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.     

A 322 MHz random-cycle embedded DRAM with high-accuracy sensing and tuning

A 16 Mb embedded DRAM macro in a fully CMOS logic compatible 90 nm process with a low noise core architecture and a high-accuracy post-fabrication tuning scheme has been developed. Based on the proposed techniques, 61% improvement of the sensing accuracy is realized. Even with the smallest 5 fF/cell capacitance, a 322 MHz random-cycle access while 32 ms data retention time which contributes to save the data retention power down to 60 /spl mu/W are achieved.     

A case of macrocystic cervical neurinoma diagnosed by MRI with Gd-DTPA

The authors report a rare case of a large cystic cervical neurinoma. A 45-year-old female was admitted to our clinic because of motor weakness of the right upper extremity, numbness of the right fingers and right posterior cervical pain. Metrizamide CT myelography demonstrated the outline of a low density mass. MRI showed a mass revealing low signal intensity on T1-weighted image, high signal intensity on T2-weighted image and marginal enhancement on contrast image with Gd-DTPA. The mass which was diagnosed as cystic tumor, was located in the intradural extramedullary space between C4 to C5 segments. After C4 through C5 laminectomy, the tumor was found to originate from the C5 anterior motor root. The tumor consisted mostly of a cystic part with a very thin solid compartment beneath the capsule. Postoperative course of the patient was uneventful. Although spinal neurinoma is one of the most common spinal tumors, an almost completely degenerated large cystic spinal neurinoma is extremely rare. MRI with Gd-DTPA was useful for the diagnosis of the cystic neurinoma by clearly enhancing the margin of the tumor.     

Nigrofrontal dopaminergic function as assessed by 18F-dopa PET

The existence of the nigrofrontal dopaminergic pathway has been demonstrated in neuroanatomical studies. We evaluated the presynaptic nigrofrontal dopaminergic function using 18F-dopa (FD) positron emission tomography (PET). The multiple time PET data in the frontal cortex from 20 to 70 min post-injection for FD were evaluated by Patlak analysis using the cerebellar time-activity curve as an input function. The frontal FD uptake rate constants could not be determined in 5 of 12 normal volunteers because of large deviations in the plots. There were no significant differences between the subjects among whom the frontal FD uptake rate constants could or could not be determined regarding the amount of FD injected, the frontal 18F counts, or whether or not they were pretreated with carbidopa. The uptake constants were determined in 9 or 12 patients with parkinsonian syndrome. While the mean (+/- S.D.) uptake constants in patients with Parkinson's disease (2.89 +/- 0.06 x 10(-3), n = 4) and in patients with progressive supranuclear palsy (2.81 +/- 0.10 x 10(-3), n = 3) were not significantly different from those in the normal volunteers (2.93 +/- 0.14 x 10(-3)), those in two patients with corticobasal degeneration (2.42 and 2.46, respectively) decreased in comparison to the control values. Differences in the nigrofrontal presynaptic dopaminergic function as assessed by FD-PET may explain the different pathogenesis and also help to differentiate between corticobasal degeneration and other parkinsonian syndromes, such as Parkinson's disease and progressive supranuclear palsy.     

Effects of Environmental Temperature and Compression Energy on Polymorphic Transformation During Tabletting

The effects of temperature on the polymorphic transformation and the compression of chlorpropamlde forms A and C during tabletting were investigated by X-ray diffractometry. The X-ray diffraction profiles of the sample powders deagglomerated after compression were recorded to calculate the degree of polymorphic transformation. A single punch eccentric tabletting machine equipped with two load cells (upper and lower punches) and with a noncontact displacement transducer was used to measure the compression stress, energy and distance between punches. A heater and a liquid nitrogen pool were mounted on the die of the tabletting machine, and the die temperature was controlled with a thermocontroller. Two types of compression methods, multi-tabletting at room temperature and single tabletting at 0-45°C, were used in the present study. In the first method, the stable form A or metastable form C was loaded in to the die and the sample was compressed with a compression stress of 196 MPa. Compression was repeated from 1 to 30 times. The results for forms A and C suggested that both forms were mutually transformed, and that the content of forms A and C reached equilibrium above 100 J/g of compression energy after more than 10-times compression. After 30-times compression, the content of A, C, and the noncrystalline solid form were almost constant at about 45%, 25% and 30%, respectively. The compression energy was estimated to be 500-600 J/g. In the second method, single tabletting at 0° and 45°C, the amount of form C transformed from form A at 45°C was about two times larger than that at 0°C at the same compression energy. The amount of form A transformed from form C at 45°C was almost the same as that transformed at 0°C. This suggests that the mechanochemical stability of form A was affected by compression temperature, while that of form C was independent of temperature. The crushing strength (CS) of from A tablet was about two times higher than that of form C even at the same porosity. The relationships between log (CS) of form A tablets compressed at 0 or 45°C and porosity showed straight lines with the same slope, but the slope for form C tablets compressed at 45°C was smaller than that for those compression at 0°C. From these results it appears that the transformation mechanism of forms A and C during compression was as follows: Form A or C was converted to a noncrystalline solid by mechanical energy, and then the solid was transformed into form A or C. The transformation of every form was affected by the environmental temperature.     

Age-related accumulation of phosphatidylcholine hydroperoxide in cultured human diploid cells and its prevention by α-tocopherol

The levels of phosphatidylcholine hydroperoxide in serially cultured human fetal diploid fibroblasts at various population doubling levels were determined by high-performance liquid chromatography combined with chemiluminescence detections. This methodology utilizes a mixture of cytochromec and luminol as post-column hydroperoxide group specific luminescent reagents. The cellular hydroperoxide content increased with age from 0.34 to 27.72 pmol/10⁶ cells. At the end of the cells'in vitro lifespan (51st population doubling level), the hydroperoxide content per 10⁶ cells reached about 80 times the level found in cells of the 20th population doubling level. Supplementation of exogenous α-tocopherol to the culture medium prevented hydroperoxide accumulation, but did not extent the lifespanin vitro. The results indicate that substantial intracellular phospholipid hydroperoxide accumulation occurred in the course of aging of human fetal liploid fibroblasts.