Multifunctional magnetoliposomes as drug delivery vehicles for the potential treatment of Parkinson's disease.

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.

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.

Plataformas de secuenciación de nueva generación y proteómica aplicadas a la virología vegetal: ¿Cómo ha avanzado Colombia? / Next generation sequencing and proteomics in plant virology: how is colombia doing?

ABSTRACT 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.

RESUMEN 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.

Assembly and release of Dengue virus: Role of Alix protein / Ensamblaje y liberación del virus dengue: controversia sobre la participación de la proteína alix

RESUMEN Algunos virus envueltos usurpan la maquinaria celular ESCRT (complejo de clasificación endosomal requerido para el transporte) para llevar a cabo funciones como la transcripción, la traducción, el ensamblaje y la liberación de partículas virales desde las células huésped. Aunque esta estrategia ha sido estudiada principalmente en retrovirus, son varios los virus envueltos que la usan. El objetivo del trabajo fue explorar la participación de una proteína accesoria de ESCRT, la proteína Alix, en la transcripción, traducción, ensamblaje y liberación del virus dengue (DENV), así como su interacción con la proteína viral NS3. Células A549 infectadas con DENV2 fueron tratadas con pequeños ARN de interferencia (siRNA) para disminuir la expresión ("knock-down") de la proteína Alix. Simultáneamente, se obtuvo una línea A549 que expresaba una proteína NS3 recombinante y sobre este sistema se hicieron ensayos de inmunoprecipitación y "pull-down" para detectar interacción entre NS3 y Alix. Los resultados mostraron que el "knock-down" de Alix no tuvo efecto notable en la transcripción o la traducción viral, pero sí en el ensamblaje y la liberación de DENV2, mientras que los ensayos de "pull-down" revelaron la interacción entre NS3 y Alix. La participación de Alix en la producción de DENV2 y su interacción con NS3 constituyen un potencial blanco para el diseño de estrategias dirigidas a controlar la propagación de DENV.

ABSTRACT Since the finding that HIV recruits cellular ESCRT (endosomal sorting complexes required for transport) machinery to accomplish viral budding, this strategy has emerged as an escape route for enveloped viruses also. The work aimed to explore the participation of the cellular protein Alix (a human protein that acts as an adapter in the ESCRT pathway) on the transcription, protein expression, assembly and release of Dengue virus (DENV), and explore for its potential interaction with the viral protein NS3. To this purpose, A549 cells were infected with DENV2 and treated with small interfering RNAs (siRNA) to generate an Alix stable knockdown cells line. Also, an A549 cells line expressing a histidine-tagged NS3 protein was obtained. Both cells lines were used in immunoprecipitation and pull-down assays to assess the interaction between NS3 and Alix. The results showed that Alix knockdown had no effect on viral transcription or viral protein expression but influenced the assembly and release of DENV2 negatively. Finally, pull-down assays revealed the interaction between NS3 and Alix. The finding of an Alix participation in the production of DENV2 and its interaction with NS3 provides a potential target for the design of control/inhibition strategies against DENV spread.

Aislamiento de citomegalovirus humano a partir de fluidos corporales / Isolation of the human cytomegalovirus from bodily fluids

ABSTRACT 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.

RESUMEN 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.

In vivo infection by a neuroinvasive neurovirulent dengue virus.

Although neurological manifestations associated with dengue infections have been reported in endemic countries, the viral or host characteristics determining the infection or alteration of nervous function have not been described. In order to investigate neurobiological conditions related to central nervous system dengue virus (DENV) infection, we established a mouse model of neuroinfection. A DENV-4 isolate was first adapted to neuroblastoma cells, later inoculated in suckling mice brain, and finally, this D4MB-6 viral variant was inoculated intraperitoneally in Balb/c mice at different postnatal days (pnd). Virus-induced fatal encephalitis in 2 and 7 pnd mice but infected at 14 and 21 pnd mice survived. The younger mice presented encephalitis at the sixth day postinfection with limb paralysis and postural instability concomitant with efficient viral replication in brain. In this mice model, we found activated microglial cells positive to viral antigen. Neurons, oligodendrocytes, and endothelial cells were also infected by the D4MB-6 virus in neonatal mice, which showed generalized and local plasma leakage with blood-brain barrier (BBB) severe damage. These results suggest that there was a viral fitness change which led to neuroinfection only in immune or neurological immature mice. Infection of neurons, endothelial, and microglial cells may be related to detrimental function or architecture found in susceptible mice. This experimental neuroinfection model could help to have a better understanding of neurological manifestations occurring during severe cases of dengue infection.