Expressão da proteína recombinante Sm29 em Escherichia coli e purificação a partir dos corpúsculos de inclusão

Schistosomiasis is a neglected chronic parasitic tropical disease caused by the trematode worm Schistosoma spp., common in developing countries. Pharmacological treatments are available, but they are not effective due to the global epidemiological situation. A preventive vaccine could effectively contribute to reduce the infection. Among a number of antigens, the tegumental protein Sm29 from adult worm Shistosoma mansoni has been identified as a promising vaccine candidate. The gene Sm29 was cloned with 393 nucleotides, excluding a signal peptide and the transmembrane domain, to express the protein with 130 amino acids, approximately 18 kDa, with theoretical pI 6.5. Our objectives is to obtain the recombinant Sm29 protein expressed in Escherichia coli without histidine tag. Therefore, we must optimize the Sm29 obtaining processe, upstream and downstream, in order to design the industrial process, improving the yield and quality of the product to be used in clinical trials. Cells of E. coli BL21(DE3)_pET28_Sm29 were cultivated in bioreactor for Sm29 expression and they were separated by centrifugation. The cells were resuspended in buffer and lysed in French Press. The inclusion bodies (IB) were recovered by centrifugation and we studied the clarification of IB by washing with different solutions and using different centrifugation forces. The solubilization of IB was tested with 8 M Urea under different pH conditions. After the treatments, the protein profiles were analyzed by SDS-PAGE, to verify the presence of the expected band of ~18 kDa and the removal of contaminants. The washing solutions and the lower centrigugation speed helped to clarify the material, without causing losses. The pH of the solubilization solutions interfere with protein recovery, determining to work above pH 7.5, to seek the best balance between protein recovery and contaminant elimination. To meet regulatory requirements in the industrial process for vaccine production, we aim to replace purification by metal affinity chromatography (IMAC), allowed by the presence of the his-tag, added to the antigen as a purification strategy. Thus, the purification of rSm29 by IMAC was compared with studies of ion exchange chromatography, evaluating the binding of denatured or renatured proteins to the resins. It was found that the initial IB clarification parameters and solubilization conditions interfere in the chromatography and dialysis stages. It was determined that. to date. the best condition of binding is the renatured protein to the anion exchange resin, Q-Sepharose. As we identified an alternative purification method, we cloned the Sm29 gene without the his tag code to express the protein in BL21(DE3). Furthermore, the Sm29 gene was optimized for E. coli codon usage. The optimized gene was also cloned for expression in BL21(DE3), to evaluate whether it would modify the protein synthesis in terms of amount of expression and solubility. The first tests suggest that the amount of protein expression is similar to that obtained with the original gene. Tests of solubilization by pressurization are planned to analyze whether possible structural modifications in rSm29 may improves protein recovery.

A esquistossomose é uma doença tropical parasitária crônica negligenciada, causada pelo verme trematódeo Schistosoma spp., comum em países em desenvolvimento. Os tratamentos farmacológicos disponíveis não são eficazes devido à situação epidemiológica global. Uma vacina preventiva poderia contribuir eficazmente para a reduzir a infecção. Entre uma série de antígenos, a proteína tegumentar Sm29 do verme adulto Shistosoma mansoni foi identificada como uma promissora candidata a vacina. O gene Sm29 foi clonado com 393 nucleotídeos, excluindo um peptídeo sinal e o domínio transmembrana, para expressar a proteína com 130 aminoácidos, aproximadamente 18 KDa, com pI teórico 6,5. Nosso objetivo é obter a proteína Sm29 recombinante expressa em Escherichia coli sem cauda de histidina. Para tanto, devemos otimizar os processos de fermentação dos clones e os processos de purificação, para então, desenhar o processo industrial para melhorar o rendimento e a qualidade do produto a ser utilizado em ensaios clínicos. Células do clone E. coli BL21(DE3)_pET28_Sm29 foram cultivadas em biorreator para expressão de rSm29, separadas por centrifugação, ressuspensas em tampão e lisadas em “French Press”. Os corpos de inclusão (CI) foram recuperados por centrifugação e estudamos a etapa de clarificação dos CI por lavagens com diferentes soluções e forças de centrifugação. Testamos a solubilização dos CI com Ureia 8 M sob diferentes condições de pH. Após os tratamentos, analisamos os perfis proteicos por SDS-PAGE, verificando a banda esperada de ~18 kDa e a remoção de contaminantes. As soluções utilizadas na lavagem e a menor velocidade de centrifugação clarificam o material, sem causar perdas da proteína de interesse. O pH da solução de solubilização interfere na recuperação da proteína, determinando trabalhar com pH acima de 7,5 para buscar o melhor equilíbrio entre recuperação proteica e eliminação de contaminantes. Para atender aos requisitos regulatórios de processo industrial na produção de vacinas, temos o objetivo de substituir a purificação por cromatografia de afinidade a metal (IMAC) para poder remover a cauda de histidina, adicionada ao antígeno como estratégia de purificação. Assim, comparamos a purificação da rSm29 por IMAC com estudos de purificação por trocas iônicas, avaliando a ligação da proteína desnaturada ou renaturada com as resinas. Verificamos que os parâmetros iniciais de clarificação da CI e as condições de solubilização interferem nas etapas de cromatografia e diálise. Determinamos que a melhor condição, até o momento, é a ligação da proteína renaturada à resina de troca aniônica, Q-Sepharose. Identificado um método alternativo de purificação, clonamos o gene Sm29 sem a cauda de histidina e expressamos a proteína em BL21(DE3). Além disso, o gene Sm29 foi otimizado para uso de códons de E. coli. Um gene otimizado foi clonado para expressão em BL21(DE3), para avaliarmos se modificaria a estrutura da proteína durante a síntese da cadeia primária, em termos de quantidade de expressão e solubilidade. Os primeiros testes sugerem que a expressão é semelhante à obtida com o gene original. Testes de solubilização por pressurização estão planejados para analisar se a possível modificação estrutural na rSm29 melhoraria a recuperação do antígeno.

Challenges and opportunities for the production of lactic acid bacteria inoculants aimed for ensiling processes.

The use of bacterial inoculants for ensiling based on lactic acid bacteria (LAB) to obtain conserved forages has become an alternative for the improvement of milk and meat productivity in cattle, specifically by optimizing the nutritional and microbial quality of animal feed. LAB inoculant production involves microbial and technological aspects such as biomass obtention, the use of cocultures, the inclusion of probiotics, the production of antimicrobial peptides, operational methods used in bioreactors, and the formulation of the end product to be commercialized to farmers. This review explores the technical aspects of the manufacture of bacterial inoculants, from the main features desired in LAB for ensiling purposes to the alternatives of the bioprocess involved.

Sustentabilidad asociada al traspaso generacional en un tambo / Sustainability associated with generational transfer in dairy farm

Resumen El enfoque de un sistema productivo tanto en la dinámica económica-productiva, socio-cultural y ambiental de la sustentabilidad brinda una mirada integral de las problemáticas estableciendo las limitantes y potencialidades con las que cuenta el sistema, las cuales tienen un impacto directo sobre la productividad. La continuidad de un emprendimiento a lo largo de las generaciones presenta dificultades desde el punto de vista empresarial y sistémico. Los desafíos humanos y técnicos traen aparejados conflictos de difícil resolución que en muchas ocasiones terminan con la disolución de la empresa o una fuerte modificación en su dirección para mejorar los resultados en los pilares mencionados.

Abstract The focus of a production system on the economic, productive, socio-cultural and environmental dynamics of sustaina-bility offers a comprehensive perspective of common issues, establishing the limitations and potentialities of said system, which will impact productivity directly. Continuity of an enterprise over generations proves difficult from an entrepreneurial and systemic point of view. Human and technical challenges bring about complex conflicts which end in the dissolution of the enterprise in many instances or a considerable shift in direction in order to improve the results of the mentioned pillars.

Poro de Tabasco cheese: Chemical composition and microbiological quality during its artisanal manufacturing process.

Poro de Tabasco cheese (PTC) is one of the most popular cheeses in southern Mexico. It has been made by traditional, nonstandardized artisanal techniques dating back more than 50 yr. These techniques result in the cheese having a heterogeneous chemical and microbiological composition and, consequently, distinct organoleptic characteristics. Scientific interest in artisanal cheese is growing because it represents a source of bacteria with potential health benefits. However, the quality of raw-milk cheeses often does not comply with official sanitary standards. The objective of the present study was to explore the chemical composition and microbiological quality of PTC and to describe its production process. Based on chemical composition, this cheese can be classified as a hard, full-fat, fresh cheese, with moisture on a fat-free basis and fat in dry matter ranging from 41 to 55% and from 49 to 57%, respectively. The chemical and microbiological composition of PTC varied among the evaluated dairies due to the lack of standardization in the production process. Microbial populations decreased during production, which may be associated with high acidity and high salt and low moisture contents, the presence of lactic acid bacteria or antimicrobial substances, and the drainage of whey. However, despite the absence of Escherichia coli, Salmonella spp., and Listeria monocytogenes in final cheeses for all dairies, Staphylococcus aureus and its toxins were found in some samples from one dairy. Therefore, heat treatment for milk and good manufacturing practices should be implemented throughout the entire production process to ensure a safe product.

Trabalho, saúde e formação política na enquete operária de marx / Work, health, and political training in marx's worker's inquiry / Trabajo, salud y formación política en la encuesta obrera de marx

Resumo Em 1880, Marx redigiu e publicou a Enquete operária para investigar as condições de vida e saúde da classe trabalhadora francesa. Além da investigação em si, tratava-se de politizar e fortalecer as organizações de luta dos trabalhadores franceses. A Enquete é, portanto, um mecanismo de investigação e politização da classe trabalhadora. O objetivo deste ensaio foi analisar a atualidade da Enquete de 1880. Quatro aspectos dela foram discutidos: desenvolvimento e utilização, acidentes de trabalho, jornadas e intensidade do trabalho, salários e condições de vida. A consistência da Enquete se deve à teoria marxista que a sustenta. Afirma-se, neste ensaio, que o questionário de Marx e a forma enquete operária - entendida como mecanismo de investigação e politização - permanecem válidos.

Abstract In 1880, Marx wrote and published A Worker's Inquiry to investigate the living and health conditions of the French working class. In addition to the research itself, it had to do with politicizing and strengthening the French workers' struggle organizations. A Worker's Inquiry is, therefore, an investigation and politicization mechanism of the working class. The purpose of this assay was to analyze how current the 1880 A Worker's Inquiry is today. Four aspects of it were discussed: Development and use, labor accidents, working hours and intensity of the work, wages, and living conditions. The consistency of A Worker's is due to the Marxist theory that supports it. This essay states that Marx' questionnaire and the form of "worker's inquiry" - understood as a mechanism for research and politicization - remain valid.

Resumen En 1880, Marx redactó y publicó la Encuesta obrera para investigar sobre las condiciones de vida y salud de la clase trabajadora francesa. Además de la investigación en sí, se trataba de politizar y fortalecer las organizaciones de lucha de los trabajadores franceses. La Encuesta es, por lo tanto, un mecanismo de investigación y politización de la clase trabajadora. El objetivo de este ensayo fue analizar la actualizada de la Encuesta de 1880. Se discutieron cuatro aspectos de la misma: desarrollo y utilización, accidentes de trabajo, jornadas e intensidad del trabajo, salarios y condiciones de vida. La consistencia de la Encuesta se debe a la teoría marxista que la sostiene. En este ensayo, se afirma que el cuestionario de Marx y la forma encuesta obrera - entendida como mecanismo de investigación y politización - continúan siendo válidos.

Artisanal Sonoran cheese (Cocido cheese): an exploration of its production process, chemical composition and microbiological quality.

BACKGROUND: The objective of this study was to explore and document the production process of artisanal Cocido cheese and to determine its chemical composition and microbiological quality, considering samples from six dairies and four retailers. RESULTS: Cocido cheese is a semi-hard (506-555 g kg-1 of moisture), medium fat (178.3-219.1 g kg-1 ), pasta filata-type cheese made from raw whole cow's milk. The production process is not standardized and therefore the chemical and microbiological components of the sampled cheeses varied. Indicator microorganisms significantly decreased (P < 0.05) during the processing of Cocido cheese. Salmonella spp. were not found during the production process, and both Listeria monocytogenes and staphylococcal enterotoxin were absent in the final cheeses. CONCLUSION: This study provides more information on one of the most popular artisanal cheeses with high cultural value and economic impact in northwestern Mexico. In view of the foregoing, good manufacturing practices need to be implemented for the manufacture of Cocido cheese. Also, it is of utmost importance to make sure that the heat treatment applied for cooking the curd ensures a phosphatase-negative test, otherwise it would be necessary to pasteurize milk. Nevertheless, since Cocido cheese is a non-ripened, high-moisture product, it is a highly perishable product that could present a health risk if not properly handled. © 2017 Society of Chemical Industry.

Modifying Yeast Tolerance to Inhibitory Conditions of Ethanol Production Processes.

Saccharomyces cerevisiae strains having a broad range of substrate utilization, rapid substrate consumption, and conversion to ethanol, as well as good tolerance to inhibitory conditions are ideal for cost-competitive ethanol production from lignocellulose. A major drawback to directly design S. cerevisiae tolerance to inhibitory conditions of lignocellulosic ethanol production processes is the lack of knowledge about basic aspects of its cellular signaling network in response to stress. Here, we highlight the inhibitory conditions found in ethanol production processes, the targeted cellular functions, the key contributions of integrated -omics analysis to reveal cellular stress responses according to these inhibitors, and current status on design-based engineering of tolerant and efficient S. cerevisiae strains for ethanol production from lignocellulose.

Avaliação do processo produtivo de polpas de frutas congeladas / Evaluation of the produtive quality for processing the frozen fruit pulps

No estado de Roraima, os fabricantes de polpas de frutas empregam processos artesanais de produção, os quais não asseguram a qualidade do produto; apesar disso, esse segmento da agroindústria tem se expandido principalmente entre as microempresas. O procedimento de produção de polpas de frutas congeladas empregado em Boa Vista/RR foi avaliado por meio de visitas técnicas e aplicação de check list em quatro fabricantes selecionados que produzem polpa de fruta in natura. Constatou-se que o processo produtivo das indústrias locais é insatisfatório, sendo necessária a sua adequação às exigências da legislação em vigor.

In Roraima State, the local frozen fruit pulps producers employ artesanal procedures which do not ensure the quality of the final products; even so, this agroindustry segment has been increasing and predominalty among the small companies. The quality of frozen fruit pulps production in Boa Vista/RR was assessed by a technical visit and applying a check list at four selected companies proccessing in natura fruit pulps. Inadequate frozen fruit pulps productive processing at local industries was evidenced, thus being innoncompliace with the legislation in force.

Evaluation of the productive quality for processing the frozen fruit pulps / Avaliação do processo produtivo de polpas de frutas congeladas

In Roraima State, the local frozen fruit pulps producers employ artesanal procedures which do not ensurethe quality of the final products; even so, this agroindustry segment has been increasing and predominaltyamong the small companies. The quality of frozen fruit pulps production in Boa Vista/RR was assessedby a technical visit and applying a check list at four selected companies proccessing in natura fruit pulps.Inadequate frozen fruit pulps productive processing at local industries was evidenced, thus being innoncompliace with the legislation in force.

No estado de Roraima, os fabricantes de polpas de frutas empregam processos artesanais de produção, osquais não asseguram a qualidade do produto; apesar disso, esse segmento da agroindústria tem se expandidoprincipalmente entre as microempresas. O procedimento de produção de polpas de frutas congeladasempregado em Boa Vista/RR foi avaliado por meio de visitas técnicas e aplicação de check list em quatrofabricantes selecionados que produzem polpa de fruta in natura. Constatou-se que o processo produtivodas indústrias locais é insatisfatório, sendo necessária a sua adequação às exigências da legislação em vigor.

Estudio de la integridad mental en el trabajo: propuesta teórica, metodológia y técnica / Study of the mental integrity in the technical methodological and theorical proposal

Se presenta una propuesta teórica, metodológica y técnica para el estudio de la integridad mental de los trabajadores. La discusión de las categorías centrales que dan cuenta de manera específica de este objeto de estudio, resalta la relevancia del Proceso de Producción y la necesidad de precisar conceptualmente la "Salud-Enfermedad" mental. En esta discusión inicial se propone que los conceptos de trastorno mental vs. integridad mental definen con mayor precisión los procesos a que hacen referencia. Se enfatiza la importancia del estudio de la alienación objetiva y subjetiva como una consecuencia inmediata del proceso de producción, la cual explica el surgimiento de los trastornos mentales asociados al proceso de trabajo desde un punto de vista psicosocial. Se pone énfasis también en ubicar en su justa dimensión el estudio de la personalidad de los trabajadores, dado que en este trabajo es entendida como categoría intermedia y no como inmediata de los trastornos mentales. Se resalta la importancia del estudio de los diversos recursos individuales y grupales (apoyos sociales) con los que cuentan los trabajadores para afrontar los distintos requerimientos del medio. A nivel metodológico se plantea como fundamental la participación de los trabajadores como sujetos activos en el proceso de investigación y de generación de un conocimiento novedoso. Se recuperan los principios de la Investigación Participante y se propone la incorporación de la teoria de la representación social del trabajo como una herramienta para explorar la subjetividad obrera(AU)

A theoretical, methodological and technical proposal is made here for the study of the mental integrity of workers. The relevance of the production process is stressed and also the need for a precise conceptual definition of mental "health" or "sickness". In this initial discussion, it is suggested that the concepts "mental disorder" and "mental integrity" describe more exactly the states referred to. The importance of the study of objective and subjective alienation as a direct consequence of the production process is emphasized, and the appearance of mental disorders associated with the work process is thus explained from a psycho-social point of view. The study of the personality of the workers is considered here as an intermediate category rather than a direct cause of mental disorder. The proposal also stresses the importance of studying the individual and social support available to workers for coping with the environmental requirements made for them. Active participation of the workers in the research process is considered fundamental for the extension of knowledge. For this purpose the concept of participatory research and the theory of the social representation of work are used as tools to explore worker subjectivity(AU)