Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10 +/- 2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17 +/- 2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4 +/- 1% to 7 +/- 1% in the stable clone and from 10 +/- 2% to 16 +/- 1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. (C) 2014 Elsevier B.V. All rights reserved.Fundacao para a Ciencia e Tecnologia (FCT), Portugal [PTDC/QUI-BIQ/119677/2010, PTDC/CTM-NAN/2700/2012]; FCT [PEst-OE/EQB/LA0023/2011, SFRH/BD/48664/2008, SFRH/BPD/64932/2009]info:eu-repo/semantics/publishedVersio

Methodology evaluation of pin microrelief meter

The effects of natural weathering and different managements performed in agriculture may best be understood by studying the soil roughness. The aim of this study was to evaluate the optimization of the use of pin microrelief meter, an instrument used to determine the soil surface roughness, as the number of readings collected over traditional methodology proposed in the bibliography. The study was conducted in Rio Paranaiba (MG), in a Haplustox soil. The experimental design was completely randomized in a 2×3 factorial design with four replications. There were combined two types of primary tillage: conventional tillage with disc plow (PCAD) and harrow (PCGA), and three amounts of readings (100, 200, and 300 reading points) sampled in each experimental unit. Independently of the soil tillage, disc plow and harrow, the collection of 100 readings using a pin microrelief meter of a square meter, was sufficient to determine the surface roughness before and after soil preparation, without accuracy loss compared with the traditional method

Fontes de resistência de videira ao fungo oídio no Nordeste brasileiro.

Foram avaliadas 135 variedades distintas em três ciclos, observando-se as mesmas plantas sem nenhum oídicida

mTOR pathway in papillary thyroid carcinoma: Different contributions of mTORC1 and mTORC2 complexes for tumor behavior and SLC5A5 mRNA expression

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression.Acknowledgments: This study was supported by FCT (“Portuguese Foundation for Science and Technology”) through PhD grants to Catarina Tavares (SFRH/BD/87887/2012), Ana Pestana (SFRH/BD/110617/2015), and Rui Batista (SFRH/BD/111321/2015) and by a CNPq PhD grant (“National Counsel of Technological and Scientific Development”, Brazil), Science without Borders, Process n# 237322/2012-9 for Luciana Ferreira. Miguel Melo received a grant from Genzyme for the research project “Molecular biomarkers of prognosis and response to therapy in differentiated thyroid carcinomas”. Further funding was obtained from FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project "Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and by the project “Advancing cancer research: from basic acknowledgement to application”; NORTE-01-0145-FEDER-000029; “Projetos Estruturados de I&D&I, funded by Norte 2020-Programa Operacional Regional do Norte. This work was also financed by Sociedade Portuguesa de Endocrinologia Diabetes e Metabolismo through a grant “Prof. E. Limbert Sociedade Portuguesa de Endocrinologia Diabetes e Metabolismo/Sanofi-Genzyme in thyroid pathology”

Sobreposição do efeito dispersivo causado pela valência dos cátions no complexo de troca pela matéria orgânica de vinhaça em um Cambissolo Háplico degradado por queimadas.

A produção nacional de vinhaça é elevada e seu principal destino é o uso como fonte de nutrientes e matéria orgânica para solos cultivados. A queima de pastagens é uma prática comumente realizada na região de Nazareno ? MG. O objetivo do presente trabalho foi avaliar o efeito da vinhaça na dispersão de argila de um Cambissolo Háplico degradado por queimadas. Foi realizado uma amostragem composta por 40 pontos da camada de 0-20 cm do solo em questão. O solo coletado foi posto em vasos e doses equivalentes a 0; 18,8; 37,5; 56,3; 75,0; 112, e 150,0 m3 ha-1 foram adicionadas. A incubação durou 60 dias e cara tratamento foi composto por 3 repetições. A dispersão de argila se correlacionou negativamente (r2 < 0) com a matéria orgânica e com a proporção K+ / Ca2+ + Mg2+, com p-valor significativo (<0,05) para ambos os modelos. A expectativa de correlação positiva entre a proporção K+ / Ca2+ + Mg2+ indica que, mesmo com o aumento da presença de íons monovalentes (K+), a matéria orgânica foi capaz de aumentar a floculação das partículas primárias do solo. Conclui-se que a adição de vinhaça incrementa a floculação do solo e é potencialmente favorável à recuperação desses solos

Predictive Factors of Relapse After Methotrexate Discontinuation in Juvenile Idiopathic Arthritis Patients With Inactive Disease

Objective: To identify predictive factors of relapse after discontinuation of Methotrexate (MTX) in Juvenile Idiopathic Arthritis (JIA) patients with inactive disease. Methods: We conducted a prospective multicenter cohort study of patients diagnosed with JIA using real world data from the Portuguese national register database, Reuma.pt. Patients with JIA who have reached JADAS27 inactive disease and discontinued MTX before the age of 18 were evaluated. Results: A total of 1470 patients with JIA were registered in Reuma.pt. Of the 119 bionaive patients who discontinued MTX due to inactive disease, 32.8% have relapsed. Median time of persistence (using the Kaplan-Meier method and log-rank tests) with inactive disease was significantly higher in patients with more than two years of remission before MTX discontinuation and in those who did not use NSAIDs at time of MTX discontinuation. In Cox regression analyses and after adjustment for age at diagnosis, MTX tapering and JIA category, the use of NSAIDs at the time of MTX discontinuation (HR, 1.98 95%CI 1.03-3.82) and remission time of less than two years before suspension (HR, 3.12 95%CI 1.35-7.13) remained associated with relapse. No association was found between JIA category or the regimen of MTX discontinuation and the risk of relapse. Conclusions: In this large cohort we found that the use of NSAIDs at the time of MTX discontinuation was associated with a two times higher likelihood of relapse. In addition, longer duration of remission before MTX withdrawal reduces the chance of relapse in bionaive JIA patients.info:eu-repo/semantics/publishedVersio

Citotoxicity and acute oral toxicity tests of nanometer- and submicrometer-sized hollow spheres of chondroitin sulfate as a potential formulation strategy for anti-inflammatory encapsulation.

NanoAgri 2010. Editors: Caue Ribeiro, Odílio Benedito Garrido de Assis, Luiz Henrique Capparelli Mattoso, Sérgio Mascarenhas

Dopamine D2 receptor agonist, bromocriptine, remodels adipose tissue dopaminergic signalling and upregulates catabolic pathways, improving metabolic profile in type 2 diabetes

Funding: We thank to the at Laboratório de Biomedicina Mitocondrial e Teranóstica, Centro de Neurociências e Biologia Celular for the assistance with RNA integrity analysis. We thank the Serviço de Anatomia Patológica, Coimbra University Hospital, especially Ilda Simões, for the support with histological techniques. This work was supported by a grant from GIFT (Grupo de Investigação Fundamental e Translacional) from the Portugal Society of Diabetes and Portugal Foundation for Science and Technology (PEst UID/NEU/04539/2013 and UID/NEU/04539/2019: CNC.IBILI; PEst UIDB/04539/2020 and UIDP/04539/2020: CIBB). G.T and B.F.M. were supported by PhD Grants from the Portuguese Foundation for Science and Technology (PD/BD/127822/2016 and PD/BD/128336/2017, respectively). Bromocriptine was kindly provided by Generis Portugal.Background and objectives: The therapeutic effects of the dopamine D2 receptor (D2R) agonist, bromocriptine, in type 2 diabetes (T2D) have been attributed to central nervous system actions. However, peripheral dopamine directly modulates glucose uptake in insulin-sensitive tissues and lipid metabolism in adipose tissue (AT). We hypothesized that the dopaminergic system may be impaired in the adipose tissue of patients with T2D and that the therapeutic actions of bromocriptine could involve the modulation of metabolism in this tissue. Methods: The expression of dopamine receptors was evaluated in visceral AT samples from patients with obesity and stratified in several groups: insulin sensitive (IS); insulin resistance (IR) normoglycaemic; insulin resistant prediabetic; insulin resistant diabetic, according to Ox-HOMA2IR, fasting glycaemia and HbA1c levels. T2D Goto-Kakizaki rats (GK) were fed a high-caloric diet (HCD) for five months and treated with bromocriptine (10 mg/kg/day, i.p.) in the last month. The levels of dopaminergic system mediators and markers of insulin sensitivity and glucose and lipid metabolism were assessed in the peri-epididymal adipose tissue (pEWAT) and brown (BAT) adipose tissues, liver, and skeletal muscle. Results: Patients with IR presented a decreasing trend of DRD1 expression in the visceral adipose tissue, being correlated with the expression of UCP1, PPARA, and insulin receptor (INSR) independently of insulin resistance and body mass index. Although no differences were observed in DRD2, DRD4 expression was significantly decreased in patients with prediabetes and T2D. In HCD-fed diabetic rats, bromocriptine increased D1R and tyrosine hydroxylase (TH) levels in pEWAT and the liver. Besides reducing adiposity, bromocriptine restored GLUT4 and PPARγ levels in pEWAT, as well as postprandial InsR activation and postabsorptive activation of lipid oxidation pathways. A reduction of liver fat, GLUT2 levels and postprandial InsR and AMPK activation in the liver was observed. Increased insulin sensitivity and GLUT4 levels in BAT and an improvement of the overall metabolic status were observed. Conclusions: Bromocriptine treatment remodels adipose tissue and the liver dopaminergic system, with increased D1R and TH levels, resulting in higher insulin sensitivity and catabolic function. Such effects may be involved in bromocriptine therapeutic effects, given the impaired expression of dopamine receptors in the visceral adipose tissue of IR patients, as well as the correlation of D1R expression with InsR and metabolic mediators.publishersversionpublishe