A METABOLIC-LIKE CYCLE FOR SYNTHETIC APPLICATIONS

Systems Biocatalysis is a new approach consisting of organizing enzymes in vitro to generate an artificial metabolism for synthetic purposes. The interconversion of functional groups is the main objective of biocatalysis, and systems organizing a series of enzymes to achieve a multi-step reaction have been reported. The assembly of essentially the same enzymes utilized in Nature to drive the transformation of carbohydrates towards useful synthetic intermediates [1] has been referred to as an artificial metabolism. SysBiocat aims at a similar goal addressing the generalization and organization of group of enzymes (a tool-box) able to perform a series of reactions of general synthetic utility where the feasibility is connected with the obtainment of enzymes of wide substrate specificity or in a rich array of variable common catalytic functions. [2] As a demonstration of this concept, we have recently assembled a biochemical like cycle (Asp-cycle) connecting among them an unsaturated carboxylate (fumaric acid), an alpha-amino acid (L-aspartic acid), a keto acid (oxalacetic acid) and the corresponding alpha-hydroxyacid (D- or L-malic acid). [3] In this view, the obtained cycle may be exploited by coupling it with synthetically relevant reactions which are driven to completion thanks to one or more irreversible steps in the reaction sequence. ____ [1] W.D. Fessner, C. Walter, “Artificial metabolism”, Angew Chem Int Ed, 1992, 31, p. 614 [2] U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K. Robins, “Engineering The Third Wave Of Biocatalysis”, Nature, 2012, 485, p. 185 [3] D. Tessaro, L. Pollegioni, L. Piubelli, P. D’Arrigo, S. Servi, “Systems Biocatalysis: An Artificial Metabolism for Interconversion of Functional Groups”, ACS Catalysis, 2015, 5, p. 160

Comparison between glucose threshold and critical velocity for aerobic capacity determination in men

The blood glucose threshold (GT) and critical velocity (CV) has been used for the assessment of the aerobic capacity for trained individuals in replace the blood lactate and ventilatory parameters for anaerobic threshold determination. But, there are few studies with physically active subjects. The purpose of this study was to measure, compare and correlate the running velocities associated with the GT and CV of a group of untrained men. Fifteen adult men (23±3.74 years old; 72±10.97 kg; 1.76±0.07 m; 21±5.36 % fat mass) performed the following tests: 1) 500m and 3km time trial (Vm500 and Vm3km); 2) Incremental test on treadmill for of GT identification. The CV was obtained from linear regression (distance x time on 500m and 3km test). Normality was verified through Shapiro-Wilk, GT and CV was compared through dependent t-test and correlation by product moment Pearson. A high correlation was verified for Vm3km and CV (r=0.99 and R2=0.99), Vm3000 and GT (r=0.91 and R2=0.82), and between CV and GT (r=0.89 and R2=0.79). Differences were observed between GT and CV (138.8±19.9 and 170.6±27.8 m.min–1, respectively) (P\u3c0.001). In conclusion, the study shows that CV can not be used for anaerobic threshold estimation, because this parameter overestimated the GT, despite the high correlation with GT and Vm3000

Métodos alternativos para estimar a velocidade da máxima fase estável de lactato em adultos jovens fisicamente ativos

The aim of this study was to compare the velocities found in the protocols used to measure the indirect individual anaerobic threshold (IATind), glucose threshold (GT) and critical velocity (CV) with the gold standard, the maximum lactate steady state (MLSS) protocol. Fourteen physically active young adults (23±3.1 years; 72±10.97 kg; 176±7 cm; 21±5.36% body fat) performed a 3000-m track running test to determine IATind using the prediction equation and an incremental test on a treadmill to determine GT. The CV was identified by linear regression of the distance-time relationship based on 3000-m and 500-m running performance. The MLSS was identified using two to five tests on different days to identify the intensity at which there was no increase in blood lactate concentration greater than 1 mmol/L between the 10th and 30th minute. A significant difference was observed between mean CV and MLSS (P≤0.05) and there was a high correlation between MLSS and IATind (R2=0.82; P≤0.01) and between MLSS and GT (R2=0.72; P≤0.01). The Bland-Altman method showed agreement between MLSS and IATind [mean difference -0.24 (confidence interval -1.72 to 1.24) km/h] and between MLSS and GT [0.21 (-1.26 to 1.29) km/h]. We conclude that the IATind and GT can predict MLSS velocity with good accuracy, thus making the identification of MLSS practical and efficient to prescribe adequate intensities of aerobic exercise.O objetivo do presente estudo foi comparar as velocidades encontradas nos protocolos de Limiar Anaeróbio Individual Indireto (LAIind), Limiar Glicêmico (LG) e Velocidade Crítica (VC) com o padrão ouro, o protocolo de identificação da máxima fase estável do lactato (MFEL). Participaram 14 adultos jovens fisicamente ativos (23±3,1 anos; 72±10,97 kg; 1,76±0,07 m; 21±5,36 % gordura corporal) que realizaram um teste de 3000m em pista para determinar o LAIind através de equação de predição; teste incremental em esteira ergométrica para determinação do LG; a VC foi identificada por regressão linear através da relação distância-tempo com base no desempenho em corridas nas distâncias de 3.000m e 500m; a MFEL foi identificada utilizando de dois a cinco testes em dias distintos até encontrar a intensidade onde não houve aumento da concentração de lactato sanguíneo maior que 1 mmol.L-1 entre os minutos 10 e 30. Houve diferença estatística entre os valores médios da VC e a MFEL (P≤0,05), elevada correlação entre MFEL e LAIind (R2=0,82; P≤0.01) e MFEL e LG (R2=0,72; P≤0.01). Através do método Bland-Altman foram encontradas as concordâncias entre MFEL e LAIind [diferença média -0,24 (intervalo de confiança -1,72 a 1,24) km/h] e MFEL e LG [0,21 (-1,26 a 1,29) km/h]. Concluímos que o LAIind e o LG são testes que podem predizer com boa precisão a velocidade da MFEL, tornando sua identificação prática e eficiente para prescrição de intensidades adequadas para o treinamento aeróbio.Universidade Federal de São Paulo (UNIFESP)Faculdade Anhanguera de BauruUniversidade Federal de São Paulo (UNIFESP) Departamento de Ciências do Movimento HumanoUNIFESP, Depto. de Ciências do Movimento HumanoSciEL

Optimizing HIV-1 protease production in Escherichia coli as fusion protein

<p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the <it>pol </it>gene is responsible for proteolytic processing of the <it>gag-pol </it>polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations.</p> <p>Results</p> <p>A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in <it>Escherichia coli </it>cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for <it>E. coli </it>expression and to avoid autoproteolysis) and by screening six different <it>E. coli </it>strains and five growth media. The best expression yields were achieved in <it>E. coli </it>BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1Pr per liter.</p> <p>Conclusions</p> <p>By using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.</p

A luta dos Guarani Kaiowá do Mato Grosso do Sul pelo território : memórias e imagens do (re)existir num permanente estado de exceção no Brasil (1964-2018)

Tese (doutorado)—Universidade de Brasília, Centro de Estudos Avançados Multidisciplinares, Programa de Pós-Graduação em Desenvolvimento, Sociedade e Cooperação Internacional, 2019.Procuro analisar o processo de luta pelo território dos Guarani Kaiowá do estado do Mato Grosso do Sul (no período compreendido entre a última ditadura civil-militar – 1964-1985 - até os dias atuais, 2018), a partir de relatos orais e de fontes documentais escritas e audiovisuais, buscando compreender a criação de normas, jurisdições, ações e práticas desenvolvidas, implementadas ou impostas pelo Estado brasileiro aos povos indígenas, com as quais se buscaria instituir relações de poder vividas dentro de um estado que se caracterizaria como de exceção permanente. Esse modus operandi do Estado, de caráter quase sempre vertical, traduzido em suas políticas sociais, evidencia noções de desenvolvimento social, econômico e cultural que tensionam os modos de viver indígenas, suas culturas, cosmologias e perspectivas de organização social e cultural.I try to analyze the process of fighting for the territory of the Guarani Kaiowá of the state of Mato Grosso do Sul (in the period between the last civil-military dictatorship - 1964-1985 - to the present day, 2018), based on oral reports and sources documentaries and audiovisuals, seeking to understand the creation of norms, jurisdictions, actions and practices developed, implemented or imposed by the Brazilian State on indigenous peoples, with which it would seek to establish relations of power lived within a state that would be characterized as a permanent exception . This modus operandi of the State, which is almost always vertical, translated into its social policies, demonstrates notions of social, economic and cultural development that stress indigenous ways of living, their cultures, cosmologies and perspectives of social and cultural organization

Memórias e imagens em torno do índio pataxó hãhãhãe Galdino Jesus dos Santos (1997 a 2012)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Humanas, Programa de Pós-Graduação em História, 2012.A presente dissertação tem como objetivo identificar e interpretar memórias e imagens em torno do índio da etnia pataxó hãhãhãe Galdino Jesus dos Santos, assassinado em Brasília, em abril de 1997. Sua morte provocou, na sociedade brasileira e especialmente em Brasília, um debate acerca das sociabilidades e das sensibilidades entre indivíduos numa determinada sociedade. Buscou-se identificar inicialmente, mediante pesquisa e análise de matérias jornalísticas e relatos de entrevistados, analisar e interpretar como a morte de Galdino, na capital federal, permitiu uma identificação entre passado e presente. No segundo momento, o objetivo foi identificar e analisar temas e questões tratadas nos jornais da época acerca do assassinato, bem como, perceber as relações existentes entre os entrevistados com o espaço da praça onde aquele evento ocorreu e o monumento ali erguido em lembrança e repúdio ao fato. E, ainda, identificar imagens de Brasília, sugeridas pelos jornais pesquisados, comparando-as com as construídas pelos narradores em seus relatos, na tentativa de captar possíveis imaginários construídos e desconstruídos a respeito da cidade a partir da comoção causada pelo assassinato de Galdino. Buscou-se, assim, compreender certas relações que se estabelecem entre direitos, cultura, história e memória. E, deste modo, que referências, sentimentos e valores foram - e continuam a ser - destacados no debate que se seguiu ao fato, como também o que foi e é lembrado - e o que foi esquecido -, ontem e nos dias atuais. _______________________________________________________________________________________ ABSTRACTThis thesis aims to identify and interpret memories and images around the Indian ethnic pataxó Hãhãhãe Galdino Jesus dos Santos, murdered in Brasilia in April 1997. His death resulted in Brazilian society and especially in Brasilia, a debate about the sociability and sensitivities between individuals in a given society. First, we sought through research and analysis of news stories and reports of respondents, analyze and interpret as the death of Galdino, in the federal capital, allowed an identification between past and present. In the second phase, the objective was to identify and analyze themes and issues addressed by newspapers about the murder and also understand the relationship between the respondents with the space where the square and the monument that was erected there in memory and repudiation of fact . And also identify images of Brasilia, suggested by the newspapers surveyed, comparing them with those built by the narrators in their accounts in an attempt to capture possible constructed and deconstructed imaginary about the city from the commotion caused by the murder of Galdino. We sought to thus understand certain relationships established between rights, culture, history and memory. And thus it references, values and feelings were - and remain - highlighted in the debate that followed the incident, as well as what was and is remembered - and what has been forgotten - yesterday and today

Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in <it>Escherichia coli </it>of type II cholesterol oxidase from <it>Brevibacterium sterolicum </it>(BCO).</p> <p>Results</p> <p>Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant <it>E. coli </it>cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies.</p> <p>Conclusions</p> <p>Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in <it>E. coli </it>or <it>Streptomyces </it>spp.), shows that the fully active type II BCO can be produced in <it>E. coli </it>at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.</p