N-glycosylation in sugarcane

The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST) project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST) clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.A N-glicosilação é uma das principais modificações pós-traducionais, sendo responsável por alterações na conformação, estabilidade e conseqüentemente na funcionalidade de proteínas em eucariotos. Com a finalidade de melhor compreender a via de N-glicosilação em plantas foi realizada uma prospecção no banco de seqüências expressas do projeto genoma da cana de açúcar (SUCEST). Foram identificadas noventa seqüências cujos produtos gênicos apresentam alto grau de similaridade com enzimas envolvidas na biossíntese e processamento de N-glicanos. Dos vinte e três genes da via de N-glicosilação previamente descritos em diferentes espécies, vinte e um foram detectados em cana de açúcar.231234Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

<p>Abstract</p> <p>Background</p> <p>Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including <it>Coffea arabica</it>. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.</p> <p>Results</p> <p>The expression levels of five frequently used housekeeping genes (reference genes), namely <it>alcohol dehydrogenase </it>(<it>adh</it>), <it>14-3-3</it>, <it>polyubiquitin </it>(<it>poly</it>), <it>β-actin </it>(<it>actin</it>) and <it>glyceraldehyde-3-phosphate dehydrogenase </it>(<it>gapdh</it>) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of <it>Coffea arabica </it>plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (<it>cys</it>), a caffeine synthase (<it>ccs</it>) and the 60S ribosomal protein L7 (<it>rpl7</it>) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of <it>gapdh</it>, which showed no significant changes in expression among the investigated experimental conditions.</p> <p>Conclusion</p> <p>Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, <it>gapdh</it>, followed by <it>14-3-3 </it>and <it>rpl7 </it>were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. <it>Gapdh </it>is therefore the recommended reference gene for measuring gene expression in <it>Coffea arabica</it>. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.</p

Efeitos do sotalol sobre o eletrocardiograma de alta resolução avaliados por ensaio duplo-cego cruzado randomizado

A presente investigação teve por objetivo avaliar os efeitos do Sotalol sobre o eletrocardiograma de alta resolução (ECGAR), em uma população com arritmia ventricular idiopática. Foi estudado um grupo de 12 pacientes submetidos a um ensaio clínico do tipo duplo-cego cruzado e randomizado, para avaliaçao da eficácia da droga. Foram obtidos ECGAR em condiçoes de controle (C), uso de placebo (P) e de droga (O), confrontando os resultados entre as três situações e a eficácia medicamentosa. No vetor-magnitude foram analisados os seguintes parâmetros: voltagem média dos 40 ms terminais do complexo QRS filtrado (VM - normal > 20 µV), duração dos sinais de baixa amplitude < 40 µV no final da ativação (SBA - normal < 38 ms) e duração total do complexo QRS filtrado (OQRS - normal < 114.0 ms). Em função da resposta terapêutica, os pacientes foram divididos em responsivos (G1) e não-responsivos (G2). Não foram observadas diferenças estatisticamente significativas entre C e P. No Grupo I, composto por 5 pacientes (42% de eficácia), não foram observadas diferenças significativas nas 3 variáveis avaliadas entre as condições de P e O. No Grupo 11, composto por 7 pacientes, ocorreram modificaçoes nos SBA, cujos valores no P estavam em 24.80 ± 7.60 ms e passando com a O para 29.10 ± 14.76 ms (p < 0,01). Em 5 dos 7 pacientes deste grupo (71%), prolongaram-se no pós-droga os SBA, numa média de 11.20 ± 4.80 ms, com significância estatística em relaçao ao placebo (p < 0.04). Frente aos resultados observados com os SBA, foram obtidos sensibilidade de 71 %, especificidade de 86%, valor preditivo positivo de 83% e negativo de 75% para definir a populaçao responsiva à droga. Concluiu-se que na população estudada, o Sotalol, quando efetivo, não produziu modificações significativas nos parâmetros do ECGAR. Um incremento médio dos SBA de 11.2 ± 4.8 ms, por influência da droga, associou-se a uma ausência de resposta terapêutica

In silico evaluation of the Eucalyptus transcriptome

The expressed sequence tags (ESTs) produced in the Forests project provide an invaluable opportunity to assess the Eucalyptus transcriptome. Besides providing information on the different proteins produced by this plant, it is possible to infer gene expression profiles because non-normalized cDNA libraries were used. The EST frequency from any gene is correlated to the transcript levels in the tissues from which the cDNA libraries were constructed. The goal of this work was to identify Eucalyptus genes that showed either differential expression pattern or were ubiquitously expressed in the tissues sampled in the Forests project. Six robust statistical tests and very restrictive rules were applied to gain confidence in the in silico data aiming to avoid false positives. Several genes with interesting expression profiles were identified and some of them were validated by RT-PCR.487495Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a wellcharacterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 mu m diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 angstrom ngstrom were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.11Ysciescopu