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.     

Aminoglycosides prevent and dissociate the aggregation of platelets in patients with EDTA-dependent pseudothrombocytopenia

Although EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is of practical importance because failure to recognize this clinical entity may result in misdiagnosis and subsequent mismanagement of the patients, the pathophysiological nature of EDTA-PTCP remains unknown. To develop an effective way to evaluate the platelet counts in patients with EDTA-PTCP, we introduced aminoglycosides-supplemented anticoagulating agents. When kanamycin was pre-supplemented with EDTA for anticoagulating blood samples from EDTA-PTCP patients there was no significant change in the platelet counts and the morphology of blood cells after 150 min of incubation at room temperature. Furthermore, when kanamycin was added to EDTA-anticoagulated blood samples from EDTA-PTCP patients within 30 min after blood withdrawal, rapid dissociation of platelets without apparent morphological changes of blood cells was observed, and complete blood cell counts as well as the histogram patterns were almost the same as those examined immediately after blood sampling. The dissociation of aggregated platelets was also detected when other antibiotics were used, although it was associated with some extent of morphological changes of blood cells. These findings indicate that the supplementation of aminoglycosides either before or after blood sampling is a useful method for the diagnosis EDTA-PTCP and for the evaluation of platelet counts in patients with EDTA-PTCP.     

Electric field-assisted and field-depressed segregation of reactive metals to the bonding interface in braze alloy joining

Segregation of reactive metals at the bonding interface has been observed in various ceramic and/or metal joints bonded with reactive metal-bearing braze alloys. When a d.c. of 20 mAcm⁻² is applied to the ceramic/braze/ceramic system at a brazing temperature of, say, 1373 K, the electric field assists the segregation at the braze-ceramic interface on the cathode side and suppresses the segregation at the interface on the anode side. This may imply that reactive metal atoms in the braze can migrate as a cation. E.m.f. measurement on the ceramic (AIN or ZrB₂)-metal foil systems with increasing temperature shows that a negative e.m.f. to the ceramic pole appears from about 900 K for AIN and from 500 K for ZrB₂, as does the thermally stimulated current in polymers. These temperatures coincide well with those where the electrical conductivity of AIN and ZrB₂, respectively, begins to increase with increasing temperature. Therefore, it is considered that the polarization of the ceramics may take place and assist the migration, and consequently segregation, of reactive metals in braze alloys to the braze-ceramic interface during brazing.     

Structure and mechanical properties of polyethylene-fullerene composites

The microhardness of films of fullerene-polyethylene composites prepared by gelation from semidilute solution, using ultrahigh molecular weight polyethylene (PE) (6×10⁶), has been determined. The composite materials were characterized by optical microscopy and X-ray diffraction techniques. The microhardness of the films is shown to increase notably with the concentration of fullerene particles within the films. In addition, a substantial hardening of the composites is obtained after annealing the materials at high temperatures (T _a=130 °C) and long annealing times (t _a=10⁵s). The hardening of the composites with annealing temperature has been identified with the thickening of the PE crystalline lamellae. Comparison of X-ray scattering data and the microhardness values upon annealing leads to the conclusion of phase separation of C₆₀ molecules from the polyethylene crystals within the material. The temperature dependence is discussed in terms of the independent contribution of the PE matrix of the C₆₀ aggregates to the hardness value.     

Performance prediction of a hybrid‐excitation synchronous machine with axially arranged excitation poles and permanent‐magnet poles

This paper presents a method of predicting the steady‐state performance of a new hybrid‐excitation synchronous machine (HESM) theoretically. The field pole of this HESM is axially divided into two parts; one is an excitation part and the other a permanent‐magnet (PM) part. A nonlinear equivalent circuit, which can include the saliency of the rotor and the magnetic saturation due to the iron core, is derived. Based on this equivalent circuit, the steady‐state performance of the HESM is calculated, and the results are confirmed through experiments. © 2004 Wiley Periodicals, Inc. Electr Eng Jpn, 150(2): 43–49, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/eej.20033     

Effect of surfactants on phenol removal by the method of polymerization and precipitation catalysed by Coprinus cinereus peroxidase

The removal of phenol by peroxidase‐catalysed polymerization was examined using Coprinus cinereus peroxidase in the presence of surfactants. The non‐ionic surfactants with poly(oxyethene) residues, Triton X‐100, Triton X‐405 and Tween 20, enhanced the phenol removal efficiency at a level similar to high relative molecular mass poly(ethylene glycol) (relative molecular mass 3000). Although the improvement in the removal efficiency was less than that of Triton X‐100, Span 20, sodium lauryl sulfate (SDS) and lauryl trimethylammonium bromide (DTAB) also enhanced the removal efficiency. The requirement of the enzyme for almost 100% removal of 100 mg dm^?3 phenol decreased to one‐fourth by the addition of 30 mg dm^?3 Triton X‐100. Triton X‐100, Triton X‐405, Tween 20 and DTAB could reactivate the enzyme precipitated with the phenol polymer, leading to the restarting of the phenol removal reaction. Copyright © 2003 Society of Chemical Industry     

Electron transport in InGaO3(ZnO)m (m=integer) studied using single-crystalline thin films and transparent MISFETs

We have investigated the characteristics of transparent metal-insulator-semiconductor field-effect transistors (MISFETs) fabricated using InGaO₃(ZnO)_m (m=integer) single-crystalline thin films as n-channel layers and amorphous alumina as gate insulator films. The MISFETs exhibit good characteristics such as insensitivity to visible light illumination, off-current as low as ∼1 nA with a positive threshold voltage of ∼3 V and on/off current ratio of 10⁵. The field-effect mobility increased from ∼1 to ∼10 cm² (V s)⁻¹ as the m-value increased. Room temperature Hall mobility also increased. However, unexpectedly these values were lower than the field-effect mobility. It is explained by existence of shallow localized state in the homologous compounds.     

0.5-μm 3.3-V BiCMOS standard cells with 32-kilobyte cache andten-port register file

BiCMOS standard cell macros, including a 0.5-W 3-ns register file, a 0.6-W 5-ns 32-kbyte cache, a 0.2-W 3-ns table look-aside buffer (TLB), and a 0.1-W 3-ns adder, are designed with a 0.5-μm BiCMOS technology. A supply voltage of 3.3 V is used to achieve low power consumption. Several BiCMOS/CMOS circuits, such as a self-aligned threshold inverter (SATI) sense amplifier and an ECL HIT logic are used to realize high-speed operation at the low supply voltage. The performance of the BiCMOS macros is verified using a fabricated test chip     

Effects of nondensifying inclusions on the densification and microstructure of zinc oxide matrix composites

The densification and microstructure development of ZnO containing Zn₇Sb₂O₁₂, ZrO₂, and aggregated ZnO were investigated to elucidate the effect of nondensifying inclusions on the sintering of ceramic/ceramic composites. The inclusion retarded the densification, and the degree of retardation was found to depend on the chemical species of inclusion; Zn₇Sb₂O₁₂ had the largest effect, followed by ZrO₂ and then aggregated ZnO last. The experimental results for aggregated ZnO was explained by the theory which predicts the generation of backstresses. The backstresses give a less significant effect on the densification. For Zn₇Sb₂O₁₂ and ZrO₂, the microstructure of the matrix varied with distance from an inclusion particle; much porosity was observed in the region surrounding the inclusion. Circumferential voids, which are responsible for the suppression of densification, form during the initial stage of sintering. Inclusion particles generate an anchoring effect which retards the densification of the matrix immediately surrounding the inclusion particle during the intermediate stage.Supported by the Inamori Foundation.     

Subarachnoid haemorrhage induced proliferation of leptomeningeal cells and deposition of extracellular matrices in the arachnoid granulations and subarachnoid space. Immunhistochemical study

Subarachnoid haemorrhage (SAH) often leads to subarachnoid fibrosis and resultant normal pressure hydrocephalus; however, how subarachnoid fibrosis occurs is unknown. We examined the changes within arachnoid granulations (AGs) and the subarachnoid space (SAS) chronologically at the parasagittal region obtained from patients with SAH at autopsy and made comparison with controls by immunostaining for cytokeratin, specific marker for leptomeningeal cells and by the elastica Masson-Goldner methods. Within a week some AGs were torn, and many inflammatory cells filled the AGs and SAS. Cytokeratin positive cells were scarce. During the next two weeks cytokeratin positive cells increased. After three weeks, AGs and SAS were filled by dense deposits of extracellular matrices surrounded by multiple layers of leptomeningeal cells.