Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota-LS-(vaccine strain) and Sao Joao do Meriti-SJM-strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity. © 2012 Springer Science+Business Media B.V.

Amplifiability of mitochondrial, microsatellite and amelogenin DNA loci from fecal samples of red brocket deer Mazama americana (Cetartiodactyla, Cervidae)

We tried to amplify mitochondrial, microsatellite and amelogenin loci in DNA from fecal samples of a wild Mazama americana population. Fifty-two deer fecal samples were collected from a 600-ha seasonal semideciduous forest fragment in a subtropical region of Brazil (21°20′, 47°17′W), with the help of a detection dog; then, stored in ethanol and georeferenced. Among these samples 16 were classified as fresh and 36 as non-fresh. DNA was extracted using the QIAamp® DNA Stool Mini Kit. Mitochondrial loci were amplified in 49 of the 52 samples. Five microsatellite loci were amplified by PCR; success in amplification varied according to locus size and sample age. Successful amplifications were achieved in 10/16 of the fresh and in 13/36 of the non-fresh samples; a negative correlation (R = -0.82) was found between successful amplification and locus size. Amplification of the amelogenin locus was successful in 22 of the 52 samples. The difficulty of amplifying nuclear loci in DNA samples extractedfrom feces collected in the field was evident. Some methodological improvements, including collecting fresh samples, selecting primers for shorter loci and quantifying the extracted DNA by real-time PCR, are suggested to increase amplification success in future studies. © FUNPEC-RP.

Cooperative RNA Polymerase Molecules Behavior on a Stochastic Sequence-Dependent Model for Transcription Elongation

The transcription process is crucial to life and the enzyme RNA polymerase (RNAP) is the major component of the transcription machinery. The development of single-molecule techniques, such as magnetic and optical tweezers, atomic-force microscopy and single-molecule fluorescence, increased our understanding of the transcription process and complements traditional biochemical studies. Based on these studies, theoretical models have been proposed to explain and predict the kinetics of the RNAP during the polymerization, highlighting the results achieved by models based on the thermodynamic stability of the transcription elongation complex. However, experiments showed that if more than one RNAP initiates from the same promoter, the transcription behavior slightly changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAPs and predicts their cooperative behavior during multi-round transcription generalizing the Bai et al. stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica® and compared the results of the single and the multiple-molecule transcription with experimental results and other theoretical models. Our multi-round approach can recover several expected behaviors, showing that the transcription process for the studied sequences can be accelerated up to 48% when collisions are allowed: the dwell times on pause sites are reduced as well as the distance that the RNAPs backtracked from backtracking sites. © 2013 Costa et al.

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity. © 2012 Springer Science+Business Media B.V.

Avaliação histopatológica, imunoistoquímica e detecção molecular do DNA viral no sistema nervoso central de bovinos inoculados experimentalmente com herpesvirus bovino 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detecção da microbiota endodôntica de dentes sem vitalidade pulpar com ou sem lesão periapical radiográfica, por meio das técnicas de checkerboard DNA-DNA hybridization e cultura microbiológica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Atividade anti-tumoral e imunomodulatória de complexos de paládio (II) utilizando modelo experimental de Ehrlich

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da PCR em tempo real para detecção de Cryptosporidium parvum em amostras fecais de bezerros: Camila Guariz Homem. -

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência do padrão alimentar e dos polimorfismos dos genes GSTM1, GSTT1, GSTP1, CYP2E1, XRCC1, MTHFR e TS sobre os níveis de danos oxidativos no DNA e de uracilas incorporadas ao DNA

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência do padrão alimentar sobre os níveis de danos oxidativos no DNA em linfócitos de sangue periférico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Amplificação de DNA circulante para o diagnóstico da cisticercose bovina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação clínica de doadores de sangue portadores do vírus linfotrópico de células T humanas (HTLV - I/II)

Os vírus linfotrópicos de células T humanas do tipo I e II (HTLV-I/II) são retrovírus que podem ser transmitidos por transfusão de sangue. Estes vírus estão associados com paraparesia espástica tropical (PET), leucemia/linfoma de células T do adulto (L/LTA) e outras doenças sistêmicas imunomediadas. O presente estudo teve como objetivo investigar sinais clínicos de patologias associadas a esses vírus para utilizar na triagem clinica de candidatos à doação de sangue. Usou procedimentos padronizados para avaliação clinica de 30 doadores de sangue soropositivos para HTLV-I/II confirmados pela técnica de reação em cadeia da polimerase (PCR) matriculados no ambulatório do Núcleo de Medicina Tropical da UFPA. Paralelamente, através de interrogatório clínico complementar, estudou grupo controle com 40 candidatos à doação de sangue escolhidos aleatoriamente, que tiveram resultados sorológicos negativos. Dos 30 pacientes examinados, verificou-se que 23 eram portadores de HTLV-I e 07 HTLV-II. Na avaliação clínica, 15 pacientes (50%) não referiram queixas, sendo que 12 pacientes com queixas exclusivamente neurológicas. Observou 05 pacientes com formigamentos; 05 com diminuição da força muscular; 04 com constipação intestinal; 02 com parestesia; 02 com nódulos subcutâneos; 01 com incontinência urinária; 01 com visão borrada; 01 com diminuição do libido. No grupo controle, 05 candidatos (12,5%) referiram queixas. Os resultados indicam que diminuição da força muscular e formigamento devem ser questionados na triagem clínica prévia à doação de sangue para reduzir risco de infecção transfusional.

Influência dos caretonóides, retinol e α-Tocoferol e dos polimorfismos dos genes CYP1A1, GSTP1, MTHFR (A1298C E C6777) E XRCC1 (194Trp E 399 Gln) sobre os níveis de danos oxidativos do DNA, de uracilas incorporadas ao DNA e da capacidade de reparo do DNA

Pós-graduação em Patologia - FMB

A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes)

DNA barcoding is a recently proposed global standard in taxonomy based on DNA sequences. The two main goals of DNA barcoding methodology are assignment of specimens to a species and discovery of new species. There are two main underlying assumptions: i) reciprocal monophyly of species, and ii) intraspecific divergence is always less than interspecific divergence. Here we present a phylogenetic analysis of the family Potamotrygonidae based on mitochondrial cytochrome c oxidase I gene, sampling 10 out of the 18 to 20 valid species including two non-described species. Potamotrygonidae systematics is still not fully resolved with several still-to-be-described species while some other species are difficult to delimit due to overlap in morphological characters and because of sharing a complex color patterns. Our results suggest that the family passed through a process of rapid speciation and that the species Potamotrygon motoro, P. scobina, and P. orbignyi share haplotypes extensively. Our results suggest that systems of identification of specimens based on DNA sequences, together with morphological and/or ecological characters, can aid taxonomic studies, but delimitation of new species based on threshold values of genetic distances are overly simplistic and misleading.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb)gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.