Poliformismo e expressão gênica da leptina em bovinos superprecoces

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

Caracterização molecular de espécies da família Anaplasmataceae em leucócitos e plaquetas de cães de Jaboticabal-SP e de Campo Grande-MS

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Simbiontes do trato digestivo de formigas (Hymenoptera : Formicidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo das interações entre a proteína TRF de Leishmania amazonensis (LaTRF) e suas possíveis parcerias

Pós-graduação em Ciências Biológicas (Genética) - IBB

Análise comparativa de genes das caseínas de búfalo

Pós-graduação em Genética e Melhoramento Animal - FCAV

Resistance to QoI fungicides Is widespread in brazilian populations of the wheat blast pathogen magnaporthe oryzae

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

Molecular characterization of interferon regulatory factor 1 in Bubalus bubalis

Interferon regulatory factor 1 (IRF1) is functionally diverse in the regulation of immune response and is considered to be an important candidate gene for studying disease susceptibility in mammals. In this paper, we characterized the whole sequence of the IRF1 gene in river buffalo (Bubalus bubalis) and compared genomic and the amino acid sequences between different species. The buffalo IRF1 gene was 7099 bp long and organized into 10 exons and nine introns. Its molecular structure showed exactly the same number of exons (10) and introns (nine) in bovids, mice, horses, humans, and chickens. However, rats did not have exon 5, but had the largest exon 4, which suggests that exon 5 was incorporated into exon 4. The coding and the amino acid sequences of the gene showed that identity varied from 73 to 99% at the coding sequence level and from 61 to 100% at the amino acid level when compared with other mammals and chickens. Comparative analysis of the gene sequence between two different buffalo breeds, Murrah and Mediterranean, revealed six potential SNPs that are primarily located in the 5' and 3'UTRs.

Validation of absolute quantitative real-time PCR for the diagnosis of streptococcus agalactiae in fish

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

Avaliação da diluição do DNA extraido de material parafinado para amplificação em PCR

Introduction: Several reasons may lead to the failure of polymerase chain reaction (PCR) using DNA purified from paraffin-embedded materials: presence of inhibitors and degradation of target DNA. DNA dilution will often reduce the concentration of potential inhibitors and still contain enough DNA to allow PCR amplification. Objective: To evaluate the dilution influence of DNA purified from paraffin-embedded materials on β-globin PCR amplification. Material and Method: Paraffin-embedded blocks from 30 patients with oropharynx squamous cell carcinomas, diagnosed and treated at the Oral Oncology Center were selected. DNA extraction was performed using QIAmp minikit (Quiagen). DNA was quantified and evaluated for purity by spectrophotometer analysis. Two groups were formed with different amounts of DNA: group I had the originally extracted DNA and group II had the same DNA, however diluted with ultrapure water addition. PCR was performed in both groups using oligonucleotides for human β-globin gene. Results: For Group I, amplification of the β-globin gene sequence was successful in 33.33% of the samples and for Group II, in 23.33%. Conclusion: Dilution of the DNA extracted of paraffin-embedded materials did not modify statistically the amount of positive samples β-globin gene amplified in PCR, although the results suggest that this is a way to increase the method for efficacy amplification of PCR.

Detection of dengue virus in sera of Brazilian blood donors

BACKGROUND: Dengue is the most important arboviral disease in the world. Dengue viruses (DENVs) have produced huge outbreaks in Brazil in the past 25 years with more than 5 million reported cases. During these epidemics, asymptomatic individuals infected with DENV could donate blood and serve as a source of virus dissemination in the community. Here, we studied the circulation of DENV in healthy individuals during an epidemic outbreak. STUDY DESIGN AND METHODS: The study included 500 serum samples from healthy blood donors collected at the Hemotherapy Center of Ribeirao Preto, Brazil, during a dengue outbreak. The presence of DENV RNA in the serum samples was screened by real-time reverse transcriptionpolymerase chain reaction (PCR). The virus serotype was determined by a heminested PCR procedure. A partial fragment of the NS5 gene sequence was used for phylogenetic analysis. RESULTS: DENV RNA was detected in the serum sample of 2 of 500 (0.4%) individuals. Both of them were infected with DENV-3 Genotype III, a virus that has been circulating in Brazil in the past decade. CONCLUSION: Individuals with asymptomatic DENV infection can be blood donors and serve as a source of virus dissemination in the community. Further studies are needed to determine the risk of recipient infection by DENV as a result of transfusion in Brazil, especially during epidemic periods.

Gastrotricha: A Marine Sister for a Freshwater Puzzle

Background: Within an evolutionary framework of Gastrotricha Marinellina flagellata and Redudasys fornerise bear special interest, as they are the only Macrodasyida that inhabit freshwater ecosystems. Notwithstanding, these rare animals are poorly known; found only once (Austria and Brazil), they are currently systematised as incertae sedis. Here we report on the rediscovery of Redudasys fornerise, provide an account on morphological novelties and present a hypothesis on its phylogenetic relationship based on molecular data. Methodology/Principal Findings: Specimens were surveyed using DIC microscopy and SEM, and used to obtain the 18 S rRNA gene sequence; molecular data was analyzed cladistically in conjunction with data from 42 additional species belonging to the near complete Macrodasyida taxonomic spectrum. Morphological analysis, while providing new information on taxonomically relevant traits (adhesive tubes, protonephridia and sensorial bristles), failed to detect elements of the male system, thus stressing the parthenogenetic nature of the Brazilian species. Phylogenetic analysis, carried out with ML, MP and Bayesian approaches, yielded topologies with strong nodal support and highly congruent with each other. Among the supported groups is the previously undocumented clade showing the alliance between Redudasys fornerise and Dactylopodola agadasys; other strongly sustained clades include the densely sampled families Thaumastodermatidae and Turbanellidae and most genera. Conclusions/Significance: A reconsideration of the morphological traits of Dactylopodola agadasys in light of the new information on Redudasys fornerise makes the alliance between these two taxa very likely. As a result, we create Anandrodasys gen. nov. to contain members of the previously described D. agadasys and erect Redudasyidae fam. nov. to reflect this novel relationship between Anandrodasys and Redudasys. From an ecological perspective, the derived position of Redudasys, which is deeply nested within the Macrodasyida clade, unequivocally demonstrates that invasion of freshwater by gastrotrichs has taken place at least twice, in contrast with the single event hypothesis recently put forward.

Genotyping of Human parvovirus B19 among Brazilian patients with hemoglobinopathies

Human parvovirus B19 (B19V) infection can be a life-threatening condition among patients with hereditary (chronic) hemolytic anemias. Our objective was to characterize the infection molecularly among patients with sickle cell disease and thalassemia. Forty-seven patients (37 with sickle cell disease, and 10 with beta-thalassemia major) as well as 47 healthy blood donors were examined for B19V infection by anti-B19V IgG enzyme immunoassay, quantitative PCR, which detects all B19V genotypes, and DNA sequencing. B19V viremia was documented in nine patients (19.1%) as two displayed acute infection and the rest had a low titre viremia (mean 3.4 x 10(4) copies/mL). All donors were negative for B19V DNA. Anti-B19V IgG was detected in 55.3% of the patients and 57.4% among the donors. Based on partial NS1 fragments, all patient isolates were classified as genotype 1 and subgenotype 1A. The evolutionary events of the examined partial NS1 gene sequence were associated with a lack of positive selection. The quantification of all B19V genotypes by a single hydrolytic probe is a technically useful method, but it is difficult to establish relationships between B19V sequence characteristics and infection outcome.

Phylogenetic relationships of rock-wallabies, Petrogale (Marsupialia: Macropodidae) and their biogeographic history within Australia

The rock-wallaby genus Petrogale comprises a group of habitat-specialist macropodids endemic to Australia. Their restriction to rocky outcrops, with infrequent interpopulation dispersal, has been suggested as the cause of their recent and rapid diversification. Molecular phylogenetic relationships within and among species of Petrogale were analysed using mitochondrial (cytochrome oxidase c subunit 1, cytochrome b. NADH dehydrogenase subunit 2) and nuclear (omega-globin intron, breast and ovarian cancer susceptibility gene) sequence data with representatives that encompassed the morphological and chromosomal variation within the genus, including for the first time both Petrogale concinna and Petrogale purpureicollis. Four distinct lineages were identified, (1) the brachyotis group, (2) Petrogale persephone, (3) Petrogale xanthopus and (4) the lateralis-penicillata group. Three of these lineages include taxa with the ancestral karyotype (2n = 22). Paraphyletic relationships within the brachyotis group indicate the need for a focused phylogeographic study. There was support for P. purpureicollis being reinstated as a full species and P. concinna being placed within Petrogale rather than in the monotypic genus Peradorcas. Bayesian analyses of divergence times suggest that episodes of diversification commenced in the late Miocene-Pliocene and continued throughout the Pleistocene. Ancestral state reconstructions suggest that Petrogale originated in a mesic environment and dispersed into more arid environments, events that correlate with the timing of radiations in other arid zone vertebrate taxa across Australia. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Efficient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNai (TM)). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80 degrees C. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAi (TM) was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Strain-specific polyketide synthase genes of Aspergillus niger

In silico comparison of 34 putative pks genes in Aspergillus niger strain CBS 513.88 versus A. niger strain ATCC 1015 genome revealed significant nucleotide identity (>95% covering a minimum of 99% of the gene sequence) for 31 of these genes (approximately 91%). A. niger CBS 513.88 harbors three putative pks genes (An01g01130, An11g05940, and An15g07920), for which nucleotide identity was not found in A. niger ATCC 1015. To compare the results of the in silico analysis with the in vivo situation, experimental data were obtained for a large number of A. niger strains obtained from different substrates and geographical regions. Three putative Os genes that were found to be variable between the two A. niger strains using bioinformatics tools were in fact strain-specific genes based on experimental data. The PCR amplification signals for the An01g01130, An11g05940, and An15g07920 pks genes were detected in only 97%, 71%, and 26% of the strains, respectively. Southern blot analyses confirmed the PCR data. Because one of the strain-specific pits genes (An15g07920) is located in a putative ochratoxin cluster, we focused our investigation on that region. We assessed the ochratoxin production capability of the 119 A. niger strains and found a positive association between the presence of this pia gene and the capability of the respective strain to produce ochratoxin. (C) 2012 Elsevier B.V. All rights reserved.