Development of an HPLC method using relative molar sensitivity for the measurement of blood concentrations of nine pharmaceutical compounds.

We developed a reliable high-performance liquid chromatographic analysis method using a relative molar sensitivity (RMS) technique that does not require an authentic, identical reference analyte material to quantify blood serum carbamazepine, phenytoin, voriconazole, lamotrigine, meropenem, mycophenolic acid, linezolid, vancomycin, and caffeine levels for routine blood concentration measurements. Carbamazepine and caffeine were also used as non-analyte reference materials to calculate the RMS of each analyte. The RMS was calculated from the ratio of the slope of the calibration equation (analyte/non-analyte reference material), then used to quantify analytes in control serum samples spiked with carbamazepine, phenytoin, voriconazole, meropenem, mycophenolic acid, linezolid or vancomycin. In addition, the concentrations of these six drugs in control serum samples determined by the proposed RMS method agreed well with that obtained using a conventional method. The proposed RMS method is a promising tool for the clinical determination of nine drugs, given the accuracy, precision, and efficiency of quantifying these analytes.

Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.

First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.