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A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive inspection systems. Among these NATs, fusion-PCR (also called as overlap-extension-PCR) has been focused on this study and adopted to generate the fused amplicon composed of plural marker gene fragments for detection. Generally, conventional agarose gel electrophoresis followed by gel staining is employed to check the PCR results. However, these are time-consuming processes that use specific equipment. To overcome these disadvantages, the immunochromatographic test (ICT) for the detection of PCR amplicons with hapten-labels that were generated by PCR using hapten-labeled primers was also adopted in this study. Based on these concepts, we constructed the systems of hapten-labeled fusion-PCR (HL-FuPCR) followed by ICT (HL-FuPCR-ICT) for the two and three marker genes derived from pathogenic microbe. As a result, we successfully developed a two marker genes system for the pathogenic influenza A virus and a three marker genes system for the penicillin-resistant Streptococcus pneumoniae. These detection systems of HL-FuPCR-ICT are characterized by simple handling and rapid detection within few minutes, and also showed the results as clear lines. Thus, the HL-FuPCR-ICT system introduced in this study has potential for use as a user-friendly inspection tool with the advantages especially in the detection of specific strains or groups expressing the characteristic phenotype(s) such as antibiotic resistance and/or high pathogenicity even in the same species.

Small-angle X-ray scattering (SAXS) coupled with computed tomography (CT), denoted SAXS-CT, enables the spatial distribution of the characteristic parameters of nanoscale structures inside samples to be visualized. In this work, a new scheme with Tikhonov regularization was developed to remove the effects of artifacts caused by streak scattering originating from contour regions of the sample., Small-angle X-ray scattering (SAXS) coupled with computed tomography (CT), denoted SAXS-CT, has enabled the spatial distribution of the characteristic parameters (e.g. size, shape, surface, length) of nanoscale structures inside samples to be visualized. In this work, a new scheme with Tikhonov regularization was developed to remove the effects of artifacts caused by streak scattering originating from the reflection of the incident beam in the contour regions of the sample. The noise due to streak scattering was successfully removed from the sinogram image and hence the CT image could be reconstructed free from artifacts in the contour regions.

We attempted to produce three dimensional (3D) aero-sponges using liquid silk (LS) extracted from the silk glands of fully grown larvae of Bombyx mori silkworms by combining an ice-crystal-sublimat...

We attempted to produce three dimensional (3D) aero-sponges using liquid silk (LS) extracted from the silk glands of fully grown larvae of Bombyx mori silkworms by combining an ice-crystal-sublimation method and a water-annealing treatment. The pore structure of the 3D LS-aero-sponge was constructed by the branched fibrous substrates and the films with small holes. It was assumed that the branched fibrous structure was mainly comprised of fibroin whereas the films were comprised of the fibroin interacting with the sericin fractions. Most of the WAXD peaks from the as-molded 3D LS-aerosponge could be assigned to the silk1 crystal diffraction peaks of fibroin. The silk1 crystal modification of fibroin formed in the course of the production of the 3D LS-aero-sponge could bring about a good thermal shrinkproof property. Furthermore, a gamma ray irradiation treatment applied prior to the cell proliferation tests of the 3D LS-aero-sponges was found to be effective to form cross-linking in the sericin fractions. As a result, the yield stress in the compression tests of the 3D LS-aero-sponges was increased. The 3D LS-aerosponge exhibited a good living cell proliferation properly in the culture tests using NH3T3 cells (Swiss albino mice embryonic fibroblasts) although that of the 2D LS-cast films was very poor. We suggest the good cell proliferation property of the 3D LS-aerosponge was due to the continuous branched fibrous structure comprised of fibroin.

Many insects use tactics to protect their eggs from predation, infection, and water loss, among other risks. Some assassin bugs only in the New World tribe Apiomerini are known to use plant substances, such as resin, instead of secretions to protect their eggs. Here, we report a novel storage mechanism and the utilization of plant resin for egg protection in Asian assassin bugs, Velinus nodipes and Agriosphodrus dohrni (tribe: Harpactorini). Adult females of both species were observed to take in plant resin through their genitalia. We reported on the handling of resin by these species and confirmed that A. dohrni adult females stored resin in their subrectal glands and that the substance covering their egg masses was derived from plant resin. This is the first report on the storage of plant resin inside the bodies of assassin bugs and on resin-protected eggs in Harpactorini. Such internal resin storage was not accompanied by external morphological features, as observed in some Apiomerini assassin bugs, thereby suggesting that this tactic has been overlooked and may have evolved in broader tribes of assassin bugs than previously thought.

Grazing-incidence small-angle X-ray scattering coupled with computed tomography (CT) has enabled the visualization of the spatial distribution of nanostructures in thin films. In this study, to optimize the CT image quality, total variation regularization is introduced to minimize sinogram image noise and artifacts., Grazing-incidence small-angle X-ray scattering (GISAXS) coupled with computed tomography (CT) has enabled the visualization of the spatial distribution of nanostructures in thin films. 2D GISAXS images are obtained by scanning along the direction perpendicular to the X-ray beam at each rotation angle. Because the intensities at the q positions contain nanostructural information, the reconstructed CT images individually represent the spatial distributions of this information (e.g. size, shape, surface, characteristic length). These images are reconstructed from the intensities acquired at angular intervals over 180°, but the total measurement time is prolonged. This increase in the radiation dosage can cause damage to the sample. One way to reduce the overall measurement time is to perform a scanning GISAXS measurement along the direction perpendicular to the X-ray beam with a limited interval angle. Using filtered back-projection (FBP), CT images are reconstructed from sinograms with limited interval angles from 3 to 48° (FBP-CT images). However, these images are blurred and have a low image quality. In this study, to optimize the CT image quality, total variation (TV) regularization is introduced to minimize sinogram image noise and artifacts. It is proposed that the TV method can be applied to downsampling of sinograms in order to improve the CT images in comparison with the FBP-CT images.

An attempt has been made to combine small-angle scattering of X-rays or neutrons with scanning electron microscopy in reciprocal space, in order to establish a structural analysis method covering a wide range of sizes from micro- to macro-scales. A system with a binary contrast, in which scattering objects with a homogeneous density are dispersed in vacuum (or air), is considered. A topological surface image, detected by secondary electron emission, is converted by means of a Fourier transform into a two-dimensional scattering amplitude in reciprocal space. The method was first tested by studying a dilute system of monodisperse SiO2 particles, with respect to calibrations for brightness inversion, noise reduction and two-dimensional Fourier transform, to obtain a scattering amplitude that agrees well with the analytical amplitude for a spherical particle. Secondly, the microstructure of a carbon-supported Pt catalyst for polymer electrolyte fuel cell applications was examined with the combined method, covering length scales from 10 µm down to nanometres. After two-dimensional Fourier transformation, the secondary electron emission images with low magnification are able to overcome the limitation of the minimum wavenumber (q min) detectable by ultra-small-angle scattering.

Injection-molding was performed using ethylene–propylene copolymer as the matrix by varying the physicochemical treatment of the reinforcement, i.e., core portion of kenaf stem that was dry-distilled and coated with or without β-sheet protein derivatives. Although dry distillation reduced the moisture content, the Charpy impact strength of the molded composites was low. When molding was performed using the fillers coated with β-sheet protein derivatives, especially coated with liquid silk (LS), a satisfactory Charpy impact strength of the molded composites was markedly gained. Thus, the β-sheet protein derivatives had potential to work as a compatibilizer. Coating the fillers with β-sheet proteins was also effective in suppressing the generation of silver streaks on the surface of composites and in preventing the occurrence of defective injection molding.

Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers.

Injection molding was performed using poly(l-lactide) (PLLA) as a matrix and by varying the reinforcements, i.e., dry-distilled kenaf core (D-core) or untreated kenaf bast fibers, and the physical properties for these composites were subsequently compared. The dry-distillation was able to reduce the moisture content of D-core by a maximum of 4.2% as compared with untreated control core. As a result, the hydrophobicity was increased, which contributed to favorable Charpy impact strength and tensile properties of the D-core/PLLA composite relative to the kenaf bast fiber/PLLA composite. Moreover, the puffing phenomenon, which arises when heating with a microwave oven due to the vaporization of water dispersed within the interfacial regions and associated softening of the PLLA matrix by the heated vapor, was completely suppressed for the D-core/PLLA composite, whereas a large puffing ratio was observed for the kenaf bast fiber/PLLA composite. Reducing the moisture content also effectively suppressed ...

Cholesteric liquid crystal elastomer (CLCE) films, whose helical axis is parallel to a direction in the film plane, exhibit a periodic surface undulation with a period corresponding to a half-pitch (about 100 μm) of the helical director configuration. Temperature variation drives not only a finite macroscopic uniaxial deformation along the helical axis but also a marked variation in the surface undulation. AFM results demonstrate that the surface undulation varies thermoreversibly like a stationary wave with an amplitude of about 600 nm. The top and bottom positions at 40 °C become the bottom and top ones, respectively, at 130 °C, while the undulation disappears near 100 °C. Microspectroscopy reveals the periodic spatial variation in optical birefringence, which reflects the quasi-helical superstructure of local directors in the CLCE films. The combined analysis of the results of AFM and microspectroscopy indicates that the thermally induced variation in the surface undulation becomes largest at positions...

Nonlinear stress-strain behavior of the elastomer foams of high porosity is investigated in several types of biaxial tension, and uniaxial tension and compression. The values of Poisson's ratio for the open- and closed-cell foams are estimated to be about 0.24 almost independently of the imposed tensile strain, indicating that the foams undergo finite volume increase under tension. Unequal biaxial tensile experiments reveal no explicit cross-effect of strains between different axes in the elastomer foams: The deduced strain energy density function ( W ) has no appreciable contribution from the term of I 2 which is the second invariant of deformation gradient tensor representing the explicit cross-effect of strains, while in general W for conventional non-cellular elastomers involves finite contributions from I 2 -term. No explicit cross-effect of strains result in a unique feature in biaxial tensile behavior that the effect of the strain in one direction on the stress in the other direction is considerably small. These features in tensile behavior are commonly observed for the open- and closed-cell foams. In contrast, the cell type has a pronounced effect on the compression behavior accompanying the buckling and collapsing processes of the cells, as observed in earlier studies. The stress-strain relations in compression accompanying the cell buckling are markedly different from the expectation of W obtained from the data of biaxial and uniaxial tension where the cell bucking is not possible.

Images of the spatial distribution of nanostructures in thin films were successfully reconstructed by grazing-incidence small-angle X-ray scattering (GISAXS) coupled with computed tomography (CT) measurements. As a model sample of inhomogeneous thin films, a thin film was patterned with four characters (F, B, S and L) consisting of nanoparticles of gold (Au), platinum (Pt), Au/Pt and Pt/Au, respectively, on a silicon substrate. The characters each produced respective two-dimensional GISAXS images which reflect the nanoparticle structures and their correlations in the thin film. The application of the GISAXS-CT technique to the characteristic scattering GISAXS intensity of each component enables one to reconstruct the images of each character independently. Moreover, it was found that the patterned images could be reconstructed even from very weak scattered intensities at higherqpositions and the diffuse intensities. These results indicate that the GISAXS-CT method is a powerful tool to obtain distinct reconstruction images detailing the particle size, shape and surface roughness.

Using grazing-incidence small-angle scattering (GISAXS) with computed tomography (CT), we have individually reconstructed the spatial distribution of a thin gold (Au) layer buried under a thin poly(styrene-b-2-vinylpyridine) (PS-b-P2VP) layer. Owing to the difference between total reflection angles of Au and PS-b-P2VP, the scattering profiles for Au nanoparticles and self-assembled nanostructures of PS-b-P2VP could be independently obtained by changing the X-ray angle of incidence. Reconstruction of scattering profiles allows one to separately characterize spatial distributions in Au and PS-b-P2VP nanostructures.

Tomographic images constructed from small-angle X-ray scattering (SAXS) intensities utilizing microbeams have been examined for a model multilayered aluminum alloy sample heat-treated to form spatially modulated composition and microstructures. Spatial distributions of composition and precipitation microstructure have been examined by one-dimensional scanning SAXS measurements and two-dimensional tomographic images of attenuation parameters and integrated intensity of SAXS intensity. Reconstructed images using the integrated intensity reflected the distribution of precipitates in terms of their volume fraction over the sample inside the sample, and agreed with more detailed analysis made in the one-dimensional case.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B’γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.