Kaketsuken (Japan)

Metastatic colorectal cancer (mCRC) differs clinically based on RAS and BRAF mutations or DNA methylation. However, in mCRC, the prognostic value and biological characteristics of genome-wide DNA methylation status (GWMS) in each RAS/BRAF genotype is unknown. To clarify this, primary tumor samples from patients with informed consent in the phase III TRICOLORE study were collected, and RAS and BRAF mutations, immunohistochemical staining of mismatch repair-related proteins, comprehensive gene expression, and genome-wide DNA methylation were analyzed. The tumor samples were classified into high-methylated colorectal cancer (HMCC) and low-methylated colorectal cancer (LMCC). Gene set enrichment analysis (GSEA) was conducted using microarray data. A total of 226 patients were included and classified into the RAS/BRAF wild-type (wt) (n = 125), RAS mutant (mt) (n = 87), and BRAF mt (n = 14), of whom 22 (17.6%), 30 (34.5%), and 14 (100%) had HMCC. HMCC exhibited significantly worse overall survival (OS) than that of LMCC in the RAS/BRAF wt group but not in the RAS mt group. In GSEA, RAS/BRAF wt HMCC was associated with microsatellite instability and BRAF V600E mutation. The poor prognosis of RAS/BRAF wt HMCC may be attributed to gene expression patterns associated with microsatellite instability and BRAF V600E mutation.

ObjectiveAntithrombotic therapy is essential for patients with atrial fibrillation (AF) and stable coronary artery disease (CAD) because of the high risk of thrombosis, whereas a combination of antiplatelets and anticoagulants is associated with a high risk of bleeding. We sought to develop and validate a machine-learning-based model to predict future adverse events.MethodsData from 2215 patients with AF and stable CAD enrolled in the Atrial Fibrillation and Ischaemic Events With Rivaroxaban in Patients With Stable Coronary Artery Disease trial were randomly assigned to the development and validation cohorts. Using the random survival forest (RSF) and Cox regression models, risk scores were developed for net adverse clinical events (NACE) defined as all-cause death, myocardial infarction, stroke or major bleeding.ResultsUsing variables selected by the Boruta algorithm, RSF and Cox models demonstrated acceptable discrimination and calibration in the validation cohort. Using the variables weighted by HR (age, sex, body mass index, systolic blood pressure, alcohol consumption, creatinine clearance, heart failure, diabetes, antiplatelet use and AF type), an integer-based risk score for NACE was developed and classified patients into three risk groups: low (0–4 points), intermediate (5–8) and high (≥9). In both cohorts, the integer-based risk score performed well, with acceptable discrimination (area under the curve 0.70 and 0.66, respectively) and calibration (p>0.40 for both). Decision curve analysis showed the superior net benefits of the risk score.ConclusionsThis risk score can predict the risk of NACE in patients with AF and stable CAD.Trial registration numbersUMIN000016612, NCT02642419.

BackgroundSpontaneous pneumothorax is a major and frequently recurrent complication of lymphangioleiomyomatosis (LAM). Despite the customary use of pleurodesis to manage pnenumothorax, the recurrence rate remains high, and accompanying pleural adhesions cause serious bleeding during subsequent lung transplantation. Therefore, we have developed a technique of total pleural covering (TPC) for LAM to wrap the entire visceral pleura with sheets of oxidized regenerated cellulose (ORC) mesh, thereby reinforcing the affected visceral pleura and preventing recurrence.MethodsSince January 2003, TPC has been applied during video-assisted thoracoscopic surgery for the treatment of LAM. The medical records of LAM patients who had TPC since that time and until August 2014 are reviewed.ResultsTPC was performed in 43 LAM patients (54 hemithoraces), 11 of whom required TPC bilaterally. Pneumothorax recurred in 14 hemithoraces (25.9%) from 11 patients (25.6%) after TPC. Kaplan-Meier estimates of recurrence-free hemithorax were 80.8% at 2.5 years, 71.7% at 5 years, 71.7% at 7.5 years, and 61.4% at 9 years. The recurrence-free probability was significantly better when 10 or more sheets of ORC mesh were utilized for TPC (P = 0.0018). TPC significantly reduced the frequency of pneumothorax: 0.544 ± 0.606 episode/month (mean ± SD) before TPC vs. 0.008 ± 0.019 after TPC (P<0.0001). Grade IIIa postoperative complications were found in 13 TPC surgeries (24.1%).ConclusionsTPC successfully prevented the recurrence of pneumothorax in LAM, was minimally invasive and rarely caused restrictive ventilatory impairment.

In the prenatal diagnosis of Gaucher disease (GD), glucocerebrosidase (GBA) activity is measured with fetal cells, and gene analysis is performed when pathogenic mutations in GBA are identified in advance. Herein is described prenatal diagnosis in a family in which two children had GD. Although prior genetic information for this GD family was not obtained, next-generation sequencing (NGS) was carried out for this family because immediate prenatal diagnosis was necessary. Three mutations were identified in this GD family. The father had one mutation in intron 3 (IVS2+1), the mother had two mutations in exons 3 (I[-20]V) and 5 (M85T), and child 1 had all three of these mutations; child 3 had none of these mutations. On NGS the present fetus (child 3) was not a carrier of GD-related mutations. NGS may facilitate early detection and treatment before disease onset.

Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, β and γ). There have been no reports of high-level expression of fibrinogen at commercial levels using mammalian cultured cells such as CHO cells because of the difficulty in highly expressing a protein with such a complex structure. We achieved high-level (1.3 g/l or higher) expression of recombinant human fibrinogen using CHO DG44 cells by optimizing the expression system and culture conditions. We also succeeded in establishing a high-recovery preparation method for recombinant fibrinogen that rarely yields degraded products. To characterize the properties of the recombinant human fibrinogen, we performed SDS-PAGE; western blotting of the α, β and γ chains using specific antibodies and scanning electron microscopy observations of fibrin fibres. We also evaluated the functional equivalence between recombinant fibrinogen and plasma fibrinogen with respect to the release of fibrinopeptides initiated by thrombin and its cross-linking properties. The basic properties of recombinant fibrinogen showed no apparent differences from those of plasma fibrinogen. Here, we report the development of methods for the culture and preparation of recombinant human fibrinogen of satisfactory quality that can be scaled up to the commercial level.

A healthy 40-year-old man was admitted with severe coagulopathy that developed after Rhabdophis tigrinus bites. On admission, he showed significantly elevated levels of thrombin-antithrombin III complex (60 ng/mL), plasmin-alpha 2-plasmin inhibitor complex (22.3 μg/mL), and fibrinogen degradation products (592 μg/mL). He subsequently developed severe hypofibrinogenemia (50 mg/dL). Antivenom was given 28 h after the patient was bitten, following which his hemorrhagic symptoms resolved. By day 3 of admission, scabs had formed over the bite wounds. Furthermore, his fibrinogen levels increased to >100 mg/dL, while his thrombin-antithrombin III complex, plasmin-alpha 2-plasmin inhibitor complex, and fibrinogen degradation product levels normalized. He was discharged on day 6 of admission. Rhabdophis tigrinus bites induced disseminated intravascular coagulation with a fibrinolytic phenotype, which completely recovered with antivenom treatment.

This study evaluates the efficiency of novel non-viral vectors consisting of super paramagnetic iron oxide nanoparticles functionalized with the chemically tunable cationic polymer for in vitro gene magnetofection. The cationic polymer, poly(vinyl pyridinium alkyl halide), with a reactive alkoxysilyl group at one terminal of the polymer (VPCmn, m = length of the side chain and n = polymerization degree), was grafted onto the surface of iron oxide nanoparticles through a silane coupling reaction. The VPCmn grafted-magnetic nanoparticles (Mag-VPCmn) were quaternized with various alkyl halides such as methyl iodide (m = 1), ethyl bromide (m = 2), butyl bromide (m = 4), hexyl bromide (m = 6) and octyl bromide (m = 8). Mag-VPCmn quaternized with a shorter alkyl chain (m = 1, 2, 4 and 6) were water dispersible, but that quaternized with a longer alkyl chain (m = 8) was precipitated in water. The surface of water dispersible Mag-VPCmns was positively charged in pH ranging from 2 to 11, and is stable for more than one month in this pH range. The complexes of Mag-VPCmns and nucleoside molecules with various N/P ratios were evaluated using gel electrophoresis, surface charge (ζ-potential) measurement, and particle size measurement. In vitro transfection experiments were assayed in human embryonic kidney 293 cells (HEK293 cells) using pmaxGFP plasmid as a reporter gene. Gene expression was found to be strongly influenced by the length of the side alkyl chains. Higher transfection efficiencies were observed with longer alkyl chains (C6 > C4 > C2 ≥ C1), indicating that hydrophobic side chains were effective in increasing the transfection efficiency.