Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

Quantitative risk assessment using a Japanese hydrogen refueling station model

Although hydrogen refueling stations (HRSs) are becoming widespread across Japan and are essential for the operation of fuel cell vehicles, they present potential hazards. A large number of accidents such as explosions or fires have been reported, rendering it necessary to conduct a number of qualitative and quantitative risk assessments for HRSs. Current safety codes and technical standards related to Japanese HRSs have been established based on the results of a qualitative risk assessment and quantitative effectiveness validation of safety measures over ten years ago. In the last decade, there has been much development in the technologies of the components or facilities used in domestic HRSs and much operational experience as well as knowledge to use hydrogen in HRSs safely have been gained through years of commercial operation. The purpose of the present study is to conduct a quantitative risk assessment (QRA) of the latest HRS model representing Japanese HRSs with the most current information and to identify the most significant scenarios that pose the greatest risks to the physical surroundings in the HRS model. The results of the QRA show that the risk contours of 10^?3 and 10^?4 per year were confined within the HRS boundaries, whereas the risk contours of 10^?5 and 10^?6 per year are still present outside the HRS. Comparing the breakdown of the individual risks (IRs) at the risk ranking points, we conclude that the risk of jet fire demonstrates the highest contribution to the risks at all of the risk ranking points and outside the station. To reduce these risks and confine the risk contour of 10^?6 per year within the HRS boundaries, it is necessary to consider risk mitigation measures for jet fires.     

A wafer level monitoring method for plasma-charging damage usingantenna PMOSFET test structure

A novel monitoring method for plasma-charging damage is proposed. This method performs a quick and accurate evaluation using antenna PMOSFET. It was found that not only hot-carrier (HC) lifetime but transistor parameters such as initial gate current and substrate current were changed according to the degree of plasma-charging damage. However, the present work suggests that monitoring the shift of drain current after a few seconds of HC stress is a more accurate method to indicate plasma-charging damage. The monitoring method using the present test structure is demonstrated to be useful for realizing highly reliable devices     

Studies on chitin. IX. Crosslinking of water-soluble chitin and evaluation of the products as adsorbents for cupric ion

Water-soluble chitin was successfully crosslinked to varying extents with glutaraldehyde in homogeneous aqueous solutions to improve the properties as an adsorbent for metal cations, and the effects of crosslinking were discussed. Complete insolubilization was achieved with the fivefold excess aldehyde, but, in terms of adsorptivity of Cu²⁺, the chitin crosslinked at an aldehyde/amino group ratio of 1.0 was found to exhibit remarkable capacity and was much superior to others. The desorption of Cu²⁺ from the adsorption complex was also attained effectively at pH 2.0. These results indicated that the loose crosslinking was quite simple and efficient to produce high capacity adsorbents for practical use. Thermal behavior of the crosslinked chitin was examined by TMA and TGA; a softening phenomenon was observed at 145°C.     

Theoretical model for flash generation

Reduction of flash generated in a gas vent is of great concern for manufacturers of electronic parts. The present study proposes a theoretical model for flash generation through consideration of flow characteristics in a gas vent. The model predicts the factors controlling flash, i.e., material parameters such as zero‐shear viscosity, crystallization temperature, thermal conductivity, and heat capacity, and process parameters such as injection and mold wall temperatures, packing pressure, and the clearance of a gas vent. On the other hand, we measure the amount of flash generated in the molding of poly(phenylene sulfide) (PPS) composites containing glass fiber and spherical fillers (CaCO₃ or Al₂O₃). Flash reduces with decreasing size of spherical fillers. These experimental data are successfully interpreted using the flash model. Polym. Eng. Sci., 45:198–206, 2005. © 2005 Society of Plastics Engineers     

Transmittance and cathodoluminescence of AlN ceramics sintered with Ca3Al2O6 as sintering additive

Aluminum nitride ceramics were prepared by sintering with 0–4.8 mass% of Ca₃Al₂O₆ (C₃A) as a sintering additive. The transmittance in the range of 260–550 nm increased with increasing amount of C₃A. The cathodoluminescence intensity attributed to oxygen-induced defects decreased with increasing amount of C₃A. From the results, the increase of the transmittance in the range of 260–550 nm was considered to be related to the decrease of the oxygen-induced defect density.     

Evaluation of myocardial damage using 123I-BMIPP imaging in patients with vasospastic angina

The purpose of this study is to determine whether left ventricular dysfunction following coronary artery spasm by 123I-BMIPP myocardial imaging. To reveal the clinical efficacy of 123I-BMIPP SPECT, 20 patients with vasospastic angina were studied using resting, 3-hour delayed image with 123I-BMIPP and exercise, 3-hour delayed image with 201Tl SPECT. 123I-BMIPP uptake was decreased compared to 201Tl (discordant) in 12 patients (60%) and in 49/100 myocardial segments (49%). The extent and severity score in resting image with 123I-BMIPP were significantly larger than that in delayed image with 201Tl (p < 0.01). In 123I-BMIPP SPECT, the severity score in the latest ischemia were significantly larger than that in others. The incidence of a complete agreement of decreased 123I-BMIPP uptake and coronary artery spasm was significantly higher (75%) than that in 201Tl (28%, p < 0.01). Furthermore, compared to 201Tl uptake, decreased 123I-BMIPP uptake much more corresponded to reduced wall motion in 9 of patients with mismatching. The severity of regional wall motion abnormality was significantly correlated with severity score of 123I-BMIPP. Late redistribution in delayed image with 123I-BMIPP was seen in 6 patients. The regional washout rate and the severity of regional wall motion abnormality in 6 patients was significantly lower than that in others (p < 0.05). Thus, metabolic abnormality assessed by 123I-BMIPP is well associated with left ventricular asynergy and spastic region in patients with vasospastic angina. In conclusion, 123I-BMIPP SPECT may sensitively delineate the impaired myocardium following coronary artery spasm, and it is very useful in diagnosing and estimating the severity of vasospastic angina.     

Effect of immunosuppression on composition of quasispecies population of hepatitis C virus in patients with chronic hepatitis C coinfected with human immunodeficiency virus

BACKGROUND/AIMS: To study the effects of the immunosuppression caused by the reduction of CD4 activity on the composition of hepatitis C virus (HCV) populations, we analyzed the number of HCV quasispecies clones and the nucleotide diversity of the hypervariable region 1 (HVR1) of HCV in 37 patients with hemophilia with persistent HCV infection, with or without human immunodeficiency virus (HIV). METHODS: The numbers of HCV quasispecies clones were measured by fluorescence single-strand conformation polymorphism analysis. Direct sequencing was used to analyze the degree of diversity of HVR1. We compared these values according to coinfection with HIV, and CD4 counts of patients. RESULTS: There were no differences in either the number of HCV clones or the diversity between patients with and without HIV coinfection. In HIV coinfected patients the diversity decreased in association with the decrease in CD4 count while the number of HCV clones did not. The diversity of HVR1 was 3.64 +/- 5.03% in patients with a CD4 count < 50/microliters and 14.92 +/- 6.03% in patients with a CD4 count > or = 50/microliters; it was significantly lower in the former (p = 0.0002). CONCLUSIONS: A severe reduction in the CD4 count, which is considered to cause a decline in the activity of helper T-lymphocytes, induced changes in the composition of HCV populations; one or a few quasispecies clones are predominant in the HCV population in the serum of individual patients.     

The temperature dependence of luminescence from a long-lasting phosphor exposed to ionizing radiation

The temperature dependence of luminescence from a long-lasting phosphor (LLP), SrAl₂O₄ : Eu²⁺,Dy³⁺, exposed to ionizing radiation has been measured to understand the LLP luminescence mechanism. Evaluation of the decay constants of the LLP exposed to -, β- or γ-rays at temperatures from 200 to 390 K showed that the decay constant is divided into four components ranging from 10⁻⁴ to 10⁻¹ s⁻¹ with activation energies of 0.02–0.35 eV.

Total luminous intensity from the LLP with changing irradiation temperature has its maximum value around the room temperature. Irradiation at elevated temperature (390 K) has the total luminescence pattern with monotonous decrease as temperature rises. As a result of evaluating the temperature dependence of luminescence, the luminescence mechanism is considered as follows:

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