Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Baroreflex function in conscious rats submitted to iron overload

Our hypothesis is that iron accumulated in tissue, rather than in serum, may compromise cardiovascular control. Male Fischer 344 rats weighing 180 to 220 g were divided into 2 groups. In the serum iron overload group (SIO, N = 12), 20 mg elemental iron was injected ip daily for 7 days. In the tissue iron overload group (TIO, N = 19), a smaller amount of elemental iron was injected (10 mg, daily) for 5 days followed by a resting period of 7 days. Reflex heart rate responses were elicited by iv injections of either phenylephrine (0.5 to 5.0 µg/kg) or sodium nitroprusside (1.0 to 10.0 µg/kg). Baroreflex curves were determined and fitted to sigmoidal equations and the baroreflex gain coefficient was evaluated. To evaluate the role of other than a direct effect of iron on tissue, acute treatment with the iron chelator deferoxamine (20 mg/kg, iv) was performed on the TIO group and the baroreflex was re-evaluated. At the end of the experiments, evaluation of iron levels in serum confirmed a pronounced overload for the SIO group (30-fold), in contrast to the TIO group (2-fold). Tissue levels of iron, however, were higher in the TIO group. The SIO protocol did not produce significant alterations in the baroreflex curve response, while the TIO protocol produced a nearly 2-fold increase in baroreflex gain (-4.34 ± 0.74 and -7.93 ± 1.08 bpm/mmHg, respectively). The TIO protocol animals treated with deferoxamine returned to sham levels of baroreflex gain (-3.7 ± 0.3 sham vs -3.6 ± 0.2 bpm/mmHg) 30 min after the injection. Our results indicate an effect of tissue iron overload on the enhancement of baroreflex sensitivity.

Prediction of genetic gain from selection indices for disease resistance in papaya hybrids

In order to select superior hybrids for the concentration of favorable alleles for resistance to papaya black spot, powdery mildew and phoma spot, 67 hybrids were evaluated in two seasons, in 2007, in a randomized block design with two replications. Genetic gains were estimated from the selection indices of Smith & Hazel, Pesek & Baker, Williams, Mulamba & Mock, with selection intensity of 22.39%, corresponding to 15 hybrids. The index of Mulamba & Mock showed gains more suitable for the five traits assessed when it was used the criterion of economic weight tentatively assigned. Together, severity of black spot on leaves and on fruits, characteristics considered most relevant to the selection of resistant materials, expressed percentage gain of -44.15%. In addition, there were gains for other characteristics, with negative predicted selective percentage gain. The results showed that the index of Mulamba & Mock is the most efficient procedure for simultaneous selection of papaya hybrid resistant to black spot, powdery mildew and phoma spot.

Comparison of methods for classification of the coefficient of variation in papaya

ABSTRACT The objective of this work was to study the distribution of values of the coefficient of variation (CV) in the experiments of papaya crop (Carica papaya L.) by proposing ranges to guide researchers in their evaluation for different characters in the field. The data used in this study were obtained by bibliographical review in Brazilian journals, dissertations and thesis. This study considered the following characters: diameter of the stalk, insertion height of the first fruit, plant height, number of fruits per plant, fruit biomass, fruit length, equatorial diameter of the fruit, pulp thickness, fruit firmness, soluble solids and internal cavity diameter, from which, value ranges were obtained for the CV values for each character, based on the methodology proposed by Garcia, Costa and by the standard classification of Pimentel-Gomes. The results obtained in this study indicated that ranges of CV values were different among various characters, presenting a large variation, which justifies the necessity of using specific evaluation range for each character. In addition, the use of classification ranges obtained from methodology of Costa is recommended.

Obesity and gestational weight gain: cesarean delivery and labor complications

OBJECTIVE: To assess the association between pre-gestational obesity and weight gain with cesarean delivery and labor complications. METHODS: A total of 4,486 women 20-28 weeks pregnant attending general prenatal care clinics of the national health system in Brazil from 1991 to 1995 were enrolled and followed up through birth. Body mass index categories based on prepregnancy weight and total weight gain were calculated. Associations between body mass index categories and labor complications were adjusted through logistic regression analysis. RESULTS: Obesity was present in 308 (6.9%) patients. Cesarean delivery was performed in 164 (53.2%) obese, 407 (43.1%) pre-obese, 1,045 (35.1%) normal weight and 64 (24.5%) underweight women. The relative risk for cesarean delivery in obese women was 1.8 (95% CI: 1.5-2.0) compared to normal weight women. Greater weight gain was particularly associated with cesarean among the obese (RR 4th vs 2nd weight gain quartile 2.2; 95% CI: 1.4-3.2). Increased weight at the beginning of pregnancy was associated with a significantly higher adjusted risk of meconium with vaginal delivery and perinatal death and infection in women submitted to cesarean section. Similarly, greater weight gain during pregnancy increased the risk for meconium and hemorrhage in women submitted to vaginal delivery and for prematurity with cesarean. CONCLUSIONS: Pre-gestational obesity and greater weight gain independently increase the risk of cesarean delivery, as well as of several adverse outcomes with vaginal delivery. These findings provide further evidence of the negative effects of prepregnancy obesity and greater gestational weight gain on pregnancy outcomes.

Body weight gain during adulthood and uterine myomas: Pró-Saúde Study

This study intended to investigate whether body weight gain during adulthood is associated with uterine myomas. 1,560 subjects were evaluated in a Pró-Saúde Study. Weight gain was evaluated in a continuous fashion and also in quintiles. Odds ratios and 95% confidence intervals were estimated through logistic regression models that were adjusted for education levels, color/race, body mass indices at age 20, age of menarche, parity, use of oral contraceptive methods, smoking, health insurance, and the Papanicolaou tests. No relevant differences were observed regarding the presence of uterine myomas among weight gain quintiles in that studied population.

Analysis of the detection coefficient for the association between leprosy and pregnancy in the integration region of Carajas, State of Para, Brazil

Introduction The association between leprosy and pregnancy is currently poorly understood and has been linked to serious clinical consequences. Methods A retrospective study between 2007 and 2009 was performed in the integration region of Carajás, Brazil on a population of pregnant lepers, with non-lepers of ages 12-49 years serving as the reference population. Results Twenty-nine pregnant lepers were studied during the study period. The detection rates (DRs) for the studied association were 4.7 in 2007, 9.4 in 2008, and 4.3 in 2009. Conclusions The Carajás region presented a medium pattern of endemicity during most of the study period, with a high DR found in 2008.

Leprosy and pregnancy: detection coefficient and proposal for a new index

Introduction Our study presents a method to generate a novel detection coefficient for the association between leprosy and pregnancy (DCLP). Methods The DCLP was calculated for women from the State of Pará (2007-2009), Brazil. Data were ordered, divided into five equal parts (corresponding to the P20, P40, P60, and P80 percentiles), and classified as low, medium, high, very high, or hyperendemic. Results Using the new index, we established the DCLP parameters for low (<0.36), medium (0.36-0.69), high (0.70-1.09), very high (1.10-1.50), and hyperendemic (>1.50). Conclusions The new DCLP is more appropriate than the overall detection coefficient (DC), which does not take into account the particularities of the interaction between a disease and a specific physiological state.

Genetic breeding on the bee Melipona scutellaris (Apidae, Meliponinae)

A selection of queens of Melipona scutellaris through the most productive colonies were carried out during eight months in an orange honeyflow. Each of the colonies was evaluated by its production, that is, the gross weight production ( pollen, brood, geopropolis and wax of each hive). With this data a coefficient of repeatability was estimated by the intraclass correlation method, obtained r = 0.835 ± 0.071. The repeatibility is very high showing that the analysed data (production) is repeatable. Selection was then carried out using the regression coefficient of each colony and the respective production gain. Using these data the colonies were divided into three groups according to the method Vencovsky and Kerr (1982): a with the colonies of highest productivity, b of least productivity, and c of intermediary productivity. Colonies with the highest production (Group a) gave their queens to those of the lowest production (Group b) after their queens were taken out and killed; while those of intermediate (Group c) stayed with the same queens during the entire experiment both before and after the selection. The modifications in weight, that is, the genetic response was (R)= 7.98 gr per day which indicated a selection gain. The estimate of the realized herdability is twice the rate of the response to selection (R) by the selection differential (S2). That is then h²R=2(R/S2) then h²R= 0.166

Time-variations of equivalent water heights'from Grace Mission and in-situ river stages in the Amazon basin

Gravity Recovery and Climate Experiment (GRACE) mission is dedicated to measuring temporal variations of the Earth's gravity field. In this study, the Stokes coefficients made available by Groupe de Recherche en Géodésie Spatiale (GRGS) at a 10-day interval were converted into equivalent water height (EWH) for a ~4-year period in the Amazon basin (from July-2002 to May-2006). The seasonal amplitudes of EWH signal are the largest on the surface of Earth and reach ~ 1250mm at that basin's center. Error budget represents ~130 mm of EWH, including formal errors on Stokes coefficient, leakage errors (12 ~ 21 mm) and spectrum truncation (10 ~ 15 mm). Comparison between in situ river level time series measured at 233 ground-based hydrometric stations (HS) in the Amazon basin and vertically-integrated EWH derived from GRACE is carried out in this paper. Although EWH and HS measure different water bodies, in most of the cases a high correlation (up to ~80%) is detected between the HS series and EWH series at the same site. This correlation allows adjusting linear relationships between in situ and GRACE-based series for the major tributaries of the Amazon river. The regression coefficients decrease from up to down stream along the rivers reaching the theoretical value 1 at the Amazon's mouth in the Atlantic Ocean. The variation of the regression coefficients versus the distance from estuary is analysed for the largest rivers in the basin. In a second step, a classification of the proportionality between in situ and GRACE time-series is proposed.

Atrial activation during sinus rhythm in patients with rheumatic and non-rheumatic paroxysmal atrial fibrillation. Frequency-domain analysis using signal-averaged electrocardiography

OBJECTIVE: Using P-wave signal-averaged electrocardiography, we assessed the patterns of atrial electrical activation in patients with idiopathic atrial fibrillation as compared with patterns in patients with atrial fibrillation associated with structural heart disease. METHODS: Eighty patients with recurrent paroxysmal atrial fibrillation were divided into 3 groups as follows: group I - 40 patients with atrial fibrillation associated with non-rheumatic heart disease; group II - 25 patients with rheumatic atrial fibrillation; and group III - 15 patients with idiopathic atrial fibrillation. All patients underwent P-wave signal-averaged electrocardiography for frequency-domain analysis using spectrotemporal mapping and statistical techniques for detecting and quantifying intraatrial conduction disturbances. RESULTS: We observed an important fragmentation in atrial electrical conduction in 27% of the patients in group I, 64% of the patients in group II, and 67% of the patients in group III (p=0.003). CONCLUSION: Idiopathic atrial fibrillation has important intraatrial conduction disturbances. These alterations are similar to those observed in individuals with rheumatic atrial fibrillation, suggesting the existence of some degree of structural involvement of the atrial myocardium that cannot be detected with conventional electrocardiography and echocardiography.

Heart Weight and Heart Weight/Body Weight Coefficient in Malnourished Adults

OBJECTIVE: To compare the heart weight and the heart weight/body weight coefficient of adults with and without chronic malnutrition. METHODS: In an initial case series of 210 autopsies performed in adults, we recorded body and heart weights and calculated the heart weight/body weight coefficients (HW/BW x 100). The exclusion criteria were as follows: positive serology for Chagas' disease, edema, obesity, heart diseases, hepatopathies, nephropathies, and systemic arterial hypertension. Malnutrition was characterized as a body mass index <18.5kg/m². Differences with p<0.05 were considered significant. RESULTS: Individuals in the malnourished (n=15) and control (n=21) groups were statistically different, respectively, in regard to body mass index (15.9±1.7 versus 21.3±2.5kg/m²), heart weight (267.3±59.8 versus 329.1±50.4g), and the HW/BW coefficient (0.64±0.12 versus 0.57±0.09%). A positive and significant correlation was observed between heart weight and body mass index (r=0.52), and between heart weight and body weight (r=0.65). CONCLUSION: Malnourished individuals have lighter hearts and a greater HW/BW coefficient than non-malnourished individuals do. These findings indicate a possible preservation of the myocardium in relation to the intensity of weight loss associated with the probable relative increase in cardiac connective tissue and heart blood vessels.

Detection of incipient left ventricular hypertrophy in mild to moderate arterial hypertension with normal electrocardiogram and echocardiogram: a new use for signal-averaged electrocardiography

OBJECTIVE: To assess signal-averaged electrocardiogram (SAECG) for diagnosing incipient left ventricular hypertrophy (LVH). METHODS: A study with 115 individuals was carried out. The individuals were divided as follows: GI - 38 healthy individuals; GII - 47 individuals with mild to moderate hypertension and normal findings on echocardiogram and ECG; and GIII - 30 individuals with hypertension and documented LVH. The magnitude vector of the SAECG was analyzed with the high-pass cutoff frequency of 40 Hz through the bidirectional four-pole Butterworth high-pass digital filter. The mean quadratic root of the total QRS voltage (RMST) and the two-dimensional integral of the QRS area of the spectro-temporal map were analyzed between 0 and 30 Hz for the frequency domain (Int FD), and between 40 and 250 Hz for the time domain (Int TD). The electrocardiographic criterion for LVH was based on the Cornell Product. Left ventricular mass was calculated with the Devereux formula. RESULTS: All parameters analyzed increased from GI to GIII, except for Int FD (GII vs GIII) and RMST log (GII vs GIII). Int TD showed greater accuracy for detecting LVH with an appropriate cutoff > 8 (sensitivity of 55%, specificity of 81%). Positive values (> 8) were found in 56.5% of the G II patients and in 18.4% of the GI patients (p< 0.0005). CONCLUSION: SAECG can be used in the early diagnosis of LVH in hypertensive patients with normal ECG and echocardiogram.

Signal transduction and activation of the NADPH oxidase in eosinophils

Activation of the eosinophil NADPH oxidase and the subsequent release of toxic oxygen radicals has been implicated in the mechanism of parasite killing and inflammation. At present, little is known of the signal transduction pathway that govern agonist-induced activation of the respiratory burst and is the subject of this review. In particular, we focus on the ability of leukotrine B4 to activate the NADPH oxidase in guinea-pig peritoneal eosinophils which can be obtained in sufficient number and purity for detailed biochemical experiments to be performed.