Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

Abstract Background The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Results Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Conclusion Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Effects of EDTA and Sodium Citrate on hormone measurements by fluorometric (FIA) and immunofluorometric (IFMA) methods

Abstract Background Measurements of hormonal concentrations by immunoassays using fluorescent tracer substance (Eu3+) are susceptible to the action of chemical agents that may cause alterations in its original structure. Our goal was to verify the effect of two types of anticoagulants in the hormone assays performed by fluorometric (FIA) or immunofluorometric (IFMA) methods. Methods Blood samples were obtained from 30 outpatients and were drawn in EDTA, sodium citrate, and serum separation Vacutainer®Blood Collection Tubes. Samples were analyzed in automatized equipment AutoDelfia™ (Perkin Elmer Brazil, Wallac, Finland) for the following hormones: Luteinizing hormone (LH), Follicle stimulating homone (FSH), prolactin (PRL), growth hormone (GH), Sex hormone binding globulin (SHBG), thyroid stimulating hormone (TSH), insulin, C peptide, total T3, total T4, free T4, estradiol, progesterone, testosterone, and cortisol. Statistical analysis was carried out by Kruskal-Wallis method and Dunn's test. Results No significant differences were seen between samples for LH, FSH, PRL and free T4. Results from GH, TSH, insulin, C peptide, SHBG, total T3, total T4, estradiol, testosterone, cortisol, and progesterone were significant different between serum and EDTA-treated samples groups. Differences were also identified between serum and sodium citrate-treated samples in the analysis for TSH, insulin, total T3, estradiol, testosterone and progesterone. Conclusions We conclude that the hormonal analysis carried through by FIA or IFMA are susceptible to the effects of anticoagulants in the biological material collected that vary depending on the type of assay.

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

Anderson MIYOSHI, Daniela FREITAS, Luciana RIBEIRO, Jane E. GABRIEL, Sophie LECLERCQ, Maricê N. OLIVEIRA, and Valeria D. GUIMARÃES were recipients of a CAPES fellowship (project CAPESCOFECUB #319II). Luis BERMUDEZ and Sébastien NOUAILLE were recipients of a fellowship from the French Ministry of Education and Research. INRA and Région IledeFrance also financed L. BERMUDEZ and V. GUIMARAES. Cathy CHARLIER is recipient of a fellowship from INRA and Région Bretagne.

Advanced Glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1

Abstract Background Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Methods Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. Results In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. Conclusion In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1

Abstract Background In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.

Uso do bivalve límnico Anodontites tenebricosus (LEA, 1834) no biomonitoramento de metais do Rio Ribeira de Iguape

This study investigated the contamination of the Ribeira de Iguape River - RIR by Cd, Zn, Cr and Pb, using the bivalve Anodontites tenebricosus as a biomonitor. Metal concentrations in tissue samples were measured by HR-ICPMS. Bivalve tissues exhibited mean levels of 1.00 µg/g Cd; 152.89 µg/g Zn; 14.79 µg/g Cr and 4.40 µg/g Pb. Lead concentrations were comparable to those reported for moderately contaminated sites. The results showed that Pb is bioavailable to the bivalves, exhibiting high concentrations and exceeding both natural and reference values for human consumption. The freshwater bivalve Anodontites tenebricosus is a suitable biomonitor of contamination by metals.

Estado nutricional e consumo de energia e nutrientes de pré-escolares que frequentam creches no município de Manaus, Amazonas: existem diferenças entre creches públicas e privadas?

OBJETIVO: Verificar o estado nutricional e o consumo alimentar de crianças assistidas em creches públicas e privadas no município de Manaus, Amazonas. MÉTODOS: Foram avaliadas crianças entre 24 e 72 meses de duas creches públicas (n=217) e duas creches privadas (n=91) de Manaus. O estado nutricional foi classificado pelos índices peso para idade, peso para estatura, estatura para idade e índice de massa corporal (IMC) para idade, em valores de escores Z. O consumo alimentar na creche foi avaliado pelo método da pesagem direta individual dos alimentos e, no domicílio, pelo registro alimentar de um dia aplicado aos responsáveis. Estimou-se a frequência de crianças com ingestão de nutrientes acima ou abaixo dos pontos de corte de Estimated Average Intake (EAR) ou Adequate Intake (AI). RESULTADOS: Verificou-se maior frequência de crianças com excesso de peso nas creches privadas segundo os índices peso para estatura e IMC para idade. As crianças das instituições públicas, quando comparadas àquelas das privadas, consumiram mais gorduras poli-insaturadas, trans, ácido graxo ômega-6, vitamina C e sódio, e menos zinco. Em ambos os tipos de creches observaram-se consumo elevado de energia e proporção elevada de crianças com consumo de vitaminas A e C, zinco e sódio acima do limite superior tolerável de ingestão. A proporção de crianças com inadequação de consumo de cálcio foi maior nas creches públicas do que nas privadas (27,6 versus 7,9%; p<0,001). CONCLUSÕES: O consumo de energia e de sódio foi elevado em ambos os tipos de creche e o consumo de cálcio, insuficiente, principalmente nas creches públicas. Recomenda-se melhor monitoramento da dieta infantil para prevenção de doenças futuras.

Personalidade paterna como fator prognóstico no tratamento da tendência antissocial

A maior parte dos estudos, sobre a importância do ambiente familiar no desenvolvimento emocional e no tratamento psicológico de crianças, busca associar as características da mãe à patologia da criança, em detrimento da figura do pai. Tendo isso em vista, este estudo teve como objetivo investigar as características de personalidade de pais de crianças com tendência antissocial, submetidas ao psicodiagnóstico interventivo, e sua possível relação com os resultados terapêuticos dos filhos. Seis pais de sete crianças foram avaliados pelo Teste de Rorschach, e o follow-up dos casos indicou a ocorrência de cinco sucessos e dois fracassos terapêuticos. As características paternas associadas ao sucesso foram ausência de comprometimentos severos no teste da realidade, controle pulsional e relacionamentos interpessoais, além de uma organização neurótica de personalidade. Sendo assim, os resultados mostram a importância de incluir informações sobre a personalidade paterna na realização de indicação terapêutica de crianças e na proposição de prognósticos.

Caminhos da integralidade: adolescentes e jovens na atenção primária à saúde

Adolescentes e jovens constituem importante desafio para a construção da integralidade na Atenção Primária à Saúde (APS). Isso se deve à complexa apreensão e resposta ao conjunto de suas necessidades de saúde, decorrentes do processo de crescimento e desenvolvimento, próprio da fase, mas, sobretudo, dos aspectos socioculturais relacionados. O presente estudo buscou reconhecer alcances e limites em como o princípio da integralidade vem sendo operado em uma Unidade Básica de Saúde. Embora tenha sido possível identificar uma efetiva percepção da especificidade necessária à atenção à saúde desse grupo, também se verificaram importantes limitações relacionadas à construção de projetos de cuidado capazes de integrar as diversas finalidades do trabalho no cotidiano da Unidade, com destaque para a insuficiência de interações profissionais e setoriais, e fragilidades na comunicação de profissionais entre si e de profissionais e usuários.

Gestão do empréstimo unificado nas bibliotecas da Universidade de São Paulo

Apresenta a implantação do empréstimo unificado nas Bibliotecas do Sistema Integrado de Bibliotecas da Universidade de São Paulo (SIBiUSP), coordenado pelo Grupo de Gestão do Empréstimo Unificado. Introduz o cenário inicial das bibliotecas da Universidade, destacando a autonomia para definir políticas locais. Descreve as iniciativas isoladas que culminaram com unificação da política do empréstimo utilizada por todas as bibliotecas. Estabelece comparação entre os procedimentos adotados antes e depois da implantação e conclui relatando os benefícios obtidos pelos 138 mil usuários das bibliotecas USP e na padronização de procedimentos e rotinas adotadas pelas bibliotecas em seus 1,7 milhões de empréstimos domiciliares.

Uso de carotenoides en dietas para reproductores de dorada (Sparus aurata): relaciones con otros nutrientes y su efecto en la calidad de la puesta

Las necesidades nutricionales de los reproductores de dorada han sido poco estudiadas, esto unido al comportamiento reproductor imprevisible y variable de las especies en cautividad, constituyen un importante factor limitante para la producción masiva de semilla. La adecuada alimentación de los reproductores no solo puede mejorar la calidad de huevos y espermatozoides sino también aumentar la producción de semilla. El objetivo general de este trabajo fue el de realizar una aproximación al efecto de la inclusión de sustancias bioactivas como los carotenoides, en la dieta de los reproductores de dorada (Sparus aurata),y su relación con otros micronutrientes (n-3 HUFA y vitamina E) sobre la calidad de la puesta de esta especie bajo condiciones de cultivo. Se evaluaron los efectos de dietas experimentales, sobre el número de huevos producidos, morfometría de los mismos, viabilidad, número de larvas eclosionadas y supervivencia larvaria, entre otros. En forma paralela se estudió, la composición bioquímica de los huevos y su relación con la dieta suministrada, y con la calidad de la puesta. Se realizaron una serie de experimentos, todos ellos con un diseño experimental muy similar entre sí, los objetivos específicos de los experimentos realizados fueron: evaluar el efecto de la utilización de carotenoides de distinto origen; determinar el requerimiento de carotenoides en dietas isoprotéicas e isolipídicas; evaluar el efecto de la inclusión de carotenoides y distintos niveles de n-3 HUFA y evaluar el efecto de la inclusión de Vitamina E y carotenoides en la dieta. Los resultados de este trabajo sugieren que la calidad de la puesta de la dorada puede mejorarse por la inclusión de carotenoides dietéticos. La inclusión de carotenoides, como el pimentón en polvo, de origen vegetal, produjo mejores resultados en la calidad de la puesta que la harina de langostino, de origen animal. Esto se podría deber al tipo de carotenoides que determinan también la calidad nutricional. La calidad de la puesta de la dorada puede mejorarse con la inclusión de entre un 1 y un 2 % de carotenoides, provenientes de pimentón comercial, en la dieta de los reproductores. Los niveles de n-3 HUFA en dietas para reproductores de esta especie podrían elevarse hasta un 3,5% cuando se complementan conjuntamente con carotenoides provenientes de oleorresina del pimentón y con vitamina E, mejorando así la calidad de la puesta.

Combined levels of carotenoids and vitamin E in diets for gilthead seabream broodstocks (Sparus aurata L.): effect on the quality of egg spawning and composition

Watanabe et al. (1991 a,b) state that, vitamin E and carotenoids perform an essential role on the quality of egg spawning. Vitamin E is one of the main nutrients for the reproduction of fish (Izquierdo et al., 2001), and it has been proved that its inclusion in diets for broodstocks favors the quality of egg spawning in several species of fish (Watanabe and Takashima,1977; Takeuchi et al., 1981; Watanabe et al., 1985, 1991 a,b; Sutjaritvongsanon, 1987; Watanabe, 1990; Schimittou, 1993; Mushiake et al., 1993; Dube, 1996; Shiranee and Natarajan, 1996; Izquierdo et al., 2001; Morehead et al., 2001; Fernández- Palacios et al., 2005). On the other hand, the carotenoids which also perform an antioxidizing function (including the protection of lipids from oxidation), have been involved in the reproductive processes of marine organisms: crustaceans (Liñan-Cabello et al., 2002), marine fish (Watanabe y Kiron, 1995; Verakunpiriya et al., 1997 a,b; Vassallo-Agius et al., 2001 a,b,c, 2002; Watanabe and Vassallo-Agius 2003) and fresh water fish (Ahmadi et al., 2006). The results of this study suggest that the recommended levels of n-3 HUFA in diets for gilthead sea bream broodstocks could be increased up to 3,5 % when supplemented jointly with carotenoids from paprika oleoresin and vitamin E, thus favoring the quality of spawning.