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Abstract Background In-hospital therapeutic drug monitoring (TDM) requires a suitable quantification method for target drugs from the viewpoint of precision, throughput, and testing costs. We previously developed a practical HPLC-UV platform for quantification of serum levels of various drugs. In this report, the platform was effectively applied to the quantification of patient serum levels of five different drugs by clinical professionals in our hospital during their daily work. Methods The residual sera of patients receiving carbamazepine (CBZ), phenytoin (PHT), lamotrigine (LTG), vancomycin (VCM), or voriconazole (VRCZ) were used in the present clinical study. The quantification method for each drug consisted of rapid solid-phase extraction (SPE) of each drug in the patient serum, followed by optimized HPLC-UV analysis of the drug in the SPE eluate. Furthermore, patient serum levels of PHT, CBZ, and VCM were also measured by ligand-binding assay using a cobas® analyzer in our hospital, and those of LTG and VRCZ were measured by HPLC-MS/MS at an outsourced provider. Passing–Bablok regression analysis and Bland–Altman analysis were employed to analyze the agreement of drug levels in patient sera, which was separately quantified using two different methods—our HPLC-UV platform and the cobas analyzer, or HPLC-UV and HPLC-MS/MS. Results All analytical conditions of the present method using our HPLC-UV platform were well optimized for each target drug quantification in the patient’s serum, and the quantification method for each drug was fully validated for accuracy, precision and reproducibility. Furthermore, Passing–Bablok regression analysis and Bland–Altman analysis revealed that patient serum levels of PHT, CBZ, and VCM quantified by our HPLC-UV platform were closely correlated with those quantified by the cobas® analyzer, and the levels of LTG and VRCZ quantified by our HPLC-UV platform were also correlated with those quantified by HPLC-MS/MS. Conclusions Our HPLC-UV platform can be performed without requiring special analytical techniques. This platform is expected to be used for the measurement of blood levels of multiple drugs for in-hospital routine TDM.

Micrococcal nuclease (MNase) has been widely used for analyses of nucleosome locations in many organisms. However, due to its sequence preference, the interpretations of the positions and occupancies of nucleosomes using MNase have remained controversial. Next-generation sequencing (NGS) has also been utilized for analyses of MNase-digests, but some technical biases are commonly present in the NGS experiments. Here, we established a gel-based method to map nucleosome positions in Saccharomyces cerevisiae, using isolated nuclei as the substrate for the histone H4 S47C-site-directed chemical cleavage in parallel with MNase digestion. The parallel mapping allowed us to compare the chemically and enzymatically cleaved sites by indirect end-labeling and primer extension mapping, and thus we could determine the nucleosome positions and the sizes of the nucleosome-free regions (or nucleosome-depleted regions) more accurately, as compared to nucleosome mapping by MNase alone. The analysis also revealed that the structural features of the nucleosomes flanked by the nucleosome-free region were different from those within regularly arrayed nucleosomes, showing that the structures and dynamics of individual nucleosomes strongly depend on their locations. Moreover, we demonstrated that the parallel mapping results were generally consistent with the previous genome-wide chemical mapping and MNase-Seq results. Thus, the gel-based parallel mapping will be useful for the analysis of a specific locus under various conditions.

In this study, we investigated the interaction between apple polyphenols (AP; mainly consisting of procyanidin (PC) from an apple) and staphylococcal enterotoxin A (SEA), and the inhibitory effects of AP on SEA activity. According to the degree of polymerization, in particularly highly polymerized PC (more than pentamer) strongly interacted with SEA. The binding affinity of AP with SEA molecules was determined using Biacore analysis. AP reacted with SEA immobilized on a Biacore sensor chip. After treatment with pepsin and pancreatin, to examine the changes of binding affinity of AP in intragastric conditions, AP maintained interaction with SEA. We examined whether AP inhibits the proliferation and interferon-γ (IFN-γ) production induced by SEA in mouse spleen cells. AP strongly inactivated the proliferation and IFN-γ production induced by SEA. These results suggest that AP, which has a higher degree of polymerization, inactivates stronger biological activity of SEA through interaction with SEA. Our studies are the first to demonstrate the relationship between the degree of polymerization of AP and the inhibitory effects on SEA activities.