Automatic contour extraction from 2D neuron images

This work describes a novel methodology for automatic contour extraction from 2D images of 3D neurons (e.g. camera lucida images and other types of 2D microscopy). Most contour-based shape analysis methods cannot be used to characterize such cells because of overlaps between neuronal processes. The proposed framework is specifically aimed at the problem of contour following even in presence of multiple overlaps. First, the input image is preprocessed in order to obtain an 8-connected skeleton with one-pixel-wide branches, as well as a set of critical regions (i.e., bifurcations and crossings). Next, for each subtree, the tracking stage iteratively labels all valid pixel of branches, tip to a critical region, where it determines the suitable direction to proceed. Finally, the labeled skeleton segments are followed in order to yield the parametric contour of the neuronal shape under analysis. The reported system was successfully tested with respect to several images and the results from a set of three neuron images are presented here, each pertaining to a different class, i.e. alpha, delta and epsilon ganglion cells, containing a total of 34 crossings. The algorithms successfully got across all these overlaps. The method has also been found to exhibit robustness even for images with close parallel segments. The proposed method is robust and may be implemented in an efficient manner. The introduction of this approach should pave the way for more systematic application of contour-based shape analysis methods in neuronal morphology. (C) 2008 Elsevier B.V. All rights reserved.

Mutagenicity and DNA adduct formation of PAH, nitro-PAH, and oxy-PAH fractions of atmospheric particulate matter from Sao Paulo, Brazil

Urban particulate matter (UPM) contributes to lung cancer incidence. Here, we have studied the mutagenic activity and DNA adduct-forming ability of fractionated UPM extractable organic matter (EOM). UPM was collected with a high-volume sampler in June 2004 at two sites, one at street level adjacent to a roadway and the other inside a park within the urban area of the city of Sao Paulo, Brazil. UPM was extracted using dichloromethane, and the resulting EOM was separated by HPLC to obtain PAH, nitro-PAH, and oxy-PAH fractions which were tested for mutagenicity with the Salmonella strains TA98 and YG1041 with and without S9 metabolic activation. The PAH fraction from both sites showed negligible mutagenic activity in both strains. The highest mutagenic activity was found for the nitro-PAH fraction using YG1041 without metabolic activation; however, results were comparable for both sites. The nitro-PAH and oxy-PAH fractions were incubated with calf thymus DNA under reductive conditions appropriate for the activation of nitro aromatic compounds, then DNA adduct patterns and levels were determined with thin-layer chromatography (TLC) (32)p-postlabeling method using two enrichment procedures-nuclease PI digestion and butanol extraction. Reductively activated fractions from both sites produced diagonal radioactive zones (DRZ) of putative aromatic DNA adducts on thin layer plates with both enrichment procedures. No such DRZ were observed in control experiments using fractions from unexposed filters or from incubations without activating system. Total adduct levels produced by the nitro-PAH fractions were similar for both sites ranging from 30 to 45 adducts per 10(8) normal nucleotides. In contrast, the DNA binding of reductively activated oxy-PAH fractions was three times higher and the adduct pattern consisted of multiple discrete spots along the diagonal line on the thin layer plates. However, DNA adduct levels were not significantly different between the sampling sites. Both samples presented the same levels of mutagenic activity. The response in the Salmonella assay was typical of nitroaromatics. Although, more mutagenic activity was related to the nitro-PAH fraction in the Salmonella assay, the oxy-PAH fractions showed the highest DNA adduct levels. More studies are needed to elucidate the nature of the genotoxicants occurring in Sao Paulo atmospheric samples. (C) 2008 Elsevier B.V. All rights reserved.

Pendimethalin determination in natural water, baby food and river sediment samples using electroanalytical methods

This work describes the electroanalytical determination of pendimethalin herbicide levels in natural waters, river sediment and baby food samples, based on the electro-reduction of herbicide on the hanging mercury drop electrode using square wave voltammetry (SWV). A number of experimental and voltammetric conditions were evaluated and the best responses were achieved in Britton-Robinson buffer solutions at pH 8.0, using a frequency of 500 s(-1). a scan increment of 10 mV and a square wave amplitude of 50 mV. Under these conditions, the pendimethalin is reduced in an irreversible process, with two reduction peaks at -0.60 V and -0.71 V. using a Ag/AgCl reference system. Analytical curves were constructed and the detection limit values were calculated to be 7.79 mu g L(-1) and 4.88 mu g L(-1), for peak 1 and peak 2, respectively. The precision and accuracy were determinate as a function of experimental repeatability and reproducibility, which showed standard relative deviation values that were lower than 2% for both voltammetric peaks. The applicability of the proposed methodology was evaluated in natural water, river sediments and baby food samples. The calculated recovery efficiencies demonstrate that the proposed methodology is suitable for determining any contamination by pendimethalin in these samples. Additionally, adsorption isotherms were used to evaluate information about the behavior of pendimethalin in river sediment samples. (C) 2010 Elsevier B.V. All rights reserved.

Characterization of organic matter from composting of different residues by physicochemical and spectroscopic methods

Chemical and spectroscopic methods were used to characterize organic matter transformations during the composting process. Four different residue mixtures were studied: P1 - garden trimmings (GT) only, P2 - GT plus fresh cattle manure, P3 - GT plus orange pomace and P4 - GT plus filter cake. The thermophilic phase was not reached in PI compost, but the P2, P3 and P4 composts showed all three typical process phases. The thermophilic phase and CEC/C ratio stabilized after 90 days, while C/N ratio and the ash content stabilized after 60 days. The increasing E(4)/E(6) ratio indicated oxidation reactions occurring during the process in the material from P2, P3 and P4. The (13)C NMR and FTIR results suggested extraction of both pectin and lignin in the HA-like fraction. The CEC/C ratio, temperature and E(4)/E(6) ratio showed that within 90 days P2, P3 and P4 composts were humified. However, material from P1 did not show characteristics of humified compost. From these data, it is apparent that C/N ratio and ash content are not reliable methods for monitoring the composting process. (C) 2009 Elsevier Ltd. All rights reserved.

Application of chemometric methods in the evaluation of chemical and spectroscopic data on organic matter from Oxisols in sewage sludge applications

Chemometric methods can contribute to soil research by permitting the extraction of more information from the data. The aim of this work was to use Principal Component Analysis to evaluate data obtained through chemical and spectroscopic methods on the changes in the humification process of soil organic matter from two tropical soils after sewage sludge application. In this case, humic acids extracted from Typic Eutrorthox and Typic Haplorthox soils with and without sewage sludge application for 7 consecutive years were studied. The results obtained for all of the samples and methods showed two clusters: samples extracted from the two soil types. These expected results indicated the textural difference between the two soils was more significant than the differences between treatments (control and sewage sludge application) or between depths. In this case, an individual chemometric treatment was made for each type of soil. It was noted that the characterization of the humic acids extracted from soils with and without sewage sludge application after 7 consecutive years using several methods supplies important results about changes in the humification degree of soil organic matter, These important result obtained by Principal Component Analysis justify further research using these methods to characterize the changes in the humic acids extracted from sewage sludge-amended soils. (C) 2009 Elsevier B.V. All rights reserved.

Prevalência da deleção 4977pb do DNA mitocondrial em pacientes com doença renal crônica em tratamento conservador ou submetidos a hemodiálise no sul do Brasil

Introdução. Danos no DNA mitocondrial (DNAmt) têm sido descritos em pacientes com doença renal crônica (DRC). Estes danos podem ser avaliados através da deleção 4977pb do DNAmt em diversos tecidos. Métodos. Identificamos a prevalência da deleção 4977pb do DNAmt através da técnica da reação em cadeia da polimerase (PCR) no sangue de pacientes com DRC em tratamento conservador (creatinina >2mg/dl) ou submetidos a hemodiálise. Resultados. A freqüência da ocorrência da deleção do DNAmt foi de 73.1% (38/52) nos pacientes com DRC submetidos a hemodiálise, 57.1% (27/42) nos pacientes com DRC em tratamento conservador e 27.8% (15/54) nos controles (P< 0.001). Não encontramos aumento da freqüência desta deleção em relação a idade dos pacientes com DRC (P= 0.54) ou ao tempo de diálise (P= 0.70). Conclusão. Danos no DNAmt podem ser induzidos pela DRC em especial nos pacientes submetidos a hemodiálise. Desta forma, a deleção 4977pb do DNAmt pode servir como um marcador de danos moleculares em pacientes com DRC.

Potentialities of two solventless extraction approaches—Stir bar sorptive extraction and headspace solid-phase microextraction for determination of higher alcohol acetates, isoamyl esters and ethyl esters in wines

A stir bar sorptive extraction with liquid desorption followed by large volume injection coupled to gas chromatography–quadrupole mass spectrometry (SBSE-LD/LVI-GC–qMS) was evaluated for the simultaneous determination of higher alcohol acetates (HAA), isoamyl esters (IsoE) and ethyl esters (EE) of fatty acids. The method performance was assessed and compared with other solventless technique, the solid-phase microextraction (SPME) in headspace mode (HS). For both techniques, influential experimental parameters were optimised to provide sensitive and robust methods. The SBSE-LD/LVI methodology was previously optimised in terms of extraction time, influence of ethanol in the matrix, liquid desorption (LD) conditions and instrumental settings. Higher extraction efficiency was obtained using 60 min of extraction time, 10% ethanol content, n-pentane as desorption solvent, 15 min for the back-extraction period, 10 mL min−1 for the solvent vent flow rate and 10 °C for the inlet temperature. For HS-SPME, the fibre coated with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) afforded highest extraction efficiency, providing the best sensitivity for the target volatiles, particularly when the samples were extracted at 25 °C for 60 min under continuous stirring in the presence of sodium chloride (10% (w/v)). Both methodologies showed good linearity over the concentration range tested, with correlation coefficients higher than 0.984 for HS-SPME and 0.982 for SBES-LD approach, for all analytes. A good reproducibility was attained and low detection limits were achieved using both SBSE-LD (0.03–28.96 μg L−1) and HS-SPME (0.02–20.29 μg L−1) methodologies. The quantification limits for SBSE-LD approach ranging from 0.11 to 96.56 μg L−and from 0.06 to 67.63 μg L−1 for HS-SPME. Using the HS-SPME approach an average recovery of about 70% was obtained whilst by using SBSE-LD obtained average recovery were close to 80%. The analytical and procedural advantages and disadvantages of these two methods have been compared. Both analytical methods were used to determine the HAA, IsoE and EE fatty acids content in “Terras Madeirenses” table wines. A total of 16 esters were identified and quantified from the wine extracts by HS-SPME whereas by SBSE-LD technique were found 25 esters which include 2 higher alcohol acetates, 4 isoamyl esters and 19 ethyl esters of fatty acids. Generally SBSE-LD provided higher sensitivity with decreased analysis time.

Application of chemometric methods in the evaluation of chemical and spectroscopic data on organic matter from Oxisols in sewage sludge applications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Non invasive methods for genetic analysis applied to ecological and behavioral studies in Latino-America

Documenting the presence and abundance of the neotropical mammals is the first step for understanding their population ecology, behavior and genetic dynamics in designing conservation plans. The combination of field research with molecular genetics techniques are new tools that provide valuable biological information avoiding the disturbance in the ecosystems, trying to minimize the human impact in the process to gather biological information. The objective of this paper is to review the available non invasive sampling techniques that have been used in Neotropical mammal studies to apply to determine the presence and abundance, population structure, sex ratio, taxonomic diagnostic using mitochondrial markers, and assessing genetic variability using nuclear markers. There are a wide range of non invasive sampling techniques used to determine the species identification that inhabit an area such as searching for tracks, feces, and carcasses. Other useful equipment is the camera traps that can generate an image bank that can be valuable to assess species presence and abundance by morphology. With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in feces and amplify it to analyze the species diversity in an area, and the genetic variability at intraspecific level. This is particularly helpful in cases of sympatric and cryptic species in which morphology failed to diagnose the taxonomic status of several species of brocket deer of the genus Mazama.

Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum é um importante agente causal de dermatomicose. Os métodos de tipagem molecular têm sido recentemente desenvolvidos para responder questões sobre epidemiologia e auxiliar no esclarecimento de recidivas, após o tratamento. As seqüências aleatórias 1- (5'-d[GGTGCGGGAA]-3') e 6- (5'-d[CCCGTCAGCA]-3') foram usadas para tipagem molecular deste fungo por RAPD produzindo variabilidade intraespecífica. Cinco padrões foram observados entre os 10 isolados de T. rubrum, com ambas as seqüências. Foi concluído que a análise por RAPD pode ser utilizada para estudos epidemiológicos.

Drug resistance in Mycobacterium tuberculosis clinical isolates from Brazil: Phenotypic and genotypic methods

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5'-d[GGTGCGGGAA]-3' and 5'-d[CCCGTCAGCA]-3', as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPID products showed a high degree of similarity (> 90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60% similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPID analysis is especially suitable for molecular typing in T. rubrum.

Analytical Methods for the Determination of Chlorhexidine: A Review

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Methacycline: A Review of Analytical Methods

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)