Novel zebrafish polycystic kidney disease models reveal functions of the Hippo pathway in renal cystogenesis.

The Hippo signaling pathway is a kinase cascade that plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription coactivators Yap1/Wwtr1 are regulated by the upstream kinase Stk3. Recent studies in mammals have implicated the Hippo pathway in kidney development and kidney diseases. To further illustrate its roles in vertebrate kidney, we generated a series of zebrafish mutants targeting stk3, yap1 and wwtr1 genes. The stk3-/- mutant exhibited edema, formation of glomerular cysts and pronephric tubule dilation during the larval stage. Interestingly, disruption of wwtr1, but not yap1, significantly alleviated the renal phenotypes of the stk3-/- mutant, and overexpression of Wwtr1 with the CMV promoter also induced pronephric phenotypes, similar to those of the stk3-/- mutant, during larval stage. Notably, adult fish with Wwtr1 overexpression developed phenotypes similar to those of human polycystic kidney disease (PKD). Overall, our analyses revealed roles of Stk3 and Wwtr1 in renal cyst formation. Using a pharmacological approach, we further demonstrated that Stk3-deficient zebrafish could serve as a PKD model for drug development.

Simultaneous Determination of Coumarin and Its Derivatives in Tobacco Products by Liquid Chromatography-Tandem Mass Spectrometry.

In this paper an analytical method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the determination of coumarin and its derivatives in tobacco products was developed. The MS/MS fragmentation pathways of the eight coumarins were elucidated. The new analytical method was defined based on two main axes, an extraction procedure with acetonitrile and analyte detection performed by HPLC-MS/MS in electron impact mode. The excellent selectivity and sensitivity achieved in multiple reaction monitoring (MRM) mode allowed satisfactory confirmation and quantitation for the coumarin flavor additives. Under the optimized gradient elution conditions, it took only 4.5 min to separate all eight coumarins. Good linearity for all the analytes were confirmed by the correlation coefficient r², ranging from 0.9987 to 0.9996. The limits of detection (LODs) and limits of quantitation (LOQs) of these compounds were in the range of 0.5-1.7 µg/kg and 1.7-5.2 µg/kg, respectively. The average recoveries at three spiked levels (LOQ, 1.5LOQ, 2LOQ) were all in the range of 69.6%-95.1% with RSDs (n = 6) lower than 5.3%. The method of HPLC-MS/MS developed in this study was initially applied to the research of coumarin flavor additives in tobacco products collected from the located market in Beijing from China and proved to be accurate, sensitive, convenient and practical.

[Determination of catechol in tobacco by high performance liquid chromatography-tandem mass spectrometry].

An analytical method for the determination of catechol in tobacco by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed. A Sep-Park-C18 solid phase extraction cartridge was used for the enrichment of the analyte for HPLC-MS/MS analysis. The mobile phase was methanol-0.2% (v/v) formic acid with gradient elution. The sample was analyzed by HPLC-MS/MS in the ESI-scanning mode with multi-reaction monitoring (MRM) for qualitative and quantitative analyses. The linear range of calibration curve was 0.5-200 µg/kg with good correlation coefficients (r2 = 0.998 9). The recoveries of catechol spiked in three levels were in the range of 83.1%-98.6%, with the relative standard deviations of 1.9%-5.8%. This method was initially applied to the research of catechol as a flavor additive in six retail tobacco samples and proved to be accurate, sensitive, convenient and practical. The analyte was found in the six retail tobacco samples, and in some cases, the presence of quite high concentrations of catechol in tobacco should be a matter of concern.