The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with few effective therapeutic options for advanced disease. At least 40% of HCCs are clonal, potentially arising from STAT3 þ , NANOG þ and OCT3/4 þ liver progenitor/stem cell transformation, along with inactivation of transforming growth factor-beta (TGF-b) signaling. Here we report significantly greater signal transducer and activator of transcription 3 (STAT3) and tyrosine phosphorylated STAT3 in human HCC tissues (Po0.0030 and Po0.0455, respectively) than in human normal liver. Further, in HCC cells with loss of response to TGF-b, NSC 74859, a STAT3-specific inhibitor, markedly suppresses growth. In contrast, CD133 þ status did not affect the response to STAT3 inhibition: both CD133 þ Huh-7 cells and CD133 -Huh-7 cells are equally sensitive to NSC 74859 treatment and STAT3 inhibition, with an IC 50 of 100 lM. Thus, the TGF-b/beta2 spectrin (b2SP) pathway may reflect a more functional 'stem/progenitor' state than CD133. Furthermore, NSC 74859 treatment of Huh-7 xenografts in nude mice significantly retarded tumor growth, with an effective dose of only 5 mg/kg. Moreover, NSC 74859 inhibited tyrosine phosphorylation of STAT3 in HCC cells in vivo. We conclude that inhibiting interleukin 6 (IL6)/STAT3 in HCCs with inactivation of the TGF-b/b2SP pathway is an effective approach in management of HCCs. Thus, IL6/STAT3, a major signaling pathway in HCC stem cell renewal and proliferation, can provide a novel approach to the treatment of specific HCCs.

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