ICT, Innovation and Knowledge Challenges as seen by a World Conference of Experts

The World Conference on Computers in Agriculture (WCCA), held in San Jose Costa Rica in 2014, provided a forum where professionals from different disciplines exchanged research finding and experience on the use of ICT in a wide variety of contexts. This special issue is a collection of manuscripts by experts in agriculture and food related disciplines presenting the results of their research and experience on uses of ICT, which although not comprehensive in their scope, partially address the issues discussed in the editorial

Salt tolerant endonucleases for the removal of host cell DNA in Downstream Processing of enveloped viruses

Host cell DNA is a critical impurity in downstream processing of enveloped viruses. For vaccine applications, host cell DNA content should be below 10 ng per dose in the final product. Enveloped viruses exhibit an overall negative net charge on their surface and therefore have binding properties similar to DNA. Consequently, separation of virus from DNA can be cumbersome. In addition to DNA in its naked form, host cell DNA is present in virus preparations in form of chromatin. Chromatin (Figure 1) consists of complex and large structures which include DNA and highly positively charged histones. Therefore, different types of interactions of chromatin with chromatographic material and membranes can be observed, electrostatic interaction through negative charges of DNA and positive charges histones and hydrophobic interaction through hydrophobic patches of histones. Moreover, chromatin is often similar in size to viruses, further complicating their separation. We evaluated the performance of four different endonucleases, two salt tolerant endonucleases and two sensitive to salt, in the downstream processing of recombinant Measles virus. Endonuclease treatment was performed after clarification and followed by a purification step using flowthrough chromatography with Capto™ Core 700 resin. Nanoparticle tracking analysis (NTA) was used to determine size and particle concentration and TCID50 to determine the infectivity of the viruses. DNA and histones presence (in process and purified samples) were determined using PicoGreen™ assay and Western blot analysis using detecting anti-histone antibodies. The salt tolerant endonucleases are more efficient in the removal of chromatin and consequently in the removal of host cell DNA. A 97 % reduction of DNA could be observed. Please click Download on the upper right corner to see the full abstract

Virus-like particles adsorption in anion exchange chromatography media

Biotechnological and pharmaceutical industries have the development of modern vaccines and novel drug delivery systems as one of their main focus. At this point, Virus-Like Particles (VLPs) are key candidates once they have the ability to stimulate humoral and cellular immune responses combined with the inability to replicate or proliferate. VLPs are non-infectious self-assembled protein structures which mimic native viruses (lacking any viral genetic material). However great developments in VLPs manufacturing have already been achieved, their purification is still a complex process, usually slow and with low productivity. Accordingly, there is a demand for new purification strategies and unit operations. Anion exchange chromatography is well established and widely used in industry for the purification all sorts of biomolecules. It is already known that polymer-grafted media in form of charged hydrogels and/or chromatography beads have a very high protein binding capacity and they also bind large biomolecules such as plasmids and viruses. However, the separation mechanism of large biomolecules is still not well understood and this lack of knowledge hinders the development and optimization of the purification processes. To overcome this, our aim is to elucidate the adsorption mechanisms of VLPs, large proteins and protein superstructures into different types of anion exchange chromatography media including highly charged hydrogels and polymer-grafted media. The binding kinetics and equilibria of HIV-1 VLPs expressed in CHO cells and Influenza VLPs expressed in Baculovirus-Insect cell system have been measured for polymer grafted media to elucidate the effect of the charged polymer. Adsorption isotherms were measured in microtiter plates and kinetics in batch mode. Gerster, P., Kopecky, E.-M., Hammerschmidt, N., Klausberger, M., Krammer, F., Grabherr, R., Mersich, C., Urbas, L., Kramberger, P., Paril, T., Schreiner, M., Nöbauer, K., Razzazi-Fazeli, E., Jungbauer, A. Purification of infective baculoviruses by monoliths (2013) Journal of Chromatography A, 1290, 36-45. Jungbauer, A., Hahn, R. Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies (2008) Journal of Chromatography A, 1184 (1-2), 62-79. Jungbauer, A. Chromatographic media for bioseparation (2005) Journal of Chromatography A, 1065 (1), 3-12

Polymer grafted chromatography media for direct capture and high-resolution purification of enveloped virus-like particles

Usually, the downstream processing of viruses and virus-like particles (VLP) does not include conventional chromatography media (beads) in the capture and/or purification steps. For large biomolecules, the binding capacity of conventional resins is limited to the outer surface of the beads. We developed a purification process based on polymer-grafted media, which allows a swift purification of HIV-1 VLP from CHO cell culture supernatant. The dynamic binding capacity is one order of magnitude lower than convective media but still in the range of 5-7x1011 particles/ml packed bed, which is unexpectedly high. For that reason, the binding mechanism was studied in detail. As expected, transmission electron micrographs showed that the VLPs only adsorb at the outer surface of the beads. This was corroborated by confocal microscopy using florescence labelled VLPs by incorporating cell membrane label. In batch update experiments, we observed a biphasic behavior with a fast uptake within minutes followed by a slow adsorption within hours. Desorption was also occurring very fast within minutes. Modeling linear gradient elutions with different gradient slopes showed that the number of effective charges involved in the adsorption is in the range of 30 and the adsorption is not really affected by salt. This explains why VLPs can be directly loaded from culture supernatant without further preconditioning. Scalability is not an issue, because these polymer grafted media can be packed in any scale from less than 0.5 ml to several hundred liters and in any column geometry

First records of Pantophthalmidae (Insecta: Diptera) for the state of Tocantins, Brazil

Pantophthalmidae (Diptera) are recorded exclusively in the Neotropical Region. Despite the large size of adults, their species are often rare and poorly represented in entomological collections. Only two genera and 20 species are known, of which 12 are recorded in the five regions of Brazil. In the North region, the family is reported from all states, except in Tocantins. The present work provides the first records of the family for Tocantins, expanding the distribution of two species, Pantophthalmus kerteszianus (Enderlein, 1914) and P. tabaninus Thunberg, 1819. Both species are recorded for the first time in the Cerrado biome. In addition, we provide photographs of the species and a distribution map

Understanding the interaction between pDNA and anion exchange supports under linear and overloaded chromatographic conditions

O elevado potencial da utilização de moléculas de DNA, na forma de plasmídeos, em terapias cujo objetivo é o tratamento ou a cura de várias doenças, tem sido comprovado por inúmeros estudos desenvolvidos recentemente. No entanto, a utilização deste tipo de moléculas em terapias como Vacinas de DNA e na Terapia Génica, exige que a sua produção seja realizada até se atingir a escala da grama e garantindo elevados níveis de pureza do plasmídeo. Para além disso, pretende-se que o processo seja realizado da forma mais económica possível, cumprindo sempre as diretrizes das agências reguladoras como por exemplo a FDA (Food and Drug Administration) e a EMEA (European Agency for the Evaluation of Medical Products). Vários processos têm sido utilizados no processo de purificação de plasmídeos, quer para a sua separação de impurezas como restos celulares, proteínas, DNA genómico, etc., quer para separar as diferentes isoformas em que um plasmídeo se pode encontrar (linear, circular aberto ou superenrolado). Uma das técnicas que tem sido utilizado com sucesso é a cromatografia de troca aniónica. No entanto, o mecanismo de separação de plasmídeos por esta técnica não é, ainda, totalmente compreendido. Para além disso, devido a razões económicas, sugere-se que os processos cromatográficos sejam conduzidos em modo de sobrecarga, ou seja através da sobrecarga da coluna de cromatografia. Contudo, trabalhar em sobrecarga é consideravelmente mais complexo do que trabalhar em cromatografia linear e, até ao momento, não existem modelos adequados que prevejam o mecanismo do processo cromatográfico nestas condições. Deste modo, a previsão do comportamento das moléculas neste caso, bem como do resultado da separação através de cromatografia não é viável, sendo um dos principais obstáculos na conceção e implementação de unidades preparativas à escala industrial. Assim, uma melhor compreensão dos mecanismos subjacentes à cromatografia não linear tem um elevado interesse prático. A análise termodinâmica dos processos de adsorção através da Microcalorimetria de Fluxo (FMC) tem vindo a ser utilizada para obter uma melhor compreensão das forças motrizes, dos mecanismos e das cinéticas envolvidas no processo de adsorção de biomoléculas em diferentes sistemas cromatográficos. Por isso, neste trabalho, através da utilização da microcalorimetria de fluxo, pretende-se compreender o mecanismo de adsorção de um plasmídeo na sua forma linear (ln pVAX1-LacZ) sobre um suporte cromatográfico de troca aniónica (Fast Flow Q-sepharose). Os ensaios foram levados a cabo de modo a que o processo possa ser avaliado em condições lineares e em condições de sobrecarga. Para além do estudo do processo de adsorção tendo em conta a interação entre a biomolécula e o suporte cromatográfico, foi também considerada e avaliada a presença e a influência de efeitos não específicos como por exemplo a reorientação da biomolécula. Para além da microcalorimetria de fluxo, para obter uma melhor compreensão do mecanismo de adsorção, foram também realizados estudos da capacidade de ligação em modo estático (static binding capacity), microcalorimetria de titulação (Isothermal Titration Microcalorimetry) bem como estudos cromatográficos (fast performance chromatography). Os resultados obtidos nos estudos de capacidade de ligação revelaram que o processo de adsorção do plasmídeo segue uma isotérmica de Langmuir, em que a capacidade de ligação da Q-sepharose aumenta com o aumento da concentração de plasmídeo em equilíbrio até se atingir um patamar em que a saturação do suporte cromatográfico é atingida. Os estudos realizados utilizando a microcalorimetria de fluxo foram efetuados nas zonas linear, de transição e de sobrecarga tendo em conta a isotérmica construída. A injeção das amostras for realizada de dois modos diferentes, através da introdução de um pulso de amostra na célula do microcalorímetro ou através da alimentação contínua de amostra na célula. Estes modos de injeção foram conseguidos utilizando loops de diferentes volumes. Todos os termogramas obtidos são compostos por um primeiro pico endotérmico seguido de um pico exotérmico, o qual, em alguns casos específicos (sobrecarga de volume) resulta de picos sobrepostos. Para os picos endotérmicos o processo de dessolvatação foi sugerido como sendo o principal contribuinte enquanto que para o caso dos picos exotérmicos a interação entre o DNA e o suporte cromatográfico e a adsorção secundária foram apontados como os principais contribuidores. Para além disso, a microcalorimetria de fluxo sugere que o processo global de adsorção é exotérmico, o que é esperado uma vez que se trata de uma interação de troca aniónica. No entanto, foram também demonstradas evidências da presença de efeitos não específicos do processo de adsorção, nomeadamente a reorientação e a repulsão electroestática entre as moléculas adsorvidas.The potential of plasmid DNA as a therapeutic molecule in the cure of several diseases has been sustained by innumerous studies. The use of pDNA in therapies (DNA Vaccines and Gene Therapy) requires its production at the gram scale, with high purity levels and in an economic way, always taking into account the guidelines from the regulatory agencies. Anion-exchange chromatography have been successfully used in pDNA purification. Nevertheless, the mechanism of pDNA separation using this technique is still not completely understood. Additionally, due to economic reasons, it may be interesting to run the chromatographic processes in the overloaded mode. However, the overloaded mode is considerably more complex than linear chromatography, and suitable models do not exist. Thus, the prediction of separation behavior is generally unreliable, being a major impediment in the design and implementation of scaled-up units. Therefore, a better understanding of the mechanisms underlying non-linear chromatography of biomolecules has considerable practical interest. Flow Microcalorimetry (FMC) has proven its ability to provide an improved understanding of the driving forces, mechanisms and kinetics involved in the adsorption process of biomolecules onto several chromatographic systems. Thus, using FMC as a central technique, this study aims to understand the adsorption mechanism of pDNA (pVAX1-LacZ) onto the anion-exchange support Fast Flow Q-sepharose, considering linear and overloaded conditions and showing the role of nonspecific effects at the adsorptive process. Static binding capacity, Isothermal Titration Microcalorimetry (ITM) and fast performance chromatography were also accomplished to obtain a better understanding of the adsorption mechanism. Static binding capacity studies revealed that the mechanism of pDNA adsorption onto Q-sepharose follows a Langmuir-type isotherm profile. FMC experiments were performed in the linear, transition and overloaded zones of the isotherm, through a pulse injection or a continuous feed. All obtained thermograms comprised a first endothermic peak followed by an exothermic peak which, in some cases (volume overloading), resulted from overlapped peaks. Endothermic heat major contributor was suggested to be the desolvation process. Exothermic heats were related to the interaction between pDNA and Q-sepharose primary and secondary adsorption. Furthermore, FMC revealed that the overall adsorption process is exothermic, as expected for an anion-exchange interaction. Nevertheless, there are evidences of the presence of nonspecific effects, such as reorientation and electrostatic repulsive forces

Renovación de área administrativa, investigación y recreación del Biotopo Chocón Machacas, Livingston, Izabal.

Propone la readecuación de los ambientes del Biotopo Chocón Machacas, y la incorporación de las garitas de control, para revertir el daño a las instalaciones a causa del paso del tiempo, y de las malas prácticas ambientales de personas inescrupulosas. La propuesta plantea la aplicación de las técnicas constructivas más apropiadas, el uso de los materiales más adecuados, con el fundamento teórico de la arquitectura tropical moderna, y de la arquitectura regenerativa; se integrará al entorno natural, en el marco de respeto al medio ambiente para minimizar el impacto humano

Evolución de las cooperativas de ahorro y crédito en ecuador, 2000-2015

Este artículo evalúa la evolución de las Cooperativas de Ahorro y Crédito controladas por la Superintendencia de Economía Popular y Solidaria de Ecuador en el período 2000-2015.En la metodología se calculó un índice basado en los factores clave del negocio (cartera, captaciones y resultados) ajustados por inflación (año base 2007), cuyos resultados fueron aplicados a la teoría Fisher y comparados con la variación porcentual del Producto Interno Bruto. Del análisis se concluye que las Cooperativas de Ahorro y Crédito que forman parte del Sistema Financiero Popular y Solidario crecieron en términos reales el 18,18% lo que las convierte en las Instituciones Financieras de mejor desempeño en el Sistema Financiero Nacional

Unveiling early childhood health inequities by age five through the national neighborhood equity index and the early development instrument.

There is growing public urgency to close equity gaps in health and development by addressing inequities at multiple levels of childrens developmental ecosystems. Current measurement strategies obscure the dynamic structural and relational patterns of oppression, adversity, and disadvantage that children can experience in their local intimate developmental ecosystem, as well as the leverage points that are necessary to change them. The purpose of this study is to examine the relationship between a universally available measure of neighborhood socio-economic context, the National Neighborhood Equity Index (NNEI), and a population measure of early child development and well-being, the Early Development Instrument (EDI). Data from a convenience sample of 144,957 kindergarteners in neighborhoods across the US demonstrate that children living in neighborhoods with more equity barriers are more likely to be on vulnerable developmental trajectories than those who reside in neighborhoods without any equity barriers. A multi-dimensional measurement approach that incorporates both the EDI and the NNEI can be used to quantify ethnoracialized patterns of structural disadvantage during critical periods of health development. These measures can inform community action to intervene early in the lifecourse to optimize childrens health development trajectories at a population level