Prediction of p53 target genes based on integrative analysis of chromatin immunoprecipitated and sequenced tags，by using Galaxy，a web-based interactive platform for large-scale genome analysis

Chromatin immunoprecipitation (ChIP) followed by sequencing of immunoprecipitated DNA fragments is the high throughput method for identifying transcription factor binding sites. In one such method, ChIP PET, paired end ditags (PETs) derived from both ends of the immunoprecipitated DNA fragments are sequenced and mapped to the genome. We report here the prediction of p53 target genes by meta analyzing tags of p53 ChIP PET and by combining with other genomic annotations, using Galaxy, a web based platform for large scale genome analysis. We found 327 of p53 binding sites on the genome of 5-fluorouracil (5-FU)-treated HCT116 colon cancer cells by searching the total 65,509 PETs for PET clusters. The search for p53 target gene, which focused on PET clusters with computationally-predicted p53 binding motif, identified 20 of putative p53 target genes as well as 11 of known p53 targets. Another search for p53 target genes, which focused on PET clusters located within 50-kb flanking regions of transcription start sites of genes, identified 278 of Refseq genes, 79 of non-coding RNAs and 5 of microRNAs as p53 targets which included lots of known validated targets. Our results indicate that sequencing-based ChIP analysis combined with the existing genome annotation is effective method to predict p53 binding loci and target genes, and also show that the Galaxy platform is well-suited for multiple-type analyses and visualization of ChIP data, leading to functional annotation of transcription factor binding sites

Cross-enhancement of ANGPTL4 transcription by HIF1 alpha and PPAR beta/delta is the result of the conformational proximity of two response elements

BACKGROUND: Synergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated. RESULTS: We present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) β/δ and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARβ/δ agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements. CONCLUSIONS: To the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes

Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

<p>Abstract</p> <p>Background</p> <p>Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of <it>KRAS </it>are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.</p> <p>Methods</p> <p>DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for <it>KRAS </it>amplification by quantitative PCR, and investigated <it>KRAS </it>amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of <it>KRAS </it>knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.</p> <p>Results</p> <p>DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the <it>KRAS </it>gene locus. Amplification of the <it>KRAS </it>locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). <it>KRAS </it>mutations were identified in two of the three cell lines in which <it>KRAS </it>was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased <it>KRAS </it>copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type <it>KRAS</it>, but not in cells with amplified mutant <it>KRAS</it>. Knock-down of <it>KRAS </it>in gastric cancer cells that carried amplified wild-type <it>KRAS </it>resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.</p> <p>Conclusion</p> <p>Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified <it>KRAS </it>as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of <it>KRAS </it>amplification and overexpression.</p

Bitstream encryption and authentication with AES-GCM in dynamically reconfigurable systems

A high-speed and secure dynamic partial reconfiguration (DPR) system is realized with AES-GCM that guarantees both confidentiality and authenticity of FPGA bitstreams. In DPR systems, bitstream authentication is essential for avoiding fatal damage caused by unintended bitstreams. An encryption-only system can prevent bitstream cloning and reverse engineering, but cannot prevent erroneous or malicious bitstreams from being configured. Authenticated encryption is a relatively new concept that provides both message encryption and authentication, and AES-GCM is one of the latest authenticated encryption algorithms suitable for hardware implementation. We implemented the AES-GCMbased DPR system targeting the Virtex-5 device on an offthe-shelf board, and evaluated its throughput and hardware resource utilization. For comparison, we also implemented AES-CBC and SHA-256 modules on the same device. The experimental results showed that the AES-GCM-based system achieved higher throughput with less resource utilization than the AES/SHA-based system. The AES-GCM module achieved more than 1 Gbps throughput and the entire system achieved about 800 Mbps throughput with reasonable resource utilization. This paper clarifies the advantage of using AES-GCM for protecting DPR systems. 1

Preparation of Pickering emulsions through interfacial adsorption by soft cyclodextrin nanogels

Background: Emulsions stabilized by colloidal particles are known as Pickering emulsions. To date, soft microgel particles as well as inorganic and organic particles have been utilized as Pickering emulsifiers. Although cyclodextrin (CD) works as an attractive emulsion stabilizer through the formation of a CD–oil complex at the oil–water interface, a high concentration of CD is normally required. Our research focuses on an effective Pickering emulsifier based on a soft colloidal CD polymer (CD nanogel) with a unique surface-active property.Results: CD nanogels were prepared by crosslinking heptakis(2,6-di-O-methyl)-β-cyclodextrin with phenyl diisocyanate and subsequent immersion of the resulting polymer in water. A dynamic light scattering study shows that primary CD nanogels with 30–50 nm diameter assemble into larger CD nanogels with 120 nm diameter by an increase in the concentration of CD nanogel from 0.01 to 0.1 wt %. The CD nanogel has a surface-active property at the air–water interface, which reduces the surface tension of water. The CD nanogel works as an effective Pickering emulsion stabilizer even at a low concentration (0.1 wt %), forming stable oil-in-water emulsions through interfacial adsorption by the CD nanogels.Conclusion: Soft CD nanogel particles adsorb at the oil–water interface with an effective coverage by forming a strong interconnected network and form a stable Pickering emulsion. The adsorption property of CD nanogels on the droplet surface has great potential to become new microcapsule building blocks with porous surfaces. These microcapsules may act as stimuli-responsive nanocarriers and nanocontainers

of the G7 Information Society-Global Interoperability for Broadband Network (GIBN) Projects. Aiming at a world-wide Visible Human Anatomical Co-laboratory, an

application (VHP Viewer) was developed, which was used for data transmission testing (Trans-Pacific Demonstration of Visible Human) through broadband satellite links between the US and Japan. The demonstration includes (1) remote VH database access and (2) network multi-parallel computing access. It is shown that wide-area database access and high-speed multi-parallel computing could be effectively demonstrated via broadband satellite networks by circumventing a large time-delay by using the Mentat SkyX Gateway system and Personal File System (PFS). Elements of the demonstration verified here could be also applied to distance education and telemedicine as well as a postgenome project.