High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

<p>Abstract</p> <p>Background</p> <p>During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective.</p> <p>Results</p> <p>In this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the <it>p53 </it>gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in <it>p53</it>, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the <it>atr </it>and <it>atm </it>genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature.</p> <p>Conclusions</p> <p>These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.</p

Intraoperative HFJV support during bronchoplasty

High frequency jet ventilation (HFTV) was experimentally studied to ensure intraoperative respiratory support at tracheo-bronchoplasty. On the condition of driving pressure of 5 to 15 PSI and frequency of 100 to 400 during bronch-plastic procedure, HFJV is of great help to shorter the operation time and to secure the anastomosis. And the arterial Po2 and Pco2, and pulmonary hemodynamics were kept satisfactory in the circumstances of intraoperative respiratory suppor

Benefit from omentopexy on bronchial wound healing in performing concurrent esophagectomy

The healing process of bronchial wound was compared among wrapping tissues such as pedicled omentum, pericardium, and parietal pleura in terms of the degrees of revascularization of the bronchial artery interrupted by bronchoplasty itself by microangiography, including the circumstances of performing a procedure of esophagectomy. The development of neovascularity was marked and facilitated by omentopexy. The procedure of wrapping by pedicled pericardium and pleura was not so useful for promoting neovascularity as would be expected, and it was almost the same as non-wrapping one. Meanwhile, recanalization by wrapping with free pleura was delayed. When esophagectomy was combined with bronchoplasty, revascularization was apparently retarded. In conclusion, wound healing at bronchial anastomosis was markedly impaired so that omentopexy was recommended for facilitating wound healing at anastomosis

Generation of medaka gene knockout models by target-selected mutagenesis

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes

Phase II study of ifosfamide, cisplatin, and vindesine combination in advanced non-small cell lung cancer.

Twenty-seven previously untreated patients with unresectable non-small cell lung cancer were treated with a 3-drug combination of ifosfamide, cisplatin, and vindesine as a phase II study. Patients received ifosfamide, 1.3g/m2, on days 1 to 5; cisplatin, 20mg/m2, on days 1 to 5; and vindesine, 3mg/m2, on days 1 and 8; with a sufficient parenteral hydration. Courses were repeated every 4 weeks. Twenty males and seven females with a median age of 61 years were treated and fully evaluated. Five patients had stage IIIA, seven had stage IIIB, and 15 had stage IV disease. One patient with adenocarcinoma achieved a complete response and 16 achieved a partial response, for an overall response rate of 63% (95% confidence limit: 45% to 81%). The median duration of response was 34 weeks (range: 9 to 52 weeks). The median survival time was 58 weeks for patients with IIIA/B disease, and 33 weeks for those with IV disease. The major toxicity was myelosuppression, however, it was generally well-tolerated. These results indicate that the 3-drug combination is active against non-small cell lung cancer and warrants further clinical trials.</p

Mortality and morbidity in two-year disease-free survivors of small cell lung cancer after treatment with combination chemotherapy with or without irradiation.

We evaluated the long-term outcome of 148 patients with small cell lung cancer (SCLC) who had been entered into clinical trials of chemotherapy with or without thoracic and prophylactic cranial irradiation (PCI) between 1981 and 1987. Eighteen patients (12%) survived for 2 or more years. With a minimum follow-up of 4.5 years, 10 of the 18 patients who remained disease-free at 2 years are currently alive and free of SCLC. Seven of these 10 patients currently function as they did before diagnosis. However, three suffer from central nervous system changes of varying degrees in severity which appeared 2-3 years after PCI. Eight of the 18 patients who were disease-free at 2 years have died. Two died of isolated relapse in the brain at 3.6 and 4.2 years after initiation of chemotherapy. Five died of other malignancies while continuing their complete response to SCLC; two of non-small cell lung cancer, two of acute myelogenous leukemia, and one of hepatocellular carcinoma. Another patient died of an unrelated disease without any evidence of SCLC. A small but substantial proportion of patients who underwent intensive treatment will achieve long-term survival; however, these patients remain at higher risk for second cancers and late toxicities. Therefore, attention must be directed to defining the safest way to employ such treatment in the management of SCLC.</p

Quantitation of Pyrrole-Imidazole Polyamide in Rat Plasma by High-Performance Liquid Chromatography Coupled with UV Detection

A simple and robust method using high-performance liquid chromatography with UV detection was developed and validated for the determination of six pyrrole-imidazole (PI) polyamides (HN.49, TGF-β1f, TGF-β1t, HN.50f, HN.50t, and LOX-1) in rat plasma. After the plasma proteins were precipitated with methanol containing phenacetin as an internal standard, the analytes were separated on a Luna C18 (2) (5 μm, 4.6×150 mm). Calibration curves were linear over the range of 0.5 to 200 μg/mL for HN.49, 0.25 to 200 μg/mL for TGF-β1f, TGF-β1t, HN.50t, and LOX-1, 1 to 200 μg/mL for HN.50f in rat plasma. The inter- and intraday precision were below 15%, and the accuracy was within 15% at the quality controls. The validated method was successfully applied to sample analysis for the pharmacokinetic study