NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs

AbstractP75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism

Structural Basis of p75 Transmembrane Domain Dimerization

Dimerization of single span transmembrane receptors underlies their mechanism of activation. p75 neurotrophin receptor plays an important role in the nervous system, but the understanding of p75 activation mechanism is still incomplete. The transmembrane (TM) domain of p75 stabilizes the receptor dimers through a disulfide bond, essential for the NGF signaling. Here we solved by NMR the three-dimensional structure of the p75-TM-WT and the functionally inactive p75-TM-C257A dimers. Upon reconstitution in lipid micelles, p75-TM-WT forms the disulfide-linked dimers spontaneously. Under reducing conditions, p75-TM-WT is in a monomer-dimer equilibrium with the Cys(257) residue located on the dimer interface. In contrast, p75-TM-C257A forms dimers through the AXXXG motif on the opposite face of the α-helix. Biochemical and cross-linking experiments indicate that AXXXG motif is not on the dimer interface of p75-TM-WT, suggesting that the conformation of p75-TM-C257A may be not functionally relevant. However, rather than mediating p75 homodimerization, mutagenesis of the AXXXG motif reveals its functional role in the regulated intramembrane proteolysis of p75 catalyzed by the γ-secretase complex. Our structural data provide an insight into the key role of the Cys(257) in stabilization of the weak transmembrane dimer in a conformation required for the NGF signaling.This work was supported in part by Russian Science Foundation Project 14-50-00131 (to A. S. A.) (NMR structural studies) and Spanish Ministry of Economy and Competitiveness (MINECO) Project BFU2013-42746-P (to M. V.). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.S

Antibacterial and antioxidant potential of aqueous extracts of bio-residues from agrocybe cylindracea and pleurotus ostreatus mycelium

The production of fresh mushrooms results in a large quantity of bio-residues, which may account for more than 20% of a crop volume and contributes to production costs and environmental impact, if not used. These products consist mainly of stalks, mushrooms of irregular dimensions and shape and basal material. Thus, the main objective of MicoBioExtract project is the valorisation of these bio-residues and the development of add-value by-products with bioactive compounds such as polysaccharides and phenolic compounds produced by mushrooms. In the present work, the aqueous extracts from Agrocybe cylindracea byproducts (ACB) and Pleurotus ostreatus mycelium (POM) were evaluated for potential antioxidant and antimicrobial activities. Water soluble substances were extracted from mushrooms according to two different methods. In method 1 it was performed a hot extraction (extract 1A; 90 °C; 1 h; 500 rpms). In method 2, the biomass was submitted to an extraction at room temperature (extract 2B) and the resulting residue was extracted with hot water (extract 2C; 90 °C; 1 h; 500 rpms). The aqueous extracts from POM were obtained only by method 1. Extracts were lyophilized and their bioactivity evaluated measuring the antioxidant (ABTS, DPPH and ORAC) and antimicrobial (determination of the minimum inhibitory concentration – MIC) activities. The mutagenicity was also tested (Ames assay with and without S9). Moreover, it was determined the total phenolics content of the extracts and α and β – glucans (Folin Ciocalteu and Beta-Glucan Assay methods, respectively). The results obtained from antioxidant methods showed that all extracts from both mushrooms are a potential source of natural antioxidant and phenolic compounds. According to ABTS, extract 2B from ACB showed the best value with 8.48±0.33 mg ascorbic acid equivalent (AAE)/g dry extract and 4.14±0.52 mg AAE/g dry extract for POM. Regarding the antimicrobial activity, MIC results showed that ACB and POM extracts inhibited the growth of Gram- (Salmonella enteritidis) and Gram+ (Bacillus cereus and Listeria monocytogenes). Concerning the mutagenicity, the results showed no toxic effect except for extract 1A from ACB. Regarding the β-glucan content, the extracts from ACB presented higher content of β-glucan than the C extracts from POM. Thus, aqueous extracts from mushroom bio-residues showed potential antioxidant and antimicrobial properties and to develop a circular bioeconomy approach.info:eu-repo/semantics/publishedVersio

Custo de produção de mudas clonais de Café Arábica produzidas por embriogênese somática

A produção de mudas clonais de café pode ser feita de diferentes formas. Entretanto, a produção por embriogênese somática é a que tem despontado como a mais promissora. A embriogênese somática permite a produção de mudas em larga escala a partir de folhas usadas como explantes. Plantas obtidas por esse processo apresentam comportamento semelhante ao de plantas oriundas de sementes, não havendo limitação para a sua utilização comercial. A produção de mudas clonais de café é realizada em laboratórios de cultura de tecidos adaptados para a produção em escala industrial, denominados de biofábricas. Todavia, a produção de mudas clonais via embriogênese somática é uma técnica que ainda não foi explorada comercialmente no Brasil e não existe uma estimativa do custo de produção de mudas clonais de café arábica. Assim, o objetivo no presente trabalho foi calcular o custo de produção de mudas clonais de café em uma biofábrica de pequeno porte, tendo-se como base o protocolo desenvolvido pela Fundação Procafé/Embrapa Café, com a colaboração de pesquisadores da Embrapa Recursos Genéticos e Biotecnologia e do Centro de Cooperação Internacional em Pesquisa Agronômica para o Desenvolvimento, CIRAD, França. Este trabalho apresenta também uma relação dos equipamentos e materiais necessários para a produção de mudas de café in vitro. O custo de produção de uma muda foi de R$0,97 para uma produção anual de 400.000 mudas, utilizando-se o plantio de embriões pré-germinados diretamente em tubetes. Caso a etapa de produção de plântulas em potes in vitro seja utilizada, o custo de produção passa a ser de R$1,34

Crystallization and preliminary X-ray characterization of 1,3-propanediol dehydrogenase from the human pathogen Klebsiella pneumoniae

1,3-Propanediol dehydrogenase from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.7 Å resolution

Linking ecosystem pressures and marine macroinvertebrate ecosystem services in mangroves and seagrasses

African coastal ecosystems encompass high biodiversity that provides crucial ecosystem services (ES). However, the supply of these ES is threatened due to ecosystem degradation, which threatens human well-being and livelihoods. This study investigated the link between pressures and the ES provided by marine macroinvertebrates (MMIs) in mangroves and seagrasses. We assessed ecosystem condition (marine protected areas, MPAs), pressures, namely climate change (sea surface temperature and sea level), land-use and land-cover changes, overexploitation (mangrove deforestation and overfishing), and core MMI ES (provisioning, regulation, cultural). Our results revealed a low ratio of MPAs compared to the Aichi Target 11, emphasizing the need for a comprehensive conservation strategy. Sea temperature and level showed an increasing trend, indicating the vulnerability of coastal ecosystems to climate change. The decline in mangrove forest cover highlights the need to mitigate adverse effects of land-use change. The increasing number of artisanal fishery licences suggests increased pressure on MMIs, which can have severe consequences for local communities. MMI food production, particularly shrimp, and recreational fishing increased in the last 2 decades. Regulation services and cultural services related to research and education varied through time due to the limited availability of data. This information was used to develop an exploratory conceptual model illustrating the complex relationships among pressures, condition, MMI ES, and management goals for the sustainable use of marine resources and their connection with food security. Our findings underscore the importance of preserving MMI populations and habitats while addressing knowledge gaps to enhance the resilience of coastal ecosystems

Comportamento agronômico de clones de café arábica produzidos por embriogêneses somática.

O desenvolvimento de cultivares de Coffea arabica L. é um processo bastante longo, normalmente demandando cerca de 30 anos de trabalho para a liberação comercial de uma nova cultivar. Uma forma de reduzir consideravelmente esse tempo é o desenvolvimento de cultivares clonais, cuja multiplicação é feita por propagação vegetativa via embriogênese somática. Essa técnica permite a utilização comercial de plantas que ainda não possuem todas as características de interesse fixadas, tais como híbridos. Com o objetivo de desenvolver cultivares clonais a Fundação Procafé selecionou plantas matrizes com resistência ao bicho-mineiro e à ferrugem, boa qualidade de bebida e alta produtividade, visando reduzir a utilização de agrotóxicos e o custo da produção de café. Este trabalho relata o comportamento agronômico de clones com resistência ao bicho-mineiro e à ferrugem avaliados em Varginha e Boa Esperança, MG. Os resultados evidenciram que é possível obter produtividades superiores à de cultivares comerciais propagadas por sementes mediante a utilização de clones propagados por embriogênese somática. A qualidade da bebida dos clones 3, 5, 12, 13, 14 e 18 foi considerada muito boa, com valores acima de 80 pontos na escala da BSCA. Todos os clones apresentaram alta resistência ao bicho-mineiro e à ferrugem. Não foram observadas plantas com variações somaclonais que afetassem significativamente a produtividade das plantas propagadas por embriogênese somática, evidenciando que é possível a utilização comercial de plantas propagadas por esta técnica