a moderate effect on control cells, but greatly disturbed the metabolome of CDA-deficient HeLa cells, worsening the deregulation of many metabolites. In particular, the levels of the three oncometabolites, fumarate, succinate and 2-hydroxyglutarate, were significantly lower in CDA-depleted cells, and this decrease in levels was exacerbated by X55 treatment, revealing an unexpected link between CDA deficiency, mitochondrial function and X55 response. Finally, we identified strong downregulation of MAPT (encoding Tau, a microtubule associated protein) expression as a reliable predictive marker for tumor cell X55 sensitivity

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+

International audienceCytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity

A Recombinant omega-Gliadin-like D-Type Glutenin and an alpha-Gliadin from Wheat (Triticum aestivum): Two Immunoglobulin E Binding Proteins, Useful for the Diagnosis of Wheat-Dependent Allergies

Among the wheat prolamins, D-type glutenins display a highly repetitive sequence similar to omega-gliadins, but they contain a cysteine, that allows them to be included in the gluten macropolymers. An omega-gliadin-like D-type glutenin, an alpha-gliadin, and an omega 5-gliadin-like D-type glutenin were obtained as recombinant proteins and compared using synchrotron radiation circular dichroism. This technique evidenced the strong thermostability of the omega 5-gliadin-like protein. The IgE reactivity of recombinant proteins was evaluated using 45 sera from wheat-allergic patients. The sera from patients diagnosed with cutaneous hypersensitivity to hydrolyzed wheat proteins often reacted with the omega-gliadin-like D-type glutenin and alpha-gliadin, whereas the IgE reaction was less frequent after dietary sensitization. So, these two proteins could be useful to diagnose these diseases. The sera from patients with exercise-induced anaphylaxis recognized the omega 5-gliadin-like protein as a positive control and, less frequently, the other proteins tested. Only some sera from patients with baker's asthma reacted with the proteins tested