Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis.

[Waiting list in a public health facility in Santiago, Chile]. / Situación y abordaje de lista de espera en un establecimiento de salud público en Santiago de Chile.

INTRODUCTION: Waiting lists are a well-known problem in public healthcare systems worldwide. For instance, England had over one million people in waiting lists for elective surgical procedures in 2000. Spain had over 360 000 patients in surgical waiting lists in 2007. Chile has been trying to manage waiting times through the GES (Explicit Guarantees in Healthcare) plan, which was established by the Chilean government in 2005. Waiting lists for the guaranteed-care diseases in the GES plan had 380 000 patients at the beginning of 2010, and that number was reduced to zero in 2011. Internationally, there are some descriptive studies about waiting lists that focus on variables such as waiting times and number of patients in the list. In Chile, however, this type of study is lacking. PURPOSE: This study aims to describe the characteristics of waiting lists for medical specialties between April and October 2011. It also aims to identify the components of management models in public healthcare centers, and to identify and analyze waiting-time frames of patients referred to a secondary or tertiary healthcare public center from a public primary healthcare center. METHODS: A descriptive cross-sectional study of the waiting list for first-time consultations for medical specialties was carried out. Referred patients were described and grouped using indicators of access to healthcare and waiting time between April and October 2011. Each consultation request or referral of a new patient was included in the waiting list and analyzed. RESULTS: There were 15 935 requests for consultations; 5 717 requests were resolved, and 8 544 were not (54% of the total requests for consultation). There was a mean waiting time of 498 days for non-resolved requests for consultation, and a mean of 141 days for resolved requests. The specialties in highest demand were orthopedic surgery and ophthalmology. The main waiting-list management processes were referral and reception of requests for consultation, contact of the patient, schedule of the consultation, and the medical appointment itself. RESULTS: There was low level of resolution of the waiting list, with completion of only 35% of the requests for consultation. There was also evidence of incomplete administrative procedures, as well as long waiting periods for first-time consultations on patients who were seen, up to 150 days. Patients who were not seen had a mean waiting period of almost 500 days. The management model was insufficient during the period of the study. Resolution was not timely; there was no increase in human resources. There was neither standardization of processes nor effective biomedical prioritization.

INTRODUCCIÓN: Es conocido que el problema de las listas de espera es común en los sistemas sanitarios de financiamiento público en distintas partes del mundo. Por ejemplo, Inglaterra tenía cerca de un millón de personas en lista de espera para tratamientos electivos hospitalarios en el año 2000 y España contaba con más de 360 mil pacientes en lista de espera quirúrgica en 2007. En Chile se ha trabajado en aplicar tiempos de atención garantizados con el Plan de Garantías Explicitas en Salud (GES), impulsado por el Gobierno en el año 2005, junto con el término de la lista de espera de dicho plan durante 2011 que tenía a 380.000 pacientes en espera de atención a comienzos de 2010. A nivel internacional existen diversos estudios descriptivos de las listas de espera que abordan diferentes variables, principalmente enfocados en tiempos de espera y número de pacientes en espera. No obstante, en Chile estos estudios son claramente escasos. OBJETIVOS: Caracterizar las listas de esperas de atención ambulatoria de especialidades médicas, desde abril hasta octubre de 2011; identificar y caracterizar los componentes del modelo de gestión de lista de espera en establecimiento de salud público; y realizar medición y análisis de los distintos tramos de tiempos de espera de los usuarios derivados a un establecimiento de salud público con especialidades médicas desde la atención pública en salud. MÉTODOS: Se realizó un estudio descriptivo de corte transversal de la lista de espera de consultas nuevas de especialidades médicas, se caracterizó el listado de pacientes derivados, junto a un levantamiento de indicadores de acceso y tiempo de espera, efectuado entre los meses de abril y octubre de 2011. Se consideró cada ingreso o derivación de paciente nuevo, que fue aumentando la lista de espera y se describió su comportamiento. RESULTADOS: Se registraron 15.935 ingresos, 5.717 ingresos atendidos, 8.544 ingresos sin atender que corresponden al 54% del total ingresado, 498 días promedio de espera para ingresos sin atender y 141 días promedio de espera para ingresos atendidos. Las especialidades médicas con mayor demanda fueron traumatología y oftalmología. Los macro procesos del modelo interno de gestión en la lista de espera fueron derivación y recepción de la solicitud, contacto y agendamiento del usuario, y atención médica del usuario. CONCLUSIONES: Se observó un bajo nivel de resolución de la lista de espera, en el que se resolvió el 35% del total de solicitudes de interconsultas ingresadas. Además, se evidenció una incompleta definición de procesos administrativos, así como un alto tiempo de espera en pacientes finalmente atendidos para su primera cita que llegó hasta los 150 días. Los pacientes sin atención promediaron cerca de 500 días de espera. El modelo de gestión fue insuficiente para el periodo evaluado. No se observó oportunidad en la resolución, ni aumento de recursos humanos, ni estandarización de procesos, tampoco se evidenció priorización biomédica efectiva y constante.

A cis-acting element present within the Gag open reading frame negatively impacts on the activity of the HIV-1 IRES.

Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1.

Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors.

The 5' leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA harbors an internal ribosome entry site (IRES) that is functional during the G2/M phase of the cell cycle. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. We used 'standard' chemical and enzymatic probes and an 'RNA SHAPE' analysis to model the structure of the HIV-1 5' leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. We also assessed the impact of mutations on IRES function. Strikingly, mutations did not significantly affect IRES activity suggesting that the requirement for pre-formed stable secondary or tertiary structure within the HIV-1 IRES may not be as strict as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5' leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins.

Translation initiation of viral mRNAs.

Viruses depend on cells for their replication but have evolved mechanisms to achieve this in an efficient and, in some instances, a cell-type-specific manner. The expression of viral proteins is frequently subject to translational control. The dominant target of such control is the initiation step of protein synthesis. Indeed, during the early stages of infection, viral mRNAs must compete with their host counterparts for the protein synthetic machinery, especially for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition viruses use diverse strategies so that ribosomes can be recruited selectively to viral mRNAs. In this review we focus on the initiation of protein synthesis and outline some of the strategies used by viruses to ensure efficient translation initiation of their mRNAs.

Mutational screening of the APC gene in Chilean families with familial adenomatous polyposis: nine novel truncating mutations.

PURPOSE: Familial adenomatous polyposis is characterized by the development of hundreds of adenomatous polyps located mainly in the colon and rectum. Patients with familial adenomatous polyposis who do not receive treatment will develop cancer before aged 40 years. This disease is caused by germline mutations in the adenomatous polyposis coli gene. Different studies have shown a correlation between the location of the mutation and the clinical phenotype, such as the grade of severity and/or the presence of extracolonic manifestations, such as desmoid tumors. This study was designed to identify germline mutation in the adenomatous polyposis coli gene in Chilean families with familial adenomatous polyposis. METHODS: We examined the adenomatous polyposis coli gene in 24 Chilean families with familial adenomatous polyposis. The adenomatous polyposis coli gene was screened for mutations combining single strand conformation polymorphism technique, protein truncation test, and DNA sequencing. RESULTS: We detected 17 different truncating mutations in 21 of 24 families (87.5 percent); 9 of these were novel. Fourteen mutations were detected in exon 15, being the most frequent c.3,927_3,931delAAAGA, found in 3 of 21 families (14 percent). Eight families (33 percent) showed at least one patient affected with desmoid tumors, presenting mutations between codons 849 and 1,533. Interestingly, two mutations, c.3,632dupA and c.3,783_3,784delTT, leading into a truncating protein at codons 1,216 and 1,274, were associated with almost 100 percent penetrance for desmoid tumors among relatives. CONCLUSIONS: We achieved 87 percent mutation detection rate in adenomatous polyposis coli gene; more than 50 percent of them were novel. The high percentage of novel mutations found may be because of the genetic composition of the Chilean population, which is an admixture of Amerindian and Spaniards, and the scarce information in the literature about similar populations.