Identificação de polimorfismos em genótipos de Coffea arabica de uma coleção da Etiópia.

Os marcadores moleculares são ferramentas importantes para acelerar os programas de melhoramento. Para o cafeeiro, uma espécie perene, o uso de marcadores é particularmente desejável devido ao tempo e recursos gastos para o lançamento de uma nova cultivar. Duas espécies do gênero Coffea são responsáveis por quase toda a produção de café: Coffea arabica e C. canephora. Contudo, para C. arabica, o número de marcadores polimórficos é relativamente baixo comparado a C. canephora e outras culturas, uma vez que a espécie apresenta baixa diversidade genética. Muitos estudos com marcadores genéticos foram feitos para analisar a diversidade da C. arabica, mas os resultados não foram eficientes para a discriminação genotípica detalhada e mapeamento genético. O Instituto Agronômico do Paraná (IAPAR) possui uma coleção de 132 acessos de C. arabica originários da Etiópia, que apresentam variabilidade fenotípica com potencial para serem utilizados para exploração da diversidade. Neste sentido, este estudo buscou analisar a diversidade nucleotídica pela identificação de polimorfismos, SNPs e INDELs, de uma população do centro de origem de C. arabica, associado com o sequenciamento de nova geração. O RNA-seq de dois tecidos, frutos e folhas, de quatro genótipos de C. arabica de uma população da Etiópia, C. arabica cv. Mundo Novo e de um dos seus ancestrais de C. arabica ? C. eugenioides, foram sequenciados pela metodologia Illumina HiSeq2000. Os reads obtidos foram processados e posteriormente as sequências foram mapeadas em uma referência de C. canephora para identificação dos polimorfismos. Foram feitas duas estratégias: i) na primeira estratégia, foi utilizado uma ferramenta chamada SNiPloid com critérios de cobertura para o polimorfismo identificado e ii) uma segunda estratégia que considera os polimorfismos encontrados diretamente dos arquivos de detecção dos polimorfismos. Os resultados identificaram um número grande de polimorfismos. Na primeira estratégia, foram encontrados pelo menos 5.500 SNPs potenciais para a genotipagem e na segunda, 103.791 SNPs potenciais. Para essa última, ainda é necessário estabelecer critérios e filtros para escolher os polimorfismos que serão inicialmente genotipados. Os dados também mostraram a importância de utilizar um grupo mais diverso de genótipos associado com o sequenciamento de nova geração para detecção de SNPs. Este trabalho será importante para direcionar futuros trabalhos na caracterização da diversidade genética em C. arabica, além de estudos de mapeamento genético por associação

Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities

Protein-tyrosine phosphatases (PTPases) have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible) fraction of the endogenous PTPase pool

Burden, coping, physical symptoms and psychological morbidity in caregivers of functionally dependent family members

Objectives: this study assessed burden, coping, physical symptoms and psychological morbidity in caregivers of functionally dependent family members. Methods: fifty family caregivers completed self-reported measures of burden, physical symptoms, psychological morbidity and coping strategies. Results: there was a significant negative correlation between coping strategies and the different clinical variables, as well as a significant positive correlation between coping strategies and duration of care. It appears that the stronger bond between caregiver and family member leads to a poorer use of adaptive coping strategies. It also appears that the deterioration of the relationship between them and the lower perceived self-efficacy are more prominente in caregivers of family members with cognitive impairment, indicating that caregivers with family members without cognitive impairment face fewer difficulties. Conclusion: these results emphasize the need for interventions to include coping strategies, since they are important in reducing caregivers’ burden, psychological morbidity and physical symptoms

Genomic organization and alternative splicing of the human and mouse RPTPρ genes

BACKGROUND: Receptor protein tyrosine phosphatase rho (RPTPρ, gene symbol PTPRT) is a member of the type IIB RPTP family. These transmembrane molecules have been linked to signal transduction, cell adhesion and neurite extension. The extracellular segment contains MAM, Ig-like and fibronectin type III domains, and the intracellular segment contains two phosphatase domains. The human RPTPρ gene is located on chromosome 20q12-13.1, and the mouse gene is located on a syntenic region of chromosome 2. RPTPρ expression is restricted to the central nervous system. RESULTS: The cloning of the mouse cDNA, identification of alternatively spliced exons, detection of an 8 kb 3'-UTR, and the genomic organization of human and mouse RPTPρ genes are described. The two genes are comprised of at least 33 exons. Both RPTPρ genes span over 1 Mbp and are the largest RPTP genes characterized. Exons encoding the extracellular segment through the intracellular juxtamembrane 'wedge' region are widely spaced, with introns ranging from 9.7 to 303.7 kb. In contrast, exons encoding the two phosphatase domains are more tightly clustered, with 15 exons spanning ∼ 60 kb, and introns ranging in size from 0.6 kb to 13.1 kb. Phase 0 introns predominate in the intracellular, and phase 1 in the extracellular segment. CONCLUSIONS: We report the first genomic characterization of a RPTP type IIB gene. Alternatively spliced variants may result in different RPTPρ isoforms. Our findings suggest that RPTPρ extracellular and intracellular segments originated as separate modular proteins that fused into a single transmembrane molecule during a later evolutionary period

Collecting self-report data with people with dementia: Benefits, challenges, and best-practice

One-third of people with dementia live in care home settings and in order to deliver better evidence-based care, robust research including clinical trials is required. Concerns have been raised by researchers about the capacity of care home residents with dementia to participate in clinical trials. This includes self-report measures, completion of which researchers have suggested may be unreliable or impossible and may cause distress for residents. Many trials, therefore, utilise only proxy completed outcome measures. This is despite evidence that individuals with mild through to advanced dementia can reliably report on outcomes, if appropriate measures and approaches to data collection are used. However, little has been written about best practice in data collection with this group. This study aimed to explore the experiences of researchers working on dementia trials in care homes and identify best practices to assist design of future trials. Thirty-three researchers completed an online, qualitative questionnaire outlining their experiences and the perceived benefits and challenges of data collection with people with dementia. We identified five main benefits; [1] improving the delivery of person-centred care, [2] hearing the voice of people with dementia, [3] residents spending time with researchers, [4] improving researcher understanding, and [5] having an evidence base from multiple sources. We also identified five main challenges; [1] effective communication, [2] fluctuating capacity, [3] causing distress to residents, [4] time pressures, and [5] staff availability. Researchers also made suggestions about how these can be overcome. We recommend that the challenges identified could be overcome using appropriate methods for collecting data. Thorough training for researchers on data collection with people with dementia was identified as important for ensuring successful data collection

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3+ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103+ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103+ DCs from Il10−/−, Tlr2−/−, and Myd88−/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103+ DCs failed to induce IL-10 production from co-cultured Il27ra−/− T cells. B. breve treatment of Tlr2−/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103+ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4+ T cells from wild-type mice, but not Il10−/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells

A Key Role of Dendritic Cells in Probiotic Functionality

BACKGROUND: Disruption of the intestinal homeostasis and tolerance towards the resident microbiota is a major mechanism involved in the development of inflammatory bowel disease. While some bacteria are inducers of disease, others, known as probiotics, are able to reduce inflammation. Because dendritic cells (DCs) play a central role in regulating immune responses and in inducing tolerance, we investigated their role in the anti-inflammatory potential of probiotic lactic acid bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Selected LAB strains, while efficiently taken up by DCs in vitro, induced a partial maturation of the cells. Transfer of probiotic-treated DCs conferred protection against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Protection was associated with a reduction of inflammatory scores and colonic expression of pro-inflammatory genes, while a high local expression of the immunoregulatory enzyme indolamine 2, 3 dioxgenase (IDO) was observed. The preventive effect of probiotic-pulsed DCs required not only MyD88-, TLR2- and NOD2-dependent signaling but also the induction of CD4+ CD25+ regulatory cells in an IL-10-independent pathway. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that selected probiotics can stimulate DC regulatory functions by targeting specific pattern-recognition receptors and pathways. The results not only emphasize the role of DCs in probiotic immune interactions, but indicate a possible role in immune-intervention therapy for IBD

The coffee genome provides insight into the convergent evolution of caffeine biosynthesis.

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