A novel HS-SBSE system coupled with gas chromatography and mass spectrometry for the analysis of organochlorine pesticides in water samples

A methodology to analyze organochlorine pesticides (OCPs) in water samples has been accomplished by using headspace stir bar sorptive extraction (HS-SBSE). The bars were in house coated with a thick film of PDMS in order to properly work in the headspace mode. Sampling was done by a novel HS-SBSE system whereas the analysis was performed by capillary GC coupled mass spectrometric detection (HS-SBSE-GC-MS). The extraction optimization, using different experimental parameters has been established by a standard equilibrium time of 120 min at 85 degrees C. A mixture of ACN/toluene as back extraction solvent promoted a good performance to remove the OCPs sorbed in the bar. Reproducibility between 2.1 and 14.8% and linearity between 0.96 and 1.0 were obtained for pesticides spiked in a linear range between 5 and 17 ng/g in water samples during the bar evaluation.

Anti-CD25 Treatment Depletes Treg Cells and Decreases Disease Severity in Susceptible and Resistant Mice Infected with Paracoccidioides brasiliensis

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4(+)CD25(+)Foxp3(+) Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-beta. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4(+)CD25(+)Foxp3(+) Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4(+) and CD8(+) T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25(+) cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.

Lipase-Catalyzed Kinetic Resolution of (+/-)-Mandelonitrile under Conventional Condition and Microwave Irradiation

The kinetic resolution of (+/-)-mandelonitrile was carried out using lipase from Candida antarctica under conventional condition (orbital shaker) and microwave irradiation in toluene, producing the (S)-mandelonitrile acetate with high selectivity (up to >98% ee, enantiomeric excess). The unreacted (R)-mandelonitrile under microwave irradiation and conventional condition was partially converted into benzaldehyde by spontaneous chemical equilibrium. The (S)-mandelonitrile acetate under microwave irradiation was produced with 92% ee and 35% yield for 8 h of reaction. Conventional transesterification of (+/-)-mandelonitrile in an orbital shaker produced unreacted (R) -mandelonitrile (51% ee) and (S)-mandelonitrile acetate (98% ee) in accordance with Kazlauskas rule for 184 h of reaction.

Long-Term Occupational Exposure to Organic Solvents Affects Color Vision, Contrast Sensitivity and Visual Fields

The purpose of this study was to evaluate the visual outcome of chronic occupational exposure to a mixture of organic solvents by measuring color discrimination, achromatic contrast sensitivity and visual fields in a group of gas station workers. We tested 25 workers (20 males) and 25 controls with no history of chronic exposure to solvents (10 males). All participants had normal ophthalmologic exams. Subjects had worked in gas stations on an average of 9.6 +/- 6.2 years. Color vision was evaluated with the Lanthony D15d and Cambridge Colour Test (CCT). Visual field assessment consisted of white-on-white 24-2 automatic perimetry (Humphrey II-750i). Contrast sensitivity was measured for sinusoidal gratings of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 cycles per degree (cpd). Results from both groups were compared using the Mann-Whitney U test. The number of errors in the D15d was higher for workers relative to controls (p<0.01). Their CCT color discrimination thresholds were elevated compared to the control group along the protan, deutan and tritan confusion axes (p<0.01), and their ellipse area and ellipticity were higher (p<0.01). Genetic analysis of subjects with very elevated color discrimination thresholds excluded congenital causes for the visual losses. Automated perimetry thresholds showed elevation in the 9 degrees, 15 degrees and 21 degrees of eccentricity (p<0.01) and in MD and PSD indexes (p<0.01). Contrast sensitivity losses were found for all spatial frequencies measured (p<0.01) except for 0.5 cpd. Significant correlation was found between previous working years and deutan axis thresholds (rho = 0.59; p<0.05), indexes of the Lanthony D15d (rho = 0.52; p<0.05), perimetry results in the fovea (rho = -0.51; p<0.05) and at 3, 9 and 15 degrees of eccentricity (rho = -0.46; p<0.05). Extensive and diffuse visual changes were found, suggesting that specific occupational limits should be created.

Human Endometrial-Derived Mesenchymal Stem Cells Suppress Inflammation in the Central Nervous System of EAE Mice

FAPESP [2009/13109-5]

Tributyltin in Crustacean Tissues: Analytical Performance and Validation of Method

The hermit crab Clibanarius vittatus is a typical organism from intertidal regions being considered as a good bioindicator of tributyltin presence at these environments. Thus this study presents the analytical performance and validation method for TBT quantification in tissues of C. vittatus by gas chromatography with pulsed flame photometric detector (GC-PFPD) after extraction with an apolar solvent (toluene) and Grignard derivatization. The limits of detection of the method (LOD) were 2.0 and 2.8 ng g(-1) for TBT and DBT (dibutyltin), respectively, and its limits of quantification (LOQ) were 6.6 and 8.9 ng g(-1) for TBT and DBT, respectively. The method was applied to samples from Santos Estuary, Sao Paulo State, Brazil. TBT and DBT concentrations ranged from 26.7 to 175.0 ng g(-1) and from 46.2 to 156.0 ng g(-1), respectively. These concentrations are worrisome since toxic effects (such as endocrine disruption) have been reported for other organisms even under lower levels of registred at this study.

Solvent Effect in Reactions Using Stryker's Reagent

The solvent has a significant influence in the rate of reactions promoted by Stryker's reagent The reactions performed in THF were, in most cases, faster than in toluene.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Optical chemical sensors using polythiophene derivatives as active layer for detection of volatile organic compounds

Several studies on polythiophene gas sensors, based mainly on electrochemical and gravimetric principles can be found in the literature. However, other principles of gas detection, such as optical and thermal, are still little studied. Optical sensing is suitable for remote detection and offers great versatility at low cost. Here,we report on the use of thin films of seven polythiophene derivatives as active layer in optical sensors for the detection of six volatile organic compounds (n-hexane, toluene, tetrahydrofuran, chloroform, dichloromethane and methanol) and water vapor, in concentration range of 500-30,000 ppm. The results showed that it is possible to use different polythiophene derivatives to differentiate VOCs by optical sensing. Differentiation can be performed based on the presence or not of response to an analyte and the sensitivity value of the sensors for the analytes. Another important feature is the lack of the effect of humidity on the response of most films, which could be a major drawback in the application of these sensors. (C) 2011 Elsevier B.V. All rights reserved.

Oxidation of lysergic acid diethylamide (LSD) by peroxidases: a new metabolic pathway

Lysergic acid diethylamide (LSD) is a potent hallucinogen that is primarily metabolized to 2-oxo-3-hydroxy-LSD (O-H-LSD) and N-desmethyl-LSD (nor-LSD) by cytochrome P450 complex liver enzymes. Due to its extensive metabolism, there still is an interest in the identification of new metabolites and new routes of its metabolism in humans. In the present study, we investigated whether LSD could be a substrate for horseradish peroxidase or myeloperoxidase (MPO). Using liquid chromatography coupled to UV detection and electrospray ionization mass spectrometry (LC-UV-ESI-MS), we found that both peroxidases were capable of metabolizing LSD to the same compounds that have been observed in vivo (i.e., O-H-LSD and nor-LSD). In addition, we found another major metabolite, N,N-diethyl-7-formamido-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide (FOMBK), which is an opened indolic ring compound. Hydrolysis of FOMBK led to the deformylated compound 7-amino-N,N-diethyl-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide. The reactions of LSD with the peroxidases were chemiluminescent and sensitive to inhibition by reactive oxygen scavengers, which indicated that the classic peroxidase cycle is involved in this new alternative metabolic pathway. Considering that MPO is abundant in immune cells and also present in the central nervous system, the degradation pathway described in this study suggests a possible route of LSD metabolism that may occur concurrently with the in vivo reaction catalyzed by the cytochrome P450 system.

Integration of processes induced air flotation and photo-Fenton for treatment of residual waters contaminated with xylene

Produced water in oil fields is one of the main sources of wastewater generated in the industry. It contains several organic compounds, such as benzene, toluene, ethyl benzene and xylene (BTEX), whose disposal is regulated by law. The aim of this study is to investigate a treatment of produced water integrating two processes, i.e., induced air flotation (IAF) and photo-Fenton. The experiments were conducted in a column flotation and annular lamp reactor for flotation and photodegradation steps, respectively. The first order kinetic constant of IAF for the wastewater studied was determined to be 0.1765 min(-1) for the surfactant EO 7. Degradation efficiencies of organic loading were assessed using factorial planning. Statistical data analysis shows that H2O2 concentration is a determining factor in process efficiency. Degradations above 90% were reached in all cases after 90 min of reaction, attaining 100% mineralization in the optimized concentrations of Fenton reagents. Process integration was adequate with 100% organic load removal in 20 min. The results of the integration of the IAF with the photo-Fenton allowed to meet the effluent limits established by Brazilian legislation for disposal. (C) 2011 Elsevier B.V. All rights reserved.

Tributyltin in crustacean tissues: analytical performance and validation of method

The hermit crab Clibanarius vittatus is a typical organism from intertidal regions being considered as a good bioindicator of tributyltin presence at these environments. Thus this study presents the analytical performance and validation method for TBT quantification in tissues of C. vittatus by gas chromatography with pulsed flame photometric detector (GC-PFPD) after extraction with an apolar solvent (toluene) and Grignard derivatization. The limits of detection of the method (LOD) were 2.0 and 2.8 ng g-1 for TBT and DBT (dibutyltin), respectively, and its limits of quantification (LOQ) were 6.6 and 8.9 ng g-1 for TBT and DBT, respectively. The method was applied to samples from Santos Estuary, São Paulo State, Brazil. TBT and DBT concentrations ranged from 26.7 to 175.0 ng g-1 and from 46.2 to 156.0 ng g-1, respectively. These concentrations are worrisome since toxic effects (such as endocrine disruption) have been reported for other organisms even under lower levels of registred at this study.

Lipase-catalyzed kinetic resolution of (±)-mandelonitrile under conventional condition and microwave irradiation

The kinetic resolution of (±)-mandelonitrile was carried out using lipase from Candida antarctica under conventional condition (orbital shaker) and microwave irradiation in toluene, producing the (S)-mandelonitrile acetate with high selectivity (up to > 98% ee, enantiomeric excess). The unreacted (R)-mandelonitrile under microwave irradiation and conventional condition was partially converted into benzaldehyde by spontaneous chemical equilibrium. The (S)-mandelonitrile acetate under microwave irradiation was produced with 92% ee and 35% yield for 8 h of reaction. Conventional transesterification of (±)-mandelonitrile in an orbital shaker produced unreacted (R)-mandelonitrile (51% ee) and (S)-mandelonitrile acetate (98% ee) in accordance with Kazlauskas rule for 184 h of reaction.

Heme oxygenase-1 activity is involved in the control of Toxoplasma gondii infection in the lung of BALB/c and C57BL/6 and in the small intestine of C57BL/6 mice

Heme oxygenase-1 (HO-1) is an enzyme that catabolizes free heme, which induces an intense inflammatory response. The expression of HO-1 is induced by different stimuli, triggering an anti-inflammatory response during biological stress. It was previously verified that HO-1 is able to induce indoleamine 2,3-dioxygenase (IDO), an enzyme that is induced by IFN-γ in Toxoplasma gondii infection. To verify the role of HO-1 during in vivo T. gondii infection, BALB/c and C57BL/6 mice were infected with the ME49 strain and treated with zinc protoporphyrin IX (ZnPPIX) or hemin, which inhibit or induce HO-1 activity, respectively. The results show that T. gondii infection induced high levels of HO-1 expression in the lung of BALB/c and C57BL6 mice. The animals treated with ZnPPIX presented higher parasitism in the lungs of both lineages of mice, whereas hemin treatment decreased the parasite replication in this organ and in the small intestine of infected C57BL/6 mice. Furthermore, C57BL/6 mice infected with T. gondii and treated with hemin showed higher levels of IDO expression in the lungs and small intestine than uninfected mice. In conclusion, our data suggest that HO-1 activity is involved in the control of T. gondii in the lungs of both mouse lineages, whereas the hemin, a HO-1 inducer, seems to be involved in the control of parasitism in the small intestine of C57BL/6 mice.

Theoretical studies of mononuclear non-heme iron active sites

The quantum chemical investigations presented in this thesis use hybrid density functional theory to shed light on the catalytic mechanisms of mononuclear non-heme iron oxygenases, accommodating a ferrous ion in their active sites. More specifically, the dioxygen activation process and the subsequent oxidative reactions in the following enzymes were studied: tetrahydrobiopterin-dependent hydroxylases, naphthalene 1,2-dioxygenase and α-ketoglutarate-dependent enzymes. In light of many experimental efforts devoted to the functional mimics of non-heme iron oxygenases, the reactivity of functional analogues was also examined. The computed energetics and the available experimental data served to assess the feasibility of the reaction mechanisms investigated. Dioxygen activation in tetrahydrobiopterin- and α-ketoglutarate-dependent enzymes were found to involve a high-valent iron-oxo species, which was then capable of substrate hydroxylation. In the case of naphthalene 1,2-dioxygenase, the reactivity of an iron(III)-hydroxperoxo species toward the substrate was investigated and compared to the biomimetic counterpart.