Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

Presença de substâncias fisològicamente ativas em culturas de E. Coli isolados das fezes de pacientes alérgicos pela técnica de Heist-Cohen

Four patients with several allergic symptoms have been studied. Heist-Cohen pathogen selective method was positive in feces of all these patients for E. coli. Other causes that could be responsible for the allergic symptoms were discarded. Samplets after seeding in a medium physiologically inactive have been tested in guinea pig illea following Cohnheim-Magnus technique. It was demonstrated that all samples isolated by the Heist-Cohen technique were physiologically active.(Fig.1). Ten other strains of E. coli taken at random from the collection of I.O.C. and tried according similar technique, brought evidence that only three samples possessed such activity. This confirms the works of KOESSLER-HANKE (1922). In order to obtain a quick relief from allergic symptoms, one of us had employed in some others, identical cases an antibiotic (Teramycin, Chas Pfeizer) before the specific disesitization was done (Figs. 2, 3, 4, 5). Such method were choice since LABORDE, PARROT, and URQUIA (1953) have demonstrated the importance of production of histamine, from components of the bowel's flora in the production of allergic symptoms.

Influência da Matéria Orgânica no Diâmetro Médio de Minerais da Fração Argila de Solos Desenvolvidos de Basalto no Sul do Brasil

RESUMO A interação de compostos orgânicos com minerais de argila pode alterar o tamanho dos cristais. No entanto, em solos, esse efeito é ainda pouco claro por causa das limitações na separação de fases puras de minerais para realizar experimentos de dissolução. Neste estudo, a relação entre a matéria orgânica do solo (MOS) e diâmetro médio do cristal (DMC) de minerais de argila de horizontes superficiais e subsuperfíciais de solos de uma topossequência no sul do Brasil foi avaliada. Os teores de C e N foram determinados, e a natureza dos grupos funcionais da MOS foi avaliada por espectroscopia de FTIR. O DMC dos minerais foi avaliado por difração de raios X, em umas amostras desferrificadas e outras com óxidos de Fe concentrados. Os teores de C e N e as intensidades relativas dos espectros de FTIR foram considerados como variáveis preditoras; e o DMC de hematita, goethita, caulinita e gibbsita, como variáveis preditas. Os teores de C e N e os grupos carboxílicos e C-O-Alquil evidenciaram efeito significativo sobre a variação do DMC. A dimensão dos cristais de óxidos de Fe e de caulinita foi inversamente correlacionada com esses atributos da MOS. Em contraste, o DMC de gibbsita não foi influenciado pelos atributos dae MOS. A influência da MOS sobre o DMC dos óxidos de Fe foi atribuída às reações de complexação de superfície e de redox, que promovem um processo contínuo de dissolução-precipitação.

Genetic diversity of a brazilian wine grape germplasm collection based on morphoagronomic traits

The objectives of this study were to evaluate the performance of cultivars, to quantify the variability and to estimate the genetic distances of 66 wine grape accessions in the Grape Germplasm Bank of the EMBRAPA Semi-Arid, in Juazeiro, BA, Brazil, through the characterization of discrete and continuous phenotypic variables. Multivariate statistics, such as, principal components, Tocher's optimization procedure, and the graphic of the distance, were efficient in grouping more similar genotypes, according to their phenotypic characteristics. There was no agreement in the formation of groups between continuous and discrete morpho-agronomic traits, when Tocher's optimization procedure was used. Discrete variables allowed the separation of Vitis vinifera and hybrids in different groups. Significant positive correlations were observed between weight, length and width of bunches, and a negative correlation between titratable acidity and TSS/TTA. The major part (84.12%) of the total variation present in the original data was explained by the four principal components. The results revealed little variability between wine grape accessions in the Grape Germplasm Bank of Embrapa Semi-Arid.

Agronomic and molecular traits of the 'Italia Muscat' clone in Sao Francisco River Valley

The objective of this study was to evaluate agronomic and molecular traits of the 'Italia Muscat' clone and compare it with the cv. 'Italia', providing information to support the cultivation of 'Italia Muscat' this cultivar in the São Francisco River Valley. Agronomic characteristics of both clones were evaluated for two seasons in 2004. The characteristics were phenology, bud break (%), bud fertility (%), yield (kg) mass of bunches (g), length and width of bunches (cm), mass of berries (g), length and diameter of berries (mm), TSS (ºBrix), ATT (% titratable acidity) and TSS/TTA. Molecular analysis of seven SSR markers were carried out. The clone 'Italia Muscat' showed larger berries, mass of bunches and better TSS/TA ratio than 'Italia'. The molecular analysis resulted in the same allelic profile in both clones, highlighting the need to use a larger number of microsatellite markers or other molecular technique to allow their discrimination. Based on their morpho-agronomic characteristics, 'Italia Muscat' seems to be a good table grape cultivar alternative for grape growers of São Francisco River Valley.

Ciclização do lapachol induzida por sais de tálio III

This work describes the cyclization of lapachol (1) induced by thallium triacetate (TTA) and thallium trinitrate (TTN) in several solvents using magnetic stirring and under microwave irradiation. alpha-Xyloidone (2) - dehydro-alpha-lapachone - was obtained as the main product in these reactions in 20 75% yield. However, rhinacanthin-A (4) was isolated as main product in a 40% yield, using TTA and acetic anhydride:water (1:1) as solvent, and dehydro-iso-alpha-lapachone (3) in 21% yield, using TTA and dichloromethane as solvent. The reaction time decreased drastically under microwave conditions, but the yields of these reactions were not the expected.

A NOVEL METHOD FOR THE DETERMINATION OF TRACE THORIUM BY DISPERSIVE LIQUID-LIQUID MICROEXTRACTION BASED ON SOLIDIFICATION OF FLOATING ORGANIC DROP

In this study, dispersive liquid-liquid microextraction based on the solidification of floating organic droplets was used for the preconcentration and determination of thorium in the water samples. In this method, acetone and 1-undecanol were used as disperser and extraction solvents, respectively, and the ligand 1-(2-thenoyl)-3,3,3-trifluoracetone reagent (TTA) and Aliquat 336 was used as a chelating agent and an ion-paring reagent, for the extraction of thorium, respectively. Inductively coupled plasma-optical emission spectrometry was applied for the quantitation of the analyte after preconcentration. The effect of various factors, such as the extraction and disperser solvent, sample pH, concentration of TTA and concentration of aliquat336 were investigated. Under the optimum conditions, the calibration graph was linear within the thorium content range of 1.0-250 µg L-1 with a detection limit of 0.2 µg L-1. The method was also successfully applied for the determination of thorium in the different water samples.

Adaptation of "Niagara Rosada" grape must to winemaking by partial cluster dehydration

This study aimed to verify the influence of partial dehydration of "Niagara Rosada" grape clusters in physicochemical quality of the pre- fermentation must. In Brazil, during the winemaking process it is common to need to adjust the grape must when the physicochemical characteristics of the raw material are insufficient to produce wines in accordance with the Brazilian legislation for classification of beverages, which establishes the minimum alcohol content of 8.6 % for the beverage to be considered wine. Therefore, given that the reduction in the water content of grape berries allows the concentration of chemical compounds present in its composition, especially the concentration of total soluble solids, we proceeded with the treatments that were formed by the combination of two temperatures (T1-37.1ºC and T2-22.9 ºC) two air speeds (S1: 1.79 m s-1 and S2: 3.21 m s-1) and a control (T0) that has not gone through the dehydration treatment. Analysis of pH, Total Titratable Acidity (TTA) were performed in mEq L-1, Total Soluble Solids (TSS) in ºBrix, water content on a dry basis and Concentration of Phenolic Compounds (CPC) in mg of gallic acid per 100g of must. The average comparison test identified statistically significant modifications for the adaptation of must for winemaking purposes, having the treatment with 22.9 ºC and air speed of 1.79 m s-1 shown the largest increase in the concentration of total soluble solids, followed by the second best result for concentration of phenolic compounds.

Ripening pattern of guava cv. Pedro Sato

Guava is a fruit with high respiration rates and a very short shelf life. Since information on its respiration pattern is contradictory, the objective was to study the changes occurring in the fruit during ripening and to relate them to the respiration behavior of this fruit. Guavas were picked at the half-ripe stage and stored for 8 days at 22 ± 1 ºC and 78 ± 1% relative humidity. The analyses conducted were: peel and pulp coloration, firmness, total soluble solids (TSS), total titratable acidity (TTA), and ethylene production. According to the results, it was verified that the parameters analyzed apparently do not coincide and are ethylene-independent. There was an accentuated ethylene production during ripening, starting from the 4th day. The ethylene synthesis continued increasing up to the 8th day, when the fruits were already decomposing. It was observed that the firmness decreased sharply in the first three days of ripening, and the skin and pulp color changed during ripening. The TSS, total soluble solids, and the TTA, total titratable acidity, practically did not change during the ripening, even with the increased ethylene production. It can be concluded that guava is a fruit that presents characteristics of climacteric and non-climacteric fruits.

Chemical and physical characterization of mume fruit collected from different locations and at different maturity stages in São Paulo State

Prunus mume is widely studied due to its health benefits regarding increase of blood fluidity and consequent improvement of the cardiovascular system and the prevention or even the fight against different types of cancer. However, in Brazil this culture is found only among oriental descendants. The present study aimed to characterize mume fruit collected from three different locations in the State of São Paulo regarding general aspects such as pH, total titratable acidity (TTA), total soluble solids (TTS), pectin content and yield of pulp and chemical characteristics: total phenolic compounds (TPC) and antioxidant capacity. Mume fruit were collected unripe and analyzed until maturation about 88 days after flowering. Fruit collected in Botucatu came from a commercial mume fruit producer and had average weight of 16.9 g, while in fruit from other locations weight varied from 5.7-6.9 g. TSS ranged from 9.5 to 10.0 Brix, total solids was 10.2-12.2% and pH showed values between 2.5 and 2.7 for all locations. TTA expressed in citric acid decreased from 4.0-5.7 g (100g- 1) at unripe stage to 2.0-3.8 g (100g- 1) in mature-stage fruit. Pectin content decreased from 11.2 to 10.8% during fruit maturation, TPC content was 147-226 mg catechin (g- 1) on a dry matter basis and the antioxidant capacity was 96-169 µMol Trolox (g- 1) on a dry matter basis or 21-34 µMol Trolox (g- 1) on a wet matter basis.