Design, construction and evaluation of a simple pressurized solvent extraction system

This work describes the construction and testing of a simple pressurized solvent extraction (PSE) system. A mixture of acetone:water (80:20), 80 ºC and 103.5 bar, was used to extract two herbicides (Diuron and Bromacil) from a sample of polluted soil, followed by identification and quantification by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD). The system was also used to extract soybean oil (70 ºC and 69 bar) using pentane. The extracted oil was weighed and characterized through the fatty acid methyl ester analysis (myristic (< 0.3%), palmitic (16.3%), stearic (2.8%), oleic (24.5%), linoleic (46.3%), linolenic (9.6%), araquidic (0.3%), gadoleic (< 0.3%), and behenic (0.3%) acids) using high-resolution gas chromatography with flame ionization detection (HRGC-FID). PSE results were compared with those obtained using classical procedures: Soxhlet extraction for the soybean oil and solid-liquid extraction followed by solid-phase extraction (SLE-SPE) for the herbicides. The results showed: 21.25 ± 0.36% (m/m) of oil in the soybeans using the PSE system and 21.55 ± 0.65% (m/m) using the soxhlet extraction system; extraction efficiency (recovery) of herbicides Diuron and Bromacil of 88.7 ± 4.5% and 106.6 ± 8.1%, respectively, using the PSE system, and 96.8 ± 1.0% and 94.2 ± 3.9%, respectively, with the SLP-SPE system; limit of detection (LOD) and limit of quantification (LOQ) for Diuron of 0.012 mg kg-1 and 0.040 mg kg-1, respectively; LOD and LOQ for Bromacil of 0.025 mg kg-1 and 0.083 mg kg-1, respectively. The linearity used ranged from 0.04 to 1.50 mg L-1 for Diuron and from 0.08 to 1.50 mg L-1 for Bromacil. In conclusion, using the PSE system, due to high pressure and temperature, it is possible to make efficient, fast extractions with reduced solvent consumption in an inert atmosphere, which prevents sample and analyte decomposition.

Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.

Eletroforese capilar acoplada à espectrometria de massas (CE-MS): vinte anos de desenvolvimento

CE-MS has been increasingly used for analysis of a vast array of compounds. This article reviews the different electrophoretic modes, interfaces and mass analyzers that are commonly used in the CE-MS coupling, as well as the technique advantages and performance characteristics. A large compilation of CE-MS applications is also presented. Therefore, this review is both a guide for beginners and a collection of key references for people who are familiar to the technique. Furthermore, this is the first CE-MS review published in a Brazilian journal and marks the installation of the first two commercial CE-MS units in Sao Paulo State.

Métodos para fabricação de microeletrodos visando a detecção em microambientes

Analysis at microenvironments, like single cells or in minute volumes (nL), is an area of great interest for analytical and biological sciences. Measurements at these experimental conditions demand analytical tools (microelectrodes) capable of monitoring with rapid response, good resolution and minimal perturbation of the system. The major drawbacks in producing these microscopic electrodes have been largely overcome, principally due to the development of new fabrication methods. In this review, these procedures are described with emphasis to those devoted to the construction of microelectrodes for application in microenvironments. Examples of our efforts to use these devices as effective electrochemical sensors are also addressed.

Redescription of two neotropical species of Toxophora Meigen (Diptera, Bombyliidae, Toxophorinae)

Two neotropical species of Toxophora Meigen, 1848 are redescribed (T. aurea Macquart, 1848 and T. leucon Séguy, 1930) and the male terminalia, female spermathecae, and the eggs are described and illustrated. Both species can be easily segregated from the other congeners by the following features: T. leucon: body covered with dark brown scales, longitudinal stripe formed by yellow scales on center of mesonotum, scutellum and abdomen, and abdomen slender; T. aurea: antenna with short dark brown scales, body covered with yellow scales and spots of dark brown scales with greenish reflex, wings without inter-radial vein, femora with yellow scales and without setae on males, and abdomen stout.

Imagem por ressonância magnética na investigação da cabeça de cães

A imagem por ressonância magnética (IRM) é o método de diagnóstico por imagem não invasivo mais sensível para avaliar as partes moles, particularmente o encéfalo, porém trata-se de uma técnica onerosa. O método fundamenta-se no fenômeno da ressonância magnética nuclear que ocorre quando núcleos atômicos com propriedades magnéticas presentes no corpo são submetidos a um campo magnético intenso, sendo posteriormente excitados por energia de radiofrequência e gerando, por sua vez, um sinal de onda de radiofrequência capaz de ser captado por uma antena receptora, passando por um processo matemático, chamado Transformada de Fourier, para posterior formação da imagem. Esse estudo objetivou realizar 10 exames completos da cabeça em cadáveres de cães normais à IRM e confeccionar um Atlas com as estruturas identificadas. As imagens foram adquiridas em um aparelho de ressonância magnética Gyroscan S15/HP Philips com campo magnético de 1,5Tesla. Os cadáveres foram posicionados com a cabeça no interior de uma bobina de cabeça humana e foram submetidos a cortes iniciais sagitais a partir de onde se planejou os cortes transversais e dorsais nas sequências de pulso spin-eco T1, T2 e DP. Em T1 utilizou-se TR=400ms e TE=30ms, T2 utilizou-se TR=2000ms e TE=80ms e na DP utilizou-se TR=2000ms e TE=30ms. A espessura do corte foi de 4mm, o número de médias foi igual a 2, a matriz foi de 256x256, o fator foi igual a 1,0 e o campo de visão foi de 14cm. A duração do exame completo da cabeça foi de 74,5minutos. As imagens obtidas com as sequências utilizadas e com a bobina de cabeça humana foram de boa qualidade. Em T1 a gordura tornou-se hiperintensa e o líquido hipointenso. Em T2 a gordura ficou menos hiperintensa e o líquido hiperintenso. A cortical óssea e o ar foram hipointensos em todas as sequências utilizadas devido a baixa densidade de prótons. A sequência DP mostrou o melhor contraste entre a substância branca e cinzenta quando comparada a T2 e a T1. T2 evidenciou o líquido cefalorraquidiano tornando possível a distinção dos sulcos e giros cerebrais. Através do exame de IRM foi possível, pelo contraste, identificar as estruturas ósseas componentes da arquitetura da região, músculos, grandes vasos venosos e arteriais e estruturas do sistema nervoso central, além de elementos do sistema digestório, respiratório e estruturas dos olhos entre outras. Nesse estudo as IRM adquiridas nas sequências T1, DP e T2 foram complementares para o estudo dos aspectos anatômicos da cabeça de cães demonstrando-os com riqueza de detalhes. O tempo requerido para o exame completo da cabeça é compátivel para uso em animais vivos desde que devidamente anestesiados e controlados. Os resultados obtidos por esse trabalho abrem caminho em nosso meio, para o estudo de animais vivos e para o início da investigação de doenças, principalmente as de origem neurológica, visto ser esta técnica excelente para a visibilização do encéfalo.

Imagem por ressonância magnética: princípios básicos

A ressonância magnética é a propriedade física exibida por núcleos de determinados elementos que, quando submetidos a um campo magnético forte e excitados por ondas de rádio em determinada freqüência (Freqüência de Larmor), emitem rádio sinal, o qual pode ser captado por uma antena e transformado em imagem. A imagem por ressonância magnética (IRM) é o método de diagnóstico por imagem não-invasivo mais sensível para avaliar partes moles, particularmente o encéfalo, porém trata-se de uma técnica onerosa. Ela apresenta grande potencial diagnóstico, poucos efeitos deletérios e muitos benefícios a serem obtidos com o seu uso. Além disso, a IRM fornece informações anatômicas acuradas, imagens em qualquer plano do corpo, bom contraste e resolução espacial e por si só pode sugerir um diagnóstico. Porém, não permite um diagnóstico histológico específico e deve ser interpretada em contexto com outros achados clínicos e patológicos. Esta revisão teve como objetivos mostrar as bases físicas da ressonância magnética e propiciar mais conhecimento aos veterinários.

Formulação de rações para leitões com base nos nutrientes digestíveis da silagem de grãos úmidos de milho

O estudo foi realizado com o objetivo de avaliar o resultado econômico e o desempenho de leitões na fase de creche alimentados com rações formuladas com base nos nutrientes digestíveis da silagem de grãos úmidos de milho. Foram utilizados 18 leitões mestiços (Landrace × Large White) com peso médio inicial de 16,01 kg no experimento de digestibilidade e 60 leitões mestiços com peso médio inicial de 7,11 kg no experimento de desempenho. O delineamento experimental nos dois experimentos foi em blocos ao acaso. No experimento de digestibilidade, avaliou-se uma ração-referência contendo ou não 30% de milho seco moído (30%) e silagem de grãos úmidos de milho e, no experimento de desempenho, avaliaram-se quatro rações: à base de grãos de milho seco moídos; à base de silagem de grãos úmidos de milho (SGUM) em substituição a 100% do milho seco, com base na matéria seca; à base de SGUM, considerando o valor de energia digestível da silagem determinado no experimento de digestibilidade; e à base de SGUM, considerando os valores de energia digestível, proteína digestível e fósforo disponível da silagem, determinados no experimento de digestibilidade. A digestibilidade aparente de matéria seca (MS), proteína bruta (PB), fósforo (P) e cálcio (Ca) e o nível de energia digestível foram maiores para a ração à base de SGUM formulada considerando os valores nutricionais determinados no experimento de digestibilidade. Durante o período total do experimento de desempenho (0 a 32 dias), a SGUM proporcionou melhor conversão alimentar e os menores custos/kg de peso ganho, mas não foram observadas diferenças no ganho diário de peso e no consumo diário de ração. Na formulação de rações com silagem de grãos úmidos de milho para suínos, deve-se considerar o valor nutricional, principalmente o teor de energia digestível, desse alimento.

Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae)

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Hematological profile of Chelonia mydas (Testudines, Cheloniidae) according to the severity of fibropapillomatosis or its absence

The green turtle Chelonia mydas feeds and nests in the Brazilian coastal area and is considered an endangered species by the World Conservation Union (IUCN 2009) and threatened by the Red List of Brazilian Fauna (Ministério do Meio Ambiente 2009). Fibropapillomatosis is a disease characterized by benign skin tumors (fibropapillomas), and it is one of the main threats to the survival of this species. Studies suggest the involvement of viruses as infectious agents associated with environmental and genetic factors. Blood samples were collected from 45 turtles captured in the coastal area of the state of Sao Paulo, Brazil. From these, 27 were affected by fibropapillomas and 18 were tumor free. Biometrical data on the turtles, size, location and quantity of tumors were recorded. The area occupied by fibropapillomas per animal was calculated and four groups were determined according to severity of the disease or its absence. The objective of the study was to compare hemogram results of the sea turtles classified in these four groups. The lowest hematocrit value was observed in severely affected animals. In the hemoglobin assay, the highest value was observed in the group of tumor free turtles and the lowest, in animals severely affected. Lymphocyte counts and curved carapace length were on the verge of statistical significance.

The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense

Background: Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks. Results: cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology. Conclusions: We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.

Free expanding cloud of cold atoms as an atomic standard: Ramsey fringes contrast

A compact frequency standard based on an expanding cold (133)CS cloud is under development in our laboratory. In a first experiment, Cs cold atoms were prepared by a magneto-optical trap in a vapor cell, and a microwave antenna was used to transmit the radiation for the clock transition. The signal obtained from fluorescence of the expanding cold atoms cloud is used to lock a microwave chain. In this way the overall system stability is evaluated. A theoretical model based on a two-level system interacting with the two microwave pulses enables interpretation for the observed features, especially the poor Ramsey fringes contrast. (C) 2008 Optical Society of America.

FILTERING TECHNIQUES APPLIED ON RAW CARRIER PHASE FOR GPS DETECTING SMALL DYNAMIC DISPLACEMENTS

This work is part of a research under construction since 2000, in which the main objective is to measure small dynamic displacements by using L1 GPS receivers. A very sensible way to detect millimetric periodic displacements is based on the Phase Residual Method (PRM). This method is based on the frequency domain analysis of the phase residuals resulted from the L1 double difference static data processing of two satellites in almost orthogonal elevation angle. In this article, it is proposed to obtain the phase residuals directly from the raw phase observable collected in a short baseline during a limited time span, in lieu of obtaining the residual data file from regular GPS processing programs which not always allow the choice of the aimed satellites. In order to improve the ability to detect millimetric oscillations, two filtering techniques are introduced. One is auto-correlation which reduces the phase noise with random time behavior. The other is the running mean to separate low frequency from the high frequency phase sources. Two trials have been carried out to verify the proposed method and filtering techniques. One simulates a 2.5 millimeter vertical antenna displacement and the second uses the GPS data collected during a bridge load test. The results have shown a good consistency to detect millimetric oscillations.

Chicken skeletal muscle-associated macroarray for gene discovery

Macro- and microarrays are well-established technologies to determine gene functions through repeated measurements of transcript abundance. We constructed a chicken skeletal muscle-associated array based on a muscle-specific EST database, which was used to generate a tissue expression dataset of similar to 4500 chicken genes across 5 adult tissues (skeletal muscle, heart, liver, brain, and skin). Only a small number of ESTs were sufficiently well characterized by BLAST searches to determine their probable cellular functions. Evidence of a particular tissue-characteristic expression can be considered an indication that the transcript is likely to be functionally significant. The skeletal muscle macroarray platform was first used to search for evidence of tissue-specific expression, focusing on the biological function of genes/transcripts, since gene expression profiles generated across tissues were found to be reliable and consistent. Hierarchical clustering analysis revealed consistent clustering among genes assigned to 'developmental growth', such as the ontology genes and germ layers. Accuracy of the expression data was supported by comparing information from known transcripts and tissue from which the transcript was derived with macroarray data. Hybridization assays resulted in consistent tissue expression profile, which will be useful to dissect tissue-regulatory networks and to predict functions of novel genes identified after extensive sequencing of the genomes of model organisms. Screening our skeletal-muscle platform using 5 chicken adult tissues allowed us identifying 43 'tissue-specific' transcripts, and 112 co-expressed uncharacterized transcripts with 62 putative motifs. This platform also represents an important tool for functional investigation of novel genes; to determine expression pattern according to developmental stages; to evaluate differences in muscular growth potential between chicken lines, and to identify tissue-specific genes.

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.