Single-molecular real-time deep sequencing reveals the dynamics of multi-drug resistant haplotypes and structural variations in the hepatitis C virus genome

While direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have dramatically progressed, patients still suffer from treatment failures. For the radical eradication of HCV, a deeper understanding of multiple resistance-associated substitutions (RASs) at the single-clone level is essential. To understand HCV quasispecies and their dynamics during DAA treatment, we applied single-molecule real-time (SMRT) deep sequencing on sera from 12 patients with genotype-1b HCV infections with DAA treatment failures, both pre- and post-treatment. We identified >3.2 kbp sequences between NS3 and NS5A genes of 187, 539 clones in total, classifying into haplotype codes based on the linkage of seven RAS loci. The number of haplotype codes during the treatment, per sample, significantly decreased from 14.67 ± 9.12 to 6.58 ± 7.1, while the number of nonsynonymous codons on the seven RAS loci, per clone, significantly increased from 1.50 ± 0.92 to 3.64 ± 0.75. In five cases, the minority multi-drug resistant haplotypes at pre-treatment were identical to the major haplotypes at relapse. Moreover, various structural variations (SVs) were detected and their dynamics analysed. These results suggest that SMRT deep sequencing is useful for detecting minority haplotypes and SVs, and to evaluate the dynamics of viral genomes at the single-clone level

Mutational spectrum of hepatitis C virus in patients with chronic hepatitis C determined by single molecule real-time sequencing

The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure

アートプロジェクトによる地域・建築リノベーションの継続的発展性/「沙弥島アートプロジェクト by神戸芸術工科大学」の実践を通して

近年、アートプロジェクトを媒介とした地域活性化が盛んである。本学でも2013年3月から開催された「瀬戸内国際芸術祭2013」以来、「沙弥島アートプロジェクト」として香川県坂出市を対象地として活動してきている。これまでの活動については本学紀要「芸術工学 2013」「芸術工学 2014」においても報告してきているので、本稿は2014年度の活動について報告するものである。2014年度は主として2つのプロジェクトを実施した。今年度は、芸術祭自体の開催年ではないので、芸術性の高い作家の作品を展示するのではなく、市民参加型の創作として、アートに関心を持ってもらうことを主眼にしている。「アートのちからで壁画創生プロジェクト2014」では、坂出市民美術館敷地内の庭園の塀にワークショップ形式で壁画を創作した。「ART SETOUCHI 2014 沙弥島アートプロジェクト」は瀬戸内国際芸術祭の一環に位置付けられるものであるが、芸術祭主会場である旧沙弥小中学校を会場に、市民が栽培したコスモスの鉢植えをモチーフとしたインスタレーションを制作した。　これまでのプロジェクトの参加者は、どうしても児童や高年齢者などの時間に余裕のある人が中心だったが、今回は仕事をしている層を取り込むことが出来たことに意義を感じている。Since the participation to the Setouchi Triennale 2013, we continue the art project activities ‘Shamijima Art Project by Kobe Design University’ . Achievements of these project were reported on bulletin of ‘2012’ and ‘2013’. 　In 2014, we planned a couple of work shop projects and consider the way of continuous community design by arts and architectural works.‘SAKAIDE no　Shio’　the tide of SAKAIDE.Wall painting project at Sakaide citizen museum. Old concrete block fence of museum garden was recoating by blue motif graphic. These motifs were abstracted from sea side scape of Sakaide. ‘ART SETOCHI 2014’ Installation art works.‘Cosmos project’ and ‘Together man project’. Innumerable potted cosmos flowers were grown by Sakaide community residents and installed in the former Shami elementary school’s classroom. Collaborators also create ‘together man’ figure and note a message on together man’s body

Dose Intensity/Body Surface Area Ratio is a Novel Marker Useful for Predicting Response to Lenvatinib against Hepatocellular Carcinoma

Lenvatinib was recently approved as a novel first-line molecular targeted agent (MTA) for treating hepatocellular carcinoma (HCC). The importance of relative dose intensity (RDI) has been shown in the treatment of various types of cancers. However, RDI may not accurately reflect the treatment intensity of lenvatinib, as it is the first oral MTA where the dose is based on the patient’s weight. We aimed to evaluate the utility of 2M-DBR (the delivered dose intensity/body surface area ratio at 60 days) by comparing the relationship between 2M-DBR, 2M-RDI (RDI at 60 days), and the therapeutic response. The therapeutic response to lenvatinib was evaluated in 45 patients who underwent computed tomography 8–12 weeks after treatment initiation. We also investigated the clinical factors associated with high 2M-DBR. The area under the receiver operating characteristic of 2M-DBR that predicts the response to lenvatinib was higher than that of 2M-RDI (0.8004 vs. 0.7778). Patients with high 2M-DBR achieved significantly better objective responses and disease control rates than those with low 2M-DBR (p < 0.0001 and 0.0008). Patients with high 2M-DBR experienced significantly longer progression-free survival (PFS) than those with low 2M-DBR (p = 0.0001), while there was no significant correlation between 2M-RDI levels and PFS (p = 0.2198). Patients who achieved higher levels of 2M-DBR had a significantly better modified ALBI grade (p = 0.0437), better CONUT score (p = 0.0222), and higher BTR (p = 0.0281). Multivariate analysis revealed that high 2M-DBR was the only significant factor associated with longer PFS. In conclusion, 2M-DBR could be an important factor that reflects treatment intensity and useful for predicting the response to lenvatinib against HCC, instead of 2M-RDI

Helper T cell-dominant tertiary lymphoid structures are associated with disease relapse of advanced colorectal cancer

Tertiary lymphoid structures (TLSs), clusters of immune cells found around tumor tissue, have been shown to be associated with anti-tumor immunity, but the cellular composition within each TLS and whether the cellular composition of a TLS affects a patient’s prognosis are poorly understood. In the present study, each TLS was categorized according to its cellular composition determined by a system of multiplex immunohistochemical staining and quantitative analysis, and the correlation between the category and prognosis was examined. Sixty-seven patients with curatively resected stage II/III colorectal cancer (CRC) were enrolled. A TLS, consisting of germinal center B cells, follicular dendritic cells, T helper (Th) cells, B cells, cytotoxic T cells, and macrophages, was confirmed in the tumor tissue of 58 patients (87%). The densities of Th cells and macrophages were significantly higher in relapsed patients than in not-relapsed patients (p = .043 and p = .0076). A higher ratio of Th cells was the most significant independent risk factor for disease relapse on multivariate analysis. The subset increasing in Th cells was GATA3+ Th2. A total of 353 TLSs was divided into five clusters according to immune cell composition. Among them, the Th-rich type TLS was significantly increased (p = .0009) in relapsed patients. These data suggest the possibility that Th cell-dominant composition might disturb the anti-tumor immune response, and the function of each TLS might differ depending on its composition