Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

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.

Plasmodium falciparum CS protein - prime malaria vaccine candidate: definition of the human CTL domain and analysis of its variation

Studies in mice have shown that immunity to malaria sporozoites is mediated primarily by citotoxic T lymphocytes (CTL) specific for epitopes within the circumsporozoite (CS) protein. Humans, had never been shown to generate CTL against any malaria or other parasite protein. The design of a sub-unit vaccine for humans ralies on the epitopes recognized by CTL being identified and polymorphisms therein being defined. We have developed a novel technique using an entire series of overlapping synthetic peptides to define the epitopes of the Plasmodium falciparum CS protein recognized by human CTL and have analyzed the sequence variation of the protein with respect to the identified CTL epitopic domain. We have demonstrated that some humans can indeed generate CTL. against the P. falciparum CS protein. Furthermore, the extent of variation observed for the CTL recognition domain is finite and the combination of peptides necessary for inclusion in a polyvalent vaccine may be small. If ways can be found to increase immune responsiveness, then a vaccine designed to stimulate CS protein-specific CTL activity may prevent malaria.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Assessing protein stability of the dimeric DNA-binding domain of E2 human papillomavirus 18 with molecular dynamics

The objective of this study is to understand the structural flexibility and curvature of the E2 protein of human papillomavirus type 18 using molecular dynamics (6 ns). E2 is required for viral DNA replication and its disruption could be an anti-viral strategy. E2 is a dimer, with each monomer folding into a stable open-faced β-sandwich. We calculated the mobility of the E2 dimer and found that it was asymmetric. These different mobilities of E2 monomers suggest that drugs or vaccines could be targeted to the interface between the two monomers.

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Worker morphology of the ant Gnamptogenys striatula Mayr (Formicidae, Ectatomminae) in different landscapes from the Atlantic Forest domain

Morphological traits, such as size and shape, may reflect a combination of ecological and evolutionary responses by organisms. Ants have been used to evaluate the relationship between the environment and species coexistence and morphology. In the present study, we analyzed the morphology of workers of Gnamptogenys striatula Mayr in different landscapes from the Atlantic Domain in southeastern Brazil, focusing on the variation in the morphological attributes of these populations compared to those from a dense ombrophilous forest. Eighteen morphological traits of functional importance for interactions between workers and the environment were measured to characterize the size and shape of the workers. In general, the results show that ants of urban areas possess some morphological attributes of smaller size, with highly overlapped morphological space between the populations in forested ecosystems. Further, some of the traits related to predation were relatively smaller in modified land areas than in the populations from preserved areas of dense ombrophilous forest. These results help broaden the knowledge regarding morphological diversity in G. striatula, suggesting that the characterization of the morphology may be important to quantify the effects of land use on morphological diversity, and presumably, to facilitate the use of ants as biological indicators.

Electrical conductivity of soil irrigated with swine wastewater estimated by time-domain reflectometry¹

Wastewater application to soil is an alternative for fertilization and water reuse. However, particular care must be taken with this practice, since successive wastewater applications can cause soil salinization. Time-domain reflectometry (TDR) allows for the simultaneous and continuous monitoring of both soil water content and apparent electrical conductivity and thus for the indirect measurement of the electrical conductivity of the soil solution. This study aimed to evaluate the suitability of TDR for the indirect determination of the electrical conductivity (ECse) of the saturated soil extract by using an empirical equation for the apparatus TDR Trase 6050X1. Disturbed soil samples saturated with swine wastewater were used, at soil proportions of 0, 0.45, 0.90, 1.80, 2.70, and 3.60 m³ m-3. The probes were equipped with three handmade 0.20 cm long rods. The fit of the empirical model that associated the TDR measured values of electrical conductivity (EC TDR) to ECse was excellent, indicating this approach as suitable for the determination of electrical conductivity of the soil solution.

Water percolation estimated with time domain reflectometry (TDR) in drainage lysimeters

Due to the difficulty of estimating water percolation in unsaturated soils, the purpose of this study was to estimate water percolation based on time-domain reflectometry (TDR). In two drainage lysimeters with different soil textures TDR probes were installed, forming a water monitoring system consisting of different numbers of probes. The soils were saturated and covered with plastic to prevent evaporation. Tests of internal drainage were carried out using a TDR 100 unit with constant dielectric readings (every 15 min). To test the consistency of TDR-estimated percolation levels in comparison with the observed leachate levels in the drainage lysimeters, the combined null hypothesis was tested at 5 % probability. A higher number of probes in the water monitoring system resulted in an approximation of the percolation levels estimated from TDR - based moisture data to the levels measured by lysimeters. The definition of the number of probes required for water monitoring to estimate water percolation by TDR depends on the soil physical properties. For sandy clay soils, three batteries with four probes installed at depths of 0.20, 0.40, 0.60, and 0.80 m, at a distance of 0.20, 0.40 and 0.6 m from the center of lysimeters were sufficient to estimate percolation levels equivalent to the observed. In the sandy loam soils, the observed and predicted percolation levels were not equivalent even when using four batteries with four probes each, at depths of 0.20, 0.40, 0.60, and 0.80 m.

Public-domain software for root image analysis

In the search for high efficiency in root studies, computational systems have been developed to analyze digital images. ImageJ and Safira are public-domain systems that may be used for image analysis of washed roots. However, differences in root properties measured using ImageJ and Safira are supposed. This study compared values of root length and surface area obtained with public-domain systems with values obtained by a reference method. Root samples were collected in a banana plantation in an area of a shallower Typic Carbonatic Haplic Cambisol (CXk), and an area of a deeper Typic Haplic Ta Eutrophic Cambisol (CXve), at six depths in five replications. Root images were digitized and the systems ImageJ and Safira used to determine root length and surface area. The line-intersect method modified by Tennant was used as reference; values of root length and surface area measured with the different systems were analyzed by Pearson's correlation coefficient and compared by the confidence interval and t-test. Both systems ImageJ and Safira had positive correlation coefficients with the reference method for root length and surface area data in CXk and CXve. The correlation coefficient ranged from 0.54 to 0.80, with lowest value observed for ImageJ in the measurement of surface area of roots sampled in CXve. The IC (95 %) revealed that root length measurements with Safira did not differ from that with the reference method in CXk (-77.3 to 244.0 mm). Regarding surface area measurements, Safira did not differ from the reference method for samples collected in CXk (-530.6 to 565.8 mm²) as well as in CXve (-4231 to 612.1 mm²). However, measurements with ImageJ were different from those obtained by the reference method, underestimating length and surface area in samples collected in CXk and CXve. Both ImageJ and Safira allow an identification of increases or decreases in root length and surface area. However, Safira results for root length and surface area are closer to the results obtained with the reference method.

Photomultiplier nonlinear response in time-domain laser-induced luminescence spectroscopy

A new procedure to find the limiting range of the photomultiplier linear response of a low-cost, digital oscilloscope-based time-resolved laser-induced luminescence spectrometer (TRLS), is presented. A systematic investigation on the instrument response function with different signal input terminations, and the relationship between the luminescence intensity reaching the photomultiplier and the measured decay time are described. These investigations establish that setting the maximum intensity of the luminescence signal below 0.3V guarantees, for signal input terminations equal or higher than 99.7 ohm, a linear photomultiplier response.

Be-Fe coupled method for the analysis of 3D elastodynamic problems in the time domain

By coupling the Boundary Element Method (BEM) and the Finite Element Method (FEM) an algorithm that combines the advantages of both numerical processes is developed. The main aim of the work concerns the time domain analysis of general three-dimensional wave propagation problems in elastic media. In addition, mathematical and numerical aspects of the related BE-, FE- and BE/FE-formulations are discussed. The coupling algorithm allows investigations of elastodynamic problems with a BE- and a FE-subdomain. In order to observe the performance of the coupling algorithm two problems are solved and their results compared to other numerical solutions.

A frequency-domain pseudo-force method for dynamic structural analysis nonlinear systems and nonproportional damping

A frequency-domain method for nonlinear analysis of structural systems with viscous, hysteretic, nonproportional and frequency-dependent damping is presented. The nonlinear effects and nonproportional damping are considered through pseudo-force terms. The modal coordinates uncoupled equations are iteratively solved. The treatment of initial conditions in the frequency domain which is necessary for the treatment of the uncoupled equations is initially adressed.

A Well Stated Time Domain Integral Representation for Elastodynamic Analysis and Applications

This article discusses three possible ways to derive time domain boundary integral representations for elastodynamics. This discussion points out possible difficulties found when using those formulations to deal with practical applications. The discussion points out recommendations to select the convenient integral representation to deal with elastodynamic problems and opens the possibility of deriving simplified schemes. The proper way to take into account initial conditions applied to the body is an interesting topict shown. It illustrates the main differences between the discussed boundary integral representation expressions, their singularities and possible numerical problems. The correct way to use collocation points outside the analyzed domain is carefully described. Some applications are shown at the end of the paper, in order to demonstrate the capabilities of the technique when properly used.