Insect cell-based production processes intensified via high cell density perfusion and continuous culture

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Bioprocess engineering of insect cells for accelerating vaccines development

Insect cells emerged as a powerful and versatile platform for vaccines production, mostly using the lytic baculovirus expression vector system (BEVS). Stable expression in such hosts has been increasingly explored to circumvent BEVS-related drawbacks, but protein titers achieved to date are still seemingly low. The design of new or improved cell factories and bioprocess intensification strategies are therefore necessary to increase productivities and thus accelerate implementation of stable insect cell lines as a fast, cost-effective platform for vaccines manufacturing. In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect (Sf-9 and High Five) cell platforms for fast production of enveloped virus-like particles (VLPs). Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept demonstrated using two membrane proteins: the influenza HA protein (for vaccines) and the human beta-2 adrenergic receptor (for drug screening or antibody discovery). Aiming to improve production yields in developed stable cell lines, two bioprocess engineering schemes were evaluated (either individually or in combination): (i) adaptive laboratory evolution of insect cells to hypothermic culture conditions, and (ii) supplementation of insect cell cultures with productivity enhancers. The stable cell line expressing HIV Gag-VLPs was used as model. Under hypothermic culture conditions, adapted Sf-9 cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the latter alone leads to lower production yields. A more modest increase in productivity (up to 7-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. No synergistic effect was observed when combining adapted cells and supplementation with productivity enhancers. Production of HIV Gag-VLPs was successfully scaled-up to stirred-tank bioreactors. The adapted cell line was then pseudo-typed with influenza HA protein for production of Gag-HA VLPs, and their performance benchmarked against (i) parental Sf-9 cells stably expressing Gag-HA VLPs and (ii) insect cells-BEVS, both cultured under standard temperature conditions (27C). Adapted cells showed increased production of Gag-HA VLPs when compared to parental/stable cells, corroborating previously obtained data, but still lower when compared to insect cells-BEVS. Bioprocess intensification strategies are currently under in-house testing to further improve yields of adapted cells and thus shorten the gap between stable insect cells and IC-BEVS. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist and accelerate vaccines development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBB-BIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

a double health threat to Cape Verdeans

BACKGROUND: Cape Verde presents a high rate of cardiovascular diseases. Low potassium and high sodium intakes are related to cardiovascular diseases. However, studies regarding these two micronutrients continue to be rare in African urban settings. This work aims to estimate potassium and sodium intakes and to analyse the self-reported salt intake by gender and by type of urban area in the city of Praia - the capital of Cape Verde. METHODS: In the first stage (n = 1912), an intra-urban study was designed in two types of urban areas (formal and informal), using a sampling strategy based on random selection of geographical coordinates, in order to apply a questionnaire. In a second stage, a 24-h dietary recall and anthropometric measurements were performed by local nutritionists. Potassium and sodium intakes were estimated for 599 participants (149 men and 450 women). Non-parametric methods (including quantile regression) were used in the statistical analysis. RESULTS: In informal areas, a higher percentage of women reported having hypertension (31.0%) compared to formal areas (19.7%). Based on 24-h dietary recall, median potassium intake for men was 2924.2 mg/day and for women and 2562.6 mg/day. Almost 70.0% of men and 80.0% of women ingested less than the recommended 3510 mg/day of potassium. In informal areas, men and women presented high medians of sodium intakes compared to formal areas (men: 4131.2 vs 3014.6 mg/day and women: 3243.4 vs 2522.4 mg/day). On the other hand, the percentage of participants exceeding 2000 mg/day for sodium was high (≥70.8%), even for participants that self-reported low-salt intake. Quantile regression models revealed effects of the type of urban area and gender in the potassium and sodium intakes, at least, in some quartiles, accounting for age, academic qualifications, and professional situation. CONCLUSIONS: A low potassium intake and a high sodium intake were found in Praia. Thus, efficient health education campaigns and health promotion are needed and should be tailored considering gender and urban areas.publishersversionpublishe

Leveraging single-cell and bulk transcriptomics towards improved insect cell factories for biopharmaceuticals

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Looking in a Small-Scale in a City of Cape Verde

PMID: 26599004 WOS:000365853900049Background The lack of high-quality data to support evidence-based policies continues to be a concern in African cities, which present marked social, economic and cultural disparities that may differently impact the health of the groups living in different urban contexts. This study explores three urban units-formal, transition and informal-of the capital of Cape Verde, in terms of overweight/obesity, cardiometabolic risk, physical activity and other aspects related to the urban environment. Methods Quantitative and qualitative research methods were used in this intra-urban study. A proportional stratified random sample (n = 1912 adults), based on geographical coordinates of private households, was selected to apply the UPHI-STAT questionnaire. In a second stage (n = 599), local nutritionists collected anthropometric measurements (e.g., height, waist circumference) and body composition by bioelectric impedance (e.g., body weight, body fat, muscle mass). In a third stage, pedometers were used to count study participants' steps on working and non-working days for one week (n = 118). After a preliminary statistical analysis, a qualitative study was developed to complement the quantitative approach. Generalized linear models, among others, were used in the multivariate analysis. Results Insecurity was the main concern among survey respondents in the three units, notwithstanding with significant differences (p <0.001) among units. About three-quarters (76.6%) of the participants of the informal unit emphasised the need for more security. The formal unit presents an older age structure (61.3% above 40 years old) and the transition unit a younger age structure (only 30.5% above 40 years old). Some health-related variables were analysed in each unit, revealing an excess of chronic conditions reported by inhabitants of informal unit, compared with the formal unit despite the informal unit's younger age profile. The self-reported hypertension varied significantly among urban units (p <0.001), with 19.3% in the formal unit, 11.4% in the transition unit and 22.5% in the informal unit. Women of the urban units present significant differences (5% level) for body mass index calculated from self-reported measures (p <0.001), fat mass (p = 0.005), waist circumference (p = 0.046) and waist-to-height ratio (p = 0.017). For women, overall physical activity was 67.4% (95%CI [64.8,70.0]), with differences among urban units (p = 0.025). For men it was of 85.2% (95%CI [82.3,87.6]), without significant differences among urban units (p = 0.266). The percentage of women and men who reported physical activity in leisure time was discrepant, with 95%CI [22.6, 27.4] and [53.2, 60.2], respectively. The results of pedometers also indicated that men walk significantly more than women (p <0.001), with a difference of approximately 2000 steps/day. Conclusions The data collection process itself also gave us some clues on the involvement of local communities, exploring the potential of social capital of these settings and the role of the woman in family and society in Cape Verde. The higher participation of women and residents of informal unit (the most disadvantaged groups) suggests these as the priority target groups for health promotion campaigns. The link between health planning, urban planning and security of the city needs to be reinforced to minimize health, social and gender inequalities.publishersversionpublishe

Pathogenic activity of Isaria spp. for control of Dactylopius opuntiae (Hemiptera: Dactylopiideae) and the effects of selected insecticides

We analyzed the activity of the entomopathogenic fungi Isaria farinosa, I. fumosorosea and I. javanica against the cochineal Dactylopius opuntiae, as well as the effects of the insecticides clorpirifos, acetamiprid, thiamethoxam and lambda-cyhalothrin on the conidia germination, mycelium growth and a sporulation of the fungi. These fungi were not pathogenic to cochineal. This the first report of the efficiency of Isaria spp. against this the insect. The bioassays of the compatibility of the insecticides acetamiprid and thiametoxam with species of Isaria indicate the possibility of the joint use of these products in studies on the control of D. opuntiae.El trabajo analizó las acciones de los hongos entomopatógenos Isaria farinosa, I. fumosorosea y I. javanica contra la cochonilla Dactylopius opuntiae, así como el efecto de los insecticidas clorpirifos, acetamiprid, tiametoxam y lambda-cialotrina sobre la germinación, el crecimiento micelial y esporulación del mismo. Los hongos no fueron patógenos a D. opuntiae, siendo éste el primer reporte de la eficiencia de Isaria spp., sobre este insecto. Los bioensayos de la compatibilidad de los insecticidas acetamiprid y thiametoxam con especies de Isaria indican la posibilidad de uso conjunto de estos productos en estudios para el control de D. opuntiae

Instrumentos para avaliação da Atenção Primária à Saúde: revisão integrativa

A avaliação da Atenção Primária à Saúde (APS) busca evidências científicas sobre sua real efetividade. Traz consequências importantes, como a definição de políticas públicas. O objetivo foi analisar a produção científica resultante da aplicação de instrumentos de avaliação à área da Atenção Primária à Saúde. Esta revisão integrativa utiliza o protocolo PRISMA nas bases de dados LILACS, Scopus, PubMed, BIREME e Scielo. Um total de 2.785 manuscritos foram identificados, critérios de elegibilidade selecionados 46 e, no final, incluídos 20 manuscritos na revisão. O PCATool-Brasil foi o instrumento mais utilizado, seguido pelo Sistema de Monitoramento e Avaliação da Atenção Básica – QualiAB, entrevistas não diretivas, coleta e análise de dados secundários. Dada a complexidade da APS, recomenda-se que seus processos de avaliação considerem o contexto de participação de instituições de ensino, pesquisa, profissionais, gestores e usuários

Diagnóstico fitossanitário e de práticas associadas ao uso de agrotóxicos nas hortas em ambiente protegido em Boa Vista - Roraima

Objetivou-se com esse trabalho fazer o diagnóstico das pragas, doenças, plantas daninhas e das práticas associadas ao uso dos agrotóxicos utilizados na produção de hortaliças, em ambiente protegido, no município de Boa Vista, Roraima. O estudo descritivo foi realizado em área urbana e rural, nos meses de fevereiro a abril de 2008, nas hortas atendidas pelo Projeto Estufa, administrado pela Secretaria Municipal de Desenvolvimento Agrícola (SMDA) da Prefeitura. O instrumento de coleta de dados foi um questionário semi-estruturado com 35 questões, aplicado in loco sem aviso prévio a 75 horticultores (47% da população em estudo). Os dados foram analisados pela estatística descritiva. Após análise dos dados constatou-se que a moscabranca, pulgão, tripes e lagartas são os insetos-pragas que mais precisam ser controlados. Em relação às doenças, as principais foram a mela e a queima das folhas. Quanto às plantas daninhas, destacaram-se as espécies das famílias Poaceae, Cyperaceae, Portulaceae e Amaranthaceae. Quanto ao uso de agrotóxicos, os resultados mostraram que 68% dos horticultores utilizam inseticidas, 24% fungicidas e 8% herbicidas; 92% disseram já ter recebido algum tipo de orientação técnica e 41,3% buscam um técnico quando precisam adquirir um agrotóxico. Portanto, conclui-se que há alta incidência de problemas fitossanitários nas hortas, uso intensivo de agrotóxicos, grande diversidade de formulações de inseticidas, fungicidas e herbicidas, associados à adoção de práticas e atitudes de riscos pelos horticultores, que pode agravar os problemas de intoxicação direta e indireta, além de causar a contaminação ao ambiente

Prevalence and determinants of child undernutrition and stunting in semiarid region of Brazil

OBJETIVO : Analisar tendências na prevalência e determinantes da desnutrição em crianças na região semiárida do Brasil. MÉTODOS : Foram analisados dados de duas pesquisas transversais domiciliares de base populacional que utilizaram a mesma metodologia. A amostragem por conglomerados foi utilizada para coletar os dados de 8.000 famílias, do estado do Ceará, Nordeste do Brasil, para os anos de 1987 e 2007. A desnutrição aguda foi calculada como peso/idade < -2 desvios padrão; nanismo como altura/idade < -2 desvios padrão; e emaciação como peso/altura < -2 desvios padrão. Os dados sobre os determinantes biológicos e sociodemográficos foram analisados por meio de análises multivariadas com base em um modelo teórico hierarquizado. RESULTADOS : Amostras de 4.513 e 1.533 crianças menores de três anos de idade, em 1987 e 2007, respectivamente, foram incluídas nas análises. A prevalência de desnutrição aguda foi reduzida em 60,0%, passando de 12,6% em 1987, para 4,7% em 2007, enquanto a prevalência de nanismo foi reduzida em 50,0%, passando de 27,0% em 1987 para 13,0% em 2007. A prevalência de emaciação teve pouca alteração no período. Em 1987, as características socioeconômicas e biológicas (renda familiar, escolaridade da mãe, disponibilidade de latrina e água potável, consulta médica e hospitalização da criança, idade, sexo e peso ao nascer) foram fatores significativamente associados à desnutrição, ao nanismo e à emaciação. Em 2007, os determinantes da desnutrição ficaram restritos às características biológicas (idade, sexo e peso ao nascer). Apenas uma característica socioeconômica, a disponibilidade de latrina, permaneceu significantemente associada ao nanismo. CONCLUSÕES : O desenvolvimento socioeconômico, além de intervenções de saúde, parecem ter efetivamente contribuído para a melhoria do estado nutricional das crianças. Peso ao nascer, especialmente o peso extremamente baixo (< 1.500 g), aparece como o fator de risco mais importante para a desnutrição na primeira infância.OBJECTIVE : To analyze the evolution in the prevalence and determinants of malnutrition in children in the semiarid region of Brazil. METHODS : Data were collected from two cross-sectional population-based household surveys that used the same methodology. Clustering sampling was used to collect data from 8,000 families in Ceará, Northeastern Brazil, for the years 1987 and 2007. Acute undernutrition was calculated as weight/age < -2 standard deviation (SD); stunting as height/age < -2 SD; wasting as weight/height < -2 SD. Data on biological and sociodemographic determinants were analyzed using hierarchical multivariate analyses based on a theoretical model. RESULTS : A sample of 4,513 and 1,533 children under three years of age, in 1987 and 2007, respectively, were included in the analyses. The prevalence of acute malnutrition was reduced by 60.0%, from 12.6% in 1987 to 4.7% in 2007, while prevalence of stunting was reduced by 50.0%, from 27.0% in 1987 to 13.0% in 2007. Prevalence of wasting changed little in the period. In 1987, socioeconomic and biological characteristics (family income, mother’s education, toilet and tap water availability, children’s medical consultation and hospitalization, age, sex and birth weight) were significantly associated with undernutrition, stunting and wasting. In 2007, the determinants of malnutrition were restricted to biological characteristics (age, sex and birth weight). Only one socioeconomic characteristic, toilet availability, remained associated with stunting. CONCLUSIONS : Socioeconomic development, along with health interventions, may have contributed to improvements in children’s nutritional status. Birth weight, especially extremely low weight (< 1,500 g), appears as the most important risk factor for early childhood malnutrition.OBJETIVO : Objetivo: Analizar tendencias en la prevalencia y determinantes de la desnutrición en niños en la región semiárida de Brasil. MÉTODOS : Se analizaron datos de dos investigaciones transversales domiciliares de base poblacional que utilizaron la misma metodología. El muestreo por conglomerados se utilizó para colectar los datos de 8.000 familias, del estado de Ceará, Noreste de Brasil, para los años de 1987 y 2007. La desnutrición aguda fue calculada como peso/edad < -2 desviaciones estándar; enanismo como altura/edad < -2 desviaciones estándar; y demacración como peso/altura < -2 desviaciones estándar. Los datos sobre los determinantes biológicos y sociodemográficos se analizaron por medio de análisis multivariados con base en un modelo teórico jerarquizado. RESULTADOS : Muestras de 4.513 y 1.533 niños menores de tres años de edad, en 1987 y 2007, respectivamente, se incluyeron en los análisis. La prevalencia de desnutrición aguda fue reducida en 60,0%, pasando de 12,6% en 1987, a 4,7% en 2007, mientras que la prevalencia del enanismo fue reducida en 50,0% pasando de 27,0% en 1987 a 13,0% en 2007. La prevalencia de demacración tuvo poca alteración en el período. En 1987, las características socioeconómicas y biológicas (renta familiar, escolaridad de la madre, disponibilidad de letrina y agua potable, consulta médica y hospitalización del niño, edad, sexo y peso al nacer) fueron factores significativamente asociados con la desnutrición, el nanismo y la demacración. En 2007, los determinantes de la desnutrición quedaron restringidos a las características biológicas (edad, sexo y peso al nacer). Sólo una característica socioeconómica, la disponibilidad de letrina, permaneció significativamente asociada con el enanismo. CONCLUSIONES : El desarrollo socioeconómico, así como las intervenciones de salud, parecen haber contribuido efectivamente en la mejoría del estado nutricional de los niños. Peso al nacer, especialmente el peso extremadamente bajo

INFLUÊNCIA DO MANEJO QUÍMICO NO CAPIM AMARGOSO EM CULTIVO DE SOJA

A soja é uma cultura de elevado potencial produtivo, entretanto sua produção é limitada pelas plantas daninhas, devido a competição por fatores abióticos, além de produzir substancias alelopáticas capazes de interferir na qualidade e produtividade da cultura. Objetivou-se avaliar a eficiência do herbicida glyphosate, isolado ou em mistura com haloxifop-p-metílico, no controle do capim-amargoso (Digitaria insularis). O trabalho foi desenvolvido em área experimental com a cultura da soja e desenvolvimento espontâneo de plantas daninhas. Os tratamentos foram: dose recomendada de glyphosate (720 g ha-1 e.a.); dose recomendada de haloxyfop (60 g ha-1 i.a.); meia dose de glyphosate + meia dose de haloxyfop (360 g ha-1 e.a. + 30 g ha-1 i.a., respectivamente); dose recomendada de glyphosate + haloxyfop; meia dose de glyphosate + dose recomendada de haloxyfop; dose recomendada de glyphosate + meia dose de haloxyfop; testemunha sem a presença de plantas daninhas e com presença de daninhas. Foram avaliados os componentes produtivos da soja, fitotoxicidade do herbicida na cultura e na espécie invasora, e a massa vegetal da planta daninha. Os tratamentos com haloxyfop isolado ou em mistura com glyphosate promovem amarelecimento evoluindo para necrose do capim-amargoso, entretanto, em aplicação de glyphosate isoladamente, este apresentou pouco ou nenhum efeito sobre as plantas daninhas, indicando possível tolerância do biótipo do capim amargoso ao glyphosate. A produtividade da soja apresenta decréscimo de até 3.059 kg ha-1 (90,10%) quando comparado com áreas onde houve aplicação. A população de plantas de soja por metro linear é reduzida significativamente quando não há o controle químico das plantas daninhas.Palavras-chave: Digitaria insularis; fitointoxicação; herbicida. INFLUENCE OF CHEMICAL MANAGEMENT IN THE BITTER CAPIM IN SOYBEAN CULTIVATION ABSTRACT: Soybean is a crop with high productive potential, but its production is limited by weeds due to competition for abiotic factors, as well as producing allelopathic substances capable of interfering with the quality and productivity of the crop. The objective of this study was to evaluate the efficiency of the glyphosate herbicide, isolated or in combination with haloxyfop-p-methyl, in the control of bittergrass (Digitaria insularis). The work was developed in an experimental area with the soybean crop and spontaneous development of weeds. The treatments were: recommended dose of glyphosate (720 g ha-1 e.a.); recommended dose of haloxyfop (60 g ha-1 a.i.); half dose of glyphosate + half dose of haloxyfop (360 g ha-1 e.a. + 30 g ha-1 a.i., respectively); recommended dose of glyphosate + haloxyfop; half dose of glyphosate + recommended dose of haloxyfop; recommended dose of glyphosate + half dose of haloxyfop; control without the presence of weeds. The productive components of soybean, phytotoxicity of the herbicide in the crop and the invasive species, and the plant mass of the weed were evaluated. The treatments with haloxyfop alone or in combination with glyphosate promote yellowing evolving towards amargoso necrosis; however, in application of glyphosate alone, this showed little or no effect on the weeds, indicating a possible tolerance of the biotype of the bitter grass to the glyphosate. Soybean yield decreased by up 3,059 kg ha-1 (90.10%) when compared to areas where there was application. The population of soybean plants per linear meter is significantly reduced when there is no chemical control of weeds.Keywords: Digitaria insularis; phytointoxication; herbicide