Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

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

Molecular detection of in-vivo microbial contamination of metallic orthodontic brackets by checkerboard DNA-DNA hybridization

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparison Between the Polymerase Chain Reaction-Based Screening and the Southern Blot Methods for Identification of Fragile X Syndrome

The fragile X syndrome (FXS), the most common cause of hereditary mental retardation, is caused by expansions of CGG repeats in the FMR1 gene. The gold-standard method to diagnose FXS is the Southern blot (SB). Because SB is laborious and costly, some adaptations in the polymerase chain reaction (PCR) method have been utilized for FXS screening. A previous PCR-based screening method for FXS identification utilizing small amounts of DNA was reported as simple and efficient. The aim of this study was to reproduce the mentioned PCR-based screening method for identification of expanded alleles of the FMR1 gene in Brazilian individuals and to investigate the efficiency of this method in comparison with SB. Utilizing the enzyme Expand Long Template PCR System, 78 individuals were investigated by that PCR-based screening method for FXS identification. Conclusive results were obtained for 75 samples. Considering all the allelic forms of FXS (normal [NL], premutation [PM], and full-mutation [FM]), the comparison of the PCR-based screening method with SB demonstrated 100% of accuracy, sensitivity, and specificity. However, when the PM and the FM were analyzed separately from each other, but together with the NL allele, the accuracy, sensitivity, and specificity decreased (to 42.9%-97.4%). We concluded that the PCR-based screening method was reproducible and capable of identifying all different FXS alleles, but because the differentiation between the PM and the FM alleles was not accurate, SB is still the gold-standard method for the molecular diagnosis of FXS.

Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A(2) from Bothrops jararacussu Venom

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

Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling

Two L-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni (BmooLAAO) and Bothrops jararacussu (Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action. (c) 2006 Elsevier B.V. All rights reserved.

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Strand Analysis, a free online program for the computational identification of the best RNA interference (RNAi) targets based on Gibbs free energy

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

Boron transport in Eucalyptus. 2. Identification in silico of a putative boron transporter for xylem loading in eucalypt

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

Identification of a microRNA signature associated with progression of leukoplakia to oral carcinoma

MicroRNAs (miRs) are non-coding RNA molecules involved in cancer initiation and progression. Deregulated miR expression has been implicated in cancer; however, there are no studies implicating an miR signature associated with progression in oral squamous cell carcinoma (OSCC). Although OSCC may develop from oral leukoplakia, clinical and histological assessments have limited prognostic value in predicting which leukoplakic lesions will progress. Our aim was to quantify miR expression changes in leukoplakia and same-site OSCC and to identify an miR signature associated with progression. We examined miR expression changes in 43 sequential progressive samples from 12 patients and four non-progressive leukoplakias from four different patients, using TaqMan Low Density Arrays. The findings were validated using quantitative RT-PCR in an independent cohort of 52 progressive dysplasias and OSCCs, and five non-progressive dysplasias. Global miR expression profiles distinguished progressive leukoplakia/OSCC from non-progressive leukoplakias/normal tissues. One hundred and nine miRs were highly expressed exclusively in progressive leukoplakia and invasive OSCC. miR-21, miR-181b and miR-345 expressions were consistently increased and associated with increases in lesion severity during progression. Over-expression of miR-21, miR-181b and miR-345 may play an important role in malignant transformation. Our study provides the first evidence of an miR signature potentially useful for identifying leukoplakias at risk of malignant transformation.

Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

Background: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.Results: The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), beta-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.Conclusion: Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/ organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

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

A Streamlined DNA Tool for Global Identification of Heavily Exploited Coastal Shark Species (Genus Rhizoprionodon)

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

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

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

Identification and description of distinct B chromosomes in Cyphocharax modestus (Characiformes, Curimatidae)

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

Genetic identification of lamniform and carcharhiniform sharks using multiplex-PCR

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