歯の形態形成に関与する新規遺伝子の解明

Tooth development is controlled by body plan during the fetal period, the generation of teeth from tooth germ is induced by the epithelial-mesenchymal interaction. Spatiotemporal regulation of tooth morphogenesis is supported by gene expression. Although many of the genes involved in tooth development are known, the molecular mechanism underlying tooth morphogenesis is not completely understood. For a comprehensive understanding of tooth development, the elucidation of unknown genes is necessary. In this study, to identify unknown genes involved in tooth development, we performed genome-wide analysis at each stage of tooth development and identified 17 genes with high levels of expression and large changes in expression. In addition, we performed qPCR and in situ hybridization analyses to elucidate the spatiotemporal regulation, such as the regulation that occurs around or in the entire tooth germ, enamel knots, epithelium, and mesenchyme. These results show that these characteristic genes may play important roles in each time period or region of tooth development, and the elucidation of the functions of these genes will lead to an integrated understanding of the process of tooth development.博士（医学）・甲第790号・令和3年3月15日発行元である日本再生歯科医学会の許諾を得て登録（2021年6月29日付）ジャーナル公式サイト（日本再生歯科医学会HP内）：http://www.jarde.jp/zasshi/e/18-2-1.htm

Efficacy and Safety of Intravitreal Aflibercept Treat-and-Extend Regimens in Exudative Age-Related Macular Degeneration: 52- and 96-Week Findings from ALTAIR : A Randomized Controlled Trial.

PURPOSE:To evaluate efficacy and safety of intravitreal injections of aflibercept (IVT-AFL) treat-and-extend (T&E) dosing regimens in treatment-naïve patients with exudative age-related macular degeneration (AMD).METHODS:Adults aged at least 50 years old with exudative AMD and best-corrected visual acuity (BCVA) of 73-25 Early Treatment Diabetic Retinopathy Study (ETDRS) letters were included. Patients received three monthly doses of IVT-AFL 2 mg. At week 16, patients were randomized 1:1 to IVT-AFL T&E with either 2- or 4-week adjustments. The primary endpoint was mean change in BCVA from baseline to week 52. Outcomes were assessed at weeks 52 and 96.RESULTS:Baseline characteristics were comparable between the groups (n = 123 each). Over 52 weeks, mean number of injections was 7.2 and 6.9 and mean last injection interval was 10.7 and 11.8 weeks, for the 2- and 4-week groups, respectively. From baseline, mean change in BCVA was + 9.0 and + 8.4 letters (week 52) and + 7.6 and + 6.1 letters (week 96); mean change in central retinal thickness was - 134.4 µm and - 126.1 µm (week 52) and - 130.5 µm and - 125.3 µm (week 96). Last injection interval before week 52 was at least 12 weeks in 42.3% and 49.6% of patients and 56.9% and 60.2% before week 96. Over 96 weeks, mean number of injections was 10.4 (both groups). The safety profile of IVT-AFL was consistent with previous reports.CONCLUSIONS:IVT-AFL administered using two different T&E regimens for treatment-naïve exudative AMD improved functional and anatomic outcomes at week 52 and outcomes were maintained to week 96. Outcomes were similar between the 2- and 4-week groups.TRIAL REGISTRATION:ClinicalTrials.gov identifier, NCT02305238

Functional Evolution of Duplicated Odorant-Binding Protein Genes, Obp57d and Obp57e, in Drosophila

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\OBP57de, Dmel\OBP57d was more specialized to tridecanoic acid while Dmel\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection

Quantitative Analysis of Serum Procollagen Type I C-Terminal Propeptide by Immunoassay on Microchip

BACKGROUND: Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. METHODS AND FINDINGS: The microchip was made of cyclic olefin copolymer with straight microchannels that were 300 µm wide and 100 µm deep. For the construction of a sandwich ELISA for procollagen type I C-peptide (PICP), a biomarker for bone formation, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-PICP antibody on the surface of the microchannel. After the infusion of the mixture of 2.0 µl of peroxidase-labeled 2nd anti-PICP antibody and 0.4 µl of sample to the microchannel and a 30-min incubation, the substrate for peroxidase was infused into the microchannel; and the luminescence intensity of each spot of 1st antibody was measured by CCD camera. A linear relationship was observed between PICP concentration and luminescence intensity over the range of 0 to 600 ng/ml (r(2) = 0.991), and the detection limit was 4.7 ng/ml. Blood PICP concentrations of 6 subjects estimated from microchip were compared with results obtained by the conventional method. Good correlation was observed between methods according to simple linear regression analysis (R(2) = 0.9914). The within-day and between-days reproducibilities were 3.2-7.4 and 4.4-6.8%, respectively. This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. CONCLUSION: This assay enabled us to determine serum PICP with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis

Microfluidic Separation of Blood Cells Based on the Negative Dielectrophoresis Operated by Three Dimensional Microband Electrodes

A microfluidic device is presented for the continuous separation of red blood cells (RBCs) and white blood cells (WBCs) in a label-free manner based on negative dielectrophoresis (n-DEP). An alteration of the electric field, generated by pairs of slanted electrodes (separators) that is fabricated by covering parts of single slanted electrodes with an insulating layer is used to separate cells by their sizes. The repulsive force of n-DEP formed by slanted electrodes prepared on both the top and bottom substrates led to the deflection of the cell flow in lateral directions. The presence of gaps covered with an insulating layer for the electric field on the electrodes allows the passing of RBCs through gaps, while relatively large WBCs (cultured cultured human acute monocytic leukemia cell line (THP-1 cells)) flowed along the slanted separator without passing through the gaps and arrived at an edge in the channel. The passage efficiency for RBCs through the gaps and the arrival efficiency for THP-1 cells to the upper edge in the channel were estimated and found to be 91% and 93%, respectively

Particle Patterning Based on Positive Dielectrophoresis Using a Scanning Microelectrode

Positioning and patterning of polystyrene particles on a silicon nitride (SiN) membrane with a microhole array has been demonstrated by positive dielectrophoresis (p-DEP). A chamber with an SiN membrane with the well-aligned microholes as the bottom substrate was positioned on an indium-tin-oxide (ITO) electrode with a 1 mm space between the bottom substrate and the ITO electrode. The chamber and the space were filled with water and a suspension of particles (10 µm diameter) in water, respectively. An AC electric signal was then applied to a microelectrode positioned at 10 µm above the SiN membrane, while the ITO electrode was connected to the ground. Particles present in the space between the SiN membrane and ITO electrode gradually moved toward the lower surface of the SiN membrane directly under the microelectrode owing to the strong electric field generated on and in localized microholes and accumulated at this position to form aggregates. For particles of 3 µm diameter, one particle was deposited in each hole (2 µm diameter) in the region directly under the microelectrode. The horizontal movement of the microelectrode gave rise to the formation of a line pattern of particles along the trail of the microelectrode because of the shift of the region with the strong electric field. These demonstrations could be applicable to arranging the particles at desired positions and in desired holes, and form particle patterns with highly flexible designs

Particle Patterning Based on Positive Dielectrophoresis Using a Scanning Microelectrode

Positioning and patterning of polystyrene particles on a silicon nitride (SiN) membrane with a microhole array has been demonstrated by positive dielectrophoresis (p-DEP). A chamber with an SiN membrane with the well-aligned microholes as the bottom substrate was positioned on an indium-tin-oxide (ITO) electrode with a 1 mm space between the bottom substrate and the ITO electrode. The chamber and the space were filled with water and a suspension of particles (10 µm diameter) in water, respectively. An AC electric signal was then applied to a microelectrode positioned at 10 µm above the SiN membrane, while the ITO electrode was connected to the ground. Particles present in the space between the SiN membrane and ITO electrode gradually moved toward the lower surface of the SiN membrane directly under the microelectrode owing to the strong electric field generated on and in localized microholes and accumulated at this position to form aggregates. For particles of 3 µm diameter, one particle was deposited in each hole (2 µm diameter) in the region directly under the microelectrode. The horizontal movement of the microelectrode gave rise to the formation of a line pattern of particles along the trail of the microelectrode because of the shift of the region with the strong electric field. These demonstrations could be applicable to arranging the particles at desired positions and in desired holes, and form particle patterns with highly flexible designs