RESUMEN
In this direct replication of Mueller and Oppenheimer's (2014) Study 1, participants watched a lecture while taking notes with a laptop (n = 74) or longhand (n = 68). After a brief distraction and without the opportunity to study, they took a quiz. As in the original study, laptop participants took notes containing more words spoken verbatim by the lecturer and more words overall than did longhand participants. However, laptop participants did not perform better than longhand participants on the quiz. Exploratory meta-analyses of eight similar studies echoed this pattern. In addition, in both the original study and our replication, higher word count was associated with better quiz performance, and higher verbatim overlap was associated with worse quiz performance, but the latter finding was not robust in our replication. Overall, results do not support the idea that longhand note taking improves immediate learning via better encoding of information.
Asunto(s)
Aprendizaje , Microcomputadores , HumanosRESUMEN
Human immunodeficiency virus (HIV)-1 remains as a global health issue that is primarily treated with highly active antiretroviral therapy, a combination of drugs that target the viral life cycle. One class of these drugs are non-nucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral reverse transcriptase (RT). First generation NNRTIs were troubled with poor pharmacological properties and drug resistance, incentivizing the development of improved compounds. One class of developed compounds are the 2-naphthyl phenyl ethers, showing promising efficacy against the Y181C RT mutation. Further biochemical and structural work demonstrated differences in potency against the Y181C mutation and binding mode of the compounds. This work aims to understand the relationship between the binding mode and ability to overcome drug resistance using macromolecular x-ray crystallography. Comparison of 2-naphthyl phenyl ethers bound to Y181C RT reveal that compounds that interact with the invariant W229 are more capable of retaining efficacy against the resistance mutation. Additional modifications to these compounds at the 4-position, computationally designed to compensate for the Y181C mutation, do not demonstrate improved potency. Ultimately, we highlight important considerations for the development of future HIV-1 drugs that are able to combat drug resistance.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , VIH-1/enzimología , Mutación Missense , Éteres Fenílicos/química , Inhibidores de la Transcriptasa Inversa/química , Sustitución de Aminoácidos , Sitios de Unión , Transcriptasa Inversa del VIH/genética , VIH-1/genéticaRESUMEN
A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Recent reports of tenofovir toxicity in patients taking ART for HIV cannot be explained solely on the basis of off-target inhibition of mitochondrial DNA polymerase gamma (Polγ). PrimPol was discovered as a primase-polymerase localized to the mitochondria with repriming and translesion synthesis capabilities and, therefore, a potential contributor to mitochondrial toxicity. We established a possible role of PrimPol in tenofovir-induced toxicity in vitro and show that tenofovir-diphosphate incorporation by PrimPol is dependent on the n-1 nucleotide. We identified and characterized a PrimPol mutation, D114N, in an HIV+ patient on tenofovir-based ART with mitochondrial toxicity. This mutant form of PrimPol, targeting a catalytic metal ligand, was unable to synthesize primers, likely due to protein instability and weakened DNA binding. We performed cellular respiration and toxicity assays using PrimPol overexpression and shRNA knockdown strains in renal proximal tubular epithelial cells. The PrimPol-knockdown strain was hypersensitive to tenofovir treatment, indicating that PrimPol protects against tenofovir-induced mitochondrial toxicity. We show that a major cellular role of PrimPol is protecting against toxicity caused by ART and individuals with inactivating mutations may be predisposed to these effects.
Asunto(s)
ADN Primasa/genética , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , Mutación , Tenofovir/toxicidad , Animales , Biocatálisis , ADN Primasa/química , ADN Primasa/deficiencia , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/deficiencia , Estabilidad de Enzimas , Técnicas de Silenciamiento del Gen , Humanos , Riñón/efectos de los fármacos , Cinética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Moleculares , Enzimas Multifuncionales/química , Enzimas Multifuncionales/deficiencia , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.