JSPS KAKENHI Grant Number Research Articles

How to design artificial protein surpassing native enzyme function ~ Design and multidisciplinary analysis of artificial L‐threonine 3‐dehydrogenase ~

IntroductionCurrently, we can easily obtain a large amount of protein sequence data through database, such as PubMed. The data are being used for protein engineering to reconstruct sequence of target protein, like full‐consensus protein design (FPD) and ancestral sequence reconstruction (ASR). However, redesign of highly functional protein is challenging tasks. In this study, we attempt to get a clue how to succeed in the redesign through protein sequence reconstruction of native l‐threonine 3‐dehydrogenase from Cupriavidus necator (CnTDH) (1).MethodsFirstly, total 5000 of homologous sequences with CnTDH were obtained by submitting sequence of CnTDH to Blastp: the sequences were called “library”. Artificial TDH, FpdTDH‐N0, was designed by the FPD method using the library. Next, utilizing INTMSAlign (2, 3), the curated library was generated to maximize frequency of each consensus residues of artificial TDH. Two artificial TDHs were designed by FPD (FpdTDH‐N1) and ASR (AsrTDH) method, respectively, utilizing the curated library.Results & DiscussionSpecific activity was measured for the three artificial TDHs; and the activity could be confirmed for FpdTDH‐N1 and AsrTDH which designed using the curated library. FpdTDH‐N1 and AsrTDH share 69 and 65% of sequence identity with CnTDH, respectively. Enzyme properties of CnTDH, FpdTDH‐N1 and AsrTDH were estimated by the following multidisciplinary analysis. Thermal stability of both FpdTDH‐N1 and AsrTDH was better than that of CnTDH: Tm values of FpdTDH‐N1 and AsrTDH were higher about 10 and 5 °C than that of CnTDH. Enzyme kinetics parameters of FpdTDH‐N1 and AsrTDH (kcat/Km value) were comparable to CnTDH. Furthermore, dissociation constant (Kd) for AsrTDH and FpdTDH‐N1 toward NAD+ which measured by ITC were about 8‐ and 2‐fold lower than that of CnTDH. Structural comparison among CnTDH, FpdTDH‐N1 (2.9 Å resolution) and ArsTDH (2.2 Å resolution) indicated that there is no mutation around 7 Å of oxygen atom of catalytic Tyr. The mutation was assembled at NAD+ binding site and protein surface.Taken together, we succeeded in design artificial TDHs bearing high stability, activity and affinity toward NAD+ compared with native TDH, CnTDH. This result also inferred that curated library designed by maximizing frequency of amino acids for consensus residues of target protein is effective to design “super enzyme” surpassing native enzyme function.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Number 16K18688, 17KT0010, 17H06169 and JST by ERATO grant number JPMJER1102.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High Intensity Muscle Metaboreflex Activation Blunts Cardiopulmonary Baroreflex Control of Sympathetic Vasomotor Outflow

Muscle sympathetic nerve activity (MSNA) is reduced during low intensity dynamic leg exercise. This reduction in MSNA has been attributed to loading of the cardiopulmonary baroreflex with increased venous return and it has been suggested that such inhibition is overridden with higher intensity exercise due to muscle metaboreflex activation. However, limited data are available regarding the interaction between the cardiopulmonary baroreflex and the muscle metaboreflex. Herein, we tested the hypothesis that cardiopulmonary baroreflex control of MSNA is attenuated when the muscle metaboreflex is strongly activated. In eight healthy young men, MSNA (right peroneal nerve), mean arterial blood pressure (MAP, Finometer), heart rate (ECG), and thoracic impedance were recorded. Graded isolation of the muscle metaboreflex was achieved by post‐exercise ischemia (PEI) following low‐ (PEI‐L), moderate‐ (PEI‐M) and high‐ (PEI‐H) intensity isometric handgrip performed at 20%, 30% and 40% maximum voluntary contraction, respectively. Lower‐body positive pressure (LBPP, +10 mmHg), to load the cardiopulmonary baroreflex, was applied at rest and during PEI. Handgrip exercise elicited intensity‐dependent increases in MSNA and MAP that were maintained during PEI indicating a graded muscle metaboreflex activation. LBPP at rest significantly decreased MSNA burst frequency and total activity (total activity: −39.5 ± 5.7%, mean ± SE, P < 0.05), while MAP was unchanged. When LBPP was applied during PEI, MSNA burst frequency and total activity decreased significantly at PEI‐L (total activity: −41.8 ± 10.6%, P < 0.05) and PEI‐M (total activity: −34.7 ± 8.6%, P < 0.05), but not at PEI‐H (total activity: −11.8 ± 8.6%, n.s., one‐way repeated measures ANOVA: F = 5.07, P < 0.05). Thoracic admittance, which was calculated from thoracic impedance as an index of central blood volume, was not changed during handgrip or PEI alone, whereas LBPP evoked similar increases in thoracic admittance during all conditions (rest: +5.4±0.8, PEI‐L: +5.8±0.5, +PEI‐M: +5.4±0.5, PEI‐H: +5.8±0.5 S·10−4, one‐way repeated measures ANOVA: F = 0.38, n.s.). These preliminary findings suggest that high intensity muscle metaboreflex activation attenuates cardiopulmonary baroreflex control of sympathetic vasomotor outflow.Support or Funding InformationNIH R01 HL127071 JSPS KAKENHI Grant Number JP16KK0201This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of the laser-induced water bubble actuator

Aiming to realize the newly actuator without any electric cable, the use of laser-induced water vaper bubble has been proposed and performed the principle confirmation experiment. The laser induced water bubble has been applied for the liquid jet therapeutic catheter in medical field. The liquid jet system use physiologic saline for the liquid and a Ho: YAG laser of 2.1μm wavelength that has highly absorption of water. Instead of this combination, we propose the use of carbon powder suspension for working liquid and an infrared Laser Diode (LD) of 970nm wavelength. As the 970nm LD light is not absorbed by water but carbon, the water of the working liquid could be vaporized by heated carbon. LD is considered as a suitable laser source for the actuator in the viewpoint of output controllability by the drive-current modulation. Figure 1 shows the conceptual image of the laser-induced water bubble actuator. It is constructed with a 970nm LD, an optical fiber and an actuator unit with working liquid for producing bubble. An optical fiber connector (SMA connector) has been used for the bubble generator unit in our principle confirmation experiment. We use the inlet space for inserting and fixing fiber cable as the actuator cylinder. The working liquid was prepared by suspending carbon powder of 20μm particle size in water with surface active agent. After the working liquid has been injected in the cylinder, a stainless steel rod of Φ2.3mm diameter and 30mm length inserted as the actuator piston. When the laser irradiation was performed at the LD power of 160-180W and the pulse duration of 2msec, the reciprocating motions of the stainless steel rod have been observed. For the next step, we are planning to evaluate the operating characteristic and also try to maximize the generating force. This work was supported by JSPS KAKENHI Grant Number JP16K12914.

Phenomenological aspects of possible vacua of a neutrino flavor model**Supported by JSPS KAKENHI Grant Number JP17K05418 (T.M.). This work is also supported in part by Grants-in-Aid for Scientific Research [No. 16J05332 (Y.S.), Nos. 24540272, 26247038, 15H01037, 16H00871, and 16H02189 (H.U.)] from the Ministry of Education, Culture, Sports, Science and Technology in Japan. H.O. is also supported by Hiroshima Univ. Alumni

We discuss a supersymmetric model with discrete flavor symmetry . The additional scalar fields which contribute masses of leptons in the Yukawa terms are introduced in this model. We analyze their scalar potential and find that they have various vacuum structures. We show the relations among 24 different vacua and classify them into two types. We derive expressions of the lepton mixing angles, Dirac CP violating phase and Majorana phases for the two types. The model parameters which are allowed by the experimental data of the lepton mixing angles are different for each type. We also study the constraints on the model parameters which are related to Majorana phases. The different allowed regions of the model parameters for the two types are shown numerically for a given region of two combinations of the CP violating phases.

Fabrication of the Sulfur Lithium Ion Secondary Battery with Different Li Ion Source

Introduction Sulfur, with the theoretical specific capacity of 1672mAhg-1 undergoing the rocking chair reaction (S8+16Li++16e-↔8Li2S), is the most promising cathode material candidates for future Li ion batteries excepting two main drawbacks hindered its application.1 On the one hand, during cycling, the Li deposited on Li foil is readily to form Li dendrite, resulting in the internal short-circuit of the battery.2 On the other hand, the dissolution of polysulfide species in electrolyte will lead to fast capacity fading. However, most papers reported are focused on the S cathode problems, less researchers paid attention to the drawbacks caused by the Li metal. In order to resolve the safety issue, a surface contact pre-lithiation method was used to introduce the Li ion in the S electrode.3 In this way, the Li metal can be replaced by other anode forming Li metal free full cell. Experimental The sulfur/ Ketjen Black (S/KB) composite was mixed in a weight ratio of 1:1 after a heating process at 155 ℃. The S cathode was prepared by mixing S/KB composite:PVdF= 90:10 wt%. The KB can provide conductivity and physical confinement for the sulfur species, alleviating the dissolution of polysulfides. Moreover, the ionic liquid electrolyte, triglyme (G3) was used in the full cell which showed good polysulfide suppression ability.4 Because of the compatibility of the G3 electrolyte for the graphite anode, the graphite electrode (made in graphite:AB:PVdF=90:5:5 wt%) with an areal capacity of 0.9 mAh cm-2 (~300 mAhg-1) was used to pair with the pre-lithiated S/KB cathode (Li2S/KB). The graphite anode was pre-lithiated at the same condition to fabricate LixC anode. The graphite electrode with a solid electrolyte interphase layer (CSEI) anode was fabricated by disassembling the C-Li cell after 5 cycles. All the electrodes used were cut into 1 cm2size. Results and Discussion The cycling performance of the difference cells were tested at 0.1 C-rate (based on the weight of sulfur), showed in Fig. 1. Firstly, it is observed that the S-Li cell showed fast capacity fading using G3 electrolyte. It has been proved that the increased internal resistance give rise to the fast capacity decay in the S-Li half cell. In the initial cycle, the S-Li cell showed high specific capacity higher than 1600 mAhg-1 (0.8 mAh cm-2) due to the abundant Li ion source from Li metal. The S-Li cell turned to internal short in 80 cycle due to the formation of Li dendrite, indicating the safety issue of the Li metal containing sulfur battery.Secondly, Li2S-C cell showed over half amount of capacity (about 53.3%) decay in the first cycle, leading to only 673 mAhg-1 discharge capacity. In order to study to reason caused the irreversible capacity lost in the first cycle, the Li2S-CSEI was assembled showing higher reversible capacity in the first cycle than that of the Li2S-C cell, indicating the irreversible capacity lost in the first cycle was mainly caused by the formation of the SEI layer on the graphite anode. Under this context, the surface contact pre-lithaition method was applied to graphite anode to introduce Li ion. The SEI layer was expected to be formed on the graphite surface at the same time. As a result, the S-LixC full cell showed increased capacity retention than the Li2S-C cell in Fig. 1. In conclusion, it is convictive to say that the lithium metal free S-LixC cell showed high irreversible capacity and better safety. We believe that this work will make contribution to future sulfur lithium ion secondary battery. Fig. 1 Cycling performance of different sulfur lithium secondary batteries at 0.1 C-rate. Acknowledgement This work is supported by Advanced Low Carbon Technology Research and Development Program (JST-ALCA) Special Priority Research Area "Next-generation Rechargeable Battery”, and JSPS KAKENHI Grant Numbers JP17J09973.

(Invited) Nanostructure Design for Control of Phonon and Electron Transports

Thermoelectric materials are required to high electrical conductivity and low thermal conductivity for high performance. The independent control of electrons and phonons is difficult due to their correlation resulting in the difficulty of the enhancement of thermoelectric performance. Recently, nanostructures have drawn much attention in thermoelectric material study. One of the mechanisms of its enhancement is the thermal conductivity reduction due to the phonon scattering at the interfaces introduced by nanostructuring [1]. In some case, however, its interfaces can scatter the carriers leading to the reduction of the carrier conductivity. In this study, we are proposing the novel nanostructure for independent control of electrons and phonons using nanodots. The nanostructure is ultra-small epitaxial Ge nanodots are embedded in Si layers. It is expected that carrier transports in Si layers with high electrical conductivity and high Seebeck coefficient, while phonons are scattered at the interfaces between nanodots and Si layers [2,3]. Ultra-small Ge nanodots in this nanostructure were grown by our original technique: ultrathin SiO2 film technique. First, we formed the ultrathin SiO2 films with one monolayer thickness by oxidizing clean Si surfaces at~500ºC after introducing the Si substrates into molecular beam epitaxy chamber at the base pressure of ~10-8 Pa. We deposited Ge on the ultrathin SiO2 film to form Ge nanodtos. At the first stage, of Ge deposition the reaction of Ge+SiO2 →GeO↑+SiO↑ occurred to form nanowindows in the ultrathin SiO2 films. Subsequently deposited Ge atoms were trapped at the nanowindows, which worked as nucleation sites. Then, ultras-mall nanodots were formed. The nanodots contacted with Si substrates through nanowindows leading to the epitaxial growth. Si was deposited on the epitaxial Ge nanodots to form Si layers. These formations of the ultrathin SiO2 films, epitaxial Ge nanodots, and epitaxial Si layers were repeated to form the nanostructure that is Si films including Ge nanodots. The thermal conductivity measurements of the nanostructure showed the drastic reduction of thermal conductivity. Hall effect measurements exhibited a similar high electrical conductivity to that of bulk Si. Electrons transports through the interfaces easily by band engineering. On the other hand, ultras-mall nanodots work as phonon scatterers leading to the drastic reduction of thermal conductivity. This phonon scattering is consistent with wave scattering theory. These results demonstrated nanostructure design brought to the independent control of carrier and phonon transport using the Si layers with ultrasmall epitaxial Ge nanodots This work was partially supported by JSPS KAKENHI Grant Number 16H02078 for Scientific Research (A) and 15K13276 for Challenging Exploratory Research. In part, this work was supported by CREST-JST program. Reference [1] Y. Nakamura, et al., Nano Energy 12, 845 (2015). [2] S. Yamasaka, et al., Sci. Rep. 5, 14490 (2015). [3] S. Yamasaka, et al., Sci. Rep. 6, 22838 (2016).

Analysis of Ionomer Distribution in Catalyst Layers by Two-Stage Ion-Beam Processing

Mass transport properties of catalyst layers (CLs) for proton exchange membrane fuel cells (PEMFCs) strongly depends on its structure. The ionomer is a proton-carrier material in the CLs. On the other hand, the gas transfer is prevented by the ionomer coated on a catalyst. Ionomer distribution in the CLs, therefore, have gathered much attention. Some papers have suggested methods to analyze the ionomer distribution [1-4]. However, quantitative validation is not enough so far. In addition, these methods especially focused on a microscopic local ionomer distribution. From the point of view of mass transfer, overall through-plane ionomer distribution is important as well as the microscopic distribution. In this study, a novel visualization method of the overall through-plane ionomer distribution in the CLs by using a two-stage ion-beam processing is suggested. The first stage of the two-stage ion-beam processing is the formation of flat and smooth cross-section by a broad ion beam. The second stage is the selective removal of the ionomer in the catalyst layer by a focused ion beam. Scanning ion microscopic (SIM) images were obtained after the first and second stage. Smoothing filter was applied on the obtained SIM images. The SIM image after the second stage was then conducted threshold by Otsu method and a carbon distribution map was obtained. A pores distribution map is obtained from a combined image of the SIM image after the first stage and the carbon distribution map. The threshold was determined by a p-tile method with ionomer to carbon ratio (I/C). Ionomer distribution was extracted from the carbon and pores distribution maps. The ionomer distribution of the catalyst layer with I/C 2.0 was analyzed as shown in Figure 1. A catalyst coated membrane was fabricated by a doctor-blading and decal-transfer method [5]. Lower side in the figures is the interface with a polymer electrolyte membrane (PEM) and upper edge is the bottom in the fabrication process before decal-transferring. The through-plane ionomer distribution is not uniform. Thin ionomer layer was formed at the interface with the PEM. This can be because of deposition of ionomer during the drying process. Acknowledgments This work was supported by JSPS KAKENHI Grant Number 15H03932.

Oxygen Concentration Measurement in the Porous Cathode of Aqueous Lithium–Air Battery By Fine Optical Fiber

Spatial and temporal oxygen concentration distribution in the porous cathode of discharging aqueous lithium-air secondary battery was successfully measured. Lithium-air battery attracts great attention because of its high energy density. In this study, we focus on the aqueous lithium–air secondary battery. The aqueous electrolyte realizes cost reduction and ensures the safe use of a battery. On the other hand, low solubility of oxygen in aqueous electrolyte limits oxygen transport to the reaction site. To realize a more powerful aqueous lithium–air battery, it is necessary to clarify and improve the oxygen transport phenomena in the porous cathode. In this study, we developed the measurement technique of oxygen concentration in the porous cathode under operating condition by inserting a fine optical fiber oxygen sensor. In this study, platinum tetrakis pentrafluoropheny porphine (PtTFPP) was employed as oxygen indicator [1]. When the PtTFPP is exposed to excitation light (λ = 405 nm), phosphorescence emission (λ= 650 nm) is produced, and its intensity depends on oxygen partial pressure. Thus, quantitative oxygen concentration can be obtained by measuring phosphorescence intensity and using calibration data. The PtTFPP was painted at the edge of optical fiber of which diameter was 110 μm. In order to measure the phosphorescence intensity, bifurcated optical fibers were connected to excitation light source and spectrometer, respectively. As shown in Figure 1, phosphorescence intensity changed at 650 nm depending on oxygen concentration. The experimental setup is shown in Figure 2. The porous cathode (SIGRACET® 10AA, SGL Group, USA) was fixed near electrolyte interface horizontally. The tiny optical fiber sensor was moved up and inserted in the cathode. The temporal oxygen concentration change from the start of discharge was measured at arbitrary positions in the porous cathode. The composite anode was water-stable and had a multilayer structure for the aqueous lithium air battery [2]. Measurement of spatial and temporal distribution of oxygen concentration has been conducted under the assumption that discharge experiment had reproducibility. Initial position (z = 0 μm) was defined arbitrary, and the sensor was moved up each 70 μm. Time variation of oxygen concentration was measured at each point. Finally, spatiotemporal distribution of oxygen concentration for each current density was obtained. As shown in Figure 3, the spatial and temporal oxygen concentration distribution at 0.10 mA/cm2 was successfully observed. When the discharge was started, the oxygen concentration gradually decreased in the cathode, and finally, oxygen concentration reached considerably low value even in the condition of 0.10 mA/cm2. In this experiment, oxygen concentration gradients are present at both ends of the atmosphere side (0-140 μm) and electrolyte side (490-910 μm) of the cathode. Flux for both side of cathode, upper side and lower side, was discussed and comprehensible interpretation was obtained. Although dissolved oxygen that forms flux from the lower side of cathode has an effective supply to the porous electrode, electrolyte layer formed at between the atmosphere and the cathode causes oxygen transport resistance. Acknowledgement: This work was supported by JSPS KAKENHI Grant Number 15K13881, 15H02347. Reference: [1] CS Chu., CA Lin, Sensors and Actuators B: Chemica, 2014, 195, 259-265. [2] T. Zhang, N. Imanishi, S. Hasegawa, A. Hirano, J. Xie, Y. Takeda, O. Yamamoto, N. Sammes, J. Electrochem. Soc., 2008, 155, A965. Figure 1

Reconsideration of Metal Work Function at Metal/Semiconductor Interface

It is known that vacuum work function (WF) of metal is composed of bulk and surface terms [1]. The former is characterized by bulk properties of many-body effect and kinetic energy of electrons. The latter is by potential drop through the surface dipole induced by wave function tailing of electron from metal to vacuum through the potential barrier from Fermi-level to vacuum level. This fact implicates that the surface term of WF is sensitive to a counterpart of interface. Namely, the surface term at semiconductor or insulator interface should be different from that at vacuum surface, though vacuum WF is commonly used to discuss band alignment at metal/semiconductor (M/S) or metal/insulator/semiconductor interfaces. On the other hand, Metal induced gap states (MIGS) is the intrinsic Fermi level pinning (FLP) mechanism at M/S interface, and it is also described as a dipole induced by wave function tail into the band gap of semiconductor [2]. We note that the MIGS and surface term of WF at M/S interface are described by common physics by considering that both of hole and electron tailing from metal as shown in Fig. 1. In other words, MIGS seems just corresponded to surface term at M/S interface. Schottky barrier height (SBH) for conduction band of semiconductor at M/S interface is commonly described as shown in Eq. (1). However, considered with above discussion, the FLP caused by MIGS should rather be described as a modulation of WF. Therefore, if the FLP in Eq. (1) is caused by MIGS, the Eq. (1) should be rewritten with a definition of interface WF as shown in Eq. (2), and then from Eqs. (1) and (2), the interface WF can be written as shown in Eq. (3). This indicates that the S parameter of MIGS is a weight average parameter. Next, we would like to discuss how to suppress the WF modulation and MIGS. We think there are two approaches. The one is passive approach from semiconductor side. As shown in Fig. 1, the modulation of wave function tailing is enhanced by narrowing the gap of semiconductor, which is consistent with general trend discussed in MIGS model [2]. Therefore, for example, effective gap widening by inserting ultra-thin dielectrics between metal and semiconductor is effective. The other is active approach from metal side. Since surface term is only modulated in WF, WF modulation should be suppressed by reducing a weight of surface term in WF. Considering the result of first principle calculation that the surface term is drastically decreased by reducing free electron density n in metal [1], we noticed low n metal seems effective. It is also consistent with Heine’s original idea [3]. Here, we emphasize that MIGS can be controlled from both semiconductor and metal sides independently, and that it is different from common other FLP models based on interface gap states only in semiconductor side. We think that metal/germanium (Ge) interface is well understandable based on this view, which means that MIGS is a dominant origin of strong FLP at metal/Ge interface in Eq.(1) [4]. It is well known that the passive approach is effective to increase S parameter [5]. Recently we also demonstrated deviation from the strong FLP trend and S parameter increasing at low n metal/Ge interface by the active approach [6,7]. In that work, interface structure dependence on SBH was also observed at low n metal/Ge interface [6,7]. It might be due to an appearance of surface structure dependence on WF and various local dipoles by suppression of strong WF modulation to align Fermi-level with FLP energy level characterized by bulk Ge. We discussed M/S interface WF in terms of the wave function tailing from metal. We proposed that interface WF is different from vacuum WF in principle and MIGS is corresponded to surface term of interface WF. Furthermore, the strong FLP at metal/Ge interface is reasonably understandable based on the view. Acknowledgement This work was supported by JST CREST Grant Number JPMJCR14F2, Japan, and partly by JSPS KAKENHI Grant Number JP17K06374. Reference [1] N. D. Lang, and W. Kohn, Phys. Rev. B 3, 1215 (1971). [2] W. Mönch, Appl. Surf. Sci. 92, 367 (1996). [3] V. Heine, Phys. Rev. 138, A1689 (1965). [4] T. Nishimura, et al., Appl. Phys. Lett. 91, 123123 (2007). [5] e.g. T. Nishimura, et al., Appl. Phys. Express 1, 051406 (2008). [6] T. Nishimura, et al., Appl. Phys. Express 9, 081201 (2016). [7] T. Nishimura, et al., Ext. Abs. IEEE Semiconductor Interface Specialist Conference 2016. Figure 1

Observation of the vocal tract configuration while playing a woodwind instrument

Skillful players of brass instruments and reed woodwinds are able to control the vocal tract effectively and perform with intended pitches and sound qualities. The player’s vocal tract is, to a first approximation, connected with the instrument bore acoustically in series. If the magnitude of the vocal tract impedance is comparable with the bore impedance, the acoustic pressure in the mouth can then exert influences with a vibrating reed. However, studies reliant enough to confirm the configuration of the vocal tract while playing are limited. In this study, a magnetic resonance imaging (MRI) device was used to scan the vocal tract in three dimensions. The participant was an amateur saxophone player and the instrument was made from reed, mouthpiece, and a short uniform tube. Image data revealed how the vocal tract was formed when she blew a number of different notes. They were also used to determine the cross-sectional area and the input impedance of the vocal tract. The results were compared with trumpeter data taken and published previously, and also with data taken for the act of whistling. [This work was supported by JSPS KAKENHI Grant Number JP16K00242.]

Investigation on Crack Suppression by Thermal-Plasma-Jet Crystallization of Amorphous Silicon Films on Flexible Glass Substrate

Introduction.Ultra-thin glass substrate is attracting much attention as flexible substrates for curved display applications. In fabrication of thin-film transistors (TFTs) on flexible glass, crystallization of amorphous Si (a-Si) is one of the key process technologies [1-3]. Rapid-thermal-annealing (RTA) in microsecond regime is necessary to form high crystallinity Si without thermal damage to glass. We have proposed the application of atmospheric pressure DC ark discharge thermal-plasma-jet (TPJ) to the crystallization of a-Si films [4]. Because of its simple structure and atmospheric pressure process, TPJ enables low cost crystallization compared with excimer laser annealing (ELA) technique. High speed lateral crystallization (HSLC) is induced by sweeping a molten region at a speed as high as 4000 mm/s. However, residual tensile stress is generated because of the densification of glass surface after RTA process. As a result, significant number of cracks are generated on the surface of glass substrate in cases of sever annealing conditions. In this study, we applied µ-TPJ crystallization of a-Si films on ultra-thin glass substrate and investigated the effect of glass thickness on residual stress and crack generation. Experimental.After RCA cleaning and HF treatment of 100-µm-thick flexible glass and 500-µm-thick conventional glass substrates, 100-nm-thick a-Si films were deposited by plasma-enhanced chemical vapor deposition (PECVD) at a substrate temperature of 250 °C. Dehydrogenation was carried at 450 °C in N2 ambient for 1 h. TPJ irradiation set up is shown in Fig. 1. µ-TPJ was generated by DC arc discharge under atmospheric pressure with supplying power (P) of 1.7 kW between electrodes. The Ar gas flow rate (f) was 1.0 L/min. µ-TPJ was generated by blowing out the thermal plasma through an orifice with its diameter (ϕ) of 600 µm. The distances between the plasma source and glass substrates (d) were fixed at 1.0 mm and the substrates were linearly moved by a motion stage in front of the µ-TPJ with scanning speed (v) of 2200 mm/s. For the measurement of temperature profile in the glass substrate, transient reflectivity during TPJ annealing was measured by irradiating bare conventional glass substrate with a He-Ne laser (632.8nm) from the back surface. Details of the non-contact temperature measurement are described in Ref [5]. Results and discussion.The microscope images of crystallized Si films on each substrate are shown in Fig. 2. Many cracks were observed in conventional glass substrate. On the other hand, no crack was observed in flexible glass substrate, although both glass substrates were annealed at the same time. Crystallization was conducted under various µ-TPJ irradiation conditions, and almost no cracks were found in flexible glass and it showed significant resistance against RTA. This result suggests that the tensile stress at glass surface is significantly reduced in the case of ultra-thin flexible glass. Previous study calculated residual stress profile in glass substrate on the basis of multilayered beam theory [6]. To obtain stress profile, we measured transient temperature profile during TPJ irradiation by optical probe (Fig .3). We assumed surface densification of glass when the temperature exceeded softening point (1258 K). We conducted residual stress analysis of glass substrate as the residual stress is shown in Fig. 4 [6]. Strong tensile stress is introduced in glass surface due to the shrinkage by RTA. At the same time, compressive stress is introduced inside of glass to compensate the tensile stress at the surface. It should be noted that the depth of stress compensation is in the range of ~400 µm as shown in Fig. 4, which is larger than the thickness of flexible glass. It is expected that the back side of thin glass substrate is free to relax the stress and plays a roll in suppression of residual stress in the surface of glass. Conclusions.We have crystallized a-Si films on flexible glass substrate by µ-TPJ without crack generation. Flexible glass substrate is useful to relieve surface tensile stress and as the result, crack generation is significantly suppressed. Acknowledgments. A part of this work was supported by Research Institute for Nanodevice and Bio Systems (RNBS) Hiroshima University, and JSPS KAKENHI Grant Number (B)(16H0433400).

(Invited) The Evolution of W-Co-C Catalyst during the CVD Growth of Chirality-Selective Growth of Single-Walled Carbon Nanotubes

Bimetallic catalysts such as Fe-Co and Co-Mo have been used for efficient growth of bulk single-walled carbon nanotube (SWNTs) and vertically aligned SWNTs [1]. Recently, bimetallic catalysts such as Co-W [2] or Co-Cu [3] are employed for structure controlled growth of SWNTs. Here, roles of bimetallic catalysts are explored by newly proposed SiO2 grid based in-plane TEM technique, which enables a direct TEM characterization of catalysts on SiO2, CVD growth of SWNTs in normal furnace, and TEM characterization of catalysts and grown nanotubes after CVD. The in-plane TEM results of the traditional Co-Mo bimetallic catalysts were consist with our previous report [1]; metallic Co nanoparticle embedded in Co-Mo oxide is responsible for the nucleation of SWNTs. By using Co-Cu catalysts, we can synthesize vertically aligned SWNTs with subnanometer diameters on quartz (and SiO2/Si) substrates [3]. EDS-STEM and HAADF-STEM imaging of the Co-Cu bimetallic catalyst system showed that small Co catalysts were captured and anchored by adjacent Cu nanoparticles, which grew small diameter of SWNTs. High-melting point W6Co7 alloy produced from metal cluster precursors is reported to grow a single chirality (12,6) with over 90 % abundance [2]. Here, we show that a sputtered Co-W catalyst can selectively grow (12,6) SWNTs by CVD at lower reduction and growth temperatures [4]. Statistical Raman mapping analysis and optical absorption spectrum of the as-grown SWNTs reveal that the abundance of (12,6) is over 60 %. The morphology and structure of catalyst is investigated by the in-plane TEM before and after CVD growth. The catalyst alloy we produced was W6Co6C and the only metallic Co was left after the CVD of 5 min. By comparing the catalyst TEM images with variable CVD period, we can discuss the evolution of the alloy catalysts. The W rich initial catalyst is composed of bcc W bound with W6Co6C. Once ethanol is introduced, additional W will be gradually removed to the gas phase product of WO3 and the ratio of W decreases. It is speculated that after the formation of pure W6Co6C, further loss of W will result the precipitation of Co; nucleation of Co cluster which is primary responsible for growth of an SWNT. This work was supported by JSPS KAKENHI Grant Numbers JP25107002, JP15H05760, and IRENA Project by JST-EC DG RTD, Strategic International Collaborative Research Program, SICORP. Part of this work is based on results obtained from a project commissioned by the New Energy and Industrial Technology Development Organization (NEDO). We also acknowledge supports from Advanced Characterization Nanotechnology Platform of the University of Tokyo, supported by "Nanotechnology Platform" of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Room-Temperature Atomic Layer Deposition of Al2O3 for Anticorrosion Coating on Metal Surfaces

A room-temperature atomic layer deposition (RT-ALD) of Al2O3 as an anticorrosion coating for metal surfaces was developed using trimethylaluminum (TMA) and a plasma excited humidified argon. The schematic of ALD system is shown in Fig.1. TMA as the aluminum precursor was introduced with a mass flow controller. A remote plasma system generating excited humidified argon was installed to the ALD chamber. The source gas for the oxidizing gas is a mixture of water vapor and argon. The plasma was generated in a glass tube with an induction coil with a frequency of 13.56 MHz and a RF power of 100 W. For the Al2O3 deposition, we repeated the cycle of TMA saturation and oxidizing gas treatment at RT. The TMA exposure was set at 5.86×10-3 Torr×40 sec. The saturation of TMA on the hydroxylated surface was confirmed by the IR absorption spectroscopy. The plasma excited oxidizing gas was injected for 2 min. As a result, we show the anticorrosion test of ALD coated metal in a HCl solution. We used SUS430 plates with a size of 20×50×0.5 mm3 as the sample. In this test, we used a not-attenuated HCl solution with a concentration of 35 %. All the samples were immersed in the HCl solution for a certain amount of minutes at room temperature and we observed its surface coloring. Fig.2 shows a picture of the tested samples. The not-treated SUS430 sample clearly exhibits a white stain by the HCl corrosion. On the other hand, the Al2O3 coated sample maintained a mirror surface, which indicates that the RT-ALD coated film works as the anti-corrosion film against the acid solution. In the conference, we discuss the applicability of the RT-ALD to the anticorrosion coating for metal components. Acknowledgements This work was partly supported by JST-CREST, JSPS KAKENHI Grant Numbers 15H03536 and 15K13299. Figure 1

Revealling the Structure of Catalyst for Diameter and Chirality Selective Growth of Single-Walled Carbon Nanotube Directly on SiO2

Here we present our recent progress on controlled growth of single-walled carbon nanotubes (SWNTs) by our alcohol catalytic chemical vapor deposition (ACCVD),[1] and a technique for revealling the structure of catalyst directly on SiO2 film.[2] With Co-Cu binary catalyst, a novel combination recently proposed, we are able to synthesize vertically aligned (VA-) SWNTs with diameter tunable down to 1 nm, which is so far smallest for VA-SWNT arrays.[3] Chirality selective growth of SWNTs achieved in ACCVD from sputtered Co-W catalyst. This sputtered Co-W catalyst is able to grow SWNTs uniformly on entire substrate with a (12,6) enrichment of 70%.[4] To clarify to growth mechanism in binary catalyst, we propose an in-plane TEM approach, which enables a direct characterization of catalyst particles with atomic resolutions on a MEMS-fabricated thin SiO2 film. Since no transfer or any post treatments are needed, the most original morphology is preserved. By this technique we confirm that, with the addition of Cu, much smaller Co particles form epitaxially on Cu and produce all sub-nm vertically aligned SWNTs. In Co-W, selective area electron diffraction (SAED) suggests an intermediate Co6W6C structure that is possibly associated with growth selectivity. High-angle annular dark-field imaging (HAADF) reveals the the junction between metallic W and Co-W. [1] S. Maruyama*, R. Kojima, Y. Miyauchi, S. Chiashi and M. Kohno, Chem. Phys. Lett., (2002), 360-3-4, 229-234.[2] R. Xiang, A. Kumamoto, Y. Ikuhara, S. Maruyama, et al., to be submitted.[3] K. Cui, A. Kumamoto, R. Xiang, H. An, B. Wang, T. Inoue, S. Chiashi,Y. Ikuhara, S. Maruyama*, Nanoscale, (2016), 8-3, 1608-1617.[4] H. An, A. Kumamoto, H. Takezaki, S. Ohyama, Y. Qian, T. Inoue, Y. Ikuhara, S. Chiashi, R. Xiang*, S. Maruyama*, Nanoscale, (2016), 8, 14523-14529. This work was supported by JSPS KAKENHI Grant Numbers JP25107002, JP15H05760, JP15K17984, and IRENA Project by JST-EC DG RTD, Strategic International Collaborative Research Program, SICORP. Part of this work is based on results obtained from a project commissioned by the New Energy and Industrial Technology Development Organization (NEDO). We also acknowledge supports from Advanced Characterization Nanotechnology Platform of the University of Tokyo, supported by "Nanotechnology Platform" of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

(Invited) Carbon Nanotube Films for Organic-Inorganic Hybrid Perovskite Solar Cells

A film of single-walled carbon nanotubes (SWNTs) can be a dual-functional layer as electron-blocking-layer and transparent-conductive electrode in various solar cells. We have demonstrated efficient SWNT/Si solar cells [1,2] and organic solar cells [3,4] using dry-deposited high-quality SWNTs and honeycomb-structured SWNTs. For organic solar cells, the SWNT doped by MoOxand PEDOT:PSS demonstrated a dual function, which can replace both ITO and electron-blocking-layer [3]. Using this advantage of the SWNT film, flexible organic solar cells were fabricated with ease [3]. Also, it was possible to replace electron-blocking-layer and metal electrode for inverted-type organic solar cells [4]. The removal of metal electrode enabled flexible, the semi-transparent window-like solar cells, which was inexpensive in their production process [4]. Here, this dual functional application of SWNT film is also demonstrated for organic-inorganic hybrid perovskite solar cells. Replacement of ITO in inverted-type perovskite solar cells, SWNTs/PEDOT:PSS/CH3NH3PbI3/PCBM/Al, is demonstrated by adjusting the surface energy of PEDOT:PSS [5]. The flexible application on polyethylene terephthalate (PET) is also demonstrated [5]. Furthermore, replacement of electron-blocking-layer and metal electrode in normal-type perovskite solar cells is demonstrated as well. They show high power conversion efficiency (PCE), cost-efficiency, and higher stability. Those devices can have comparable PCE as the conventional design with organic electron-blocking layer and top metal electrode. In addition to the expected lower cost and improved stability, These solar cells can be potentially semi-transparent when transparent SWNT films are used. This means that they can be illuminated from either cathode or anode side. The normal-type perovskite solar cell, composed of ITO/TiO2/CH3NH3PbI3/SWNTs, can achieve a PCE of 10 % without doping of SWNTs [6]. The PCE can be as high as 17 % with the preliminary doping of the film of SWNTs using Spiro-MeOTAD, which is the typical electron-blocking-layer used for the normal type perovskite solar cells. The PMMA layer on top of the film of SWNTs can also contribute as doping and protection layer. Furthermore, the modified structure with a perovskite layer sandwiched by C60 and SWNTs, i.e. ITO/C60/CH3NH3PbI3/SWNTs, can lead to the solar cells without hysteresis and with much improved air-stability [7]. The effective passivation of the degradation of perovskite material by moisture can be achieved with C60and SWNTs [7]. This device show about 13 % PCE without hole-transporting layer and 17% with spiro-MeOTAD this far [7]. This can be a good candidate for scale-up demonstration of practical perovskite solar cells. This work was supported by JSPS KAKENHI Grant Numbers JP25107002, JP15H05760, and IRENA Project by JST-EC DG RTD, Strategic International Collaborative Research Program, SICORP. Part of this work is based on results obtained from a project commissioned by the New Energy and Industrial Technology Development Organization (NEDO).

Dietary effect of Kaempferia parviflora, a PPARγ agonist, on glucose tolerance in non‐obese type diabetic mice

Type 2 diabetes mellitus (T2DM) in Asian countries is often characterized as non‐obese type. PPARγ agonists are known to reduce adipocyte size and improve adiponectin secretion and insulin resistance, but to induce abdominal fat accumulation as an adverse effect. Search for PPARγ agonists which have little adverse effects from natural food resources is desirable for people with non‐obese T2DM. We previously showed that Kaempferia parviflora (black ginger, BG) and its ethanol extract (BGE) had PPARγ agonistic activities in a binding study, and exerted a beneficial impact on adiponectin secretion in an animal study. Although some studies showed anti‐T2DM effects of BG in diet‐induced obesity animal model, little is known to have suppressive effect of BG and BGE on T2DM in non‐obese T2DM model. We here investigated the effect of BG and BGE in diet on glucose tolerance in NSY mice with non‐obese T2DM. Male NSY mice were divided into five dietary groups and fed each diet for 8 weeks; mice fed an AIN‐93G diet (N‐LF), high‐fat and high‐sucrose diet (HF), HF diet supplemented with BG (1%) (BG), and HF diet supplemented with BGE (0.19%) (BGE). Half of mice in HF group were treated with pioglitazone (3 mg/kg) as PPARγ agonistic positive control (PIO). ICR mice fed the LF diet are used as non‐diabetic control (I‐LF). At the end of 6th and 7th week, oral glucose and insulin tolerance tests (OGTT and ITT) were respectively carried out. In OGTT, elevation of plasma glucose level was significantly suppressed in the BG and BGE groups. In ITT, plasma glucose level was significantly lowered in the BG and BGE groups. These effects were equivalent to those of pioglitazone. In conclusion, BG and its methanol extract can be effective for prevention of insulin resistance and T2DM.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Number 16K21349 and Mishima Kaiun Memorial Fundation.

Cultural heritage protection system in Japan: Current issues and prospects for the future

Japan has been modernizing for the past 150 years, wiping out the old for the sake of socio-economic progress. Since the end of World War II (WWII) in particular, Japan’s new constitution renounced war and Japan placed a strong focus on economic development. In many cases, the protection of cultural heritage was marginalized. However, in recent years, Japan’s society and economy have matured, and the national demand for a better quality of life has increased. Demand for non-material satisfaction has led to growing concern about social cohesion, local identity, and culture. On the other hand, due to structural changes in the economy and production, industries have also increased attention to the importance of utilizing culture for the creation of economic value. Thus, cultural heritage has been integrated into local communities and has gradually come to be considered an important component of a high-quality lifestyle as well as a precious resource for regional development through cultural tourism and the creation of high value-added products. Accordingly, the cultural heritage protection system has evolved to meet these socio-economic changes. The main purpose of this paper is to overview the major achievements of cultural heritage protection in general, which comprises the core of Japanese cultural policy. Also the underlying socio-economic changes will be addressed. Then, current issues and prospects for the future will be considered. This paper is largely based on GRIPS Discussion Paper 14-10, written by the author, and was revised for this journal with the support of JSPS KAKENHI Grant Number 26380292. Except as otherwise cited, data used in this paper are based on Agency for Cultural Affairs (2001, 2016).

39 THE EFFECTS OF RESVERATROL DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES VITRIFIED AT THE IMMATURE STAGE

Previously, live offspring have been produced from porcine oocytes vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731); however, their embryo developmental rates remain low. The aim of our current research was to test the effects of resveratrol, an antioxidant and anti-apoptotic agent on the developmental competence of immature vitrified oocytes during in vitro maturation (IVM) after warming. Follicular porcine cumulus-oocyte complexes (COC) were vitrified on Cryotop® sheets (Kitazato Corp. Shizuoka, Japan) using the cryoprotectant treatment and warming method of Somfai et al. (2015 J. Reprod. Dev. 61, 571–579). After warming, the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium (POM) enriched with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 hCG. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cAMP. The following 24 h of IVM was performed in POM without dibutyryl cAMP. Vitrified/warmed COC (vitrified group) and freshly collected COC (control group) were matured either in the absence or presence of 2 µM resveratrol (RES− and RES+, respectively) throughout the entire IVM. At the end of IVM, oocytes were denuded and their survival was evaluated. Then, those with 1 polar body (PB1+) were selected for parthenogenetic activation (Day 0). Activated oocytes were cultured for 7 days in PZM-3. Survival, nuclear maturation, cleavage, and blastocyst rates were assessed. The experiment was replicated 5 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. Vitrification reduced the percentage of live oocytes after IVM both in RES− and RES+ groups in a similar manner (47.9 and 51.8%, respectively) compared with control RES− and RES+ groups (99.4 and 100%, respectively; P < 0.05) There was no statistical difference among groups in the percentage of PB1+ oocytes (ranging between 76.1 and 90.2%). On Day 2, the cleavage rate in vitrified RES− group was lower than those in control RES− and RES+ groups (55.9 v. 78.5% and 79.2%, respectively) whereas the vitrified RES+ group did not differ from the others (72.1%). The blastocyst developmental rate calculated from total cultured oocytes on Day 7 in vitrified RES+ group was significantly higher (P < 0.05) than that in the vitrified RES− group (26.2% v. 6.9%, respectively) and did not differ significantly from those of control RES− and RES+ groups (32.1 and 36.0%, respectively). Blastocyst rates in control RES− and RES+ groups were significantly higher (P < 0.05) than that in vitrified RES− group but did not differ from one another. In conclusion, supplementation of IVM medium with resveratrol improved the developmental competence of vitrified, but not freshly collected oocytes. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS. E.C.S. Santos was supported by a CNPq-Brasil fellowship.

256P Recombination signal binding protein for immunoglobulin- kappa-J region (RBPJ) and Mastermind-like 3 (MAML3) are potential therapeutic targets for pancreatic cancer

Background We have reported that Hypoxia induces Smo transcription, which is driver gene of Hh signaling, and that hypoxia enhances invasion and proliferation of pancreas cancer cells through increased Smo transcription. We have also reported protein-bound polysaccharide-K (PSK) can reduce Smo transcription under hypoxia. However, the precise mechanism of hypoxia-induced activation of Smo transcription remains unclear. Our goal is to find pivotal factors which regulates hypoxia-induced activation of SMO transcription and to detect a new effective therapeutic target for pancreatic cancer. Methods Three pancreatic cancer cell lines (ASPC1, SUIT2 and PANC1) were cultured under normoxic and hypoxic (1% O2) conditions. DNA microarray analysis was performed using cells cultured under normoxia and hypoxia. RNA interference and plasmid were used to knock down and overexpress each genes. mRNA expression was estimated by Real time RT-PCR, and Protein expression was by Western blotting. Proliferation assay, Matrigel invasion assay were performed to investigate cell functions. We used PSK to block Smo transcription. Results 1) A transcriptional regulator, recombination signal binding protein for immunoglobulin-kappa-J region (RBPJ) and a transcriptional co-activator, Mastermind-like 3 (MAML3) were picked up for candidate genes. 2) Expressions of SMO, RBPJ and MAML3 were increased under hypoxia in pancreatic cancer cell lines. PSK reduced these expressions under hypoxia. 3) RBPJ and MAML3 inhibition under hypoxia led to decreased SMO and GLI1 expressions. 4) Overexpression of RBPJ under hypoxia led to increased SMO expression and PSK reduced this increased expression. 5) Overexpression of RBPJ under hypoxia showed increased invasiveness and proliferation. PSK reduced these invasiveness and proliferation. Conclusions These results suggest that hypoxia promotes SMO transcription through upregulation of MAML3 and RBPJ to induce proliferation, invasiveness in pancreatic cancer, and that MAML3 and RBPJ can be a new therapeutic target for pancreatic cancer. In addition to that, PSK can be a promising drug for pancreatic cancer by down regulating these new targets. Legal entity responsible for the study N/A Funding JSPS KAKENHI grant number 26293289 Disclosure All authors have declared no conflicts of interest.

Aperiodicity analysis of filled pause in the Corpus of Spontaneous Japanese database

We applied a novel aperiodicity analysis method (Kawahara et al. 2016, SSW9) to CSJ database (Maekawa, 2003, ISCA & IEEE Workshop). The applied method derives the amount of aperiodicity as a time-frequency map, using three staged procedures. The first stage derives a probability map of the fundamental component witout prior information. The second stage finds the most probable trajectory of the fundamental component based on a state transition model and calculates the initial estimate of FO. The final stage refines this initial estimat using FO adaptive time axis warping and instantaneous frequency of each harmonic component. Recursive application of this refinemet procedure provides simultaneous estimation of FO and aperiodicity map. Classification of filled pause using the refined FO trajectories and the aperiodicity maps will be discussed. [This work was supported by JSPS KAKENHI Grant Number JP26284062.]