New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

Projeto Flora Amazônica: eight years of binational botanical expeditions

A review of the history and results of the first eight years of fieldwork of Projeto Flora Amazônica is given. This binational plant collecting program, sponsored by the Conselho Nacional de Desenvolvimento Científico e Tecnológico and the National Science Foundation, has mounted 25 expeditions to many parts of Brazilian Amazonia. Expeditions have visited both areas threatened with destruction of the forest and remote areas previously unknown botanically. The results have included the collection of 32,976 members of vascular plants, 16, 482 of cryptogams, as well as quantitative inventory of 18.67 hectares of forest with the collection of 7,294*** numbers of sterile vouncher collections. The non-inventory collections have been made in replicate sets of 10-13 where possible and divided equally between Brazilian and U.S. institutions. To date, 55 botanists from many different institutions and with many different specialities have taken part with 36 different Brazilian botanists. The resulting herbarioum material is just beginning to be worked up and many nem species have been collected as well as many interesting range extensions and extra material of many rare species.

Ammonia losses estimated by an open collector from urea applied to sugarcane straw

The quantification of ammonia (NH3) losses from sugarcane straw fertilized with urea can be performed with collectors that recover the NH3 in acid-treated absorbers. Thus, the use of an open NH3 collector with a polytetrafluoroethylene (PTFE)-wrapped absorber is an interesting option since its cost is low, handling easy and microclimatic conditions irrelevant. The aim of this study was to evaluate the efficiency of an open collector for quantifying NH3-N volatilized from urea applied over the sugarcane straw. The experiment was carried out in a sugarcane field located near Piracicaba, São Paulo, Brazil. The NH3-N losses were estimated using a semi-open static collector calibrated with 15N (reference method) and an open collector with an absorber wrapped in PTFE film. Urea was applied to the soil surface in treatments corresponding to rates of 50, 100, 150 and 200 kg ha-1 N. Applying urea-N fertilizer on sugarcane straw resulted in losses NH3-N up to 24 % of the applied rate. The amount of volatile NH3-N measured in the open and the semi-open static collector did not differ. The effectiveness of the collection system varied non-linearly, with an average value of 58.4 % for the range of 100 to 200 kg ha-1 of urea-N. The open collector showed significant potential for use; however, further research is needed to verify the suitability of the proposed method.

Tabular cusum control charts of chemical variables applied to the control of surface water quality

The aim of this study was to evaluate the application possibility of tabular CUSUM control charts in the quality control of chemical variables in surface water. It was performed bibliographic and field research to collect water samples from 2003 to 2009, totaling 30 samples, some monthly and others semi-annual in order to observe the variables that regulate water quality. It was found that these charts may be applied to control the quality of river water; showing to be effective in the perception of changes during the process, especially for small samples (n=1) which there is no repetition as in this research. It was also concluded that the Mandurim River does not presents significant levels of pollution.

Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae

In order to evaluate the resolving power of several typing methods to identify relatedness among Brazilian strains of Enterobacter cloacae, we selected twenty isolates from different patients on three wards of a University Hospital (Orthopedics, Nephrology, and Hematology). Traditional phenotyping methods applied to isolates included biotyping, antibiotic sensitivity, phage-typing, and O-serotyping. Plasmid profile analysis, ribotyping, and macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) were used as genotyping methods. Sero- and phage-typing were not useful since the majority of isolates could not be subtyped by these methods. Biotyping, antibiogram and plasmid profile permitted us to classify the samples into different groups depending on the method used, and consequently were not reliable. Ribotyping and PFGE were significantly correlated with the clinical epidemiological analysis. PFGE did not type strains containing nonspecific DNase. Ribotyping was the most discriminative method for typing Brazilian isolates of E. cloacae.

Psychometric properties of the Portuguese version of the State-Trait Anxiety Inventory applied to college students: factor analysis and relation to the Beck Depression Inventory

The psychometric properties of the Portuguese version of the trait form of the State-Trait Anxiety Inventory (STAI-T) and its relation to the Beck Depression Inventory (BDI) were evaluated in a large Brazilian college student sample containing 845 women and 235 men. STAI-T scores tended to be higher for women, singles, those who work, and subjects under 30 years. Factor analysis of the STAI-T for total sample and by gender yielded two factors: the first representing a mood dimension and the second being related to worrying or cognitive aspects of anxiety. In order to study the relation between anxiety and depression measures, factor analysis of the combination of the 21 BDI items and the 20 STAI-T items was also carried out. The analysis resulted in two factors that were analyzed according to the tripartite model of anxiety and depression. Most of the BDI items (measuring positive affectivity and nonspecific symptoms of depression) were loaded on the first factor and four STAI-T items that measure positive affectivity. The remaining STAI-T items, all of them measuring negative affect, remained in the second factor. Thus, factor 1 represents a depression dimension and factor 2 measures a mood-worrying dimension. The findings of this study suggest that, although widely used as an anxiety scale, the STAI-T in fact measures mainly a general negative affect.

Effect of one stretch a week applied to the immobilized soleus muscle on rat muscle fiber morphology

We determined the effect of stretching applied once a week to the soleus muscle immobilized in the shortened position on muscle fiber morphology. Twenty-six male Wistar rats weighing 269 ± 26 g were divided into three groups. Group I, the left soleus was immobilized in the shortened position for 3 weeks; group II, the soleus was immobilized in the shortened position and stretched once a week for 3 weeks; group III, the soleus was submitted only to stretching once a week for 3 weeks. The medial part of the soleus muscle was frozen for histology and muscle fiber area evaluation and the lateral part was used for the determination of number and length of serial sarcomeres. Soleus muscle submitted only to immobilization showed a reduction in weight (44 ± 6%, P = 0.002), in serial sarcomere number (23 ± 15%) and in cross-sectional area of the fibers (37 ± 31%, P < 0.001) compared to the contralateral muscles. The muscle that was immobilized and stretched showed less muscle fiber atrophy than the muscles only immobilized (P < 0.05). Surprisingly, in the muscles submitted only to stretching, fiber area was decreased compared to the contralateral muscle (2548 ± 659 vs 2961 ± 806 µm², respectively, P < 0.05). In conclusion, stretching applied once a week for 40 min to the soleus muscle immobilized in the shortened position was not sufficient to prevent the reduction of muscle weight and of serial sarcomere number, but provided significant protection against muscle fiber atrophy. In contrast, stretching normal muscles once a week caused a reduction in muscle fiber area.

Selection of patients for myocardial perfusion scintigraphy based on fuzzy sets theory applied to clinical-epidemiological data and treadmill test results

Coronary artery disease (CAD) is a worldwide leading cause of death. The standard method for evaluating critical partial occlusions is coronary arteriography, a catheterization technique which is invasive, time consuming, and costly. There are noninvasive approaches for the early detection of CAD. The basis for the noninvasive diagnosis of CAD has been laid in a sequential analysis of the risk factors, and the results of the treadmill test and myocardial perfusion scintigraphy (MPS). Many investigators have demonstrated that the diagnostic applications of MPS are appropriate for patients who have an intermediate likelihood of disease. Although this information is useful, it is only partially utilized in clinical practice due to the difficulty to properly classify the patients. Since the seminal work of Lotfi Zadeh, fuzzy logic has been applied in numerous areas. In the present study, we proposed and tested a model to select patients for MPS based on fuzzy sets theory. A group of 1053 patients was used to develop the model and another group of 1045 patients was used to test it. Receiver operating characteristic curves were used to compare the performance of the fuzzy model against expert physician opinions, and showed that the performance of the fuzzy model was equal or superior to that of the physicians. Therefore, we conclude that the fuzzy model could be a useful tool to assist the general practitioner in the selection of patients for MPS.

Detection of patients with functional dyspepsia using wavelet transform applied to their electrogastrogram

The aim of the present study was to develop a classifier able to discriminate between healthy controls and dyspeptic patients by analysis of their electrogastrograms. Fifty-six electrogastrograms were analyzed, corresponding to 42 dyspeptic patients and 14 healthy controls. The original signals were subsampled, filtered and divided into the pre-, post-, and prandial stages. A time-frequency transformation based on wavelets was used to extract the signal characteristics, and a special selection procedure based on correlation was used to reduce their number. The analysis was carried out by evaluating different neural network structures to classify the wavelet coefficients into two groups (healthy subjects and dyspeptic patients). The optimization process of the classifier led to a linear model. A dimension reduction that resulted in only 25% of uncorrelated electrogastrogram characteristics gave 24 inputs for the classifier. The prandial stage gave the most significant results. Under these conditions, the classifier achieved 78.6% sensitivity, 92.9% specificity, and an error of 17.9 ± 6% (with a 95% confidence level). These data show that it is possible to establish significant differences between patients and normal controls when time-frequency characteristics are extracted from an electrogastrogram, with an adequate component reduction, outperforming the results obtained with classical Fourier analysis. These findings can contribute to increasing our understanding of the pathophysiological mechanisms involved in functional dyspepsia and perhaps to improving the pharmacological treatment of functional dyspeptic patients.

Ultrasound method applied to characterize healthy femoral diaphysis of Wistar rats in vivo

A simple experimental protocol applying a quantitative ultrasound (QUS) pulse-echo technique was used to measure the acoustic parameters of healthy femoral diaphyses of Wistar rats in vivo. Five quantitative parameters [apparent integrated backscatter (AIB), frequency slope of apparent backscatter (FSAB), time slope of apparent backscatter (TSAB), integrated reflection coefficient (IRC), and frequency slope of integrated reflection (FSIR)] were calculated using the echoes from cortical and trabecular bone in the femurs of 14 Wistar rats. Signal acquisition was performed three times in each rat, with the ultrasound signal acquired along the femur's central region from three positions 1 mm apart from each other. The parameters estimated for the three positions were averaged to represent the femur diaphysis. The results showed that AIB, FSAB, TSAB, and IRC values were statistically similar, but the FSIR values from Experiments 1 and 3 were different. Furthermore, Pearson's correlation coefficient showed, in general, strong correlations among the parameters. The proposed protocol and calculated parameters demonstrated the potential to characterize the femur diaphysis of rats in vivo. The results are relevant because rats have a bone structure very similar to humans, and thus are an important step toward preclinical trials and subsequent application of QUS in humans.

THE GENERALIZED MAXIMUM LIKELIHOOD METHOD APPLIED TO HIGH PRESSURE PHASE EQUILIBRIUM

The generalized maximum likelihood method was used to determine binary interaction parameters between carbon dioxide and components of orange essential oil. Vapor-liquid equilibrium was modeled with Peng-Robinson and Soave-Redlich-Kwong equations, using a methodology proposed in 1979 by Asselineau, Bogdanic and Vidal. Experimental vapor-liquid equilibrium data on binary mixtures formed with carbon dioxide and compounds usually found in orange essential oil were used to test the model. These systems were chosen to demonstrate that the maximum likelihood method produces binary interaction parameters for cubic equations of state capable of satisfactorily describing phase equilibrium, even for a binary such as ethanol/CO2. Results corroborate that the Peng-Robinson, as well as the Soave-Redlich-Kwong, equation can be used to describe phase equilibrium for the following systems: components of essential oil of orange/CO2.

Chemometrics applied to the incorporation of omega-3 in tilapia fillet feed flaxseed flour

This study evaluated the effect of adding flaxseed flour to the diet of Nile tilapia on the fatty acid composition of fillets using chemometrics. A traditional and an experimental diet containing flaxseed flour were used to feed the fish for 60 days. An increase of 18:3 n-3 and 22:6 n-3 and a decrease of 18:2 n-6 were observed in the tilapia fillets fed the experimental diet. There was a reduction in the n-6:n-3 ratio. A period of 45 days of incorporation caused a significant change in tilapia chemical composition. Principal Component Analysis showed that the time periods of 45 and 60 days positively contributed to the total content of n-3, LNA, and DHA, highlighting the effect of omega-3 incorporation in the treatment containing flaxseed flour.

Effect of modified atmosphere applied to minimally processed radicchio (Cichorium intybus L.) submitted to different sanitizing treatments

Stability of minimally processed radicchio (Cichorium intybus L.) was evaluated under modified atmosphere (2% O2, 5% CO2, and 93% N2) on 3, 5, 7 and 10 days of storage at 5°C. The samples were hygienized in sodium hypochlorite or hydrogen peroxide solutions to identify the most effective sanitizing solution to remove microorganisms. Microbiological analysis was conducted to identify the presence of coliforms at 35°C and 45°C, mesophilic microorganisms, and yeast and mold. Physicochemical analyses of mass loss, pH, soluble solids, and total acidity were conducted. The color measurements were performed using a Portable Colorimeter model CR-400. The antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic methods. The sensory evaluation was carried out using a hedonic scale to test overall acceptance of the samples during storage. The sodium hypochlorite (150 mg.L-1) solution provided greater safety to the final product. The values of pH ranged from 6.17 to 6.25, total acidity from 0.405 to 0.435%, soluble solids from 0.5 to 0.6 °Brix, mass loss from 1.7 to 7.2%, and chlorophyll from 1.068 to 0.854 mg/100g. The antioxidant activity of radicchio did not show significant changes during the first 3 days of storage. The overall acceptance of the sample stored in the sealed package without modified atmosphere was 70%, while the fresh sample was obtained 77% of approval. Although the samples packaged under modified atmosphere had a higher acceptance score, the samples in sealed packages had satisfactory results during the nine days of storage. The use of modified atmosphere, combined with cooling and good manufacturing practices, was sufficient to prolong the life of minimally processed radicchio, Folha Larga cultivar, for up to ten days of storage.

Alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) applied to dot-ELISA

The alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) was assessed in dot-ELISA for the diagnosis of Chagas' disease. Serum samples (355) from chagasic and non-chagasic patients were studied, and IgG antibodies to ASEA were found in all patients with chronic Chagas' disease. In non-chagasic patients 95.6% were negative, except for those with leishmaniasis (visceral and mucocutaneous), and some patients from control group reacted in low titers. The data indicate that dot-ELISA using ASEA is suitable for seroepidemiologic surveys to be employed in endemic areas for Chagas' disease.

Refractive index matching applied to fecal smear clearing

Thick smears of human feces can be made adequate for identification of helminth eggs by means of refractive index matching. Although this effect can be obtained by simply spreading a fleck of feces on a microscope slide, a glycerol solution has been routinely used to this end. Aiming at practicability, a new quantitative technique has been developed. To enhance both sharpness and contrast of the images, a sucrose solution (refractive index = 1.49) is used, which reduces the effect of light-scattering particulates. To each slide a template-measured (38.5 mm³) fecal sample is transferred. Thus, egg counts and sensitivity evaluations are easily made.