Expression of Warburg effect genes.

<p>(A) Expression of glycolytic genes in <i>Myc</i>, <i>xmrk</i> and <i>Myc/xmrk</i> tumors. (B) Expression of glycolytic genes splicing factors in <i>Myc</i>, <i>xmrk</i> and <i>Myc/xmrk</i> tumors. Asterisks indicate significantly changed genes (fold change>1.5, P<0.05). (C) Schematic comparison of genomic structure of human and zebrafish <i>PKM1/pkm1</i>and <i>PKM2/pkm2</i> isoforms. The primers used for RT-qPCR are indicated by arrowheads. (D) RT-qPCR quantification of zebrafish <i>pkm1</i> and <i>pkm2</i>expression in three tumor samples as compared with non-tumor controls. ***P<0.001; ****P<0.0001. (E) RT-qPCR quantification of <i>pklr</i> expression in three tumor samples as compared with non-tumor controls. ****P<0.0001.</p

Counteractive effects of pathways oppositely regulated by <i>Myc</i> and <i>xmrk</i>.

<p>All but one pathway which were oppositely regulated by <i>Myc</i> and <i>xmrk</i> were counterbalanced and did not show any significant changes in the <i>Myc</i>/<i>xmrk</i> transgenic liver cancer. Red colors indicate up-regulation while green color down-regulation. FDR values are shown in different color gradients as indicated.</p

RNA-Seq analyses of <i>Myc</i>-, <i>xmrk</i>-, <i>Myc/xmrk</i>-induced liver tumors.

<p>(A) Hierarchical clustering of the eight RNA-Seq samples.(B) Venn diagram of up- and down-regulated transcripts in the three liver tumors. (C) Venn diagram of up-and down-regulated canonical pathways in the three liver tumors. (D) Venn diagram of pathways with opposite directions between <i>Myc</i>- and <i>xmrk</i>-induced liver tumors.</p

Uniquely up- and down-regulated biological process (BP) and KEGG pathways in <i>Myc/xmrk</i> liver tumors.

<p>Uniquely up- or down-regulated genes in the <i>Myc/xmrk</i> tumors (1,114 and 359 genes respectively as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132319#pone.0132319.g002" target="_blank">Fig 2B</a>) were input into the DAVID online software and top BPs and KEGG pathways are shown. (A) Top significantly up-regulated BPs. (B) Significantly up-regulated KEGG pathway with P<0.05 and Benjamini value<0.05 cutoff. (C) Significantly down-regulated KEGG pathway with P<0.05 and Benjamini value<0.05 cutoff. Negative log P-value was plotted against different processes/pathways.</p

Synergistic Induction of Potential Warburg Effect in Zebrafish Hepatocellular Carcinoma by Co-Transgenic Expression of <i>Myc</i> and <i>xmrk</i> Oncogenes

<div><p>Previously we have generated inducible liver tumor models by transgenic expression of <i>Myc</i> or <i>xmrk</i> (activated <i>EGFR</i> homolog) oncogenes in zebrafish. To investigate the interaction of the two oncogenes, we crossed the two transgenic lines and observed more severe and faster hepatocarcinogenesis in <i>Myc</i>/<i>xmrk</i> double transgenic zebrafish than either single transgenic fish. RNA-Seq analyses revealed distinct changes in many molecular pathways among the three types of liver tumors. In particular, we found dramatic alteration of cancer metabolism based on the uniquely enriched pathways in the <i>Myc/xmrk</i> tumors. Critical glycolytic genes including <i>hk2</i>, <i>pkm</i> and <i>ldha</i> were significantly up-regulated in <i>Myc/xmrk</i> tumors but not in either single oncogene-induced tumors, suggesting a potential Warburg effect. In RT-qPCR analyses, the specific <i>pkm2</i> isoformin Warburg effect was found to be highly enriched in the <i>Myc/xmrk</i> tumors but not in <i>Myc</i> or <i>xmrk</i> tumors, consistent with the observations in many human cancers with Warburg effect. Moreover, the splicing factor genes (<i>hnrnpa1</i>, <i>ptbp1a</i>, <i>ptbp1b and sfrs3b</i>) responsible for generating the <i>pkm</i> isoform were also greatly up-regulated in the <i>Myc/xmrk</i> tumors. As Pkm2 isoform is generally inactive and causes incomplete glycolysis to favor anabolism and tumor growth, by treatment with a Pkm2-specific activator, TEPP-46, we further demonstrated that activation of Pkm2 suppressed the growth of oncogenic liver as well as proliferation of liver cells. Collectively, our <i>Myc/xmrk</i> zebrafish model suggests synergetic effect of EGFR and MYC in triggering Warburg effect in the HCC formation and may provide a promising <i>in vivo</i> model for Warburg effect.</p></div

Synergetic effects of pathways co-regulated by <i>Myc</i> and <i>xmrk</i>.

<p>44 canonical pathways were identified to be up-regulated in both <i>Myc</i>- and <i>xmrk</i>-induced zebrafish liver cancer. 32 out of them showed more significant up-regulation in the <i>Myc</i>/<i>xmrk</i> transgenic liver cancer. FDR values are shown in different color gradient as indicated.</p

Synergistic effect of <i>Myc</i> and <i>xmrk</i> oncogenes in transgenic zebrafish survival and liver tumorigenesis.

<p>(A,B) Survival curve (A) and gross morphology (B) of oncogene transgenic zebrafish following doxycycline induction at the juvenile stage (starting from 21 dpf). (C) Survival curve of oncogene transgenic zebrafish following doxycycline induction at the adult stage (starting from 3.5 mpf). (D) Gross observation of liver phenotype (left) and histological sections of livers stained by hematoxylin and eosin dyes (right). Abbreviations: X,<i>xmrk;</i> M,<i>Myc;</i> D, doxycycline treatment.</p

Ultrasensitive Liquid Chromatography–Tandem Mass Spectrometric Methodologies for Quantification of Five HIV‑1 Integrase Inhibitors in Plasma for a Microdose Clinical Trial

HIV-1 integrase strand transfer inhibitors are an important class of compounds targeted for the treatment of HIV-1 infection. Microdosing has emerged as an attractive tool to assist in drug candidate screening for clinical development, but necessitates extremely sensitive bioanalytical assays, typically in the pg/mL concentration range. Currently, accelerator mass spectrometry is the predominant tool for microdosing support, which requires a specialized facility and synthesis of radiolabeled compounds. There have been few studies attempted to comprehensively assess a liquid chromatography–tandem mass spectrometry (LC–MS/MS) approach in the context of microdosing applications. Herein, we describe the development of automated LC–MS/MS methods to quantify five integrase inhibitors in plasma with the limits of quantification at 1 pg/mL for raltegravir and 2 pg/mL for four proprietary compounds. The assays involved double extractions followed by UPLC coupled with negative ion electrospray MS/MS analysis. All methods were fully validated to the rigor of regulated bioanalysis requirements, with intraday precision between 1.20 and 14.1% and accuracy between 93.8 and 107% at the standard curve concentration range. These methods were successfully applied to a human microdose study and demonstrated to be accurate, reproducible, and cost-effective. Results of the study indicate that raltegravir displayed linear pharmacokinetics between a microdose and a pharmacologically active dose

The assembled contigs and unigenes, the GO annotations and the microsatellite genotypes from tested individuals

Aspidistra saxicola As011-annotation.xls, Aspidistra saxicola As011-Contig.fa, Aspidistra saxicola As011-Unigene.fa. The microsatellite genotypes from tested individuals in Aspidistra elatior.xls