PROCESS SCALE-UP AND OPTIMIZATION FOR PRODUCTION OF HIGH EFFICACY ORAL RABIES VACCINE

Rabies is an important causative agent of disease resulting in an acute infection of the nervous system and death of the individual. Rabies remains an important public health program in developing countries, and the indigenous threat of rabies continues in developed countries because of wildlife reservoirs. Globally, there are about 55,000 fatal human cases of rabies each year [WHO, 2007]. Control of rabies in wildlife remains an important challenge for government offices. There are numbers of rabies vaccines commercially available for controls of wildlife rabies. However, these vaccines currently distributed to wildlife do not effectively immunize all at-risk species, especially skunks. Alternative efficacious vaccines are needed. A human adenovirus rabies glycoprotein recombinant vaccine candidate (AdRG1.3), developed by the Rabies Research and Development Unit at Ontario Ministry of Natural Resources, Canada, has shown the most promising result in laboratory trials. The adenovirus used in rabies vaccine laden bait is produced using HEK 293 cell culture process. This presentation will focus on demonstrating the successful scale-up of AdRG1.3 adenovirus production from 1 liter to 500 liter to manufacture large quantities of bulk material required to support field trials and demonstrate efficacy of this new vaccine. The robustness of production process was improved through elimination of medium replacement operation at the time of virus infection, and culture titer was increased by over 3 folds through optimization of cell culture medium. The elimination of medium replacement step reduced the risk of culture contamination, and resulted in significant saving in material expenses and reduction in labor costs. Over 10,000 liters of active AdRG1.3 adenovirus cultures were manufactured so far to support field trials. AdRG1.3 adenovirus is formulated and packaged in baits using Artemis Technologies Inc. proprietary technology prior to aerial-baiting. AdRG1.3 rabies vaccine has been distributed by several provincial agencies to testing areas located in Ontario, Quebec and New Brunswick provinces, Canada, for field trials in yearly campaigns from 2006 to 2009. The field results showed that the new vaccine was more efficient than the existing ones in immunizing animals that were previously difficult to vaccinate

A novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient siRNA delivery in breast cancer therapy: preparation and in vitro analysis

Wei Shao1, Arghya Paul1, Sana Abbasi1, Parminder S Chahal2, Jimmy A Mena2, Johnny Montes2, Amine Kamen2, Satya Prakash11Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 2Animal Cell Technology, Bioprocess Centre, Biotechnology Research Institute, National Research Council, Montreal, Quebec, CanadaBackground: Systemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs).Methods: AAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions.Results: PEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection.Conclusion: The present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy.Keywords: adeno-associated virus type 2, virus-like particles, small interfering RNA delivery, breast cancer therapy, nanomedicin

La Torre de Hanoí una estrategia lúdica en el proceso enseñanza y aprendizaje de las series y sucesiones en la competencia de la resolución de problemas

El presente proyecto se basa en la aplicación de una estrategia lúdica usando como herramienta las Torres de Hanoi en la enseñanza de sucesiones y series en la resolución de situaciones problemas en estudiantes de grado noveno le la Escuela Normal Superior La Hacienda de la ciudad de Barranquilla. Al aplicar las pruebas Supérate, Colombia aprende y olimpiadas regionales y nacionales se observó que las temáticas de sucesiones son aplicadas en toda y cada una de ellas no importa el tiempo el cual se aplica dicha prueba. Sabiendo que la temática de sucesiones y series en el plan o mapas curriculares se encuentran como unidad número 8, se decidió cambiar la ubicación de dicha unidad para ser una de las primeras y así poder aplicar una estrategia lúdica como es la torre de Hanoi para que los estudiantes aprendan jugando, dando opiniones sobre qué ocurre y así crear expectativas y dar solución a los hechos. El proyecto estuvo enmarcado en una investigación de tipo cuantitativo, fundamentado en la aplicación de un pretest -postest y el uso de sus promedios aritméticos. Para la elaboración y cumplimiento de los objetivos planteados para el marco disciplinario se utilizo el autor De Guzmán (2004) cuya estructura es cumplir con cuatro pasos para enfrentarse a una situación problema(Familiarización del problema, Búsqueda de estrategia, escoger estrategia adecuada, revisión del proceso y consecuencias). De esta manera se pudo concluir que la herramienta (la torre de Hanoi) es una estrategia apropiada en el uso de la enseñanza de los conceptos de series y progresiones

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”. In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1x106 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1x106 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1x106 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5x105 IVP/mL after 48 hpt and 1x107 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5x104 IVP/mL and 2.8x107 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CVV within timelines dictated by pandemic situations and produce effective responsiveness than previous methodolog

Probing inhibitory effects of destruxins from Metarhizium anisopliae using insect cell based impedance spectroscopy : Inhibition vs chemical structure

A noninvasive technique based on electric cell-substrate impedance sensing (ECIS) was demonstrated for on-line probing inhibitory effects of five destruxins on Spodoptera frugiperda Sf9 insect cells. Such chemically structurally similar cyclic hexadepsipeptides, were isolated and purified from the fungus Metarhizium anisopliae. Based on a response function, the inhibitory effect of the destruxins was established from determining the half-inhibition concentration (ECIS50), i.e., the level at which 50% inhibition of the cell response was obtained. Probing by cell based impedance spectroscopy indicated that only a slight change in their chemical structures provoked a significant effect on inhibition. Destruxin B was most inhibitory but replacement of a single methyl group with hydrogen (destruxin B2) or addition of a hydroxylgroup (destruxin C) significantly reduced the inhibition. The removal of one methyl group and one hydrogen (destruxin A) lowered the inhibitory effect even more whereas the formation of an epoxy ring (destruxin E) in the structure nullified the inhibitory effect.NRC publication: Ye

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

ACCIÓN SIMBIÓTICA DEL Lactobacillus Acidophylus - HARINA DE MAÍZ E INULINA EN VIDA ÚTIL DEL QUESO

La presente investigación tuvo como objetivo prolongar la vida útil del queso fresco mediante la acción microbiana en la cual se emplearon dosis de 1.35, 2.35 y 3.35 g de microorganismos probióticos (Lactobacillus acidophilus) y mezclas de ingredientes prebióticos (harina de maíz + inulina) en niveles de 7.14 g (5 g + 2.14 g), 9.1g (6.4 g + 2.7 g), 11.14 g (7.8 g + 3.34 g). Sus combinaciones aplicadas sobre nueve litros de leche originaron nueve tratamientos, más uno sin aplicación de probiótico ni prebiótico (testigo); distribuidos bajo un diseño experimental completamente al azar, en arreglo factorial 3 x 3 + 1 y tres réplicas. Los resultados mostraron que la mayor dosificación (3.35 g) de probiótico combinada con el más alto nivel (11.14 g) de prebiótico (harina de maíz, 7.8 g e inulina, 3.34 g) logró conservar por 30 dias, las características microbiológicas, bromatológicas y organolépticas óptimas y aceptables del queso fresco

Material de suturas en periodoncia e implantes.

Entre los biomateriales más usados en Periodoncia e Implantología Oral las suturas constituyen parte fundamental del éxito de los tratamientos quirúrgicos. En la extensa revisión de la literatura disponible no hay una idea clara del material ideal de sutura  para la cirugía periodontal e implantológica. A pesar de varias evaluaciones comparativas que se han publicado existen controversias de las propiedades, la biodegradabilidad y el rendimiento de los materiales de sutura. El presente artículo propone revisar las propiedades de los materiales de suturas absorbibles y no absorbibles más utilizados en Periodoncia e Implantología Oral

Noninvasive cell-based impedance spectroscopy for real-time probing inhibitory effects of Graphene derivatives

Three water-dispersible graphene derivatives, graphene oxide (GO), sulfonated graphene oxide (SGO), and sulfonated graphene (SG), were prepared and probed for their plausible cytotoxicity by non-invasive electric cell-substrate impedance sensing (ECIS). With Spodoptera frugiperda S\u1929 insect cells adhered on gold microelectrodes as an active interface, it is feasible to monitor changes in impedance upon exposure to different graphene derivatives. S\u1929 insect cells were then exposed to different concentrations of graphene derivatives and their spreading and viability were monitored and quantified by ECIS in real-time. On the basis of the 50% inhibition concentration (ECIS\u2085\u2080), none of the graphene derivatives were judged to have any significant cytotoxicity with respect to the chosen cell line as the ECIS\u2085\u2080 values were all above 100 \u3bcg/mL. However, all graphene derivatives exhibited inhibitory effects on the S\u1929 response at the cell spreading level with the following order: SG (ECIS\u2085\u2080 = 121 \ub1 8 \u3bcg/mL), SGO (ECIS\u2085\u2080 = 151 \ub1 9 \u3bcg/mL), and GO (ECIS\u2085\u2080 = 232 \ub1 27 \u3bcg/mL), reflecting differences observed in their \u3b6-potential and surface area. The presence of phenyl sulfonyl groups in SGO and SG improves their aqueous dispersity which enables these materials to have a greater inhibitory effect on S\u1929 insect cells in comparison to GO. Such results were corroborated well with the cell count and viability by the Trypan Blue exclusion assay.Peer reviewed: YesNRC publication: Ye

High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: Comparison to a batch Sf-9-BEV system

DA - 20061020IS - 0006-3592 (Print)LA - ENGPT - JOURNAL ARTICLENRC publication: Ye