Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 18

Search results for: Yuta Mano

18 The Interaction between Hydrogen and Surface Stress in Stainless Steel

Authors: Osamu Takakuwa, Yuta Mano, Hitoshi Soyama

Abstract:

This paper reveals the interaction between hydrogen and surface stress in austenitic stainless steel by X-ray diffraction stress measurement and thermal desorption analysis before and after being charged with hydrogen. The surface residual stress was varied by surface finishing using several disc polishing agents. The obtained results show that the residual stress near surface had a significant effect on hydrogen absorption behavior, that is, tensile residual stress promoted the hydrogen absorption and compressive one did opposite. Also, hydrogen induced equi-biaxial stress and this stress has a linear correlation with hydrogen content.

Keywords: hydrogen embrittlement, residual stress, surface finishing, stainless steel

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17 Comparison of Two-Phase Critical Flow Models for Estimation of Leak Flow Rate through Cracks

Authors: Tadashi Watanabe, Jinya Katsuyama, Akihiro Mano

Abstract:

The estimation of leak flow rates through narrow cracks in structures is of importance for nuclear reactor safety, since the leak flow could be detected before occurrence of loss-of-coolant accidents. The two-phase critical leak flow rates are calculated using the system analysis code, and two representative non-homogeneous critical flow models, Henry-Fauske model and Ransom-Trapp model, are compared. The pressure decrease and vapor generation in the crack, and the leak flow rates are found to be larger for the Henry-Fauske model. It is shown that the leak flow rates are not affected by the structural temperature, but affected largely by the roughness of crack surface.

Keywords: crack, critical flow, leak, roughness

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16 Plasma Actuator Application to Control Surfaces of a Model Aircraft

Authors: Yuta Moriyama, Etsuo Morishita

Abstract:

Plasma actuator is very effective to recover stall flows over an upper airfoil surface. We first manufacture the actuator, test the stability of the device by trial and error basis and find the conditions for steady operations. We visualize the flow around an airfoil in the smoke tunnel and observe the stall recovery. The plasma actuator is stationary device and has no moving parts, and it might be an ideal device to control a model aircraft. We can use the actuator not only as a stall recovery device but also as a spoiler. We put the actuator near the leading edge of an elevator of a model aircraft as a spoiler, and measure the aerodynamic forces by a three-component balance. We observe the effect of the plasma actuator on the aerodynamic forces and the device effectiveness changes depending on the angle of attack whether it is positive or negative. We also visualize the flow caused by the plasma actuator by a desk-top Schlieren photography which is otherwise very difficult in a low-speed wind tunnel experiment.

Keywords: aerodynamics, plasma actuator, model aircraft, wind tunnel

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15 Indigo-Reducing Activity by Microorganisms from the Fermented Indigo Dyeing Solution

Authors: Yuta Tachibana, Ayuko Itsuki

Abstract:

The three strains of bacteria (Lysinibacillus xylanilyticus, Bacillus kochii, and Enterococcus sp.) were isolated from the fermented Indigo (Polygonum tinctorium) dyeing solution using the dilution plate method and some fermentation conditions were determined. High-Performance Liquid Chromatography (HPLC) was used to determine the indigo concentration. When the isolated bacteria were cultured in the indigo liquid culture containing various sugars, starch, and ethanol, the indigo culture solutions containing galactose, mannose, ribose, and ethanol were remarkably decreased. Comparison of decreasing indigo between three strains showed that Enterococcus sp. had the fastest growth and decrease of indigo. However, decreasing indigo per unit micro biomass did not correspond to the results of decreasing indigo―Bacillus kochii had higher indigo-reducing activity than Enterococcus sp. and Lysinibacillus xylanilyticus.

Keywords: fermentation condition, high-performance liquid chromatography (HPLC), indigo dyeing solution, indigo-reducing activity

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14 Aerodynamic Devices Development for Model Aircraft Control and Wind-Driven Bicycle

Authors: Yuta Moriyama, Tsuyoshi Yamazaki, Etsuo Morishita

Abstract:

Several aerodynamic devices currently attract engineers and research students. The plasma actuator is one of them, and it is very effective to control the flow. The actuator recovers a separated flow to an attached one. The actuator is also inversely applied to a spoiler. The model aircraft might be controlled by this actuator. We develop a model aircraft with the plasma actuator. Another interesting device is the Wells turbine which rotates in one direction. The present authors propose a bicycle with the Wells turbine in the wheels. Power reduction is measured when the turbine is driven by an electric motor at the exit of a wind tunnel. Several Watts power reduction might be possible. This means that the torque of the bike can be augmented by the turbine in the cross wind. These devices are tested in the wind tunnel with a three-component balance and the aerodynamic forces and moment are obtained. In this paper, we introduce these devices and their aerodynamic characteristics. The control force and moment of the plasma actuator are clarified and the power reduction of the bicycle is quantified.

Keywords: aerodynamics, model aircraft, plasma actuator, Wells turbine

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13 Cybernetic Modeling of Growth Dynamics of Debaryomyces nepalensis NCYC 3413 and Xylitol Production in Batch Reactor

Authors: J. Sharon Mano Pappu, Sathyanarayana N. Gummadi

Abstract:

Growth of Debaryomyces nepalensis on mixed substrates in batch culture follows diauxic pattern of completely utilizing glucose during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming xylose. The present study deals with the development of cybernetic mathematical model for prediction of xylitol production and yield. Production of xylitol from xylose in batch fermentation is investigated in the presence of glucose as the co-substrate. Different ratios of glucose and xylose concentrations are assessed to study the impact of multi substrate on production of xylitol in batch reactors. The parameters in the model equations were estimated from experimental observations using integral method. The model equations were solved simultaneously by numerical technique using MATLAB. The developed cybernetic model of xylose fermentation in the presence of a co-substrate can provide answers about how the ratio of glucose to xylose influences the yield and rate of production of xylitol. This model is expected to accurately predict the growth of microorganism on mixed substrate, duration of intermediate lag phase, consumption of substrate, production of xylitol. The model developed based on cybernetic modelling framework can be helpful to simulate the dynamic competition between the metabolic pathways.

Keywords: co-substrate, cybernetic model, diauxic growth, xylose, xylitol

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12 The Vulnerability of a Small, Open Economy in a Situation of Global Fiscal Crisis: The Impact of the Greek Debt Crisis on the Foreign Direct Investments to Macedonia

Authors: Viktorija Mano

Abstract:

The objective of my research is to critique the International Monetary Fund (IMF) stance on foreign investment and the benefits for small, open economies of allowing the free movement of capital. In my research as a whole I will explore the extent to which this stance impacted upon and influenced the economic policies of Macedonia. This will involve providing a contextualized, critical account of the policy of the IMF focusing on a comparison of its policies during the early 2000s through policy documents, political discourse and enacted policies in Macedonia. The conditionality associated with these policies, such as the enforcement of austerity measures (including cutting public spending and reducing debt) and the privatization of public institutions has provoked strong reactions in countries which receive such loans. My main focus in my research is on exploring how the process of Financial Liberalization (FL) of the Macedonian economy affected capital flows in the form of foreign direct investments (FDI) in the private sector and how the recent Greek crisis of 2008 has impacted on this. In the case of Macedonia, the reality of FL was tested by the collapse of the Greek economy. However, this paper will highlight the main duties of the IMF and the goals of the FL process implemented in various countries.Additionally, I will undertake a rhetorical documentary analysis on the IMF reports regarding the process of FL in Macedonia since its independence until today.

Keywords: FDI, financial liberalization, Greece, IMF, Macedonia

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11 Building Information Models Utilization for Design Improvement of Infrastructure

Authors: Keisuke Fujioka, Yuta Itoh, Masaru Minagawa, Shunji Kusayanagi

Abstract:

In this study, building information models of the underground temporary structures and adjacent embedded pipes were constructed to show the importance of the information on underground pipes adjacent to the structures to enhance the productivity of execution of construction. Next, the bar chart used in actual construction process were employed to make the Gantt chart, and the critical pass analysis was carried out to show that accurate information on the arrangement of underground existing pipes can be used for the enhancement of the productivity of the construction of underground structures. In the analyzed project, significant construction delay was not caused by unforeseeable existence of underground pipes by the management ability of the construction manager. However, in many cases of construction executions in the developing countries, the existence of unforeseeable embedded pipes often causes substantial delay of construction. Design change based on uncertainty on the position information of embedded pipe can be also important risk for contractors in domestic construction. So CPM analyses were performed by a project-management-software to the situation that influence of the tasks causing construction delay was assumed more significant. Through the analyses, the efficiency of information management on underground pipes and BIM analysis in the design stage for workability improvement was indirectly confirmed.

Keywords: building-information modelling, construction information modelling, design improvement, infrastructure

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10 Effectiveness of Visual Auditory Kinesthetic Tactile Technique on Reading Level among Dyslexic Children in Helikx Open School and Learning Centre, Salem

Authors: J. Mano Ranjini

Abstract:

Each and every child is special, born with a unique talent to explore this world. The word Dyslexia is derived from the Greek language in which “dys” meaning poor or inadequate and “lexis” meaning words or language. Dyslexia describes about a different kind of mind, which is often gifted and productive, that learns the concept differently. The main aim of the study is to bring the positive outcome of the reading level by examining the effectiveness of Visual Auditory Kinesthetic Tactile technique on Reading Level among Dyslexic Children at Helikx Open School and Learning Centre. A Quasi experimental one group pretest post test design was adopted for this study. The Reading Level was assessed by using the Schonell Graded Word Reading Test. Thirty subjects were drawn by using purposive sampling technique and the intervention Visual Auditory Kinesthetic Tactile technique was implemented to the Dyslexic Children for 30 consecutive days followed by the post Reading Level assessment revealed the improvement in the mean score value of reading level by 12%. Multi-sensory (VAKT) teaching uses all learning pathways in the brain (visual, auditory, kinesthetic-tactile) in order to enhance memory and learning and the ability in uplifting emotional, physical and societal dimensions. VAKT is an effective method to improve the reading skill of the Dyslexic Children that ensures the enormous significance of learning thereby influencing the wholesome of the child’s life.

Keywords: visual auditory kinesthetic tactile technique, reading level, dyslexic children, Helikx Open School

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9 A Business-to-Business Collaboration System That Promotes Data Utilization While Encrypting Information on the Blockchain

Authors: Hiroaki Nasu, Ryota Miyamoto, Yuta Kodera, Yasuyuki Nogami

Abstract:

To promote Industry 4.0 and Society 5.0 and so on, it is important to connect and share data so that every member can trust it. Blockchain (BC) technology is currently attracting attention as the most advanced tool and has been used in the financial field and so on. However, the data collaboration using BC has not progressed sufficiently among companies on the supply chain of manufacturing industry that handle sensitive data such as product quality, manufacturing conditions, etc. There are two main reasons why data utilization is not sufficiently advanced in the industrial supply chain. The first reason is that manufacturing information is top secret and a source for companies to generate profits. It is difficult to disclose data even between companies with transactions in the supply chain. In the blockchain mechanism such as Bitcoin using PKI (Public Key Infrastructure), in order to confirm the identity of the company that has sent the data, the plaintext must be shared between the companies. Another reason is that the merits (scenarios) of collaboration data between companies are not specifically specified in the industrial supply chain. For these problems this paper proposes a Business to Business (B2B) collaboration system using homomorphic encryption and BC technique. Using the proposed system, each company on the supply chain can exchange confidential information on encrypted data and utilize the data for their own business. In addition, this paper considers a scenario focusing on quality data, which was difficult to collaborate because it is a top secret. In this scenario, we show a implementation scheme and a benefit of concrete data collaboration by proposing a comparison protocol that can grasp the change in quality while hiding the numerical value of quality data.

Keywords: business to business data collaboration, industrial supply chain, blockchain, homomorphic encryption

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8 Temporal Focus Scale: Examination of the Reliability and Validity in Japanese Adolescents and Young Adults

Authors: Yuta Chishima, Tatsuya Murakami, Michael McKay

Abstract:

Temporal focus is described as one component of an individual’s time perspective and defined as the attention individuals devote to thinking about the past, present, and future. It affects how people incorporate perceptions about past experiences, current situations, and future expectations into their attitudes, cognitions, and behavior. The 12-item Temporal Focus Scale (TFS) is comprised of three-factors (past, current and future focus). The purpose of this study was to examine the reliability and validity of TFS scores in Japanese adolescents and young adults. The TFS was translated into Japanese by a professional translator, and the original author confirmed the back translated items. Study 1 involved 979 Japanese university students aged 18-25 years old in a questionnaire-based study. The hypothesized three-factor structure (with reliability) was confirmed, although there were problems with item 10. Internal consistency estimates for scores without item 10 were over .70, and test-retest reliability was also adequate. To verify the concurrent and convergent validity, we tested the relationship between TFS scores and life satisfaction, time perspective, self-esteem, and career efficacy. Results of correlational analyses supported our hypotheses. Specifically, future focus was strongly correlated to career efficacy, while past and current focus was not. Study 2 involved 1030 Japanese junior and junior high school students aged 12-18 years old in a questionnaire-based study, and results of multigroup analyses supported the age invariance of the TFS.

Keywords: Japanese, reliability, scale, temporal focus, validity

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7 Effects of Indole on Aerobic Biodegradation of Butanoic Acid by Pseudomonas aeruginosa and Serratia marcescens

Authors: J. B. J. Njalam’mano, E. M. N. Chirwa

Abstract:

In low resource settings in Africa and other developing regions, pit latrines remain the dominant basic minimum acceptable form of sanitation. However, unpleasant smells-malodours emitted from faecal sludge in the pit latrines, which elicit disgusting or repulsive response, are one of the factors that thwart people to use latrines and instead opt for open defecation as an alternative. This provides an important but often overlooked major impediment, dissuading people from adopting and using the pit latrines hence affecting successful, effective sanitation promotion. The malodours are primarily attributed to four odorants: butanoic acid (C₄H₈O₂), dimethyl trisulphide (C₂H₆S₃), indole (C₈H₇N) and para-cresol (C₇H₈O). Several pit latrine deodorisation methods such as addition of carbonous materials, use of ventilation systems and urine separation are available, and they continue to occupy their niche, but social, economic, environmental and technological shortfalls remain. Bioremediation has been gaining popularity because it is inexpensive, simple to operate and environmentally friendly. Recently, the biodegradation of butanoic acid as individual odorant has been studied. However, to the best of our knowledge, there have been no kinetic studies of the butanoic acid in the presence of other key odorous compounds. In this study, a series of experiments were conducted to investigate the effects of indole on the removal of butanoic acid under aerobic conditions using indigenous bacteria strains, Pseudomonas aeruginosa, and Serratia marcescens isolated from faecal sludge as pure cultures as well as mixed cultures. In this purpose, butanoic acid removal was performed in a batch reactor containing the bacterial strains in mineral salt medium (MSM) amended with 3000 ppm of butanoic acid at the temperature of 30°C, under continuous stirring rate of 150 rpm and the concentration of indole was varied from 50-200 ppm. The initial pH of the solution was in the range of 6.0-7.2. Overall, there were significant differences in the bacterial growth rate and total butanoic acid removal dependent on the concentration of indole in the solution.

Keywords: biodegradation, butanoic acid, indole, pit latrine

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6 The Role of General Councils in the Supervision of the Organizational Performance of Higher Education Institutions

Authors: Rodrigo T. Lourenço, Margarida Mano

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Higher Education Institutions (HEI), and other levels of Education, face important challenges. One of the most relevant one is the ability to adapt to a society that is changing over time, whilst guarantying levels of training that do not merely react to such changes. Thus, interacting with society, particularly with surrounding communities and key stakeholders, has become an essential requirement for the sustainability of these institutions. One of the formal mechanisms implemented in European educational institutions has been the design of organizational structures that include a top governance body sharing its constitution with both internal members, students and external members. Such frame holds the core mission of involving communities in the governance of educational institutions, assuming, both strategic decision-making functions, with the approval of the institutions’ strategic plans, and a supervision function, approved by activity reports. It also plays an essential role in the life of institutions by holding the responsibility of electing its top executives. In Portugal, it has been almost a decade since the publication of RJIES, the legal framework of Higher Education, such bodies being designated by General Councils. Thus, one may highlight that there has been a better understanding of the operative process of these bodies, as well as their added value to the education system. It has also been possible to analyse the extent to which their core mission has been fulfilled and to understand its growing relevance, particularly regarding the autonomy of institutions. This article aims to contribute to this theme by presenting the results of a study on the role of these bodies in the governance of Public Portuguese HEI, with a special focus on the supervisory competence of organizational performance. Through questionnaires made to board members and interviews with chairpersons of the bodies and top managers of the institutions, it was possible to conclude that there is a high concern with the connections to the external environment. However, regarding organizational performance and the role of the Council as a supervisor of that performance, the activity of the bodies has fallen short of what would be expected. Several reasons may be identified. It is important to emphasize the importance of the profile of the external members and the relationship between the organ’s standard functioning and the election of the head of the institution.

Keywords: governance, stakeholders, supervision, performance

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5 Effect of Nanostructure on Hydrogen Embrittlement Resistance of the Severely Deformed 316LN Austenitic Steel

Authors: Frank Jaksoni Mweta, Nozomu Adachi, Yoshikazu Todaka, Hirokazu Sato, Yuta Sato, Hiromi Miura, Masakazu Kobayashi, Chihiro Watanabe, Yoshiteru Aoyagi

Abstract:

Advances in the consumption of hydrogen fuel increase demands of high strength steel pipes and storage tanks. However, high strength steels are highly sensitive to hydrogen embrittlement. Because the introduction of hydrogen into steel during the fabrication process or from the environment is unavoidable, it is essential to improve hydrogen embrittlement resistance of high strength steels through microstructural control. In the present study, the heterogeneous nanostructure with a tensile strength of about 1.8 GPa and the homogeneous nanostructure with a tensile strength of about 2.0 GPa of 316LN steels were generated after 92% heavy cold rolling and high-pressure torsion straining, respectively. The heterogeneous nanostructure is composed of twin domains, shear bands, and lamellar grains. The homogeneous nanostructure is composed of uniformly distributed ultrafine nanograins. The influence of heterogeneous and homogenous nanostructures on the hydrogen embrittlement resistance was investigated. The specimen for each nanostructure was electrochemically charged with hydrogen for 3, 6, 12, and 24 hours, respectively. Under the same hydrogen charging time, both nanostructures show almost the same concentration of the diffusible hydrogen based on the thermal desorption analysis. The tensile properties of the homogenous nanostructure were severely affected by the diffusible hydrogen. However, the diffusible hydrogen shows less impact on the tensile properties of the heterogeneous nanostructure. The difference in embrittlement behavior between the heterogeneous and homogeneous nanostructures was elucidated based on the mechanism of the cracks' growth observed in the tensile fractography. The hydrogen embrittlement was suppressed in the heterogeneous nanostructure because the twin domain became an obstacle for crack growth. The homogeneous nanostructure was not consisting an obstacle such as a twin domain; thus, the crack growth resistance was low in this nanostructure.

Keywords: diffusible hydrogen, heterogeneous nanostructure, homogeneous nanostructure, hydrogen embrittlement

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4 Associations between Sleep Problems and Disordered Eating in Japanese Adolescents: A Cross-Sectional Study

Authors: Takaharu Hirai, Yuta Mitobe, Hiromi Hirai

Abstract:

Introduction: Eating disorders (ED) are serious psychiatric disorders that affect individuals, especially adolescents. It has been suggested that nonclinical ED-like characteristics are related to sleep problems. However, studies exploring the association between potential ED and sleep disorders have primarily been conducted in Europe and the United States. We conducted a survey of Japanese adolescents to investigate this claim. Method: In this cross-sectional study, 398 school-aged adolescents, aged 12–18 years old, matched for gender ratio, responded to a self-administered questionnaire survey. We used the Eating Attitudes Test-26 (EAT-26) and the Athens Insomnia Scale (AIS) to measure potential ED and sleep problems, respectively. In this study, participants with an EAT-26 total score of 0–19 points were classified as non-ED, while those with scores of 20 points or higher were classified as potential ED. Result: Of the 398 participants, 17 (4.3%) had an EAT-26 total score of 20 or higher. Among boys, the rate was 6 of 199 participants (3%), and among girls, the rate was 11 of 182 participants (6%). There were 89 participants (22.4%) with an AIS score of 6 points or higher, of which 36 (17.6%) were boys, and 53 (27.5%) were girls. Adolescents with potential ED had significantly higher rates of daytime sleep problems than those without ED. Further, while examining the types of sleep problems, adolescents with potential ED had greater problems with a sense of well-being and physical and mental functioning during the day. In contrast, no significant associations were found between potential ED and sleep initiation, awakenings during the night, early morning awakening, total sleep duration, or overall quality of sleep. Finally, nocturnal and daytime sleep scores were significantly associated with dieting, bulimia, and oral control EAT-26 sub-scores. Discussion: While Japanese adolescents with possible ED do not experience nighttime sleep problems, they do experience problems related to well-being and mental and physical functioning, which are indicators of daytime sleep problems. This may assist with early detection of disordered eating in adolescents. The study suggested that professionals working towards adolescent mental health issues need an approach that comprehensively integrates both sleep problems and potential ED.

Keywords: adolescents, potential eating disorders, sleep problems, eating attitudes test-26

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3 Acoustic Radiation Pressure Detaches Myoblast from Culture Substrate by Assistance of Serum-Free Medium

Authors: Yuta Kurashina, Chikahiro Imashiro, Kiyoshi Ohnuma, Kenjiro Takemura

Abstract:

Research objectives and goals: To realize clinical applications of regenerative medicine, a mass cell culture is highly required. In a conventional cell culture, trypsinization was employed for cell detachment. However, trypsinization causes proliferation decrease due to injury of cell membrane. In order to detach cells using an enzyme-free method, therefore, this study proposes a novel cell detachment method capable of detaching adherent cells using acoustic radiation pressure exposed to the dish by the assistance of serum-free medium with ITS liquid medium supplement. Methods used In order to generate acoustic radiation pressure, a piezoelectric ceramic plate was glued on a glass plate to configure an ultrasonic transducer. The glass plate and a chamber wall compose a chamber in which a culture dish is placed in glycerol. Glycerol transmits acoustic radiation pressure to adhered cells on the culture dish. To excite a resonance vibration of transducer, AC signal with 29-31 kHz (swept) and 150, 300, and 450 V was input to the transducer for 5 min. As a pretreatment to reduce cell adhesivity, serum-free medium with ITS liquid medium supplement was spread to the culture dish before exposed to acoustic radiation pressure. To evaluate the proposed cell detachment method, C2C12 myoblast cells (8.0 × 104 cells) were cultured on a ø35 culture dish for 48 hr, and then the medium was replaced with the serum-free medium with ITS liquid medium supplement for 24 hr. We replaced the medium with phosphate buffered saline and incubated cells for 10 min. After that, cells were exposed to the acoustic radiation pressure for 5 min. We also collected cells by using trypsinization as control. Cells collected by the proposed method and trypsinization were respectively reseeded in ø60 culture dishes and cultured for 24 hr. Then, the number of proliferated cells was counted. Results achieved: By a phase contrast microscope imaging, shrink of lamellipodia was observed before exposed to acoustic radiation pressure, and no cells remained on the culture dish after the exposed of acoustic radiation pressure. This result suggests that serum-free medium with ITS liquid inhibits adhesivity of cells and acoustic radiation pressure detaches cells from the dish. Moreover, the number of proliferated cells 24 hr after collected by the proposed method with 150 and 300 V is the same or more than that by trypsinization, i.e., cells were proliferated 15% higher with the proposed method using acoustic radiation pressure than with the traditional cell collecting method of trypsinization. These results proved that cells were able to be collected by using the appropriate exposure of acoustic radiation pressure. Conclusions: This study proposed a cell detachment method using acoustic radiation pressure by the assistance of serum-free medium. The proposed method provides an enzyme-free cell detachment method so that it may be used in future clinical applications instead of trypsinization.

Keywords: acoustic radiation pressure, cell detachment, enzyme free, ultrasonic transducer

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2 Single Cell Sorter Driven by Resonance Vibration of Cell Culture Substrate

Authors: Misa Nakao, Yuta Kurashina, Chikahiro Imashiro, Kenjiro Takemura

Abstract:

The Research Goal: With the growing demand for regenerative medicine, an effective mass cell culture process is required. In a repetitive subculture process for proliferating cells, preparing single cell suspension which does not contain any cell aggregates is highly required because cell aggregates often raise various undesirable phenomena, e.g., apoptosis and decrease of cell proliferation. Since cell aggregates often occur in cell suspension during conventional subculture processes, this study proposes a single cell sorter driven by a resonance vibration of a cell culture substrate. The Method and the Result: The single cell sorter is simply composed of a cell culture substrate and a glass pipe vertically placed against the cell culture substrate with a certain gap corresponding to a cell diameter. The cell culture substrate is made of biocompatible stainless steel with a piezoelectric ceramic disk glued to the bottom side. Applying AC voltage to the piezoelectric ceramic disk, an out-of-plane resonance vibration with a single nodal circle of the cell culture substrate can be excited at 5.5 kHz. By doing so, acoustic radiation force is emitted, and then cell suspension containing only single cells is pumped into the pipe and collected. This single cell sorter is effective to collect single cells selectively in spite of its quite simple structure. We collected C2C12 myoblast cell suspension by the single cell sorter with the vibration amplitude of 12 µmp-p and evaluated the ratio of single cells in number against the entire cells in the suspension. Additionally, we cultured the collected cells for 72 hrs and measured the number of cells after the cultivation in order to evaluate their proliferation. As a control sample, we also collected cell suspension by conventional pipetting, and evaluated the ratio of single cells and the number of cells after the 72-hour cultivation. The ratio of single cells in the cell suspension collected by the single cell sorter was 98.2%. This ratio was 9.6% higher than that collected by conventional pipetting (statistically significant). Moreover, the number of cells cultured for 72 hrs after the collection by the single cell sorter yielded statistically more cells than that collected by pipetting, resulting in a 13.6% increase in proliferated cells. These results suggest that the cell suspension collected by the single cell sorter driven by the resonance vibration hardly contains cell aggregates whose diameter is larger than the gap between the cell culture substrate and the pipe. Consequently, the cell suspension collected by the single cell sorter maintains high cell proliferation. Conclusions: In this study, we developed a single cell sorter capable of sorting and pumping single cells by a resonance vibration of a cell culture substrate. The experimental results show the single cell sorter collects single cell suspension which hardly contains cell aggregates. Furthermore, the collected cells show higher proliferation than that of cells collected by conventional pipetting. This means the resonance vibration of the cell culture substrate can benefit us with the increase in efficiency of mass cell culture process for clinical applications.

Keywords: acoustic radiation force, cell proliferation, regenerative medicine, resonance vibration, single cell sorter

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1 Membrane Permeability of Middle Molecules: A Computational Chemistry Approach

Authors: Sundaram Arulmozhiraja, Kanade Shimizu, Yuta Yamamoto, Satoshi Ichikawa, Maenaka Katsumi, Hiroaki Tokiwa

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Drug discovery is shifting from small molecule based drugs targeting local active site to middle molecules (MM) targeting large, flat, and groove-shaped binding sites, for example, protein-protein interface because at least half of all targets assumed to be involved in human disease have been classified as “difficult to drug” with traditional small molecules. Hence, MMs such as peptides, natural products, glycans, nucleic acids with various high potent bioactivities become important targets for drug discovery programs in the recent years as they could be used for ‘undruggable” intracellular targets. Cell membrane permeability is one of the key properties of pharmacodynamically active MM drug compounds and so evaluating this property for the potential MMs is crucial. Computational prediction for cell membrane permeability of molecules is very challenging; however, recent advancement in the molecular dynamics simulations help to solve this issue partially. It is expected that MMs with high membrane permeability will enable drug discovery research to expand its borders towards intracellular targets. Further to understand the chemistry behind the permeability of MMs, it is necessary to investigate their conformational changes during the permeation through membrane and for that their interactions with the membrane field should be studied reliably because these interactions involve various non-bonding interactions such as hydrogen bonding, -stacking, charge-transfer, polarization dispersion, and non-classical weak hydrogen bonding. Therefore, parameters-based classical mechanics calculations are hardly sufficient to investigate these interactions rather, quantum mechanical (QM) calculations are essential. Fragment molecular orbital (FMO) method could be used for such purpose as it performs ab initio QM calculations by dividing the system into fragments. The present work is aimed to study the cell permeability of middle molecules using molecular dynamics simulations and FMO-QM calculations. For this purpose, a natural compound syringolin and its analogues were considered in this study. Molecular simulations were performed using NAMD and Gromacs programs with CHARMM force field. FMO calculations were performed using the PAICS program at the correlated Resolution-of-Identity second-order Moller Plesset (RI-MP2) level with the cc-pVDZ basis set. The simulations clearly show that while syringolin could not permeate the membrane, its selected analogues go through the medium in nano second scale. These correlates well with the existing experimental evidences that these syringolin analogues are membrane-permeable compounds. Further analyses indicate that intramolecular -stacking interactions in the syringolin analogues influenced their permeability positively. These intramolecular interactions reduce the polarity of these analogues so that they could permeate the lipophilic cell membrane. Conclusively, the cell membrane permeability of various middle molecules with potent bioactivities is efficiently studied using molecular dynamics simulations. Insight of this behavior is thoroughly investigated using FMO-QM calculations. Results obtained in the present study indicate that non-bonding intramolecular interactions such as hydrogen-bonding and -stacking along with the conformational flexibility of MMs are essential for amicable membrane permeation. These results are interesting and are nice example for this theoretical calculation approach that could be used to study the permeability of other middle molecules. This work was supported by Japan Agency for Medical Research and Development (AMED) under Grant Number 18ae0101047.

Keywords: fragment molecular orbital theory, membrane permeability, middle molecules, molecular dynamics simulation

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