Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

Numerical analysis of the impact of charcoal production on soil hydrological behavior, runoff response and erosion susceptibility

The impact of charcoal production on soil hydraulic properties, runoff response and erosion susceptibility were studied in both field and simulation experiments. Core and composite samples, from 12 randomly selected sites within the catchment of Kotokosu were taken from the 0-10 cm layer of a charcoal site soil (CSS) and adjacent field soils (AFS). These samples were used to determine saturated hydraulic conductivity (Ksat), bulk density, total porosity, soil texture and color. Infiltration, surface albedo and soil surface temperature were also measured in both CSS and AFS. Measured properties were used as entries in a rainfall runoff simulation experiment on a smooth (5 % slope) plot of 25 x 25 m grids with 10 cm resolutions. Typical rainfall intensities of the study watershed (high, moderate and low) were applied to five different combinations of Ks distributions that could be expected in this landscape. The results showed significantly (p < 0.01) higher flow characteristics of the soil under charcoal kilns (increase of 88 %). Infiltration was enhanced and runoff volume reduced significantly. The results showed runoff reduction of about 37 and 18 %, and runoff coefficient ranging from 0.47-0.75 and 0.04-0.39 or simulation based on high (200 mm h-1) and moderate (100 mm h-1) rainfall events over the CSS and AFS areas, respectively. Other potential impacts of charcoal production on watershed hydrology were described. The results presented, together with watershed measurements, when available, are expected to enhance understanding of the hydrological responses of ecosystems to indiscriminate charcoal production and related activities in this region.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban triatominae

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 µL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved.

Multilocus enzyme electrophoresis study of Bacillus sphaericus

Multilocus enzyme electrophoresis (MLEE) has been used in the study of some Bacillus species. In this work we applied MLEE and numerical analysis in the study of the Bacillus sphaericus group. B. sphaericus can be distinguished from other entomopathogenic Bacillus by a unique allele (NP-4). Within the species, all insect pathogens were recovered in the same phenetic cluster and all of these strains have the same band position (electrophoresis migration) on the agarose gel (ADH-2). The entomopathogenic group of B. sphaericus seems to be a clonal population, having two widespread frequent genotypes (zymovar 59 and zymovar 119).

Genetic Data Showing Evolutionary Links between Leishmania and Endotrypanum

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Using numerical classification of profiles based on Vis-NIR spectra to distinguish soils from the Piracicaba Region, Brazil

Considering that information from soil reflectance spectra is underutilized in soil classification, this paper aimed to evaluate the relationship of soil physical, chemical properties and their spectra, to identify spectral patterns for soil classes, evaluate the use of numerical classification of profiles combined with spectral data for soil classification. We studied 20 soil profiles from the municipality of Piracicaba, State of São Paulo, Brazil, which were morphologically described and classified up to the 3rd category level of the Brazilian Soil Classification System (SiBCS). Subsequently, soil samples were collected from pedogenetic horizons and subjected to soil particle size and chemical analyses. Their Vis-NIR spectra were measured, followed by principal component analysis. Pearson's linear correlation coefficients were determined among the four principal components and the following soil properties: pH, organic matter, P, K, Ca, Mg, Al, CEC, base saturation, and Al saturation. We also carried out interpretation of the first three principal components and their relationships with soil classes defined by SiBCS. In addition, numerical classification of the profiles based on the OSACA algorithm was performed using spectral data as a basis. We determined the Normalized Mutual Information (NMI) and Uncertainty Coefficient (U). These coefficients represent the similarity between the numerical classification and the soil classes from SiBCS. Pearson's correlation coefficients were significant for the principal components when compared to sand, clay, Al content and soil color. Visual analysis of the principal component scores showed differences in the spectral behavior of the soil classes, mainly among Argissolos and the others soils. The NMI and U similarity coefficients showed values of 0.74 and 0.64, respectively, suggesting good similarity between the numerical and SiBCS classes. For example, numerical classification correctly distinguished Argissolos from Latossolos and Nitossolos. However, this mathematical technique was not able to distinguish Latossolos from Nitossolos Vermelho férricos, but the Cambissolos were well differentiated from other soil classes. The numerical technique proved to be effective and applicable to the soil classification process.