Functional characterization and evolutionary implication of the internal 157-amino-acid sequence of an exoinulinase from Penicillium sp. strain TN-88

An extracellular exoinulinase from the filamentous fungus Penicillium sp. strain TN-88 has a 14-fold higher specific activity of 743 U/mg toward inulin than its equivalent from the Aspergillus niger strain 12 and possesses an internal 157-amino-acid sequence whose corresponding region is absent in the A. niger enzyme. On the basis of sequence alignment, the internal region D' encoding the 157-amino-acid sequence in the Penicillium exoinulinase gene inuD cDNA was inserted into the site between the nucleotides 897 and 898 of the A. niger exoinulinase gene inuE cDNA. The resultant inuE::D' fusion was expressed in the methylotrophic yeast Pichia pastoris. The K(m) value of the secreted hybrid enzyme InuE::D' for inulin hydrolysis was about 1/15 that of the A. niger InuE, whereas its k(cat) value did not differ greatly from that of the InuE. These observations indicate that the Penicillium exoinulinase has evolved by the horizontal transfer and integration of a relevant DNA segment and that the internal sequence D' functions as an additional noncatalytic inulin-affinity region.     

Integrated protocol for optimized link state routing and localization: OLSR-L

Localization protocol is important for estimating node positions in a wireless multi-hop network. Routing protocol is also important for controlling paths. In previous research, localization and routing protocols have been discussed and evaluated separately. In this paper, we propose an integrated protocol for optimized link state routing (OLSR) and OLSR based localization (ROULA). Our protocol enables simultaneous localization and routing. ROULA’s localization is performed using OLSR overhead such as hello packets and routing tables. The routing overheads and the processing procedures can be efficiently integrated. We demonstrate that the integrated protocol for ROULA and OLSR enables simultaneous localization and routing.     

Quantitative evaluation of reheat cracking susceptibility by in situ observation and measurement using laser confocal microscope*

In the post-weld heat treatment process, the reheat cracking which might occur in the weldments of low-alloy steels has been a serious problem. So, it is considered to be important to predict the possibility of occurrence of reheat cracking in these steels. It is however recognized as a time-consuming procedure to evaluate quantitatively the susceptibility to this type of cracking. In the present study, a new quantitative evaluation method of reheat cracking susceptibility by in situ observation and measurement using a laser confocal microscope has been proposed. Through this new method, the reheat cracking susceptibility of any kind of steels can be evaluated with the same standard. Moreover, because the position of the initial crack can be focused and the critical ductility to initiate the crack is measured by in situ observation, the reheat cracking susceptibility can be evaluated using only one specimen. So the newly developed method can provide efficient quantitative assessment of the reheat cracking sensitivity with high accuracy.     

Magnetic leakage field due to sub-surface defects in ferromagnetic specimens

This paper describes analytical expressions for the magnetic leakage field of two types of internal defects: two dimensional rectangular and elliptical defects as functions of the applied magnetic field strength, the defect size and the distance below the surface. In this study, the magnetic image effects from the spatial boundary and the defect boundary are taken into consideration. That is, the leakage field of rectangular-like defects or elliptically shaped defects ‘below the surface’ are derived by using the modified dipole model and image theory. The profiles and strengths of leakage fields calculated from the proposed analytical forms are presented under various conditions and compared with experimentally measured ones.     

FATIGUE STRENGTH OF NON-LOAD-CARRYING FILLET WELDED JOINTS: EFFECTS OF WELD RESIDUAL STRESSES AND STRESS CONCENTRATION

The fatigue strength of non-load-carrying fillet welded joints of KE36(TMCP) steel was studied. Both residual stress measurements and fatigue tests were carried out, with the plate thickness, the plate width and the heat input being varied. Specimens given a Post Weld Heat Treatment (PWHT) were also prepared. The plate width had no effect on the fatigue strength, because it hardly affected the transverse residual stresses at the weld toe. However, the heat input influenced the transverse residual stress distribution, and a significant difference in fatigue strength due to the heat input was observed, especially when N≥ 10⁶ cycles. It was also found that PWHT removed almost all the residual stresses at the weld toe, improving the fatigue strength drastically. In this study, the values of stress concentration factor K₂ were estimated by Machida's method and it was concluded that the thickness effect resulted from a combination of both stress concentration and residual stresses with the contribution of the latter being particularly significant for N≥ 10⁶ cycles.     

Development of a prestressed and precast concrete segmental lining

Prestressed & precast concrete segmental lining (P&PCSL) is used for shield tunnels. Its primary feature is that it integrates segments into one ring by introducing prestress in the tunnel circumferential and longitudinal directions. Introducing prestress enables the elimination of bolt joints and reduces the volume of reinforcement, thus reducing the manufacturing cost of precast concrete segments. It also enables quality improvement and labor saving in lining and provides greater adaptability for tunnels with large diameter, where deformation due to dead load is a problem. The P&PCSL has been implemented in three construction projects after undergoing various performance tests and workability verification tests.     

Impact of Al in Cu alloy interconnects on electro and stress migration reliabilities

The reliability of Cu interconnects was successfully improved by applying a CuAl alloy seed. However, the effect of additive Al on the reliability is not fully understood. In order to reveal the reliability improvement mechanism, Cu films using CuAl alloy seed were investigated in detail. As stress induced voiding (SIV) as well as electromigration is caused by migration of vacancies and/or Cu atoms, the measured activation energy value of electromigration using CuAl indicates that the fast diffusion paths are Cu grain boundaries. The analysis using high lateral resolution scanning type secondary ion mass spectrometry (nano-SIMS) clarifies that additive Al in ECP-Cu film is mainly localized at grain boundaries. Furthermore, positron annihilation was used to probe vacancy-type defects in Cu films. The CuAl films before recrystallization contain larger and higher density vacancy-type defects. Whereas, the recrystallized CuAl films after annealing above 250 °C contain smaller and lower density defects. Furthermore, CuAl films with annealing above 350 °C contain less Al inside the grains. These results represent that Al atoms in Cu films with annealing above 350 °C are exhausted from inside grains to the grain boundaries, and the spewed Al atoms existing at Cu grain boundary effectively prevents the diffusion of Cu and/or vacancies.     

Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds

Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell–deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.     

Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose

The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16–2.5 mM glucose, with a correlation coefficient, r, of 0.998.     

Generation mechanism and prevention method of secondary molybdate phase during vitrification of PUREX wastes in liquid-fed ceramic melter

ABSTRACT

In order to develop a glass production method that inhibits formation of the Mo-bearing secondary phase, the so-called yellow phase (YP), and also to clarify its formation behavior, vitrification tests using a small-scale liquid-fed ceramic melter (LFCM) were conducted. Chemically simulated PUREX liquid waste containing Na, Mo and other fission elements was fed into a molten glass pool in the melter together with several sizes of feed glass. When the glass beads with a diameter of around 2 mm were fed, some YP was contained in the vitrified glass products at 25 wt% waste loading. While, almost no YP was detected in the glass products when glass powder with a diameter of less than 63 μm was used. The microscopic analysis of the cold-cap samples revealed that powderization of the feed glass contributed to the facilitation of Na dissolution to the feed glass in the cold-cap. This quick intake of Na to the feed glass prevented the formation of liquid Na₂MoO₄ aggregation and provided homogeneous Mo dispersion in the cold-cap with forms of alkali earth and/or rare earth molybdates. The homogeneous dispersion of Mo resulted in the fast and complete dissolution of Mo in the glass melt.