A Single Mutation at the Ferredoxin Binding Site of P450 Vdh Enables Efficient Biocatalytic Production of 25‐Hydroxyvitamin D3

Vitamin D₃ hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two‐step hydroxylation of vitamin D₃ (VD₃) to produce 25‐hydroxyvitamin D₃ (25(OH)VD₃) and 1α,25‐dihydroxyvitamin D₃ (1α,25(OH)₂VD₃). These hydroxylated forms of VD₃ are useful as pharmaceuticals for the treatment of conditions associated with VD₃ deficiency and VD₃ metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin‐binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD₃, a promising intermediate for the synthesis of various hydroxylated VD₃ derivatives, by using nisin‐treated Rhodococcus erythropolis cells containing Vdh_T107A. The gene‐expression cassette encoding Bacillus megaterium glucose dehydrogenase‐IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL^?1 25(OH)VD₃ was successfully produced by a 2 h bioconversion.     

Synthesis of an AlN Polycrystalline Bulk Layer and Nanotubes by Using NH3 and Bi

A bulk layer of aluminum nitride (AlN) polycrystals was synthesized on a boron nitride crucible surface by heating Al chunks with 5 mol% of bismuth at 1273 K for 3 h under NH₃ gas flow. The fragments of the layer were characterized by X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. The platelet grains of AlN with a size of 0.1–1.0 μm and having preferred orientation of the c -axis perpendicular to the layer were formed at the crucible side. Nanotubes 6–15 μm long and about 20–100 nm thick grew on the gas phase side of the layer.     

A three-dimensional microfluidic tumor cell migration assay to screen the effect of anti-migratory drugs and interstitial flow

Most anti-cancer drug screening assays are currently performed in two dimensions, on flat, rigid surfaces. However, there are increasing indications that three-dimensional (3D) platforms provide a more realistic setting to investigate accurate morphology, growth, and sensitivity of tumor cells to chemical factors. Moreover, interstitial flow plays a pivotal role in tumor growth. Here, we present a microfluidic 3D platform to investigate behaviors of tumor cells in flow conditions with anti-migratory compounds. Our results show that interstitial flow and its direction have significant impact on migration and growth of hepatocellular carcinoma cell lines such as HepG2 and HLE. In particular, HepG2/HLE cells tend to migrate against interstitial flow, and their growth increases in interstitial flow conditions regardless of the flow direction. Furthermore, this migratory activity of HepG2 cells is enhanced when they are co-cultured with human umbilical vein endothelial cells. We also found that migration activity of HepG2 cells attenuates under hypoxic conditions. In addition, the effect of Artemisinin, an anti-migratory compound, on HepG2 cells was quantitatively analyzed. The microfluidic 3D platform described here is useful to investigate more accurately the effect of anti-migratory drugs on tumor cells and the critical influence of interstitial flow than 2D culture models.     

Framework for Performance-Based Design of Building Structures

Abstract:   This article proposes a new framework for performance-based design (PBD) of building structures. This framework was proposed under the 3-year Japanese Government Comprehensive Research and Development Project on "Development of a New Engineering Framework for Building Structures" launched in the fiscal year of 1995. The primary objective of the project is to create a system in which the performance of buildings is clearly stated, and consumers, that is, occupants, are well informed of how their buildings will perform and how much it will cost to maintain their performance. The framework emphasizes the establishment of target performance, the performance evaluation, and the performance statement as the main three elements. It also stresses that an institutional framework and support systems need to be provided to enable PBD to be practiced efficiently. The implementation of the proposed framework is also expected to promote engineering innovation, progress in building engineering, and globalization. The new framework will also bring other benefits, such as improved design techniques, greater design flexibility, and international harmonization. It is also important for building structural performance to become one of the most important indexes for consumers to define a building's value. The Japanese building code was changed to the performance-based code, based in the clear and comprehensive manner proposed in this article. In the United States, "Vision 2000" ( SEAOC, 1995 ) was published, then many research activities were conducted simultaneously.     

Seismic damage analysis of reinforced concrete buildings based on statistics of structural lateral resistance

The largest revision of the building code in Japan was released in 1981, in which the examination of ultimate lateral strength of structure is required. As a result, the capacity of the earthquake resistance of buildings is different between those designed before and after the revision of the code.The objective of this paper is to evaluate the seismic reliability of two groups of reinforced concrete buildings; one is the building group designed by the current code (after the 1981 revision) and the other is the building group designed by the former code (before the revision). The site is assumed to be Sendai city, Japan. Seismic reliability analyses of buildings are carried out and the probability of structural responses exceeding certain response levels during 100 years is evaluated for each type of building group. In addition to the uncertainty of earthquake forces, the uncertainties of structural characteristics, such as lateral resistance and the first natural period of structures, are considered based on the statistics of existing buildings.     

Adjuvant activity of muramyl dipeptide derivatives to enhance immunogenicity of a hantavirus-inactivated vaccine

The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-1 virus (B-1 vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined. When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-1 vaccine admixed with or without 100 micrograms mouse-1 of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-1 vaccine alone. Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-1 vaccine alone during 3-9 weeks after the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-1 alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid protein (rNP) of Hantaan 76-118 strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a significantly higher proliferating activity than those treated with B-1 vaccine alone. In addition, when mice were immunized once with B-1 vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-1 vaccine on day 7, mice immunized with B-1/B30-MDP and B-1/MDP-Lys(L18) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-1 vaccine alone. These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-1 vaccine to induce a humoral and cellular response to HFRS virus.     

Synthesis of Colloidal SU‐8 Polymer Rods Using Sonication

The bulk synthesis of fluorescent colloidal SU‐8 polymer rods with tunable dimensions is described. The colloidal SU‐8 rods are prepared by shearing an emulsion of SU‐8 polymer droplets and then exposing the resulting non‐Brownian rods to ultrasonic waves, which breaks them into colloidal rods with typical lengths of 3.5–10 µm and diameters of 0.4–1 µm. The rods are stable in both aqueous and apolar solvents, and by varying the composition of apolar solvent mixtures both the difference in refractive index and mass density between particles and solvent can be independently controlled. Consequently, these colloidal SU‐8 rods can be used in both 3D confocal microscopy and optical trapping experiments while carefully tuning the effect of gravity. This is demonstrated by using confocal microscopy to image the liquid crystalline phases and the isotropic–nematic interface formed by the colloidal SU‐8 rods and by optically trapping single rods in water. Finally, the simultaneous confocal imaging and optical manipulation of multiple SU‐8 rods in the isotropic phase is shown.     

Robust bin-picking system using tactile sensor

ABSTRACT

This paper presents a robust bin-picking system utilizing tactile sensors and a vision sensor. The object position and orientation are estimated using a fast template-matching method through the vision sensor. When a robot picks up an object, the tactile sensors detect the success or failure of the grasping, and a force sensor detects the contact with the environment. A weight sensor is also used to judge whether the lifting of the object has been successful. The robust and efficient bin-picking system presented herein is implemented through the integration of different sensors. In particular, the tactile sensors realize rope-shaped object picking that has yet to be made possible with conventional picking systems. The effectiveness of the proposed method was confirmed through grasping experiments and in a competitive event at the World Robot Challenge 2018.     

Low‐frequency sound absorption of organic hybrid comprised of chlorinated polyethylene and N,N′‐dicyclohexyl‐2‐benzothiazolyl sulfenamide

We investigated the sound absorption characteristics of an organic hybrid material comprised of chlorinated polyethylene (CPE) as the matrix polymer and N,N′‐dicyclohexyl‐2‐benzothiazolyl sulfenamide (DBS) as the second component of an organic low‐molecular‐weight compound. We found specific crystallites, obtained by annealing, that generated new absorption for a low‐frequency sound in a CPE/DBS blend. We observed two sound absorption peaks, around 300 and 1000 Hz, in the annealed CPE/DBS (50 : 50 w/w) blends, whereas those peaks were not observed in the untreated sample. There were two kinds of crystals with different melting points in the annealed samples. It was confirmed that the crystals with the lower melting point brought about sound absorption at a low frequency. The crystals that had the lower melting point were smaller and/or more disordered than the crystals that had the higher melting point. We calculated the fraction of these two types of crystals from differential scanning calorimetry and wide‐angle X‐ray diffraction measurements. The annealing or reannealing temperature specified the fraction of the crystal with the lower melting point, and the obtained crystal fraction characterized sound absorption frequency. Therefore, it is possible to control the sound absorption frequency of an organic hybrid by heat treatment such as annealing. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006