AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo

Nonviral Approaches for Neuronal Delivery of Nucleic Acids

The delivery of therapeutic nucleic acids to neurons has the potential to treat neurological disease and spinal cord injury. While select viral vectors have shown promise as gene carriers to neurons, their potential as therapeutic agents is limited by their toxicity and immunogenicity, their broad tropism, and the cost of large-scale formulation. Nonviral vectors are an attractive alternative in that they offer improved safety profiles compared to viruses, are less expensive to produce, and can be targeted to specific neuronal subpopulations. However, most nonviral vectors suffer from significantly lower transfection efficiencies than neurotropic viruses, severely limiting their utility in neuron-targeted delivery applications. To realize the potential of nonviral delivery technology in neurons, vectors must be designed to overcome a series of extra- and intracellular barriers. In this article, we describe the challenges preventing successful nonviral delivery of nucleic acids to neurons and review strategies aimed at overcoming these challenges

An overview of tissue engineering approaches for management of spinal cord injuries

Severe spinal cord injury (SCI) leads to devastating neurological deficits and disabilities, which necessitates spending a great deal of health budget for psychological and healthcare problems of these patients and their relatives. This justifies the cost of research into the new modalities for treatment of spinal cord injuries, even in developing countries. Apart from surgical management and nerve grafting, several other approaches have been adopted for management of this condition including pharmacologic and gene therapy, cell therapy, and use of different cell-free or cell-seeded bioscaffolds. In current paper, the recent developments for therapeutic delivery of stem and non-stem cells to the site of injury, and application of cell-free and cell-seeded natural and synthetic scaffolds have been reviewed

The Digital Fish Library: Using MRI to Digitize, Database, and Document the Morphological Diversity of Fish

Museum fish collections possess a wealth of anatomical and morphological data that are essential for documenting and understanding biodiversity. Obtaining access to specimens for research, however, is not always practical and frequently conflicts with the need to maintain the physical integrity of specimens and the collection as a whole. Non-invasive three-dimensional (3D) digital imaging therefore serves a critical role in facilitating the digitization of these specimens for anatomical and morphological analysis as well as facilitating an efficient method for online storage and sharing of this imaging data. Here we describe the development of the Digital Fish Library (DFL, http://www.digitalfishlibrary.org), an online digital archive of high-resolution, high-contrast, magnetic resonance imaging (MRI) scans of the soft tissue anatomy of an array of fishes preserved in the Marine Vertebrate Collection of Scripps Institution of Oceanography. We have imaged and uploaded MRI data for over 300 marine and freshwater species, developed a data archival and retrieval system with a web-based image analysis and visualization tool, and integrated these into the public DFL website to disseminate data and associated metadata freely over the web. We show that MRI is a rapid and powerful method for accurately depicting the in-situ soft-tissue anatomy of preserved fishes in sufficient detail for large-scale comparative digital morphology. However these 3D volumetric data require a sophisticated computational and archival infrastructure in order to be broadly accessible to researchers and educators

MR-guided parenchymal delivery of adeno-associated viral vector serotype 5 in non-human primate brain.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with AAV5 viral vector after parenchymal delivery. AAV5-CAG-GFP (1 × 1013 vector genomes per milliliter (vg ml-1)) was bilaterally infused either into putamen, thalamus or with the combination left putamen and right thalamus. Robust expression of GFP was seen throughout infusion sites and also in other distal nuclei. Interestingly, thalamic infusion of AAV5 resulted in the transduction of the entire corticospinal axis, indicating transport of AAV5 over long distances. Regardless of site of injection, AAV5 transduced both neurons and astrocytes equally. Our data demonstrate that AAV5 is a very powerful vector for the central nervous system and has potential for treatment of a wide range of neurological pathologies with cortical, subcortical and/or spinal cord affection

Practical Wisdom in Ancient Rome: Public Action and Contemplative Life

Producción CientíficaThis chapter covers the main moments and authors of Roman thought in the field of practical wisdom. The contents of this proposal integrate a rich legacy within coexist, not without difficulties, two ways of understanding life. The sophisticated Hellenistic philosophy, which since the end of the Second Macedonian War and after the fall of Carthage got to become more and more present in the cultural scenes of the republican Rome; and the traditional Roman perspective, more pragmatic than reflexive, more agrarian than navigating, more realistic than idealistic. After analyzing both perspectives, we present the main proposals on practical wisdom during the republican period until the Principate: the traditionalist, from the hand of Cato the Censor; the epicurean, by Lucretius; the eclectic conciliation, developed by Cicero; and, finally, the Roman Stoicism, for which we will rely on the figure of Seneca, as one of his three top representatives and also for writing in Latin, unlike Epictetus and Marcus Aurelius. We intend to offer not so much an exhaustive work, but rather a map that allows to recognize the main veins of Roman practical thought

Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Transduction patterns in the CNS following various routes of AAV-5-mediated gene delivery

Various administration routes of adeno-associated virus (AAV)-based gene therapy have been examined to target the central nervous system to answer the question what the most optimal delivery route is for treatment of the brain with certain indications. In this study, we evaluated AAV5 vector system for its capability to target the central nervous system via intrastriatal, intrathalamic or intracerebroventricular delivery routes in rats. AAV5 is an ideal candidate for gene therapy because of its relatively low level of existing neutralizing antibodies compared to other serotypes, and its broad tissue and cell tropism. Intrastriatal administration of AAV5-GFP resulted in centralized localized vector distribution and expression in the frontal part of the brain. Intrathalamic injection showed transduction and gradient expression from the rostral brain into lumbar spinal cord, while intracerebroventricular administration led to a more evenly, albeit relatively superficially distributed, transduction and expression throughout the central nervous system. To visualize the differences between localized and intra-cerebral spinal fluid administration routes, we compared intrastriatal to intracerebroventricular and intrathecal administration of AAV5-GFP. Together, our results demonstrate that for efficient transgene expression, various administration routes can be applied.Cellular mechanisms in basic and clinical gastroenterology and hepatolog