Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 A resolution.

Interference with Hemozoin Formation Represents an Important Mechanism of Schistosomicidal Action of Antimalarial Quinoline Methanols

Background: The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches. Methodology/Principal Findings: Treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11(th) to 17(th) day after infection caused significant decreases in worm burden (39%-61%) and egg production (42%-98%). Hz formation was significantly inhibited (40%-65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms. Conclusions: The overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.

Rapid and sensitive measurements of nitrate ester explosives using microchip electrophoresis with electrochemical detection

This article describes an effective microchip protocol based on electrophoretic-separation and electrochemical detection for highly sensitive and rapid measurements of nitrate ester explosives, including ethylene glycol dinitrate (EGDN), pentaerythritol tetranitrate (PETN), propylene glycol dinitrate (PGDN) and glyceryl trinitrate (nitroglycerin, NG). Factors influencing the separation and detection processes were examined and optimized. Under the optimal separation conditions obtained using a 15 mM borate buffer (pH 9.2) containing 20 mM SDS, and applying a separation voltage of 1500 V, the four nitrate ester explosives were separated within less than 3 min. The glassy-carbon amperometric detector (operated at -0.9 V vs. Ag/AgCl) offers convenient cathodic detection down to the picogram level, with detection limits of 0.5 ppm and 0.3 ppm for PGDN and for NG, respectively, along with good repeatability (RSD of 1.8-2.3%; n = 6) and linearity (over the 10-60 ppm range). Such effective microchip operation offers great promise for field screening of nitrate ester explosives and for supporting various counter-terrorism surveillance activities.

Exploitation of a single interface flow system for on-line aqueous biphasic extraction

The exploitation of aqueous biphasic extraction is proposed for the first time in flow analysis This extraction strategy stands out for being environmentally attractive since it is based in the utilization of two immiscible phases that are intrinsically aqueous The organic solvents of the traditional liquid-liquid extractions ale no longer used, being replaced by non-toxic, non-flammable and non-volatile ones. A single interface flow analysis (SIFA) system was implemented to carry out the extraction process due to its favourable operational characteristics that include the high automation level and simplicity of operation, the establishment of a dynamic interface where the mass transfer occurred between the two immiscible aqueous phases, and the versatile control over the extraction process namely the extraction time The application selected to demonstrate the feasibility of SIFA to perform this aqueous biphasic extraction was the pre-concentration of lead. After extraction, lead reacted with 8-hydroxyquinoline-5-sulfonic acid and the resulting product was determined by a fluorimetric detector included in the flow manifold. Therefore, the SIFA single interface was used both as extraction (enrichment) and reaction interface. (C) 2010 Elsevier B.V All rights reserved.

Preliminary studies of laser-induced breakdown spectrometry for the determination of Ba, Cd, Cr and Pb in toys

The performance of laser-induced breakdown spectrometry (LIBS) for the determination of Ba, Cd, Cr and Pb in toys has been evaluated by using a Nd:YAG laser operating at 1064 nm and an Echelle spectrometer with intensified charge-coupled device detector. Samples were purchased in different cities of Sao Paulo State market and analyzed directly without sample preparation. Laser-induced breakdown spectrometry experimental conditions (number of pulses, delay time. integration time gate and pulse energy) were optimized by using a Doehlert design. Laser-induced breakdown spectrometry signals correlated reasonably well with inductively coupled plasma optical emission spectrometry (ICP OES) concentrations after microwave-assisted acid digestion of selected samples. Thermal analysis was used for polymer identification and scanning electron microscopy to Visualize differences in crater geometry of different polymers employed for toy fabrication. Results indicate that laser-induced breakdown spectrometry can be proposed as a rapid screening method for investigation of potentially toxic elements in toys. The unique application of laser-induced breakdown spectrometry for identification of contaminants in successive layers of ink and polymer is also demonstrated. (C) 2009 Elsevier B.V. All rights reserved.

Evaluation of laser induced breakdown spectroscopy for the determination of micronutrients in plant materials

Laser induced breakdown spectroscopy (LIBS) has been evaluated for the determination of micronutrients (B, Cu, Fe, Mn and Zn) in pellets of plant materials, using NIST, BCR and GBW biological certified reference materials for analytical calibration. Pellets of approximately 2 mm thick and 15 mm diameter were prepared by transferring 0.5 g of powdered material to a 15 mm die set and applying 8.0 tons cm(-2). An experimental setup was designed by using a Nd:YAG laser operating at 1064 nm (200 mJ per pulse, 10 Hz) and an Echelle spectrometer with ICCD detector. Repeatability precision varied from 4 to 30% from measurements obtained in 10 different positions (8 laser shots per test portion) in the same sample pellet. Limits of detection were appropriate for routine analysis of plant materials and were 2.2 mg kg(-1) B, 3.0 mg kg(-1) Cu, 3.6 mg kg(-1) Fe, 1.8 mg kg(-1) Mn and 1.2 mg kg(-1) Zn. Analysis of different plant samples were carried out by LIBS and results were compared with those obtained by ICP OES after wet acid decomposition. (C) 2009 Elsevier B.V. All rights reserved.

INAA with gamma-gamma coincidence for selenium determination in food

Selenium detection limits of INAA are normally above its concentration in most biological materials. Gamma-gamma coincidence methodology can be used to improve the detection limits and uncertainties in the determination of selenium. Here, some edible parts of plants were measured using a HPGe detector equipped with a NaI(Tl) active shielding, producing spectra both in normal and coincidence modes. The results presented the reduction of the detection limits of selenium by a factor of 2 to 3 times and improvement in the uncertainty of up to 2 times.

Collimated scanning LS-INAA for testing trace elements homogeneity in Brazilian coffee beans

The degree of homogeneity is normally assessed by the variability of the results of independent analyses of several (e.g., 15) normal-scale replicates. Large sample instrumental neutron activation analysis (LS-INAA) with a collimated Ge detector allows inspecting the degree of homogeneity of the initial batch material, using a kilogram-size sample. The test is based on the spatial distributions of induced radioactivity. Such test was applied to samples of Brazilian whole (green) coffee beans (Coffea arabica and Coffea canephora) of approximately I kg in the frame of development of a coffee reference material. Results indicated that the material do not contain significant element composition inhomogeneities between batches of approximately 30-50 g, masses typically forming the starting base of a reference material.

A critical evaluation of a flow-cell based on a liquid core waveguide for chemiluminescence measurements

Liquid-core waveguides (LCWs), devices that constrain the emitted radiation minimizing losses during the transport, are an alternative to maximize the amount of detected radiation in luminescence. In this work, the performance of a LCW flow-cell was critically evaluated for chemiluminescence measurements, by using as model the oxidation of luminol by hydrogen peroxide or hypochlorite. An analytical procedure for hypochlorite determination was also developed, with linear response in the range 0.2-3.8 mg/L (2.7-51 mu mol/L), a detection limit estimated as 8 mu g/L (0.64 mu mol/L) at the 99.7% confidence level and luminol consumption of 50 mu g/determination. The coefficients of variation were 3.3% and 1.6% for 0.4 and 1.9 mg/L CIO(-), respectively, with a sampling rate of 164 determinations/h. The procedure was applied to the analysis of Dakin`s solution samples, yielding results in agreement with those obtained by iodometric titration at the 95% confidence level. Copyright (c) 2008 John Wiley & Sons, Ltd.

Evaluation of laser induced breakdown spectroscopy for the determination of macronutrients in plant materials

Laser induced breakdown spectroscopy (LIBS) has become an analytical tool for the direct analysis of a large variety of materials in order to provide qualitative and/or quantitative information. However, there is a lack of information for LIBS analysis of agricultural and environmental samples. In this work a LIBS system has been evaluated for the determination of macronutrients (P, K, Ca, Mg) in pellets of vegetal reference materials. An experimental setup was designed by using a Nd:YAG laser operating at 1064 nm and an Echelle spectrometer with ICCD detector. The plasma temperature was estimated by Boltzmann plots and instrumental paragmeters such as delay time, lens-to-sample distance and pulse energy were evaluated. Certified reference materials as well as reference materials were used for analytical calibrations of P, K, Ca, and Mg. Most results of the direct analysis of plant samples by LIBS were in reasonable agreement with those obtained by ICP OES after wet acid decomposition. (C) 2008 Elsevier B.V. All rights reserved.

A flow-based analytical procedure for salbutamol determination exploiting chemiluminescence in a liquid-core waveguide

Salbutamol is a bronchodilator whose use is restricted due to its anabolic effects. A flow-based procedure for salbutamol determination based on the inhibition of chemiluminescence of the luminol/hypochlorite system was developed. A flow cell constructed with a liquid-core waveguide was employed to constrain the emitted radiation, minimizing losses during transport to detector. Linear response was observed within 2.5 x 10(-6) and 1.0 x 10(-5) mol L-1 with a detection limit estimated as 1 x 10(-7) mol L-1 at the 99.7% confidence level. The coefficient of variation (n = 20), sampling rate, and luminol consumption per determination were estimated as 2.8%, 164 determinations h(-1), and 50 mu g, respectively. Results for pharmaceutical samples were in agreement with those obtained by reference procedures at the 95% confidence level.

Sequential injections as an alternative to gradient exploitation for implementing differential kinetic analysis in a flow injection system

A novel flow-based strategy for implementing simultaneous determinations of different chemical species reacting with the same reagent(s) at different rates is proposed and applied to the spectrophotometric catalytic determination of iron and vanadium in Fe-V alloys. The method relies on the influence of Fe(II) and V(IV) on the rate of the iodide oxidation by Cr(VI) under acidic conditions, the Jones reducing agent is then needed Three different plugs of the sample are sequentially inserted into an acidic KI reagent carrier stream, and a confluent Cr(VI) solution is added downstream Overlap between the inserted plugs leads to a complex sample zone with several regions of maximal and minimal absorbance values. Measurements performed on these regions reveal the different degrees of reaction development and tend to be more precise Data are treated by multivariate calibration involving the PLS algorithm The proposed system is very simple and rugged Two latent variables carried out ca 95% of the analytical information and the results are in agreement with ICP-OES. (C) 2010 Elsevier B V. All rights reserved.

Zone trapping/merging zones in flow analysis: A novel approach for rapid assays involving relatively slow chemical reactions

A novel strategy for accomplishing zone trapping in flow analysis is proposed. The sample and the reagent solutions are simultaneously inserted into convergent carrier streams and the established zones merge together before reaching the detector, where the most concentrated portion of the entire sample zone is trapped. The main characteristics, potentialities and limitations of the strategy were critically evaluated in relation to an analogous flow system with zone stopping. When applied to the spectrophotometric determination of nitrite in river waters, the main figures of merit were maintained, exception made for the sampling frequency which was calculated as 189h(-1), about 32% higher relatively to the analogous system with zone stopping. The sample inserted volume can be increased up to 1.0 mL without affecting sampling frequency and no problems with pump heating or malfunctions were noted after 8-h operation of the system. In contrast to zone stopping, only a small portion of the sample zone is halted with zone trapping, leading to these beneficial effects. (C) 2011 Elsevier B.V. All rights reserved.

Improvements on ICA mixture models for image pre-processing and segmentation

Today several different unsupervised classification algorithms are commonly used to cluster similar patterns in a data set based only on its statistical properties. Specially in image data applications, self-organizing methods for unsupervised classification have been successfully applied for clustering pixels or group of pixels in order to perform segmentation tasks. The first important contribution of this paper refers to the development of a self-organizing method for data classification, named Enhanced Independent Component Analysis Mixture Model (EICAMM), which was built by proposing some modifications in the Independent Component Analysis Mixture Model (ICAMM). Such improvements were proposed by considering some of the model limitations as well as by analyzing how it should be improved in order to become more efficient. Moreover, a pre-processing methodology was also proposed, which is based on combining the Sparse Code Shrinkage (SCS) for image denoising and the Sobel edge detector. In the experiments of this work, the EICAMM and other self-organizing models were applied for segmenting images in their original and pre-processed versions. A comparative analysis showed satisfactory and competitive image segmentation results obtained by the proposals presented herein. (C) 2008 Published by Elsevier B.V.

Cellulose modified fibres in cement based composites

The objective of the present work is to evaluate the effect of surface modification of cellulose pulp fibres on the mechanical and microstructure of fibre-cement composites. Surface modification of the cellulose pulps was performed with Methacryloxypropyltri-methoxysilane (MPTS) and Aminopropyltri-ethoxysilane (APTS) in an attempt to improve their durability into fibre-cement composites. The surface modification showed significant influence on the microstructure of the composites on the fibre-matrix interface and in the mineralization of the fibre lumen as seen by scanning electron microscopy (SEM) with back-scattered electron (BSE) detector. Accelerated ageing cycles decreased modulus of rupture (MOR) and toughness (TE) of the composites. Composites reinforced with MPTS-modified fibres presented fibres free from cement hydration products, while APTS-modified fibres presented accelerated mineralization. Higher mineralization of the fibres led to higher embrittlement of the composite after accelerated ageing cycles. These observations are therefore very useful for understanding the mechanisms of degradation of fibre-cement composites. (C) 2009 Elsevier Ltd. All rights reserved.