Search

The vibration characteristics of a bladed disk with the continuous ring type structure have been studied extensively, and the vibration response analyses considering the friction damping between adjacent shrouds have been performed in the recent blade design. The effect of the friction damping on the self-excited vibration also has been studied by several researchers. However, because the most of these studies have been performed using the simple macroslip model, the damping characteristics of bladed disks with the friction damping are not yet clarified sufficiently. In this paper, the damping characteristics of the bladed disks with the continuous ring type structure is studied using the conventional macroslip model and the sophisticated microslip model for the forced and self-excited vibration and the results calculated by both models are compared. From the analysis results, it is concluded that, in the forced vibration, the damping characteristics predicted by both models are almost the same if the excitation force is extremely large or small. However, when the excitation force is medium, the damping characteristics predicted by both models are different. In the self-excited vibration, the stability predicted by both models are qualitatively the same. However, the amplitude of the limit cycle oscillation and the critical aerodynamic damping predicted by both models are quantitatively different.

Hard carbon is a promising negative electrode material for sodium-ion batteries that operate at low potentials. However, reversible and high-capacity charging and discharging in all-solid-state sodium batteries with hard carbon electrodes using sulfide solid electrolytes have not been reported. This study reports that reductive decomposition of the sulfide solid electrolyte occurs at both the negative composite electrode and the interface between the negative electrode layer and the solid electrolyte layer. In the first cycle, the all-solid-state cell with a composite electrode containing a Na3PS4 solid electrolyte exhibited a large irreversible capacity of 561 mAh g−1 because of the reductive decomposition of Na3PS4 to Na2S and Na3P. The use of a Na3BS3 glass electrolyte with reduction stability can lead to the successful charging and discharging of all-solid-state cell that utilizes hard carbon. This glass electrolyte can serve as a solid electrolyte for the negative composite electrode and as a buffer layer between the negative electrode layer and the solid electrolyte layer. Hence, it can also help suppress the irreversible capacity of the cell to 122 mAh g−1.

Each growth factor (GF) has different effects and targets, and plays a critical role in periodontal healing. Dehydrated human amnion-chorion membrane (dHACM) contains various GFs and has been used to enhance wound healing. The purpose of this study was to evaluate the effects of dHACM on periodontal healing, using in vitro and in vivo experimental approaches. Standardized periodontal defects were created in rats. The defects were randomly divided into three groups: Unfilled, filled with hydroxypropyl cellulose (HPC), and dHACM+HPC. At 2 and 4 weeks postoperatively, periodontal healing was analyzed by microcomputed tomography (micro-CT), and histological and immunohistochemical analyses. In vitro, periodontal ligament-derived cells (PDLCs) isolated from rat incisors were incubated with dHACM extract. Cell proliferation and migration were evaluated by WST-1 and wound healing assay. In vivo, micro-CT examination at 2 weeks revealed enhanced formation of new bone in the dHACM+HPC group. At 4 weeks, the proportions of vascular endothelial growth factor (VEGF)-positive cells and α-smooth muscle actin (α-SMA)-positive blood vessels in the dHACM+HPC group were significantly greater than those in the Unfilled group. In vitro, dHACM extracts at 100 µg/mL significantly increased cell proliferation and migration compared with control. These findings suggest that GFs contained in dHACM promote proliferation and migration of PDLCs and angiogenesis, which lead to enhanced periodontal healing.

In genomic DNA, G-quadruplex (G4)-forming DNA can form either a duplex or G4 structure, suggesting that understanding the factors regulating G4 formation is important for revealing the cellular functions controlled by G4 formation. Cytosine DNA methylation in the CpG islands is known to play an important role in transcriptional regulation. Additionally, CpG methylation increases the thermal stability of G4 structures such as BCL2 and VEGF G4. In this study, we evaluated the effects of CpG methylation in three G4 structures (c-kit2, c-kit*, and c-kit1) produced by the c-KIT promoter. Each was analyzed using circular dichroism (CD) melting analysis. The results demonstrate that CpG methylation does not alter the thermal stability of c-kit2 G4 structure when formed in the presence of K+; a single-CpG methylation at C1 or C11 decreases the thermal stability of any c-kit2 G4 structure formed in the presence of Na+ and Mg2+ while methylation at C5 increases the thermal stability; CpG methylation does not alter the thermal stability of c-kit1 or c-kit* G4 structures formed in the presence of K+; and the c-kit1 and c-kit* G4-forming oligonucleotides do not form G4 structures in the presence of Na+ and Mg2+. These results provide important clues for understanding the regulatory mechanisms underlying the formation of CpG methylation-induced G4 structures.

The aim of this study was to investigate the effects of fibroblast growth factor (FGF)-2 used in combination with deproteinized bovine bone mineral (DBBM) on the healing of experimental periodontal defects. Periodontal defects created in rats were treated by FGF-2, DBBM, FGF-2 + DBBM, or left unfilled. Microcomputed tomography, histological, and immunohistochemical examinations were used to evaluate healing. In vitro cell viability/proliferation on DBBM with/without FGF-2 was assessed by WST-1. Cell behavior was analyzed using scanning electron and confocal laser scanning microscopy. Osteogenic differentiation was evaluated by staining with alkaline phosphatase and alizarin red. Bone volume fraction was significantly greater in FGF-2 and FGF-2 + DBBM groups than in other groups at 2 and 4 weeks postoperatively. In histological assessment, newly formed bone in FGF-2 and FGF-2 + DBBM groups appeared to be greater than other groups. Significantly greater levels of proliferating cell nuclear antigen-, vascular endothelial growth factor-, and osterix-positive cells were observed in FGF-2 and FGF-2 + DBBM groups compared to Unfilled group. In vitro, addition of FGF-2 to DBBM promoted cell viability/proliferation, attachment/spreading, and osteogenic differentiation. The combination therapy using FGF-2 and DBBM was similarly effective as FGF-2 alone in the healing of experimental periodontal defects. In certain bone defect configurations, the combined use of FGF-2 and DBBM may enhance healing via promotion of cell proliferation, angiogenesis, and osteogenic differentiation.

Summary: As an essential step for brain morphogenesis, neurons migrate via mechanical interactions with components of their environment such as neighboring cells and the extracellular matrix. However, the molecular mechanism by which neurons exert forces on their environment during migration remains poorly understood. Here, we show that shootin1b is expressed in migrating mouse olfactory interneurons and accumulates at their leading process growth cone. We demonstrate that shootin1b, by binding to cortactin and L1-CAM, couples F-actin retrograde flow and the adhesive substrate as a clutch molecule. Shootin1b-mediated clutch coupling at the growth cone generates traction force on the substrate, thereby promoting leading process extension and subsequent somal translocation of olfactory interneurons. Furthermore, loss of shootin1 causes abnormal positioning of the interneurons and dysgenesis of the olfactory bulb. Our findings indicate that shootin1b plays a key role in force-driven leading process extension, which propels the migration of olfactory interneurons during olfactory bulb formation. : Minegishi et al. use gene knockout, protein interaction assays, force microscopy, speckle imaging, and migration assays to analyze the molecular mechanics driving neuronal migration in vivo. Their data demonstrate that shootin1b mediates a mechanical clutch to produce force for the migration of olfactory interneurons in the brain. Keywords: neuronal migration, olfactory bulb, interneuron, leading process, growth cone, shootin1, L1-CAM, clutch molecule

N6-methyladenine modification (m6dA) has recently been identified in eukaryote genomic DNA. The methylation destabilizes the duplex structure when the adenine forms a Watson–Crick base pair, whereas the methylation on a terminal unpaired adenine stabilizes the duplex structure by increasing the stacking interaction. In this study, the effects of m6dA modification on the thermal stability of four distinct telomeric G-quadruplex (G4) structures were investigated. The m6dA-modified telomeric oligonucleotide d[AGGG(TTAGGG)3] that forms a basket-type G4 in Na+, d[(TTAGGG)4TT] that forms a hybrid-type G4 in K+ (Form-2), d[AAAGGG(TTAGGG)3AA] that forms a hybrid-type G4 in K+ (Form-1), and d[GGG(TTAGGG)3T] that forms a basket-type G4 with two G-tetrads in K+ (Form-3) were analyzed. Circular dichroism melting analysis demonstrated that (1) A7- and A19-methylation destabilized the basket-type G4 structure that formed in Na+, whereas A13-methylation stabilized the structure; (2) A15-methylation stabilized the Form-2 G4 structure; (3) A15- and A21-methylations stabilized the Form-1 G4 structure; and (4) A12-methylation stabilized the Form-3 G4 structure. These results suggest that m6dA modifications may affect the thermal stability of human telomeric G4 structures in regulating the biological functions.

G-quadruplexes (G4s) are noncanonical DNA/RNA structures formed by guanine-rich sequences. Recently, G4s have been found not only in aptamers but also in the genomic DNA and transcribed RNA. In this study, we identified new RNA oligonucleotides working as aptamers by focusing on G4-forming RNAs located within the pre-mRNA. We showed that the G4 in the 5′ UTR and first intron of VEGFA bound to the protein encoded in VEGFA gene, VEGF165, with high affinity. Moreover, G4-forming RNAs located within the PDGFA and the PDGFB introns bound to PDGF-AA and PDGF-BB, respectively, indicating that G4 in the pre-mRNA could be an aptamer. It had been reported that the putative G4-forming RNA sequences are located in some parts of most genes, thus our strategy for aptamer identification could be applicable to other proteins. It has been reported that some G4-forming RNAs in 5′ UTRs are involved in translation control; however, G4-forming excised intronic RNA function has not been revealed previously. Therefore, these findings could not only contribute to the identification of RNA aptamers but also provide new insights into the biological functioning of G4-forming RNAs located within intronic RNA sequences.

Growth cones navigate axonal projection in response to guidance cues. However, it is unclear how they can decide the migratory direction by transducing the local spatial cues into protrusive forces. Here we show that knockout mice of Shootin1 display abnormal projection of the forebrain commissural axons, a phenotype similar to that of the axon guidance molecule netrin-1. Shallow gradients of netrin-1 elicited highly polarized Pak1-mediated phosphorylation of shootin1 within growth cones. We demonstrate that netrin-1–elicited shootin1 phosphorylation increases shootin1 interaction with the cell adhesion molecule L1-CAM; this, in turn, promotes F-actin–adhesion coupling and concomitant generation of forces for growth cone migration. Moreover, the spatially regulated shootin1 phosphorylation within growth cones is required for axon turning induced by netrin-1 gradients. Our study defines a mechano-effector for netrin-1 signaling and demonstrates that shootin1 phosphorylation is a critical readout for netrin-1 gradients that results in a directional mechanoresponse for axon guidance.

We have developed a novel method, antagonistic template-based biopanning, for screening peptide ligands specifically recognizing local tertiary protein structures. We chose water-soluble pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH-B) as a model enzyme for this screening. Two GDH-B mutants were constructed as antagonistic templates; these have some point mutations to induce disruption of local tertiary structures within the loop regions that are located at near glucose-binding pocket. Using phage display, we selected 12-mer peptides that specifically bound to wild-type GDH-B but not to the antagonistic templates. Consequently, a peptide ligand showing inhibitory activity against GDH-B was obtained. These results demonstrate that the antagonistic template-based biopanning is useful for screening peptide ligands recognizing the specific local tertiary structure of proteins.

G-quadruplex (G4) is a DNA/RNA conformation that consists of two or more G-tetrads resulting from four-guanine bases connected by Hoogsteen-type hydrogen bonds, which is often found in the telomeres of chromatin, as well as in the promoter regions of genes. The function of G4 in the genomic DNA is being elucidated and some G4-protein interactions have been reported; these are believed to play a role in vital cellular functions. In this study, we focused on CpG methylation, a well-known epigenetic modification of the genomic DNA, especially found in the promoter regions. Although many G4-forming sequences within the genomic DNA harbor CpG sites, the relationship between CpG methylation and the binding properties of associated proteins remains unclear. We demonstrated that the binding ability of vascular endothelial growth factor (VEGF) G4 DNA to VEGF165 protein was significantly decreased by CpG methylation. We identified the binding activity of G4 DNA oligonucleotides derived from gene promoter regions to SP1, a transcription factor that interacts with a G4-forming DNA and is also altered by CpG methylation. The effect of methylation on binding affinity was accompanied by changes in G4 structure and/or topology. Therefore, this study suggested that CpG methylation might be involved in protein binding to G4-forming DNA segments for purposes of transcriptional regulation.

Insulators are cis-elements that control the direction of enhancer and silencer activities (enhancer-blocking) and protect genes from silencing by heterochromatinization (barrier activity). Understanding insulators is critical to elucidate gene regulatory mechanisms at chromosomal domain levels. Here, we focused on a genomic region upstream of the mouse Ccnb1ip1 (cyclin B1 interacting protein 1) gene that was methylated in E9.5 embryos of the C57BL/6 strain, but unmethylated in those of the 129X1/SvJ and JF1/Ms strains. We hypothesized the existence of an insulator-type element that prevents the spread of DNA methylation within the 1.8 kbp segment, and actually identified a 242-bp and a 185-bp fragments that were located adjacent to each other and showed insulator and enhancer activities, respectively, in reporter assays. We designated these genomic regions as the Ccnb1ip1 insulator and the Ccnb1ip1 enhancer. The Ccnb1ip1 insulator showed enhancer-blocking activity in the luciferase assays and barrier activity in the colony formation assays. Further examination of the Ccnb1ip1 locus in other mammalian species revealed that the insulator and enhancer are highly conserved among a wide variety of species, and are located immediately upstream of the transcriptional start site of Ccnb1ip1. These newly identified cis-elements may be involved in transcriptional regulation of Ccnb1ip1, which is important in meiotic crossing-over and G2/M transition of the mitotic cell cycle.

We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as "G4 promoter-derived aptamer selection (G4PAS)." Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10(-7) M, 6.3 × 10(-9) M, and 4.4 × 10(-7) M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.