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Although RNA interference (RNAi) is a popular technique, no method for simultaneous silencing of multiple targets by small-hairpin RNA (shRNA)-expressing RNAi vectors has yet been established. Although gene silencing can be achieved by synthetic small-interfering RNA (siRNA) duplexes, the approach is transient and largely dependent on the transfection efficiency of the host cell. We offer a solution: a simple, restriction enzyme-generated stable RNAi technique that can efficiently silence multiple targets with a single RNAi vector and a single selection marker. In this study, we succeeded in simultaneous stable knockdown of transforming growth factor beta (TGF-beta) pathway-related Smads--Smad2, Smad3 and Smad4--at the cellular level. We observed distinct phenotypic changes in TGF-beta-dependent cellular functions such as invasion, wound healing and apoptosis. This method is best suited for an analysis of complex signal transduction pathways in which silencing of a single gene cannot account for the whole process.

Prospective Study on Usefulness of Hood Attached to the Tip of Colonoscope Shintaro Kondo, Miki Ohta, Hirotsugu Watabe, Tsuneo Ikenoue, Takayuki Kawakami, Yutaka Yamaji, Makoto Okamoto, Haruhiko Yoshida, Takao Kawabe, Masao Omata Background: A hood attached to the tip of a colonoscope theoretically makes it possible to keep distance between lens of colonoscope and colonic mucosa and to obtain a better view. Recently, a variety of hoods have become commercially available. However, their usefulness has not been fully evaluated. Patients ,d Methods: A total of 294 patients were enrolled into this study. They were randomly allocated to transparent hood group, conventional hood group and non-hood group. Evaluated outcomes were completeness of the examination to the cecum, insertion time to the cecum and complications. No sedative medicine was administrated. PCF230I or PCF240I (Olympus, Tokyo, Japan) were used. Colonoscopists were all trainees who had experienced less than 1000 examinations. Results: Out of eligible 279 patients, 93, 91 and 95 patients were allocated to the transparent hood group, the conventional hood group and the non-hood group, respectively. No significant difference was found in patient’s background between the groups. Total colonoscopy were successful in 259 cases (92.8%). Failure were 5 cases (5.4%) in the transparent hood group, 6 cases (6.6%) in the conventional hood group and 9 cases (9.5%) in the non-hood group. No significant difference was found in unsuccessful rate between the groups. The median insertion time to the cecum was 11.0 minutes in the transparent hood group, 13.0 minutes in the conventional hood group and 12.0 minutes in the non-hood group (p Z 0.10). In the analysis of female cases, however, median insertion time was significantly shorter in the transparent hood group (11.0 minutes, 34 cases) than in the conventional hood group (16.0 minutes, 34 cases, p ! 0.05) and in the non-hood group (17.5 minutes 26 cases, p ! 0.05). No serious complications have occurred. Conclusion: A transparent hood may contribute to facilitate total colonoscopy for female patients.

AIM: Vascular endothelial growth factor (VEGF) is a potent mediator of peritoneal fluid accumulation following tumor progression. This study investigated the role of VEGF secreted by cancerous cells in the formation of malignant ascites. METHODS: VEGF expression was eliminated by knockdown in the pancreas cancer cell-line PancO2 using vector-based short-hairpin type RNA interference (RNAi). Malignant ascites formation in the mouse was analyzed by intraperitoneal injection of PancO2 cells expressing VEGF or with expression knockdown. RESULTS: The VEGF knockdown PancO2 cell was successfully established. Knockdown of VEGF did not affect cancer cell proliferation in vitro or in vivo. The volume of ascites following peritoneal expansion of the tumor in VEGF knockdown cells and control cells did not differ statistically in this in vivo study. Moreover, the VEGF concentration in the ascites did not differ statistically. CONCLUSION: Malignant ascites formation might be mediated by VEGF production in noncancerous tissues, such as stromal compartments. An anti-VEGF strategy against malignant ascites could be applied to various tumors regardless of whether they secrete VEGF.