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.

Genetics of resistance to powdery mildew (Microsphaera diffusa) in Brazilian soybean populations

Crosses between resistant and susceptible soybean cultivars were performed and the F2 populations were obtained to study the inheritance of soybean resistance to powdery mildew and to estimate the number and action of genes related to resistance. The reaction to powdery mildew was studied in a greenhouse and pots carrying plants with symptoms were distributed among the pots carrying the genotypes to be tested as a source of inoculum. Individual plants were scored according to the method of Yorinori (1997), with modifications, and classified as resistant or susceptible. The results showed that adult soybeans plants can present resistance to powdery mildew, which is controlled by one major gene with a dominant effect.

Selection and validation of reference genes for gene expression studies by reverse transcription quantitative PCR in Xanthomonas citri subsp citri during infection of Citrus sinensis

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

Xylella fastidiosa gene expression analysis by DNA microarrays

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

Comparative Studies of the (Anti) Mutagenicity of Baccharis dracunculifolia and Artepillin C by the Bacterial Reverse Mutation Test

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

A new algorithm for Reverse Monte Carlo simulations

We present a new algorithm for Reverse Monte Carlo (RMC) simulations of liquids. During the simulations, we calculate energy, excess chemical potentials, bond-angle distributions and three-body correlations. This allows us to test the quality and physical meaning of RMC-generated results and its limitations. It also indicates the possibility to explore orientational correlations from simple scattering experiments. The new technique has been applied to bulk hard-sphere and Lennard-Jones systems and compared to standard Metropolis Monte Carlo results. (C) 1998 American Institute of Physics.

A study of reverse logistics flow management in vehicle battery industries in the midwest of the state of São Paulo (Brazil)

Having the objective of minimizing costs and improving their image in the consumer and export market, car battery industries began to seek environmental alternatives geared towards sustainable development. Reverse logistics flow represents an unprecedented tool for the economic and operational development of company activities, as well as a differential in the search for competitive advantages through environmentally correct practices. The aim of this paper is to describe the reverse logistics chain adopted by automotive battery industries in the midwest of the state of São Paulo, proposing a reverse logistics framework for small manufacturers that creates actions aimed at not harming the environment. (C) 2010 Elsevier Ltd. All rights reserved.

The genetics of hypertension modifies the renal cell replication response induced by experimental diabetes

To investigate whether the genetics of hypertension modifies renal cell responses in experimental diabetes, we studied the renal cell replication and its regulation by two cyclin-dependent kinase (Cdk) inhibitors, p27(Kip1) and p21(Cip1), in prehypertensive spontaneously hypertensive rats (SUR) and their genetically normotensive counterparts, Wistar Kyoto (WKY) rats, with and without streptozotocin-induced diabetes. In diabetic SIIR, the number of proliferating glomerular (0.6 +/- 0.3 positive cells/50 glomeruli) and tubulointerstitial (2.8 +/- 0.6 positive tubulointerstitial cells/50 grid fields) cells assessed by the bromodeoxyuridine technique was significantly (P = 0.0002) lower than in control SIIR (13.2 +/- 1.7 and 48.6 +/- 9.7, respectively) and control (14.0 +/- 1.8 and 63.9 +/- 10.6) and diabetic (14.3 +/- 3.5 and 66.4 +/- 11.5) WKY rats. Proliferating cell nuclear antigen, another marker of cell proliferation, was significantly reduced in replicating glomerular (P = 0.0002) and tubulointerstitial (P < 0.0001) cells in diabetic SHR. In freshly isolated glomeruli, the level of p27(Kip1) detected by Western blotting was significantly higher In diabetic SIIR than in nondiabetic SHR (1.52 +/- 0.14 vs. 1.00 +/- 0.10% of control, P = 0.014). The expression of p21(Cip1) in isolated glomeruli did not differ among the groups of rats. In conclusion, the response of renal cell replication to diabetes differs markedly between prehypertensive SIIR and their WKY control rats. The decreased glomerular cell proliferation in prehypertensive diabetic SIIR is at least partly mediated by a higher expression of the Cdk inhibitor p27(Kip1).

Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

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

Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction

The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.

Phenylalanine ammonia-lyase activity in soybean roots extract measured by reverse-phase high performance liquid chromatography

The high performance liquid chromatography (HPLC) technique was applied to measure phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity in soybean (Glycine max L. Merril cv. BR16) roots. t-Cinnamate, the catalytic product of the PAL reaction was quantified at 275 nm by isocratic elution with methanol:water through an ODS(M) column. Comparative experiments were carried out with 1.0 mM ferulic acid, an inducer of PAL activity. The results suggest that liquid chromatography is a rapid and sensitive method to analyze PAL activity in non-purified extract.

Leishmania amazonensis: Partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage

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