Magnetoliposomas multifuncionales como vehículos de administración de fármacos para el tratamiento potencial de la enfermedad de Parkinson

La enfermedad de Parkinson (EP) es el segundo trastorno neurodegenerativo más frecuente después de la enfermedad de Alzheimer. Por ello, el desarrollo de nuevas tecnologías y estrategias para tratarla es una prioridad sanitaria mundial. Los tratamientos actuales incluyen la administración de levodopa, inhibidores de la monoaminooxidasa, inhibidores de la catecol-O-metiltransferasa y fármacos anticolinérgicos. Sin embargo, la liberación efectiva de estas moléculas, debido a la limitada biodisponibilidad, es un reto importante para el tratamiento de la EP. Como estrategia para resolver este desafío, en este estudio desarrollamos un novedoso sistema de liberación de fármacos multifuncional magnético y sensible a estímulos redox, basado en nanopartículas de magnetita funcionalizadas con la proteína translocadora de alto rendimiento OmpA y encapsuladas en liposomas de lecitina de soja. Los magnetoliposomas multifuncionales (MLP) obtenidos se ensayaron en neuroblastoma, glioblastoma, astrocitos primarios humanos y de rata, células endoteliales de rata de barrera hematoencefálica, células endoteliales microvasculares primarias de ratón y en un modelo celular inducido por EP. Los MLP demostraron un excelente rendimiento en ensayos de biocompatibilidad, incluyendo hemocompatibilidad (porcentajes de hemólisis por debajo del 1%), agregación plaquetaria, citocompatibilidad (viabilidad celular por encima del 80% en todas las líneas celulares probadas), potencial de membrana mitocondrial (alteraciones no observadas) y producción intracelular de ROS (impacto insignificante en comparación con los controles). Además, las nanovehículas mostraron una aceptable internalización celular (área cubierta cercana al 100% a los 30 min y a las 4 h) y capacidad de escape endosomal (disminución significativa de la colocalización lisosomal tras 4 h de exposición). Además, se emplearon simulaciones de dinámica molecular para comprender mejor el mecanismo de translocación subyacente de la proteína OmpA, mostrando hallazgos clave relativos a interacciones específicas con fosfolípidos. En general, la versatilidad y el notable rendimiento in vitro de este novedoso nanovehículo lo convierten en una tecnología de administración de fármacos adecuada y prometedora para el tratamiento potencial de la EP.Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. Therefore, development of novel technologies and strategies to treat PD is a global health priority. Current treatments include administration of Levodopa, monoamine oxidase inhibitors, catechol-O-methyltransferase inhibitors, and anticholinergic drugs. However, the effective release of these molecules, due to the limited bioavailability, is a major challenge for the treatment of PD. As a strategy to solve this challenge, in this study we developed a novel multifunctional magnetic and redox-stimuli responsive drug delivery system, based on the magnetite nanoparticles functionalized with the high-performance translocating protein OmpA and encapsulated into soy lecithin liposomes. The obtained multifunctional magnetoliposomes (MLPs) were tested in neuroblastoma, glioblastoma, primary human and rat astrocytes, blood brain barrier rat endothelial cells, primary mouse microvascular endothelial cells, and in a PD-induced cellular model. MLPs demonstrated excellent performance in biocompatibility assays, including hemocompatibility (hemolysis percentages below 1%), platelet aggregation, cytocompatibility (cell viability above 80% in all tested cell lines), mitochondrial membrane potential (non-observed alterations) and intracellular ROS production (negligible impact compared to controls). Additionally, the nanovehicles showed acceptable cell internalization (covered area close to 100% at 30 min and 4 h) and endosomal escape abilities (significant decrease in lysosomal colocalization after 4 h of exposure). Moreover, molecular dynamics simulations were employed to better understand the underlying translocating mechanism of the OmpA protein, showing key findings regarding specific interactions with phospholipids. Overall, the versatility and the notable in vitro performance of this novel nanovehicle make it a suitable and promising drug delivery technology for the potential treatment of PD

Next generation sequencing and proteomics in plant virology: how is Colombia doing?

Crop production and trade are two of the most economically important activities in Colombia, and viral diseases cause a high negative impact to agricultural sector. Therefore, the detection, diagnosis, control, and management of viral diseases are crucial. Currently, Next-Generation Sequencing (NGS) and ‘Omic’ technologies constitute a right-hand tool for the discovery of novel viruses and for studying virus-plant interactions. This knowledge allows the development of new viral diagnostic methods and the discovery of key components of infectious processes, which could be used to generate plants resistant to viral infections. Globally, crop sciences are advancing in this direction. In this review, advancements in ‘omic’ technologies and their different applications in plant virology in Colombia are discussed. In addition, bioinformatics pipelines and resources for omics data analyses are presented. Due to their decreasing prices, NGS technologies are becoming an affordable and promising means to explore many phytopathologies affecting a wide variety of Colombian crops so as to improve their trade potential

Plataformas de secuenciación de nueva generación y proteómica aplicadas a la virología vegetal: ¿Cómo ha avanzado Colombia?

Crop production and trade are two of the most economically important activities in Colombia, and viral diseases cause a high negative impact to agricultural sector. Therefore, the detection, diagnosis, control, and management of viral diseases are crucial. Currently, Next-Generation Sequencing (NGS) and ‘Omic’ technologies constitute a right-hand tool for the discovery of novel viruses and for studying virus-plant interactions. This knowledge allows the development of new viral diagnostic methods and the discovery of key components of infectious processes, which could be used to generate plants resistant to viral infections. Globally, crop sciences are advancing in this direction. In this review, advancements in ‘omic’ technologies and their different applications in plant virology in Colombia are discussed. In addition, bioinformatics pipelines and resources for omics data analyses are presented. Due to their decreasing prices, NGS technologies are becoming an affordable and promising means to explore many phytopathologies affecting a wide variety of Colombian crops so as to improve their trade potential.La producción y el comercio de cultivos es una de las actividades económicas más importantes para el país. Las enfermedades causadas por virus ocasionan graves pérdidas económicas en el sector, por lo tanto, la detección, diagnóstico y diseño de estrategias para su control y manejo es crucial. Las tecnologías de secuenciación masiva (NGS por sus siglas en ingles) y las ciencias Ómicas constituyen hoy, una herramienta para el descubrimiento de nuevos virus y para el estudio de la interacción entre los virus y su hospedero vegetal. Este conocimiento no solo permite el desarrollo de nuevos métodos de diagnóstico, sino también permite el descubrimiento de componentes claves en la infección, los cuales podrían usarse para obtener plantas resistentes a los virus. En el mundo, el manejo de cultivos se está trabajando con ese enfoque. Por lo tanto, en esta revisión se presentan las diferentes aplicaciones de las tecnologías ómicas en la virología de plantas y el avance que ha alcanzado Colombia. Adicionalmente, se muestran los diferentes recursos y programas usados para el análisis bioinformático de datos ómicos. Debido a su costo cada vez más reducido, las tecnologías NGS son una excelente oportunidad para explorar fitopatologías en una gran diversidad de productos agrícolas y para mejorar su potencial comercial

Isolation of the Human Cytomegalovirus from bodily fluids

In vitro studies on the pathogenesis of the human cytomegalovirus (HCMV) are conducted regularly using laboratory adapted strains that lose some characteristics during the adaptation process. Since HCMV is excreted from bodily fluids during infection or reactivation, this work aimed to isolate and culture HCMV from the MRC-5 human cells found in the urine, bronchoalveolar lavage, saliva, and plasma samples of pediatric patients with probable or confirmed infection. The samples were inoculated on cell cultures either for 14 days or until a cytopathic effect (CPE) of 80 % was observed. The cell lysates and supernatants were used to perform successive viral passages. Besides HCMV, the herpes simplex virus was detected from all the saliva samples. Inoculation of the HCMV positive sera induced cell clustering and immediate monolayer damage that restricted their use. One sample of bronchoalveolar lavage induced a CPE after inoculation like that of the HCMV reference strains (Towne and Merlin), which was consequently propagated and titrated. A second viral isolate derived from the urine sample of a patient with congenital infection did not demonstrate a CPE, although presence of the virus had been confirmed using PCR. The viral isolates were examined and found to be negative for adenoviruses or enteroviruses. Despite the evident difficulty encountered for the isolation and harvesting of the HCMV, this work shows that it was possible to obtain a low passage viral strain using a modified shell vial method and inoculation protocol with extended follow-up and confirmation

Aislamiento de citomegalovirus humano a partir de fluidos corporales

In vitro studies on the pathogenesis of the human cytomegalovirus (HCMV) are conducted regularly using laboratory adapted strains that lose some characteristics during the adaptation process. Since HCMV is excreted from bodily fluids during infection or reactivation, this work aimed to isolate and culture HCMV from the MRC-5 human cells found in the urine, bronchoalveolar lavage, saliva, and plasma samples of pediatric patients with probable or confirmed infection. The samples were inoculated on cell cultures either for 14 days or until a cytopathic effect (CPE) of 80 % was observed. The cell lysates and supernatants were used to perform successive viral passages. Besides HCMV, the herpes simplex virus was detected from all the saliva samples. Inoculation of the HCMV positive sera induced cell clustering and immediate monolayer damage that restricted their use. One sample of bronchoalveolar lavage induced a CPE after inoculation like that of the HCMV reference strains (Towne and Merlin), which was consequently propagated and titrated. A second viral isolate derived from the urine sample of a patient with congenital infection did not demonstrate a CPE, although presence of the virus had been confirmed using PCR. The viral isolates were examined and found to be negative for adenoviruses or enteroviruses. Despite the evident difficulty encountered for the isolation and harvesting of the HCMV, this work shows that it was possible to obtain a low passage viral strain using a modified shell vial method and inoculation protocol with extended follow-up and confirmation.Estudios in vitro de la patogénesis del citomegalovirus humano (HCMV) se hace empleando cepas adaptadas de laboratorio que han perdido algunas de sus características durante ese proceso. En vista que el HCMV se excreta en distintos fluidos corporales, dependiendo de la condición clínica del paciente, en este trabajo se propuso aislar y propagar HCMV en fibroblastos MRC-5 usando muestras de orina, lavado broncoalveolar, saliva y plasma de pacientes pediátricos. Estas muestras fueron inoculadas sobre los cultivos celulares por 14 días o hasta alcanzar un efecto citopático en el 80 % de la monocapa. El lisado celular y el sobrenadante del aislamiento se usaron para hacer pasajes virales sucesivos. Además de HCMV, el virus de herpes simple se aisló en todas las muestras de saliva. Con el empleo de los sueros positivos para HCMV se observó la formación de agregados y daño inmediato en la monocapa que impidieron su uso. Una muestra de lavado broncoalveolar indujo ECP desde la inoculación, similar al control positivo para HCMV (cepas Towne y Merlin), por lo que fue propagada y se tituló. Un segundo aislamiento viral obtenido de la orina de un paciente con infección congénita no produjo ECP a pesar de ser confirmado por PCR. En los aislamientos llevados hasta el pasaje 1, se descartó la presencia de enterovirus y adenovirus. A pesar de la evidente dificultad para aislar y propagar el HCMV, fue posible obtener un aislamiento usando un protocolo de Shell vial e inoculación modificado, y con un seguimiento prolongado del proceso

Extracellular vesicles of U937 macrophage cell line infected with DENV-2 induce activation in endothelial cells EA.hy926.

Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus