Development and Validation of an RNA Sequencing Assay for Gene Fusion Detection in Formalin-Fixed, Paraffin-Embedded Tumors.

RNA sequencing (RNA-seq) is a well-validated tool for detecting gene fusions in fresh-frozen tumors; however, RNA-seq is much more challenging to use with formalin-fixed, paraffin-embedded (FFPE) tumor samples. We evaluated the performance of RNA-seq to detect gene fusions in clinical FFPE tumor samples. Our assay identified all 15 spiked-in NTRK fusions from RNA reference material and six known fusions from five cancer cell lines. Limit of detection for the assay was assessed with a series of dilutions of RNA from the cell line H2228. These fusions can be detected when the dilution is down to 10%. Good intra-assay and interassay reproducibility was observed in three specimens. For clinical validation, the assay detected 10 of 12 fusions initially identified by a DNA panel (covering 23 gene fusions) in clinical specimens (83.3% sensitivity), whereas one fusion (MET fusion) was identified in another 34 fusion-negative tumor specimens as determined by the DNA panel (negative prediction value of 94.3%). This MET fusion was confirmed by RT-PCR and Sanger sequencing, which found that this is a false-negative result for the DNA panel. The assay also identified 26 extra fusions not covered by the DNA panel, 20 (76.9%) of which were validated by RT-PCR and Sanger sequencing. Therefore, this RNA assay has reasonable performance and could complement DNA-based next-generation sequencing assays.

Two new isoflavones from the seeds of Psoralea corylifolia with diacylglycerol acyltransferase inhibitory activity.

Two new compounds 7-hydroxy-4'-methoxy-3'-(7''-hydroxy-3'',7''-dimethyl-octa-2'',5''-dienyl)-isoflavone (1) and 7-hydroxy-4'-methoxy-3'-(6''-hydroxy-3'',7''-dimethyl-octa-2'',7''-dienyl)-isoflavone (2), together with five known compounds (3-7), were isolated from EtOAc-soluble extract of the seeds of Psoralea corylifolia. Their structures were elucidated on the basis of detailed spectroscopic and physico-chemical analyses. All the isolates were evaluated for in vitro inhibitory activity against diacylglycerol acyltransferase (DGAT). And compounds 1-7 displayed significant inhibitory activity on DGAT1 with IC50 values ranging from 51.2 ± 1.1 to 116.4 ± 1.3 µM.

Simultaneous determination of idelalisib and its metabolite GS-563117 in dog plasma by LC-MS/MS: Application to a pharmacokinetic study.

The purpose of this study was to develop and validate an LC-MS/MS method for simultaneous determination of idelalisib and GS-563117 in dog plasma. The analytes were extracted using ethyl acetate and then separated on a Waters Acquity UPLC BEH C18 column (50 × 2.1 mm, i. d., 1.7 µm) using 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.3 mL/min in gradient elution mode. The analytes were quantified using selected reaction monitoring with precursor-to-product transitions at m/z 416.2 → 176.1, m/z 432.2 → 192.1 and m/z 421.2 → 176.1 for idelalisib, GS-563117 and [2 H5 ]-idelalisib (internal standard). The assay showed good linearity (r > 0.9992) over the tested concentration range of 0.1-600 ng/mL for idelalisib and 0.1-300 ng/mL for GS-563117. The intra- and inter-day RSD values for idelalisib and GS-563117 were <8.84 and 12.41%, respectively. The intra- and inter-day RE values were within the range of -7.21-8.52%, and -6.44-14.23%, respectively. The extraction recovery was found to be >84.59% and no matrix effects were observed. The validated LC-MS/MS method has been successfully applied for the simultaneous determination of idelalisib and GS-563117 in a pharmacokinetic study in dogs. Our results suggested that idelalisib was rapidly metabolized into its metabolite GS-563117 in dog and the in vivo exposure of GS-563117 was 17.59% of that of idelalisib.

Effects of miR-101, miR-345 on HBV replication regulation and on the growth of liver cancer cells.

The aim of the study was to investigate the effects of miRNA-101 and miRNA-345 on HBV replication and liver cancer cell growth. qPCR was performed to detect the expression of miRNA-101 and miRNA-345. The expression of HBV RNA was detected by PCR. The expression of HbsAg was detected using ELISA. BEL-7404 cell line proliferation was detected by MTT assay. The expression levels of miR-101 and miR-345 in BEL-7404 pSUPER.neo-miR-101 group and BEL-7404 pSUPER.neo-miR-345 group were significantly higher than those in BEL-7404 pSUPER.neo group (P<0.05). The expression levels of miR-101 and miR-345 in MHCC97-L pSUPER.neo-miR-101 group and MHCC97-L pSUPER.neo-miR-345 group were significantly higher than those in MHCC97-L pSUPER.neo group (P<0.05). The expression of HBV DNA in MHCC97-L pSUPER.neo-miR-101 group was significantly lower than that in MHCC97-L pSUPER.neo group (P<0.05), and the expression of HBV DNA in MHCC97-L pSUPER.neo-miR-345 group was significantly higher than that in MHCC97-L pSUPER.neo group (P<0.05). The expression of HbsAg in MHCC97-L pSUPER.neo-miR-101 group was significantly lower than that in MHCC97-L pSUPER.neo group (P<0.05), and the expression of HbsAg in MHCC97-L pSUPER.neo-miR-345 group was significantly higher than that in MHCC97-L pSUPER.neo group (P<0.05). There was a significant difference in terms of HbsAg expression between the MHCC97-L pSUPER.neo-miR-101 and MHCC97-L pSUPER.neo-miR-345 groups (P<0.05). The proliferation of BEL-7404 cells in the BEL-7404 pSUPER.neo-miR-101 group was significantly lower than that in the BEL-7404 pSUPER.neo group (P<0.05). The proliferation of BEL-7404 cells in the BEL-7404 pSUPER.neo-miR-345 group was significantly higher than that in the BEL-7404 pSUPER.neo group (P<0.05). The proliferation of BEL-7404 cells in BEL-7404 pSUPER.neo-miR-101 group was different from that in BEL-7404 pSUPER.neo-miR-345 group (P<0.05). miR-101 reduced the level of HBV replication, and inhibited the proliferation of liver cancer cells. miR-345 also upregulated the level of HBV replication, and promoted the proliferation of liver cancer cells.

Simultaneous determination of evodiamine and its four metabolites in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

A simple and sensitive liquid chromatography tandem mass spectrometry method was validated for simultaneous quantification of evodiamine and its metabolites 10-hydroxyevodiamine (M1), 18-hydroxyevodiamine (M2), 10-hydroxyevodiamine-glucuronide (M3) and 18-hydroxy- evodiamine-glucuronide (M4) in rat plasma for the first time. The analytes were extracted with acetonitrile and separated on a C18 column within 3 min. The detection was achieved in positive selected reaction monitoring mode with precursor-to-product transitions at m/z 304.1 → 161.1 for evodiamine, m/z 320.1 → 134.1 for M1, m/z 320.1 → 150.1 for M2, m/z 496.2 → 134.1 for M3, m/z 496.2 → 171.1 for M4 and m/z 349.2 → 305.1 for camptothecin (internal standard). The linearity was evident over the tested concentration ranges with correlation coefficients >0.9991. The lower limits of quantification for evodiamine, M1, M2, M3 and M4 were 0.1, 0.1, 0.1, 0.25 and 0.25 ng mL-1 , respectively. Extraction recoveries and matrix effects of the analytes were within the ranges of 84.51-97.21 and 90.13-103.30%, respectively. The accuracy (relative error) ranged from -8.14 to 7.23% while the intra- and inter-day precisions (relative standard deviation) were < 9.31%. The validated assay was successfully applied for the pharmacokinetic study of evodiamine, M1, M2, M3 and M4 in rat. The current study will be helpful in understanding the in vivo disposition of evodiamine.

The cell death and DNA damages caused by the Tet-On regulating HSV-tk/GCV suicide gene system in MCF-7 cells.

Ganciclovir (GCV) affects the molecular mechanism of cell death and DNA damage by the rAAV (recombinant adeno-associated virus)-mediated Tet-On/HSV-tk/GCV suicide gene system in human breast cancer cell line MCF-7. A rAAV/TRE/Tet-On/HSV-tk combining a Tet-On regulating system and a suicide gene HSV-tk was used to transfect human breast cancer cell line MCF-7, and therapeutic effects on this system were studied. Afterwards, we used RT-PCR, western blotting, and a modified comet-assay to explore the potential mechanism of the HSV-tk/GCV suicide gene system in breast cancer treatments. MTT assay has shown that the cell number of GCV+rAAV+Dox group was significantly decreased compared with that of other groups after treatment and flow cytometric analysis detected that there was a substantial increase of S phase cells in this group, which means the HSV-tk/GCV suicide gene system probably works on the cell cycle. RT-PCR detected the expression level of p21 increased and PCNA had an opposite trend. Western blotting detected the protein expression of p21 and p53 increased and PCNA, CDK1, cyclin B decreased in GCV+rAAV+Dox group. The modified comet-assay shown that the very small extra fragments generated by the GCV+rAAV+Dox group treatment are visible as a small cloud extending from the comet in the direction of electrophoresis. The therapeutic mechanism of the HSV-tk/GCV suicide gene system on human breast cancer cell line MCF-7 is probably by upregulating the expression of p21 through a p53-dependent DNA damage signalling pathway, leading the decrease of protein expression of PCNA, cyclin B, CDK1 in MCF-7 cells and promoting the cell cycle arrest at G1/S phase. In summary, the HSV-tk/GCV suicide gene system arouses the death of MCF-7 cells from blocking the cell cycle and DNA damage.