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.     

Identification of stress-induced leakage current components and thecorresponding trap models in SiO2 films [MOStransistors]

Time-decay stress-induced leakage current (SILC) has been systematically investigated for the cases of both Fowler-Nordheim (FN) stress and substrate hot-hole stress. From the three viewpoints of the reproducibility of the current component for the gate voltage scan, the change of oxide charge during the gate voltage scan, and the resistance of the current component to thermal annealing, it has been found that time-decay stress-induced leakage current is composed of five current components, regardless of stress type. Trap models corresponding to each current component have been proposed. In addition, it has also been proven that holes generate the electron traps related to one of those current components     

High-dose intravenous steroid pulse therapy in thyroid-associated ophthalmopathy

MIcrospheres containing diclofenac sodium (DS) were prepared using carboxymethylcellulose (CMC) as the main support material (1.0, 2.0, 3.0% (w/v)) and aluminum chloride as the crosslinker. Drug to polymer ratios of 1:1, 1:2 and 1:4 were used to obtain a range of microspheres. The microspheres were then coated with an enteric coating material, Eudragit S-100, efficiency, % yield value, particle sizes an in-vitro dissolution behaviour were investigated. The surface of the enteric coated microspheres seemed to be all covered with Eudragit S-100 from scanning electron microscopy observation. It was also observed that increasing the CMC concentration led to an increase in the encapsulation efficiency, % yield value and particle size and decreased the release rate. Eudragit S-100 coating did not significantly alter the size but the release rate was significantly lower even when the lower concentration solution was used.     

Enhancement by ganglioside GT1b of annexin I phosphorylation in bovine mammary gland in the presence of phosphatidylserine and Ca2+

Ganglioside GT1b and, to a lesser extent, GD3, enhanced phosphorylation of a 36 kDa protein (the substrate of protein kinase C) in the particulate fraction from bovine mammary gland. Sialic acids, asialogangliosides, and GM3 were without effect, and GD1a conversely inhibited phosphorylation of the 36 kDa protein. The enhanced phosphorylation by GT1b required the simultaneous presence of phosphatidylserine (PS) and Ca²⁺. The 36 kDa protein reacted with anti-annexin I in Western blot analysis. Addition of purified annexin I to the reaction mixture containing the particulate fraction increased the extent of phosphorylated 36 kDa protein, and the phosphorylation was further enhanced by GT1b. The enhanced phosphorylation of annexin I by GT1b was also dependent on PS and Ca²⁺. When annexin I was phosphorylated by purified protein kinase C, GT1b inhibited the annexin I phosphorylation. Addition of epidermal growth factor or insulin to the particulate fraction had little effect on the enhancement. These results suggest that an enzyme or enzymes other than protein kinase C, epidermal growth factor receptor kinase, or insulin receptor kinase is responsible for the GT1b- and GD3-enhanced phosphorylation of annexin I in the presence of PS and Ca²⁺.     

Fundamental solutions for half plane with an oblique edge crack

An elastic half plane with an oblique edge crack is considered in this paper. A pair of concentrated forces or point dislocations is assumed to act at an arbitrary point in the half plane. The half plane with an edge crack is first mapped into a unit circle by a rational mapping function so that the following analysis can be carried out on the mapped plane analytically. Then the complex stress functions are derived by separating the whole problem into two parts; one is the principal part corresponding to the infinite plane acted on by concentrated forces or dislocations, the other is the holomorphic part, which can be determined by making use of the property of regularity of complex stress functions. The stress intensity factors of the crack can be calculated with different inclined angles of the crack, and the displacement and stress components at an arbitrary position in the half plane can be expressed explicitly.     

The effects of R-75,317 on antiglomerular basement membrane glomerulonephritis in rats

Platelet-activating factor (PAF) is a potent inflammatory mediator which is released by various inflammatory cells and produced by certain tissues, including the kidney. PAF has been shown to increase glomerular permeability to protein and to decrease glomerular filtration rate (GFR) by contracting mesangium. On the basis of these observations, it has been suspected that PAF may play a role as mediator of glomerular damage in glomerular nephritis. To examine this possibility, we studied the effects of a specific PAF antagonist, R-75,317, on the development of an experimental model of anti-glomerular basement membrane (anti-GBM) glomerulonephritis. Glomerulonephritis was initiated by injecting rabbit anti-rat GBM serum into rats. Proteinuria gradually developed after serum injection, plateaued at week 2, and remained at the high level of week 2 throughout the experimental period (6 wk). Chronic treatment with R-75,317 (10 mg/kg/day i.p.) tended to delay the onset of proteinuria and significantly accelerated the recovery phase. Creatinine clearance (Ccr) fell to 40% at week 3. R-75,317 treatment completely prevented this decline of Ccr. Histological changes in this model (glomerular hypertrophy, proliferation of mesangial matrix and interstitial fibrosis) were also ameliorated by the R-75,317 treatment. The results suggest that PAF may play a role in the development of glomerulonephritis and that PAF antagonists could be used in the treatment of human renal disease. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, May 1989.     

On-Line Shape recognition with incremental training using binary synaptic weights algorithm

Recognition of hand drawn shapes is beneficial in drawing packages and automated sketch entry in handheld computers. In this paper, we propose a new approach to on-line geometric shape recognition with incremental training function, which utilizes a heuristic function to reduce noise and a neural network for classification and on-line training. Instead of recognizing segments of a drawing and then performing syntactical analysis to match with a predefined shape, which is weak in terms of generalization and dealing with noise, we examine the shape as a whole. The main concept of the recognition method is derived from the fact that internal angles are very important in the perceived shape of outlines. Our application's aim is to recognize elliptic, rectangular, and triangular shapes in a way similar to human cognition of these shapes. Human beings recognize such basic shapes regardless of the variations in size, noise on the shape border, rotation and in the case of triangles, regardless of the type of the triangle. The key concept is that the neural network learns the relationships between the internal angles of a shape and its classification, therefore only a few training samples which represent the class of the shape are sufficient. Fast meremental training, which is performed on-line, is accomplished by the use of the Binary Synaptic Weights algorithm, a one pass, feedforward neural network training algorithm. Incremental training offers the advantage of adjusting the recognition capability of the system to the user's drawings. the results are very successful, such that the neural network correctly classified shapes that did not have any resemblance to the shapes in the initial training set.     

Selective oxidation of Fe-30Ni alloy in a low-temperature range (433–473 K)

Fe-30Ni alloy specimens were oxidized for 10–240 min at 433–473 K in pure oxygen at a pressure of 1.33×10⁴ Pa. The thickness of oxide films was measured by a multiple-angle-of-incidence ellipsometer. The kinetics of film growth were found to obey a parabolic rate law. The depth-profiling of oxidized surfaces, performed with simultaneous use of Auger electron spectroscopy (AES) and argon-ion sputter-etching technique, reveals that iron component is selectively oxidized and an iron-depeletion zone is formed in the underlying alloy. The thickness of the iron depletion zone increases with increasing oxidation time or oxidation temperature. During surface oxidation of the alloy, the transport rate of iron component in the film is almost equal to the interdiffusion rate in the underlying alloy, indicating the establishment of a steady state. The values of the interdiffusion coefficient, , of the underlying alloy estimated from the depth-composition profiles are more than 10 orders of magnitude as large as the values extrapolated from the lattice diffusion data of the corresponding alloy obtained at high temperature. The enormously large values of may be explained in terms of the vacancy (monovacancy or divacancy)-enhanced lattice diffusion mechanism.     

Preparation and characterization of TiO2 and polymer nanocomposite films with high refractive index

Novel nanocomposite films of TiO₂ nanoparticles and hydrophobic polymers having polar groups, poly (bisphenol‐A and epichlorohydrin) or copolymer of styrene and maleic anhydride, with high refractive indices, high transparency, no color, solvent‐resistance, good thermal stability, and mechanical properties were prepared by incorporating surface‐modified TiO₂ nanoparticles into polymer matrices. In the process of preparing colloidal solution of TiO₂ nanoparticles, severe aggregation of particles can be reduced by surface modification using carboxylic acids and long‐chain alkyl amines. These TiO₂ nanoparticles dispersed in solvents were found not to aggregate after mixing with polymer solutions. Transparent colorless free‐standing films were obtained by drying a mixture of TiO₂ nanoparticles colloidal solution and polymer solutions in vacuum. Transmission electronic microscopic studies of the films suggest that the TiO₂ nanoparticles of 3–6 nm in diameter were dispersed in polymer matrices while maintaining their original size. Thermogravimetric analysis results indicate that the nanocomposite film has good thermal stability and the weight fraction of observed TiO₂ nanoparticles in the film is in good accordance with that of theoretical calculations. The refractive index of nanocomposite films of TiO₂ and poly(bisphenol‐A and epichlorohydrin) was in the range of 1.58–1.81 at 589 nm, which linearly increased with the content of TiO₂ nanoparticles from 0 to 80 wt %. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007