A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression

Regulation of functional KCNQ1OT1 lncRNA by β-catenin.

Long noncoding RNAs (lncRNAs) have been implicated in many biological processes through epigenetic mechanisms. We previously reported that KCNQ1OT1, an imprinted antisense lncRNA in the human KCNQ1 locus on chromosome 11p15.5, is involved in cis-limited silencing within an imprinted KCNQ1 cluster. Furthermore, aberration of KCNQ1OT1 transcription was observed with a high frequency in colorectal cancers. However, the molecular mechanism of the transcriptional regulation and the functional role of KCNQ1OT1 in colorectal cancer remain unclear. Here, we show that the KCNQ1OT1 transcriptional level was significantly increased in human colorectal cancer cells in which β-catenin was excessively accumulated in the nucleus. Additionally, overexpression of β-catenin resulted in an increase in KCNQ1OT1 lncRNA-coated territory. On the other hand, knockdown of β-catenin resulted in significant decrease of KCNQ1OT1 lncRNA-coated territory and an increase in the mRNA expression of the SLC22A18 and PHLDA2 genes that are regulated by KCNQ1OT1. We showed that β-catenin can promote KCNQ1OT1 transcription through direct binding to the KCNQ1OT1 promoter. Our evidence indicates that β-catenin signaling may contribute to development of colorectal cancer by functioning as a novel lncRNA regulatory factor via direct targeting of KCNQ1OT1

Effects of indole-3-carbinol and phenethyl isothiocyanate on bile and pancreatic juice excretion in rats

Bile and pancreatic juice contain a number of parameters for cancer chemoprevention. Indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC), which are hydrolytic products of brassica plants, have been established to be anti-cancer agents. Here, we developed a method for the continuous and selective sampling of bile and pancreatic juice, and the effects of I3C and PEITC on bile and pancreatic excretion and γ-glutamyl transpeptidase (γ-GTP) activity in the samples were investigated. Male Fisher 344 rats (eight weeks of age) were challenged intragastrically with I3C (150 mg/kg) or PEITC (160 mg/kg) for five days. Twenty-four hours after the final administration, cannulation was undertaken into the rats’ bile and pancreatic ducts, and the bile and pancreatic juice were separately collected for 48 h. In this rat model, bile was stably excreted, and the bile and pancreatic excretion of the control rats was 21.9±1.4 ml/48 h and 12.8±1.7 ml/48 h, respectively. Bile excretion for the first 24 h significantly increased in the I3C- or PEITC-treated rats compared with the control rats. In the case of pancreatic juice, excretion during the first 24 h significantly increased in the PEITC-treated rats. In bile, γ-GTP activity was significantly increased for the first 24 h in the I3C- and PEITC-treated rats, but no difference was observed in the pancreatic juice. Increases of bile excretion and γ-GTP activity in bile might be a factor involved in the anti-cancer effect of I3C and PEITC. Our rat model described here is a useful tool for the study of cancer chemoprevention

黒色腫細胞において、miR-19bはPITX1 mRNAを標的としてhTERT mRNAの発現を制御する

Purpose: This article sheds light onto the increasing problem of product returns, which is exacerbated by growing e-commerce. Many retailers and academics are oblivious to the nature and scale of this challenge. Interdisciplinary research is needed to develop supporting theory, and cross-functional teams are required to implement measures addressing economic, ecological and social sustainability issues. Design/methodology/approach: The initial project adopted a multi-case study approach, whereby returns processes were mapped, vulnerabilities identified and a returns cost calculator was developed. Findings: Product returns processes are usually complicated, prone to internal and external fraud, inefficient and lack sustainability. They can generate considerable losses to the business, especially as returns data are often not systematically collected, monitored or reported to senior management. There are important implications for strategic and operational management, namely the need to develop a concept for Lean returns systems. Originality/value: Product returns are a unique and understudied but growing field in academic research, with only few publications over the last two decades. Yet the phenomenon is causing increasing problems in business and society. Robust solutions could achieve great financial and non-financial impacts.</p

A Case of Pulmonary Sarcoma with Significant Extension into the Right Lung

A female patient in her 30s was referred to us with a mass approximately 8 centimeters in diameter in right lung segment 6. Bronchoscopy was done, and a tumorous lesion obstructing right B6 was found. Biopsy of this lesion supported suspicions of sarcoma or spindle cell carcinoma. Contrast-enhanced CT showed that the mass extended to and obstructed the right main pulmonary artery. A skip lesion was also suspected in the periphery of pulmonary artery trunk. The tumor was removed by right pneumonectomy accompanied by resection of the main and left pulmonary arteries under cardiopulmonary bypass. The pulmonary artery trunk and the left pulmonary artery were reconstructed with a vascular graft. Collectively, intimal sarcoma originating from the right main pulmonary artery with extension into the right lung was diagnosed. Significant extension of pulmonary artery sarcoma into the lung, as was observed in the present case, is considered to be rare, and to our knowledge this is the first report in which the primary lesion was biopsied by bronchoscopy

Titanium peroxide nanoparticles enhanced cytotoxic effects of X-ray irradiation against pancreatic cancer model through reactive oxygen species generation in vitro and in vivo

Details of the synthetic protocol for PAA-TiOxNPs formation from TiO2NPs. (PPTX 151 kb

Effect of Kampo medicine “Dai-kenchu-to” on microbiome in the intestine of the rats with fast stress

[Purpose] Diversity of gut microbiome has been recently reported to be lost in inflammatory bowel disease. We have previously reported that the Dai-kenchu-to (DKT) prevented the bacterial translocation through suppression of cytokine and apoptosis in rat’s fast stress model. The aim of this study was to evaluate the effect of DKT on maintenance of microbial diversity in rat’s intestine with inflammation. [Method] Wister rats were received the fast stress for 5 days. In DKT group, rats were administered with DKT (300 mg/kg/day) during the fast stress (DKT-group). The gut microbiomes were analyzed at before- and after- fast stress, and the effect of DKT for on microbial diversities of the gut were evaluated by the PCR-clone library method targeting the 16 S ribosomal RNA gene. [Result] In Control-group, Erysipelotrichaceae increased to 86% in after fast stress, OTU of before-fast stress was 111 and after fast stress was only 9 (changing rate : 58%). The diversity of microbiome was severely decreased. On the other hand, in DKT-group, diversity of microbiome was kept after fast stress (Lachnospiraceae : Ruminococcaceae : Coriobacteriales 54%, 22%, 5%), Operational taxonomic units of before fast stress was 52 and after fast stress was 55 (changing rate : 6%). Family Lachnospiraceae which includes butyrate-producing Clostridia (Clostridium IV and XIVa). [Conclusion] DKT prevented the reduction of diversity of microbiome in rat’s fast stress model. Our data suggested the new anti-inflammatory mechanism of DKT through gut microbiome