Method for rapid synchronization of different growth cycles of Plasmodium falciparum in vitro and application in differential gene expression profile of 3D7 after dihydroartemisinin treatment / 中国中药杂志

Plasmodium culture in vitro is often used as an antimalarial drug evaluation model, but the lifecycle of P. falciparum culture in vitro tends to be disordered, which affects the research and evaluation of antimalarial drug mechanism in vitro. By combining magnetic bead separation method with sorbitol synchronization method, a synchronization method was constructed to quickly acquire different lifecycles of P. falciparum and obtain large amounts of parasite with a narrow synchronization window in a short period. Furthermore, the dihydroartemisinin(DHA) was used to treat the early trophozoite phase of P. falciparum 3 D7 for 4 h. Then mRNA was extracted and RNA-seq was conducted to analyze the differential expression of mRNA after drug treatment and obtain the differential gene expression profile. Differential expression of up-regulated genes and down-regulated genes was analyzed according to the screening criteria of |log_2FC|>1 and P<0.05. There, 262 genes were up-regulated and 77 genes were down-regulated. GO functional enrichment analysis of all the differentially expressed genes showed that the enrichment items mainly included cell membrane components, transporter activity, serine/threonine kinase activity, Maurer's clefts(MCs), rhoptry, antigen variation and immune evasion. The enrichment of KEGG pathway included malaria, fatty acid metabolism and peroxisome. Protein-protein interaction(PPI) analysis showed that the down-regulated genes in the modules with high degree of association included rhoptry, myosin complex, transporter and other genes related to the important life activities of malaria invasion and immune escape; the up-regulated genes were mainly related to various toxic exportins of malaria, such as PfSBP1 of MCs. qRT-PCR was used to verify the expression level of some genes, and most of the results were the same as the sequencing results. SBP1 was significantly up-regulated, while some antigenic protein expression levels were down-regulated. Above all, key molecules of DHA therapy were mainly involved in the parasites' rhoptry, transporter, antigenic variation, plasmodium exportin. These results offer us many hints to guide the further studies on mechanism of artemisinin and provide a new way for development of new antimalarial drugs.

Simultaneous determination of metformin and glipizide in human plasma by liquid chromatography-tandem mass spectrometry / 药学学报

To develop a sensitive and rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for simultaneous quantitation of metformin and glipizide in human plasma, metformin, glipizide and internal standard diphenhydramine were separated from plasma by protein precipitation with acetonitrile (containing 0.3% formic acid), then chromatographed by using a Zorbax Extend C18 column. The mobile phase consisted of acetonitrile-water-formic acid (70:30: 0.3, v/v/v), at a flow rate of 0.50 mL x min(-1). A tandem mass spectrometer equipped with atmospheric pressure chemical ionization source was used as detector and operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor/production combinations of m/z 130-->m/z 60, m/z 446-->m/z 321 and m/z 256-->m/z 167 were used to quantify metformin, glipizide and diphenhydramine, respectively. The linear concentration ranges of the calibration curves for metformin and glipizide were 2.00 - 2000 ng x mL(-1) and 1.00 - 1000 ng x mL(-1), respectively. The lower limits of quantitation of metformin and glipizide were 2.00 ng x mL(-1) and 1.00 ng x mL(-1), respectively. The method proved to be sensitive, simple and rapid, and suitable for clinical investigation of compound preparation containing metformin and glipizide.

Metabolism of roxithromycin in dogs / 药学学报

<p><b>AIM</b>To investigate the metabolic profile of roxithromycin in dogs and the effects of oral and intravenous administrations on the metabolism of roxithromycin.</p><p><b>METHODS</b>Liquid chromatography-tandem mass spectrometry (LC-MSn) was used for separation and analysis of roxithromycin and its metabolites in dog bile after an oral dose or intravenous dose of roxithromycin. The metabolites were identified by comparisons of their mass spectra and LC behaviors with the references.</p><p><b>RESULTS</b>Totally 13 metabolites were detected in dog bile, including N-demethylated derivatives, N, N-didemethylated derivatives, O-dealkylether derivatives, decladinose derivatives, and the geometric isomers of parent drug and its metabolites.</p><p><b>CONCLUSION</b>Roxithromycin underwent 4 metabolic pathways in which geometric isomerization and decladinose metabolism were found to be markedly different between the two administration routes.</p>