Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

Background　 　 The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. 　 Methodology/Principal Findings　 　 Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5′-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0–9.0×10−3 and 2.7–4.0×10−3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. 　 Conclusions/Significance　 　 Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis

Molecular Mechanism Underlying the Suppression of CPB2 Expression Caused by Persistent Hepatitis C Virus RNA Replication

The mechanisms of hepatitis C virus (HCV)-associated hepatocarcinogenesis and disease progression are unclear. We previously observed that the expression level of carboxypeptidase B2 (CPB2) gene was remarkably suppressed by persistent HCV RNA replication in human hepatoma cell line Li23-derived cells. The results of the present study demonstrated that the CPB2 expression in patients with chronic hepatitis C was inversely correlated with several risk factors of hepatic fibrosis or steatosis, although ectopic CPB2 expression did not suppress the expression of fibrogenic or lipogenic genes. The suppressed CPB2 expression was restored by treatment with 5-azacytidine. To clarify the mechanism underlying this phenomenon, we analyzed the CPB2 promoter, and the results revealed that (1) hepatocyte nuclear factor 1 (HNF1), especially HNF1α, was essential for the CPB2 promoter, and (2) CPB2 promoter was not methylated by persistent HCV RNA replication. The expression levels of HNF1α and HNF1β were also not changed by persistent HCV RNA replication. These results suggest the existence of 5-azacytidine-inducible or -reducible unknown factor(s) that can control the CPB2 expression. To evaluate this idea we performed a microarray analysis, and several gene candidates corresponding to the suggested factor(s) were identified

Identification of host genes showing differential expression profiles with cell-based long-term replication of hepatitis C virus RNA

　Persistent hepatitis C virus (HCV) infection frequently causes hepatocellular carcinoma. However, the mechanisms of HCV-associated hepatocarcinogenesis and disease progression are unclear. Although the human hepatoma cell line, HuH-7, has been widely used as the only cell culture system for robust HCV replication, we recently developed new human hepatoma Li23 cell line-derived OL, OL8, OL11, and OL14 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates. OL, OL8, OL11, and OL14 cells were cultured for more than 2 years. We prepared cured cells from OL8 and OL11 cells by interferon-γ treatment. The cured cells were also cultured for more than 2 years. cDNA microarray and RT-PCR analyses were performed using total RNAs prepared from these cells. We first selected several hundred highly or moderately expressed probes, the expression levels of which were upregulated or downregulated at ratios of more than 2 or less than 0.5 in each set of compared cells (e.g., parent OL8 cells versus OL8 cells cultured for 2 years). From among these probes, we next selected those whose expression levels commonly changed during a 2-year culture of genome-length HCV RNA-replicating cells, but which did not change during a 2-year culture period in cured cells. We further examined the expression levels of the selected candidate genes by RT-PCR analysis using additional specimens from the cells cultured for 3.5 years. Reproducibility of the RT-PCR analysis using specimens from recultured cells was also confirmed. Finally, we identified 5 upregulated genes and 4 downregulated genes, the expression levels of which were irreversibly altered during 3.5-year replication of HCV RNA. These genes may play roles in the optimization of the environment in HCV RNA replication, or may play key roles in the progression of HCV-associated hepatic diseases

A New Strategy for Surgical Intervention of Bisphosphonate-Related Osteonecrosis of the Jaw : A retrospective study.

Bisphosphonates (BPs) are now widely used to treat various skeletal complications. Although the number of reported cases ofbisphosphonate-related osteonecrosis of the jaw (BRONJ) is rapidly increasing worldwide, therapeutic strategies remain controversial.Conservative treatments including antibacterial mouth rinses, the systemic administration of antibiotics, and superficial debridement in stage II BRONJ have been recommended by the American Association of Oral and Maxillofacial Surgeons position paper. However, these treatments are only partially successful. We performed a surgical intervention that consisted of osteotomy and primary wound closure in patients with stages II and III BRONJ. Forty-three out of 44 cases were treated effectively by this strategy, leading to improvements in quality of life. All BRONJ patients treated with oral BPs were treated successfully by the surgical intervention. We also proposed a surgical intervention for patients with stage II BRONJ

保育者評定による最近の幼児に見られる変化 : 小1プロブレムの背景要因

近年の初等教育界の大きな問題として「小1プロブレム」が取り上げられている。新入学児に対して経験豊かな教師でさえ指導に窮している現象であり、こうした問題への対処として幼保小連携、環境移行、交流などの幼保小接続や、基本的生活習慣、ソーシャル・スキルの形成等で移行をスムーズにするという視点から、現在多くの方策が模索されている。しかし、その背後には単に小学校という場への慣れという問題では解決できない別の複数の要因が関わっていることが考えられる。近年の社会の急激な変化に伴い必然的に子どもを取り巻く環境自体も大きく変化し、子どもの育ちに直接的影響を及ぼしている。従って滑らかな移行といった対症療法では背後に潜む問題自体を解決することはできないであろうし、また環境移行以外の問題に対処することも困難であろう。本研究では、現在の日本社会で生じている子どもたちの問題を幼児期に遡って原因を探り、解決のためには幼児期に何が必要であるのかを解明する。首都圏の保育士および幼稚園教諭を対象に、近年の幼児や保護者に見られるようになった現象について質問紙調査を行い、今回はその第一報として最近の幼児の特徴と傾向を探った。その結果、「目的的調整力の低位」、「内発的活力の低位」、「行動・防衛体力の低位」、「生命維持力の低位」の4因子が抽出され、すでに幼児期において、多くの子どもに生来備わっているはずの健やかな育ちを求める機能が十分に育まれず心身共に脆弱性を抱えている傾向が明らかになった

Present Status in the Development of 6 MeV Heavy Ion Beam Probe on LHD

In order to measure the potential in Large Helical Device (LHD), we have been developing a heavy ion beam probe (HIBP). For probing beam, gold beam is used, which is accelerated by a tandem accelerator up to the energy of 6 MeV. The experiments for calibration of beam orbit were done, and experimental results were compared with orbit calculations. The experimental results coincided fairly with the calculation results. After the calibration of the beam orbit, the potential in plasma was tried to measure with the HIBP. The experimental data showed positive potential in a neutral beam heating phase on the condition of ne ? 5 × 10^18 m^-3, and the increase of potential was observed when the additional electron cyclotron heating was applied to this plasma. The time constant for this increase was about a few tens ms, which was larger than a theoretical expectation. In the spatial position of sample volume, we might have an ambiguity in this experiment

Extension of operational regime in high-temperature plasmas and effect of ECRH on ion thermal transport in the LHD

A simultaneous high ion temperature (Ti) and high electron temperature (Te) regime was successfully extended due to an optimized heating scenario in the LHD. Such high-temperature plasmas were realized by the simultaneous formation of an electron internal transport barrier (ITB) and an ion ITB by the combination of high power NBI and ECRH. Although the ion thermal confinement was degraded in the plasma core with an increase of Te/Ti by the on-axis ECRH, it was found that the ion thermal confinement was improved at the plasma edge. The normalized ion thermal diffusivity ${{\chi}_{\text{i}}}/T_{\text{i}}^{1.5}$ at the plasma edge was reduced by 70%. The improvement of the ion thermal confinement at the edge led to an increase in Ti in the entire plasma region, even though the core transport was degraded

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

Background　 　 The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. 　 Methodology/Principal Findings　 　 Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5′-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0–9.0×10−3 and 2.7–4.0×10−3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. 　 Conclusions/Significance　 　 Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis