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.     

Identification of Multiple Domains of Entamoeba histolytica Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica have roles in the pathogenicity of intestinal amoebiasis. Igl1, the intermediate subunit lectin-1 of E. histolytica, has been shown to have both hemolytic and cytotoxic activities that reside in the C-terminus of the protein. To identify the amino acid regions responsible for these activities, recombinant proteins were prepared and used in hemolytic and cytotoxic assays. The results revealed that Igl1 has multiple domains with hemolytic and cytotoxic activities and that amino acids 787-846, 968-1028 and 1029-1088 are involved in these activities. The hemolytic activities of the fragments were partly inhibited by mannose, galactose and N-acetylgalactosamine, and glucose showed lower or negligible inhibitory effects for the activities. This is the first report of a protozoan protein with hemolytic and cytotoxic activities in multiple domains.     

Subcellular localization of fructosyl amino acid oxidases in peroxisomes of Aspergillus terreus and Penicillium janthinellum

Fructosyl amino acid oxidase (FAOD) is the enzyme catalyzing the oxidative deglycation of Amadori compounds, such as fructosyl amino acids, yielding the corresponding amino acids, glucosone, and H(2)O(2). In a previous report, we determined the primary structures of cDNAs coding for FAODs from two fungal strains Aspergillus terreus AP1 and Penicillium janthinellum and we found that both fungal FAODs included the putative peroxisome targeting signal 1 (PTS1) at the carboxyl terminal (Yoshida, N. et al., Eur. J. Biochem., 242, 499-505, 1996). In this study, we determined the intracellular localization of FAODs in these two fungi. Subcellular fractionation experiments and immuno-electronmicroscopic observations, together with the previous findings indicated that the FAODs were localized in peroxisomes of A. terreus AP1 and P. janthinellum. These FAODs were also found to belong to a new member of "peroxisomal sarcosine oxidase family protein" in eucaryotic cells.     

Alcohol dehydrogenases that catalyse methyl formate synthesis participate in formaldehyde detoxification in the methylotrophic yeast Candida boidinii

Methyl formate synthesis during growth on methanol by methylotrophic yeasts has been considered to play a role in formaldehyde detoxification. An enzyme that catalyses methyl formate synthesis was purified from methylotrophic yeasts, and was suggested to belong to a family of alcohol dehydrogenases (ADHs). In this study we report the gene cloning and gene disruption analysis of three ADH-encoding genes in the methylotrophic yeast Candida boidinii (CbADH1, CbADH2 and CbADH3) in order to clarify the physiological role of methyl formate synthesis. From the primary structures of these three genes, CbAdh1 was shown to be cytosolic and CbAdh2 and CbAdh3 were mitochondrial enzymes. Gene products of CbADH1, CbADH2 and CbADH3 expressed in Escherichia coli showed both ADH- and methyl formate-synthesizing activities. The results of gene-disruption analyses suggested that methyl formate synthesis was mainly catalysed by a cytosolic ADH (CbAdh1), and this enzyme contributed to formaldehyde detoxification through glutathione-independent formaldehyde oxidation during growth on methanol by methylotrophic yeasts.     

SnO-SnO2 modified two-dimensional MXene Ti3C2Tx for acetone gas sensor working at room temperature

Acetone,as widely used reagents in industry and laboratories,are extremely harmful to the human.So the detection of acetone gas concentrations and leaks in special environments at room temperature is essential.Herein,the nanocomposite combining SnO-SnO2 (p-n junction) and Ti3C2Tx MXene was successfully synthesized by a one-step hydrothermal method.Because of the existence of a small amount of oxygen during the hydrothermal conditions,part of the p-type SnO was oxidized to n-type SnO2,forming in-situ p-n junctions on the surface of SnO.The hamburger-like SnO-SnO2/Ti3C2Tx sensor exhibited improved acetone gas sensing response of 12.1 (Rg/Ra) at room temperature,which were nearly 11 and 4 times higher than those of pristine Ti3C2Tx and pristine SnO-SnO2,respectively.Moreover,it expressed a short recovery time (9 s) and outstanding reproducibility.Because of the different work functions,the Schottky barrier was formed between the SnO and the Ti3C2Tx nanosheets,acting as a hole accumulation layer (HALs) between Ti3C2Tx and tin oxides.Herein,the sensing mechanism based on the formation of hetero-junctions and high conductivity of the metallic phase of Ti3C2Tx MXene in SnO-SnO2/Ti3C2Tx sensors was discussed in detail.     

Oxidation of doped europium in BaMgAl10O17 by annealing studied by x‐ray‐absorption fine‐structure measurements

Abstract— We studied the influence of annealing in air on doped europium in BaMgAl₁₀O₁₇ by performing x‐ray absorption fine‐structure measurements. We determined the oxidation of doped divalent europium by annealing in air at over 500°C. The interatomic distance between the europium and the surrounding oxygen atoms was compressed by oxidation. It also appears that the oxidation process of europium is determined by the diffusion of oxygen into BaMgAl₁₀O₁₇.     

Prediction of Human Behavior in Human--Robot Interaction Using Psychological Scales for Anxiety and Negative Attitudes Toward Robots

When people interact with communication robots in daily life, their attitudes and emotions toward the robots affect their behavior. From the perspective of robotics design, we need to investigate the influences of these attitudes and emotions on human-robot interaction. This paper reports our empirical study on the relationships between people's attitudes and emotions, and their behavior toward a robot. In particular, we focused on negative attitudes, anxiety, and communication avoidance behavior, which have important implications for robotics design. For this purpose, we used two psychological scales that we had developed: negative attitudes toward robots scale (NARS) and robot anxiety scale (RAS). In the experiment, subjects and a humanoid robot are engaged in simple interactions including scenes of meeting, greeting, self-disclosure, and physical contact. Experimental results indicated that there is a relationship between negative attitudes and emotions, and communication avoidance behavior. A gender effect was also suggested.     

Mutants of the nematode Caenorhabditis elegans that are defective specifically in their attraction to cycloheximide

The nematode Caenorhabditis elegans exhibits chemotaxis toward a wide variety of chemicals including water-soluble molecules and volatile organic compounds. We have previously discovered that C. elegans wild-type strain N2 is strongly attracted by cycloheximide which has long been known as a bitter tastant for humans and other mammals. We describe here the isolation and initial characterization of the first mutants which were defective specifically in their attraction to cycloheximide. In our screenings, we selected two mutants that were defective in chemotaxis to cycloheximide but normal in their attraction to NH4Cl and histidine. These mutants also avoided quinine hydrochloride, CuSO4 and high concentrations of NaCl similar to the wild-type strain N2. Furthermore, no observable defect was detected in their attraction to volatile odorants such as isoamyl alcohol and diacetyl. Dye-filling experiments suggested that they have no morphological defect in the sensory endings of the amphid neurons.