Elliptic neutron-focusing supermirror for illuminating small samples in neutron reflectometry.

This paper details the development of a precise assembly of two supermirrors for neutron-focusing, designed for installation in neutron reflectometer SOFIA at BL16 in J-PARC MLF to intensify the illumination for small samples. The supermirrors are sputtered on two metal substrates, whose surfaces are coated with amorphous Ni-P plating, and are figured by diamond cutting and polished to subnanometer roughness. Special care is taken while polishing the substrates to reduce waviness and surface roughness for achieving a sharp focusing spot and uniform neutron reflectivity. The supermirror could converge the neutrons into a focal spot with a width of 0.13 mm in the full width at half maximum.

Development of precision elliptic neutron-focusing supermirror.

This paper details methods for the precision design and fabrication of neutron-focusing supermirrors, based on electroless nickel plating. We fabricated an elliptic mirror for neutron reflectometry, which is our second mirror improved from the first. The mirror is a 550-millimeter-long segmented mirror assembled using kinematic couplings, with each segment figured by diamond cutting, polished using colloidal silica, and supermirror coated through ion-beam sputtering. The mirror was evaluated with neutron beams, and the reflectivity was found to be 68-90% at a critical angle. The focusing width was 0.17 mm at the full width at half maximum.

A pivot-hinge-style DNA immobilization method with adaptable surface concentration based on oligodeoxynucleotide-phosphorothioate chemisorption on gold surfaces.

The chemisorption of oligodeoxynucleotide phosphorothioate (s-oligo) is reported. A series of s-oligo DNAs was designed for use as capture probe DNA molecules. The s-oligo DNAs consist of the K-ras gene (5'-GGA GCT GGT GGC-3') and a dodecamer deoxyriboadenosine, both of which lie on either side of an s-oligo DNA sequence. By primarily focusing on the capture probe DNA having five-successive s-oligo sequences, e37, the immobilization chemistry of e37 was examined; atomic force microscopy achieved the direct visualization of individual molecules on Au(111) substrates, while a series of surface analyses, including IR, ellipsometry, and microgravimetry, showed that the s-oligo functional groups played a pivotal role in the surface-adlayer through the gold-thiol interaction. Interestingly, the amount of immobilization showed a definite relationship with the number of s-oligo linkages introduced, which should be important to regulate the concentration of the capture probe DNA molecules on the surface. Some preliminary studies using ferrocene-modified complementary sequences indicated that electrochemical labeling and readouts were possible.

AFM-imaging diagnosis method for single nucleotide polymorphism using molecular beacon DNA as an intramolecular ligation template of target DNA and a viewable indicator.

An AFM-imaging-based method for single nucleotide polymorphism (SNP) analysis is described. A stem-loop-forming 34-mer oligonucleotide (p34s) was designed. p34s contains the complementary sequence for K-ras (5'-GGT GGC-3', t6G), one of the human oncogenes, at the 5'-end for target-recognition and five successive phosphorothioate linkages in the loop. The functional probe, either alone or hybridized with target DNA (p34s/t6G), relaxed upon treatment with "opener" DNA. The template/target DNA interstrand hybridization product is covalently connected by ligase if the correct target is used, but not hybridized species including mismatches. With these results, developed was a solid-phase SNP assay by transferring an aliquot of the product onto an Au(111) substrate for self-assembly, followed by AFM imaging. Clear contrasts that allow the detection of SNPs, were observed for the ligated and non-ligated species representing the loop-to-linear conformational change. Simple statistical surface-roughness analysis determined the lowest concentration of the sample to be 5 × 10(-10) M, whose necessary sample quantity was 5 fmol.

Transglutaminase-mediated in situ hybridization (TransISH) system: a new methodology for simplified mRNA detection.

Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA-enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site-specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen-antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection.

Programmable protein-protein conjugation via DNA-based self-assembly.

Protein molecules were precisely arrayed on a designable DNA scaffold close to each other using a DNA aptamer. By adding a chemical cross-linker, the neighboring protein molecules were effectively and covalently cross-linked to each other without losing their activities.

Prediction of groundwater contamination with 137Cs and 131I from the Fukushima nuclear accident in the Kanto district.

We measured the concentrations of (131)I, (134)Cs, and (137)Cs released from the Fukushima nuclear accident in soil and rainwater samples collected March 30-31, 2011, in Ibaraki Prefecture, Kanto district, bordering Fukushima Prefecture to the south. Column experiments revealed that all (131)I in rainwater samples was adsorbed onto an anion-exchange resin. However, 30% of (131)I was not retained by the resin after it passed through a soil layer, suggesting that a portion of (131)I became bound to organic matter from the soil. The (137)Cs migration rate was estimated to be approximately 0.6 mm/y in the Kanto area, which indicates that contamination of groundwater by (137)Cs is not likely to occur in rainwater infiltrating into the surface soil after the Fukushima accident.

Label-free DNA detection platform based on atomic force microscopy visualisation: characterising the molecular-recognition-triggered conformational changes of an immobilised receptor oligonucleotide probe.

Using atomic microscopy imaging, probe DNA sequence self-assemblies developed on Si(100) substrates undergo a conformational transition from an extended stem-loop structure to a double helix; such assemblies readily report on DNA molecular recognition events and should be suitable as a label-free, DNA hybridisation assay platform.

A culture device demonstrates that hydrostatic pressure increases mRNA of RGS5 in neuroblastoma and CHC1-L in lymphocytic cells.

Previous studies demonstrated that mechanical forces affect a wide range of cellular behaviors. These forces regulate important cellular responses in the human body and consist of gravity, hydrostatic pressure, stretch, and shear stress, which is exerted on the vascular system by the passage of blood flow. We reasoned that these forces might be significant and dynamic regulators of cellular functions within the human body. While cellular effects of stretch and shear stress have been studied particularly with endothelial cells, little is known about the effects of gravity and hydrostatic pressure to cells. To examine the direct effect of hydrostatic pressure, we developed a culture device to confer hydrostatic pressures to cells ranging from 0 to 1,000 psi. We subjected human neuroblastoma cells and rIL-2-activated lymphocytes to a constant pressure of 20 or 100 psi for 48 h and attempted to identify genes regulated by hydrostatic pressure. Genes of regulator of G-protein signaling 5 in neuroblastoma cells and CHC1-L in lymphocytes increased after exposure to hydrostatic pressure. The results demonstrated that hydrostatic pressure directly regulates the expression of specific genes in mammalian cells. Moreover, there may be some underlying mechanisms that have common effects in altered physical environments. Our in vitro culture system may provide some insight into the mechanisms through the intracellular processes affected by mechanical forces.

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture.

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.

Adenoviral transfer of antisenses or ribozyme to O6-methylguanine-DNA methyltransferase mRNA in brain-tumor model resistant to chloroethyl-nitrosourea.

Chloroethyl-nitrosourea is a potent chemotherapeutic agent for brain tumors. However, acquired resistance to this drug has become a serious problem for the treatment of patients. Previously, we established an animal model resistant to nitrosourea (Anticancer Res 19: 5313-5318, 1999). In this study, we evaluate the efficacies of antisense sequences and ribozyme transduction by an adenoviral vector utilizing this model. Adenoviral vectors encoding antisense sequences or ribozyme to MGMT mRNA were constructed, then MGMT-expressing glioma cells were infected with these viruses and 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) sensitivities were quantified. The adenoviral transfer of antisense RNA and ribozyme down-regulated the transcription and expression of MGMT in vitro. It also conferred sensitivity to nitrosourea in vitro and in vivo. However, the effect was minimal. These data suggest that incomplete depletion of MGMT is not sufficient to overcome the resistance and that additional optimization will be required for the complete reversion of drug resistance.

Numerical simulation for electrochemical cultivation of iron oxidizing bacteria.

A numerical simulation model was constructed for electrochemical cultivation of iron oxidizing bacterium, Thiobacillus ferrooxidans, based on Monod's dual limitation equation. In this model, two limiting factors were examined, low supply of Fe(II) ion and dissolved oxygen, from empirical viewpoints. The simulation model was constructed taking into consideration the energy balance based on the amount of the electronic flow from the electrode to bacteria via an iron ion, and then to oxygen. The model consisted of a logarithmic bacterial growth phase during the first three days, followed by a plateau and growth limitation thereafter. The predicted results were in agreement with the actual growth under electrochemical cultivation. It was predicted the growth limiting factor would be changed from insufficient supply of Fe(II) ions to that of oxygen by decreasing the value of oxygen transfer constant K, which correlated with the aeration rate. The optimum aeration rate was determined for the ideal electrochemical cultivation. The algorithm described here can be used in any electrochemical cultivation by modifying the parameters for each system.