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Bactericidal Action of Photogenerated Singlet Oxygen from Photosensitizers Used in Plaque Disclosing Agents
Kirika Ishiyama, Keisuke Nakamura, Hiroyo Ikai, Taro Kanno, Masahiro Kohno, Keiichi Sasaki, Yoshimi Niwano
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0037871
Abstract: Background Photodynamic therapy (PDT) has been suggested as an efficient clinical approach for the treatment of dental plaque in the field of dental care. In PDT, once the photosensitizer is irradiated with light of a specific wavelength, it transfers the excitation energy to molecular oxygen, which gives rise to singlet oxygen. Methodology/Principal Findings Since plaque disclosing agents usually contain photosensitizers such as rose bengal, erythrosine, and phloxine, they could be used for PTD upon photoactivation. The aim of the present study is to compare the ability of these three photosensitizers to produce singlet oxygen in relation to their bactericidal activity. The generation rates of singlet oxygen determined by applying an electron spin resonance technique were in the order phloxine > erythrosine ≒ rose bengal. On the other hand, rose bengal showed the highest bactericidal activity against Streptococcus mutans, a major causative pathogen of caries, followed by erythrosine and phloxine, both of which showed activity similar to each other. One of the reasons for the discrepancy between the singlet oxygen generating ability and bactericidal activity was the incorporation efficiency of the photosensitizers into the bacterial cells. The incorporation rate of rose bengal was the highest among the three photosensitizers examined in the present study, likely leading to the highest bactericidal activity. Meanwhile, the addition of L-histidine, a singlet oxygen quencher, cancelled the bactericidal activity of any of the three photoactivated photosensitizers, proving that singlet oxygen was responsible for the bactericidal action. Conclusions It is strongly suggested that rose bengal is a suitable photosensitizer for the plaque disclosing agents as compared to the other two photosensitizers, phloxine and erythrosine, when used for PDT.
In Vitro Evaluation of the Risk of Inducing Bacterial Resistance to Disinfection Treatment with Photolysis of Hydrogen Peroxide
Hiroyo Ikai, Yu Odashima, Taro Kanno, Keisuke Nakamura, Midori Shirato, Keiichi Sasaki, Yoshimi Niwano
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081316
Abstract: The purpose of the present study was to evaluate the risk of inducing bacterial resistance to disinfection treatment with photolysis of H2O2 and comparing this with existing antibacterial agents. We tested seven antibacterial agents, including amoxicillin, cefepime hydrochloride, erythromycin, ofloxacin, clindamycin hydrochloride, ciprofloxacin hydrochloride, and minocycline hydrochloride, as positive controls for validation of the assay protocol. For all of the agents tested, at least one of the four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Streptococcus salivarius) was resistant to these agents by repeated exposure to subinhibitory concentrations of the agents up to 10 times. In contrast, antibacterial activity against any of the bacterial species tested (S. aureus, E. faecalis, E. coli, S. salivarius, Pseudomonas aeruginosa, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans) was not affected by repeated exposure to the disinfection treatment up to 40 times. This finding suggested that the risk of inducing bacterial resistance by disinfection treatment was low. The active ingredient of this disinfection treatment is hydroxyl radicals generated by photolysis of H2O2. Therefore, hydroxyl radicals interact with several cell structures and different metabolic pathways in microbial cells, probably resulting in a lack of development of bacterial resistance. In conclusion, disinfection treatment with photolysis of H2O2 appears to be a potential alternative for existing antimicrobial agents in terms of a low risk of inducing bacterial resistance.
Photo-Irradiation of Proanthocyanidin as a New Disinfection Technique via Reactive Oxygen Species Formation
Keisuke Nakamura, Midori Shirato, Hiroyo Ikai, Taro Kanno, Keiichi Sasaki, Masahiro Kohno, Yoshimi Niwano
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0060053
Abstract: In the present study, the bactericidal effect of photo-irradiated proanthocyanidin was evaluated in relation to reactive oxygen species formation. Staphylococcus aureus suspended in proanthocyanidin aqueous solution was irradiated with light from a laser at 405 nm. The bactericidal effect of photo-irradiated proanthocyanidin depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power. When proanthocyanidin was used at the concentration of 1 mg/mL, the laser irradiation of the bacterial suspension could kill the bacteria with a >5-log reduction of viable cell counts. By contrast, bactericidal effect was not observed when proanthocyanidin was not irradiated. In electron spin resonance analysis, reactive oxygen species, such as hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide, were detected in the photo-irradiated proanthocyanidin aqueous solution. The yields of the reactive oxygen species also depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power as is the case with the bactericidal assay. Thus, it is indicated that the bactericidal effect of photo-irradiated proanthocyanidin is exerted via the reactive oxygen species formation. The bactericidal effect as well as the yield of the oxygen radicals increased with the concentration of proanthocyanidin up to 4 mg/mL, and then decreased with the concentration. These findings suggest that the antioxidative activity of proanthocyanidin might prevail against the radical generation potency of photo-irradiated proanthocyanidin resulting in the decreased bactericidal effect when the concentration is over 4 mg/mL. The present study suggests that photo-irradiated proanthocyanidin whenever used in an optimal concentration range can be a new disinfection technique.
Fabrication of two-dimensional periodic TiO2 pillar arrays by multi-beam laser interference lithography  [PDF]
Hiroyo Segawa, Hiroaki Misawa
Natural Science (NS) , 2009, DOI: 10.4236/ns.2009.13022
Abstract: Two-dimensional (2D) periodic TiO2 pillar arrays, applicable to photonic crystals and micro-channels, were fabricated by direct patterning of a TiO2-organic hybrid material by multi-beam laser interference lithography and calcination of hybrid patterns. 2D periodic pillars of a TiO2– organic hybrid material were prepared by irra-diation with the interference pattern of femto-second laser beams and removal of the non- irradiated portions. Two types of periodic pillar arrays, standing pillars and top-gathering pillars (four pillars gathered at the top), were obtained, depending on laser irradiation conditions. After calcination of TiO2–organic hybrid pillars, TiO2 pillar arrays were obtained without collapse.
Interaction between Peptide Pheromone or Its Truncated Derivatives and Pheromone Receptor of the Fission Yeast Schizosaccharomyces pombe Examined by a Force Spectroscopy Study and a GFP Reporter Assay  [PDF]
Sho Hidaka, Osamu Nikaido, Shoichi Kiyosaki, Atsushi Ikai, Toshiya Osada
Journal of Surface Engineered Materials and Advanced Technology (JSEMAT) , 2013, DOI: 10.4236/jsemat.2013.34A1005

In our previous study, the specific interaction between P-factor, a peptide pheromone and its receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was investigated by two methods, an atomic force microscope (AFM) and a GFP reporter assay. The removal of Leu at C-terminal of P-factor resulted in an inactivation of P-factor function to bind Mam2 and induce the signal transduction pathway. Here, we used truncated P-factor derivatives lacking N-terminal of P-factor (P12 ~ P22: 12 ~ 22 amino acid residues from C-terminal) as ligands for Mam2. From the dose-dependent analysis of the GFP reporter assay ranging from 1 nM to 100 μM of the peptide concentration, the peptides can be classified into three groups based on EC50 and maximal GFP production level, group1 (P-factor), group2 (P17 ~ 22), and group3 (P12 ~ P16). At 0.1 μM, only P-factor induced the signal transduction pathway. At 1 μM, peptides from group2 partially induced the pathway and peptides from group3 induced the pathway a little. At 10 μM, all peptides induced the pathway mostly depending on the length of peptides. We also performed AFM experiments using P-factor and peptides from group3 to investigate the interaction between the peptides and Mam2 for comparison between the two methods.

Simultaneous removal of cesium and strontium using a photosynthetic bacterium, Rhodobacter sphaeroides SSI immobilized on porous ceramic made from waste glass  [PDF]
Ken Sasaki, Hiroyo Morikawa, Takashi Kisibe, Kenji Takeno, Ayaka Mikami, Toshihiko Harada, Masahiro Ohta
Advances in Bioscience and Biotechnology (ABB) , 2013, DOI: 10.4236/abb.2013.41002
Abstract: This study investigated practical and simultaneous removal of cesium (Cs, initial concentration of 5 mg/L) and strontium (Sr, initial concentration of 5 mg/L) using a photosynthetic bacterium, Rhodobacter sphaeroides SSI, immobilized on recovery-type porous ceramic made from glass waste. When 4 - 8 pieces /L of SSI immobilized ceramic were added to synthetic sewage wastewater containing glucose, almost 100% of Cs and 57% - 61% removal of Sr was observed after 3 day’s aeration treatment. The high potassium (K) concentration in wastewater suppressed Cs removal, but did not affect Sr removal. Other substrates such as lactic, acetic, and propionic acids were useful for Cs and Sr removal. But, removal efficiencies were lower than about 50%. When the practical outdoor removal experiment carried out using1 m3 vessel, almost 100% of Cs and 51% of Sr were removed like a laboratory experiment after 3 day’s aerobic treatment. After treatment, the SSI immobilized ceramic was recovered easily from water using an electromagnet. This SSI immobilized ceramic seem to remove radioactive Cs and Sr from water environments of Fukushima,Japan.
Adverse Effects of Dietary Habits on Menstrual Disorders in Young Women
Tomoko Fujiwara, Natsuyo Sato, Hiroyo AwajiRieko Nakata
The Open Food Science Journal , 2007, DOI: 10.2174/1874256400701010024]
Abstract: It has been accepted that food customs are closely associated with quality of life in women of the reproductive age. Food customs are speculated to not only influence the present life style but also to induce gynecological disorders such as dysmenorrhea and irregular menstruation. Although there is no constant definition of regular or normal menstruation, epidemiologic evaluation of menstrual cycle has been becoming an important issue. In addition, latent development of organic diseases such as endometriosis, which are accompanied by dysmenorrhea, is a concern under the current nutritional environment in young women. Thus, it is an important issue to evaluate the present situation of eating habits in young women and estimate the influence of these habits on the quality of reproductive functions. Therefore, in this review, recent articles that are concerned with these issues have been reevaluated.
The antithrombotic activity of mini-type tomatoes is dependent on the particular variety and the stage of harvest. Lycopene content does not contribute to antithrombotic activity  [PDF]
Junichiro Yamamoto, Hiroyo Ohno, Kanae Hyodo, Masahiko Onishi, John C. Giddings
Health (Health) , 2013, DOI: 10.4236/health.2013.54090

The prevention of arterial thrombotic disease has a high priority in developed countries. We have focused our studies on the antithrombotic activity of those fruits and vegetables with the potential to prevent the disease, and the present study was undertaken as part of a series of investigations to examine beneficial fruits and vegetables. For this purpose, suitable laboratory tests as well as diets have been devised. In the current investigation, we have classified various tomato varieties with antithrombotic properties, and we now have extended our overall data to include more than ten antithrombotic varieties of fruits and vegetables. A method designed to measure shear-induced platelet activity (the Global Thrombosis Test, GTT) was used to assess haemostasis in vitro and a He-Ne laser-induced thrombosis technique was utilized to examine arterial thrombogenesis in vivo. Concentrations of the antioxidant, lycopene, were also measured. Three mini-type tomato varieties, coded “Cin”, “Pik” and “Caec”, and one mediumtype variety, coded “K”, were harvested at different stages of maturity. All mini-type varieties demonstrated antithrombotic activity at an early (green) stage. The antithrombotic activity decreased with the maturation of “Cin” and “Caec” but remained constant at all stages of maturity with “Pik”. The medium variety, “K”, did not possess antithrombotic activity. Lycopene was not detected at any stage in any of the tomato varieties, suggesting that this antioxidant did not contribute to antithrombotic activity. The present results indicated that the antithrombotic activity of tomatoes is dependent on the particular variety and stage of maturity, and that this activity is not due to lycopene.

Two-Fingered Haptic Device for Robot Hand Teleoperation
Futoshi Kobayashi,George Ikai,Wataru Fukui,Fumio Kojima
Journal of Robotics , 2011, DOI: 10.1155/2011/419465
Abstract: A haptic feedback system is required to assist telerehabilitation with robot hand. The system should provide the reaction force measured in the robot hand to an operator. In this paper, we have developed a force feedback device that presents a reaction force to the distal segment of the operator's thumb, middle finger, and basipodite of the middle finger when the robot hand grasps an object. The device uses a shape memory alloy as an actuator, which affords a very compact, lightweight, and accurate device. 1. Introduction Various humanoid robot hands have been developed so far. The Utah/MIT dexterous hand has four fingers with four joints driven by tendon cables and tactile sensors over the entire surface [1, 2]. The Gifu hand has five fingers and 20 joints with 16 degrees of freedom (DOF) [3], and the KH hand type S has five fingers and 20 joints with 15?DOF [4]. More recently manufactured, robot hands incorporate multiaxis/force torque sensors and tactile sensors with conductive ink and are relatively lightweight. The TWENDY-ONE hand has four fingers and 16 joints with 13?DOF [5]. This robot hand is equipped with the six-axis force sensors and array-type tactile sensors. Honda Motor Co., Ltd., has developed a multifingered robot hand, which has five fingers and 20 joints with 13?DOF [6]. Each DOF is hydraulically actuated, and the robot hand has tactile sensors on the entire surface. AIST also developed a multifingered robot hand with 4 fingers and 17 joints with 13?DOF that are actuated by an electrical servomotor [7]. The AIST robot hand also employed multi-axis force/torque sensors in the fingertips. Many other robot hands have been developed and researched [8, 9]. We have also reported the universal robot hands I [10] and II [11]. A tele-rehabilitation system receives attention for medical care because of a shortage of rehabilitation therapists [12]. The tele-rehabilitation system with a robot hand allows a rehabilitation therapist to care intuitively. However, typically a tele-operation system is unable to give tactile and haptic information to the human operator because conventional teleoperation systems lack a feedback system. It is difficult to complete tasks or control various operations without dexterous tactile and haptic information; thus human operators make errors because there is no tactile feedback. Therefore, many haptic devices were developed to enable the human operator to feel the force. The haptic devices are classified into three types according to their mechanical grounding configuration [13] including the grounded type [14–19],
mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization
Hironori Uehara, Yuji Kunitomi, Atsushi Ikai, Toshiya Osada
Journal of Nanobiotechnology , 2007, DOI: 10.1186/1477-3155-5-7
Abstract: In order to evaluate the SCN method, we compared the SCN method with in situ hybridization (ISH). First, we examined spatial β-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for β-actin mRNA. In the SCN method, quantity of β-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of β-actin mRNA. We showed that intensity of ISH is higher; quantity of β-actin mRNA detected by the SCN method increased more.In this study, we compare the SCN method with the ISH. We examined β-actin mRNA expression in single cells using both methods. We picked up β-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level.In situ hybridization (ISH) is a powerful molecular tool used to visualize nucleic acids, and it has attributed significantly to the advancement of the study of gene expression in cells and tissues. ISH was invented by two groups in 1969 [1,2]. Around that time, only radioisotope (RI) was available to label nucleic acids. But nowadays, non-RI ISH can be preformed based on synthesis of nucleotides containing certain functional groups and synthesis of a modified oligonucleotide by Digoxigenin (DIG) system [3-6]. Its primary advantage over the Northern blot and reverse transcription polymerase chain reaction (RT-PCR) is its ability to detect localization of specific mRNA to a particular cell or a particular region in a cell. So ISH are applied for bacteria, culture cells, tissue section and whole mount embryo [7-11]. However, ISH cannot examine time-lapse change of identical cells because the cells have to be fixed.We reported a single cell nanoprobe (SCN) method to examine mRNA expression without killing cells in a previous repor
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