Phos-tag analysis of Rab10 phosphorylation by LRRK2:a powerful assay for assessing kinase function and inhibitors

Autosomal dominant mutations that activate the leucine-rich repeat kinase-2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved Thr/Ser residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen derived B Cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2 phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase inactive LRRK2[D2017A] knock-in MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knock-in mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A, S935A] knock-in MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aide with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway

Efficacy and safety of cumaru syrup as complementary therapy in mild persistent asthma: a double-blind, randomized, placebo-controlled study

Amburana cearensis is a medicinal plant known as "cumaru". It is used in Northeast Brazil in the treatment of respiratory diseases. This was a randomized, double-blind, placebo-controlled study, with the aim of evaluating the efficacy and safety of cumaru syrup as complementary therapy in mild persistent asthma. The study consisted of 3 phases, pre-treatment, treatment and post-treatment. The primary efficacy outcome was comparison of the changes reported by patients of the cumaru and placebo groups after treatment, using the "Asthma Quality of Life Questionnaire" (AQLQ). The secondary outcome was the effect of cumaru syrup on lung function based on spirometry. The results showed that in the cumaru group, the proportion of patients who had global improvement in asthma symptoms was significantly greater (61.90%, P=0.0009) than in the placebo group (9.52%). Only the spirometric parameters Forced Vital Capacity (FVC) and Forced Expiratory Volume in 1 second (FEV1) showed significant intergroup differences in post-treatment (P0.05). Adverse events were reported by 3 patients (14.29%) in the cumaru group and 3 patients (14.29%) in the placebo group. All adverse events were considered non-serious and mild.Amburana cearensis é uma planta medicinal conhecida como "cumaru". No Nordeste do Brasil é usada no tratamento de doenças respiratórias. Este é um estudo randomizado, duplo-cego e controlado por placebo, com o objetivo de avaliar a eficácia e segurança do xarope de cumaru como terapia complementar da asma persistente leve. O estudo consistiu de três fases, pré-tratamento, tratamento e pós-tratamento. A variável primária para determinação da eficácia foi a comparação das mudanças referidas pelos pacientes dos grupos cumaru e placebo após o tratamento, usando o "Questionário sobre Qualidade de Vida na Asma" (QQVA). A variável secundária foi o efeito do xarope de cumaru na função pulmonar baseado na espirometria. Os resultados mostraram que no grupo cumaru, a proporção de pacientes com melhora global dos sintomas da asma foi significativamente maior (61,90%, P=0.0009) que no grupo placebo (9,52%). Somente os parâmetros espirométricos, capacidade vital forçada (CVF) e volume expiratório forçado no primeiro segundo (VEF1), mostraram diferença intergrupo significtivas no pós-tratamento (P0.05). Eventos adversos foram reportados por 3 pacientes (14,29%) no grupo cumaru e 3 (14,29%) no grupo placebo. Todos os eventos adversos foram não sérios e leves

Transfer of passive immunity and serum proteinogram in the first six months of life of Criollo Lageano and black and white holstein calves

The objective of this study was to evaluate and compare the transfer of passive immunity and the proteinogram in Criollo Lageano (CL) and Black and White Holstein (BWH) calves. Two groups were utilized with 13 Criollo Lageano and 10 BWH calves. Blood samples were collected for the measurement of total serum protein, electrophoresis of serum proteins, activity of the gamma glutamyl transferase, and concentration of IgG by the method of the zinc sulfate turbidity in periods between 24 and 36 hours of life, 15, 30, 60, 90, 120, 150 and 180 days. Statistical analysis was performed by ANOVA and Tukey test at 5% significance level, and correlations between variables were calculated. Variations of serum proteins followed a pattern of physiological behavior over the first six months of life and production of immunoglobulins was active earlier in BWH calves and slower in the Criollo Lageano, without causing any impact on their health. Gamma globulin in the first days of life (24-36h) was correlated with IgG (r=0.87 for CL and r=0.89 for BWH), PTS (r=0.91 for CL and r=0.92 for BWH), Glob (r=0.99 for CL and r=0.98 for BWH) and GGT (r=0.14 for CL and r=0.83 for BWH). It was concluded that there was no failure in the transfer of passive immunity in Criollo Lageano calves but this failure occurred in the BWH calves. IgG values estimated by the zinc sulfate turbidity and serum proteins were considered good indicators of the transfer of passive immunity in calves between 24 and 36 hours of life

Influence of scrotal bipartition on spermatogenesis yield and sertoli cell efficiency in sheep

Abstract With the objective to assess the effect of scrotal bipartition on spermatogenesis in sheep, the testes were used from 12 crossbred rams of sheep farms in the municipality of Patos, Paraíba, Brazil, distributed into two groups: GI with six rams with scrotal bipartition, and GII with six rams without scrotal bipartition. The testicular biometry was measured and the testes were collected, fixed in Bouin and fragments were processed to obtain histological slides. The spermatogenesis yield and the Sertoli cell efficiency was estimated by counting the cells of the spermatogenetic line at stage one of the seminiferous epithelium cycle and the Sertoli cells. The results were submitted to analysis of variance with the ASSISTAT v.7.6 program and the mean values were compared by the Student-Newman-Keuls test (SNK) at 5% significance. The testicular biometric parameters did not show statistical difference (p>0.05) between the groups. The meiotic, spermatogenetic and Sertoli cell efficiency were higher in bipartitioned rams (p0.05) between GI and GII. The results indicated that there is superiority in the spermatogenetic parameters of bi-partitioned rams, suggesting that these sheep present, as reported in goats, indication of better reproductive indices

Determination of metals in medicinal plants highly consumed in Brazil

In this work, samples of the medicinal plants: Boldo (Peumus boldus), Castanha da Índia (Aesculus hippocastanum), Chá Verde (Camelia sinensis), Erva Cidreira (Melissa officinalis), Espinheira Santa (Maytenus ilicifolia), Guaraná (Paullinia cupana), Maracujá (Passiflora sp.), Mulungu (Erythrina velutina), Sene (Cassia angustifolia) and Valeriana (Valeriana officinalis) were evaluated BY using the Neutron Activation Analysis technique (NAA- k0) in order to determine the levels of metals and other chemical contaminants. The results showed the presence of non essential elements to the human body. The diversity of chemical impurities found even at low concentration levels, considering the potential for chronic toxicity of these elements, reinforces the need to improve the implementation of good practices by growers and traders, and the hypothesis of lack of quality control in plant products

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas