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São Carlos, Brazil

Amaral C.D.B.,Federal University of Sao Carlos | Nobrega J.A.,Federal University of Sao Carlos | Nogueira A.R.A.,Embrapa Southeast Livestock
Microchemical Journal

In studies of speciation, the conservation of species in their original forms is a very challenging issue, and the reliability of the results deeply depends on it. Preliminary steps of sample preparation and storage temperature may influence the conservation of species and extraction efficiency. Plant sample (Brachiaria brizantha Stapf. cv. Marandu) grown in As-containing soil was harvested and divided into four portions. The first portion was frozen-dried, milled with liquid N2 and stored at room temperature; the second portion was lyophilized and stored at 4°C; and the remaining portions were kept at two storage temperatures (-18 and -80°C) without any prior procedure. Aliquots of samples stored under different conditions were extracted every 2months for a 12-month period. The total content of As was 2.30±0.27mg kg-1 of dried and ground sample and As species were determined by HPLC-ICP-MS. The extraction was more efficient in sample aliquots that were lyophilized and ground (87-90%) than those only stored under different temperatures (53-66%). Furthermore, only for lyophilized and ground samples dimethylarsenic acid (DMA) was extracted. Concerning the storage time under the two tested temperatures, no significant differences on species were found and they remained stable. Standard deviations for replicates of samples that were not frozen-dried and ground were higher, and this behavior can be explained by sample heterogeneity. For the As species here studied it can be concluded that lyophilization and cryogenic grinding strategies were the most suitable sample pretreatments for As speciation in plant tissue. © 2014 Elsevier B.V. Source

Amaral C.D.B.,Federal University of Sao Carlos | Amais R.S.,Federal University of Sao Carlos | Amais R.S.,University of Sao Paulo | Fialho L.L.,Federal University of Sao Carlos | And 5 more authors.
Analytical Methods

This work demonstrates the potential of inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) in trace element analysis. Aiming Hg determination in water, sample preservation requires chloride addition, which presents formation of polyatomic interfering ions as a drawback for the determination of other analytes (e.g. As and V). Thus, the effect of chloride on As, Cr, V and Hg determination and the feasibility of using ICP-MS/MS to circumvent the interference were evaluated. A standard reference material (NIST SRM 1643e) and recoveries from tap water spiked with As, Cr, Hg and V were used for accuracy assessment and for the evaluation of matrix effects. The reaction between Hg and oxygen gas is not favorable in the octopole reaction system (ORSsup3/sup). Therefore, Hg was accurately determined as a single atom ion (sup202/supHgsup+/sup) under the same analytical conditions adopted for As, Cr and V determination by monitoring sup75/supAssup16/supOsup+/sup, sup52/supCrsup16/supOsup+/sup and sup51/supVsup16/supOsup+/sup while the ORSsup3/sup was pressurized with oxygen gas. The oxygen reaction mode significantly improved the accuracy for As, Cr and V, especially at the lowest spiked level, which is strongly affected by matrix effects. The signal gain in m/z 75 for a solution containing 1 μg Lsup-1/sup As in 0.48 mol L sup-1/sup chloride medium was 1974% when employing single quadrupole mode. On the other hand, the signal increased only 7% when 0.75 mL minsup-1/sup Oinf2/inf was used in mass shift mode proving the efficiency of the MS/MS mode. Thus, ICP-MS/MS and ORSsup3/sup pressurized with Oinf2/inf gas effectively reduced the interference, and enhanced the accuracy and sensitivity for some specific isotopes in a complex medium. The limits of detection for V (sup51/supVsup16/supOsup+/sup), Cr (sup52/supCrsup16/supOsup+/sup), As (sup75/supAssup16/supOsup+/sup) and Hg (sup202/supHgsup+/sup) were estimated to be 2, 3, 1.6 and 38 ng Lsup-1/sup at the 99.7% confidence level, respectively. © 2015 The Royal Society of Chemistry. Source

de Alvarenga Mudadu M.,Embrapa Southeast Livestock | Carvalho V.,Research and Development Center | Leclercq S.Y.,Research and Development Center
Applied Biochemistry and Biotechnology

Reverse vaccinology strategies have already been applied to a variety of microorganisms and have contributed significantly to vaccine development. However, most of the studies focused on an individual organism or on proteins with signature sequence motifs commonly found in known secreted proteins from bacteria. In this work, we applied a reverse vaccinology strategy based on conservation, virulence, and nonclassically surface exposure criterions to identify potential antigens in two microorganisms with significant degree of genomic plasticity among isolates (Streptococcus pneumoniae and Leptospira spp.), which imposes a major limitation to the production of a multistrain component vaccine. PSORTb 3.0.2 was run to predict the subcellular localization of the proteins. OrthoMCL was run to identify groups of the most conserved proteins between strains. Virulence prediction was done for the most conserved proteins, and SecretomeP was run to predict the nonclassically secreted proteins among the potential virulence factors. Based on the above criteria, we identified 37 proteins conserved between 16 genomes of S. pneumoniae and 12 proteins conserved between 5 leptospiral genomes as potential vaccine candidates. © 2015, Springer Science+Business Media New York. Source

Castelani L.,Institute of Animal Science | Silva Santos A.F.,Institute of Animal Science | Miranda M.S.,Institute of Animal Science | Zafalon L.F.,Embrapa Southeast Livestock | And 2 more authors.
International Journal of Molecular Sciences

Staphylococcus aureus is among the main etiologic agents of bovine mastitis. A total of 83 isolates of S. aureus from mammary glands of primiparous heifers were collected in the prepartum, calving and during lactation. For lactating cows, a total of 27 isolates of S. aureus from mammary glands were collected during lactation. The samples were taken in two dairy farms located in Sao Paulo State, Brazil. The highest frequency of S. aureus isolation in heifers was at the end of lactation. Strains were typified through Pulsed-field gel electrophoresis (PFGE) and grouped according to patterns of restriction enzyme SmaI. PFGE generated seven clonal profiles that were grouped into three different lineages, with the LA lineage being predominant and identified in heifers, as well as in the cows from the two regions studied. It was concluded that the cows showed a significant source of dispersion of S. aureus. At the first lactation the heifers were infected by the same clonal profiles of S. aureus which were isolated from multiparous lactating cows. The heifers were infected during milking over the months of lactation. © 2013 by the authors; licensee MDPI, Basel, Switzerland. Source

Amaral C.D.B.,Federal University of Sao Carlos | Dionisio A.G.G.,Federal University of Sao Carlos | Santos M.C.,Sao Paulo State University | Donati G.L.,Federal University of Sao Carlos | And 2 more authors.
Journal of Analytical Atomic Spectrometry

Different conditions of extraction using water, a methanol-water mixture and nitric acid solutions were evaluated for speciation of As(iii), As(v), DMA and MMA in plant samples that previously received As(v) after being sown and emergence was investigated. Microwave-assisted extraction (MAE) using diluted nitric acid solutions was also performed for arsenic extraction from chicken feed samples. The separation and determination of arsenic species were performed using HPLC-ICP-MS. The interference standard method (IFS) using 83Kr+ as the IFS probe was employed to minimize spectral interferences caused by polyatomic species, such as 40Ar 35Cl+. The extraction procedures tested presented adequate extraction efficiencies (90%), and the four arsenic species evaluated were found in plant samples. Extractions with diluted nitric acid solution at 90 °C were the most efficient strategy, with quantitative recoveries for all four As species in plant tissues. On the other hand, the methanol-water mixture was the solvent with the lowest extraction efficiency (50-60%). For chicken feed samples, MAE at 100 °C for 30 min resulted in an extraction efficiency of 97% and only As(v) was found, without any species interconversion. The IFS method contributed to improving precision and limits of detection and quantification for all tested extraction procedures. Significant improvements on accuracy were obtained by applying the IFS method and recoveries between 77 and 94%, and 82 and 93% were obtained for plant extracts and chicken feed samples, respectively. This journal is © 2013 The Royal Society of Chemistry. Source

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