A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Morphological changes of the intestinal mucosa of broilers and layers as affected by fasting before sample collection

The present study aimed at evaluating the histo-morphological changes resulting from different fasting periods before the collection of tissue samples in different segments of the small intestine (duodenum, jejunum and ileum) of 7-d-old male chicks of a broiler and a layer strain. A completely randomized experimental design in in a 2x7 factorial arrangement, being two strains with different growth rates (Ross 308 and HyLine® W36) and seven fasting periods (0, 2, 4, 6, 8, 10 and 12 hours ), with six replicates, totaling 84 birds. The comparison of the morphometrics of the duodenum, jejunum and ileum of broiler and layer chicks demonstrated faster digestive tract development in broilers relative to layers. The fasting period caused morphological changes in the liver and small and large intestines in both strains. Therefore, it must be highlighted that in studies involving organ weights and intestinal morphometrics, birds must not be submitted to fasting before tissue collection.

Beryllium sampling methods: Comparison of two personal sample collection methods with the AEC sample collection method as used for one year in a beryllium production facility. /

Item 499-F-2

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Rapid LC-MS/MS quantification of the major benzodiazepines and their metabolites on dried blood spots using a simple and cost-effective sample pretreatment.

BACKGROUND: Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC-MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. RESULTS: The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected compounds. CONCLUSION: The presented strategy proved to be efficient for the rapid analysis of the selected drugs. Indeed, the offline sample preparation was reduced to a minimum, using a small amount of organic solvent and consumables, without affecting the accuracy of the method. Thus, this approach enables simple and rapid DBS analysis, even when using a non-DBS-dedicated autosampler, while lowering the costs and environmental impact.

Planetary Penetrators for Sample Return Missions

Thesis (Master's)--University of Washington, 2016-06

Dna Extraction From Filter-paper Spots Of Vaginal Samples Collected After Sexual Violence.

To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human β-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. β-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

[health Protection For Rural Workers: The Need To Standardize Techniques For Quantifying Dermal Exposure To Pesticides].

Quantification of dermal exposure to pesticides in rural workers, used in risk assessment, can be performed with different techniques such as patches or whole body evaluation. However, the wide variety of methods can jeopardize the process by producing disparate results, depending on the principles in sample collection. A critical review was thus performed on the main techniques for quantifying dermal exposure, calling attention to this issue and the need to establish a single methodology for quantification of dermal exposure in rural workers. Such harmonization of different techniques should help achieve safer and healthier working conditions. Techniques that can provide reliable exposure data are an essential first step towards avoiding harm to workers' health.

Concentrações plasmáticas de testosterona, triiodotironina (T3) e tiroxina (T4) em bodes submetidos ao estresse calórico

Para verificar o efeito do estresse calórico (EC) nas concentrações plasmáticas de testosterona, triiodotironina (T3) e tiroxina (T4), oito bodes, das raças Saanen (n=4) e Alpina (n=4), foram mantidos em câmara bioclimática, sob condições de termoneutralidade (13,0ºC a 26,7ºC) durante 30 dias e, após um período (60 dias) de descanso, submetidos ao EC (23,7ºC a 34,0ºC) por 30 dias. Para minimizar as variações sazonais nos perfis hormonais devido ao fotoperíodo, durante toda fase experimental, incluindo a de adaptação em condições de termoneutralidade (30 dias), o fotoperíodo foi controlado utilizando-se alternância de dias longos (16h de luz e 8h de escuro) e de dias curtos (8h de luz e 16h de escuro) a cada 30 dias. As amostras de sangue foram coletadas duas vezes por semana durante cinco semanas. No conjunto das raças, o EC não influenciou (P>0,05) as concentrações de testosterona (1,8±0,2 vs 1,3±0,2ng/ml) e nem a de T4 (52,7±2,8 vs 50,0±2,8ng/ml). Houve declínio (P<0,01) das concentrações de T3 nos animais submetidos ao experimento (1,3±0,1 vs 1,0±0,1ng/ml), mas a redução foi observada somente nos bodes Saanen. Em ambas as raças, as concentrações de T3 e T4 variaram (P<0,01) conforme o dia da coleta das amostras de sangue. O EC foi suficiente para produzir uma resposta fisiológica com redução das concentrações plasmáticas de T3 em bodes das raças Saanen, mas não da raça Alpina, assim como não foi capaz de alterar os níveis plasmáticos de testosterona e nem de T4.

Biologia reprodutiva de Heros efasciatus Heckel, 1840 (Pisces, Cichlidae) na Reserva de Desenvolvimento Sustentável Amanã-AM, visando seu manejo sustentável

Com o principal objetivo de fornecer ferramentas para auxiliar na implementação do manejo sustentável de peixes ornamentais na Reserva de Desenvolvimento Sustentável Amanã, Amazonas, foi realizado o estudo da biologia reprodutiva de Heros efasciatus Heckel, 1840, um ciclídeo com potencial ornamental e com poucos trabalhos sobre a sua biologia e ecologia, apesar de já ser comercializado em algumas regiões amazônicas. Coletas bimestrais foram realizadas de fevereiro de 2006 a janeiro de 2007 em dez igarapés contribuintes do Lago Amanã e Urini, sendo utilizados três aparelhos de pesca (rede de arrasto, rapiché e armadilha tipo matapi) e ainda galhadas artificiais nas amostragens realizadas próximas aos lagos. Foram capturados 140 exemplares de H. efasciatus, sendo 50 fêmeas, 42 machos, e 46 indivíduos cujo sexo não foi identificado devido ao pequeno tamanho. O tipo de crescimento encontrado foi isométrico, sendo que o maior indivíduo observado apresentava 174 mm e o menor 14 mm. Os resultados encontrados auxiliarão na adoção de medidas de manejo, como a determinação de tamanhos mínimos de captura, superiores aos tamanhos médios de maturação (97 mm para as fêmeas) e o estabelecimento de períodos de defeso durante a época de sua reprodução (outubro a janeiro). A pequena abundância de indivíduos da espécie, quando comparada com o total de exemplares capturados (apenas 0,07%) e a baixa fecundidade média, de 2502 ovócitos, indica que se deve trabalhar anualmente apenas com um pequeno número de indivíduos, a fim de garantir a continuidade do estoque.

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence

Background: The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum. Results: Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, interpopulation differentiation, and the degree to which allele frequencies are correlated between populations. Conclusions: The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.

Study of soils by thermal analysis

The study of soils is very important in the geological and geological engineering researches. A study of ten samples of soils was carried out by thermal analysis, and X-Ray Fluorescence Spectrometry to understand soil evolution in Angra dos Reis region, Rio de Janeiro State, Brazil. The sample collection sites were chosen based on geological characteristics, the soil layer thickness, the soil composition pattern, and whether or not it was moved either by erosion or by gravitational shifts. Because of the humid tropical climatic condition, natural soils tend to show great thickness of weathered mantles with formation of saprolites and saprolite soils. Kaolinite is an important secondary mineral which can be formed from many different minerals, like k-mica and k-feldspar and can be weathered to gibbsite. The results from TG/DTG and DTA indicated which soils had more weathering, and the same results were obtained by XRF, when silica/aluminum ratios from samples are compared with thermal analysis results.

Methylenedioxymethamphetamine (Ecstasy) Decreases Neutrophil Activity and Alters Leukocyte Distribution in Bone Marrow, Spleen and Blood

Objective: Looking for possible neuroimmune relationships, we analyzed the effects of methylenedioxymethamphetamine (MDMA) administration on neuroendocrine, neutrophil activity and leukocyte distribution in mice. Methods: Five experiments were performed. In the first, mice were treated with MDMA (10 mg/kg) 30, 60 min and 24 h prior to blood sample collection for neutrophil activity analysis. In the second experiment, the blood of nave mice was collected and incubated with MDMA for neutrophil activity in vitro analysis. In the third and fourth experiments, mice were injected with MDMA (10 mg/kg) and 60 min later, blood and brain were collected to analyze corticosterone serum levels and hypothalamic noradrenaline (NA) levels and turnover. In the last experiment, mice were injected with MDMA 10 mg/kg and 60 min later, blood, bone marrow and spleen were collected for leukocyte distribution analysis. Results: Results showed an increase in hypothalamic NA turnover and corticosterone serum levels 60 min after MDMA (10 mg/kg) administration, a decrease in peripheral blood neutrophil oxidative burst and a decrease in the percentage and intensity of neutrophil phagocytosis. It was further found that MDMA (10 mg/kg) treatment also altered leukocyte distribution in blood, bone marrow and spleen. In addition, no effects were observed for MDMA after in vitro exposure both in neutrophil oxidative burst and phagocytosis. Conclusion: The effects of MDMA administration (10 mg/kg) on neutrophil activity and leukocyte distribution might have been induced indirectly through noradrenergic neurons and/or hypothalamic-pituitary-adrenal axis activations. Copyright (C) 2009 S. Karger AG, Basel

A PCR-based method of screening individuals of all ages, from neonates to the elderly, for familial hyperaldosteronism type I

Aim: Unless specifically treated (glucocorticoids in low doses), Familial Hyperaldosteronism Type I(FH-I) may result in early death from stroke. We report the successful application of a rapid, polymerase chain reaction (PCR)-based method of detecting the 'hybrid' 11 beta-hydroxylase (11 beta-OHase)/aldosterone synthase (AS) gene as a screening test for FH-I. Methods: 'Long-PCR' was used to amplify, concurrently, a 4 kb fragment of AS gene (both primers AS-specific) and a 4 kb fragment of the hybrid gene (5' primer 11 beta-OHase-specific, 3'primer AS-specific) from DNA extracted from blood either collected locally or transported from elsewhere. Sample collection and transport were straightforward. This 4 kb fragment contains all the currently recognised hybrid gene 'crossover' points. Results: Within a single family, long-PCR identified all 21 individuals known to have FH-I. Hypertension was corrected in all 11 treated with glucocorticoids. Nine with normal blood pressure are being closely followed for development of hypertension. Long-PCR cord blood analysis excluded FH-I in three neonates born to affected individuals. Long-PCR newly identified two other affected families: (1) a female (60 years) with a personal and family history of stroke and her normotensive daughter (40 years), and (2) a female (51 years) previously treated for primary aldosteronism with amiloride, her two hypertensive sons (14 and 16 years) and her hypertensive mother (78 years). No false negative or false positive results have yet been encountered. At least seven other centres have successfully performed this test. Conclusion: Long-PCR is a reliable method of screening individuals of all ages for FH-I.