Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.

Oxidative DNA damage induced by S(IV) in the presence of Cu(II) and Cu(I) complexes

The DNA damage induced by S(IV) in the presence of some Cu(II) complexes in air saturated solution was investigated. The addition of S(IV) to an air saturated solution containing CuII GGA (GGA = glycylglycyl-L-alanine), CuII G3 (G3 = triglycine) or CuII G4 (G4 = tetraglycine) and Ni(II) traces, causes rapid formation of the respective Cu(III) complex, with simultaneous O2 uptake and S(IV) oxidation. SO3•- and HO• were detected by EPR-spin trapping experiments. The DNA strand breaks were attributed to the oxysulfur radicals formed. In the reduction of Cu(II)/BCA (BCA = 4,4' dicarboxy-2-2'-biquinoline) by S(IV), with CuI BCA complex formation, there is the possible formation of carbon centered radical of BCA or peroxyl radical (ROO•) capable of oxidizing DNA bases. The intensity of DNA damage in the presence of these Cu(II) complexes and S(IV) (10-300 µmol L-1) followed the order: CuII BCA ∼ CuII G4 ∼ Cu(II) (added as Cu(NO3)2) > CuII G3 ∼ CuII GGA. Specifically for CuII BCA the damage occurred even at lower S(IV) concentration (0.1 µmol L-1). For the Cu(II) complexes with glycylglycylhistidine, glycylhistidylglycine, glycylhistidyllysine and glycylglycyltyrosylarginine the Cu(III) formation and the DNA damage was not observed.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.

Atenção domiciliar como mudança do modelo tecnoassistencial

OBJETIVO: Analisar práticas de atenção domiciliar de serviços ambulatoriais e hospitalares e sua constituição como rede substitutiva de cuidado em saúde. PROCEDIMENTOS METODOLÓGICOS: Estudo qualitativo que analisou, com base na metodologia de caso traçador, quatro serviços ambulatoriais de atenção domiciliar da Secretaria Municipal de Saúde e um serviço de um hospital filantrópico do município de Belo Horizonte, MG, entre 2005 e 2007. Foram realizadas entrevistas com gestores e equipes dos serviços de atenção domiciliar, análise de documentos e acompanhamento de casos com entrevistas a pacientes e cuidadores. A análise foi orientada pelas categorias analíticas integração da atenção domiciliar na rede de saúde e modelo tecnoassistencial. ANÁLISE DOS RESULTADOS: A implantação da atenção domiciliar foi precedida por decisão político-institucional tanto com orientação racionalizadora, buscando a diminuição de custos, quanto com vistas à reordenação tecnoassistencial das redes de cuidados. Essas duas orientações encontram-se em disputa e constituem dificuldades para conciliação dos interesses dos diversos atores envolvidos na rede e na criação de espaços compartilhados de gestão. Pôde-se identificar a inovação tecnológica e a autonomia das famílias na implementação dos projetos de cuidado. As equipes mostraram-se coesas, construindo no cotidiano do trabalho novas formas de integrar os diferentes olhares para transformação das práticas em saúde. Foram observados desafios na proposta de integrar os diferentes serviços de caráter substitutivo do cuidado ao limitar a capacidade da atenção domiciliar de mudar o modelo tecnoassistencial. CONCLUSÕES: A atenção domiciliar possui potencial para constituição de uma rede substitutiva ao produzir novos modos de cuidar que atravessam os projetos dos usuários, dos familiares, da rede social e dos trabalhadores da atenção domiciliar. A atenção domiciliar como modalidade substitutiva de atenção à saúde requer sustentabilidade política, conceitual e operacional, bem como reconhecimento dos novos arranjos e articulação das propostas em curso.

Modelo de indução de diarréia osmótica em bezerros holandeses

Com o objetivo de avaliar um protocolo de diarréia osmótica induzida, foram utilizados 18 bezerros hígidos, com idade entre oito e 30 dias de vida, e peso variando de 37 a 50kg. A diarréia e a desidratação foram induzidas por meio da administração de leite integral (16,5mL kg-1), sacarose (4g kg-1), espirolactona e hidroclorotiazida (2mg kg-1), a cada oito horas, durante dois dias. O exame físico e as coletas de sangue para determinações de componentes do hemograma, hemogasometria e de constituintes bioquímicos foram realizados em T0 (0h), T1 (24hi) e T2 (48hi). O protocolo de indução da diarréia obteve 100% de eficiência, produzindo diarréia aquosa e desidratação intensa (13% do peso corpóreo) acompanhadas de azotemia pré-renal, aumento nos valores do hematócrito, hemoglobina e proteína total, hipercalemia, hiperlactemia, hiperfosfatemia, acidose metabólica e diminuição do défict de volume plasmático e da pressão venosa central.

Aplicação experimental de um modelo de conduta de inspeção sanitária no comércio varejista de alimentos

Este trabalho teve por objetivo aplicar um procedimento para diagnóstico e modelo de inspeção higiênico-sanitária em estabelecimentos varejistas de alimentos, no Município de Ibiúna, Estado de São Paulo, Brasil, e analisar os resultados com vistas a atingir os objetivos da vigilância sanitária. O trabalho foi desenvolvido durante seis meses em 19 estabelecimentos varejistas de alimentos classificados como padarias, restaurantes e açougues, sendo que na primeira inspeção realizou-se o diagnóstico da situação, com a identificação das não conformidades e avaliação das condições higiênico-sanitárias dos estabelecimentos e foi obtida a pontuação média de 64. Após esta etapa iniciaram-se retornos programados aos estabelecimentos pela equipe da vigilância sanitária a fim de verificar a evolução das adequações sanitárias. No diagnóstico e em todos os retornos programados foi aplicado roteiro de inspeção e produção de relatórios detalhados destinados aos proprietários. Todos os manipuladores de alimentos dos estabelecimentos inspecionados foram capacitados em relação a conceitos básicos de higiene em alimentos. Os resultados mostraram que, após três retornos de inspeções em cada estabelecimento, 100% apresentaram-se como satisfatórios, com pontuação média de 90. O modelo de inspeção sanitária aplicado mostrou-se viável para a promoção da segurança alimentar.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Modelo de suplementação nutricional com fatores hepatotróficos aumenta proliferação celular em fígado de ratos sadios

Foram avaliados dois protocolos de administração, em ratos sadios, de uma solução de fatores hepatotróficos (FH), composta por aminoácidos, vitaminas, sais minerais, glicose, insulina, glucagon e triiodotironina (T3). A solução foi administrada durante 10 dias, 40mg/kg/dia, i.p., em duas, grupo 2xFH (n=15), ou três doses, grupo 3xFH (n=15), diárias. Foram observados os efeitos na proliferação celular dos hepatócitos, na angiogênese e na matriz extracelular hepática, assim como as possíveis reações adversas. Os animais dos grupos 2xFH e 3xFH apresentaram aumento da massa hepática de 30,1% e 22,5%, respectivamente, em relação ao grupo-controle (CT; n=15). O índice de proliferação hepatocelular foi maior nos grupos 2xFH (1,4%) e 3xFH (1,2%) em relação ao grupo CT (0,53%), e a densitometria relativa do fator de crescimento do endotélio vascular pelo imunoblot não revelou diferença estatística entre os três grupos. Nos grupos 2xFH e 3xFH, houve redução do colágeno intersticial em relação ao grupo CT. A solução de FH estimulou o crescimento hepático e reduziu o volume de colágeno perissinusoidal. A administração em três doses diárias resultou em mortalidade de 26,7%, possivelmente pelo excessivo estresse da manipulação e pela menor adaptação fisiológica dos ratos, o que não ocorreu nos grupos 2xFH e CT. Para esse tipo de abordagem em ratos, o procedimento experimental mais apropriado, seguro, com melhor chance de adaptação dos animais e com resultados significativos é a aplicação dos FH em duas doses diárias.

Modelo de suprimento sanguíneo do intestino grosso do tamanduá bandeira (Myrmecophaga tridactyla)

Em seis animais adultos Myrmecophaga tridactyla estudou-se o modelo de suprimento sanguíneo do intestino grosso, que é dependente das artérias mesentérica cranial (AMCr) e caudal (AMC). Os espécimes coletados conforme as normas do IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) foram perfundidos com água (40 C), injetados com látex corado, fixados em formol e conservados em solução alcoólica (50%). O mesocólon está disposto na linha sagital mediana e fixa o intestino grosso à parede dorsal do abdome. Derivaram da AMC: a artéria retal cranial, sete a 14 artérias cólicas e uma ou duas artérias ileocólicas, que apresentaram anastomoses de irrigação com a AMCr. A AMC finaliza-se na borda mesocólica das alças intestinais, emitindo ramos cólicos retos a partir das arcadas justacólicas, que penetram na intimidade da musculatura longitudinal. Ao longo do trajeto da AMC foram observadas ilhas arteriais, e a região ileocólica apresentou maior densidade vascular.

Protective effects of mate tea (Ilex paraguariensis) on H2O2-induced DNA damage and DNA repair in mice

Yerba mate (Ilex paraguariensis) is rich in several bioactive compounds that can act as free radical scavengers. Since oxidative DNA damage is involved in various pathological states such as cancer, the aim of this study was to evaluate the antioxidant activity of mate tea as well as the ability to influence DNA repair in male Swiss mice. Forty animals were randomly assigned to four groups. The animals received three different doses of mate tea aqueous extract, 0.5, 1.0 or 2.0 g/kg, for 60 days. After intervention, the liver, kidney and bladder cells were isolated and the DNA damage induced by H2O2 was investigated by the comet assay. The DNA repair process was also investigated for its potential to protect the cells from damage by the same methodology. The data presented here show that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H2O2-induced DNA strand breaks and improved the DNA repair after H2O2 challenge in liver cells, irrespective of the dose ingested. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be afforded by the antioxidant activity of the mate tea's bioactive compounds

Modelo de programação linear para otimização econômica do projeto de irrigação Baixo Acaraú - CE

The present work had as objective uses a model of lineal programming algorithm to optimize the use of the water in the District of Irrigation Baixo Acarau-CE proposing the best combination of crop types and areas established of 8,0 ha. The model aim maximize the net benefit of small farmer, incorporating the constraints in water and land availability, and constraints on the market. Considering crop types and the constraints, the study lead to the following conclusions: 1. The water availability in the District was not a limiting resources, while all available land was assigned in six of the seven cultivation plans analyzed. Furthermore, water availability was a restrictive factor as compared with land only when its availability was made to reduce to 60% of its actual value; 2. The combination of soursop and melon plants was the one that presented the largest net benefit, corresponding to R$ 5,250.00/ha/yr. The planting area for each crop made up to 50% of the area of the plot; 3. The plan that suggests the substitution of the cultivation of the soursop, since a decrease in annual net revenue of 5.87%. However, the plan that contemplates the simultaneous substitution of both soursop and melon produced the lowest liquid revenue, with reduction of 33.8%.

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes

Background: The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally. Results: We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 mu g of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-gamma and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 mu g). Conclusion: Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

Background: Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings: Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-gamma production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-beta and IL-10 production, which could be associated with long-term protection. Conclusions/Significance: We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections.

DNA extraction from hair shafts of wild Brazilian felids and canids

Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the Sao Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol: chloroform: isoamyl alcohol general method, with proteinase K as digestive enzyme.

Non-Detection of Human Herpesvirus 8 (HHV-8) DNA in HHV-8-Seropositive Blood Donors from Three Brazilian Regions

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (Sao Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140).