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)

Paracellular Absorption: A Bat Breaks the Mammal Paradigm

Bats tend to have less intestinal tissue than comparably sized nonflying mammals. The corresponding reduction in intestinal volume and hence mass of digesta carried is advantageous because the costs of flight increase with load carried and because take-off and maneuverability are diminished at heavier masses. Water soluble compounds, such as glucose and amino acids, are absorbed in the small intestine mainly via two pathways, the transporter-mediated transcellular and the passive, paracellular pathways. Using the microchiropteran bat Artibeus literatus (mean mass 80.6 +/- 3.7 g), we tested the predictions that absorption of water-soluble compounds that are not actively transported would be extensive as a compensatory mechanism for relatively less intestinal tissue, and would decline with increasing molecular mass in accord with sieve-like paracellular absorption. Using a standard pharmacokinetic technique, we fed, or injected intraperitonealy the metabolically inert carbohydrates L-rhamnose (molecular mass = 164 Da) and cellobiose (molecular mass = 342 Da) which are absorbed only by paracellular transport, and 3-O-methyl-D-glucose (3OMD-glucose) which is absorbed via both mediated (active) and paracellular transport. As predicted, the bioavailability of paracellular probes declined with increasing molecular mass (rhamnose, 90 +/- 11%; cellobiose, 10 +/- 3%, n = 8) and was significantly higher in bats than has been reported for laboratory rats and other mammals. In addition, absorption of 3OMD-glucose was high (96 +/- 11%). We estimated that the bats rely on passive, paracellular absorption for more than 70% of their total glucose absorption, much more than in non-flying mammals. Although possibly compensating for less intestinal tissue, a high intestinal permeability that permits passive absorption might be less selective than a carrier-mediated system for nutrient absorption and might permit toxins to be absorbed from plant and animal material in the intestinal lumen.

Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the "Mfold Web Server" simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of "Dinamelt Web Server". The results were evaluated considering the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors. (C) 2011 Elsevier B.V. All rights reserved.

Differential susceptibility to chromatid breaks induced by bleomycin in sub-fertile and fertile bovines

The rate of chromatid breaks was studied in cows with a history of sub-fertility by means of a test based on measurement of the average of breaks induced in lymphocytes of peripheral blood cultures. Fourteen female specimens were divided into two groups: fertile and sub-fertile. Peripheral blood lymphocytes were cultured and prepared for cytogenetic analysis. Two types of culture were established for each animal to evaluate the response of peripheral blood lymphocyte cultures to the genotoxic effects of bleomycin. The first culture did not receive bleomycin treatment (spontaneous chromosome aberrations). Our results showed that median breaks per cell (b/c) (+/-semirange) for spontaneous culture of the fertile and sub-fertile animals and bleomycin sensitivity assay for fertile and sub-fertile animals were 0.00 +/- 0.06, 0.02 +/- 0.03, 0.08 +/- 0.05 and 0.22 +/- 0.09, respectively. There was no significant difference (P > 0.05) in the chromosomal breakage in lymphocytes not exposed to bleomycin; however, in comparing the number of chromatid breaks per cell in cultures treated with bleomycin, the sub-fertile group showed a significantly higher (P < 0.05) level than the fertile group. These findings have implications both for identifying cattle with less than optimum fertility as well as for providing potential avenues to study the origins of sub-fertility. (C) 2004 Elsevier B.V. All rights reserved.

Chromosome sensitivity to bleomycin in G2 lymphocytes from Down syndrome patients

Several studies have demonstrated that lymphocytes from patients with Down syndrome (DS) exhibit an increased frequency of chromosome aberrations when they are exposed to ionizing radiation or to chemicals at the G0 or G1 phases of the cell cycle, but not at G2 when compared to normal subjects. To determine the susceptibility of DS lymphocytes at G2 phase, bleomycin, a radiomimetic agent, was used to induce DNA breaks in blood cultures from 24 Down syndrome patients. All the patients with DS showed free trisomy 21 (47,XX + 21 or 47,XY + 21). Individuals that showed an average number of chromatid breaks per cell higher than 0.8 were considered sensitive to the drug. No control child showed susceptibility to bleomycin, and among the 24 patients with DS, only one was sensitive to the drug. No significant difference was observed between the two groups, regarding chromatid break frequencies in treated G2 lymphocytes. The distribution of bleomycin-induced breaks in each group of chromosomes was similar for DS and controls. No significant difference was found in the response to bleomycin between male and female subjects. Probably, the main factor involved in chromosome sensitivity of lymphocytes from patients with DS is the phase of the cell cycle in which the cell is treated.

Assessment of DNA damage by extracts and fractions of Strychnos pseudoquina, a Brazilian medicinal plant with antiulcerogenic activity

Strychnos pseudoquina St. Hil. is a native plant of the Brazilian Savannah, used in popular medicine to treat a number of conditions. Since it contains large quantities of alkaloids with proven antiulcer activity, we tested the genotoxic potential of crude extracts and fractions containing alkaloids and flavonoids from the leaves of this plant, on Salmonella typhimurium and performed the micronucleus test on peripheral blood cells of mice treated in vivo. The results showed that the methanol extract of the leaves of S. pseudoquina is mutagenic to the TA98 (-S9) and TA100 (+S9, -S9) strains of Salmonella. The dichloromethane extract was not mutagenic to any of the tested strains. Fractions enriched with alkaloids or flavonoids were not mutagenic. In vivo tests were done on the crude methanol extract in albino Swiss mice, which were treated, by gavage, with three different doses of the extract. The highest dose tested (1800 mg/kg b.w.) induced micronuclei after acute treatment, confirming the mutagenic potential of the methanol extract of the leaves of S. pseudoquina. In high doses, constituents of S. pseudoquina compounds act on DNA, causing breaks and giving rise to micronuclei in the blood cells of treated animals. © 2006 Elsevier Ltd. All rights reserved.

In vitro cytotoxic and non-genotoxic effects of gutta-percha solvents on mouse lymphoma cells by single cell gel (comet) assay

Chloroform and eucalyptol are widely used in clinical dentistry as gutta-percha solvents. However, these compounds may represent a hazard to human health, especially by causing injury to genetic apparatus and/or inducing cellular death. In this study, the genotoxic and cytotoxic potentials associated with exposure to chloroform and eucalyptol were assessed on mouse lymphoma cells in vitro by the single cell gel (comet) assay and trypan blue exclusion test, respectively. Both gutta-percha solvents proved to be cytotoxic at the same levels in concentrations of 2.5, 5 and 10 μL/mL (p<0.05). On the other hand, neither of the solvents induced DNA breakage. Taken together, these results suggest that although both tested compounds (chloroform and eucalyptol) are strong cytotoxicants, it seems that they are not likely to increase the level of DNA damage on mammalian cells.

Study of Brazilian commercial Oriented Strand Board panels using stress wave

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Investigation of single-strand conformational polymorphism of the TP53 gene in women with a family history of breast cancer

Breast cancer in families with germ line mutations in the TP53 gene has been described in the medical literature. Mutation screening for susceptibility genes should allow effective prophylactic and preventive measures. Using single-strand conformational polymorphism, we screened for mutations in exons 5, 6, 7 and 8 of gene TP53 in the peripheral blood of 8 young non-affected members (17 to 36 years old) of families with a history of breast cancer. Studies of this type on young patients (mean age, 25 years) are very rare in the literature. The identification of these mutations would contribute to genetic counseling of members of families with predisposition to breast cancer. The results obtained did not show any polymorphism indicating mutation. In our sample, the familial tumorigenesis is probably related to other gene etiologies.

Construction and validation of an instrument that breaks the silence: the impact of domestic and/or sexual violence on women's health, as shown during climacterium

Objective: The aim of this study was to construct and to validate a measure of the consequences of domestic violence on women's health during climacterium. Methods: A questionnaire was administered at the Outpatient Climacterium Clinic to 124 women aged 40 to 65 years who were the victims of domestic and/or sexual violence (experimental group). They were divided into three groups: (1) those who were victims of violence exclusively during childhood/adolescence, (2) those who were victims of violence exclusively during adulthood, and (3) those who were victims of violence throughout their lives. The instrument included 34 items evaluating the beginning, frequency, and type of violence; the search for health assistance and reporting of the violence; the violence and the number of comorbidities; and violence and the Kupperman Menopausal Index. We also included a control group composed of perimenopausal and postmenopausal women who did not experience any violence (n = 120). Results: The instrument presented a Cronbach alpha = 0.82, good reliability among the examiners (+0.80), and a good possibility of reproducibility. The mean age of menopause was 45.4 years, and the mean age in the control group was 48.1 years. Group 1 showed a mean of 5.1 comorbidities, Group 2 had 4.6, and Group 3 had 4.4. Sexual violence (43.5%) and other types of violence both presented average comorbidities (4.60) but represented a significant impairment in the victim's sexual life. There were significant associations in group 3 and a high Kupperman Menopausal Index score. In the experimental group, 80.6% did not seek health services for the violence they experienced. Conclusions: The questionnaire presented good internal consistency and a validated construction. It can be easily reproduced and is indicated to evaluate the consequences of domestic and/or sexual violence on women's health during climacterium.

Are there genetic breaks between Atlantic and Pacific Yellowfin tuna (Thunnus albacares) populations? A preliminary study based on microsatellites gene variation

Yellowfin tuna (Thunnus albacares, YFT, Bonnaterre 1788) is one of the most important market tuna species in the world. The high mortality of juveniles is in part caused by their bycatch. Indeed, if unregulated, it could permanently destabilize stocks health. For this reason investigating and better knowing the stock boundaries represent a crucial concern. Aim of this thesis was to preliminary investigate the YFT population structure within and between Atlantic and Pacific Oceans through the analysis of genetic variation at eight microsatellite loci and assess the occurrence of barriers to the gene flow between Oceans. For this propouse we collected 4 geographical samples coming from Atlantic and Pacific Ocean and selected a panel of 8 microsatellites loci developped by Antoni et al., (2014). Samples 71-2-Y and 77-2-Y, came from rispectively west central pacific ocean (WCPO) and east central pacific ocean (ECPO), instead samples 41-1-Y and 34-2-Y derive from west central atlantic ocean (WCAO) and east central atlantic ocean (ECAO). Total 160 specimens were analyzed (40 per sample) and were carried out several genetic information as allele frequencies, allele number, allelic richness, HWE (using He and Ho) and pairwise Fst genetic distance. Results obtained, may support the panmictic theory of this species, only one of pairwise Fst obtained is statistically significant (Fst= 0.00927; pV= 0.00218) between 41-1-Y and 71-2-Y samples. Results suggest low genetic differentiation and consequent high level of gene flow between Atlantic and Pacific populations. Furthermore, we performed an analysis of molecular taxonomy through the use of ATCO (the flaking region between ATPse6 and cytochrome oxidase subunit III genes mt DNA, to discriminate within the gener Thunnus two of the related species (Yellofin and bigeye tuna) according with their difficult recognition at certain size (<40 cm). ATCO analysis in this thesis, has provided strong discriminate evidence between the target species proving to be one of the most reliable genetic tools capable to indagate within the genus Thunnus. Thus, our study has provided useful information for possible use of this protocol for conservation plans and management of this fish stocks.

Life history, ecology and the biogeography of strong genetic breaks among 15 species of PaciWc rockWsh, Sebastes

Strong genetic change over short spatial scales is surprising among marine species with high dispersal potential. Concordant breaks among several species signals a role for geographic barriers to dispersal. Along the coast of California, such breaks have not been seen across the biogeographic barrier of Point Conception, but other potential geographic boundaries have been surveyed less often.;We tested for strong-population structure in 11 species of Sebastes sampled across two regions containing potential dispersal barriers, and conducted a meta-analysis including four additional species. We show two strong breaks north of Monterey Bay, spanning an oceanographic gradient and an upwelling jet. Moderate genetic structure is just as common in the north as it is in the south, across the biogeographic break at Point Conception. Gene Xow is generally higher among deep-water species, but these conclusions are confounded by phylogeny. Species in the subgenus Sebastosomus have higher structure than those in the subgenus;Pteropodus, despite having larvae with longer pelagic phases. DiVerences in settlement behavior in the face of ocean currents might help explain these diVerences. Across similar species across the same coastal environment, we document a wide variety of patterns in gene Xow, suggesting that interaction of individual species traits such as settlement behavior with environmental factors such as;oceanography can strongly impact population structure

HIV-1 p24 may persist during long-term highly active antiretroviral therapy, increases little during short treatment breaks, and its rebound after treatment stop correlates with CD4(+) T cell loss

The dynamics of HIV-1 RNA during structured treatment interruptions (STIs) are well established, but little is known about viral proteins like p24. We studied 65 participants of an STI trial. Before the trial, continuous highly active antiretroviral therapy (HAART) had suppressed their viral load to <50 copies/mL during 6 months. They then interrupted HAART during weeks 1 through 2, 11 through 12, 21 through 22, 31 through 32, and 41 through 52. The p24 was measured by boosted enzyme-linked immunosorbent assay of plasma pretreated by efficient virus disruption and heat denaturation. At time point 0, p24 was measurable in 22 patients (34%), who had maintained a viral load <50 copies/mL for 25.4 months (median, range: 6.2-38.9 months) under HAART. Viral rebounds during 2-week STIs led to a mean p24 increase of only 0.08 to 0.19 log10 (ie, 20%-60%). Pre-HAART viral load and p24 at time 0 independently predicted p24 rebounds during the 4 2-week STIs. The p24 at time 0 and HIV-1 RNA rebound during weeks 41 through 52 independently determined the concomitant p24 rebound. An increase of p24 but not viral load during the first 8 weeks of the long STI correlated significantly with concomitant CD4(+) T cell loss. Persisting p24 despite successful HAART may reflect virus replication in reservoirs not represented by plasma viral load and has implications for the concept of therapeutic vaccination.