Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil

The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundiaí-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundiaí river

Avaliação de produtos naturais no controle de biofilmes formados em sistema dinâmico

The biofilms microbial forms of association are responsible for generating, accelerating and / or induce the process of corrosion. The damage generated in the petroleum industry for this type of corrosion is significatives, representing major investment for your control. The aim of this study was to evaluate such tests antibiograms the effects of extracts of Jatropha curcas and essential oil of Lippia gracilis Schauer on microrganisms isolated from water samples and, thereafter, select the most effective natural product for further evaluation of biofilms formed in dynamic system. Extracts of J. curcas were not efficient on the complete inhibition of microbial growth in tests type antibiogram, and essential oil of L. gracilis Schauer most effective and determined for the other tests. A standard concentration of essential oil of 20 μL was chosen and established for the evaluation of the biofilms and the rate of corrosion. The biocide effect was determined by microbial counts of five types of microorganisms: aerobic bacteria, precipitating iron, total anaerobic, sulphate reducers (BRS) and fungi. The rate of corrosion was measured by loss of mass. Molecular identification and scanning electron microscopy (SEM) were performed. The data showed reduction to zero of the most probable number (MPN) of bacteria precipitating iron and BRS from 115 and 113 minutes of contact, respectively. There was also inhibited in fungi, reducing to zero the rate of colony-forming units (CFU) from 74 minutes of exposure. However, for aerobic and anaerobic bacteria there was no significant difference in the time of exposure to the essential oil, remaining constant. The rate of corrosion was also influenced by the presence of oil. The essential oil of L. gracilis was shown to be potentially effective

Biodegradação de hidrocarbonetos aromáticos policíclicos: prospecção metagenômica e modelagem computacional 3-D de proteínas

Knowledge of the native prokaryotes in hazardous locations favors the application of biotechnology for bioremediation. Independent strategies for cultivation and metagenomics contribute to further microbiological knowledge, enabling studies with non-cultivable about the "native microbiological status and its potential role in bioremediation, for example, of polycyclic aromatic hydrocarbons (HPA's). Considering the biome mangrove interface fragile and critical bordering the ocean, this study characterizes the native microbiota mangrove potential biodegradability of HPA's using a biomarker for molecular detection and assessment of bacterial diversity by PCR in areas under the influence of oil companies in the Basin Petroleum Geology Potiguar (BPP). We chose PcaF, a metabolic enzyme, to be the molecular biomarker in a PCR-DGGE detection of prokaryotes that degrade HPA s. The PCR-DGGE fingerprints obtained from Paracuru-CE, Fortim-CE and Areia Branca-RN samples revealed the occurrence of fluctuations of microbial communities according to the sampling periods and in response to the impact of oil. In the analysis of microbial communities interference of the oil industry, in Areia Branca-RN and Paracuru-CE was observed that oil is a determinant of microbial diversity. Fortim-CE probably has no direct influence with the oil activity. In order to obtain data for better understanding the transport and biodegradation of HPA's, there were conducted in silico studies with modeling and simulation from obtaining 3-D models of proteins involved in the degradation of phenanthrene in the transport of HPA's and also getting the 3-D model of the enzyme PcaF used as molecular marker in this study. Were realized docking studies with substrates and products to a better understanding about the transport mechanism and catalysis of HPA s

Metagenômica: busca de novos genes envolvidos com a biodegradação de hidrocarbonetos

Industrial activities, oil spills and its derivatives, as well as the incomplete combustion of fossil fuels have caused a great accumulation of hydrocarbons in the environment. The number of microorganisms on the planet is estimated at 1030 and prokaryotes the most abundant. They colonized diverse environments for thousands of years, including those considered extreme and represent an untapped source of metabolic and genetic diversity with a large biotechnological potential. It is also known that certain microorganisms have the enzymatic capacity to degrade petroleum hydrocarbons and, in many ecosystems, there is an indigenous community capable of performing this function. The metagenomic has revolutionized the microbiology allowing access uncultured microbial communities, being a powerful tool for elucidation of their ecological functions and metabolic profiles, as well as for identification of new biomolecules. Thus, this study applied metagenomic approaches not only for functional selection of genes involved in biodegradation and emulsification processes of the petroleum-derived hydrocarbons, but also to describe the taxonomic and metabolic composition of two metagenomes from aquatic microbiome. We analyzed 123.116 (365 ± 118 bp) and 127.563 sequences (352 ± 120 bp) of marine and estuarine metagenomes, respectively. Eight clones were found, four involved in the petroleum biodegradation and four were able to emulsify kerosene indicating their abilities in biosurfactants synthesis. Therefore, the metagenomic analyses performed were efficient not only in the search of bioproducts of biotechnological interest and in the analysis of the functional and taxonomic profile of the metagenomes studied as well

Avaliação do desenvolvimento neuropsicomotor em crianças na rede básica de saúde utilizando o teste de Denver II: identificação de fatores de risco materno

Alterations in the neuropsychomotor development of children are not rare and can manifest themselves with varying intensity at different stages of their development. In this context, maternal risk factors may contribute to the appearance of these alterations. A number of studies have reported that neuropsychomotor development diagnosis is not an easy task, especially in the basic public health network. Diagnosis requires effective, low-cost, and easy - to-apply procedures. The Denver Developmental Screening Test, first published in 1967, is currently used in several countries. It has been revised and renamed as the Denver II Test and meets the aforementioned criteria. Accordingly, the aim of this study was to apply the Denver II Test in order to verify the prevalence of suspected neuropsychomotor development delay in children between the ages of 0 and 12 months and correlate it with the following maternal risk factors: family income, schooling, age at pregnancy, drug use during pregnancy, gestational age, gestational problems, type of delivery and the desire to have children. For data collection, performed during the first 6 months of 2004, a clinical assessment was made of 398 children selected by pediatricians and the nursing team of each public health unit. Later, the parents or guardians were asked to complete a structured questionnaire to determine possible risk indicators of neuropsychomotor development delay. Finally the Denver II Developmental Screening Test (DDST) was applied. The data were analyzed together, using Statistical Package for Social Science (SPSS) software, version 6.1. The confidence interval was set at 95%. The Denver II Test yielded normal and questionable results. This suggests compromised neuropsychomotor development in the children examined and deserves further investigation. The correlation of the results with preestablished maternal risk variables (family income, mother s schooling, age at pregnancy, drug use during the pregnancy and gestational age) was strongly significant. The other maternal risk variables (gestational problems, type of delivery and desire to have children) were not significant. Using an adjusted logistic regression model, we obtained the estimate of the greater likelihood of a child having suspected neuropsychomotor development delay: a mother with _75 4 years of schooling, chronological age less than 20 years and a drug user during pregnancy. This study produced two manuscripts, one published in Acta Cirúrgica Brasileira , in which an analysis was performed of children with suspected neuropsychomotor development delay in the city of Natal, Brazil. The other paper (to be published) analyzed the magnitude of the independent variable maternal schooling associated to neuropsychomotor development delay, every 3 months during the first twelve months of life of the children selected.. The results of the present study reinforce the multifactorial characteristic of development and the cumulative effect of maternal risk factors, and show the need for a regional policy that promotes low-cost programs for the community, involving children at risk of neuropsychomotor development delay. Moreover, they suggest the need for better qualified health professionals in terms of monitoring child development. This was an inter- and multidisciplinary study with the integrated participation of doctors, nurses, nursing assistants and professionals from other areas, such as statisticians and information technology professionals, who met all the requirements of the Postgraduate Program in Health Sciences of the Federal University of Rio Grande do Norte

Identificação e avaliação de propriedades de polissacarídeos sulfatados de diferentes fontes naturais que possibilitem sua aplicabilidade biotecnológica

Sulfated polysaccharides (SP) are widely distributed in animals and seaweeds tissues. These polymers have been studied in light of their important pharmacological activities, such as anticoagulant, antioxidant, antitumoral, anti-inflammatory, and antiviral properties. On other hand, SP potential to synthesize biomaterials like as nanoparticules has not yet been explored. In addition, to date, SP have only been found in six plants and all inhabit saline environments. However, the SP pharmacological plant activities have not been carrying out. Furthermore, there are no reports of SP in freshwater plants. Thus, do SP from marine plants show pharmacological activity? Do freshwater plants actually synthesize SP? Is it possible to synthesize nanoparticles using SP from seaweed? In order to understand this question, this Thesis was divided into tree chapters. In the first chapter a sulfated polysaccharide (SPSG) was successfully isolated from marine plant Halodule wrightii. The data presented here showed that the SPSG is a 11 kDa sulfated heterogalactan contains glucose and xylose. Several assays suggested that the SPSG possessed remarkable antioxidant properties in different in vitro assays and an outstanding anticoagulant activity 2.5-fold higher than that of heparin Clexane® in the aPTT test; in the next chapter using different tools such as chemical and histological analyses, energy-dispersive X-ray analysis (EDXA), gel electrophoresis and infra-red spectroscopy we confirm the presence of sulfated polysaccharides in freshwater plants for the first time. Moreover, we also demonstrate that SP extracted from E. crassipes root has potential as an anticoagulant compound; and in last chapter a fucan, a sulfated polysaccharide, extracted from the brown seaweed was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution for hydrophobic chains of 1H NMR was approximately 93%. SNFfuc-TBa125 in aqueous media had a mean diameter of 123 nm and zeta potential of -38.3 ± 0.74 mV, measured bydynamic light scattering. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0 43.7% at SNFuc concentrations of 0.05 0.5 mg/ mL and RAEC non-tumor cell line proliferation displayed inhibition of 8.0 22.0%. On the other hand, nanogel improved CHO and RAW non-tumor cell line proliferation in the same concentration range. Flow cytometric analysis revealed that this fucan nanogel inhibited 786 cell proliferation through caspase and caspaseindependent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle

Metacromasia para identificação de basófilos sanguíneos em surubim híbrido: contribuição metodológica

Padua S.B. & Ishikawa M.M. [Metachromasia for blood basophils identification in hybrid surubim catfish: methodological contribution]. Metacromasia para identificacao de basofilos sanguineos em surubim hibrido: contribuicao metodologica. Revista Brasileira de Medicina Veterinaria, 33(3):147-150, 2011. Centro de Aquicultura da Unesp/Jaboticabal Rod. Paulo Donato Castellane, s/n Bairro Rural, Jaboticabal, 14884-900, SP. Brasil. E-mail: santiagopadua@live.comDifferent protocols of fixation and hydrolysis of blood smears of hybrid surubim catfish for metachromasia to marking blood basophils were evaluated. For this reason, methanol, acid-alcohol and formalin vapor were tested as fixatives. For hydrolysis of blood smears, HCl, citric acid and 2-Mercaptoethanol + Urea + NaCl solutions was evaluated. After procedures, the blood smears was stained with toluidine blue (0.025%) diluted in McIlvaine buffer (pH 4). The different protocols for fixation and hydrolysis of blood smears for metachromasia influenced the quality of the reaction. Hydrolysis with 2-Mercaptoethanol + Urea + NaCl solutions provided negative results when using methanol and acid-alcohol as fixatives. The fixation with acid-alcohol associated with citric acid hydrolysis provides highest quality reason.

Identificação morfológica de organismos semelhantes à anaplasmataceae em monócitos de surubim híbrido (Pseudoplatystoma reticulatum x P. corruscans) – relato de caso

The aim of this study was to report the occurrence of Anaplasmataceae-like organisms in monocytes from the hybrid surubim catfish. During the hematological evaluation of fish infected by Pseudomonas sp. in a fish farm located in the State of Mato Grosso do Sul we observed into the monocytes the presence of numerous pleomorphic inclusions of various sizes, rough in appearance which presented basophilic staining. These inclusions were similar to elementary bodies, initial bodies and morule of the bacteria from Anaplasmataceae family, often diagnosed in domestic mammals. This is the first report of its occurrence possibly belonging to the family of Anaplasmataceae in cultured fish in Brazil. Additional studies are necessary for molecular characterization of this bacteria, pathogenic potential, life cycle and impact on the intensive production of fish.

Identificação e caracterização de genes vip e cry coleóptero-específicos em isolados de Bacillus thuringiensis

O objetivo deste trabalho foi identificar e caracterizar os genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de 1.078 isolados de Bacillus thuringiensis potencialmente tóxicos para larvas de coleópteros. Foram utilizados pares de oligonucleotídeos iniciadores gerais obtidos a partir de regiões conservadas dos genes e do alinhamento de sequências consenso. Posteriormente, os isolados positivos foram caracterizados por meio da técnica de PCR‑RFLP, tendo-se utilizado enzimas de restrição específicas, para identificar novas subclasses de genes nos isolados. Cento e cinquenta e um isolados foram positivos para os genes avaliados, com maior frequência para o gene vip1/vip2 (139 isolados). Pela técnica de PCR‑RFLP, foram observados 14 perfis polimórficos, o que indica a presença de diferentes alelos e, consequentemente, de distintas subclasses desses genes.

Identificação dos princípios ativos presentes no extrato etanólico de cereus jamacaru e avaliação em ratos dos possíveis efeitos tóxicos e/ou comportamentais da exposição prolongada

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Identificação e fatores de virulência de Candida spp isoladas da cavidade bucal de transplantados renais do hospital universitário Onofre Lopes em Natal-RN

Despite Candida species are often human commensals isolated from various oral sites such as: tongue, cheek and palatal mucosa plus subgingival region, there are some properties linked to the organism commonly known as virulence factors which confer them the ability to produce disease. Oral candidiasis is one of the main oral manifestations reported in literature related to kidney transplant patients. The objectives of the present study were to identify and investigate virulence factors of yeasts isolated from the oral cavity of kidney transplant recipients admitted at the Hospital Universitário Onofre Lopes, in Natal RN. Seventy Candida species isolated from 111 kidney transplant recipients were investigated in this study. Identification of the isolates was performed by using the evidence of germ tube formation, hypertonic broth, tolerance to grow at 42°C, micromorphology and biochemical profiles. We observed a high rate of isolation of yeasts from the oral cavity of kidney transplant recipients (63.1%) being C. albicans was the most prevalent species. Oral candidiasis was diagnosed in 14.4% of transplant recipients. We evaluated virulence properties of the isolates regarding to: biofilm formation on polystyrene microplates as well as XTT reduction, adherence to acrylic resin and human buccal epithelial cells and proteinase activity. Most isolates were able to form biofilm by the method of adhesion to polystyrene. All isolates of Candida spp. remained viable during biofilm formation when analyzed by the method of XTT reduction. The number of CFU attached to the acrylic resin suggested high adherence for C. parapsilosis. C. albicans isolates showed higher median adherence to human buccal epithelial cells than non-C. albicans Candida isolates. Nevertheless, this difference was not statistically significant. C. dubliniensis showed low ability to adhere to plastic and epithelial cells and biofilm formation. Proteolytic activity was observed for all the isolates investigated, including the unique isolate of C. dubliniensis. There was a statistically significant association between proteinase production and the presence of oral candidiasis. Studies related to oral candidiasis in renal transplant recipients are limited to clinical and epidemiological data, but investigations concerning Candida spp. virulence factor for this group of individuals are still scarce. We emphasize the importance of studies related to virulence factors of yeasts isolated from this population to contribute to the knowledge of microbiological aspects of oral candidiasis

Análises espaciais na identificação das áreas de risco para a esquistossomose mansônica no município de Lauro de Freitas, Bahia, Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Atividade microbiana do solo em pomar de laranja em resposta ao cultivo de adubos verdes

Adubos verdes (AVs) são condicionadores do solo utilizados para a melhoria da estrutura e fertilidade do solo. Este estudo avaliou o efeito de Crotalaria spectabilis Roth (crotalária), Cajanus cajan (L) Millsp (guandu) e Brachiaria decumbens Stapf (braquiária) nas propriedades microbiológicas e bioquímicas do solo sob laranjal. As leguminosas foram plantadas nas entrelinhas do laranjal e, após 3 meses, cortadas e lançadas nas entrelinhas. A braquiária, já estabelecida, foi roçada e lançada nas entrelinhas. Após 5 meses, as amostras de solo foram coletadas na entrelinha e na linha, na profundidade de 0-20 cm. O delineamento experimental foi em parcela subdividida. Efeito significativo (p< 0,05%) da aplicação dos AV sobre as contagens de bactérias, as atividades da desidrogenase e nitrificante foram obtidas e aumentaram, em média, de 20, 39 e 190%, respectivamente. O número de fungos diminuiu de 57%. A atividade da urease e a solubilizadora, e os conteúdos de matéria orgânica e umidade não foram influenciados pela aplicação dos AVs. Dentre as plantas, respostas significativas foram obtidas decorrentes do estímulo da atividade da desidrogenase em 57% pelo guandu, da urease em 58% pela braquiária, solubilizadora em 346% pela crotalária e nitrificante em 236% pelo guandu ou braquiária em relação aos demais AVs. Estes resultados sugerem que a aplicação de AVs durante anos consecutivos pode estimular a ação dos microrganismos e melhorar a qualidade do solo.

Efetividade de métodos de identificação sexual em tilápias-do-nilo (oreochromis niloticus) revertidas sexualmente com hormônio em ração com diferentes granulometrias

Three methods of sexual identification were evaluated in Nile tilapia (Oreochromis niloticus) at ages of 30, 60 and 90 days that were submitted to sexual reversion under administration of androgen hormone 17 alpha-methyltestosterone added to crumble diets with different granules sizes (0.25, 0.35 and 0.50 mm) in dosage of 60mg/kg. The techniques of sexual identification employed were: a) Macroscopic examination of urogenital papilla; b) Microscopic examination of gonads through stain with carmine-acetate and c) Microscopic examination of gonads through histological routine. In the presence of three methods of sexual identification evaluated, histology of gonads has shown the most confident diagnosis. The grain diameters of rations did not interfere in morphological characteristics of gonads, neither in secondary sexual characters of fish.

Identificação de genes cry de Bacillus thuringiensis no controle de Sphenophorus levis, o bicudo da cana-de-açúcar

A bactéria B. thuringiensis caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos, as quais são codificadas por genes cry. Este trabalho foi realizado com objetivo de selecionar isolados de B. thuringiensis, por meio da caracterização morfológica e molecular, identificando as diferentes subclasses dos genes cry3 e cry35 e determinar a patogenicidade contra Sphenophorus levis, uma das mais importantes pragas da cultura da cana-de-açúcar. Foram utilizados 1163 isolados de B. thuringiensis e com a observação em microscópio com contraste de fases foram confirmadas como pertencentes à espécie de B. thuringiensis. O material genético foi purificado pela matriz de troca iônica Instagene Matrix e submetido a PCR com iniciadores gerais cry3 e cry35 identificando-se 30 isolados contendo genes com potencial para o controle de coleópteros, os quais juntamente com as linhagens-padrão de B. thuringiensis var. tenebrionis, B. thuringiensis var. morrissone e B. thuringiensis var. tolworthi foram utilizados para a realização do bioensaio. Através de análise discriminante alocaram-se os isolados em quatro grupos quanto à toxicidade de B. thuringiensis. Os grupos ficaram assim definidos: um grupo que promovem até 10% de mortalidade contendo as testemunhas e duas linhagens;um grupo que causou 39% de mortalidade contendo três linhagens padrão e dez isolados; um grupo com 52% de mortalidade contendo treze isolados e um grupo com 70% de mortalidade contendo cinco isolados, os quais devem ser considerados promissores no controle biológico de S. levis.