BackgroundThe success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.MethodologyWe developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.SignificanceCombined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.
myo-Inositol is being investigated as a biomarker to monitor disease states involving the central nervous system. We have developed and validated a quantitative method to study endogenous myo-inositol metabolism in rat brain tissue. Tissue samples were homogenized, and their myo-inositol content was determined using spiked calibration curves and mass spectrometry. The assay was validated on an LC/MS/MS platform, and specificity was evaluated using accurate mass measurements. A novel chiral LC/MS/MS method was also developed to resolve myo-inositol from other endogenous inositol epimers and confirm the selectivity of the quantitative procedure. The validated method is selective, convenient, precise (<15% RSD), accurate (<15% RE), and sensitive over a linear range of 0.100-100 microg/mL. This method could potentially be used as an instrument for monitoring pathological conditions related to psychotherapeutics, as well as a tool for screening curative pharmaceuticals for efficacy.
Differences in flavor attributable to age of animal and to sex were detected when broth from samples of lamb and yearling mutton meat were served to panel members in triangle tests. No differences were detected in slices of roasted, broiled, or braised meat scored by the panel. Broth from the lean meat of lamb (7 to 8 months) was preferred to that prepared from lean of 15 to 16-month-old yearling mutton carcasses. Differences in flavor intensity of wether and ram meat served as patties in triangle tests were present only in patties containing 20% added fat. Full natural flavor of slices of cooked meat was not associated with either the cover fat thickness of the cut or with the fat content of the muscle.
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