Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus

In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.     

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.     

Coupled simulation of BES-CFD and performance assessment of energy recovery ventilation system for office model

Thermal comfort and indoor air quality as well as the energy efficiency have been recognized as essential parts of sustainable building assessment. This work aims to analyze the energy conservation of the heat recovery ventilator and to investigate the effect of the air supply arrangement. Three types of mixing ventilation are chosen for the analysis of coupling ANSYS/FLUENT (a computational fluid dynamics (CFD) program) with TRNSYS (a building energy simulation (BES) software). The adoption of mutual complementary boundary conditions for CFD and BES provides more accurate and complete information of indoor air distribution and thermal performance in buildings. A typical office-space situated in a middle storey is chosen for the analysis. The office-space is equipped with air-conditioners on the ceiling. A heat recovery ventilation system directly supplies fresh air to the office space. Its thermal performance and indoor air distribution predicted by the coupled method are compared under three types of ventilation system. When the supply and return openings for ventilation are arranged on the ceiling, there is no critical difference between the predictions of the coupled method and BES on the energy consumption of HVAC because PID control is adopted for the supply air temperature of the occupied zone. On the other hand, approximately 21% discrepancy for the heat recovery estimation in the maximum between the simulated results of coupled method and BES-only can be obviously found in the floor air supply ventilation case. The discrepancy emphasizes the necessity of coupling CFD with BES when vertical air temperature gradient exists. Our future target is to estimate the optimum design of heat recovery ventilation system to control CO₂ concentration by adjusting flow rate of fresh air.     

Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan

A strong fibrinolytic enzyme (nattokinase) was purified from the vegetable cheese natto. Nattokinase was extracted from natto with saline and isolated by sequential use of hydrophobic chromatography on Butyl-Toyopearl, ion-exchange chromatography on CM-Toyopearl, and gel-filtration on Sephadex G-50. The isolated protein gave a single sharp band on SDS-PAGE either before or after reduction. The sequence, as determined by automated Edman degradation of the uncleaved molecule and its enzymatically derived peptide, consisted of a total 275 amino acid residues (M.W = 27,728) and exhibited a high homology with the subtilisins. The purified nattokinase digested not only fibrin but also several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was Suc-Ala-Ala-Pro-Phe-pNA for subtilisin. PMSF inhibited both the fibrinolytic activity and the amidolytic activity. The results indicate that nattokinase is a subtilisin-like serine protease.     

Performance-aware server architecture recommendation and automatic performance verification technology on IaaS cloud

In this paper, we propose a server architecture recommendation and automatic performance verification technology, which recommends and verifies appropriate server architecture on Infrastructure as a Service (IaaS) cloud with bare metal servers, container-based virtual servers and virtual machines. Recently, cloud services are spread, and providers provide not only virtual machines but also bare metal servers and container-based virtual servers. However, users need to design appropriate server architecture for their requirements based on three types of server performances, and users need much technical knowledge to optimize their system performance. Therefore, we study a technology that satisfies users’ performance requirements on these three types of IaaS cloud. Firstly, we measure performance and start-up time of a bare metal server, Docker containers, KVM (Kernel-based Virtual Machine) virtual machines on OpenStack with changing number of virtual servers. Secondly, we propose a server architecture recommendation technology based on the measured quantitative data. A server architecture recommendation technology receives an abstract template of OpenStack Heat and function/performance requirements and then creates a concrete template with server specification information. Thirdly, we propose an automatic performance verification technology that executes necessary performance tests automatically on provisioned user environments according to the template. We implement proposed technologies, confirm performance and show the effectiveness.     

Role of Heavy Meromyosin in Heat‐Induced Gelation in Low Ionic Strength Solution Containing l‐Histidine

The gelation of myosin has a very important role in meat products. We have already shown that myosin in low ionic strength solution containing l ‐histidine forms a transparent gel after heating. To clarify the mechanism of this unique gelation, we investigated the changes in the nature of myosin subfragments during heating in solutions with low and high ionic strengths with and without l ‐histidine. The hydrophobicity of myosin and heavy meromyosin (HMM) in low ionic strength solution containing l ‐histidine was lower than in high ionic strength solution. The SH contents of myosin and HMM in low ionic strength solution containing l ‐histidine did not change during the heating process, whereas in high ionic strength solution they decreased slightly. The heat‐induced globular masses of HMM in low ionic strength solution containing l ‐histidine were smaller than those in high ionic strength solution. These findings suggested that the polymerization of HMM molecules by heating was suppressed in low ionic strength solution containing l ‐histidine, resulting in formation of the unique gel.     

A novel quarter‐wave retardation film for improving viewing angle properties in time‐sequential stereoscopic 3D‐LCDs

We have successfully developed a quarter‐wave retardation film (QWF) for wide viewing angle 3D liquid crystal displays (3D‐LCDs) that provides high luminance, low crosstalk, low color change, and low head‐tilt‐angle dependency. It was found that the out‐of‐plane retardation (Rth) of the QWF in the LCD needs to be close to 0 nm in order to improve the 3D display properties at an off‐axis position and that the in‐plane retardation (Re) needs to be adjusted from 120 to 130 nm to achieve low color change with head tilting. We adopted a coating process for making our QWF because of its potential for retardation control. 3D‐LCDs with this QWF whose Rth was nearly zero had high performance and allowed off‐axis other than on‐axis.     

Electrochemical formation of Sm-Co alloys by codeposition of Sm and Co in a molten LiCl-KCl-SmCl3-CoCl2 system

Electrocodeposition of Sm and Co on a Cu substrate was investigated in a molten LiCl-KCl-SmCl₃ (0.5 mol.%)-CoCl₂ (0.1 mol.%) system at 723 K. Phase of the deposited Sm-Co alloys could be controlled by electrolysis potential. SmCo₃ was formed on a Cu substrate by potentiostatic electrolysis in the potential range of 0.20-0.90 V (vs. Li⁺/Li). Sm₂Co₁₇ was obtained in the potential range of 0.90-1.50 V.     

Analysis of low condensed components in condensates of mixtures of β-napthalenesulfonic and β-methylnaphthalenesulfonic acids with formaldehyde

The low condensed components in the condensates of mixtures of β-naphthalenesulfonic acid and β-methylnaphthalenesulfonic acid with formaldehyde were analyzed by high speed liquid chromatography. The main components of monomer and dimer were β-naphthalenesulfonic acid and the dimeric condensate of β-naphthalenesulfonic acid, respectively.     

Novel flexible network polymers consisting of oligomeric primary polymer chains originated in the mechanistic discussion of multiallyl crosslinking polymerization

Summary It is well known that allyl monomers polymerize only with difficulty and yield polymers having low molecular weights, i.e., oligomers. Inevitably, free-radical multiallyl crosslinking polymerization provides network polymers consisting of oligomeric primary polymer chains, i.e., having abundant dangling chains. This led to the development of novel flexible network polymers such as amphiphilic network polymers (I) consisting of short primary polymer chains and long crosslink units with opposite polarities, simultaneous interpenetrating networks (II) consisting of both polyurethane (PU) and polymethacrylate (PM) networks with oligomeric primary polymer chains, and network polymers (III) consisting of centipede-type primary polymer chains. Thus, the solution copolymerizations of benzyl methacrylate with tricosaethylene glycol dimethacrylate in the presence of lauryl mercaptan yielded I consisting of nonpolar, short primary polymer chains and polar, long crosslink units. The opposite type of I was prepared by the copolymerization of 2-hydroxyethyl methacrylate, a polar monomer having a hydroxyl group, with heneicosapropylene glycol dimethacrylate, a nonpolar monomer having a poly(oxypropylene) unit. The equimolar polyaddition crosslinking reaction of poly(methyl methacrylate-co-2-methacryloyloxyethyl isocyanate) with tri(oxytetramethylene) glycol, leading to PU networks, and the free-radical crosslinking copolymerization of methyl methacrylate with tri(oxytetramethylene) dimethacrylate in the presence of CBr₄, leading to PM networks, were progressed simultaneously, providing II formed via the topological crosslink between PU and PM network structures. The post-copolymerizations of oligomeric allyl methacrylate/alkyl methacrylate precopolymers, having different amounts of pendant allyl groups and different molecular weights, with allyl benzoate/vinyl benzoate monomer mixtures were conducted to give III.