YOSHIKAWA INTERNATIONAL SYMPOSIUM (2ND INTL. SYMP. ON OXIDATIVE STRESS FOR SUSTAINABLE DEVELOPMENT OF HUMAN BEINGS)

Voice and swallowing function are critical functions for human life which is supported by intriguing motion of pharynx and larynx. Swallowing function is complicated consisting of the motion of tongue, soft palate, pharyngeal muscles, laryngeal elevation, and the vocal folds.
Reactive oxygen species (ROS) affects the whole organs and their functions, which deteriorate vocal and swallowing function with age or diseases. Dysphagia causes sarcopenia, frail, and aspiration pneumonia which occasionally causes death. It is important to maintain swallowing function as well as vocal function to keep the body in healthy status.
Twendee X, the strongest anti-oxidant, can maintain the function of the pharynx and larynx by reducing ROS. Our previous data indicated that reduction of ROS leads to maintenance of the vocal folds against aging or injury. We have also confirmed that Twendee X can maintain the vocal function of professional singers.
To date, we have established a dysphagia model of guinea pig by resecting nerve branches to the thyropharyngeal muscle. This model represents motor-related dysphagia which is often observed in elderly or patients with neuromuscular diseases. In this model, the animals became unable to eat immediately after the surgery, and lost weight for about 1 week, but they recovered by compensation. When the animals were fed with Twendee x, the immediate reduction of food intake was prevented possibly because of maintenance of the muscles.
Twendee X is thought to be effective for maintenance of voice and swallowing function.

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 Eyes Co., Ltd., 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 (Tmt) 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 Tmt and their pregnancy was monitored. As a result, 9 pairs became pregnant, and the rate was 32% (9/28). Pregnancy rate is about 11% for a couple of more than two years of infertility when they receive infertility treatment at hospital. Tmt showed almost 3 times higher pregnancy rate than infertility treatment at hospital. The possibility of antioxidant treatment for infertility was suggested.

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.

[Purpose]
Many studies have shown the importance of keeping oral health to prevent various diseases. Neutrophils (PMNs) play an important role to keep oral health by its bactericidal activity such as reactive oxygen species (ROS) production and phagocytosis. We investigated the effects of lysed and heat-treated Enterococcus faecalis FK-23 (LFK) on ROS production and phagocytosis of human PMNs and its mechanism.
[Method]
I. PMNs (1x10⁶ cells/ml) isolated from peripheral blood were stimulated with water-soluble LFK (5 mg/ml) and/or phorbol 12-myristate 13-acetate (PMA). Furthermore, the mechanism of ROS production was examined using each inhibitor. Then, ROS production was measured by MCLA-dependent chemiluminescence for a total 300s time. II. PMNs (1x10⁶ cells/ml) were incubated for 1h at 37℃ with/without LFK. Then, carboxylate-modified fluorescent beads were added to PMNs suspension and incubated for 30 min at 37℃. After that, phagocytosis was measured by flow cytometry.
[Results and Conclusion]
LFK transiently increased ROS production from PMNs without change in total amounts. This effect was decreased by some inhibitors of mitochondrial complex and protein kinase C (PKC) inhibitor. Thus, LFK might increase the reactivity of PMNs via PKC by enhancing mitochondrial functional ability or phagocytosis ability. These results suggest that LFK may increase bactericidal activity of PMNs to improve oral health.

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.

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g., phagocytosis) and pathological conditions (e.g., cancer) during episodes involving Oxidative Stress conditions. 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. Nanometer-sized electrochemical probes are 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 and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.
Application of this method will be presented for detection of ROS/RNS in human cancer cells and for demonstrating the existence of homeostatic control in phagolysosomes during phagocytosis by macrophages.
We will also show 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 mitochondrions.

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.

COVID-19 is the shocking viral pandemics of this year which affected the health, economy, communications, and all aspects of social activities all over the world. Early diagnosis of this viral disease is very important since it can prevent lots of mortalities and care consumption.
The functional similarities between COVID-19 and COVID-2 in inducing acute respiratory syndrome lightened our mind to find a diagnostic mechanism based on early traces of mitochondrial reactive oxygen species (ROS) overproduction as lung cells’ dysfunctions induced by the virus. We designed a simple electrochemical sensor to selectively detect the intensity of ROS in the sputum sample (with a volume of less than 500µl). Comparing the results of the sensor with clinical diagnostics of more than 140 normal and involved cases resulted in a response calibration with accuracy and sensitivity both 97%. Testing the sensor in more than 4 hospitals shed promising lights in ROS based real-time tracing of COVID-19 from the sputum sample.

Sigma S (s^s) controls the synthesis of resistance proteins in stationary pathogenic bacteria [(e.g., Escherichia coli (UPEC)]. Deletion of the rpoS gene rendered E. coli more sensitive to bactericidal antibiotics (BAs): gentamicin, nnorfloxacin and ampicillin. Proteomic analysis implicated a weakened antioxidant defense (AD). Use of the psfiA genetic reporter, 3-(p-hydroxyphenyl) fluorescein (HPF) dye, and Amplex Red showed that BAs generated more oxidative stress (OS) in the mutant. Co-administration of the antioxidant N-acetyl cysteine (NAC) and treatment under anaerobic conditions decreased drug lethality of the mutant, further indicating AD involvement. The greater OS in this strain results from impaired capacity to quench endogenous ROS, e.g, respiration- linked electron leakage. Infection by UPEC in mice showed that AD was important for UPEC antibiotic reistance also in vivo. Disruption of AD by eliminating quencher proteins, or those of pentose phosphate pathway (which provides NADPH for quenching oxygen radicals) also generated greater OS and killing by BAs. Thus, BAs kill stationary-phase bacteria also by generating OS, and targeting AD can therefore enhance their efficacy. Using bioinformatics, small molecule compounds were identified towards this end, and initial results have given promising results. In space flights, astronauts often suffer from UPEC infection. The EcAMSat mission, using a highly sophisticated microfluidic system showed that UPEC missing s^s had increased sensitivity to gentamicin also in space. We have also developed method for determining resistance at single cell level. Together, these results promise to provide powerfull means to combat bacterial antibiotic resistance.

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.

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