Longitudinal assessment of B-RAF V595E levels in the peripheral cell-free tumor DNA of a 10-year-old spayed female Korean Jindo dog with unresectable metastatic urethral transitional cell carcinoma for monitoring the treatment response to a RAF inhibitor (sorafenib).

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract. In this case study, a dog with metastatic urethral TCC was treated with sorafenib. The tumor expression levels of receptor tyrosine kinase genes, including VEGFR-1, VEGFR-2, PDGFR-α, PDGFR-ß, ALK, EGFR, ErbB2, and B-RAF, were analyzed. VEGFR was overexpressed in tumor tissues compared to the normal tissues. Considering the high frequency of B-RAF mutation in canine urological tumors, the B-RAF gene was examined, and the B-RAF V595E mutation was detected in the tumor tissue. Therefore, the antitumor effect of sorafenib, a multi-tyrosine kinase inhibitor, on unresectable metastatic urethral TCC characterized by B-RAF V595E was evaluated and circulating cell-free tumor DNA (ctDNA) was assessed for monitoring the treatment response. After the initiation of oral sorafenib therapy (4 mg/kg/day escalated to 10 mg/kg/day), the dysuria was alleviated gradually, and the patient remained stable for 3 months. During that treatment period, the patient showed various levels of changes associated with B-RAF V595E mutation in ctDNA as evident from longitudinal plasma samples after initiation of sorafenib therapy. The findings of this study suggest that ctDNA may serve as a useful non-invasive tool for monitoring the treatment response to anticancer drugs.

Comparative evaluation of canine cadaver embalming methods for veterinary anatomy education.

Formalin-embalmed cadavers have been extensively used to teach anatomy. Although they ensure the preservation of anatomical structures without microbial contamination, they are considerably rigid and cannot be used to study the joint and muscle movements. Moreover, formalin irritates the eyes and airways and is carcinogenic on chronic exposure. To overcome the disadvantages of formalin-fixed cadavers, we investigated the usefulness of alternative embalming methods using saturated salt solution (SS) and Thiel's solution (TS). We compared the three solutions based on the following parameters: cost of the embalming solution; preservation of anatomical structure, color, flexibility, and texture; and microbial contamination. Convenience of anatomical structure identification and preferences in anatomical laboratory practice were evaluated using questionnaires answered by veterinary undergraduate students. Cost of the embalming solution was the lowest for formalin solution (FS) and most expensive for TS. All cadavers were successfully preserved without significant putrefaction and were useful for teaching veterinary anatomy. Cadavers embalmed with SS or TS were superior in facilitating joint and muscle movement. Compared to FS, the color and texture of muscles and internal organs were similar to those of living animals and there was no irritating and offensive smell in SS and TS cadavers. Students preferred the SS and TS cadavers for their usefulness in identification of anatomical structures, highlighting their usefulness in veterinary anatomy education.

The isolation, characterization, and expression of a novel GDF11 gene and a second myostatin form in zebrafish, Danio rerio.

In the current study, the first non-mammalian growth/differentiation factor (GDF) 11-like homolog was cloned from zebrafish. At the nucleotide level, zebrafish GDF11 is most similar to human GDF11 (79%), while the peptide is most similar to mouse GDF11 (78%). Phylogenetic analysis showed that the zebrafish GDF11 clusters with mammalian GDF11s. This study also cloned a second MSTN form in zebrafish most similar to Salmonid MSTN2 forms. Based on real time PCR, GDF11 is expressed in multiple adult tissues, with levels highest in whole heads and gonads, and expression is less ubiquitous when compared to MSTN expression. During embryonic development, real time PCR demonstrated increasing GDF11 mRNA levels 10 h post-fertilization (hpf), while MSTN mRNA levels remain low until 48 hpf. This is the first report of a transforming growth factor (TGF)-beta superfamily member in a non-mammalian species that is more closely related to GDF11 than MSTN, and also a second form of MSTN in zebrafish; suggesting that a more complex TGF-beta superfamily array exists in primitive vertebrates than previously thought.

Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.