Wen-Shyong Tzou

Molecular detection of APC, K- ras, and p53 mutations in the serum of colorectal cancer patients as circulating biomarkers.

Early detection of tumor DNA in serum/plasma prior to the development of recurrence or metastases could help improve the outcome of patients with colorectal cancer (CRC) after tumor resection. Recent advances in the detection of tumor DNA in the serum/plasma has opened up numerous new areas for investigation and new possibilities for molecular diagnosis. APC and K-ras mutations are considered to be early-stage developments of CRCs, whereas p53 mutations are thought to be relatively late events in the tumorigenesis of CRCs. The aim of this study was to search for the presence of genetic mutations in the DNA extracted from the serum of CRC patients and healthy subjects. We simultaneously evaluate the significance of APC, K-ras, and p53 gene mutations in cancer tissues and their paired serum samples of 104 CRC patients by polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) followed by direct sequencing. Additionally, analysis was carried out to detect the serum carcinoembryonic antigen (CEA) levels in CRC patients. Overall, we found at least one of the gene mutations in tumor tissues from 75% (78/104) of the CRC patients. Comparison of the three molecular markers showed that the detection rates in the serum were 30.4%, 34.0%, and 34.2% for APC, K-ras, and p53 genes, respectively. Of these patients, 46.2% (36/78) were identified as having positive serum results, whereas all healthy controls remained negative. The overall positive tumor DNA detection rates in the serum were 0% (0/7) for Dukes’ A classification, 22.4% (11/49) for Dukes’ B, 48.7% (19/39) for Dukes’ C, and 66.7% (6/9) for Dukes’ D. The detection rate increased as the tumor stage progressed (p = 0.012). Concurrently, a significant difference was observed between lymph node metastases and positive serum tumor DNA detection (p < 0.001). A significantly higher postoperative metastasis/recurrence rate in patients harboring gene mutations with serum tumor DNA than those without serum tumor DNA was also demonstrated (p < 0.001). However, no significant correlation between the postoperative metastasis/recurrence and serum CEA levels was observed (p = 0.247). These data suggest that the identification of circulating tumor DNA using the molecular detection of APC, K-ras, and p53 gene mutations is a potential tool for early detection of postoperative recurrence/metastases. Moreover, these genes may be potential molecular markers of poor clinical outcome in CRC patients.

Influence of TCDD on Zebrafish CYP1B1 Transcription during Development

Cytochrome P450 1B1 (CYP1B1) is a heme-containing monooxygenase that metabolizes various polycyclic aromatic hydrocarbons and aryl amines, as well as retinoic acid and steroid hormones. Here we report the cloning of an ortholog of CYP1B1 from zebrafish and the demonstration that transcription of zebrafish CYP1B1 was modulated by two types of mechanisms during different developmental stage. First in late pharyngula stage before hatching, CYP1B1 was constitutively transcribed in retina, midbrain-hindbrain boundary and diencephalon regions through a close coordination between aryl hydrocarbon receptor 2 (AHR2)-dependent and AHR2-independent pathways. After hatching, the basal transcription was attenuated and it could not be elicited upon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. In contrast, TCDD exposure induced de novo CYP1B1 transcription in larval branchial arches and heart tissues via an AHR2-dependent pathway. Blocking AHR2 translation completely eliminated the TCDD-mediated CYP1B1 transcription. However, we did not detect any types of CYP1B1 transcription in liver and kidney tissues through the developmental stage. It suggests that the constitutive and TCDD-inducible types of CYP1B1 transcriptions are modulated by distinct pathways with different tissue specificities. Finally, we investigated the role of CYP1B1 in TCDD-mediated embryonic toxicity. Because knockdown of CYP1B1 did not prevent TCDD-induced pericardial edema and cranial defects, it suggests that CYP1B1 is not involved in the developmental toxicity of dioxin.

Characterization of a Thermophilic L-Rhamnose Isomerase from Thermoanaerobacterium saccharolyticum NTOU1

L-rhamnose isomerase (EC 5.3.1.14, L-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-Rhi gene encoding L-Rhi was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.

Integration of CNS survival and differentiation by HIF2α

Hypoxia-inducible factor (HIF) 1α and HIF2α and the inhibitor of apoptosis survivin represent prominent markers of many human cancers. They are also widely expressed in various embryonic tissues, including the central nervous system; however, little is known about their functions in embryos. Here, we show that zebrafish HIF2α protects neural progenitor cells and neural differentiation processes by upregulating the survivin orthologues birc5a and birc5b during embryogenesis. Morpholino-mediated knockdown of hif2α reduced the transcription of birc5a and birc5b, induced p53-independent apoptosis and abrogated neural cell differentiation. Depletion of birc5a and birc5b recaptured the neural development defects that were observed in the hif2α morphants. The phenotypes induced by HIF2α depletion were largely rescued by ectopic birc5a and birc5b mRNAs, indicating that Birc5a and Birc5b act downstream of HIF2α. Chromatin immunoprecipitation assay revealed that HIF2α binds to birc5a and birc5b promoters directly to modulate their transcriptions. Knockdown of hif2α, birc5a or birc5b reduced the expression of the cdk inhibitors p27/cdkn1b and p57/cdkn1c and increased ccnd1/cyclin D1 transcription in the surviving neural progenitor cells. The reduction in elavl3/HuC expression and enhanced pcna, nestin, ascl1b and sox3 expression indicate that the surviving neural progenitor cells in hif2α morphants maintain a high proliferation rate without terminally differentiating. We propose that a subset of developmental defects attributed to HIF2α depletion is due in part to the loss of survivin activity.

Hypoxia-Inducible Factor 2 Alpha Is Essential for Hepatic Outgrowth and Functions via the Regulation of leg1 Transcription in the Zebrafish Embryo

The liver plays a vital role in metabolism, detoxification, digestion, and the maintenance of homeostasis. During development, the vertebrate embryonic liver undergoes a series of morphogenic processes known as hepatogenesis. Hepatogenesis can be separated into three interrelated processes: endoderm specification, hepatoblast differentiation, and hepatic outgrowth. Throughout this process, signaling molecules and transcription factors initiate and regulate the coordination of cell proliferation, apoptosis, differentiation, intercellular adhesion, and cell migration. Hifs are already recognized to be essential in embryonic development, but their role in hepatogenesis remains unknown. Using the zebrafish embryo as a model organism, we report that the lack of Hif2-alpha but not Hif1-alpha blocks hepatic outgrowth. While Hif2-alpha is not involved in hepatoblast specification, this transcription factor regulates hepatocyte cell proliferation during hepatic outgrowth. Furthermore, we demonstrated that the lack of Hif2-alpha can reduce the expression of liver-enriched gene 1 (leg1), which encodes a secretory protein essential for hepatic outgrowth. Additionally, exogenous mRNA expression of leg1 can rescue the small liver phenotype of hif2-alpha morphants. We also showed that Hif2-alpha directly binds to the promoter region of leg1 to control leg1 expression. Interestingly, we discovered overrepresented, high-density Hif-binding sites in the potential upstream regulatory sequences of leg1 in teleosts but not in terrestrial mammals. We concluded that hif2-alpha is a key factor required for hepatic outgrowth and regulates leg1 expression in zebrafish embryos. We also proposed that the hif2-alpha-leg1 axis in liver development may have resulted from the adaptation of teleosts to their environment.

Biochemical Characterization of Two Truncated Forms of Amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 to Identify Its Enzymatically Active Region

The enzymatically active region of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 (TsaNTOU1Apu) was identified by truncation mutagenesis. Two truncated TsaNTOU1Apu enzymes, TsaNTOU1ApuM957 and TsaNTOU1ApuK885, were selected and characterized. Both TsaNTOU1ApuM957 and TsaNTOU1ApuK885 showed similar specific activities toward various substrates. The overall catalytic efficiency (kcat/apparent Km) for the soluble starch or pullulan substrate, however, was 20–25% lower in TsaNTOU1ApuK885 than in TsaNTOU1ApuM957. Both truncated enzymes exhibited similar thermostability and substrate-binding ability against the raw starch. The fluorescence and circular dichroism spectrometry studies indicated that TsaNTOU1ApuK885 retained an active folding conformation similar to that of TsaNTOU1ApuM957. These results indicate that a large part of the TsaNTOU1Apu, such as the C-terminal carbohydrate-binding module family 20, the second fibronectin type III, and a portion of the first FnIII motifs, could be removed without causing a serious aberrant structural change or a dramatic decrease in hydrolysis of soluble starch and pullulan.

REMUS: A tool for identification of unique peptide segments as epitopes

We provide a ‘REMUS’ (reinforced merging techniques for unique peptide segments) web server for identification of the locations and compositions of unique peptide segments from a set of protein family sequences. Different levels of uniqueness are determined according to substitutional relationship in the amino acids, frequency of appearance and biological properties such as priority for serving as candidates for epitopes where antibodies recognize. REMUS also provides interactive visualization of 3D structures for allocation and comparison of the identified unique peptide segments. Accuracy of the algorithm was found to be 70% in terms of mapping a unique peptide segment as an epitope. The REMUS web server is available at and the PC version software can be freely downloaded either at or . User guide and working examples for PC version are available at , and details of the proposed algorithm can be referred to the documents as described previously [H. T. Chang, T. W. Pai, T. C. Fan, B. H. Su, P. C. Wu, C. Y. Tang, C. T. Chang, S. H. Liu and M. D. T. Chang (2006) BMC Bioinformatics, 7, 38 and T. W. Pai, B. H. Su, P. C. Wu, M. D. T. Chang, H. T. Chang, T. C. Fan and S. H. Liu (2006) J. Bioinform. Comput. Biol., 4, 75–92].

Ethanol inhibits retinal and CNS differentiation due to failure of cell cycle exit via an apoptosis-independent pathway

Alcohol exposure during embryogenesis results in a variety of developmental disorders. Here, we demonstrate that continuous exposure to 1.5% ethanol causes substantial apoptosis and abrogated retinal and CNS development in zebrafish embryos. Chronic exposure to ethanol for 24h before hatching also induces apoptosis and retinal disorder. After the 2-day post-fertilization (dpf) stage, chronic exposure to ethanol continued to induce apoptosis, but did not block retinal differentiation. Although continuous ethanol exposure induces substantial accumulation of reactive oxygen species (ROS) and increases p53 expression, depletion of p53 did not eliminate ethanol-induced apoptosis. On the other hand, sequestering ROS with the antioxidant reagent N-acetylcysteine (NAC) successfully inhibited ethanol-associated apoptosis, suggesting that the ethanol-induced cell death primarily results from ROS accumulation. Continuous ethanol treatment of embryos reduced expression of the mature neural and photoreceptor markers elavl3/huC, rho, and crx; in addition, expression of the neural and retinal progenitor markers ascl1b and pax6b was maintained at the undifferentiated stage, indicating that retinal and CNS neural progenitor cells failed to undergo further differentiation. Moreover, ethanol treatment enhanced BrdU incorporation, histone H3 phosphorylation, and pcna expression in neural progenitor cells, thereby maintaining a high rate of proliferation. Ethanol treatment also resulted in sustained transcription of ccnd1/cyclin D1 and ccne/cyclin E throughout development in neural progenitor cells, without an appropriate increase of cdkn1b/p27 and cdkn1c/p57 expression, suggesting that these cells failed to exit from the cell cycle. Although NAC was able to mitigate ethanol-mediated apoptosis, it was unable to ameliorate the defects in visual and CNS neural differentiation, suggesting that abrogated neural development in ethanol-exposed embryos is unlikely to arise from excessive apoptosis. In conclusion, we demonstrate that the pathological effect of ethanol on zebrafish embryos is partially attributable to cell death and inhibition of visual and CNS neuron differentiation. Excessive apoptosis largely results from the accumulation of ROS, whereas abrogated neural development is caused by failure of cell cycle arrest, which in turn prevents a successful transition from proliferation to differentiation.

An online conserved SSR discovery through cross-species comparison.

Simple sequence repeats (SSRs) play important roles in gene regulation and genome evolution. Although there exist several online resources for SSR mining, most of them only extract general SSR patterns without providing functional information. Here, an online search tool, CG-SSR (Comparative Genomics SSR discovery), has been developed for discovering potential functional SSRs from vertebrate genomes through cross-species comparison. In addition to revealing SSR candidates in conserved regions among various species, it also combines accurate coordinate and functional genomics information. CG-SSR is the first comprehensive and efficient online tool for conserved SSR discovery.

UniQua: A Universal Signal Processor for MS-Based Qualitative and Quantitative Proteomics Applications

Recent developments in high resolution mass spectrometry (HR-MS) technology have ushered proteomics into a new era. However, the importance of using a common, open data platform for signal processing of HR-MS spectra has not been sufficiently addressed. In this study, a MS signal processor was developed to facilitate data integration from different instruments and different proteomics approaches into a unified platform without compromising protein identification and quantitation performance. This processor supports parallel processing capability which allows full utilization of computing resources to speed up signal processing performance to >1 gigabytes/min. The storage space occupied by the processed MS data can be reduced to ~10%, which helps the analysis and management of large quantities of data from comprehensive proteomics studies. For quantitation at the MS level, processing accuracy is improved and processing time for ASAPRatio is reduced to ~50%. For quantitation at the MS/MS level, accurate reporter ion ratios from different instruments can be directly determined by the processed MS/MS spectra and reported in the Mascot search result directly without using specialized iTRAQ software.