Accumulative gene integration into a pre-determined site using Cre/loxP

Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.     

PEI-assisted preparation of Au nanoparticles via reductive crystallization process

Nano-sized gold particles were prepared in a solution containing polyethylenimine (PEI) utilizing a batch reactor. PEI acts as a reducing agent as well as a stabilizing agent of nanoparticles. The effects of initial concentration of PEI on the mean particle diameter, coefficient of variation (C.V.), growth rate, nucleation rate and the number of nuclei were studied. The particle diameter of gold decreased markedly with increasing initial concentration of PEI, from 3.3 μm to a minimum value of about 5 nm. The observed decrease of particle diameter was considered to be caused by the growth-inhibiting effect of PEI, which affects the conclusive number of nuclei.     

Molded micro- and mesoporous carbon/silica composite from rice husk and beet sugar

A molded carbon/silica composite with high micro- and mesoporosity, as well as a high bulk density, was fabricated by activating a disk-molded precursor made from carbonized rice husk (RH) and beet sugar (BS) at 875 °C in CO₂. The pore structure of the RH- and BS-based carbon/silica composite (RBC) was analysed in relation to the bulk density. An activation time of 2.0 h provided the largest BET specific surface area (1027 m²/g) and total pore volume (0.68 cm³/g) and a low bulk density (0.54 g/cm³). An RBC that was first activated for 1 h was immersed again in BS syrup and then activated in CO₂ for 1 h. This two-step activation process provided both a high bulk density (0.69 g/cm³) and a highly textured structure (BET specific surface area, 943 m²/g; total pore volume, 0.56 cm³/g). The immersion in BS syrup was useful for improving the texture without reducing the bulk density, in comparison to one-step activation for 1.0 h. The suspension of the RBCs was basic because of the residual inorganic compounds of potassium and calcium. However, the basicity of the suspension was alleviated by washing the RBCs with water.     

Fast drive of displacement magnification mechanism with flexure hinge using loading type impact damper

A displacement magnification mechanism which uses flexure motion guide using elastic hinges can realize smooth frictionless motion but has poor vibration damping capability. An impact damper is a damping mechanism which uses collision energy to dissipate vibration energy. If the damper is used for vibration control of the flexure mechanism, it may be able to dissipate unexpected vibration without killing the merits of the flexure mechanism. In the paper, a loading type impact damper is applied to settle down transient vibration of a displacement magnification mechanism. We investigate differences of damping effect by setting conditions of the damper. It is shown that the impact damper can eliminate residual vibration at step response effectively without steady state error. The experimental displacement magnification mechanism with impact damper can settle down less than 1/5 of the response without the damper under appropriate setting conditions. Influence of natural frequency ratio between damper and displacement magnification mechanism is investigated. Influences of indentation at impact point are also examined.     

Reaction of layered carbon fluorides CxF (x = 2.5-3.6) and hydrogen

The layered carbon fluorides C_xF (x = 2.5, 2.8, 3.6), generally classified as fluorine-graphite intercalation compounds, were heat-treated in hydrogen gas. These fluorides are more reactive with hydrogen compared to (CF)_n and (C₂F)_n. Reduction of C_xF to graphite-like carbon starts at about 573 K, and proceeds gradually along with the elevation of temperature. Fluorine atoms in C_xF are eliminated as HF in the reduction process without being substituted by hydrogen atoms. Systematic difference was not found in the average crystallite sizes of the carbon material prepared from C_xF by the reduction with hydrogen and that by the pyrolysis in vacuum. On the other hand, interlayer distance and fluorine content of the former are smaller than those of the latter. In the case that the C_xF precursor maintains a large particle size, the reduced carbon as well as the pyrolytically prepared carbon possesses a foam-like shape due to the exfoliation during the heat treatment.     

Gate effect of vacancy-type defect of fullerene cages on metal-atom migrations in metallofullerenes

Metal-atom migration outside from a defective fullerene cage of a metallofullerene Gd@C(82) (Ca@C(82)), where the Gd(3+) (Ca(2+)) ion is incorporated inside the C(2)(v)()-C(82) cage, is discussed in detail at the B3LYP DFT level of theory. The metal-atom migrations are initiated by the formation of vacancy-type defects involving two coordinatively unsaturated C atoms. This step, which is assumed to proceed due to energy-particle irradiation, leads to the formation of antibonding orbitals between the two C atoms. Since the antibonding orbitals can interact with vacant d-orbitals of the Gd(3)(+)() ion in an in-phase fashion, the attractive interactions allow the Gd ion to insert into the two C atoms in the defect. As a result, the metal ion passes through the defect under energy-particle irradiation. In contrast, the Ca(2+) ion with the vacant s-orbitals does not have such orbital interactions, and thus, a C-C bond is reformed between the two C atoms, which prohibits the Ca ion from penetrating the defected C(82) cage. DFT calculations nicely demonstrate that the orbital interactions control metal-atom migration around the defect site using their orbital symmetries, and therefore, the vacancy-type defect acts as a "gate" that permits a specific atom to go out from a defected fullerene cage.