RESUMEN
In genome-scale RNA interference (RNAi) screens, it is critical to control false positives and false negatives statistically. Traditional statistical methods for controlling false discovery and false nondiscovery rates are inappropriate for hit selection in RNAi screens because the major goal in RNAi screens is to control both the proportion of short interfering RNAs (siRNAs) with a small effect among selected hits and the proportion of siRNAs with a large effect among declared nonhits. An effective method based on strictly standardized mean difference (SSMD) has been proposed for statistically controlling false discovery rate (FDR) and false nondiscovery rate (FNDR) appropriate for RNAi screens. In this article, the authors explore the utility of the SSMD-based method for hit selection in RNAi screens. As demonstrated in 2 genome-scale RNAi screens, the SSMD-based method addresses the unmet need of controlling for the proportion of siRNAs with a small effect among selected hits, as well as controlling for the proportion of siRNAs with a large effect among declared nonhits. Furthermore, the SSMD-based method results in reasonably low FDR and FNDR for selecting inhibition or activation hits. This method works effectively and should have a broad utility for hit selection in RNAi screens with replicates.
Asunto(s)
Genoma/genética , Genómica/métodos , Interferencia de ARN , Línea Celular Tumoral , Diabetes Mellitus/genética , Reacciones Falso Negativas , Reacciones Falso Positivas , Humanos , Enfermedades del Sistema Nervioso/genética , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los ResultadosRESUMEN
A longitudinal study of viral reservoirs in children initiating highly active antiretroviral therapy (HAART) in early infancy was undertaken to test the hypothesis that early effective treatment affects the persistence of replication-competent viral latency and the evolution of HIV-1 in resting CD4(+) T cells. An end point dilution culture assay was used to measure the frequencies of latently-infected resting CD4(+) T cells harboring replication-competent virus in early and late treated children. Gag, pol, and env also were sequenced and compared to pretreatment sequences. HIV-1-specific humoral and cellular immune responses were also assessed. Blood samples were obtained from 12 HIV-1-infected children who started HAART at a median of 1.9 months of age and who maintained suppression of HIV-1 replication for up to 5.5 years. Replication-competent HIV-1 was recovered from 10/12 (84%) subjects. Evolution in gag, pol, and env was restricted for years in early-treated children. HAART initiated from early infancy does not prevent the establishment of a reservoir of latent provirus, but does significantly limit the evolution of HIV-1 in viral reservoirs. The effect of early therapy on HIV-1 evolution may have implications for long-term pharmacologic control of HIV-1.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/virología , Evolución Molecular , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Latencia del Virus/fisiología , Replicación Viral/fisiología , Preescolar , Esquema de Medicación , Genes env/genética , Genes pol/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Lactante , Recién Nacido , Estudios Longitudinales , Filogenia , ARN Viral/sangre , Carga Viral , Latencia del Virus/genéticaRESUMEN
Highly active antiretroviral therapy (HAART) can suppress plasma human immunodeficiency virus type 1 (HIV-1) levels to below the detection limit of ultrasensitive clinical assays. However, HIV-1 persists in cellular reservoirs, and in adults, persistent low-level viremia is detected with more sensitive assays. The nature of this viremia is poorly understood, and it is unclear whether viremia persists in children on HAART, particularly those who start therapy shortly after birth. We therefore developed a reverse transcriptase PCR (RT-PCR) assay that allows genotyping of HIV-1 protease even when viremia is present at levels as low as 5 copies of HIV-1 RNA/ml. We demonstrated that viremia persists in children with plasma virus levels below the limit of detection of clinical assays. Viremia was detected even in children who began HAART in early infancy and maintained such strong suppression of viremia that HIV-1-specific antibody responses were absent or minimal. The low-level plasma virus lacked protease inhibitor resistance mutations despite the frequent use of nelfinavir, which has a low mutational barrier to resistance. Protease sequences resembled those of viruses in the latent reservoir in resting CD4(+) T cells. Thus, in most children on HAART with clinically undetectable viremia, there is continued virus production without evolution of resistance in the protease gene.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Carga Viral , Enfermedad Aguda , Adolescente , Secuencia de Aminoácidos , Niño , Preescolar , Enfermedad Crónica , Femenino , Infecciones por VIH/virología , Proteasa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Viremia/tratamiento farmacológico , Viremia/virologíaRESUMEN
Latent HIV-1 persists in resting memory CD4+ T cells, even in patients receiving highly active antiretroviral therapy (HAART). It has been unclear how stable this latent reservoir is and whether its persistence reflects replenishment by low-level viremia. Here we show that even in treated patients who have had no detectable viremia for as long as 7 years, the reservoir decays so slowly (t(1/2) = 44 months) that eradication is unlikely.