292. Towards Large-Scale Manufacturing of Adeno-Associated Virus by Transient Transfection of HEK293 Suspension Cells in a Stirred Tank Bioreactor Using Serum-Free Medium

Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by transient transfection in a serum-free medium using NRC's patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based transient transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of transient transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application

Production of rVSV-ZEBOV in serum-free suspension culture of HEK 293SF cells.

Abstract Ebola virus disease is an urgent international priority. Promising results for several vaccine candidates have been reported in non-human primate studies and clinical trials with the most promising being the rVSV-ZEBOV vaccine. In this study, we sought to produce rVSV-ZEBOV in HEK 293SF cells in suspension and serum-free media. The purpose of this study was to establish a process using the HEK 293SF production platform, optimise the production titre, demonstrate scalability and the efficiency of the generated material to elicit an immune reaction in an animal model. Critical process parameters were evaluated to maximize production yield and process robustness and the following operating conditions: 1–2 × 106 cells/mL grown in HyClone HyCell TransFx-H media infected at an MOI of 0.001 with a temperature shift to 34 °C during the production phase and a harvest of the product after 48 h. Using these conditions, scalability in a 3.5 L controlled bioreactor was shown reaching a titre of 1.19 × 108 TCID50/mL at the peak of production, the equivalent of 4165 doses of vaccine per litre. The produced virus was shown to be thermostable in the culture media and, when concentrated, purified and administered to mice, demonstrated the ability to induce a ZEBOV-specific immune response

Socially Communicative Eye Contact and Gender Affect Memory

Because of their value as a socially communicative cue, researchers have strived to understand how the gaze of other people influences a variety of cognitive processes. Recent work in social attention suggests that the use of images of people in laboratory studies, as a substitute for real people, may not effectively test socially communicative aspects of eye gaze. As attention affects many other cognitive processes, it is likely that social attention between real individuals could also affect other cognitive processes, such as memory. However, from previous work alone, it is unclear whether, and if so how, socially communicative eye gaze affects memory. The present studies test the assumption that socially communicative aspects of eye gaze may impact memory by manipulating the eye gaze of a live speaker in the context of a traditional recognition paradigm used frequently in the laboratory. A female (Experiment 1) or male (Experiment 2) investigator read words aloud and varied whether eye contact was, or was not, made with a participant. With both female and male investigators, eye contact improved word recognition only for female participants and hindered word recognition in male participants. When a female investigator prolonged their eye contact (Experiment 3) to provide a longer opportunity to both observe and process the investigator’s eye gaze, the results replicated the findings from Experiments 1 and 2. The findings from Experiments 1–3 suggest that females interpret and use the investigator’s eye gaze differently than males. When key aspects from the previous experiments were replicated in a noncommunicative situation (i.e., when a video of a speaker is used instead of a live speaker; Experiment 4), the memory effects observed previously in response to eye gaze were eliminated. Together, these studies suggest that it is the socially communicative aspects of eye gaze from a real person that influence memory. The findings reveal the importance of using social cues that are communicative in nature (e.g., real people) when studying the relationship between social attention and memory

Reflexiones de estudiantes universitarios, docentes de Agroecología (U.N.Cuyo) y agentes de extensión del INTA en prácticas de intervención socioproductivas en las zonas de Luján de Cuyo y Valle de Uco, Mendoza

En el marco de la asignatura Agroecología y Ambientes Rurales de la carrera en Ingeniería en Recursos Naturales Renovables (FCA- UNCuyo), se han realizado durante 10 años académicos consecutivos, dos actividades consistentes en visitar a diferentes tipos sociales agrarios ubicados en Luján de Cuyo y el Valle de Uco. En las nombradas actividades, los estudiantes realizan una visita extendida a productores que integran diferentes cadenas productivas, con el objetivo de caracterizar el ecosistema, sociosistema y tecnosistema en cada caso, además de diferenciar manejos convencionales y agroecológicos. Con la información recabada y reflexionada, los estudiantes realizan un trabajo integrador, con carácter de condición obligatoria para rendir el examen final de la asignatura, que rescata una problemática detectada y propone una solución socio tecnológica con base agroecológica, ajustada a las características de tipo social agrario abordado. El nombrado material queda a disposición de los actores involucrados para ser utilizado como una herramienta de comprensión de la dinámica socio-productiva y territorial. En el presente relato realizamos una breve síntesis sobre las experiencias de cada visita a campo y se presentan las reflexiones de los actores involucrados acerca de esta experiencia.In the framework of agroecology and rural environments race course in Engineering in Renewable Natural Resources (FCA UNCuyo) were performed for 10 consecutive school years, two consistent activities visit different agricultural social types are located in Lujan de Cuyo and Uco Valley. In the named activities, students made an extended visit to producers that integrate different production chains, in order to characterize the ecosystem, sociosistema and Technosystem in each case, in addition to conventional agro differentiate and handling. With the information gathered and reflected, students take an integrative work, as an obligatory condition to take the final exam of the subject, who rescues a detected problem and proposes a solution partner agroecologically technological basis and adjusted to the characteristics of a social nature agricultural addressed. The named material is available to the actors involved to be used as a tool for understanding the socio-productive and territorial dynamics. In this story is a brief summary of experiences of each field visit and reflections of stakeholders about this experience are presented.Eje: A2: Paisajes, Territorios y AgroecologíaFacultad de Ciencias Agrarias y Forestale

Reflexiones de estudiantes universitarios, docentes de Agroecología (U.N.Cuyo) y agentes de extensión del INTA en prácticas de intervención socioproductivas en las zonas de Luján de Cuyo y Valle de Uco, Mendoza

En el marco de la asignatura Agroecología y Ambientes Rurales de la carrera en Ingeniería en Recursos Naturales Renovables (FCA- UNCuyo), se han realizado durante 10 años académicos consecutivos, dos actividades consistentes en visitar a diferentes tipos sociales agrarios ubicados en Luján de Cuyo y el Valle de Uco. En las nombradas actividades, los estudiantes realizan una visita extendida a productores que integran diferentes cadenas productivas, con el objetivo de caracterizar el ecosistema, sociosistema y tecnosistema en cada caso, además de diferenciar manejos convencionales y agroecológicos. Con la información recabada y reflexionada, los estudiantes realizan un trabajo integrador, con carácter de condición obligatoria para rendir el examen final de la asignatura, que rescata una problemática detectada y propone una solución socio tecnológica con base agroecológica, ajustada a las características de tipo social agrario abordado. El nombrado material queda a disposición de los actores involucrados para ser utilizado como una herramienta de comprensión de la dinámica socio-productiva y territorial. En el presente relato realizamos una breve síntesis sobre las experiencias de cada visita a campo y se presentan las reflexiones de los actores involucrados acerca de esta experiencia.In the framework of agroecology and rural environments race course in Engineering in Renewable Natural Resources (FCA UNCuyo) were performed for 10 consecutive school years, two consistent activities visit different agricultural social types are located in Lujan de Cuyo and Uco Valley. In the named activities, students made an extended visit to producers that integrate different production chains, in order to characterize the ecosystem, sociosistema and Technosystem in each case, in addition to conventional agro differentiate and handling. With the information gathered and reflected, students take an integrative work, as an obligatory condition to take the final exam of the subject, who rescues a detected problem and proposes a solution partner agroecologically technological basis and adjusted to the characteristics of a social nature agricultural addressed. The named material is available to the actors involved to be used as a tool for understanding the socio-productive and territorial dynamics. In this story is a brief summary of experiences of each field visit and reflections of stakeholders about this experience are presented.Eje: A2: Paisajes, Territorios y AgroecologíaFacultad de Ciencias Agrarias y Forestale

Reflexiones de estudiantes universitarios, docentes de Agroecología (U.N.Cuyo) y agentes de extensión del INTA en prácticas de intervención socioproductivas en las zonas de Luján de Cuyo y Valle de Uco, Mendoza

En el marco de la asignatura Agroecología y Ambientes Rurales de la carrera en Ingeniería en Recursos Naturales Renovables (FCA- UNCuyo), se han realizado durante 10 años académicos consecutivos, dos actividades consistentes en visitar a diferentes tipos sociales agrarios ubicados en Luján de Cuyo y el Valle de Uco. En las nombradas actividades, los estudiantes realizan una visita extendida a productores que integran diferentes cadenas productivas, con el objetivo de caracterizar el ecosistema, sociosistema y tecnosistema en cada caso, además de diferenciar manejos convencionales y agroecológicos. Con la información recabada y reflexionada, los estudiantes realizan un trabajo integrador, con carácter de condición obligatoria para rendir el examen final de la asignatura, que rescata una problemática detectada y propone una solución socio tecnológica con base agroecológica, ajustada a las características de tipo social agrario abordado. El nombrado material queda a disposición de los actores involucrados para ser utilizado como una herramienta de comprensión de la dinámica socio-productiva y territorial. En el presente relato realizamos una breve síntesis sobre las experiencias de cada visita a campo y se presentan las reflexiones de los actores involucrados acerca de esta experiencia.In the framework of agroecology and rural environments race course in Engineering in Renewable Natural Resources (FCA UNCuyo) were performed for 10 consecutive school years, two consistent activities visit different agricultural social types are located in Lujan de Cuyo and Uco Valley. In the named activities, students made an extended visit to producers that integrate different production chains, in order to characterize the ecosystem, sociosistema and Technosystem in each case, in addition to conventional agro differentiate and handling. With the information gathered and reflected, students take an integrative work, as an obligatory condition to take the final exam of the subject, who rescues a detected problem and proposes a solution partner agroecologically technological basis and adjusted to the characteristics of a social nature agricultural addressed. The named material is available to the actors involved to be used as a tool for understanding the socio-productive and territorial dynamics. In this story is a brief summary of experiences of each field visit and reflections of stakeholders about this experience are presented.Eje: A2: Paisajes, Territorios y AgroecologíaFacultad de Ciencias Agrarias y Forestale

Sustentabilidad vitivinícola para diferentes tipos de agricultores vitivinícolas

Un Desarrollo Rural Sustentable requiere de sistemas agrícolas sustentables, que sean ecológicamente adecuados, económicamente viables y socio culturalmente aceptables Abbona et al, (2004). Sarandon, (2010), define la agricultura sustentable como aquella que es: suficientemente productiva, económicamente viable, ecológicamente adecuada y cultural y socialmente aceptable. En el contexto de cambio de la vitivinicultura, se hace necesario conocer la sustentabilidad de los mismos. En este caso se usó la tipología que propuesta por Martín, (2009) que plantea tres tipos sociales: productores descapitalizados pluriactivos, productores familiares capitalizados y empresarios. El objetivo de este trabajo fue: analizar, mediante el método propuesto por Sarandón (2002) y el uso de indicadores, la sustentabilidad predial de tres modelos productivos vitivinícolas: productores descapitalizados pluriactivos, productores familiares capitalizados y empresarios, de la 1º zona de Mendoza. La propuesta de metodológica estuvo basada en la metodología de Sarandón (2002) y estaba propuesto realizar tres etapas, de 2 años cada una. En una primera etapa se desarrollaron dos acciones. Lo primero fué buscar y analizar antecedentes e información regional sobre el tema y la segunda acción fue buscar y seleccionar 2 fincas del departamento de Lujan de Cuyo que fueran representativas, para cada tipo social, factibilidad de acceso y predisposición de los agricultores. En una segunda etapa se caracterizaría, a través del enfoque sistémico, el funcionamiento de los agroecosistemas en tres dimensiones: ambiental-ecológica, tecnológica, y socioeconómica. Y en una tercera etapa: se construirán indicadores para evaluar la sustentabilidad de los sistemas agrícolas, Se logró la primera etapa y se pudo: conceptualizar y profundizar el concepto de sustentabilidad integral y su aplicación en la agronomía, proponer variables con potencial de indicadores de sustentabilidad y seleccionar las fincas.A Sustainable Rural Development requires sustainable agricultural systems that are environmentally suitable, economically viable and culturally acceptable (Abbona et al, 2004). Sarandon (2010) defines sustainable agriculture as one that is: productive enough, affordable, ecologically appropriate and culturally and socially acceptable. In the context of the viticulture changes, it is necessary to study their sustainability. In this case we take the typology proposed by Martin, 2009 that summarizes it in three social types: pluriactive undercapitalized producers, family farmers and entrepreneurs capitalized. The objective of this work was to analyze, through the method proposed by Sarandón (2002) and the use of indicators, the property sustainability of three vitivinícolas production models: pluriactively de-capitalized producers, capitalized family producers and entrepreneurs, from the first zone of Mendoza. The methodological proposal was based on the methodology of Sarandon (2002) and was proposed to perform three stages, each of 2 years. In the first stage two actions were developed. The first was to seek and analyze regional background and information on the subject; And the second action was to search and select 2 farms in the department of Lujan de Cuyo that were representative for each social type, feasibility of access and predisposition of farmers. In a second stage, the systemic approach would characterize the functioning of agroecosystems in three dimensions: environmental-ecological, technological, and socioeconomic. And in a third stage: indicators will be constructed to evaluate the sustainability of agricultural systems, The first stage was achieved and it was possible to: conceptualize and deepen the concept of integral sustainability and its application in agronomy, propose variables with potential for sustainability indicators and successful selection of farms.Trabajo publicado en Filippini, María Flavia (comp.). Congreso Internacional Aguas, Ambiente y Energía de la Asociación de Universidades Grupo Montevideo: Resúmenes de trabajos. Mendoza: Universidad Nacional de Cuyo, 2017.Facultad de Ciencias Agrarias y Forestale

Letter processing and font information during reading: beyond distinctiveness, where vision meets design

Letter identification is a critical front end of the reading process. In general, conceptualizations of the identification process have emphasized arbitrary sets of distinctive features. However, a richer view of letter processing incorporates principles from the field of type design, including an emphasis on uniformities across letters within a font. The importance of uniformities is supported by a small body of research indicating that consistency of font increases letter identification efficiency. We review design concepts and the relevant literature, with the goal of stimulating further thinking about letter processing during reading