A protocol to construct RNA-protein devices for photochemical translational regulation of synthetic mRNAs in mammalian cells

Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis

NC-6301, a polymeric micelle rationally optimized for effective release of docetaxel, is potent but is less toxic than native docetaxel in vivo

Drug release rate is an important factor in determining efficacy and toxicity of nanoscale drug delivery systems. However, optimization of the release rate in polymeric micellar nanoscale drug delivery systems has not been fully investigated. In this study NC-6301, a poly(ethylene glycol)-poly(aspartate) block copolymer with docetaxel (DTX) covalently bound via ester link, was synthesized with various numbers of DTX molecules bound to the polymer backbone. The number of DTX molecules was determined up to 14 to achieve an optimal release rate, based upon the authors’ own pharmacokinetic model using known patient data. Efficacy and toxicity of the formulation was then tested in animals. When administered three times at 4-day intervals, the maximum tolerated doses of NC-6301 and native DTX were 50 and 10 mg/kg, respectively, in nude mice. Tissue distribution studies of NC-6301 in mice at 50 mg/kg revealed prolonged release of free DTX in the tumor for at least 120 hours, thus supporting its effectiveness. Furthermore, in cynomolgus monkeys, NC-6301 at 6 mg/kg three times at 2-week intervals showed marginal toxicity, whereas native DTX, at 3 mg/kg with the same schedule, induced significant decrease of food consumption and neutrophil count. NC-6301 at 50 mg/kg in mice also regressed a xenografted tumor of MDA-MB-231 human breast cancer. Native DTX, on the other hand, produced only transient and slight regression of the same tumor xenograft. NC-6301 also significantly inhibited growth of OCUM-2MLN human scirrhous gastric carcinoma in an orthotopic mouse model. Total weight of metastatic lymph nodes was also reduced. In conclusion, NC-6301 with an optimized release rate improved the potency of DTX while reducing its toxicity

Observation of the Pharynx to the Cervical Esophagus Using Transnasal Endoscopy with Blue Laser Imaging

Background In 2014, the new transnasal endoscopy with Blue laser Imaging (BLI) has been developed. Aim We present the usefulness of the observation of from the pharynx to the cervical esophagus using transnasal endoscopy with BLI. Patients and Methods This study was conducted between June 2014 and October 2014. During this period, 70 consecutive patients (60 men, 10 women; mean age 67.9 years old) with esophageal or head and neck cancer underwent endoscopic screening at the oropharynx and hypopharynx by transnasal endoscopy with BLI system We performed this endoscopic observation from oral cavity to pharynx before inserting into the cervical esophagus.The visibility of subsites of the hypopharynx and the orifice of the esophagus was evaluated. The extent of the view of hypopharyngeal opening was classified into 3 categories (excellent, good, poor). Then, the diagnostic accuracy of transnasal endoscopy with BLI system was estimated. Our screening is as follows. First, the patient is asked to bow their head deeply in the left lateral position. We put a hand on the back of the patient’s head and push it forward. The patient is then asked to lift the chin as far as possible. In order to inspect the oral cavity, we insert an endoscope without a mouthpiece. After observation of the oral cavity, the endoscope was inserted through the nose. When the tip of the endoscope reached caudal to the uvula, the patient opened his mouth wide, stuck his tongue forward as much as possible and made a vocal sound like “ayyy”. The endoscopist caused the endoscope to U-turn and observed the oropharynx, in particular the radix linguae (Intra-oropharyngeal U-turn method). For examination of the hypopharynx and the orifice of the esophagus, the patient is asked to blow hard and puff their cheeks while the mouth remains closed (Trumpet maneuver). Results 8 elderly cases were excluded because they could not perform the adequate ballooning. Finally, 62 cases were investigated. The ballooning the pyriform sinus and posterior wall not only allows accurate assessment of the stretched pharyngeal mucosa but also gives a view of postcricoid subsite and the orifice of the esophagus. The wide endoscopic view of the pharynx was obtained in a series of the procedures (excellent=53/62, 85.4%; good=7/52, 4.5%; and poor=2/62, 7.6%). Among 70 patients, 6 superficial lesions (8.6%) at the oropharynx(n=1) and hypopharynx (n=5) were discovered with BLI system. Mucosal redness, a pale thickened mucosa, white deposits or loss of a normal vascular pattern, well demarcated areas covered with scattered dots are important characteristics to diagnose superficial carcinoma. Conclusion The more progress achieved in transnasal endoscopy rapidly in the last few years, it can improve for observing the blind area using trans-oral endoscopy, therefore the trans-nasal endoscope will be a standard tool for the screening of the upper gastrointestinal tract in the near future

Factors Predicting Difficult Biliary Cannulation during Endoscopic Retrograde Cholangiopancreatography for Common Bile Duct Stones

Background/Aims Difficult biliary cannulation is an important risk factor for post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP). Therefore, this study aimed to identify the factors that predict difficult cannulation for common bile duct stones (CBDS) to reduce the risk for PEP. Methods This multicenter retrospective study included 1,406 consecutive patients with native papillae who underwent ERCP for CBDS. Factors predicting difficult cannulation for CBDS were identified using univariate and multivariate analyses. Results Univariate analysis showed that six factors significantly predicted difficult cannulation: ERCP performed by non-expert endoscopists, low-volume center, absence of acute cholangitis, normal serum bilirubin, intradiverticular papilla, and type of major duodenal papilla. Multivariate analysis identified ERCP performed by non-expert endoscopists (odds ratio [OR], 2.5; p<0.001), low-volume center (OR, 1.6; p<0.001), intradiverticular papilla (OR, 1.3; p=0.007), normal serum bilirubin (OR, 1.3; p=0.038), and absence of acute cholangitis (OR, 1.3; p=0.049) as factors significantly predicting difficult cannulation for CBDS. Conclusions Initial cannulation by an experienced endoscopist, early rescue cannulation, or early takeover by an experienced endoscopist should be considered when performing ERCP for CBDS in the presence of factors predicting difficult cannulation