Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

Background: Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results: The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion: In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.

Cross-National Analysis of the Associations between Traumatic Events and Suicidal Behavior: Findings from the WHO World Mental Health Surveys

Background: Community and clinical data have suggested there is an association between trauma exposure and suicidal behavior (i.e., suicide ideation, plans and attempts). However, few studies have assessed which traumas are uniquely predictive of: the first onset of suicidal behavior, the progression from suicide ideation to plans and attempts, or the persistence of each form of suicidal behavior over time. Moreover, few data are available on such associations in developing countries. The current study addresses each of these issues. Methodology/Principal Findings: Data on trauma exposure and subsequent first onset of suicidal behavior were collected via structured interviews conducted in the households of 102,245 (age 18+) respondents from 21 countries participating in the WHO World Mental Health Surveys. Bivariate and multivariate survival models tested the relationship between the type and number of traumatic events and subsequent suicidal behavior. A range of traumatic events are associated with suicidal behavior, with sexual and interpersonal violence consistently showing the strongest effects. There is a dose-response relationship between the number of traumatic events and suicide ideation/attempt; however, there is decay in the strength of the association with more events. Although a range of traumatic events are associated with the onset of suicide ideation, fewer events predict which people with suicide ideation progress to suicide plan and attempt, or the persistence of suicidal behavior over time. Associations generally are consistent across high-, middle-, and low-income countries. Conclusions/Significance: This study provides more detailed information than previously available on the relationship between traumatic events and suicidal behavior and indicates that this association is fairly consistent across developed and developing countries. These data reinforce the importance of psychological trauma as a major public health problem, and highlight the significance of screening for the presence and accumulation of traumatic exposures as a risk factor for suicide ideation and attempt.

Comparative Analysis of Activation Phenotype, Proliferation, and IFN-gamma Production by Spleen NK1.1(+) and NK1.1(-) T Cells During Plasmodium chabaudi AS Malaria

The NK1.1 molecule participates in NK, NKT, and T-cell activation, contributing to IFN-gamma production and cytotoxicity. To characterize the early immune response to Plasmodium chabaudi AS, spleen NK1.1(+) and NK1.1(-) T cells were compared in acutely infected C57BL/6 mice. The first parasitemia peak in C57BL/6 mice correlated with increase in CD4(+)NK1.1(+)TCR-alpha beta(+), CD8(+)NK1.1(+)TCR-alpha beta(+), and CD4(+)NK1.1(-)TCR-alpha beta(+) cell numbers per spleen, where a higher increment was observed for NK1.1(+) T cells compared to NK1.1(-) T cells. According to the ability to recognize the CD1d-alpha-GalCer tetramer, CD4(+)NK1.1(+) cells in 7-day infected mice were not predominantly invariant NKT cells. At that time, nearly all NK1.1(+) T cells and around 30% of NK1.1(-) T cells showed an experienced/activated (CD44(HI)CD69(HI)CD122(HI)) cell phenotype, with high expression of Fas and PD-L1 correlating with their low proliferative capacity. Moreover, whereas IFN-gamma production by CD4(+)NK1.1(+) cells peaked at day 4 p.i., the IFN-gamma response of CD4(+)NK1.1(-) cells continued to increase at day 5 of infection. We also observed, at day 7 p.i., 2-fold higher percentages of perforin(+) cells in CD8(+)NK1.1(+) cells compared to CD8(+)NK1.1(-) cells. These results indicate that spleen NK1.1(+) and NK1.1(-) T cells respond to acute P. chabaudi malaria with different kinetics in terms of activation, proliferation, and IFN-gamma production.

Theory of small-signal ac response of a dielectric liquid containing two groups of ions

The analysis of Macdonald for electrolytes is generalized to the case in which two groups of ions are present. We assume that the electrolyte can be considered as a dispersion of ions in a dielectric liquid, and that the ionic recombination can be neglected. We present the differential equations governing the ionic redistribution when the liquid is subjected to an external electric field, describing the simultaneous diffusion of the two groups of ions in the presence of their own space charge fields. We investigate the influence of the ions on the impedance spectroscopy of an electrolytic cell. In the analysis, we assume that each group of ions have equal mobility, the electrodes perfectly block and that the adsorption phenomena can be neglected. In this framework, it is shown that the real part of the electrical impedance of the cell has a frequency dependence presenting two plateaux, related to a type of ambipolar and free diffusion coefficients. The importance of the considered problem on the ionic characterization performed by means of the impedance spectroscopy technique was discussed. (c) 2008 American Institute of Physics.

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 angstrom resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 angstrom. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

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.

KeaA, a Dictyostelium kelch-domain protein that regulates the response to stress and development

Background: The protein kinase YakA is responsible for the growth arrest and induction of developmental processes that occur upon starvation of Dictyostelium cells. yakA-cells are aggregation deficient, have a faster cell cycle and are hypersensitive to oxidative and nitrosoative stress. With the aim of isolating members of the YakA pathway, suppressors of the death induced by nitrosoative stress in the yakA-cells were identified. One of the suppressor mutations occurred in keaA, a gene identical to DG1106 and similar to Keap1 from mice and the Kelch protein from Drosophila, among others that contain Kelch domains. Results: A mutation in keaA suppresses the hypersensitivity to oxidative and nitrosoative stresses but not the faster growth phenotype of yakA-cells. The growth profile of keaA deficient cells indicates that this gene is necessary for growth. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. A morphological analysis of keaA deficient cells during multicellular development indicated that, although the mutant is not absolutely deficient in aggregation, cells do not efficiently participate in the process. Gene expression analysis using cDNA microarrays of wild-type and keaA deficient cells indicated a role for KeaA in the regulation of the cell cycle and pre-starvation responses. Conclusions: KeaA is required for cAMP signaling following stress. Our studies indicate a role for kelch proteins in the signaling that regulates the cell cycle and development in response to changes in the environmental conditions.

Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Background: Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings: We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-gamma, TNF-alpha and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. Conclusion: This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.

Complex Oscillatory Response of a PEM Fuel Cell Fed with H(2)/CO and Oxygen

Oscillatory kinetics is commonly observed in the electrocatalytic oxidation of most species that can be used in fuel cell devices. Examples include formic acid, methanol, ethanol, ethylene glycol, and hydrogen/carbon monoxide mixtures, and most papers refer to half-cell experiments. We report in this paper the experimental investigation of the oscillatory dynamics in a proton exchange membrane (PEM) fuel cell at 30 degrees C. The system consists of a Pt/C cathode fed with oxygen and a PtRu (1:1)/C anode fed with H(2) mixed with 100 ppm of CO, and was studied at different cell currents and anode flow rates. Many different states including periodic and nonperiodic series were observed as a function of the cell current and the H(2)/CO flow rate. In general, aperiodic/chaotic states were favored at high currents and low flow rates. The dynamics was further characterized in terms of the relationship between the oscillation amplitude and the subsequent time required for the anode to get poisoned by carbon monoxide. Results are discussed in terms of the mechanistic aspects of the carbon monoxide adsorption and oxidation. (C) 2010 The Electrochemical Society. [DOI: 10.1149/1.3463725] All rights reserved.

Flow injection analysis of paracetamol using a biomimetic sensor as a sensitive and selective amperometric detector

This work describes the coupling of a biomimetic sensor to a flow injection system for the sensitive determination of paracetamol. The sensor was prepared as previously described in the literature (M. D. P. T. Sotomayor, A. Sigoli, M. R. V. Lanza, A. A. Tanaka and L. T. Kubota, J. Braz. Chem. Soc., 2008, 19, 734) by modifying a glassy carbon electrode surface with a Nafion (R) membrane doped with iron tetrapyridinoporphyrazine (FeTPyPz), a biomimetic catalyst of the P450 enzyme. The performance of the sensor for paracetamol detection was investigated and optimized in a flow injection system (FIA) using a wall jet electrochemical cell. Under optimized conditions a wide linear response range (1.0 x 10(-5) to 5.0 x 10(-2) mol L(-1)) was obtained, with a sensitivity of 2579 (+/- 129) mu A L mu mol(-1). The detection and quantification limits of the sensor for paracetamol in the FIA system were 1.0 and 3.5 mu mol L(-1), respectively. The analytical frequency was 51 samples h(-1), and over a period of five days (320 determinations) the biosensor maintained practically the same response. The system was successfully applied to paracetamol quantification in seven pharmaceutical formulations and in water samples from six rivers in Sao Paulo State, Brazil.

Mechanistic studies of the 'blue' Cu enzyme, bilirubin oxidase, as a highly efficient electrocatalyst for the oxygen reduction reaction

The 'blue copper' enzyme bilirubin oxidase from Myrothecium verrucaria shows significantly enhanced adsorption on a pyrolytic graphite 'edge' (PGE) electrode that has been covalently modified with naphthyl-2-carboxylate functionalities by diazonium coupling. Modified electrodes coated with bilirubin oxidase show electrocatalytic voltammograms for the direct, four-electron reduction of O(2) by bilirubin oxidase with up to four times the current density of an unmodified PGE electrode. Electrocatalytic voltammograms measured with a rapidly rotating electrode (to remove effects of O(2) diffusion limitation) have a complex shape (an almost linear dependence of current on potential below pH 6) that is similar regardless of how PGE is chemically modified. Importantly, the same waveform is observed if bilirubin oxidase is adsorbed on Au(111) or Pt(111) single-crystal electrodes (at which activity is short-lived). The electrocatalytic behavior of bilirubin oxidase, including its enhanced response on chemically-modified PGE, therefore reflects inherent properties that do not depend on the electrode material. The variation of voltammetric waveshapes and potential-dependent (O(2)) Michaelis constants with pH and analysis in terms of the dispersion model are consistent with a change in rate-determining step over the pH range 5-8: at pH 5, the high activity is limited by the rate of interfacial redox cycling of the Type 1 copper whereas at pH 8 activity is much lower and a sigmoidal shape is approached, showing that interfacial electron transfer is no longer a limiting factor. The electrocatalytic activity of bilirubin oxidase on Pt(111) appears as a prominent pre-wave to electrocatalysis by Pt surface atoms, thus substantiating in a single, direct experiment that the minimum overpotential required for O(2) reduction by the enzyme is substantially smaller than required at Pt. At pH 8, the onset of O(2) reduction lies within 0.14 V of the four-electron O(2)/2H(2)O potential.

Downscaling a multicommuted flow injection analysis system for the photometric determination of iodate in table salt

In this work a downscaled multicommuted flow injection analysis setup for photometric determination is described. The setup consists of a flow system module and a LED based photometer, with a total internal volume of about 170 mu L The system was tested by developing an analytical procedure for the photometric determination of iodate in table salt using N,N-diethyl-henylenediamine (DPD) as the chromogenic reagent. Accuracy was accessed by applying the paired r-test between results obtained using the proposed procedure and a reference method, and no significant difference at the 95% confidence level was observed. Other profitable features, such as a low reagent consumption of 7.3 mu g DPD per determination: a linear response ranging from 0.1 up to 3.0 m IO(3)(-), a relative standard deviation of 0.9% (n = 11) for samples containing 0.5 m IO(3)(-), a detection limit of 17 mu g L(-1) IO(3)(-), a sampling throughput of 117 determination per hour, and a waste generation 600 mu L per determination, were also achieved. (C) 2010 Elsevier B.V. All rights reserved.

A greener and highly sensitive flow-based procedure for carbaryl determination exploiting long pathlength spectrophotometry and photochemical waste degradation

An environmentally friendly analytical procedure with high sensitivity for determination of carbaryl pesticide in natural waters was developed. The flow system was designed with solenoid micro-pumps in order to improve mixing conditions and minimize reagent consumption as well as waste generation. A long pathlength (100 cm) flow cell based on a liquid core waveguide (LCW) was employed to increase the sensitivity in detection of the indophenol formed from the reaction between carbaryl and p-aminophenol (PAP). A clean-up step based on cloud-point extraction was explored to remove the interfering organic matter, avoiding the use of toxic organic solvents. A linear response was observed within the range 5-200 mu g L(-1) and the detection limit, coefficient of variation and sampling rate were estimated as 1.7 mu g L(-1) (99.7% confidence level), 0.7% (n=20) and 55 determinations per hour, respectively. The reagents consumption was 1.9 mu g of PAP and 5.7 mu g of potassium metaperiodate, with volume of 2.6 mL of effluent per determination. The proposed procedure was selective for the determination of carbaryl, without interference from other carbamate pesticides. Recoveries within 84% and 104% were estimated for carbaryl spiked to water samples and the results obtained were also in agreement with those found by a batch spectrophotometric procedure at the 95% confidence level. The waste of the analytical procedure was treated with potassium persulphate and ultraviolet irradiation, yielding a colorless residue and a decrease of 94% of total organic carbon. In addition, the residue after treatment was not toxic for Vibrio fischeri bacteria. (c) 2010 Elsevier B.V. All rights reserved.

A flow-based procedure with solenoid micro-pumps for the spectrophotometric determination of uric acid in urine

The determination of uric acid in urine shows clinical importance, once it can be related to human organism dysfunctions, such as gout. An analytical procedure employing a multicommuted flow system was developed for the determination of uric acid in urine samples. Cu(II) ions are reduced by uric acid to Cu(I) that can be quantified by spectrophotometry in the presence of 2,2`-biquinoline 4,4`-dicarboxylic acid (BCA). The analytical response was linear between 10 and 100 mu mol L(-1) uric acid with a detection limit of 3.0 mu mol L(-1) (99.7% confidence level). Coefficient of variation of 1.2% and sampling rate of 150 determinations per hour were achieved. Per determination, 32 mu g of CuSO(4) and 200 mu g of BCA were consumed, generating 2.0 mL of waste. Recoveries from 91 to 112% were estimated and the results for 7 urine samples agreed with those obtained by the commercially available enzymatic kit for determination of uric acid. The procedure required 100-fold dilution of urine samples, minimizing sample consumption and interfering effects. In order to avoid the manual dilution step, on-line sample dilution was achieved by a simple system reconfiguration attaining a sampling rate of 95 h(-1). (C) 2009 Elsevier B.V. All rights reserved.