Locking the GFP Fluorophore to Enhance Its Emission Intensity

Funding Information: Thanks are due to the University of Aveiro, FCT/MEC, Centro 2020 and Portugal2020, the COMPETE program, and the European Union (FEDER program) via the financial support to the LAQV-REQUIMTE (UIDB/50006/2020 and UIDP/50006/2020), to the CICECO-Aveiro Institute of Materials (UID/CTM/50011/2019, UIDB/50011/2020 and UIDP/50011/2020), financed by national funds through the FCT/MCTES, to the Portuguese NMR Network. SG is supported by national funds (OE), through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. JRMF. Thanks FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for her PhD grant (UI/BD/151272/2021). Publisher Copyright: © 2022 by the authors.The Green Fluorescent Protein (GFP) and its analogues have been widely used as fluorescent biomarkers in cell biology. Yet, the chromophore responsible for the fluorescence of the GFP is not emissive when isolated in solution, outside the protein environment. The most accepted explanation is that the quenching of the fluorescence results from the rotation of the aryl–alkene bond and from the Z/E isomerization. Over the years, many efforts have been performed to block these torsional rotations, mimicking the environment inside the protein β-barrel, to restore the emission intensity. Molecule rigidification through chemical modifications or complexation, or through crystallization, is one of the strategies used. This review presents an overview of the strategies developed to achieve highly emissive GFP chromophore by hindering the torsional rotations.publishersversionpublishe

A new tetra-primer ARMS-PCR for genotyping bovine kappa-casein polymorphisms

[EN] Kappa-casein (κ-casein) is one of the most abundant milk proteins. Its main function is to avoid the aggregation of casein micelles, keeping them, and therefore calcium phosphate, in pockets in solution. In bovines, a κ-casein functional polymorphism has been associated with fat, calcium, and protein milk contents and faster curd contraction in cheese production. Quicker curd contraction reduces the loss of milk solids, enhancing cheese yield. This polymorphism induces a double amino acid substitution (Thr136Ile and Ala148Asp). The polymorphism is normally detected by PCR-RFLP, which is a laborious method. An interesting methodological alternative is the tetra-primer amplification refractory mutation system PCR (tetra-primer ARMS-PCR). A tetra-primer ARMS-PCR for the detection of this κ-casein polymorphism has been described. However, specificity was not achieved, probably due to problems with primer design. We developed a new tetra-primer ARMS-PCR for the detection of the κ-casein polymorphism. This new method was validated in a double-blind test, by comparison with the results obtained for 50 Guzerá bulls formerly genotyped by PCR-RFLP. This new method achieved 100% sensitivity and specificity. We conclude that this method is a useful, cost-efficient alternative for the detection of functional κ-casein polymorphismsSIResearch supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Pesquisa (CNPq), Pró-Reitoria de Pesquisa de Universidade Federal de Minas gerais (PRPq/UFMG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). MRSC, PASF, MAM, and RSV have CNPq fellowships. IRC has a CAPES fellowship. We are grateful to Professor Vânia Maldini Pena and Ms. Ariane Figueiredo Menicucci from Colégio Brasileiro de Criadores do Guzerá, for providing biological sample

Avaliação do curso de Engenharia de das Ciências Agrárias - Opção Engenharia Rural

O presente relatório resulta do trabalho de avaliação ao curso de Engenharia das Ciências Agrárias opção Engenharia Rural levado a cabo pela equipa de Auto-Avaliação da Escola Superior Agrária. Esta equipa, designada pelo conselho científico com a finalidade de proceder a todas as solicitações de avaliação tanto dos cursos como da instituição, é basicamente composta por um coordenador e um elemento de cada unidade departamental. Conforme o curso que se está a avaliar juntam-se à equipa o respectivo coordenador de curso e colaboradores na compilação do relatório. Além destes elementos participaram também neste trabalho um representante dos alunos (designado pela associação de estudantes) e dois funcionários representantes do pessoal administrativo e do pessoal não docente. Em termos metodológicos optou-se pelo seguimento mais ou menos fiel do guião proposto pelo CNAVES, tendo-se recolhido informação de diversos modos: pesquisa documental e bases de dados nos serviços administrativos, inquéritos auto administrados a alunos, docentes e funcionários, inquérito postal aos diplomados e entrevistas directas (por telefone) às entidades empregadoras. Uma vez recolhida e tratada a informação procedeu-se à sua análise crítica tendo como referência os trabalhos de avaliação que até então decorreram relativamente na Escola

Effect of several calcium sources on cation leaching using soil columns

Soil columns under controlled conditions were used to determine the movement of calcium and other cations after the application of lime, calcium oxide, gypsum and a mixture of Ume and gypsum, compared with a control treatment. Two Oxisols with different textures were used: clayey and silty. Rigid polyvinyl chloride (PVC) columns (length, 45cm; diam, 5cm) were used, applying 1.8 1 of water to each divided into four applications. Exchangeable cations were determined in the drainage water in 4 periods and in 5 dephts of the soil columns at the end of the experiment. The results showed that losses of Ca2+, Mg2+, K+ and A1(3+), were higher in the treatments with gypsum and lime plus gypsum. Amendments h'ke lime and calcium oxide did not promote significant losses of cations. Large losses occured in the silty and clayey soils in the first and second drainage times respectively. Gypsum did not modify the pH of both soils, but reduced cation amounts mainly in the clayey soil. Lime and calcium oxide raised the pH only near the application layer.No estudo realizado em colunas de solo montadas em laboratório, procurou-se avaliar o movimento do cálcio, e de outros cátions, após aplicação de calcário agrícola, gesso, calcário calcinado e uma mistura de calcário agrícola e gesso, comparados com um tratamento testemunha, em dois latossolos vermelho escuros de texturas diferentes: média e argilosa. Utilizaram-se colunas de PVC, com 5cm de diâmetro e 45cm de altura, e aplicaram-se em cada coluna 1,8 litros de água, parcelados em quatro vezes. Determinaram-se os cátions trocáveis presentes na água percolada e, no final do experimento, em cinco profundidades de cada solo. Os resultados mostraram que nos tratamentos gesso e calcário mais gesso as quantidades de Ca2+, Mg2+, K+ e Al3+ na solução percolada foram maiores, enquanto que os tratamentos calcário agrícola e calcário calcinado não promoveram perdas significativas de cátions. As maiores perdas ocorreram na primeira percolação no solo de textura média e na segunda no solo de textura argilosa. O gesso não modificou o pH dos solos, mas reduziu teores de bases no solo argiloso, enquanto que os calcários corrigiram o solo apenas próximo à camada de incorporação

Is type of work associated with physical activity and sedentary behaviour in women with fibromyalgia A cross-sectional study from the al-Ándalus project

Objectives To analyse the association between the type of work (productive vs reproductive work) and the levels of physical activity and sedentary behaviour in women with fibromyalgia. Method This cross-sectional study involved 258 women with fibromyalgia from southern Spain. Of them, 55% performed reproductive work (unpaid, associated with caregiving and domestic roles) exclusively, while 45% had productive job (remunerated, that results in goods or services). Physical activity of light, moderate and vigorous intensity in the leisure time, at home, at work, and totally were measured through the leisure time physical activity instrument and with the physical activity at home and work instrument, respectively. Sedentary behaviour was measured by the Sedentary Behaviour Questionnaire. Results After adjusting for age, fat percentage, education level and marital status, the multivariate analysis of covariance model informed the existence of significant differences between type of work groups (p<0.001). Women with productive work engaged in more light physical activity at work (mean difference =448.52 min; 95 % CI 179.66 to 717.38; p=0.001), and total physical activity of light (809.72 min; 535.91 to 1085.53; p<0.001) and moderate (29

Estudos sobre a nutrição mineral do arroz: XXV. Exigências nutricionais da variedade Dourado Precoce

Rice plants, Dourado Precoce, were grown in nutrient solution in order to study its nutritional requirement as well as the accumulation of dry matter and macro and micronutrients (excet Mo) during the life cycle. Demand for mineral elements followed the following decreasing order: macronutrients -K, N, Ca, Mg and S; micronutrients - Fe, Mn, Cu, Zn and B. Dry matter yield reached a maximun 100 days after germination (DAG), whereas accumulation of elements showed the geghest values at largest (140 DAG).Em condições de solução nutritiva foi cultivado o arroz Dourado Precoce com a finalidade de se estudar a marcha de absorção de nutrientes e as exigências minerais. Verificou-se que as exigências minerais obedecem a seguinte ordem decrescente: macronutrientes -K, N, Ca, Mg e S; micronutrientes -Fe, Mn, Cu, Zn e B. A produção de matéria seca atingiu o pico aos 100 dias após a germinação (DAG). A acumulação de macro e micronutrientes na planta foi, entretanto, máxima no fim do ciclo (140 DAG)