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The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.
Assuntos
Baculoviridae , Proteínas Recombinantes , Baculoviridae/genética , Animais , Proteínas Recombinantes/genética , Vetores Genéticos/genética , Transfecção/métodos , Recombinação Homóloga , Células Sf9 , Linhagem Celular , Spodoptera/virologia , Insetos/genética , Insetos/virologiaRESUMO
Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).
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Bromovirus , Bromovirus/genética , Animais , Baculoviridae/genética , Vetores Genéticos/genética , Cromatografia por Troca Iônica/métodos , Vírion/isolamento & purificação , Vírion/genética , Vírion/metabolismoRESUMO
The interest in plant-derived virus-like particles (pVLPs) for the design of a new generation of nanocarriers is based on their lack of infection for humans, their immunostimulatory properties to fight cancer cells, and their capability to contain and release cargo molecules. Asparaginase (ASNase) is an FDA-approved drug to treat acute lymphoblastic leukemia (LLA); however, it exhibits high immunogenicity which often leads to discontinuation of treatment. In previous work, we encapsulated ASNase into bacteriophage P22-based VLPs through genetic-directed design to form the ASNase-P22 nanobioreactors. In this work, a commercial ASNase was encapsulated into brome mosaic virus-like particles (BMV-VLPs) to form stable ASNase-BMV nanobioreactors. According to our results, we observed that ASNase-BMV nanobioreactors had similar cytotoxicity against MOLT-4 and Reh cells as the commercial drug. In vivo assays showed a higher specific anti-ASNase IgG response in BALB/c mice immunized with ASNase encapsulated into BMV-VLPs compared with those immunized with free ASNase. Nevertheless, we also detected a high and specific IgG response against BMV capsids on both ASNase-filled capsids (ASNase-BMV) and empty BMV capsids. Despite the fact that our in vivo studies showed that the BMV-VLPs stimulate the immune response either empty or with cargo proteins, the specific cytotoxicity against leukemic cells allows us to propose ASNase-BMV as a potential novel formulation for LLA treatment where in vitro and in vivo evidence of functionality is provided.
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Resumen Introducción: La psicología positiva ha señalado la importancia del bienestar subjetivo de los adolescentes, por ser un precursor del desarrollo positivo de los jóvenes. La investigación resalta la importancia del apoyo social percibido y el ajuste escolar como determinantes del bienestar adolescente. Por ello, este artículo tiene como finalidad analizar las relaciones de la percepción de apoyo social (de familia, profesorado, amigos) con el bienestar subjetivo de los adolescentes, mediado por su ajuste escolar. Método: Participaron 1035 estudiantes peruanos de educación secundaria, con edades entre 12 y 16 años. Se probaron dos modelos teóricos con variables latentes, uno con mediación total y otro con mediación parcial. Resultados: Los resultados del modelo final mostraron una alta capacidad predictiva del apoyo social sobre el ajuste escolar y de este sobre el bienestar de los adolescentes. Además, se encontró un potente efecto del apoyo de la familia sobre el bienestar subjetivo de los adolescentes peruanos. Conclusión: Estos resultados contribuyen a resaltar que el bienestar subjetivo es un constructo central en el contexto de la psicología positiva y en el desarrollo positivo de los jóvenes, por lo que debería cultivarse más abundantemente en las escuelas.
Abstract Introduction: Positive psychology has highlighted the importance of adolescents' subjected well-being as a precursor of the positive youth development. Research in this context highlights the importance of perceived social support and school adjustment as determinants of adolescent well-being. Therefore, the objective of this work has been to analyze the relationships of the perception of social support (from family, teachers, and friends) and the adolescents' subjective well-being, mediated by their school adjustment. Method: Participants were 1035 Peruvian Secondary Education students, aged between 12 and 16 years old. Two theoretical models with latent variables have been estimated and tested, one with total mediation and the other with partial mediation. Results: The results of the final model, with partial mediation, have shown a high predictive capacity of social support on school adjustment and, in turn, on the adolescents' well-being. In addition, a powerful effect of family support on subjective well-being of Peruvian adolescents has also been found. Conclusion: These results contribute to highlighting that subjective well-being is a central construct in the context of Positive Psychology and in the positive development of young people, which is why it should be cultivated more abundantly in schools.
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Humanos , Masculino , Feminino , Adolescente , Apoio Social , Psicologia Positiva , Inclusão Escolar , Família , EmoçõesRESUMO
BACKGROUND: The aim of this study was to evaluate quinoa protein (Q), chitosan (CH) and sunflower oil (SO) as edible film material as well as the influence of this coating in extending the shelf-life of fresh blueberries stored at 4 °C and 75% relative humidity. These conditions were used to simulate the storage conditions in supermarkets and represent adverse conditions for testing the effects of the coating. The mechanical, barrier, and structural properties of the film were measured. The effectiveness of the coating in fresh blueberries (CB) was evaluated by changes in weight loss, firmness, color, molds and yeast count, pH, titratable acidity, and soluble solids content. RESULTS: The tensile strength and elongation at break of the edible film were 0.45 ± 0.29 MPa and 117.2% ± 7%, respectively. The water vapor permeability was 3.3 × 10(-12) ± 4.0 × 10(-13) g s(-1) m(-1) Pa(-1). In all of the color parameters CB presented significant differences. CB had slight delayed fruit ripening as evidenced by higher titratable acidity (0.3-0.5 g citric acid 100 g(-1)) and lower pH (3.4-3.6) than control during storage; however, it showed reduced firmness (up to 38%). CONCLUSION: The use of Q/CH/SO as a coating in fresh blueberries was able to control the growth of molds and yeasts during 32 days of storage, whereas the control showed an increasing of molds and yeast, between 1.8 and 3.1 log cycles (between 20 and 35 days).
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Mirtilos Azuis (Planta) , Embalagem de Alimentos , Conservação de Alimentos/métodos , Frutas , Chenopodium quinoa/química , Quitosana , Embalagem de Alimentos/instrumentação , Frutas/microbiologia , Fungos/crescimento & desenvolvimento , Microscopia Eletrônica de Varredura , Permeabilidade , Óleos de Plantas , Proteínas de Plantas , Óleo de Girassol , Resistência à Tração , Fatores de Tempo , Água , LevedurasRESUMO
Rotavirus is the most common cause of severe diarrhea in many animal species of economic interest. A simple, safe and cost-effective vaccine is required for the control and prevention of rotavirus in animals. In this study, we evaluated the use of Saccharomyces cerevisiae extracts containing rotavirus-like particles (RLP) as a vaccine candidate in an adult mice model. Two doses of 1mg of yeast extract containing rotavirus proteins (between 0.3 and 3 µg) resulted in an immunological response capable of reducing the replication of rotavirus after infection. Viral shedding in all mice groups diminished in comparison with the control group when challenged with 100 50% diarrhea doses (DD50) of murine rotavirus strain EDIM. Interestingly, when immunizing intranasally protection against rotavirus infection was observed even when no increase in rotavirus-specific antibody titers was evident, suggesting that cellular responses were responsible of protection. Our results indicate that raw yeast extracts containing rotavirus proteins and RLP are a simple, cost-effective alternative for veterinary vaccines against rotavirus.