Search results for: S. Morita

Authors: Nompumelelo P. Mathebula, Roger A. Sheldon, Daniel P. Pienaar, Moira L. Bode

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The preparation of enantiopure Morita-Baylis-Hillman (MBH) adducts remains a challenge in organic chemistry. MBH adducts are highly functionalised compounds which act as key intermediates in the preparation of compounds of medicinal importance. MBH adducts are prepared in racemic form by reacting various aldehydes and activated alkenes in the presence of DABCO. Enantiopure MBH adducts can be obtained by employing Enzymatic kinetic resolution (EKR). This technique has been successfully demonstrated in our group, amongst others, using lipases in either hydrolysis or transesterification reactions. As these methods only allow 50% of each enantiomer to be obtained, our interest grew in exploring other enzymatic methods for the synthesis of enantiopure MBH adducts where, theoretically, 100% of the desired enantiomer could be obtained.Dehydrogenase enzymes can be employed on prochiral substrates to obtain optically pure compounds by reducing carbon-carbon double bonds or carbonyl groups of ketones. Ketoreductases have been used historically to obtain enantiopure secondary alcohols on an industrial scale. Ketoreductases are NAD(P)H-dependent enzymes and thus require nicotinamide as a cofactor. This project focuses on employing ketoreductase enzymes to selectively reduce ketones derived from Morita-Baylis-Hillman (MBH) adducts in order to obtain these adducts in enantiopure form.Results obtained from this study will be reported. Good enantioselectivity was observed using a range of different ketoreductases, however, reactions were complicated by the formation of an unexpected by-product, which was characterised employing single crystal x-ray crystallography techniques. Methods to minimise by-product formation are currently being investigated.

Keywords: ketoreductase, morita-baylis-hillman, selective reduction, x-ray crystallography

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Authors: Satoshi Furukawa, Satomu Morita, Katsuji Nishi, Masahito Hitosugi

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A 75-year-old woman took thyroidectomy forty years previously. Enlarged masses were seen at autopsy just above and below the left clavicle. We proved the diagnosis of papillary thyroid cancer (PTC) and lung metastasis by histological examinations. The prognosis of PTC is excellent; the 10-year survival rate ranges between 85 and 99%. Lung metastases may be found in 10% of the patients with PTC. We report an unusual case of recurrence of PTC with metastasis to the lung.

Keywords: papillary thyroid cancer, lung metastasis, autopsy, histopathological findings

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Authors: Valencia P. L. Rodrigo, Morita A. Adelina, Vargas V. Martin

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This proposal consists of an analysis of macro concepts involved within an organization growth using educational technologies, which will relate each concept, in a mathematical way with a vision of harmonic work. Working collaboratively, competitively and cooperatively so that this growth is harmonious and homogenous, coining a new term, Harmonic Work. The Harmonic Work ensures that the organization grows in all business directions, allowing managers to project a much more accurate growth, making clear the contribution of each department, resulting in an algorithm that analyzes each of the variables both endogenous and exogenous, establishing different performance indicators in its process of growth.

Keywords: business projection, collaboration, competitiveness, educational technology, harmonious growth

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Authors: Yuichi Morita, Yoshiteru Nakamori

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In car dealer business of Japan, a sale professional is a key factor of company’s success. We hypothesize that, if a corporation knows what is the sales professionals’ trait of its corporation’s business field, it will be easier for a corporation to secure and nurture sales persons effectively. The lean human resources management will ensure business success and good performance of corporations, especially small and medium ones. The goal of the paper is to determine the traits of sales professionals for small-and medium-size car dealers, using chi-square test and the variable rough set model. As a result, the results illustrate that experience of job change, learning ability and product knowledge are important, and an academic background, building a career with internal transfer, experience of the leader and self-development are not important to be a sale professional. Also, we illustrate sales professionals’ traits are persistence, humility, improvisation and passion at business.

Keywords: traits of sales professionals, variable precision rough sets theory, sales professional, sales professionals

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Authors: Satoshi Furukawa, Satomu Morita, Lisa Wingenfeld, Katsuji Nishi, Masahito Hitosugi

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We studied the expression of hypoxia-related antigens (e.g., cold-inducible antigens and apoptotic antigens) in the myocardium and the cerebellumthat were obtained from individuals after death by carbon monoxide or hypothermia. The immunohistochemistry results revealed that expression of cold-inducible RNA binding protein (CIRBP) and RNA-binding protein 3 (RBM3) may be associated with hpyothermic and the hypoxic conditions. The expression of CIRBP and RBM3 in the myocardium was different from their expression in the cerebellum, especially in the Purkinje cells. The results indicate that agonal duration influences antigen expression. In the hypothermic condition, the myocardium uses more ATP since the force of the excitation-contraction coupling of the myocardium increases by more than 400% when the experimental temperature is reduced from 35°C to 25°C. The results obtained in this study indicate that physicians should pay attention to the myocardium when cooling the patient’s body to protect the brain.

Keywords: carbon monoxide death, cerebellum, CIRBP, hypothermic death, myocardium, RBM3

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Authors: Luis Rodrigo Valencia Pérez, Juan Manuel Peña Aguilar, Adelina Morita Alexander, Alberto Lamadrid Alvarez, Héctor Fernando Valencia Pérez

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Financial education is among the areas of opportunity in the Spanish-speaking from an early age to high school, through mobile devices such as cell phones and tablets using ludic and fun applications like interactive games, children can learn money management and investment through time, thereby fostering the habit of saving and/or sound management of cash and family business resources, having interaction with an uncontrolled environment such as the involvement of other players in the external decisions of the environment in which the game is play. The application proposed in Phase 1 (design and development) was designed in multi-user environments, under methodologies of hybrid programming for any platform on the market and designed under CMMI standards that allow for quality production over time, following up on these improvements counting with continuous user feedback and usage statistics.

Keywords: mobile educational games, ludic games, children, multiuser, design and software development

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Authors: Satoshi Furukawa, Satomu Morita, Katsuji Nishi, Masahito Hitosugi

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Coronary artery dissection leading to acute myocardial infarction after blunt chest trauma is extremely rare. A 67-year-old woman suffered blunt chest trauma following a traffic accident. The electrocardiogram revealed acute posterior ST-segment elevation and myocardial infarction and coronary angiography demonstrated acute right coronary artery dissection. Following the death of the victim an autopsy was performed after cardiopulmonary support had been carried out. In this case report, we describe the case of a woman with blunt chest trauma, who developed an acute myocardial infarction secondary to right coronary artery dissection. Although there was additional the blunt chest trauma due to chest compression, we confirmed the injury at autopsy and by histological findings.

Keywords: blunt chest trauma, right coronary artery dissection, coronary angiography, autopsy, histological examination

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Authors: M. Sumida, S. Morita

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In this research, the effect of unsteady flows acting on road vehicles was experimentally investigated, using an advanced and recently introduced wind tunnel. The aims of this study were to extract the characteristics of fluid forces acting on road vehicles under unsteady wind conditions and obtain new information on drag forces in a practical on-road test. We applied pulsating wind as a representative example of the atmospheric fluctuations that vehicles encounter on the road. That is, we considered the case where the vehicles are moving at constant speed in the air, with large wind oscillations. The experimental tests were performed on the Ahmed-type test model, which is a simplified vehicle model. This model was chosen because of its simplicity and the data accumulated under steady wind conditions. The experiments were carried out with a time-averaged Reynolds number of Re = 4.16x10⁵ and a pulsation period of T = 1.5 s, with amplitude of η = 0.235. Unsteady fluid forces of drag and lift were obtained utilizing a multi-component load cell. It was observed that the unsteady aerodynamic forces differ significantly from those under steady wind conditions. They exhibit a phase shift and an enhanced response to the wind oscillations. Furthermore, their behavior depends on the slant angle of the rear shape of the model.

Keywords: Ahmed body, automotive aerodynamics, unsteady wind, wind tunnel test

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Authors: Regina Maria M. Castilho, Isabela M. Morita, Ana Carolina T. Malavolta, Maximiliano K. Pagliarini

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An essential factor for flower production is solar radiation, which is part of plant vital processes. As excess as shortage of light can harm the development of the culture leading to loss in product quality, Unfeasible or decreasing their commercial value. The objective of this research was to evaluate different light levels and their influence on Amaryllis (Hippeastrum X hybridum Hort.) development and flowering. The experiment was conducted at UNESP, São Paulo State, Brazil from August to October 2014. The bulbs were placed in black vases of 1.2 L filled with commercial substrate and divided into 4 different lighting environments (treatments): T1–greenhouse, T2–greenhouse with shade cloth (50%), T3–low lights indoor (until 500 lx) and T4–medium lights indoor (between 500–1000 lx). The used design was completely randomized with ten repetitions and three vessels (bulbs), totalling 30 vessels (bulbs) per treatment. The evaluated characteristics were: Chlorophyll content, number of leaves, length of leaf, number of simultaneous rods, rod length, rod diameter, number of flowers, flowers diameter, beginning of flowering and flowering duration. The results showed that in greenhouse provided Amaryllis better quality plants.

Keywords: açucena, bulbs, light, ornamental plants

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Authors: Satoshi Furukawa, Satomu Morita, Katsuji Nishi, Masahito Hitosugi

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We experience the autopsy cases that the deceased was alive in emergency room on arrival. Bleeding is the leading cause of preventable death after injury. This retrospective study aimed to characterize opportunities for performance improvement identified in patients who died from traffic trauma and were considered by the quality improvement of education system. The Japan Advanced Trauma Evaluation and Care (JATEC) education program was introduced in 2002. We focused the abdominal traffic trauma injury. An autopsy-based cross-sectional study conducted. A purposive sampling technique was applied to select the study sample of 41 post-mortems of road traffic accident between April 1999 and March 2014 subjected to medico-legal autopsy at the department of Forensic Medicine, Shiga University of Medical Science. 16 patients (39.0%) were abdominal trauma injury. The mean period of survival after meet with accident was 13.5 hours, compared abdominal trauma death was 27.4 hours longer. In road traffic accidents, the most injured abdominal organs were liver followed by mesentery. We thought delayed treatment was associated with immediate diagnostic imaging, and so expected to expand trauma management examination.

Keywords: abdominal traffic trauma, preventable death, autopsy, emergency medicine

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Authors: Nivon P. Alejandra, Valencia P. L. Rodrigo, Vivanco V. Martin, Morita A. Adelina

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Technological innovation is fundamental to reach and maintain the levels of competitiveness of agricultural producers, the detection of actors, their activities, resources and capacities of an innovation system is needed for the development of technological packages that adapt to each type of crops, local circumstances and characteristics of the producer. The growing development of the viticulture and wine sector in Mexico prospects an increase in its national market participation for 2020, this is the reason to consider it a fertile field for the technological packages adoption that promote Precision Agriculture (PA) in a harmonic and sustainable development. A viability inspection of technological packages adoption by viticulture and wine sector is made following the methodology proposed by SAGARPA in 2015 and the World Bank in 2008: the history, actors, strengths and opportunities are analyzed in this particular agroindustrial sector, also its technological innovation system is inspected in order to improve technological capacities and innovation networks taking into account local and regional resources. PA and technological packages adoption can help improving the conditions and quality of the grape for winemaking: increasing the wine's storage potential and its nutraceutical nature. The assertive diagnosis in vineyard opportunity areas will help the management of the crop by applying natural treatments at the right time in the right place.

Keywords: technological packages, precision farming, sustainable development, innovation

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Authors: Maximiliano K. Pagliarini, Ana Carolina T. Malavolta, Isabela M. Morita, Regina Maria M. Castilho

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Among the ornamental plants, the Amaryllis (Hippeastrum X hybridum Hort.) is one of the most cultivated plants in Brazil because of their large and showy flowers. Thus, the consumer market wants better quality plants or to flourish more in less time. One of the devices that can make such improvements or accelerate the flowering process is the use of growth regulators. The objective of this research was to evaluate the use of different Stimulate® growth regulator doses and its constituents separately in the development and flowering of Hippeastrum X hybridum Hort. Cv Orange Souvereign. The experiment was conducted in a Pad & Fan greenhouse at UNESP, São Paulo State, Brazil from August to October 2014. The bulbs were placed in black vases of 1.2 L filled with commercial substrate and divided into 9 treatments: T1 – 10 mL L-1 of Stimulate®, T2 – 5 mL L-1 of Stimulate®, T3 – 0.5 mg L-1 of gibberellic acid (GA), T4 – 0.25 mg L-1 of GA, T5 – 0.45 mg L-1 of kinetin, T6 – 0.9 mg L-1 of kinetin, T7 – 0.5 mg L-1 of indolbutiric acid (IBA), T8 – 0.25 mg L-1 of IBA and T9 – distilled water (control). All treatments were diluted in water. The used design was completely randomized with six repetitions and two vessels, totalling 12 vessels per treatment. The evaluated characteristics were: number of leaves, length of leaf, number of rods, maximum height of rods, maximum diameter of rods, maximum number of flowers, beginning of flowering, flowering duration, and weight of bulbs. The results showed that the Stimulate® was not efficient in the conducted experiment conditions. However, the best treatment was 0.5 mg L-1 of IBA.

Keywords: bulbs, gibberellic acid, indolbutiric acid, kinetin, ornamental plants

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Authors: Toshifumi Hiramatsu, Satoshi Morita, Manuel Pencelli, Marta Niccolini, Matteo Ragaglia, Alfredo Argiolas

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Automation technologies for agriculture field are needed to promote labor-saving. One of the most relevant problems in automated agriculture is represented by controlling the robot along a predetermined path in presence of rough terrain or incline ground. Unfortunately, disturbances originating from interaction with the ground, such as slipping, make it quite difficult to achieve the required accuracy. In general, it is required to move within 5-10 cm accuracy with respect to the predetermined path. Moreover, lateral velocity caused by gravity on the incline field also affects slipping. In this paper, a path-tracking controller for tracked mobile robots moving on rough terrains of incline field such as vineyard is presented. The controller is composed of a disturbance observer and an adaptive controller based on the kinematic model of the robot. The disturbance observer measures the difference between the measured and the reference yaw rate and linear velocity in order to estimate slip. Then, the adaptive controller adapts “virtual” parameter of the kinematics model: Instantaneous Centers of Rotation (ICRs). Finally, target angular velocity reference is computed according to the adapted parameter. This solution allows estimating the effects of slip without making the model too complex. Finally, the effectiveness of the proposed solution is tested in a simulation environment.

Keywords: the agricultural robot, autonomous control, path-tracking control, tracked mobile robot

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Authors: Sergio Mauricio Pérez López, Luis Rodrigo Valencia Pérez, Juan Manuel Peña Aguilar, Adelina Morita Alexander

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The culture of continuous improvement in organizations is very important as it represents a source of competitive advantage. This article discusses the transfer of knowledge between companies which formed cross-functional teams and used a dynamic model for knowledge creation as a framework. In addition, the article discusses the structure of cognitive assets in companies and the concept of "stickiness" (which is defined as an obstacle to the transfer of knowledge). The purpose of this analysis is to show that an improvement in the attitude of individual members of an organization creates opportunities, and that an exchange of information and knowledge leads to generating continuous improvements in the company as a whole. This article also discusses the importance of creating the proper conditions for sharing tacit knowledge. By narrowing gaps between people, mutual trust can be created and thus contribute to an increase in sharing. The concept of adapting knowledge to new environments will be highlighted, as it is essential for companies to translate and modify information so that such information can fit the context of receiving organizations. Adaptation will ensure that the transfer process is carried out smoothly by preventing "stickiness". When developing the transfer process on cross-functional teams (as opposed to working groups), the team acquires the flexibility and responsiveness necessary to meet objectives. These types of cross-functional teams also generate synergy due to the array of different work backgrounds of their individuals. When synergy is established, a culture of continuous improvement is created.

Keywords: knowledge transfer, continuous improvement, teamwork, cognitive assets

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Authors: Gaya Bin Noon, Thoko Hanjahanja-Phiri, Laura Xavier Fadrique, Plinio Pelegrini Morita, Hélène Vaillancourt, Jennifer Teague, Tania Donovska

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Active Assisted Living (AAL) refers to systems designed to improve the quality of life, aid in independence, and create healthier lifestyles for care recipients. As the population ages, there is a pressing need for non-intrusive, continuous, adaptable, and reliable health monitoring tools to support aging in place. AAL has great potential to support these efforts with the wide variety of solutions currently available, but insufficient efforts have been made to address concerns arising from the integration of AAL into care. The purpose of this research was to (1) explore the integration of AAL technologies and data into the clinical pathway, and (2) map data access and governance for AAL technology in order to develop standards for use by policy-makers, technology manufacturers, and developers of smart communities for seniors. This was done through four successive research phases: (1) literature search to explore existing work in this area and identify lessons learned; (2) modeling of the continuum of care; (3) adapting a framework for data governance into the AAL context; and (4) interviews with stakeholders to explore the applicability of previous work. Opportunities for standards found in these research phases included a need for greater consistency in language and technology requirements, better role definition regarding who can access and who is responsible for taking action based on the gathered data, and understanding of the privacy-utility tradeoff inherent in using AAL technologies in care settings.

Keywords: active assisted living, aging in place, internet of things, standards

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Authors: Houda Mnif Sellami, Toshi Umehara, Yuriko Yamazaki, Reie Matoba, Anna Sakashita, Yoshimi Abe, Hiroyuki Otake, Satoko Morita, Yoshitaka Akiyama, Chieko Morisawa, Eiji Omura, Masako Yazawa, Yoshie Koike, Mitsugu Tokunaga, Seiki Wada, Shinya Minagawa, Masafumi Matsuda

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Childhood obesity has been, for decades, a very serious public health problem worldwide. Some Asian countries have already reached alarming rates, as lifestyle changed dramatically in this part of the world. In many concerned countries, strategies including educational, promotional and awareness-raising activities have been established to combat obesity within kids. Objective: To estimate the obesity and also the underweight trends of Japanese kids from 5 to 17 years, by single year of age and by gender, over the last decade. Methods We used the data from the cross-sectional annual Nationwide surveys (National Nutrition Survey, Japan, Ministry of education, culture, sports, science and technology) conducted from 2007 to 2016. We compared trajectories of obesity prevalence, with the data on sex and age groups. We also analyzed energy and macronutrients intakes of Japanese kids using Ministry of Health, Labor and Welfare-Japan annual data, from 2007 to 2014. Results: From 2007 to 2016, Boys obesity was higher than Girls obesity for the over 6 YO participants. Both Boys and Girls obesity trends had 2 peaks of prevalence at (11-13 YO) and then at (15-16 YO). From 2007 to 2012, Kids obesity decreased considerably in both sex and all year of age; then obesity decline was more modest till 2016.On the other side, Kids underweight prevalence increased in both sexes. The macronutrients analyze couldn’t show an evident association between obesity trends and foods intake. Conclusion: Japanese kids’ obesity has been decreased since 2007, in opposition to some other countries reports. We didn’t find an observed association with food intake using Health Ministry data; we need further investigation to estimate energy intake, lifestyle and physical activity by year of age to know whether there is any possible relation.

Keywords: childhood, Japan, obesity, underweight

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Authors: Yan Lu, Song Hong, Janakiraman Udaiyappan, Aarti Nagayach, Quoc-Viet A. Duong, Masao Morita, Shun Saito, Yuichi Kobayashi, Yuhai, Zhao, Hongying Peng, Nicholas B. Pham, Walter J Lukiw, Christopher A. Vuong, Nicolas G. Bazan

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Aims: Neurodegeneration and behavior dysfunction occurs in patients with Alzheimer's Disease (AD), and as the disease progresses many patients develop cognitive impairment. 5XFAD mouse model of AD is widely used to study AD pathogenesis and treatment. This study aimed to investigate the effect of maresin like 1 (MaR-L1) treatment in AD pathology using 5XFAD mice. Methods: We tested 12-month-old male 5XFAD mice and wild type control mice treated with MaR-L1 in a battery of behavioral tasks. We performed open field test, beam walking test, clasping test, inverted grid test, acetone test, marble burring test, elevated plus maze test, cross maze test and novel object recognition test. We also studied neuronal loss, amyloid β burden, and inflammation in the brains of 5XFAD mice using immunohistology and Western blotting. Results: MaR-L1 treatment to the 5XFAD mice showed improved cognitive function of 5XFAD mice. MaR-L1 showed decreased anxiety behavior in open field test and marble burring test, increased muscular strength in the beam walking test, clasping test and inverted grid test. Cognitive function was improved in MaR-L1 treated 5XFAD mice in the novel object recognition test. MaR-L1 prevented neuronal loss and aberrant inflammation. Conclusion: Our finding suggests that behavioral abnormalities were normalized by the administration of MaR-L1 and the neuroprotective role of MaR-L1 in the AD. It also indicates that MaR-L1 treatment is able to prevent and or ameliorate neuronal loss and aberrant inflammation. Further experiments to validate the results are warranted using other AD models in the future.

Keywords: Alzheimer's disease, motor and cognitive behavior, 5XFAD mice, Maresin Like 1, microglial cell, astrocyte, neurodegeneration, inflammation, resolution of inflammation

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Authors: Yui Okuno, Mariko Era, Takayoshi Kawahara, Takahide Kanyama, Hiroshi Morita

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Introduction: Fungus and mite are known as allergens that cause an allergic disease for example asthma bronchiale and allergic rhinitis. Cladosporium cladosporioides is one of the most often detected fungi in the indoor environment and causes pollution and deterioration. Dermatophagoides farinae is major mite allergens indoors. Therefore, the creation of antifungal agents with high safety and the antifungal effect is required. Fatty acid salts are known that have antibacterial activities. This report describes the effects of fatty acid salts against Cladosporium cladosporioides NBRC 30314 and Dermatophagoides farinae. Methods: Potassium salts of 9 fatty acids (C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C18:1, C18:2, C18:3) were prepared by mixing the fatty acid with the appropriate amount of KOH solution to a concentration of 175 mM and pH 10.5. The antifungal method, the spore suspension (3.0×104 spores/mL) was mixed with a sample of fatty acid potassium (final concentration of 175 mM). Samples were counted at 0, 10, 60, 180 min by plating (100 µL) on PDA. Fungal colonies were counted after incubation for 3 days at 30 °C. The MIC (minimum inhibitory concentration) against the fungi was determined by the two-fold dilution method. Each fatty acid salts were inoculated separately with 400 µL of C. cladosporioides at 3.0 × 104 spores/mL. The mixtures were incubated at the respective temperature for each organism for 10 min. The tubes were then contacted with the fungi incubated at 30 °C for 7 days and examined for growth of spores on PDA. The acaricidal method, twenty D. farinae adult females were used and each adult was covered completely with 2 µL fatty acid potassium for 1 min. The adults were then dried with filter paper. The filter paper was folded and fixed by two clips and kept at 25 °C and 64 % RH. Mortalities were determained 48 h after treatment under the microscope. D. farina was considered to be dead if appendages did not move when prodded with a pin. Results and Conclusions: The results show that C8K, C10K, C12K, C14K was effective to decrease survival rate (4 log unit) of the fatty acids potassium incubated time for 10 min against C. cladosporioides. C18:3K was effective to decrease 4 log unit of the fatty acids potassium incubated time for 60 min. Especially, C12K was the highest antifungal activity and the MIC of C12K was 0.7 mM. On the other hand, the fatty acids potassium showed no acaricidal effects against D. farinae. The activity of D. farinae was not adversely affected after 48 hours. These results indicate that C12K has high antifungal activity against C. cladosporioides and suggest the fatty acid potassium will be used as an antifungal agent.

Keywords: fatty acid salts, antifungal effects, acaricidal effects, Cladosporium cladosporioides, Dermatophagoides farinae

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Authors: L. R. Valencia Pérez, A. Morita Alexander, Peña A. Juan Manuel, A. Lamadrid Álvarez

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This research shows the reflection and identification of explanatory variables and their relationships between different variables that are involved with educational technology, all of them encompassed in macro-concepts which are: cognitive inequality, economy, food and language; These will give the guideline to have a more detailed knowledge of educational systems, the communication and equipment, the physical space and the teachers; All of them interacting with each other give rise to what is called educational technology management. These elements contribute to have a very specific knowledge of the equipment of communications, networks and computer equipment, systems and content repositories. This is intended to establish the importance of knowing a global environment in the transfer of knowledge in poor countries, so that it does not diminish the capacity to be authentic and preserve their cultures, their languages or dialects, their hierarchies and real needs; In short, to respect the customs of different towns, villages or cities that are intended to be reached through the use of internationally agreed professional educational technologies. The methodology used in this research is the analytical - descriptive, which allows to explain each of the variables, which in our opinion must be taken into account, in order to achieve an optimal incorporation of the educational technology in a model that gives results in a medium term. The idea is that in an encompassing way the concepts will be integrated to others with greater coverage until reaching macro concepts that are of national coverage in the countries and that are elements of conciliation in the different federal and international reforms. At the center of the model is the educational technology which is directly related to the concepts that are contained in factors such as the educational system, communication and equipment, spaces and teachers, which are globally immersed in macro concepts Cognitive inequality, economics, food and language. One of the major contributions of this article is to leave this idea under an algorithm that allows to be as unbiased as possible when evaluating this indicator, since other indicators that are to be taken from international preference entities like the OECD in the area of education systems studied, so that they are not influenced by particular political or interest pressures. This work opens the way for a relationship between involved entities, both conceptual, procedural and human activity, to clearly identify the convergence of their impact on the problem of education and how the relationship can contribute to an improvement, but also shows possibilities of being able to reach a comprehensive education reform for all.

Keywords: relationships macro-concepts, cognitive inequality, economics, alimentation and language

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Authors: Manami Masuda, Mariko Era, Takayoshi Kawahara, Takahide Kanyama, Hiroshi Morita

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Objectives: Fatty acid salts are a type of anionic surfactant and are produced from fatty acids and alkali. Moreover, fatty acid salts are known to have potent antibacterial activities. Acanthamoeba is ubiquitously distributed in the environment including sea water, fresh water, soil and even from the air. Although generally free-living, Acanthamoeba can be an opportunistic pathogen, which could cause a potentially blinding corneal infection known as Acanthamoeba keratitis. So, in this study, we evaluated the anti-amoeba activity of fatty acid salts and fatty acids to Acanthamoeba castellanii ATCC 30010. Materials and Methods: The antibacterial activity of 9 fatty acid salts (potassium butyrate (C4K), caproate (C6K), caprylate (C8K), caprate (C10K), laurate (C12K), myristate (C14K), oleate (C18:1K), linoleate (C18:2K), linolenate (C18:3K)) tested on cells of Acanthamoeba castellanii ATCC 30010. Fatty acid salts (concentration of 175 mM and pH 10.5) were prepared by mixing the fatty acid with the appropriate amount of KOH. The amoeba suspension mixed with KOH with a pH adjusted solution was used as the control. Fatty acids (concentration of 175 mM) were prepared by mixing the fatty acid with Tween 80 (20 %). The amoeba suspension mixed with Tween 80 (20 %) was used as the control. The anti-amoeba method, the amoeba suspension (3.0 × 104 cells/ml trophozoites) was mixed with the sample of fatty acid potassium (final concentration of 175 mM). Samples were incubated at 30°C, for 10 min, 60 min, and 180 min and then the viability of A. castellanii was evaluated using plankton counting chamber and trypan blue stainings. The minimum inhibitory concentration (MIC) against Acanthamoeba was determined using the two-fold dilution method. The MIC was defined as the minimal anti-amoeba concentration that inhibited visible amoeba growth following incubation (180 min). Results: C8K, C10K, and C12K were the anti-amoeba effect of 4 log-unit (99.99 % growth suppression of A. castellanii) incubated time for 180 min against A. castellanii at 175mM. After the amoeba, the suspension was mixed with C10K or C12K, destroying the cell membrane had been observed. Whereas, the pH adjusted control solution did not exhibit any effect even after 180 min of incubation with A. castellanii. Moreover, C6, C8, and C18:3 were the anti-amoeba effect of 4 log-unit incubated time for 60 min. C4 and C18:2 exhibited a 4-log reduction after 180 min incubation. Furthermore, the minimum inhibitory concentration (MIC) was determined. The MIC of C10K, C12K and C4 were 2.7 mM. These results indicate that C10K, C12K and C4 have high anti-amoeba activity against A. castellanii and suggest C10K, C12K and C4 have great potential for antimi-amoeba agents.

Keywords: Fatty acid salts, anti-amoeba activities, Acanthamoeba, fatty acids

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Authors: Yumeho Obata, Mariko Era, Takayoshi Kawahara, Takahide Kanyama, Hiroshi Morita

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Dermatophagoides farinae is major mite allergens in indoors. D. farinae is often swarm over powder products (e.g. wheat flour), because it feeds on starch or protein that are included in them. Eating powder products which are mixed D.farinae causes various allergic symptoms. Therefore, the creation of food additive agents with high safety and control of mite effect is required. Fatty acid salts and spices are known that have pesticidal activities. This study describes the effects of fatty acid salts and spices against Dermatophagoides farinae. Materials and Methods: Potassium salts of 9 fatty acids (C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C18:1, C18:2, C18:3) were prepared by mixing the fatty acid with the appropriate amount of KOH solution to a concentration of 175 mM and pH 10.5. C12Cu and C12Zn were selected as other fatty acid salts. Cayenne pepper, habanero, Japanese pepper, mustard, jalapeno pepper, curry aroma and cinnamon were selected as spices. D. farina, have been cultured in laboratory. To rear the mites, double-soled dishes containing of sterilized food were put on the big plastic container (30.0 × 20.0 × 20.0cm) which had 100% ammonium nitrate solution in the bottom. Plastic container was placed on incubator at 25 °C and 64 % relative humidity (RH) under dark condition. Sterilized food composed of dried bonito flakes and dried yeast (Ebios), 1:1 by weight. The antiproliferative method, sample and medium culture were mixed in double-soled dish and kept at 25 °C and 64 % RH. Decrease rates were determined 1 week and 4 week after treatment under microscope. D. farina was considered to be dead if appendages did not move when prodded with a pin. Results and Conclusions: The results show that the fatty acids potassium showed no antiproliferative effects against D. farinae. On the other hand, Japanese pepper, mustard, curry aroma and cinnamon were effective to decrease propagative rate (over 80 %) after treatment for 1 week against D. farina. Japanese pepper, curry aroma and cinnamon were effective to decrease propagative rate (approximately 100 %) after treatment for 4 weeks against D. farina. Especially, Japanese pepper and cinnamon showed the fasted and the most consecutive antiproliferative effects. These results indicate that Japanese pepper and cinnamon have high antiproliferative effects against D. farina and suggest spices will be used as a food additive agent.

Keywords: fatty acid salts, spices, antiproliferative effects, dermatophagoides farinae

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Authors: Shohei Matsuo, Saki Mikai, Hiroshi Morita

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Objectives: Shochu is a popular Japanese distilled spirits. In the production of shochu, the filamentous fungus Aspergillus kawachii has traditionally been used. A. kawachii produces two types of starch hydrolytic enzymes, α-amylase (enzymatic liquefaction) and glucoamylase (enzymatic saccharification). Liquid culture system is a relatively easy microorganism to ferment with relatively low cost of production compared for solid culture. In liquid culture system, acid-unstable α-amylase (α-A) was produced abundantly, but, acid-stable α-amylase (Aα-A) was not produced. Since there is high enzyme productivity, most in shochu brewing have been adopted by a solid culture method. In this study, therefore, we investigated production of Aα-A in liquid culture system. Materials and methods: Microorganism Aspergillus kawachii NBRC 4308 was used. The mold was cultured at 30 °C for 7~14 d to allow formation of conidiospores on slant agar medium. Liquid Culture System: A. kawachii was cultured in a 100 ml of following altered SLS medium: 1.0 g of rice flour, 0.1 g of K2HPO4, 0.1 g of KCl, 0.6 g of tryptone, 0.05 g of MgSO4・7H2O, 0.001 g of FeSO4・7H2O, 0.0003 g of ZnSO4・7H2O, 0.021 g of CaCl2, 0.33 of citric acid (pH 3.0). The pH of the medium was adjusted to the designated value with 10 % HCl solution. The cultivation was shaking at 30 °C and 200 rpm for 72 h. It was filtered to obtain a crude enzyme solution. Aα-A assay: The crude enzyme solution was analyzed. An acid-stable α-amylase activity was carried out using an α-amylase assay kit (Kikkoman Corporation, Noda, Japan). It was conducted after adding 9 ml of 100 mM acetate buffer (pH 3.0) to 1 ml of the culture product supernatant and acid treatment at 37°C for 1 h. One unit of a-amylase activity was defined as the amount of enzyme that yielded 1 mmol of 2-chloro-4-nitrophenyl 6-azide-6-deoxy-b-maltopentaoside (CNP) per minute. Results and Conclusion: We experimented with co-culture of A. kawachii and lactobacillus in order to get control of pH in altered SLS medium. However, high production of acid-stable α-amylase was not obtained. We experimented with yoghurt or milk made an addition to liquid culture. The result indicated that high production of acid-stable α-amylase (964 U/g-substrate) was obtained when milk made an addition to liquid culture. Phosphate concentration in the liquid medium was a major cause of increased acid-stable α-amylase activity. In liquid culture, acid-stable α-amylase activity was enhanced by milk, but Fats and oils in the milk were oxidized. In addition, Tryptone is not approved as a food additive in Japan. Thus, alter SLS medium added to skim milk excepting for the fats and oils in the milk instead of tryptone. The result indicated that high production of acid-stable α-amylase was obtained with the same effect as milk.

Keywords: acid-stable α-amylase, liquid culture, milk, shochu

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Authors: T. Hamaishi, Y. Morinaga, H. Morita

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Introduction: Rice consumption in Japan has decreased, and Japanese government has recommended use of rice flour in order to expand the consumption of rice. There are two major protein components present in flour, called gliadin and glutenin. Gluten forms when water is added to flour and is mixed. As mixing continues, glutenin interacts with gliadin to form viscoelastic matrix of gluten. Rice flour bread does not expand as much as wheat flour bread. Because rice flour is not included gluten, it cannot construct gluten network in the dough. In recent years, some food additives have been used for dough-improving agent in bread making, especially surfactants has effect in order to improve dough extensibility. Therefore, we focused to fatty acid salt which is one of anionic surfactants. Fatty acid salt is a salt consist of fatty acid and alkali, it is main components of soap. According to JECFA(FAO/WHO Joint Expert Committee on Food Additives), salts of Myristic(C14), Palmitic(C16) and Stearic(C18) could be used as food additive. They have been evaluated ADI was not specified. In this study, we investigated to improving bread-making properties of rice flour dough adding fatty acid salt. Materials and methods: The sample of fatty acid salt is myristic (C14) dissolved in KOH solution to a concentration of 350 mM and pH 10.5. Rice dough was consisted of 100 g of flour using rice flour and wheat gluten, 5 g of sugar, 1.7 g of salt, 1.7g of dry yeast, 80 mL of water and fatty acid salt. Mixing was performed for 500 times by using hand. The concentration of C14K in the dough was 10 % relative to flour weight. Amount of gluten in the dough was 20 %, 30 % relative to flour weight. Dough expansion ability test was performed to measure physical property of bread dough according to the methods of Baker’s Yeast by Japan Yeast Industry Association. In this test, 150 g of dough was filled from bottom of the cylinder and fermented at 30 °C，85 % humidity for 120 min on an incubator. The height of the expansion in the dough was measured and determined its expansion ability. Results and Conclusion: Expansion ability of rice dough with gluten content of 20 %, 30% showed 316 mL, 341 mL for 120 min. When C14K adding to the rice dough, dough expansion abilities were 314 mL, 368 mL for 120 min, there was no significant difference. Conventionally it has been known that the rice flour dough contain gluten of 20 %. The considerable improvement of dough expansion ability was achieved when added C14K to wheat flour. The experimental result shows that c14k adding to the rice dough with gluten content more than 20 % was not improving bread-making properties. In conclusion, rice bread made with gluten content more than 20 % without C14K has been suggested to contribute to the formation of the sufficient gluten network.

Keywords: expansion ability, fatty acid salt, gluten, rice flour dough

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Authors: Tatsuhiko Aizawa, Hiroshi Morita

Abstract:

The tool steels such as SKD11 and SKH51 have been utilized as punch and die substrates for cold stamping, forging, and fine blanking processes. The heat-treated SKD11 punches with the hardness of 700 HV wrought well in the stamping of SPCC, normal steel plates, and non-ferrous alloy such as a brass sheet. However, they suffered from severe damage in the fine blanking process of smaller holes than 1.5 mm in diameter. Under the high aspect ratio of punch length to diameter, an elastoplastic bucking of slender punches occurred on the production line. The heat-treated punches had a risk of chipping at their edges. To be free from those damages, the blanking punch must have sufficient rigidity and strength at the same time. In the present paper, the small-hole blanking punch with a dual toughness structure was proposed to provide a solution to this engineering issue in production. The low-temperature plasma nitriding process was utilized to form the nitrogen supersaturated thick layer into the original SKD11 punch. Through the plasma nitriding at 673 K for 14.4 ks, the nitrogen supersaturated layer, with the thickness of 50 μm and without nitride precipitates, was formed as a high nitrogen steel (HNS) layer surrounding the original SKD11 punch. In this two-zone structured SKD11 punch, the surface hardness increased from 700 HV for the heat-treated SKD11 to 1400 HV. This outer high nitrogen SKD11 (HN-SKD11) layer had a homogeneous nitrogen solute depth profile with a nitrogen solute content plateau of 4 mass% till the border between the outer HN-SKD11 layer and the original SKD11 matrix. When stamping the brass sheet with the thickness of 1 mm by using this dually toughened SKD11 punch, the punch life was extended from 500 K shots to 10000 K shots to attain a much more stable production line to yield the brass American snaps. Furthermore, with the aid of the masking technique, the punch side surface layer with the thickness of 50 μm was modified by this high nitrogen super-saturation process to have a stripe structure where the un-nitrided SKD11 and the HN-SKD11 layers were alternatively aligned from the punch head to the punch bottom. This flexible structuring promoted the mechanical integrity of total rigidity and toughness as a punch with an extremely small diameter.

Keywords: high nitrogen supersaturation, semi-dry cold stamping, solid solution hardening, tool steel dies, low temperature nitriding, dual toughness structure, extremely small diameter punch

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Authors: Aya Tanaka, Mariko Era, Shiho Sakai, Takayoshi Kawahara, Takahide Kanyama, Hiroshi Morita

Abstract:

Objectives: Aspergillus niger and Aspergillus oryzae are used as koji fungi in the spot of the brewing. Since koji-muro (room for making koji) was a low level of airtightness, microbial contamination has long been a concern to the alcoholic beverage production. Therefore, we focused on the fatty acid salt which is the main component of soap. Fatty acid salts have been reported to show some antibacterial and antifungal activity. So this study examined antimicrobial activities against Aspergillus and Bacillus spp. This study aimed to find the effectiveness of the fatty acid salt in koji-muro as antimicrobial agents. Materials & Methods: A. niger NBRC 31628, A. oryzae NBRC 5238, A. oryzae (Akita Konno store) and Bacillus subtilis NBRC 3335 were chosen as tested. Nine fatty acid salts including potassium butyrate (C4K), caproate (C6K), caprylate (C8K), caprate (C10K), laurate (C12K), myristate (C14K), oleate (C18:1K), linoleate (C18:2K) and linolenate (C18:3K) at 350 mM and pH 10.5 were used as antimicrobial activity. FASs and spore suspension were prepared in plastic tubes. The spore suspension of each fungus (3.0×104 spores/mL) or the bacterial suspension (3.0×105 CFU/mL) was mixed with each of the fatty acid salts (final concentration of 175 mM). The mixtures were incubated at 25 ℃. Samples were counted at 0, 10, 60, and 180 min by plating (100 µL) on potato dextrose agar. Fungal and bacterial colonies were counted after incubation for 1 or 2 days at 30 ℃. The MIC (minimum inhibitory concentration) is defined as the lowest concentration of drug sufficient for inhibiting visible growth of spore after 10 min of incubation. MICs against fungi and bacteria were determined using the two-fold dilution method. Each fatty acid salt was separately inoculated with 400 µL of Aspergillus spp. or B. subtilis NBRC 3335 at 3.0 × 104 spores/mL or 3.0 × 105 CFU/mL. Results: No obvious change was observed in tested fatty acid salts against A. niger and A. oryzae. However, C12K was the antibacterial effect of 5 log-unit incubated time for 10 min against B. subtilis. Thus, C12K suppressed 99.999 % of bacterial growth. Besides, C10K was the antibacterial effect of 5 log-unit incubated time for 180 min against B. subtilis. C18:1K, C18:2K and C18:3K was the antibacterial effect of 5 log-unit incubated time for 10 min against B. subtilis. However, compared to saturated fatty acid salts to unsaturated fatty acid salts, saturated fatty acid salts are lower cost. These results suggest C12K has potential in the field of koji-muro. It is necessary to evaluate the antimicrobial activity against other fungi and bacteria, in the future.

Keywords: Aspergillus, antimicrobial, fatty acid salts, koji-muro

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Authors: E. Nakamachi, K. Matsumoto, K. Yamamoto, Y. Morita, H. Sakamoto

Abstract:

In recently, electromagnetic and mechanical stimulations have been recognized as the effective extracellular environment stimulation technique to enhance the defected peripheral nerve tissue regeneration. In this study, we developed a new hybrid bioreactor by adopting 50 Hz uniform alternative current (AC) magnetic stimulation and 4% strain mechanical stimulation. The guide tube for nerve regeneration is mesh structured tube made of biodegradable polymer, such as polylatic acid (PLA). However, when neural damage is large, there is a possibility that peripheral nerve undergoes necrosis. So it is quite important to accelerate the nerve tissue regeneration by achieving enhancement of nerve axonal extension rate. Therefore, we try to design and fabricate the system that can simultaneously load the uniform AC magnetic field stimulation and the stretch stimulation to cells for enhancement of nerve axonal extension. Next, we evaluated systems performance and the effectiveness of each stimulation for rat adrenal pheochromocytoma cells (PC12). First, we designed and fabricated the uniform AC magnetic field system and the stretch stimulation system. For the AC magnetic stimulation system, we focused on the use of pole piece structure to carry out in-situ microscopic observation. We designed an optimum pole piece structure using the magnetic field finite element analyses and the response surface methodology. We fabricated the uniform AC magnetic field stimulation system as a bio-reactor by adopting analytically determined design specifications. We measured magnetic flux density that is generated by the uniform AC magnetic field stimulation system. We confirmed that measurement values show good agreement with analytical results, where the uniform magnetic field was observed. Second, we fabricated the cyclic stretch stimulation device under the conditions of particular strains, where the chamber was made of polyoxymethylene (POM). We measured strains in the PC12 cell culture region to confirm the uniform strain. We found slightly different values from the target strain. Finally, we concluded that these differences were allowable in this mechanical stimulation system. We evaluated the effectiveness of each stimulation to enhance the nerve axonal extension using PC12. We confirmed that the average axonal extension length of PC12 under the uniform AC magnetic stimulation was increased by 16 % at 96 h in our bio-reactor. We could not confirm that the axonal extension enhancement under the stretch stimulation condition, where we found the exfoliating of cells. Further, the hybrid stimulation enhanced the axonal extension. Because the magnetic stimulation inhibits the exfoliating of cells. Finally, we concluded that the enhancement of PC12 axonal extension is due to the magnetic stimulation rather than the mechanical stimulation. Finally, we confirmed that the effectiveness of the uniform AC magnetic field stimulation for the nerve axonal extension using PC12 cells.

Keywords: nerve cell PC12, axonal extension, nerve regeneration, electromagnetic-mechanical stimulation, bioreactor

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Authors: Aya Tanaka, Mariko Era, Manami Masuda, Yui Okuno, Takayoshi Kawahara, Takahide Kanyama, Hiroshi Morita

Abstract:

Objectives— Fungi and bacteria are present in a wide range of natural environments. They are breed in the foods such as vegetables and fruit, causing corruption and deterioration of these foods in some cases. Furthermore, some species of fungi and bacteria are known to cause food intoxication or allergic reactions in some individuals. To prevent fungal and bacterial contamination, various fungicides and bactericidal have been developed that inhibit fungal and bacterial growth. Fungicides and bactericides must show high antifungal and antibacterial activity, sustainable activity, and a high degree of safety. Therefore, we focused on the fatty acid salt which is the main component of soap. We focused on especially C10K and C12K. This study aimed to find the effectiveness of the fatty acid salt as antimicrobial agents for food safety. Materials and Methods— Cladosporium cladosporioides NBRC 30314, Penicillium pinophilum NBRC 6345, Aspergillus oryzae (Akita Konno store), Rhizopus oryzae NBRC 4716, Fusarium oxysporum NBRC 31631, Escherichia coli NBRC 3972, Bacillus subtilis NBRC 3335, Staphylococcus aureus NBRC 12732, Pseudomonas aenuginosa NBRC 13275 and Serratia marcescens NBRC 102204 were chosen as tested fungi and bacteria. Hartmannella vermiformis NBRC 50599 and Acanthamoeba castellanii NBRC 30010 were chosen as tested amoeba. Nine fatty acid salts including potassium caprate (C10K) and laurate (C12K) at 350 mM and pH 10.5 were used as antifungal activity. The spore suspension of each fungus (3.0×10⁴ spores/mL) or the bacterial suspension (3.0×10⁵ or 3.0×10⁶ or 3.0×10⁷ CFU/mL) was mixed with each of the fatty acid salts (final concentration of 175 mM). Samples were counted at 0, 10, 60, and 180 min by plating (100 µL) on potato dextrose agar or nutrient agar. Fungal and bacterial colonies were counted after incubation for 1 or 2 days at 30 °C. Results— C10K was antifungal activity of 4 log-unit incubated time for 10 min against fungi other than A. oryzae. C12K was antifungal activity of 4 log-unit incubated time for 10 min against fungi other than P. pinophilum and A. oryzae. C10K and C12K did not show high anti-yeast activity. C10K was antibacterial activity of 6 or 7 log-unit incubated time for 10 min against bacteria other than B. subtilis. C12K was antibacterial activity of 5 to 7 log-unit incubated time for 10 min against bacteria other than S. marcescens. C12K was anti-amoeba activity of 4 log-unit incubated time for 10 min against H. vermiformis. These results suggest C10K and C12K have potential in the field of food safety.

Keywords: food safety, microbes, antimicrobial, fatty acid salts

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Authors: I. Nanako, Mariko Era, Hiroshi Morita

Abstract:

Objectives: Japanese uses TATAMI rather than flooring at home. Igusa ( Juncus effuses var. decipiens ), which is commonly known in the forms of TATAMI. Juncus spp. grow at a relatively high humidity area (Japan, China and Southeast Asia ). Yatsushiro region in the southern part of Kumamoto prefecture is major produing area of Igusa. Igusa found to have honeycomb structure and was also shown to have the ability to control humidity. And Igusa has been used as a medicinal herb for diuretic and antiphlogistic agent. In previous study, we investigated antimicrobial effects of Igusa, and showed high antimicrobial activity against food poisoning bacteria. Therefore, the food trays blended Igusa can be kept clean by antimicrobial activity of Igusa. We focus on ‘Igusa foam materials’. In this study, we investigated the antibacterial and antifungal activity of Igusa, and new application to foam materials for food industry. Materials and method: We used Igusa foam materials (3 × 3 × 3 cm) as a sample. We set about fifteen types of samples combined with a commercial antibacterial agent A, a commercial antibacterial agent B, potassium laurate (C12K) and a commercial antifungal agent C, a commercial antifungal agent D and a commercial antifungal agent E. We selected four bacteria strains (Escherichia coli NBRC 3972, Staphylococus aureus NBRC 12732, Salmonella typhimurium NBRC 13245, Bacillus subtilis NBRC 3335 ) and three fungus strains (Penicillium pinophilum NBRC 6345, Cladosporium cladosporioides NBRC 30314, Aspergillus oryzae NBRC 5238 ). The fungus was cultured at 30 °C on Igusa foam materials after inoculation of the fungus for fourteen days. The bacteria was cultured at 30 °C on Igusa foam materials after inoculation of the bacteria for three days. And the Igusa foam materials were washed with 10 mL normal saline after three days. The normal saline washed Igusa foam materials plated the NA medium. After, It was cultured at 30 °C and used colony counting method. Result and Conclusion: The fifteen types of sample of Igusa foam materials had antifungal activity against C. cladosporioides, A. oryzae and P. pinophilum for fourteen days. The four types of sample contained potassium laurate and antibacterial agent A, sample contained antibacterial agent B and antifungal agent D, sample contained A and antifungal agent E, sample contained B and E had antibacterial activity against B. subtilis. The three types of sample contained potassium laurate and A, sample contained B and D, sample contained A and E had antibacterial activity against S. typhimurium. The five types of sample contained potassium laurate and A, sample contained B and D, sample contained A and E, sample contained B and E, sample contained B and antifungal agent C had antibacterial activity against E. coli and S. aureus. These results indicate that Igusa of Igusa foam materials had high antifungal activity. In addition, Igusa foam materials combined with a commercial antibacterial agent had antibacterial activity. In the future, we consider that use of Igusa foam materials may be spread from food industry.

Keywords: antibacterial, antifungal, foam materials, Igusa

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Authors: E. Nakamachi, S. Tanaka, K. Yamamoto, Y. Morita

Abstract:

In this study, we developed a three-dimensional (3D) direct current electric field (DCEF) stimulation bio-reactor for axonal outgrowth enhancement to generate the neural network of the central nervous system (CNS). By using our newly developed 3D DCEF stimulation bio-reactor, we cultured the rat pheochromocytoma cells (PC12) and investigated the effects on the axonal extension enhancement and network generation. Firstly, we designed and fabricated a 3D bio-reactor, which can load DCEF stimulation on PC12 cells embedded in the collagen gel as extracellular environment. The connection between the electrolyte and the medium using salt bridges for DCEF stimulation was introduced to avoid the cell death by the toxicity of metal ion. The distance between the salt bridges was adopted as the design variable to optimize a structure for uniform DCEF stimulation, where the finite element (FE) analyses results were used. Uniform DCEF strength and electric flux vector direction in the PC12 cells embedded in collagen gel were examined through measurements of the fabricated 3D bio-reactor chamber. Measurement results of DCEF strength in the bio-reactor showed a good agreement with FE results. In addition, the perfusion system was attached to maintain pH 7.2 ~ 7.6 of the medium because pH change was caused by DCEF stimulation loading. Secondly, we disseminated PC12 cells in collagen gel and carried out 3D culture. Finally, we measured the morphology of PC12 cell bodies and neurites by the multiphoton excitation fluorescence microscope (MPM). The effectiveness of DCEF stimulation to enhance the axonal outgrowth and the neural network generation was investigated. We confirmed that both an increase of mean axonal length and axogenesis rate of PC12, which have been exposed 5 mV/mm for 6 hours a day for 4 days in the bioreactor. We found following conclusions in our study. 1) Design and fabrication of DCEF stimulation bio-reactor capable of 3D culture nerve cell were completed. A uniform electric field strength of average value of 17 mV/mm within the 1.2% error range was confirmed by using FE analyses, after the structure determination through the optimization process. In addition, we attached a perfusion system capable of suppressing the pH change of the culture solution due to DCEF stimulation loading. 2) Evaluation of DCEF stimulation effects on PC12 cell activity was executed. The 3D culture of PC 12 was carried out adopting the embedding culture method using collagen gel as a scaffold for four days under the condition of 5.0 mV/mm and 10mV/mm. There was a significant effect on the enhancement of axonal extension, as 11.3% increase in an average length, and the increase of axogenesis rate. On the other hand, no effects on the orientation of axon against the DCEF flux direction was observed. Further, the network generation was enhanced to connect longer distance between the target neighbor cells by DCEF stimulation.

Keywords: PC12, DCEF stimulation, 3D bio-reactor, axonal extension, neural network generation

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Authors: Eiji Nakamachi, Ryota Sakiyama, Koji Yamamoto, Yusuke Morita, Hidetoshi Sakamoto

Abstract:

The regeneration of injured central nerve network caused by the cerebrovascular accidents is difficult, because of poor regeneration capability of central nerve system composed of the brain and the spinal cord. Recently, new regeneration methods such as transplant of nerve cells and supply of nerve nutritional factor were proposed and examined. However, there still remain many problems with the canceration of engrafted cells and so on and it is strongly required to establish an efficacious treating method of a central nerve system. Blackman proposed the electromagnetic stimulation method to enhance the axonal nerve extension. In this study, we try to design and fabricate a new three-dimensional (3D) bio-reactor, which can load a uniform AC magnetic field stimulation on PC12 cells in the extracellular environment for enhancement of an axonal nerve extension and 3D nerve network generation. Simultaneously, we measure the morphology of PC12 cell bodies, axons, and dendrites by the multiphoton excitation fluorescence microscope (MPM) and evaluate the effectiveness of the uniform AC magnetic stimulation to enhance the axonal nerve extension. Firstly, we designed and fabricated the uniform AC magnetic field stimulation bio-reactor. For the AC magnetic stimulation system, we used the laminated silicon steel sheets for a yoke structure of 3D chamber, which had a high magnetic permeability. Next, we adopted the pole piece structure and installed similar specification coils on both sides of the yoke. We searched an optimum pole piece structure using the magnetic field finite element (FE) analyses and the response surface methodology. We confirmed that the optimum 3D chamber structure showed a uniform magnetic flux density in the PC12 cell culture area by using FE analysis. Then, we fabricated the uniform AC magnetic field stimulation bio-reactor by adopting analytically determined specifications, such as the size of chamber and electromagnetic conditions. We confirmed that measurement results of magnetic field in the chamber showed a good agreement with FE results. Secondly, we fabricated a dish, which set inside the uniform AC magnetic field stimulation of bio-reactor. PC12 cells were disseminated with collagen gel and could be 3D cultured in the dish. The collagen gel were poured in the dish. The collagen gel, which had a disk shape of 6 mm diameter and 3mm height, was set on the membrane filter, which was located at 4 mm height from the bottom of dish. The disk was full filled with the culture medium inside the dish. Finally, we evaluated the effectiveness of the uniform AC magnetic field stimulation to enhance the nurve axonal extension. We confirmed that a 6.8 increase in the average axonal extension length of PC12 under the uniform AC magnetic field stimulation at 7 days culture in our bio-reactor, and a 24.7 increase in the maximum axonal extension length. Further, we confirmed that a 60 increase in the number of dendrites of PC12 under the uniform AC magnetic field stimulation. Finally, we confirm the availability of our uniform AC magnetic stimulation bio-reactor for the nerve axonal extension and the nerve network generation.

Keywords: nerve regeneration, axonal extension , PC12 cell, magnetic field, three-dimensional bio-reactor

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