RESUMEN
The K70E mutation in HIV-1 reverse transcriptase was observed in 10% of virologic non-responders of the abacavir/lamivudine/tenofovir arm of ESS30009, alone, or in mixtures with K65R by population sequencing. Clonal analysis of six ESS30009 K70E isolates failed to identify double mutants carrying K65R+K70E. Site-directed K70E mutants had a replication capacity of 97+/-29%, but only 2.4+/-0.9% for K65R+K70E and 0.01% for K65R+K70E+M184V mutants. K65R+K70E phenotypic fold changes for abacavir, lamivudine and tenofovir were comparable to reported values for K65R alone. In molecular dynamic simulations, the epsilon-amino group of K65 was positioned 2.7+/-0.1A from the gamma-phosphate of the dTTP ligand and stabilized the triphosphate. In the R65 mutant, this distance increased to 4.2+/-0.4A and the interaction energy with the ligand was less favorable, but the K70 epsilon-amino group was repositioned closer to the gamma-phosphate and had a more favorable interaction energy. In the double mutant, E70 could not stabilize the gamma-phosphate, resulting in a more severe defect. The net effect of the atomic-level changes in the double mutant may be to destabilize the pyrophosphate leaving group of the ligand, more severely affecting the catalytic rate of the polymerization reaction than the R65 single mutation.
Asunto(s)
Adenina/análogos & derivados , Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral Múltiple/genética , Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , Organofosfonatos/uso terapéutico , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Adenina/farmacología , Adenina/uso terapéutico , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Terapia Antirretroviral Altamente Activa , Simulación por Computador , Didesoxinucleósidos/farmacología , Didesoxinucleósidos/uso terapéutico , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , Humanos , Lamivudine/farmacología , Lamivudine/uso terapéutico , Mutagénesis Sitio-Dirigida , Organofosfonatos/farmacología , Inhibidores de la Transcriptasa Inversa/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , TenofovirRESUMEN
Fungal (mold) contamination is an important indicator of low-quality raw product used in food processing operations. Fluorescent-labeled lectins, specific for chitin, have been shown to be valuable for quantitative detection of mold in raw tomatoes. In this research, the response of individual fungal species to a rapid fluorescent lectin assay was investigated. Ten of the most common mold species were grown on two types of artificial broth media, and added to blended field tomatoes. The assay was conducted on each species, and linear regressions were developed, comparing the fluorescent lectin assay score with the fungal dry weight. The assay was able to detect all molds at sensitivities required for the tomato industry, and had high linearity (r2 ranging from 0.72 to 0.99) and low variability (standard error of calibration ranging from 20 to 116 microg of fungal biomass/ml of tomato juice) for individual species grown on V-8 juice broth.