Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.     

Completely Chemically Synthesized Long DNA Can be Transcribed in Human Cells

Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.     

Development of the surface displacement velocity in a full-scale loamy model slope under multistep excavation

Bulletin of Engineering Geology and the Environment - Multistep excavations were implemented at the toe of a large-scale slope model, and the surface displacements in the slope were measured to...     

In situ doping of BaTiO3 and visualization of pressure solution in flux-assisted cold sintering

Cold sintering process (CSP) has attracted great interest due to its extremely low processing temperatures, fast processing times, and simplicity to allow for the densification of ceramics and composites. Understanding the detailed mechanisms underlying low temperature densification is crucial to develop advanced materials and facilitate sustainable and cost-effective industrial implementation to come. Here, by taking BaTiO₃ powder and Sr(OH)₂·8H₂O transient chemical flux as a model system, chemical transformation at solid/flux interfaces driving the dissolution-precipitation creep mechanism were investigated. We demonstrate that Sr(OH)₂·8H₂O acts both as a sintering flux and a solid solution doping additive, resulting in the formation of BaTiO₃ - Ba_1-xSr_xTiO₃ with lower Curie temperatures. Using strontium (Sr) as a tracer chemistry, transmission electron microscopy chemical mapping with energy-dispersive X-ray analysis indicates that there is a precipitation of a Ba_1-xSr_xTiO₃ mainly at grain/grain interfaces, while grain cores remain undoped. In addition, the difference in the interfacial Sr concentration, which is influenced by the applied uniaxial pressure direction, was clearly observed. This successful visualization of compositional distribution after CSP underlines the significant role of the pressure solution creep in densification process.     

A method for estimating the errors in many-light rendering with supersampling

Computational Visual Media - In many-light rendering, a variety of visual and illumination effects, including anti-aliasing, depth of field, volumetric scattering, and subsurface scattering, are...     

Hydrogen sulfide production and fermentative gas production by Salmonella typhimurium require F0F1 ATP synthase activity

A previously isolated mutant of Salmonella typhimurium lacking hydrogen sulfide production from both thiosulfate and sulfite was shown to have a single mutation which also caused the loss of fermentative gas production and the ability to grow on nonfermentable substrates and which mapped in the vicinity of the atp chromosomal locus. The implication that F0F1 ATP synthase might be essential for H2S and fermentative gas production was explored. The phs plasmid conferring H2S production on wild-type Escherichia coli failed to confer this ability on seven of eight E. coli atp point mutants representing, collectively, the eight genes encoding the subunits of F0F1 ATP synthase. However, it did confer some thiosulfate reductase activity on all except the mutant with a lesion in the ATP synthase catalytic subunit. Localized mutagenesis of the Salmonella atp chromosomal region yielded 500 point mutants unable to reduce thiosulfate to H2S or to produce gas from glucose, but differing in the extents of their ability to grow on succinate, to perform proton translocation as measured in a fluorescence quenching assay, and to reduce sulfite to H2S. Biochemical assays showed that all mutants were completely devoid of both methyl viologen and formate-linked thiosulfate reductase and that N,N'-dicyclohexylcarbodiimide blocked thiosulfate reductase activity by the wild type, suggesting that thiosulfate reductase activity has an absolute requirement for F0F1 ATP synthase. Hydrogenase-linked formate dehydrogenase was also affected, but not as severely as thiosulfate reductase. These results imply that in addition to linking oxidation with phosphorylation, F0F1 ATP synthase plays a key role in the proton movement accompanying certain anaerobic reductions and oxidations.     

Rendering gems with aterism or chatoyancy

In this paper we describe an algorithm to render the asterism or chatoyancy effect in gems. This effect is due to light dispersion caused by inclusions consisting of vast numbers of thin fibrous needles. We develop models for distribution of the microfacet directions of the inclusions and of the light dispersion by them. Based on these models we derive an algorithm to render gems with the asterism effect. By introducting a ray distribution map, a new algorithm for tracing dispersed light rays is developed.     

Soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and perimenopausal women

The possibility that the heightened cardiovascular risk associated with the menopause, which is said to be ameliorated by soybeans, can be reduced with soy isoflavones was tested in 21 women. Although several were perimenopausal, all have been included. A placebo-controlled crossover trial tested the effects of 80-mg daily isoflavones (45 mg genistein) over 5- to 10-week periods. Systemic arterial compliance (arterial elasticity), which declined with age in this group, improved 26% (P < .001) compared with placebo. Arterial pressure and plasma lipids were unaffected. The vasodilatory capacity of the microcirculation was measured in nine women; high acetylcholine-mediated dilation in the forearm vasculature was similar with active and placebo treatments. LDL oxidizability measured in vitro was unchanged. Thus, one important measure of arterial health, systemic arterial compliance, was significantly improved in perimenopausal and menopausal women taking soy isoflavones to about the same extent as is achieved with conventional hormone replacement therapy.     

An evaluation method of PV systems

Data evaluation methods have wide adaptations, such as feedbacks to PV system operation management and design. The authors have already developed sophisticated verification method (SV method) of PV systems, which is a simple evaluation method to identify six kinds of system loss rates using basic information and simple four measurable data. This time, the authors introduced quality diagnosis to our previous model for compensating the measurement errors in field data, and improved the algorithm of the model. Consequently, validity of the evaluation result became better than the previous model.     

Antibodies against high-density lipoprotein binding proteins enhance high-density lipoprotein uptake but do not affect cholesterol efflux from rat hepatoma cells

High-density lipoprotein plays a key role in the reverse cholesterol transport pathway as well as in the delivery of cholesterol to the liver and steroidogenic tissues. Metabolism of high-density lipoprotein is determined by one of its apolipoproteins, apolipoprotein A-I; however, the identity and function of cellular protein which binds high-density lipoprotein remains unclear. The effect of antibodies against rat high-density lipoprotein binding proteins, HB1 and HB2, on high-density lipoprotein metabolism in a rat hepatoma cell line were studied. Cells were preincubated with the antibodies and 125I-labeled high-density lipoprotein binding and uptake as well as cholesterol biosynthesis and cholesterol efflux to human plasma or isolated high-density lipoprotein were studied. Both antibodies reacted specifically with HB1 and HB2 on the ligand and Western blots, but their binding was not blocked by high-density lipoprotein. Both antibodies inhibited 125I-labeled high-density lipoprotein binding to cells by 20-40%, but stimulated 125I-labeled high-density lipoprotein uptake by up to 2.5-fold. The antibodies had no effect on cholesterol efflux or on cholesterol synthesis. It is concluded that high-density lipoprotein binding proteins, HB1 and HB2, may be involved in high-density lipoprotein uptake in the liver rather than in mediating cholesterol efflux.