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.     

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     

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     

Steady-state torque control system with secondary-current feedback for three-phase squirrel-cage induction motors

This paper presents a highly stable steady-state torque control system with a feedback of a secondary current signal. The torque control system is constructed with a feedback of the secondary current (I₂) signal using a quick response magnetic-field sensor constructed with an amorphous core multivibrator bridge, which can solve the secondary resistance change problem for temperature variation in the IM. Moreover, we added a state feedback to the system to improve the response speed for the unit step function of a torque order. The I₂ sensor method has advantages especially at a low-speed range, in which conventional method using an integrator shows control errors.     

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.     

Primary hepatic carcinoid tumor

A case of primary carcinoid tumor arising in the liver of a 69 year old woman with no endocrine symptoms is reported. Histopathologically, the tumor was diagnosed initially as a hepatocellular carcinoma in the biopsy specimen, and was shown subsequently to be a carcinoid tumor, demonstrating diffuse positive staining with Grimelius method. Mucin stained with periodic acid-Schiff (PAS), alcian-blue, and mucicarmine, and was shown partially in the glandular structures. Immunohistochemically, most of the tumor cells stained positively for chromogranin-A, epithelial membrane antigen (EMA) and neuron specific enolase (NSE). Ultrastructural examination revealed electron-dense core granules, measuring 40-120 nm in diameter in some of the tumor cells. Intensive and careful searches pre- and post-operatively revealed no other primary source of tumor other than the liver. The patient was reported well with no symptoms 3 1/2 years after the operation. This case is considered to be a primary hepatic carcinoid tumor. The recent literature is reviewed, and the possible histogenesis of hepatic carcinoid tumor is discussed.     

One-stage anterior and posterior correction of severe kyphosis of the cervical spine in neurofibromatosis. A case report

A case is presented of a 29-year-old woman with neurofibromatosis, who had a 100 degrees dystrophic kyphosis of the cervical spine and was successfully treated by a one-stage anterior and posterior correction. Anterior distractive and posterior compressive corrections were simultaneously applied keeping the posterior longitudinal ligament intact. The kyphosis was corrected to 42 degrees. Safety and better results of the one-stage correction of kyphosis is based on a concept of shortening the spinal canal.