Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.     

Comparison of behavioral effects of morphine and fentanyl in dogs and cats

Behavioral effects induced by intravenous administration of morphine at 0.3, 0.6, 1.2, and 2.4 μg/kg and fentanyl at 5, 10, 20, and 40 μg/kg were evaluated in dogs and cats. In dogs, fentanyl and morphine depressed activity and level of consciousness in a dose- dependant manner. In cats, higher doses of fentanyl stimulated activity temporarily, but excitement, so-called "opioid mania," was not observed. Morphine induced distinctive behavioral changes characterized by sitting with fixed staring, and "opioid mania" was not observed in cats.     

Quantification of DNA of citrus huanglongbing pathogen in diseased leaves using competitive PCR

A quantification system for huanglongbing pathogen using a competitive polymerase chain reaction method and image-analyzing software were developed to obtain precise results. Significant differences in the quantity of pathogen were thus determined in leaves of two citrus cultivars commonly cultivated in southern Vietnam. Less pathogen-related DNA was detected from the tissue of citrus cultivars that are believed to be more tolerant than susceptible cultivars. The quantification system will be used in studies on pathogen proliferation and movement inside citrus tissue.     

Reflection and transmission of visible light by sugi wood: effects of cellular structure and densification

Transmittance and reflectance of visible light by sugi wood (Cryptomeria japonica) were investigated in the longitudinal (L) and tangential (T) directions. Transmittance was the highest in the L direction and reflectance was the highest in the T direction, suggesting that structural anisotropy influences transmittance and reflectance. Intra-ring variations observed with a microspectrometer indicated that T transmittance was higher for latewood than for earlywood, but there was no such trend in for L transmittance in which the highest levels occurred near the annual ring boundaries, on either the earlywood or latewood side, and the lowest at the transition from earlywood to latewood. Dependence of L transmittance on wavelength also showed variations according to the intra-ring position. The increasing of transmittance of earlywood at wavelengths?<?500 nm with increasing wavelength was observed, but this was not confirmed for latewood because of absorption by lignin. These observations supported a previously published finding, which was based on measurements in the radial direction, that the number of internal cell wall reflections, rather than density, determines wood lightness. Indeed, in the L direction, most of the incident light passes through lumens in earlywood and through cell walls in latewood, while it is subjected to numerous internal reflections at the interface between lumens and cell walls. This was further confirmed by the transmittance of earlywood being greatly decreased by radial compression.     

Manufacture and mechanical properties of binderless boards from kenaf core

Binderless boards were prepared from finely ground powders of kenaf (Hibiscus cannabinus L.) core under varying manufacturing conditions. This research was designed to investigate their mechanical properties and evaluate the various manufacturing conditions: pressing temperature and time, pressing pressure, board density, board thickness, grain size of raw materials, and addition of furfural. The mechanical properties (i.e., modulus of rupture and elasticity, internal bonding strength) of boards increased with increasing board density and met the requirement for 15 type medium-density fiberboard (MDF) by JIS A 5905-1994. Thickness swelling and water absorption of boards exceeded the maximum permitted levels for 15 type MDF and S20 grade hardboard by JIS A 5905-1994, which indicates the low water-resistant property of binderless boards. In contrast to that in usual wood-based materials, internal bonding strength showed significant correlations with other board properties: modulus of rupture and elasticity, thickness swelling, and water absorption. We confirmed experimentally that the best manufacturing conditions proved to be as follows: pressing temperature 180°C, time 10min; pressing pressure 5.3MPa; board thickness 5mm; board density 1.0g/cm³; average grain size 53µm; and powder with no furfural content.Part of this paper was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002     

Effects of thinning on wind damage in Pinus thunbergii plantation ——Based on theoretlcal derlvation of risk—ratios for assessing wind damage

Based on paper of “Theoretical derivation of risk-ratios for assessing wind damage in coastal forest“,wind damage in the pine coasteal forest,which was thinned at four levels in December of 1997,was investigated for four successive growing seasons.Besides wind damage,the wind profiles outside and inside the coastal forest stand and the distributions of optical stratification porosity (OSP) were also observed.Based on these data,risk-ratios of wind damage for both individual trees and stands were estimated according to the methods developed in “Theoretical derivation of risk-ratios for assessing wind damage in a coastal forest“.The results showed that risk-ratios of wind damage,which were calculated from the meen height and diameter only and from the combination of wind and stand sructure profiles,accurately predicted wind damage in the plantation.Relationships between different thinning ratios and incidence of wind damage showed that stand stability decreased soon after the thinning.This was due to the immediate effects of thinning on increasing the canopy roughness and wind load,and on decreasing the sheltering effects from surrounding trees.However,thinning strategies could improve the stability by long-term effects on growthand development of trees against extreme wind.Only canopy damage was recorded during the experimental period,no stem damage was found,even though the maximum 10-min wind speed outside the coastal forest attained 30.2m s^-1.The results obtained in this study indicate that thinning is the most effective silvicultural strategy available for managing coastal forest despite the increased probability of wind damage soon after thinning.     

Mechanical behavior of the crystal lattice of natural cellulose in wood under repeated uniaxial tension stress in the fiber direction

This study investigated the relationship between the cellulose crystal lattice strain (crystalline region) and the macroscopic surface strain in specimens of Chamaecyparis obtusa wood under repeated uniaxial tension stress in the fiber direction. Changes in the strain of the crystal lattice were measured from the peak of (004) reflection using the transit X-ray method. The macroscopic surface strain of each specimen was measured with a strain gauge. In both loading and unloading, the surface strain changed linearly with changes in stress. However, crystal lattice strain was not linear but exhibited changes along a curve with changing stress. Under stressed conditions, the crystal lattice strain was always less than the surface strain, regardless of the frequency of repetition in the loading and unloading cycle. The ratio of the crystal lattice strain to the surface strain showed a negative correlation for stress in both loading and unloading. That is, the ratio decreased with increasing stress, and finally tend to converge to a specific value. The ratio (I/I ₀) between the diffracted intensity (I ₀) in the (004) plane in the unloaded condition and the diffracted intensity (I) in the (004) plane in the loaded condition tend to converge on a specific value with increasing frequency of repetition. When the substantial tension Young’s modulus of the wood in the longitudinal direction decreased, the ratio of the strain of the crystal lattice to the surface strain also decreased. Moreover, the ratio decreased with increasing microfibril angle of the specimen.