Dicistronic MLV-retroviral vectors transduce neural precursors in vivo and co-express two genes in their differentiated neuronal progeny

Dicistronic MLV-based retroviral vectors, in which two IRESes independently initiate the translation of two proteins from a single RNA, have been shown to direct co-expression of proteins in several cell culture systems. Here we report that these dicistronic retroviral vectors can drive co-expression of two gene products in brain cells in vivo. Injection of retroviral vector producer cells leads to the transduction of proliferating precursors in the external granular layer of the cerebellum and throughout the ventricular regions. Differentiated neurons co-expressing both transgenes were observed in the cerebellum and in lower numbers in distant brain regions such as the cortex. Thus, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo and maintaining the expression of genes after cell differentiation

Mechanism of HIV-1 Tat RNA translation and its activation by the Tat protein

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) Tat protein is a major viral transactivator required for HIV-1 replication. In the nucleus Tat greatly stimulates the synthesis of full-length transcripts from the HIV-1 promoter by causing efficient transcriptional elongation. Tat induces elongation by directly interacting with the bulge of the transactivation response (TAR) RNA, a hairpin-loop located at the 5'-end of all nascent viral transcripts, and by recruiting cellular transcriptional co-activators. In the cytoplasm, Tat is thought to act as a translational activator of HIV-1 mRNAs. Thus, Tat plays a central role in the regulation of HIV-1 gene expression both at the level of mRNA and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of Tat can be made early on in HIV-1 infected cells to sustain its own synthesis. To address this issue we studied translation of the Tat mRNA <it>in vitro </it>and in human cells using recombinant monocistronic and dicistronic RNAs containing the 5' untranslated region (5'-UTR) of Tat RNA.</p> <p>Results</p> <p>This study shows that the Tat mRNA can be efficiently translated both <it>in vitro </it>and in cells. Furthermore, our data suggest that translation initiation from the Tat mRNA probably occurs by a internal ribosome entry site (IRES) mechanism. Finally, we show that Tat protein can strongly stimulate translation from its cognate mRNA in a TAR dependent fashion.</p> <p>Conclusion</p> <p>These results indicate that Tat mRNA translation is efficient and benefits from a feedback stimulation by the Tat protein. This translational control mechanism would ensure that minute amounts of Tat mRNA are sufficient to generate enough Tat protein required to stimulate HIV-1 replication.</p

Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed

Correlation between female sex, IL28B genotype, and the clinical severity of bronchiolitis in pediatric patients

Background: Single-nucleotide polymorphisms (SNPs) that impact on the differential expression of interleukin 28B (IL28B) are implicated in the progression of viral-induced diseases. In this prospective longitudinal cohort study, we evaluated the association between IL28B SNPs rs12979860 and rs8099917 and the clinical outcome of bronchiolitis in pediatric patients. Methods: A total of 682 infants suffering from bronchiolitis, categorized based on the final clinical outcome as mild or severe, were genotyped for IL28B SNPs rs12979860 and rs8099917. Results: When infants were categorized exclusively based on the final clinical outcome, no association was established between IL28B SNPs and the severity of bronchiolitis. However, when stratified by sex, the homozygotes for the minor alleles of rs12979860 (T) and rs8099917 (G) were associated with a mild disease in girls but not in boys. Conclusion: SNPs rs12979860 and rs8099917 correlate with the severity of bronchiolitis and display a sex bias, where GG rs8099917 and TT rs12979860 genotypes are associated with a mild disease in girls but not in boys. These findings suggest that innate immunity and female sex links with the outcome of the diseases induced by respiratory viruses, such as RSV. © 2019, International Pediatric Research Foundation, Inc.Indexación: Scopu

Nota a Director del Instituto Argentino de Radioastronomía (IAR), Dr. Marcelo Arnal

Nota a Director del Instituto Argentino de Radioastronomía (IAR),Dr. Marcelo Arnal acerca de la radioastronomía

Translation initiation of the HIV-1 mRNA

International audienceTranslation initiation of the full-length mRNA of the human immunodeficiency virus can occur via several different mechanisms to maintain production of viral structural proteins throughout the replication cycle. HIV-1 viral protein synthesis can occur by the use of both a cap-dependant and IRES-driven mechanism depending on the physiological conditions of the cell and the status of the ongoing infection. For both of these mechanisms there is a need for several viral and cellular co-factors for optimal translation of the viral mRNA. In this review we will describe the mechanism used by the full-length mRNA to initiate translation highlighting the role of co-factors within this process. A particular emphasis will be given to the role of the DDX3 RNA helicase in HIV-1 mRNA translation initiation

A detection method for infectious pancreatic necrosis virus (IPNV) based on reverse transcription (RT)‐polymerase chain reaction (PCR)

A rapid, sensitive and highly specific detection method for infectious pancreatic neerosis virus (IPNV), based on reverse transcription (RT) polymerase chain reaction (PCR) has been developed. The specificity of the assay is provided by the oligonucleotide primers selected from the IPNV major capsid polypeptide VP2 gene. For each primer combination only one major product is obtained when amplifieation is performed using IPNV double‐stranded RNA from two different viral strains, Sp and VR‐299, as the initial template. No products were detected when genomie nueleic acids other than IPNV RNA were used as RT‐PCR templates. The specificity of the amplification products were confirmed by Southern hybridization using a specific cDNA probe. To assess the sensitivity of the method, dilutions of purified IPNV dsRNA total genome were amplified and quantities of as little as 1 pg of purified dsRNA were detected when the amplification product was visualized by silver‐stained polyacrylamidc gel electrophoresis. This technique detected IPNV directly in infected coho salmon, Oncorhynchus kisutch (Walbaum), and rainbow trout, Oncorhynchus mykiss (Walbaum), tissues and fish egg samples, avoiding viral propagation in cell culture. The results show that this RT‐PCR amplification method is useful for the direct tissue detection of IPN

RNA-Binding Proteins as Regulators of Internal Initiation of Viral mRNA Translation

Viruses are obligate intracellular parasites that depend on the host&rsquo;s protein synthesis machinery for translating their mRNAs. The viral mRNA (vRNA) competes with the host mRNA to recruit the translational machinery, including ribosomes, tRNAs, and the limited eukaryotic translation initiation factor (eIFs) pool. Many viruses utilize non-canonical strategies such as targeting host eIFs and RNA elements known as internal ribosome entry sites (IRESs) to reprogram cellular gene expression, ensuring preferential translation of vRNAs. In this review, we discuss vRNA IRES-mediated translation initiation, highlighting the role of RNA-binding proteins (RBPs), other than the canonical translation initiation factors, in regulating their activity

Diseño del sistema de control interno para los departamentos de cartera y tesorería de la empresa ROOTT+CO S.A.S.

Un buen diseño de control interno le permitirá a la empresa ROOTT+CO medir y comprobar la eficiencia de su gestión en cuanto al logro de los objetivos planteados, aportando un nivel de seguridad prudente en la consecución de los mismos; también le permitirá optimizar sus procedimientos, salvaguardar sus recursos y garantizar el buen desempeño de sus funcionarios, razón por la cual se crea la necesidad de diseñar un sistema de Control Interno para los departamentos de cartera y tesorería, partiendo desde el diagnostico de los factores internos y externos de acuerdo a las debilidades, oportunidades, fortalezas y amenazas; seguidamente, pasando a la caracterización de estas dos áreas, la cual se realiza de acuerdo al método de investigación cualitativa, donde se inicia por medio de observación, entrevistas y checklist aplicados al gerente, jefe de cartera y tesorería y auxiliares de las áreas, luego se propone un modelo de políticas y procedimientos para estas áreas en estudio y finalmente, se presenta una herramienta de evaluación que le permitirá a la empresa gestionar y ajustar sus estrategias desde corto a largo plazo. Como resultado del análisis anterior se establece que la empresa ROOTT+CO presenta una deficiente gestión de controles en el área de cartera y tesorería, lo cual impide el cumplimientos de sus objetivos, igualmente, se identifica que existen debilidades en la capacitación del personal, no cuentan con políticas de crédito y tesorería documentadas, no hay procedimientos documentados de estas dos áreas y tampoco cuentan con un sistema de evaluación que le permita medir el cumplimiento de los objetivos o estrategias planteadas para las áreas en estudio.A good internal control design will allow the company ROOTT + CO to measure and verify the efficiency of its management in terms of achieving the objectives set, providing a level of prudent security in achieving them; It will also allow you to optimize your procedures, safeguard your resources and ensure the good performance of your employees, which is why you create the need to design an Internal Control system for the portfolio and treasury departments, starting from the diagnosis of internal factors and external according to weaknesses, opportunities, strengths and threats; then, going on to the characterization of these two areas, which is done according to the qualitative research method, where it starts by means of observation, interviews and checklists applied to the manager, portfolio manager and treasury and area assistants, then a model of policies and procedures is proposed for these areas under study and finally, an evaluation tool is presented that will allow the company to manage and adjust its strategies from short to long term. As a result of the previous analysis, it is established that the ROOTT + CO company has a poor management of controls in the portfolio and treasury area, which prevents the fulfillment of its objectives. It also identifies weaknesses in the training of personnel, not they have documented credit and treasury policies, there are no documented procedures for these two areas and they do not have an evaluation system that allows them to measure compliance with the objectives or strategies proposed for the areas under study.Introducción. -- 1. Objetivos. -- 2. Marco Referencial. -- 2.1. Marco Teórico. -- 3. Presentación del Caso. -- 4. Antecedentes del Caso. -- 5. Literatura Sobre Casos Análogos. -- 6. Análisis e Interpretación de la Información. -- 6.1. Diagnostico Sistema de Control Interno. -- 6.2. Caracterización Sistema de Control Interno. -- 6.3. Propuesta Sistema de Control Interno. -- 6.4. Presentación del Modelo de Cuadro de Mando Integral. -- 7. Conclusiones. -- 8. Bibliografí[email protected]@[email protected]