Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (beta-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarrays

Abstract Background Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard. Results We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable. Conclusion RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Spatial reference of black capuchin monkeys in Brazilian Atlantic Forest: egocentric or allocentric?

Wild primates occupy large home ranges and travel long distances to reach goals. However, how primates are able to remember goal locations and travel efficiently is unclear. Few studies present consistent results regarding what reference system primates use to navigate, and what kind of spatial information they recognize. We analysed the pattern of navigation of one wild group of black capuchin monkeys, Cebus nigritus, at Atlantic Forest for 100 days in Carlos Botelho State Park (PECB), Brazil. We tested predictions based on the alternative hypotheses that black capuchin monkeys navigate using a sequence of landmarks as an egocentric reference system or an allocentric reference system, or both, depending on availability of food resources. The group location was recorded using a GPS device collecting coordinates at 5 min intervals, and route maps were generated using ArcView v9.3.1. The study group travelled through habitual routes during less than 30% of our study sample, and revisited resources from different starting points, using different paths and routes, even when prominent landmarks near feeding locations were not visible. The study group used habitual routes more frequently when high-quality foods were scarce, and navigated using different paths when revisiting food sources. Results support the hypothesis that black capuchin monkeys at PECB navigate using both egocentric and allocentric systems of reference, depending on the quality and distribution of the food resource they find. (C) 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Delineation of vertebral area on the coronal plane using three-dimensional ultrasonography advanced volume contrast imaging (VCI) Omni view: intrarater reliability and agreement using standard mouse, high definition mouse, and pen-tablet

Objective: To assess the fetal lumbosacral spine by three-dimensional (3D) ultrasonography using volume contrast imaging (VCI) omni view method and compare reproducibility and agreement between three different measurement techniques: standard mouse, high definition mouse and pen-tablet. Methods: A comparative and prospective study with 40 pregnant women between 20 and 34+6 weeks was realized. 3D volume datasets of the fetal spine were acquired using a convex transabdominal transducer. Starting scan plane was the coronal section of fetal lumbosacral spine by VCI-C function. Omni view manual trace was selected and a parallel plane of fetal spine was drawn including interest region. Intraclass correlation coefficient (ICC) was used for reproducibility analysis. The relative difference between three used techniques was compared by chi-square test and Fischer test. Results: Pen-tablet showed better reliability (ICC = 0.987). In the relative proportion of differences, this was significantly higher for the pen-tablet (82.14%; p < 0.01). In paired comparison, the relative difference was significantly greater for the pen-tablet (p < 0.01). Conclusion: The pen-tablet showed to be the most reproductive and concordant method in the measurement of body vertebral area of fetal lumbosacral spine by 3D ultrasonography using the VCI.

Perfomance of methods for estimating reference evapotranspiration in the municipalities of Frederico Westphalen and Palmeira das Missoes, State of Rio Grande do Sul, Brazil

The estimation of reference evapotranspiration (ETo), used in water balance, allows to determine soil water content, assisting on irrigation management. The present study aimed to compare simple ETo estimating methods with the Penman-Monteith (FAO), in the folowing time scales: daily, 5, 10, 15 and 30 days and monthly in the counties of Frederico Westphalen and Palmeira das Missoes, in the Rio Grande do Sul state, Brazil. The methods tested had their efficiency improved by increasing the time scale of analysis, keeping the same performance for both locations. The highest and lowest ETo values occurred in December and June, respectively. Most methods underestimated ETo. For any of the time scales Makking and Radiaton FAO24 methods can replace the Penman-Monteith for estimating ETo.

PD controller synthesis from open-loop response measurements of rotating system

In this work, a method of computing PD stabilising gains for rotating systems is presented based on the D-decomposition technique, which requires the sole knowledge of frequency response functions. By applying this method to a rotating system with electromagnetic actuators, it is demonstrated that the stability boundary locus in the plane of feedback gains can be easily plotted, and the most suitable gains can be found to minimise the resonant peak of the system. Experimental results for a Laval rotor show the feasibility of not only controlling lateral shaft vibration and assuring stability, but also helps in predicting the final vibration level achieved by the closed-loop system. These results are obtained based solely on the input-output response information of the system as a whole.

Experimental Investigation of the Hydrodynamic Coefficients of a Remotely Operated Vehicle Using a Planar Motion Mechanism

The determination of hydrodynamic coefficients of full scale underwater vehicles using system identification (SI) is an extremely powerful technique. The procedure is based on experimental runs and on the analysis of on-board sensors and thrusters signals. The technique is cost effective and it has high repeatability; however, for open-frame underwater vehicles, it lacks accuracy due to the sensors' noise and the poor modeling of thruster-hull and thruster-thruster interaction effects. In this work, forced oscillation tests were undertaken with a full scale open-frame underwater vehicle. These conducted tests are unique in the sense that there are not many examples in the literature taking advantage of a PMM installation for testing a prototype and; consequently, allowing the comparison between the experimental results and the ones estimated by parameter identification. The Morison's equation inertia and drag coefficients were estimated with two parameter identification methods, that is, the weighted and the ordinary least-squares procedures. It was verified that the in-line force estimated from Morison's equation agrees well with the measured one except in the region around the motion inversion points. On the other hand, the error analysis showed that the ordinary least-squares provided better accuracy and, therefore, was used to evaluate the ratio between inertia and drag forces for a range of Keulegan-Carpenter and Reynolds numbers. It was concluded that, although both experimental and estimation techniques proved to be powerful tools for evaluation of an open-frame underwater vehicle's hydrodynamic coefficients, the research provided a rich amount of reference data for comparison with reduced models as well as for dynamic motion simulation of ROVs. [DOI: 10.1115/1.4004952]

Effect of EVA on the fresh properties of cement paste

The improved workability effect of latex in dry mortars has not been fully clarified. The purpose of this research was to investigate the influence cif the EVA copolymer on the cement hydration and on the rheological properties of cement pastes. The results pointed to a minor influence of EVA on cement hydration and to a ball-bearing effect. In fact, the shear thinning behavior of reference paste at 15 min after mixing changed to shear thickening owing to the EVA addition. This behavior could be explained by the decrease in the interparticle separation distance as consequence of the solid content increase in case of shearing detachment of weakly adhered EVA particles from the cement particles surfaces. The expected EVA plasticizing effect was observed at 60 min. Such behavior points to the stabilization of EVA on the cement particles surfaces, thus resulting in a steric barrier effect. (C) 2011 Elsevier Ltd. All rights reserved.

Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states

Sarmento A.M.C., Azevedo S.S., Morais Z.M., Souza G.O., Oliveira F.C.S., Goncales A.P., Miraglia F. & Vasconcellos S.A. 2012. [Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states.] Emprego de estirpes Leptospira spp. isoladas no Brasil na microtecnica de soroaglutinacao microscopica aplicada ao diagnostico da leptospirose em rebanhos bovinos de oito estados brasileiros. Pesquisa Veterinaria Brasileira 32(7);601-606. Universidade de Sao Paulo, Faculdade de Medicina Veterinaria e Zootecnia, Departamento de Medicina Veterinaria Preventiva e Saude Animal, Av. Prof. Dr. Orlando Marques de Paiva 87, Sao Paulo, SP 05508-270, Brazil. E-mail: savasco@usp.br The aim of this study was to investigate the adequacy of the use of autochthonous strains of leptospires isolated in Brazil, added to antigen collection of the microscopic agglutination test (MAT) applied to the diagnosis of bovine leptospirosis. By means of non-probability sampling, 109 farms and 9,820 cattle, females at reproductive age were chosen from 85 municipalities in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Parana, Rio Grande do Sul, Santa Catarina and Sao Paulo. Among the 9,820 examined animals, 5,806 (59.12%) were reactants at MAT for at least one serovar using the 23 reference serovars. Employing the collection of reference serovars and the ten autochthonous strains, 6,400 (65.24%) reactants and significant difference (p=0.001) was found. The most probable serovars identified by the collection of reference antigens were Hardjo (43.03%), Shermani (20%), Wolfi (9.96%), Grippothyphosa (5.42%) and Pomona (4.28%). With the collection amplified with the ten strains isolated in Brazil, the most probable serovars were Hardjo (31%), Guaricura-M4/84 (22.50%), Shermani (15.43%), Wolffi (4.76%), Grippothyphosa (3.71%) and Autumnalis (3.24%). The serovar Guaricura, strain M4/84, isolated from bovines and buffaloes in the State of Sao Paulo, was ranked as one of the three most probable serovars in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais and Sao Paulo. The addition of autochthonous strains to the MAT antigen collection provided the confirmation of the diagnosis of leptospirosis in 594 cattle (6%) which have been classified as non-reactants by the reference collection (p=0.001).

The eagle and the starlings: Galileo's argument for the autonomy of science-how pertinent is it today?

After Galileo's argument for the autonomy of science is analysed and adapted to take into account later developments of scientific practices, we conclude that, in the final analysis, it is not compelling. Nevertheless, Galileo's argument still provides a useful point of reference, for aspects of it can be interpreted to anticipate central components of the often acclaimed ideal of science as value free, so that appraising it contributes to the larger purpose of exploring how well that ideal stands up today. Finally, we will argue that residue from Galileo's struggle with the Church remains with us, making it difficult to identify the conditions that would need to be put into place today for any robust sense of the autonomy of science to be defensible. (C) 2011 Elsevier Ltd. All rights reserved.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Preparation of percentile tables through anthropometric, performance, biochemical, hematological, hormonal and psychological parameters in professional soccer players

Introduction: The lack of reference values of anthropometric, performance, biochemical, hematological, hormonal and psychological parameters is an important limitation in the investigations with soccer players. Objective: To elaborate percentile tables to be used as comparison reference for further studies. Methods: 82 professional soccer players were evaluated approximately 30 days after the beginning of the main competition played by their teams. On the first day of evaluation, fast blood samples were collected for measurement of hematological parameters (i.e. erythrocytes, hemoglobin, hematocrit, mean corpuscular volume - MCV, mean corpuscular hemoglobin - MCH, mean corpuscular hemoglobin concentration - MCHC, leukocytes, eosinophils, lymphocytes, monocytes and platelets) and of concentrations of adrenaline, cortisol, creatine kinase, creatinine, norepinephrine, testosterone and urea. Subsequently, the soccer players had their anthropometric characteristics and psychological parameters assessed. In addition, the evaluation of the lactic anaerobic system efficiency was performed on a 400-m track. On the second day, both the alactic anaerobic and aerobic system efficiency was measured. Results: The percentile distribution (P-0, P-15, P-30, P-50, P-70, P-85 e P-100) was used to present the results. Conclusion: The elaboration of the percentile tables can be used as comparison reference for further studies.

Geographical distribution of American cutaneous leishmaniasis and its phlebotomine vectors (Diptera: Psychodidae) in the state of São Paulo, Brazil

Abstract Background American cutaneous leishmaniasis (ACL) is a re-emerging disease in the state of São Paulo, Brazil. It is important to understand both the vector and disease distribution to help design control strategies. As an initial step in applying geographic information systems (GIS) and remote sensing (RS) tools to map disease-risk, the objectives of the present work were to: (i) produce a single database of species distributions of the sand fly vectors in the state of São Paulo, (ii) create combined distributional maps of both the incidence of ACL and its sand fly vectors, and (iii) thereby provide individual municipalities with a source of reference material for work carried out in their area. Results A database containing 910 individual records of sand fly occurrence in the state of São Paulo, from 37 different sources, was compiled. These records date from between 1943 to 2009, and describe the presence of at least one of the six incriminated or suspected sand fly vector species in 183/645 (28.4%) municipalities. For the remaining 462 (71.6%) municipalities, we were unable to locate records of any of the six incriminated or suspected sand fly vector species (Nyssomyia intermedia, N. neivai, N. whitmani, Pintomyia fischeri, P. pessoai and Migonemyia migonei). The distribution of each of the six incriminated or suspected vector species of ACL in the state of São Paulo were individually mapped and overlaid on the incidence of ACL for the period 1993 to 1995 and 1998 to 2007. Overall, the maps reveal that the six sand fly vector species analyzed have unique and heterogeneous, although often overlapping, distributions. Several sand fly species - Nyssomyia intermedia and N. neivai - are highly localized, while the other sand fly species - N. whitmani, M. migonei, P. fischeri and P. pessoai - are much more broadly distributed. ACL has been reported in 160/183 (87.4%) of the municipalities with records for at least one of the six incriminated or suspected sand fly vector species, while there are no records of any of these sand fly species in 318/478 (66.5%) municipalities with ACL. Conclusions The maps produced in this work provide basic data on the distribution of the six incriminated or suspected sand fly vectors of ACL in the state of São Paulo, and highlight the complex and geographically heterogeneous pattern of ACL transmission in the region. Further studies are required to clarify the role of each of the six suspected sand fly vector species in different regions of the state of São Paulo, especially in the majority of municipalities where ACL is present but sand fly vectors have not yet been identified.

Figures of equilibrium for tidally deformed non-homogeneous celestial bodies

Abstract (2,250 Maximum Characters): Several theories of tidal evolution, since the theory developed by Darwin in the XIX century, are based on the figure of equilibrium of the tidally deformed body. Frequently the adopted figure is a Jeans prolate spheroid. In some case, however, the rotation is important and Roche ellipsoids are used. The main limitations of these models are (a) they refer to homogeneous bodies; (b) the rotation axis is perpendicular to the plane of the orbit. This communication aims at presenting several results in which these hypotheses are not done. In what concerns the non-homogeneity, the presented results concerns initially bodies formed by N homogeneous layers and we study the non sphericity of each layer and relate them to the density distribution. The result is similar to the Clairaut figure of equilibrium, often used in planetary sciences, but taking into full account the tidal deformation. The case of the rotation axis non perpendicular to the orbital plane is much more complex and the study has been restricted for the moment to the case of homogeneous bodies.

Effect of EVA on the fresh properties of cement paste

The improved workability effect of latex in dry mortars has not been fully clarified. The purpose of this research was to investigate the influence of the EVA copolymer on the cement hydration and on the rheological properties of cement pastes. The results pointed to a minor influence of EVA on cement hydration and to a ball-bearing effect. In fact, the shear thinning behavior of reference paste at 15 min after mixing changed to shear thickening owing to the EVA addition. This behavior could be explained by the decrease in the interparticle separation distance as consequence of the solid content increase in case of shearing detachment of weakly adhered EVA particles from the cement particles surfaces. The expected EVA plasticizing effect was observed at 60 min. Such behavior points to the stabilization of EVA on the cement particles surfaces, thus resulting in a steric barrier effect.