Flogen SIPS

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Haruhiko Inufusa, TIMA, Gifu, Japan Title: Antioxidant treatment for infertility Abstract The decline in fertility is a major problem in many countries, especially in Japan, where the total fertility rate in 2018 is 1.42. Half of infertility couples have male causes, and the other half have female causes. Oxidative stress can be a major cause of infertility. There are many reports that the number of spermatozoa is decreased by mobile phone that emits electromagnetic waves that cause oxidative stress in the pocket near the testicle, resulting in a decrease in motility. The decline in pregnancy rates with age in women is partly due to the firmness of the egg membrane due to oxidative stress. Administration of an antioxidant composition would increase the pregnancy rate was examined. The monitor questionnaire was conducted by EYEZ,INC., which is a third-party institution, and the permission for secondary use of the results listed on the company's website (public data) was obtained. Twendee Mtcontrol (TwM) was used as antioxidant composition, which contains L-glutamine, ascorbic acid (VC), L-cystine, coenzyme Q10, succinic acid, fumaric acid, vitamin B2, niacin, Pantothenic acid, thiamine (VB1), pyridoxamine (VB6), folic acid, biotin, vitamin B12, lactoferrin. Twenty-eight couples who had been infertile for more than 2 years and wanted to raise their children joined the monitor questionnaire. For 1 year, both couples took TwM and their pregnancy was monitored. As a result, 9 pairs became pregnant, and the rate was 32% (9/28). Pregnancy rate is about 16% for a couple of more than two years of infertility when they receive infertility treatment at hospital. TwM showed almost 3 times higher pregnancy rate than infertility treatment at hospital. The possibility of antioxidant treatment for infertility was suggested. References: 1.Novel compositions and uses thereof WO 2008071790 A3 2.Antioxidant composition and its use in diabetes EP 1932542 A1 3.Pretreatment Twendee X® reduced acute ischemic brain damage. Kusaki M, Inufusa H, Abe K, JSCVD 2017; 26: 1191-1196 4.Clinical and Pathological Benefit of Twendee X in Alzheimer's Disease Transgenic Mice with Chronic Cerebral Hypoperfusion. XiaLiu, Abe K https://doi.org/10.1016/j.jstrokecerebrovasdis.2019.03.029

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Nektarios Tavernarakis, University of Crete, Heraklion, Greece Title: The Molecular Links Between Ageing And Neurodegeneration Abstract Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular yeast Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Key determinants of this capacity are the molecular mechanisms that link ageing to main stress response pathways. How each pathway contributes to modulate the ageing process is not fully elucidated. Mitochondrial impairment is a major hallmark of several age-related neurodegenerative pathologies, including Alzheimer’s and Parkinson’s diseases. Accumulation of damaged mitochondria has been observed in post-mortem brains of Alzheimer’s disease patients. Mitophagy is a selective type of autophagy mediating elimination of damaged mitochondria, and the major degradation pathway, by which cells regulate mitochondrial number in response to their metabolic state. Little is known about the role of mitophagy in the pathogenesis of Alzheimer’s disease. We find that neuronal mitophagy is impaired in animal models of Alzheimer’s disease. Indeed, mitophagy stimulation restores learning and memory capacity, in these animals. Moreover, age-dependent decline of mitophagy both inhibits removal of dysfunctional or superfluous mitochondria and impairs mitochondrial biogenesis resulting in progressive mitochondrial accretion and, consequently, deterioration of cell function. Our observations indicate that defective removal of damaged mitochondria is a pivotal event in neurodegeneration. These findings highlight mitophagy as a potential target for the development of innovative, effective therapeutic interventions towards battling human neurodegenerative disorders.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Toshikazu Yoshikawa, Louis Pasteur Center, Kyoto, Japan Title: From medicine to free radical research Abstract Free radical reactions play an important role in the biological system. In particular, oxygen-derived free radicals are sometimes used as an energy source for organisms that use oxygen to live, and also as a weapon to attack foreign objects at one stage. This reaction that works in the body is called ‘oxygen stress’, and I have devoted my life to this research. The contents of the research can be broadly divided into two. One of them is pathophysiological research as a cause of disease, and I have been researching disease prevention with antioxidants. Contrarily, the other research is to use this powerful oxidation reaction to defeat external enemies such as cancer, viruses, and bacteria. We still cannot prevent many diseases and cure them even with these series of studies. I shall continue my research toward the solution of this proposition.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Fuhua Yang, Division of Anti-Oxidant Research, Gifu University, Gifu, Japan Title: Role of Antioxidants in Oxidative Stress Induced by High Intake of Alcohol Abstract Although there are genetic differences in the metabolism of alcohol, large amounts of alcohol intake can cause hangovers and, in severe cases, death due to acute alcohol poisoning. Once alcohol enters the body, it is metabolized mainly in the liver, where it is broken down into the highly hepatotoxic acetaldehyde. It is thought that excessive alcohol intake causes insufficient metabolism in the body, and the acetaldehyde induces oxidative stress, resulting in hangovers and organ damage. Therefore, we examined the effects of large amounts of alcohol on the body using SUPALIV, an antioxidant formula consisting of coenzyme Q10, vitamin C, vitamin B2, niacin, L-cystine, succinic acid, fumaric acid, and L-glutamine. Mice (C57BL/6, male, 6 weeks old) were intraperitoneally administered 5.6 g/kg of ethanol, and blood acetaldehyde and oxidative stress were measured. The results showed that acetaldehyde in the blood increased at 2 hours after ethanol administration and was hardly detectable at 24 hours. However, oxidative stress remained high even after 72 hours of ethanol administration, and antioxidant capacity was low. In contrast, SUPALIV-treated group suppressed blood acetaldehyde and the rising oxidative stress without reducing antioxidant capacity. In the mouse acute alcohol intoxication experiment in which 6.4 g/kg of ethanol was administered intraperitoneally, all mice in the Control group, to which nothing was administered, died after 12 hours. However, in the group that received SUPALIV (60 mg/kg) orally three times, 30 minutes before ethanol administration and every 12 hours after ethanol administration, the survival rate was 100%. These results suggest that heavy alcohol intake increases oxidative stress in the body, and that this oxidative stress may be a factor in acute poisoning deaths. In contrast, the antioxidant compound SUPALIV suppressed acute poisoning deaths by speeding up the metabolism of alcohol and suppressing elevated oxidative stress. In conclusion, heavy and prolonged alcohol intake causes significant oxidative stress in the body, which may lead to aging and disease. Therefore, countermeasures against oxidative stress caused by alcohol intake are important, and SUPALIV was shown to have the potential to reduce this risk. References: 1). Markus Graf V. Matuschka-Greinffenclau, Hans Peter Jander. Alcohol metabolism moderating composition. WO 2005077464 A1. 2005-08-25. 2). Markus Graf Matuschka Von Greiffenclau, Haruhiko Inufusa. Composition for accelerating alcohol metabolism and for reducing the risk of alcohol induced diseases. WO 2012095509 A8. 2012-10-04. 3). Petrella C, Carito V Ceccanti M, et al. Oxidative stress inhibition by resveratrol in alcohol-dependent mice. Nutrition. 2020;79-80:110783.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Yoshiaki Harakawa, Division of Anti-oxidant Research, Gifu University, Gifu, Japan Title: Effect of antioxidant Twendee X on radiation-induced oxidative stress Abstract It is known that when a living organism is exposed to radiation, water molecules in the body ionize, producing reactive oxygen species, which in turn cause inflammation. Twendee X (TWX) is an antioxidant supplement consisting of eight ingredients (coenzyme Q10, vitamin C, vitamin B2, niacin, L-cystine, succinic acid, fumaric acid, and L-glutamine), and in 2019, Japan Society for Dementia Prevention-led randomized and double-blind clinical trials certified it as effective in preventing dementia. In recent years, many products claiming antioxidant effects have become available due to the anti-aging boom. Therefore, we investigated whether TWX and other substances can inhibit oxidative stress generated by radiation. [Methods] Mice (C57BL/6J, 6 weeks old, male) were orally administered the test substance TWX (15 mg/kg) for 3 days at 24-hour intervals, irradiated with 6 Gy of radiation on the fourth day, and blood samples were taken 72 hours later, and survival was observed as is. Vitamin C (15 and 150 mg/kg), Resveratrol (115 mg/kg), Astaxanthin (0.067 mg/kg), Protandim (190 mg/kg), and Pycnogenol (100mg/kg) were used as antioxidants for comparison to TWX. Serum hydroperoxide level was measured as blood oxidative stress (d-ROMs test). [Results] The oxidative stress level in mice increased with radiation dose, and a significant increase was observed from 4 Gy onward compared to the control group. The survival rate decreased from the 20th day of irradiation at 6 Gy, and all mice were confirmed dead in 14 days of irradiation at 7 Gy. In contrast, the increased d-ROMs test value by radiation was reduced by one third in the group treated with TWX (15 mg/kg) before irradiation for 3 days (t-test, p less than 0.05), and the oxidative stress level was lower in the group treated for 7 days than in normal mice. In addition, the administration of TWX suppressed radiation-induced deaths. Vitamin C (150 mg/kg) and astaxanthin (0.067 mg/kg) treatment groups reduced d-ROMs test values, but the difference was not statistically significant. The low concentrations of vitamin C (15 mg/kg), Pycnogenol, and Protandim did not change the d-ROMs test values compared to the control group, and for the Resveratrol group, the d-ROMs test values increased compared to the control group. [Discussion] TWX significantly reduced radiation-induced oxidative stress at a small dose of 15 mg/kg, while other comparators showed no reduction. In particular, the resveratrol-treated group showed elevated oxidative stress. Resveratrol is a polyphenol that is known to have antioxidant properties, however, high intake of a single polyphenol may have resulted in an increase in oxidative stress. TWX is composed only of substances that exist in the human body, and a small dose of TWX showed high antioxidant activity. This may have reduced inflammation caused by oxidative stress, ultimately leading to an increase in survival rate. The antioxidant potential of TWX in this experiment suggests the possibility of future applications for various oxidative stress-related diseases. References: [1] Tadokoro K, et al. Clinical Benefits of Antioxidative Supplement Twendee X for Mild Cognitive Impairment: A Multicenter, Randomized, Double-Blind, and Placebo-Controlled Prospective Interventional Study. J Alzheimers Dis. 2019;71(3):1063-1069. [2] Yamamoto T, Kinoshita M, Shinomiya N, Hiroi S, Sugasawa H, Matsushita Y, Majima T, Saitoh D, Seki S. Pretreatment with ascorbic acid prevents lethal gastrointestinal syndrome in mice receiving a massive amount of radiation. J. Radiat. Res., 2010. 51, 145-156. [3] Yoshikawa T, et al. [All about Free Radicals – from Health to Environmental Pollution -] Kasseisanso , Free Radical no subete – Kennkou kara kannkyouosen made – (in Japanese), Maruzen Publishing, 2000, 78-79. [4] Seki Y. [Evaluation of total oxidative stress by d-ROMs testing] d-ROMs test niyoru sankasutoresu sougouhyouka (in Japanese). Seibutsu siryou bunseki (Journal of analytical bio-science), 2010. 32(4), 301-306.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Shigeru Hirano, Kyoto Prefectural University of Medicine, Kyoto, Japan Title: Oxidative stress and hearing Abstract Hearing loss can occur by aging, ototoxicity of drugs, noise exposure, etc. It deteriorates the quality of life. Severe hearing loss causes isolation in human life. Tinnitus is an accompanying symptom with hearing loss, and is kind of torture for some people. Prevention of hearing loss is essential to keep the comfortable human life. Oxidative stress has been revealed to cause hearing loss caused by aging, noise exposure, and ototoxic drugs such as aminoglycoside and cisplatin (CDDP). We have revealed that reactive oxygen species (ROS) is produced in the vessels and hair cells of the inner ear. The hair cells are critical to maintain the hearing ability, and ROS causes severe and refractory damage to the hair cells. We have also revealed that Nox-3 derived ROS in the cochlea induces sensorineural hearing loss. Nox-3 is the key molecule for production of ROS. Nox knock-out mice showed maintenance of hearing ability under noise exposure or ototoxic agency. Anti-oxidative supplement should be useful for maintaining hearing.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Haruhiko Inufusa, TIMA, Gifu, Japan Title: Dawn Of Antioxidant Therapy Abstract Research on oxidative stress has been in full swing for about 40 years. Professor Toshikazu Yoshikawa, whose name is on the Yoshikawa International Symposium at SIPS2022, has clearly stated for 40 years that many diseases are caused and aggravated by oxidative stress, creating further oxidative stress. Professor Yoshikawa's foresight led him to predict that suppressing oxidative stress would alleviate symptoms and treat many diseases. The work of Professor Ferido Murad, who discovered that the coronary dilating effect of nitroglycerin, developed more than 100 years ago, was due to the release of Nox from the vascular wall muscle, was one of the proofs of this. This was one of the proofs of Professor Yoshikawa's work. This led to the development of Viagra, a further phosphodiesterase synthesis inhibitor with a similar effect. However, the effect could only be seen in the male function. Although recent meta-analysis of Viagra has pointed to its potential for dementia prevention, but clinical trials for dementia have not been initiated. Evidence for the efficacy of antioxidant therapy in other diseases was scarce until the development of my Twendee X. Twendee X is a dietary supplement consisting of eight active ingredients, which has already proven effective in clinical trials for dementia prevention and is being proven effective in a number of other diseases, including diabetes and allergic diseases. In recent years, the symptoms caused by COVID-19 infection, Long COVID, and mRNA vaccine drug damage are typical examples of oxidative stress disease symptoms. The large amounts of oxidative stress substances produced by inflammation of spike proteins cause systemic thrombosis and immunodeficiency, often leading to fatal reactions. This SIPS2022 Oxidative Stress Symposium will include many presentations related to Twendee X and Twendee Mtcontrol products. This will be the SIPS meeting that will signal the dawn of antioxidant therapy for the diseases foreseen by Professor Yoshikawa.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Marilia Goulart, Federal University of Alagoas, Maceio, Brazil Title: Bioanalysis of placenta and umbilical cord: cross-talk with maternal and child diseases and therapeutic potential of natural products Abstract The placenta and umbilical cord roles during pregnancy are pivotal for fetal development and maternal health. The placenta is a unique immunological site responsible for maternal tolerance to the fetus and maternal and fetal defence against possible pathogens. Alterations on these organs can lead to dramatic outcomes, including maternal and fetal death. During pregnancy, their correct functioning also influences the newborn's development and the onset of future chronic diseases. Therefore, investigating them in different diseases can give a real picture of health at the time of birth. Among the maternal diseases, pre-eclampsia (PE) is one of the most important, and despite incomplete understanding, oxidative stress and inflammation are recognised as their biochemical basis [1]. Another important aspect related to maternal-child health during pregnancy is related to ZIKA Virus (ZIKV), which can be vertically transmitted and infects the fetus, which may develop congenital Zika syndrome, characterized by stillbirth/miscarriage, fetal growth restriction, microcephaly, ocular abnormalities, ventriculomegaly and other brain malformations [2]. As such, the aims of the present work are divided into two parts. The first one is related to the comparison of the redox imbalance and inflammation biomarkers in the placenta and umbilical cords of pregnancies with and without preeclampsia (PE), while the second one addresses the investigation of natural products, which may inhibit ZIKV infection of trophoblast cells, a good model to test ZIKV molecular interactions and potential interveners [3]. In the first part, a controlled cross-sectional study was conducted in Maceió-Alagoas-Brazil with pregnant women diagnosed with PE and healthy pregnant women (control group). After delivery, placenta and umbilical cords’ samples were collected to measure redox imbalance (antioxidant defence and oxidative damage) and inflammation biomarkers: myeloperoxidase (MPO), interleukin (IL) -6, IL-8, IL-10 and tumour necrosis factor-alpha (TNF-α). A total of 100 pregnant women with PE and 50 healthy pregnant women were studied. Higher placental levels of catalase (p = 0.018), SOD (p = 0.031), the GSH/GSSG ratio (p = 0.019) and IL-6 (p = 0.010) and lower GSSG (p = 0.001) were observed in pregnant women with PE than in the control group. As results, PE-derived placentas had high concentrations of some antioxidants (enzymes and thiols), which might be a compensation mechanism against oxidative stress. In the second topic, the potential of some natural extracts against ZIKV infection was evaluated using an in vitro method. Ethanolic extracts obtained from peels and complete fruits of pink pepper (Schinus terebinthifolius) could reduce ZIKV infection and modulate placental response to protect placentas from potential harms provoked by ZIKV infection. In conclusion, both studies unveil the role of specific antioxidants in protecting maternal-child health and provide extra value to products from family farming. References: [1] M.B.Tenório et al., OxiMed & Cellular Longevity (2019) Article ID 8238727. [2] G.V França et al., Lancet 388 (2016) 891–7. [3] P. Aldo et al., Am. J. Reprod. Immunol. 76 (2016) 348–357.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Koji Abe, National Center of Neurology and Psychiatry, Tokyo, Japan Title: Anti-oxidative approach to prevent Alzheimer's type of dementia Abstract Alzheimer's disease (AD) and chronic cerebral hypoperfusion (CCH) often coexist in dementia patients in aging societies. The hallmarks of AD including amyloid-β (Aβ)/phosphorylated tau (pTau) and pathology-related events such as neural oxidative stress and neuroinflammation play critical roles in pathogenesis of AD with CCH. A large number of lessons from failures of drugs targeting a single target or pathway on this so complicated disease indicate that disease-modifying therapies targeting multiple key pathways hold potent potential in therapy of the disease. In the present study, we used a novel mouse model of AD with CCH to investigate a potential therapeutic effect of a free radical scavenger, Edaravone (EDA) on AD with CCH via examining motor and cognitive capacity, AD hallmarks, neural oxidative stress, and neuroinflammation. Compared with AD with CCH mice at 12 months of age, EDA significantly improved motor and cognitive deficits, attenuated neuronal loss, reduced Aβ/pTau accumulation, and alleviated neural oxidative stress and neuroinflammation. These findings suggest that EDA possesses clinical and pathological benefits for AD with CCH in the present mouse model and has a potential as a therapeutic agent for AD with CCH via targeting multiple key pathways of the disease pathogenesis. In a clinical setting, anti-oxidative dietary supplement TwendeeX prevented cognitive function or even improved it with MMSE (minimental score examination) in mild cognitive impairment population. These results strongly suggest a promising function of anti-oxidative approach to prevent dementia. References: 1). Tadokoro K, Morihara R, Abe K, et al. Clinical Benefits of Antioxidative Supplement Twendee X for Mild Cognitive Impairment: A Multicenter, Randomized, Double-Blind, and Placebo-Controlled Prospective Interventional Study. J Alzheimers Dis. 2019; 71(3): 1063-1069. 2.) Feng T, Yamashita T, Abe K, et al. Clinical and Pathological Benefits of Edaravon for Alzheimar”s Disease with Chronic Cerebral Hypoperfusion in a Novel Mouse Model. J Alzheimers Dis. 2019; 71(1);327-339. 3). Feng T, Yamashita T, Abe K, et al. In Vitro Free Radical Scavenging Activities of Dietary Supplements by Electron Spin Resonance. Brain Supplements. 2020; 2: 1-12. 4). Tadokoro K, Ohta Y, Abe K, et al. Prevention of Cognitive Decline in Alzheimer’s Disease by Novel Antioxidative Supplements. Int J Mol Sci. 2020 Mar 13:21(6)

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Yoshiaki Harakawa, Division of Anti-oxidant Research, Gifu University, Gifu, Japan Title: Potential for Antioxidant Therapy for Diabetes and Diabetic Complications Abstract Diabetes is known to be a disease caused by oxidative stress, and also known to generates its own oxidative stress, resulting in numerous complications. Twendee X (TWX) is an antioxidant combination supplement with international and Japanese patents, and has been certified as effective in preventing dementia from a randomized, double-blind clinical trial led by the Japan Society for Dementia Prevention in 2019. TWX has been shown in experiments on cynomolgus monkeys to lower blood sugar and reduce insulin secretion. In recent years, we have also successfully developed Twendee Mtcontrol (Tmt), an antioxidant with seven more ingredients added to TWX to increase its antioxidant effect. The antioxidants TWX and Tmt were tested in animal models to see if they can treat diabetes and diabetic complications. To examine the effect of antioxidants on diabetes, mice (C57BL/6J male, WT) were divided into three groups (n=8~10): control (non-treated), TWX 40 mg/kg/Day, and Tmt 40 mg/kg/Day, and were fed ad libitum on a high-fat diet for 3 months to develop a diabetic disease model. TWX or Tmt was orally administered daily starting 1 month after feeding. After 16 hours of fasting, a glucose load (1 g/kg glucose i.p.) was given and blood glucose levels were measured over time. In addition, insulin levels were measured 15 minutes after glucose loading under the same conditions. The results showed that the peak blood glucose level was significantly improved in the TWX group, and the peak level was also improved in the Tmt group compared to the control group. Insulin levels also tended to be lower than controls in both groups. In addition, to verify the effectiveness of the supplements in preventing actual diabetic complications, we tested it on SDT fatty rats, which develop diabetic complications early in the course of the disease. These rats develop elevated blood pressure and cataracts at a much earlier stage due to the progression of diabetes mellitus. SDT fatty rats (5-week-old males) were divided into three groups (n=8): control (non-treated), TWX 40 mg/kg/Day, and Tmt 40 mg/kg/Day, and TWX or Tmt was mixed with drinking water. At 17 weeks of age, most rats in the non-treated group showed severe cataracts, whereas those in the Tmt-treated group had relatively mild cataracts. Systolic blood pressure also tended to increase with age in the non-treated group, but the TWX and Tmt groups significantly suppressed the age-related increase in blood pressure. These results suggest that TWX and Tmt reduced blood glucose levels in an insulin-independent manner, thereby reducing diabetic complications such as hypertension and cataracts. TWX reduced blood glucose levels more, and Tmt had a pronounced cataract-preventive effect. Tmt may have a broader range of disease-preventive effects. however, further studies are required for the detailed mechanism of action. These results suggest the possibility of antioxidant therapy for diabetes mellitus and its complications. References: [1] Yamagishi S. [Diabetes and Oxidative Stress] Tounyoubyou to Sanka Sutoresu (in Japanese), Medical Review Co., Ltd., 2011, 3.\n [2] Masuyama T, Katsuda Y, and Shinohara M. A novel model of obesity-related diabetes: introgression of the Leprfa allele of the Zucker fatty rat into nonobese Spontaneously Diabetic Torii (SDT) rats. Exp. Anim. 54(1): 13-20, 2005.\n [3] Matsui K, Ota T, Matsushita M, et al. Diabetes-associated complications in Spontaneously Diabetic Torii Fatty Rats. Exp. Anim. 57(2): 111-121, 2008.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Yoichiro Sugiyama, Department of Otolaryngolgy-Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan Title: The effect of antioxidant Twendee X on swallowing function in an animal model of dysphagia in guinea pigs Abstract Dysphagia is one of the significant concerns in the aging society. In particular, the elderly with swallowing impairment caused by either central or peripheral nervous systems has difficulty in oral intake, possibly resulting in fatal pneumonia. As such, the development of a therapeutic approach that decreases in the risk of aspiration in dysphagic patients remains a pressing issue for medical doctors. The purpose of our study was to establish the animal model of dysphagia and test the hypothesis that decreased pharyngeal constriction caused by the denervation of the vagal efferent nerves can be improved by the application of the anti-oxidant agent. Thus, we developed the animal model in guinea pigs in which the pharyngeal branch of the vagus nerve was cut and initially evaluated swallowing function using videofluoroscopy. We then tested the effect of oral application of the anti-oxidant agent Twendee X on swallowing function in the dysphagia model animals. Insufficient pharyngeal constriction and pharyngeal residue were found in the denervated animals. The swallowing dysfunction was less pronounced in dysphagic animals with the administration of Twendee X. In conclusion, Twendee X could have a possible role in the improvement of swallowing function in dysphagic patients with decreased swallowing muscle strength caused by neuromuscular atrophy.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Naomi Okada, Gifu University, Gifu, Japan Title: The antioxidant Twendee X and cancer treatment Abstract As examples of cancer treatment, immunotherapy is now widely recognized as a powerful cancer therapy in addition to conventional therapies such as operation, chemotherapy and radiotherapy. Although they are all effective treatments, chemotherapy and radiotherapy, which take advantage of small differences between cancer cell (tumor cell) and normal cell, damage normal cells as well as cancer cells. As a result, adverse events are almost inevitable and are often difficult to continue treatment depending on the degree. As these treatments are administered over months or years, physical and mental damage accumulate, leading to a decline in physical strength and immunity. These adverse events are largely due to oxidative stress. That is, the tumor itself, and reactive oxygen species (ROS) generated by treatment for it, can cause damage to various cells due to oxidative stress. And oxidative stress can cause a decline in physical strength and immunity, which leads to tumor growth and a negative spiral. Thus, suppression of oxidative stress in tumor and cancer treatment may be very effective in not only alleviating symptoms but also prolonging prognosis. Since oxidative stress-mediated mechanisms of carcinogenesis also exist, suppression of oxidative stress may help to prevent cancer. An antioxidant compound Twendee X (TWX) which composed of vitamins, amino acids, and CoQ 10 is rated as the strongest and safe antioxidant in the world by ICDD (France). Cytoprotective effects of TWX against radiation exposure were examined using murine alveolar epithelial cells (LA-4). After allowing LA-4 cells to adhere, 25ug/ml of TWX was added in the medium. The cells were irradiated with 30Gy of radiation 2 hours after the addition, and after 72 hours, the effect on cell proliferative capacity was examined. The result showed that TWX significantly inhibited the radiation-induced decrease in cell proliferative capacity. In addition, TWX can be used in single and continuous doses in both healthy and pregnant mice and also increased NK activity, which is tumor immunity. In summary, TWX not only reduced adverse events during cancer treatment but also increased tumor immunity. We believe TWX can be an aid in the treatment and prevention of cancer. References: 1. Novel compositions and uses thereof WO 2008071790 A3 2. Antioxidant composition and its use in diabetes EP 1932542 A1 3. Suplatast tosilate reduces radiation-induced lung injury in mice through suppression of oxidative stress. Yusuke Izumi, Taku Nakashima, Noboru Hattori, Free Radic Biol Med.2019;136:52-59 4. Balancing efficacy of and host immune responses to cancer therapy:the yin and yang effects. Shbaked Y, Nat Rev Clin Oncol.2016 Oct;13(10):611-26. 5. Systematic Review: Generating Evidence-Based Guidelines on the Concurrent Use of Dietary Antioxidants and Chemotherapy or Radiotherapy. Akiko Nakayama, Karen P, Obianuju Lgbokwe, and Leffrey D. white. Cancer Invest. 2011 Dec:29(10): 655-667.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Hironori Nakagami, Osaka University Graduate School of Medicine, Osaka, Japan Title: A Prospective, Placebo-Controlled, Double-Blind Study to evaluate the impact of NMN (Nicotinamide Mononucleotide) on Diabetic Patients with Impaired Physical Performance Abstract Nicotinamide adenine dinucleotide (NAD+) is involved in regulating various biological processes and decreased with aging or oxidative stress [1]. Nicotinamide mononucleotide (NMN) increases intracellular NAD+ levels and counteracts age-associated changes [2,3]. We investigated the safety and efficacy of oral NMN supplementation (250 mg/day) in older diabetic patients with impaired physical performance. We conducted a 24-week placebo-controlled, double-blind study for male diabetic patients aged 65 years or older with reduced grip strength (less than 26 kg) or reduced walking speed (lenn than 1.0 m/sec). We studied 14 participants aged 81.1±6.4 years. NMN was tolerable without any related severe adverse events. The changes in grip strength and walking speed showed no difference between the two groups; However, there were trends toward an improved prevalence of frailty in the NMN group (p=0.066) and different changes in central retinal thickness between the two groups (P=0.051). In conclusion, in the present study performed on older male diabetic patients with physically impaired performance, a 24-week administration of 250 mg NMN was safe. Still, it did not affect grip strength or walking speed. However, a subanalysis indicated that NMN has the potential to improve frailty status and central retinal thickness. Directions to improve frailty and retinal thickness with NMN may be a further expected issue to be investigated. References: [1] Gomes AP, Price NL, Ling AJ, Moslehi JJ, Montgomery MK, Rajman L, White JP, Teodoro JS, Wrann CD, Hubbard BP, Mercken EM, Palmeira CM, de Cabo R, Rolo AP, Turner N, Bell EL, Sinclair DA. Cell 2013; 155: 1624-1638. [2] Yoshino J, Mills KF, Yoon MJ, Imai SI. Cell Metab 2011; 14: 528-536. [3] Yoshino M, Yoshino J, Kayser BD, Patti GJ, Franczyk MP, Mills KF, Sindelar M, Pietka T, Patterson BW, Imai SI, Klein S. Science 2021; 372: 1224-1229.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Haruhiko Inufusa, TIMA, Gifu, Japan Title: Antioxidant treatment for Sleep Apnea Abstract The number of severely ill patients requiring treatment for sleep apnea syndrome (SAS) is estimated to be over 9 million in Japan and 400 million worldwide. The Space Shuttle Challenger tragedy and the Exxon Valdez maritime accident were caused by SAS, resulting in significant global economic losses. The only effective treatment currently available is CPAP, a forced-air ventilator worn during sleep, which is often discontinued by patients due to its complexity and noise. About 70% of SAS patients have elevated mCRP and oxidative stress in their blood, indicating inflammation somewhere in the body.　Twendee X (TWX) is a very strong antioxidant composition and reduces inflammation which contains L-glutamine, ascorbic acid (VC), L-cystine, coenzyme Q10, succinic acid, fumaric acid, vitamin B2, and niacin. Clinical research of SAS using TWX for SAS was conducted at Takashima Clinic, Utsunomiya Japan. A survey of SAS patients was also conducted at Eyez Inc.The number of snoring as a major symptom of SAS was monitored. 2 weeks of treatment with TWX significantly reduced the number of snoring in 3 out of 5 patients, and 4 weeks of treatment significantly reduced the number of snoring in all 5 patients. 4 weeks of treatment with TWX showed the following effects in 30 SAS patients. TWX reduced snoring by 85%, nighttime waking phenomenon by 84%, deep sleep by 91%, daytime sleepiness by 84%, apnea by 85%, and headache upon awakening by 96%. TWX is a supplement with no side effects and can be safely taken by children, suggesting that it may be effective for about 80% of SAS patients. References: 1). Novel compositions and uses thereof WO 2008071790 A3\n 2). Antioxidant composition and its use in diabetes EP 1932542 A1\n 3). Pretreatment Twendee X® reduced acute ischemic brain damage. Kusaki M, Inufusa H, Abe K, JSCVD 2017; 26: 1191-1196\n 4). Clinical and Pathological Benefit of Twendee X in Alzheimer's Disease Transgenic Mice with Chronic Cerebral Hypoperfusion. Liu Xia, Yamashita T, Abe K, et al. J Stroke Cerebrovasc Dis. 2019 Jul;28(7):1993-2002.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Wei hua Huang, Wuhan University, Wuhan, China Title: Stretchable Electrochemical Sensor for Real Time Monitoring of ROS/RNS Signaling and Oxidative Stress in Mechanotransduction Abstract Cells in the body reside in dynamic mechanical milieus and can sense mechanical forces and then translate them into biochemical signals via mechanotransduction. Reactive oxygen/nitrogen species (ROS/RNS) are closely involved in cellular oxidative stress and physiopathological states, and real-time acquiring the information of ROS and RNS signaling during cell mechanotransduction is vitally important to reveal their complicated roles. Owing to rapid response and excellent sensitivity, electrochemical sensing has been extensively used for tracking ROS/RNS signaling in living cells. However, conventional electrochemical sensors are rigid and fail to comply with the shape changes of soft cells, and this greatly limits the accurate measurement of ROS/RNS signaling during cell mechanotransduction. To this end, we have designed a series of stretchable electrodes based on gold nanotubes, carbon nanotubes and conductive polymer nanofibers [1,2]. These sensors possess good electrochemical stability against mechanical deformations and can be easily deformed to achieve the dynamic stretching of cells. Further, we have also introduced high-performance catalysts (e.g, platinum nanoparticles and biomimetic catalysts) to confer the stretchable sensing interfaces with prominent electrocatalytic property toward ROS and RNS [3]. Based on these stretchable electrochemical sensors, we have successively achieved the real-time monitoring of stretch-induced NO and H2O2 molecules [3,4], and the simultaneous monitoring of them by a single device during endothelial mechanotransduction [5]. Besides, the developed stretchable electrochemical sensors have been also employed to explore the ROS and RNS signaling during mechanotransduction of chondrocytes and lung cells, which demonstrate excessive or acute mechanical loading can evoke severe oxidative stress [2]. These researches present efficient platforms to evaluate the oxidative stress level in dynamic mechanotransduction, which benefit to the understanding of the role of mechanical cues in cell biology. References: [1] Y.L. Liu, Z.H. Jin, Y.H. Liu, X.B. Hu, Y. Qin, J.Q. Xu, C.F. Fan, W.H. Huang. Angew. Chem. Int. Ed. 55 (2016) 4537-4541. [2] Y.L. Liu, W.H. Huang. Angew. Chem. Int. Ed. 60 (2020) 2757-2767. [3] W.T. Fan, Y. Qin, X.B. Hu, J. Yan, W.T. Wu, Yan, Y.L. Liu, W.H Huang. Anal. Chem. 92 (2020) 15639-15646. [4] Y.L. Liu, Y. Qin, Z.H. Jin, X.B. Hu, M.M. Chen, R. Liu, C. Amatore, W.H Huang. Angew. Chem. Int. Ed. 56 (2017) 9454-9458. [5] W.T. Fan, Y. Zhao, W.T. Wu, Y. Qin, J. Yan, Y.L. Liu, W.H Huang. Anal. Chem. 94 (2022) 7425-7432.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Fuhua Yang, Division of Anti-Oxidant Research, Gifu University, Gifu, Japan Title: Usefulness of Antioxidant Combination Products for Chronic Fatigue Syndrome Abstract In Chronic Fatigue syndrome (CFS), a complex combination of factors such as infections and excessive stress in daily life causes abnormalities in the immune system, endocrine system, brain, and neurological functions. The pathogenesis of the syndrome is not yet clear, and no treatment has been established. Recently, it has been reported that CFS-like symptoms are also observed in COVID-19 sequelae and SARS-CoV-2 vaccine sequelae, and there is an urgent need to clarify the pathogenesis mechanism and establish prevention and treatment methods. Abnormal energy metabolism due to mitochondrial dysfunction and inflammation in the brain have been reported as one of the causes of CFS. Since oxidative stress is highly related to both of these factors, it was thought that suppressing oxidative stress may lead to improvement of various symptoms of CFS. Therefore, we analyzed the symptom changes before and after the use of an antioxidant combination supplement, Twendee Mtcontrol (Tmt), taken by people who had been diagnosed with chronic fatigue syndrome. Participants recruited via the web were asked whether they were diagnosed as CFS, and to rate the impact of their major symptoms (fatigue, muscle pain, joint pain, sleep disturbances, memory and concentration problems, and headaches) on their daily lives on a 6-point scale from "no impact" (0) to "high impact" (5). Of these, 23 people who were actually diagnosed with CFS were asked to took 3 tablets of Tmt orally every day for 2 months, and to self-evaluate the effects on their daily lives of the main symptoms (fatigue, muscle pain, joint pain, sleep disturbance, memory and concentration loss, and headache) before and after taking the supplement on the Web, and the changes were tabulated ( Eyze, Inc.). The results showed that before taking the supplement, 96% of participants reported that CFS had a high degree of impact (score of 3 or higher) on their daily lives. For each symptom level, the percentages of participants who reported a high degree of impact were as follows: fatigue accounted for 82%, muscle pain for 52%, joint pain for 74%, sleep disturbance for 100%, poor memory and concentration for 87%, and headache for 74%. In general, the quality of daily life was not good. However, after 2 months of treatment, there was a significant (p less than 0.01) improvement in the impact of CFS on daily life and in all symptoms. In particular, the number of people who rated it as the most affecting their daily life (score 5) regarding memory and concentration loss went from 22% to 0% after taking the supplement. Other subjective symptoms included improved awakening and sleeping, feeling lighter, feeling less tired, recovering from fatigue more quickly, and feeling less mentally depressed. Although the effects of various antioxidants have been reported for CFS, most of them are mainly for fatigue, and there are no reports showing significant effects on various symptoms. This may be due to differences in the combination of antioxidants and their antioxidant capacity. The results of this study may be useful for future treatment and prevention of CFS. References: 1). Feng T, Yamashita T, Abe K, et al. In Vitro Free Radical Scavenging Activities of Dietary Supplements by Electron Spin Resonance. Brain Supplements. 2020; 2: 1-12. 2). You F, Tanaka S, Inufusa H, et al. Effects of Antioxidant composition Twendee X on side effects of SARS -COV-2 mRNA vaccine. Brain Supplement. 2022; 4: 1-6. 3). You F, Tanaka S, Inufusa H, et al. Antioxidant composition Twendee X may improve long COVID symptoms. Brain Supplement. 2022; 4: 7-12.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Fuhua Yang, Division of Anti-Oxidant Research, Gifu University, Gifu, Japan Title: Involvement of anti-oxidant Twendee X in immunity. Abstract Many diseases and physical disorders are caused by both external and internal factors, but the common denominator is inflammation and oxidative stress. Oxidative stress is elevated in people infected with COVID-19, which has recently become an urgent problem. Suppressing oxidative stress on a daily basis and maintaining a high level of self-immunity can help prevent and treat disease and maintain good health. Intestines are involved in about 60% of our immune system. There are over 100 species of, or 100 trillions of bacteria in our intestines, and their ideal balance is 20% of good, 10% of bad, and 70% of opportunistic bacteria. This balance is lost by disturbed life rhythm and various diseases, in other words “oxidative stress”, being said to decrease bacterial diversity. Reducing oxidative stress and increasing diversity of intestinal bacteria is important for maintaining healthy life. Composed of vitamins, CoQ10, and amino acids, Twendee X (TWX) is an antioxidant which is confirmed to have a dementia-prevention effect. Effects of increased bacterial diversity and NK activity by administration of TWX was examined. Rats were given an antifungal agent OPP for 2 weeks, then blood and feces samples were collected to analyze blood oxidative stress and intestinal bacterial diversity. TWX was administrated from 1 week before OPP initiation. In addition, we measured the NK activity of TWX in normal mice and compared it with the mice without any treatment. Furthermore, a questionnaire survey for 57 healthy participants was conducted to compare their defecation status and intestinal microbial flora between before and after one month TWX administration. Although OPP rat group increased blood oxidative stress by 5%, no significant change was observed in microbial flora diversity. A rat group given TWX since 1 week before decreased oxidative stress by 13.3% and increased bacterial diversity 1.5 times higher, compared to rats which wasn’t given, and the number of Akkermansia, anti-inflammatory bacteria, was increased 3.5 times more. Furthermore, administrating TWX to healthy mice increased their NK activity twice as high as that of untreated mice. In the questionnaire survey for healthy participants who were given TWX for 1 month, the number of good bacteria and intestinal bacterial diversity were increased. Their defecation status and QOL were improved with that. TWX tends to reduce oxidative stress, to increase the number of bacteria and diversity in intestinal microflora, and to enhance NK activity known as a tumor immunity. It was suggested that this enables to protect human body from harmful substances invaded inside, improve immune system, and improve daily defecation status and QOL. References: 1. Novel compositions and uses thereof WO 2008071790 A3 2. Antioxidant composition and its use in diabetes EP 1932542 A1 3. Composition for accelerating alcohol metabolism and for reducing the risk of alcohol induced diseases WO 2012095509 A8 4. Composition for moderating triglyceride and cholesterol levels WO 2009062910 A1

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Jun-Jie Zhu, Nanjing University, Nanjing, China Title: DNA Nano Self-assemblies for the Application in Cancer Diagnosis and Therapy Abstract Cancer is a conundrum in modern medicine and has become a serious threat to human health and life [1], thus the the early diagnosis and precise therapy are crucial to improve the cancer cure rates [2]. Taking advantage of the DNA nanotechnology, an RNA aptamer-based near-infrared gated nanodevice for low-background imaging of mRNA in living cells have been first developed based on the fluorescence enhancement property of RNA aptamer and the catalytic hairpin assembly reaction-mediated signal amplification strategy. Subsequently, DNA nanocarrier with excellent biological stability, strong enzymatic resistance and collaborative drugs delivery is constructed by functionalized circular DNA, which can effectively combat the multidrug-resistant human leukemia. Moreover, a multifunctional DNA-Au nanomachine integrating both diagnostic and therapeutic agents is proposed, which can be triggered by the endogenous mRNA, leading to the activation of multi-mode synergistic therapy and in situ imaging. These researches conducive to the accurate diagnosis and image-guided precise treatment of cancers, which may provide a new route for the construction of intelligent responsive theranostic nanoplatform. References: [1] H. Dong, J. Lei, L. Ding, Y. Wen, H. Ju, X. Zhang, Chem. Rev. 113 (2013) 6207. [2] X. Zhang, Z. Xi, J. O. Machuki, Y. Yang, L. Zhang, J. Tian, K. Guo, Y. Yu, F. Gao, ACS Nano 13 (2019), 5306.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Haruhiko Inufusa, TIMA, Gifu, Japan Title: Antioxidant treatment for Long- COVID and mRNA vaccine side effects Abstract COVID-19 infection produces large amounts of Reactive Oxygen Species (ROS) in the human body, and the attack on the immune system by ROS causes a cytokine storm. Therefore, COVID19 infection generally does not cause severe disease in children with low ROS levels, but often causes severe disease and death in the elderly or those with underlying disease already ROS levels are high. The major problem with after COVID-19 pandemic is not only Long-COVID, but also the large number of patients who suffer from the tremendous side effects caused by mRNA vaccines. Pfizer and other genetic vaccines produce spike proteins and antibodies against them. Not only the spike protein itself causes ROS, but also pseudouridine and Nano Lipoprotein (NLP) contained in the vaccine induce inflammation and increase ROS. In other words, both diseases are oxidative stress diseases. Twendee X (TWX) is a very strong antioxidant composition and reduces inflammation which L-glutamine, ascorbic acid (VC), L-cystine, coenzyme Q10, succinic acid, fumaric acid, vitamin B2, and niacin was used for both patients. Patients took 3 tablets of TWX once a day for 4 weeks and answered a web-based questionnaire about their symptoms before and after taking TWX. A score of 0 on the questionnaire indicated no symptoms, while a score of 6 indicated severe symptoms. A total of 126 Long-COVID patients and 149 patients with mRNA vaccine sequelae completed the monitoring questionnaire completely. The monitored symptoms were Fatigue, Breathing difficulty, Chest pain, Smell or taste disorder, Headache, Brain fog, Joint pain, and Dizziness. For both disorders, all monitored symptoms showed significant post-medication score reductions and symptom improvement. Especially in patients with moderate to severe disease with a score of 3 or higher, the mean score dropped to less than half of the pre-medication level for all items. In addition to the monitoring questionnaire, we will also present several cases who became bedridden after mRNA vaccination were treated with TWX and Twendee Mtcontrol (Tmt), which increased the effectiveness of TWX by 1.5 times, and were able to walk. TWX and Tmt were thought to be effective in relieving symptoms in these patients, many of whom suffer from Long-COVID and mRNA vaccine sequelae worldwide. References: 1). Novel compositions and uses thereof WO 2008071790 A3 2). Antioxidant composition and its use in diabetes EP 1932542 A1 3). Pretreatment Twendee X® reduced acute ischemic brain damage. Kusaki M, Inufusa H, Abe K, JSCVD 2017; 26: 1191-1196 4). Clinical and Pathological Benefit of Twendee X in Alzheimer's Disease Transgenic Mice with Chronic Cerebral Hypoperfusion. Liu Xia, Yamashita T, Abe K, et al. J Stroke Cerebrovasc Dis. 2019 Jul;28(7):1993-2002. 5). Antioxidant composition Twendee X may improve long COVID symptoms. You F, Tanaka S, Inufusa H, et al.　Brain Supplement. 2022; 4: 7-12. 6). Effects of Antioxidant composition Twendee X on side effects of SARS -COV-2 mRNA vaccine. You F, Tanaka S, Inufusa H, et al. Brain Supplement. 2022; 4: 1-6.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Haruhiko Inufusa, TIMA, Gifu, Japan Title: Oxidative stress is major cause of allergic reaction in COVID-19 infection Abstract Oxidative stress and immune activity are closely related with disease or symptoms. Allergic reaction depending on imbalance of immune system which is easy to be damaged by oxidative stress. COVID-19 pneumonia starts at the lower and back side of lung, and CT scan shows typical interstitial pneumonia. Interstitial pneumonia is kind of allergic reaction, thus pneumonia start high blood flow area of lung and very rapidly spread to the whole lung. It is well known that interstitial pneumonia shows extremely high oxidative stress, damages lung tissue, and some patients show irreversible damage of the lung. High risks of COVID-19 infection are high age with underlying disease, especially diabetes mellitus, obesity, heart disease, hypertension, after stroke, cancer, neurogenic disorder, dementia and so on. All these risk factors show extremely high oxidative stress. Oxidative stress damages vascular endothelium and inflammation of vascular wall produces thrombosis, which are reasons of Kawasaki disease like vasculitis and cerebral infraction. Oxidative stress plays a major role in these symptoms. References: 1. Novel compositions and uses thereof WO 2008071790 A3 2. Antioxidant composition and its use in diabetes EP 1932542 A1

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Koji Fukui, Shibaura Institute of Technology, Saitama, Japan Title: Twendee X improves cognitive function in vitamin E-deficient mice Abstract Reactive oxygen species may attack several living organs and gradually accumulate oxidative products such as lipid hydroperoxides. Recently, several kinds of evidence have been demonstrated that accumulation of oxidative products induces onset and progression of several severe diseases such as inflammations, cardiovascular events and more [1]. One severe disease is dementia, and it also deeply relates to senescence. To prevent age-related neurodegenerative disorders, treatment with antioxidant is effective. Twendee X, a mixed antioxidant supplement contains 8 substances [2] and anyone can buy it. However, the detailed scientific evidence is not enough. In this study, we fed Twendee X to vitamin E-deficient mice and check cognitive function. We created vitamin E-deficient mice and Twendee X treated for one month. Cognitive function was measured by the Morris Water maze task [3]. Vitamin E deficient mice were significantly decrease learning ability. Treatment with Twendee X was significantly improved it. Brain neurotrophic factors in Twendee X-treated mouse were significantly increase compared to the untreated groups. Twendee X has a potential as a powerful antioxidant supplement through protection against neuronal dysfunction. References: [1] Yoshida, N, Kato, Y, Takatsu, H, et al., Biomedicines 10 (2022) 28. [2] Tadokoro, K, Morihara, R, Ohta, Y, et al., J Alzheimer’s Dis, 71 (2019) 1063-1069. [3] Fukui, K, Onodera, K, Shinkai, T, et al., Ann NY Acad Sci, 928 (2001) 169-176.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Christian Andre Amatore, CNRS & PSL, French Academy of Sciences, Paris, France Title: Nanometer-sized electrochemical probes for intracellular measuring ROS/RNS in single cells and cellular organelles Abstract Oxidative stress conditions are encountered by all aerobic organisms during their whole life. Indeed, aerobic cells mostly derive their energy from the intracellular enzyme-catalyzed oxidation of fat and sugars to CO2. Also, metalloenzymes which are central actors of the respiratory chain in mitochondria are generally good reducing agents, prone to open side routes leading to O2 reduction to superoxide ion (O2•-) that is the precursor of a series of hazardous species collectively named as “reactive oxygen species (ROS)” and “reactive nitrogen species (RNS)” [1,2]. ROS and RNS may induce molecular damages to almost all organic compounds performing biological functions (nucleic acids, proteins, cells carbohydrates and lipids, etc.) – a situation termed “oxidative stress” when it runs out of control. Even without exposure to radiation or other photo-biological effects, oxidative stress can bring about such pathological conditions as inflammation, carcinogenesis, Parkinson and Alzheimer diseases, and various autoimmune illnesses, as well as accelerated ageing. The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques [3]. Nanometer-sized electrochemical probes with cylindrical shapes do not experience this problem since they can penetrate across the cell membranes that reseal around their shaft (7). They are then especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this paper, we will survey recent advances in localized measurements of ROS/RNS inside single cells. Application of this method will be presented for detection of ROS/RNS in phagolysosomes during phagocytosis by macrophages (4,5). We will also evidence using these methods that remediation of Oxidative Stress in neurons artificially placed under Parkinson Disease conditions avoids the impeachment of synaptic communication when the neurons are pre-treated with Harpagide, a natural sugar derivative which alleviate the oxide stress borne by mitochondria (9). References: (1) B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 3rd ed., Oxford University Press, Oxford, 1999. (2) F. Murad: Discovery of Some of the Biological Effects of Nitric Oxide and its Role in Cell Signaling. Nobel Lecture for Medicine, 1998, https://www.nobelprize.org/uploads/20... (3) C Amatore, S. Arbault, M. Guille, F. Lemaître: Electrochemical monitoring of single cell secretion: vesicular exocytosis and oxidative stress. Chem. Rev. 108 (2008) 2585–2621. (4) K. Hu, Y.L. Liu, A. Oleinick, M.V. Mirkin, W.H. Huang, C. Amatore: Nanoelectrodes for Intracellular Measurements of Reactive Oxygen and Nitrogen Species in Single Living Cells. Curr. Opin. Electrochem., 22, 2020, 44-50, and refs therein. (5) Y.T. Qi, H. Jiang, W.T. Wu, F.L. Zhang, S.Y. Tian, W.T. Fan, Y.L. Liu, C. Amatore, W.H. Huang: Homeostasis Inside Single Activated Phagolysosomes: Quantitative and Selective Measurements of sub-Millisecond Dynamics of ROS/RNS Production with a Nanoelectrochemical Sensor. J. Am. Chem. Soc., 144, 2022, 9723-9733.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Nektarios Tavernarakis, University of Crete, Heraklion, Greece Title: Cellular energy homeostasis in neurodegeneration and ageing Abstract Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Key determinants of this capacity are the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. How each pathway contributes to modulate the ageing process is not fully elucidated. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies. Mitochondria, the indispensable and highly dynamic, energy-generating organelles in all eukaryotic cells, play essential roles in fundamental cellular processes. Neuronal cells depend, perhaps more than any other cell type, on proper mitochondrial function. Mitochondrial impairment is a major hallmark of several age-related neurodegenerative pathologies, including Alzheimer’s disease. Interestingly, accumulation of damaged mitochondria has been observed in post-mortem brain of Alzheimer’s disease patients. Mitophagy is a selective type of autophagy mediating elimination of damaged mitochondria, and the major degradation pathway, by which cells regulate mitochondrial number in response to their metabolic state. However, little is known about the role of mitophagy in the pathogenesis of Alzheimer’s disease. Although disease-associated tau and amyloid β are known to deregulate mitochondrial function, it remains elusive whether they also directly influence the efficiency of mitophagy. To address this question, we developed an in vivo imaging system to monitor mitophagy in neurons. We demonstrated that neuronal mitophagy is impaired in C. elegans models of Alzheimer’s disease. Urolithin A- and nicotinamide mononucleotide-induced mitophagy ameliorates several pathological features of Alzheimer’s disease, including cognitive defects. Mitophagy stimulation restores memory impairment. Age-dependent decline of mitophagy both inhibits removal of dysfunctional or superfluous mitochondria and impairs mitochondrial biogenesis resulting in progressive mitochondrial accretion and consequently, deterioration of cell function. Our findings suggest that impaired removal of damaged mitochondria is a pivotal event in Alzheimer’s disease pathogenesis highlighting mitophagy as a potential therapeutic intervention.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Yi-Tao Long, Nanjing University, Nanjing, China Title: Nanopore Electrochemistry for Single-Molecule Analysis Abstract Nanopore electrochemistry refers to the promising measurement science based on elaborate pore structures, which offers a well-defined geometric confined space to adopt and characterize single entities including single cells, single particles, and even single molecules by electrochemical technology.1-3 The electrochemical confined effect within the nanopore displays the incredible ability to achieve single entity discrimination by focusing energy (e.g. electrochemical, light energies and et al.) into small areas, converting the intrinsic properties of single entities into visible electrochemical read-outs with ultra-high temporal-spatial resolution. Furthermore, the excellent resolution of confined nanopore technology also permits the possibility to resolve the transient signals for further revealing the information of single biomolecules dynamics. The chemical controlled confinement inside nanopore provides the advanced electrochemically confined effects to convert the transient single molecule difference into the enhancing signal with high temporal-spatial resolution. In our group, the nanopore electrochemistry has been further applied into disease diagnostics by identifying rare sub-populations, DNA/protein sensing by reading the sequential differences and uncovering the fundamental chemical reactions pathways by revealing the hidden intermediates. With the advents of advanced measurement mechanism, instrumentation and data algorithm, electrochemically confined nanopore is certainly an exciting and promising field. We expect the next avenue for the wide applications of nanopore electrochemistry in a variety of disciplines, leading us explore the new chemistry at a much smaller scale. References: 1. Ying, Y. L.; Long, Y. T., Nanopore-Based Single-Biomolecule Interfaces: From Information to Knowledge. J. Am. Chem. Soc. 2019, 141 (40), 15720-15729. 2. Li, X.-Y.; Ying, Y.-L.; Fu, X.-X.; Wan, Y.-J.; Long, Y.-T., Single‐Molecule Frequency Fingerprint for Ion Interaction Networks in a Confined Nanopore. Angew. Chem. Int. Ed., 2021, 133 (46), 24787-24792. 3. Hu, Z.-L.; Huo, M.-Z.; Ying, Y.-L.; Long, Y.-T., Biological nanopore approach for single‐molecule protein sequencing. Angew. Chem. Int. Ed., 2021, 133 (27), 14862-14873. 4. Lu, S.-M.; Peng, Y.-Y.; Ying, Y.-L.; Long, Y.-T., Electrochemical Sensing at a Confined Space. Anal. Chem. 2020, 92 (8), 5621-5644.

FLOGEN SIPS 2022: Yoshikawa International Symposium on Oxidative Stress for Sustainable Development of Human Beings (2nd international Symposium) Presenter: Prof. Fuhua Yang, Division of Anti-Oxidant Research, Gifu University, Gifu, Japan Title: Antioxidants and Cancer Cells Abstract Oxidative stress is involved in over 150 different diseases, and cancer is one of the most common. High oxidative stress can damage proteins and DNA that are essential to the body, leading to the formation of cancer over time. Oxidative stress in the body is also elevated in carcinoma carriers. When the body's oxidative stress is elevated, inflammatory substances increase, and the body's cancer immunity is reduced, which is thought to promote cancer cell growth and metastasis. Since antioxidants are thought to play an effective role in breaking this vicious cycle, we used the antioxidant compound Twendee X (TwX) to verify the effect of TwX on cancer cells through in vivo and in vitro experiments. Human colon cancer RPMI4788 cells are used as cancer cells. In in vitro experiments, the addition of 60 μg/ml of TwX to RPMI4788 cell culture medium slowed cell proliferation from 1 hour after addition, whereas the same concentration of the antioxidant Vitamin C did not inhibit cell proliferation. The inhibition of cell proliferation by the addition of TwX was not observed in normal cells. A mouse model of lung metastasis was created by transplanting RPMI cells (2 x 104 cells/ml) into nude mice via intravenous tail injection. After 6 weeks of treatment with TwX (40 mg/kg/day), blood samples were taken, and oxidative stress in the plasma, lung　metastasis, and NK activity was measured. The results showed that the oxidative stress level of the tumor-bearing mice was twice as high as that of normal mice, but TwX suppressed the increase from the normal level to less than half. Lung weights were also significantly lighter than controls. NK activity was significantly lower NK activity than normal mice, while the TWX group had twice higher NK activity as the carriers and 1.5 times higher than the normal mice. These results suggest that TwX may suppress cancer metastasis by suppressing elevated oxidative stress, thereby increasing NK activity, which is cancer immunity. However, similar results may not be obtained in the case of single-component antioxidants. We believe that the results of this study will help antioxidants in cancer treatment and prevention. References: 1. Composition for protection against cell-damaging effects. WO/2013/072441 23.05.2013 (patent) 2. Novel compositions and uses thereof WO 2008071790 A3 3. Suplatast tosilate reduces radiation-induced lung injury in mice through suppression of oxidative stress. Yusuke Izumi, Taku Nakashima, Noboru Hattori, Free Radic Biol Med.2019;136:52-59