Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarrays

Abstract Background Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard. Results We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable. Conclusion RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Epidemiologic aspects of the malaria transmission cycle in an area of very low incidence in Brazil

Abstract Background Extra-Amazonian autochthonous Plasmodium vivax infections have been reported in mountainous regions surrounded by the Atlantic Forest in Espírito Santo state, Brazil. Methods Sixty-five patients and 1,777 residents were surveyed between April 2001 and March 2004. Laboratory methods included thin and thick smears, multiplex-PCR, immunofluorescent assay (IFA) against P. vivax and Plasmodium malariae crude blood-stage antigens and enzyme-linked immunosorbent assay (ELISA) for antibodies against the P. vivax-complex (P. vivax and variants) and P. malariae/Plasmodium brasilianum circumsporozoite-protein (CSP) antigens. Results Average patient age was 35.1 years. Most (78.5%) were males; 64.6% lived in rural areas; 35.4% were farmers; and 12.3% students. There was no relevant history of travel. Ninety-five per cent of the patients were experiencing their first episode of malaria. Laboratory data from 51 patients were consistent with P. vivax infection, which was determined by thin smear. Of these samples, 48 were assayed by multiplex-PCR. Forty-five were positive for P. vivax, confirming the parasitological results, while P. malariae was detected in one sample and two gave negative results. Fifty percent of the 50 patients tested had IgG antibodies against the P. vivax-complex or P. malariae CSP as determined by ELISA. The percentages of residents with IgM and IgG antibodies detected by IFA for P. malariae, P. vivax and Plasmodium falciparum who did not complain of malaria symptoms at the time blood was collected were 30.1% and 56.5%, 6.2% and 37.7%, and 13.5% and 13%, respectively. The same sera that reacted to P. vivax also reacted to P. malariae. The following numbers of samples were positive in multiplex-PCR: 23 for P. vivax; 15 for P. malariae; 9 for P. falciparum and only one for P. falciparum and P. malariae. All thin and thick smears were negative. ELISA against CSP antigens was positive in 25.4%, 6.3%, 10.7% and 15.1% of the samples tested for "classical" P. vivax (VK210), VK247, P. vivax-like and P. malariae, respectively. Anopheline captures in the transmission area revealed only zoophilic and exophilic species. Conclusion The low incidence of malaria cases, the finding of asymptomatic inhabitants and the geographic separation of patients allied to serological and molecular results raise the possibility of the existence of a simian reservoir in these areas.

Prevalence and risk factors of Hepatitis C virus infection in Brazil, 2005 through 2009: a cross-sectional study

Abstract Background Hepatitis C chronic liver disease is a major cause of liver transplant in developed countries. This article reports the first nationwide population-based survey conducted to estimate the seroprevalence of HCV antibodies and associated risk factors in the urban population of Brazil. Methods The cross sectional study was conducted in all Brazilian macro-regions from 2005 to 2009, as a stratified multistage cluster sample of 19,503 inhabitants aged between 10 and 69 years, representing individuals living in all 26 State capitals and the Federal District. Hepatitis C antibodies were detected by a third-generation enzyme immunoassay. Seropositive individuals were retested by Polymerase Chain Reaction and genotyped. Adjusted prevalence was estimated by macro-regions. Potential risk factors associated with HCV infection were assessed by calculating the crude and adjusted odds ratios, 95% confidence intervals (95% CI) and p values. Population attributable risk was estimated for multiple factors using a case–control approach. Results The overall weighted prevalence of hepatitis C antibodies was 1.38% (95% CI: 1.12%–1.64%). Prevalence of infection increased in older groups but was similar for both sexes. The multivariate model showed the following to be predictors of HCV infection: age, injected drug use (OR = 6.65), sniffed drug use (OR = 2.59), hospitalization (OR = 1.90), groups socially deprived by the lack of sewage disposal (OR = 2.53), and injection with glass syringe (OR = 1.52, with a borderline p value). The genotypes 1 (subtypes 1a, 1b), 2b and 3a were identified. The estimated population attributable risk for the ensemble of risk factors was 40%. Approximately 1.3 million individuals would be expected to be anti-HCV-positive in the country. Conclusions The large estimated absolute numbers of infected individuals reveals the burden of the disease in the near future, giving rise to costs for the health care system and society at large. The known risk factors explain less than 50% of the infected cases, limiting the prevention strategies. Our findings regarding risk behaviors associated with HCV infection showed that there is still room for improving strategies for reducing transmission among drug users and nosocomial infection, as well as a need for specific prevention and control strategies targeting individuals living in poverty.

Certificação de fornecimento e custos de transação: resultados do Programa Integrado de Desenvolvimento e Qualificação de Fornecedores - Prodfor

O presente estudo teve dois objetivos complementares: entender o desenvolvimento da instituição privada Programa Integrado de Desenvolvimento e Qualificação de Fornecedores (Prodfor), um programa específico de padronização e certificação de fornecedores locais de produtos e serviços no estado do Espírito Santo; e testar se o programa causou redução de custos de transação para as mantenedoras nas compras de bens e serviços. Foram utilizados os conceitos da nova economia institucional para desenvolver hipóteses a respeito do impacto da certificação sobre o desempenho de compras das empresas mantenedoras. Os resultados da pesquisa de campo e dos testes de média não paramétricos indicam que houve redução nos custos de transação das empresas compradoras após a implantação do programa. Complementarmente, verificou-se que houve maior redução nos custos de transação relacionados às atividades posteriores (ex post) às transações de compra em comparação com os custos associados às atividades anteriores à transação de compra (ex ante).

Construção e Validação da Escala de Imprevisibilidade Familiar na Infância (EIFI)

Este estudo teve o objetivo de construir e validar uma medida psicológica denominada de Escala de Imprevisibilidade Familiar na Infância (EIFI) para a população brasileira. Participaram 394 pessoas, sendo 158 adultos em conflito com a lei que já haviam passado pela prisão (média de idade=34,23 anos; DP=10,17), 122 estudantes universitários (M=19,26; DP=2,06) e 114 mulheres com idade superior a 40 anos e escolaridade a partir de ensino médio (M=51,19; DP=8,64). Foram conduzidos procedimentos de validade de conteúdo, face e construto. A análise fatorial gerou como produto um instrumento com estrutura de quatro dimensões (cuidado/apoio, recursos financeiros, alimentação e disciplina) com índices de confiabilidade satisfatórios para todas elas. Concluímos que a EIFI foi validada para a população brasileira apresentando-se no cenário nacional como um instrumento para medida de imprevisibilidade familiar na infância.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Range extensions along western Atlantic for Epialtidae crabs (Brachyura, Majoidea) genera Acanthonyx Latreille, 1828 and Epialtus H. Milne Edwards, 1834

The present study provided information extending the known geographical distribution of three species of majoid crabs, the epialtids Acanthonyx dissimulatus Coelho, 1993, Epialtus bituberculatus H. Milne Edwards, 1834, and E. brasiliensis Dana, 1852. Specimens of both genera from different carcinological collections were studied by comparing morphological characters. We provide new data that extends the geographical distributions of E. bituberculatus to the coast of the states of Paraná and Santa Catarina (Brazil), and offer new records from Belize and Costa Rica. Epialtus brasiliensis is recorded for the first time in the state of Rio Grande do Sul (Brazil), and A. dissimulatus is reported from Quintana Roo, Mexico. The distribution of A. dissimulatus, previously known as endemic to Brazil, has a gap between the states of Espírito Santo and Rio de Janeiro. However, this restricted southern distribution is herein amplified by the Mexican specimens.

Políticas de formação em educação física e saúde coletiva

Este ensaio apresenta uma iniciativa de investigação interinstitucional que agrega três grupos de pesquisa vinculados a programas de pós-graduação em educação física: Universidade Federal do Rio Grande do Sul, Universidade de São Paulo e Universidade Federal do Espírito Santo. O tema articulador do projeto são as políticas de formação em educação física e saúde coletiva, cujo foco inicial é o Programa de Educação pelo Trabalho em Saúde. As investigações a serem empreendidas têm o propósito de acompanhar e analisar os processos de composição e articulação entre ensino, serviço e comunidade com vistas a constituir uma rede de saberes e práticas que responda aos desafios da formação em saúde comprometida com a defesa e consolidação do Sistema Único de Saúde.

Development of heat sink device by using topology optimization

Small scale fluid flow systems have been studied for various applications, such as chemical reagent dosages and cooling devices of compact electronic components. This work proposes to present the complete cycle development of an optimized heat sink designed by using Topology Optimization Method (TOM) for best performance, including minimization of pressure drop in fluid flow and maximization of heat dissipation effects, aiming small scale applications. The TOM is applied to a domain, to obtain an optimized channel topology, according to a given multi-objective function that combines pressure drop minimization and heat transfer maximization. Stokes flow hypothesis is adopted. Moreover, both conduction and forced convection effects are included in the steady-state heat transfer model. The topology optimization procedure combines the Finite Element Method (to carry out the physical analysis) with Sequential Linear Programming (as the optimization algorithm). Two-dimensional topology optimization results of channel layouts obtained for a heat sink design are presented as example to illustrate the design methodology. 3D computational simulations and prototype manufacturing have been carried out to validate the proposed design methodology.

A new Fenestrulina (Bryozoa, Cheilostomata) commensal with tube-dwelling anemones (Cnidaria, Ceriantharia) in the tropical southwestern Atlantic

A new species of cheilostome bryozoan, Fenestrulina commensalis n. sp., was collected in December 2008 by scuba at 5–10 meters depth at Guaibura Beach, Guarapari, Espírito Santo state, southeastern Brazil. The specimen was found associated with tubes of the cerianthid Pachycerianthus sp., representing the first commensal association between a bryozoan and a tube-dwelling anemone. Fenestrulina commensalis n. sp. is the third species of the genus found in Brazilian waters; it is distinguished from other Atlantic species of Fenestrulina by its small angular orificial condyles, a single oral spine and basal anchoring rhizoids arising from abfrontal pore chambers. Morphological adaptations to encrust the tubes of cerianthids include anchoring rootlets and weakly contiguous zooids. These morphological features allow the colony the flexibility to grow around the tube and feed relatively undisturbed by silt and detritus, being raised well above the softsediment substratum in which the tube-anemone grows.