Ozawa International Symposium (3rd Intl. Symp. on Oxidative Stress for Sustainable Development of Human Beings)

Carbon quantum dots (CQDs), depending on their surface functionalization, have found utility across a number of biological applications [1-4].
Here, we demonstrate that individual CQDs can serve multifunctional roles, which is a key requisite for the development of drugs for neurodegenerative disorders such as Parkinson’s disease (PD) [5].
Na-citrate-, phenylboronic acid-, and 4-aminophenylboronic acid-derived CQDs were evaluated for their ability to intervene in amyloid-forming trajectories and restore intracellular homeostasis under oxidative stress.
While all three CQDs were able to prevent the soluble-monomer to mature-fibril conversion of hen-egg white lysozyme, Na-citrate- and 4-aminophenylboronic acid-derived CQDs in particular were also able to scavenge reactive oxygen species when exposed to a broad spectrum of free radical generators. Furthermore, Na-citrate and 4-aminophenylboronic acid CQDs preincubated in a neuroblastoma-derived SHSY-5Y cell line were able to rescue it from rotenone (neurotoxicant)-induced cell death while demonstrating no difference in cytotoxicity up to 120 μg/mL (compared to untreated controls). The CQDs were also able to restitute cells from rotenone-elicited apoptosis and oxidative stress. Together, these results suggest that CQDs can serve as a one-pot solution for multifactorial diseases such as PD by serving as a neuroprotectant. Importantly, the use of CQDs as multifactorial interventional agents represents a sustainable mechanism by which to advance biomedicine.

There are various allergic diseases existing. In Japan, about 1 in 2 of the population suffers from some sort of allergic disease, making it a national disease. Familiar examples include asthma, rhinitis, atopic dermatitis, and hay fever. These symptoms are largely related to oxidative stress, which increases due to inflammation caused by allergens such as pollen and dust invading the body. As the number of patients increases and the burden of medical costs rises, a treatment without side effects is needed.
Twendee X (TwX) is a safety-tested, mixed antioxidant supplement composed of vitamins and amino acids. To evaluate whether TwX improves these symptoms, a questionnaire survey was conducted on the symptoms of several allergic diseases. The results showed that about 80% of the participants showed improvement in most diseases after taking TwX compared to before.
The results suggest TwX can contribute to the future treatment of allergies as well as to the reduction of health care costs.

Glutamate (Glu) is one of the most important excitatory transmitter in the nervous system. Impairment of its vesicular release due to β-amyloid (Aβ) oligomers liberation in the brain is thought to participate in pathological processes leading to Alzheimer’s disease. However, before this work [1] it was unclear whether soluble Aβ42 oligomers affect intravesicular amounts of Glu or its release in the brain, or both.
Nanoelectrochemical measurements which will be presented used amperometric nanowire Glu biosensors to test single Glu-generating neuronal varicosities. This revealed that soluble Aβ42 oligomers first caused a dramatic increase in vesicular Glu storage and stimulation-induced release, accompanied by a high level of parallel spontaneous exocytosis. This was shown to ultimately result in the depletion of intravesicular Glu content enforcing greatly reduced Glu release.
Molecular biology tools and mouse models of Aβ amyloidosis have further established that the transient hyperexcitation observed during the primary pathological stage was mediated by an altered behavior of VGLUT1 responsible for transporting Glu into synaptic vesicles. Thereafter, an overexpression of Vps10p-tail-interactor-1a, a protein that maintains spontaneous release of neurotransmitters by selective interaction with t-SNAREs, resulted in a depletion of intravesicular Glu content, triggering advanced-stage neuronal malfunction. These findings are expected to open perspectives for remediating Aβ42-induced neuronal hyperactivity and neuronal degeneration in the context of initiation of Alzheimer disease.
The authors would like to acknowledge support from joint sino-french CNRS IRP NanoBioCatEchem. CA thanks Xiamen University for his Distinguished Visiting Professor position.

Ulcerative colitis (UC) is defined as a diffuse, nonspecific inflammation of unknown origin that continuously damages the colonic mucosa from the rectal side, often leading to erosions and ulcers. It is associated with frequent abdominal pain and sometimes diarrhea with bloody stools. There are more than 160,000 cases of the disease in Japan, but no cure has yet been established.
Although the exact cause of the disease has not yet been clarified, oxidative stress has long been implicated, as it has been observed that reactive oxygen species (ROS) increase in inflamed tissues. Reduction of ROS and improvement of oxidative stress may improve UC.
Twendee X (TwX, Oxicut®︎) is an antioxidant supplement containing eight active ingredients consisting of vitamins and amino acids; Twendee Mtcontrol (TwM, Mtcontrol®︎) is a supplement with seven active ingredients added to TwX to increase its antioxidant capacity. The therapeutic effects of antioxidants were tested in a mouse model of UC with dextran sodium sulfate (DSS), a commonly used treatment in UC research. TwX and TwM were found to ameliorate the negative physical effects of UC in the DSS model.
The results suggest that antioxidant treatment may be effective for UC.

Systemic sclerosis (SSc) is a disease of hardening of the skin and internal organs which is presumed to be caused by a complex combination of autoimmunity, fibrosis, and vascular lesions, yet the detailed mechanism is unknown. There is no established treatment, and women are at higher risk of developing the disease than men.
The involvement of reactive oxygen species (ROS) in Raynaud's phenomenon and anti-PDGFR (Platelet-Derived Growth Factor Receptor)-stimulating autoantibodies in SSc has been suggested. In fact, dermal fibroblasts of SSc patients produce large amounts of ROS themselves, which induce collagen synthesis. Therefore, SSc is considered to be closely associated with elevated levels of oxidative stress.
Twendee X (TwX, Oxicut®︎) is an antioxidant compound with mitochondrial protection properties that contains eight different ingredients, and studies have shown that TwX can potentially improve intestinal microbiota, chronic fatigue syndrome, sleep apnea, and more.
We tested whether TwX has an ameliorative effect on SSc using a hypochlorous acid (HOCl)-induced SSc mouse model. Mouse SSc induced by oxidants such as HOCl is said to exhibit clinical and biological manifestations very similar to those observed in human SSc, including skin and visceral fibrosis, vascular damage, and autoimmunity.
One month prior to the 6-week HOCl administration, mice were given drinking water (Control) or TwX-containing water ad libitum, and were dissected 2 days after the last HOCl administration to compare their skin lesions, immune status, and major protein expression with the Control group. The study showed that TwX had a favorable effect on clinical, immune, and physiological parameters in HOCl-induced SSc mice.
These results suggest that TwX may aid in the treatment and prevention of SSc.

Vitamin E deficiency accelerates in vivo oxidation and is closely related to aging. Accumulation of oxidative products increases risk of developing serious disorders such as dementia and cardiovascular events [1]. Normally, our bodies maintain a balance between oxidation and reduction, but the redox balance gradually collapses as we age. To prevent oxidation, we should take supplements. The supplement market is growing year by year all over the world. One combination supplement is Twendee X, which contains 8 substances [2]. However, the detailed scientific evidence for Twendee X has not yet been elucidated. In this study, long-term vitamin E-deficient mice were fed Twendee X and measured cognitive and motor functions using the Morris water maze and rota-rod tests [3]. Vitamin E-deficient mice had significantly impaired cognitive and motor functions, learning rate was comparable to normal aged mice. Treatment with Twendee X significantly improved both dysfunctions. However, serum indices did not differ with or without Twendee X. These results indicate that Twendee X has a potential as a powerful antioxidant supplement through protection against in vitro oxidation.

Uropathogenic Escherichia coli (UPEC) causes urinary tract infections, e.g., cystitis, which are treated by gentamicin. The protein ss, encoded by the rpoS gene, controls E. coli general resistance. We discovered that rpoS deletion renders UPEC more sensitive to Gm and other bactericidal antibiotics, and proteomic analysis suggested a weakened antioxidant defense as the reason. Reactive oxygen species (ROS) detectors (psfiA gene reporter and appropriate chemicals) indicated greater ROS generation by Gm in the mutant. Gm treatment along with an antioxidant, or under anaerobic conditions (that prevent ROS formation), decreased drug lethality. Treating UPEC infection of mice bladder corroborated these findings in vivo. Thus, oxidative stress produced by insufficient quenching of metabolic ROS accounted for greater sensitivity of the mutant. E. coli strains missing antioxidant proteins also generated greater ROS and were also more sensitive to Gm. These lacked the ROS quencher proteins, (e.g., SodA/SodB; KatE/SodA), or the pentose phosphate pathway proteins, which provide NADPH (e.g., Zwf/Gnd; TalA) required by the quenchers. We have recently made similar findings with norfloxacin. Use of a microfluidic device indicated that the results applied to a single cell level. Gm is known to kill bacteria by inhibiting protein synthesis, but UPEC has developed resistance to this mode of killing. Therefore, these findings provide a timely means of restoring Gm effectiveness by curbing antioxidant proteins. Using bioinformatic approaches, we have identified several small molecules that inhibit these proteins and can enhance Gm effectiveness. In space flights, astronauts often suffer from cystitis. Bacterial gene regulation can differ in normal vs. microgravity (MG) experienced during space flights. However, the “EcAMSat” Stanford/NASA mission showed that ss-controls Gm resistance also in MG. EcAMSat employed a free- flying “nanosatellite” equipped with a technically sophisticated microfluidic system for autonomous determination of UPEC sensitivity to Gm and its telemetric transmission in real time during space flight to Earth. Bacterial multidrug resistance (MDR), such as the one regulated by the emrRAB operon and the EmrR protein is a major public health problem. Its activation is due to alteration in the EmrR protein conformation, which too can be prevented by small molecules and bioinformatic approaches that we have pursued. Another such pump along with penetration barriers contribute to bacterial biofilm antibiotic resistance. Several collaborators contributed to this work; they will be identified in the presentation.

A decreased ability to maintain consistent energy production and a low level of oxidative stress in brain cells have been identified among the underlying factors for the development of various neurodegenerative conditions, such as Alzheimer's disease (AD). Mitochondria play a central role in maintaining energy homeostasis and generating reactive oxygen species in cells. Mitochondrial dysfunction is well documented in AD patients.  However, recent evidence has revealed the important roles of mitochondria signaling in adapting to energy stress, promoting survival and function, and restoring energy levels. Mitochondria signaling coordinates several crucial cellular mechanisms, including maintaining energy balance, metabolic functions, autophagy, epigenetic modifications, regulating inflammation, providing antioxidant protection, and regulating cell death. Thus, harnessing mitochondrial signaling could represent a novel therapeutic strategy to improve and maintain multiple essential mechanisms of cell survival. We have developed small molecules that target mitochondria and mildly reduce the activity of mitochondrial complex I, resulting in the induction of mild energetic stress. This approach has proven effective in delaying the onset of AD in multiple transgenic mouse models and promoting healthy aging in wild-type mice [1-6]. The mechanism of action involves activating various neuroprotective mechanisms involved in adaptive stress response, which ultimately results in promoting health and longevity, and restoring mitochondria function and energy balance in the brain.

Reactive oxygen species (ROS) and nicotinamide adenine dinucleotide (NADH) are important intracellular redox-active molecules involved in various pathological processes including inflammation, neurodegenerative diseases, cancer, etc. However, their fast dynamic changes and mutual regulatory kinetic relationship during these biological processes were hard to simultaneously investigate intracellularly at single cells level. 

A dual-channel nanowire electrode integrating two conductive nanowires [1], one functionalized with platinum nanoparticles [2], and the other with conductive polymer [3], was nanofabricated to allow the selective and simultaneous real-time monitoring of intracellular ROS and NADH releases by mitochondria in single living MCF-7 tumoral cells stimulated by resveratrol. The release of ROS was observed to occur a few tenths of a second before that of NADH. Beyond the importance of such an information impossible to acquire by other means, this work established the feasibility of simultaneously monitoring two intracellular species and their kinetics relationships over sub-second time scales. It is expected that this concept will benefit to a deeper understanding of mutual regulatory relationship between crucial intracellular molecular markers during physiological and pathological processes as well as for evaluating medical treatments.

The authors would like to acknowledge support from joint sino-french CNRS IRP NanoBioCatEchem. CA thanks Xiamen University for his Distinguished Visiting Professor position.

The vocal fold is a vibratory mucosa essential for creating voice, but it is fragile after injury, and once it is scarred, voice becomes hoarse and harsh, which is difficult to recover. Regeneration of the mucosa after injury is important to avoid such dysphonia. Our previous research indicated that oxidative stress is one of the leading issues that causes scarring of the vocal fold, and anti-oxidant can preserve the tissue. The current clinical trial examined the effects of the strong anti-oxidant, Twendee X (TWX), on regeneration of the vocal fold after surgery.
Materials and Methods: Ten patients received surgery for vocal fold pathologies including polyp, leukoplakia, CIS, etc. They were randomly assigned to TWX group which were treated with TWX perioperatively and control group without TWX treatment. The vocal fold function after the surgery was assessed at 1 to 3 months using stroboscopy and acoustic parameters.
TWX group indicated significantly better vibratory parameters on stroboscopy at 1 month through 3 months as compared to the control group. TWX group also showed better GRBAS scale and loudness of the voice.
TWX was shown to be effective for better recovery of the vocal fold after surgery with less scarring.

Alla I. Potapovich ^a, Tatyana V. Kostyuk ^a, Tatsiana G. Shutova ^b, Vladimir A. Kostyuk^a*

^a Byelorussian state university, Niezaližnasci Avenue, 4, 220030, Minsk, Belarus

^bInstitute of Chemistry of New Materials, National Academy of Sciences of Belarus, Skaryny Street, Minsk 220141, Belarus 

The antiradical activity of flavonoids in simple biochemical systems was discovered more than 30 years ago [1]. Subsequently, the antioxidant and anti-inflammatory effects of flavonoids were demonstrated in human cells upon exposure to various inflammatory stimuli [2, 3]. However, despite their numerous biological activities, the clinical use of flavonoids is limited mainly due to their low water solubility, which results in poor cellular uptake and poor permeability of flavonoids through the skin. To overcome these limitations, potential drug molecules can be incorporated into liposomes or polymer nanoparticles, which have great potential as drug carriers [4]. In this work, we evaluated the cellular effects of native and microstructured flavonoids. Microcrystals of quercetin and resveratrol coated with (polyallylamine hydrochloride/sodium polystyrenesulfonate)₄ or (chitosan/dextran sulfate)₄ shells were prepared by layer-by-layer assembly. Cultured human HaCaT keratinocytes were exposed to UV-C, after which the cells were incubated with native and microstructured flavonoids. Phosphorylation of histones H2AX, DNA damage, cell viability and integrity were assessed. The data obtained indicate that native and microstructured flavonoids added immediately after exposure to UV-C increased cell viability in a dose-dependent manner, however, the effectiveness of microstructured quercetin was more pronounced than that of the native compound. It has been shown that flavonoids can activate the process of repairing genetic damage, while the coated quercetin (chitosan/dextran sulfate)₄ was more effective than the native compound in reducing the amount of DNA damage in the nuclei of keratinocytes exposed to UV-C radiation.

Hangovers after drinking, acne on face, constipation, lack of sleep and lethargy, worries and anxiety, and lifestyle-related diseases all have a significant impact on the quality of daily life. Quality of life is based on the individual's physical and mental health. For example, hangover reduces the quality of normal life. Improving the quality of daily routines is important for healthy longevity and a fulfilling life.
Twendee X (TwX, Oxicut®︎) and Twendee S (TwS, SUPALIV®︎) are composition of antioxidant supplements containing a total of eight vitamins, amino acids and CoQ10.TwX and TwS contain the same ingredients but in different mixture ratio and amount.
Twendee Mtcontrol (TwM, Mtcontrol®︎) is based on TwX with seven additional ingredients. In this study, consumption was monitored after 1-2 months of of TwS, TwX, and TwM.
TwS taken to prevent hangover after drinking, the unpleasant smell by themselves of the next morning disappeared at the same time. TwX taken to improve acne on the face, the patient slept well at the same time. TwM taken to improve fatigue, the patient had an improved bowel movement at the same time. As shown above, a wide range of improvements in daily life have been reported.
These results suggest that TwS, TwX, and TwM may regulate the rhythm of daily life.

Melatonin (N-acetyl-5-methoxytryptamine) was discovered as a secretory product of the mammalian pineal gland in 1958. At the time, melatonin was thought to be exclusively synthesized and released from the pineal gland at night with the duration of the nocturnal melatonin elevation being related to the length of the daily dark period. Since the duration of the night length varies seasonally, melatonin rhythm was originally believed to regulate only circadian (sleep/wake cycle) and circannual (annual reproductive fluctuations in seasonally breeding species) rhythms, i.e., to function as both a physiological clock and as a calendar. Subsequently, melatonin was also identified in non-mammalian vertebrates, all of which have a pineal gland, and soon thereafter in invertebrates including unicells and in plants, none of which have a pineal gland [1]; thus, melatonin synthesis is obviously not exclusively of pineal origin. 

Within the last three decades, melatonin or its synthesizing enzymes have been  found in prokaryotes including true bacteria and archaebacteria (archaeans). Thus, melatonin presumably evolved in prokaryotes about 3.0-2.5 billion years ago.  During eukaryogenesis, which occurred an estimated 2.5 – 2.0 billion years ago, prokaryotes were engulfed by early eukaryotes for food and energy; the prokaryotes eventually established a symbiotic relationship with the eukaryotes that had phagocytized them, and they eventually evolved into mitochondria.  The origin of mitochondria from prokaryotes is a widely accepted theory.  The melatonin-synthesizing activity of the prokaryotes was retained in all eukaryotic mitochondria [2]. As a result, the mitochondria of many cells of present-day eukaryotes may also produce melatonin; thus, vertebrates have two sources of melatonin. The pineal gland of vertebrates discharges melatonin into the blood and cerebrospinal fluid (the releasable pool) for circadian and circannual rhythm regulation.  Additionally, all plant and animal species synthesize melatonin in their mitochondria, which is used as an antioxidant in the cell of origin (the non-releasable pool).

Numerous publications have shown melatonin to be a multifaceted and highly efficient direct free radical scavenger and an indirect antioxidant. Melatonin quenches highly destructive reactive oxygen (ROS) and reactive nitrogen species as does many of its metabolites in what is referred to as the antioxidant cascade.  Additionally, melatonin promotes the activities of numerous antioxidant enzymes while inhibiting pro-oxidant enzyme activities.  Since mitochondria are a major source of ROS generation when electrons leak from complexes I and II of the electron transport chain and chemically reduce nearby ground-state oxygen to the superoxide anion radical, melatonin synthesis in mitochondria is ideally situated to reduce free radical damage [3].  Supplemental melatonin limits elevated oxidative stress in many pathologies, such as in models of ischemia/reperfusion including stroke and heart attack, exposure of animals to heavy metals, environmental particulate matter, microplastics, hyperglycemia and atherosclerosis, excessive alcohol, conventional medications including chemotherapies, illegal drugs, ionizing radiation, neurodegenerative diseases, and those caused by dysfunctional mitochondria. These studies have been extended to humans where melatonin has similar protective actions. Some of the functions of melatonin involve transmembrane receptors which are widely distributed while other actions are receptor-independent [4]. 

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Melatonin (N-acetyl-5-methoxytryptamine) was discovered as a secretory product of the mammalian pineal gland in 1958. At the time, melatonin was thought to be exclusively synthesized and released from the pineal gland at night with the duration of the nocturnal melatonin elevation being related to the length of the daily dark period. Since the duration of the night length varies seasonally, melatonin rhythm was originally believed to regulate only circadian (sleep/wake cycle) and circannual (annual reproductive fluctuations in seasonally breeding species) rhythms, i.e., to function as both a physiological clock and as a calendar. Subsequently, melatonin was also identified in non-mammalian vertebrates, all of which have a pineal gland, and soon thereafter in invertebrates including unicells and in plants, none of which have a pineal gland [1]; thus, melatonin synthesis is obviously not exclusively of pineal origin. 

Within the last three decades, melatonin or its synthesizing enzymes have been found in prokaryotes including true bacteria and archaebacteria (archaeans). Thus, melatonin presumably evolved in prokaryotes about 3.0-2.5 billion years ago.  During eukaryogenesis, which occurred an estimated 2.5 – 2.0 billion years ago, prokaryotes were engulfed by early eukaryotes for food and energy; the prokaryotes eventually established a symbiotic relationship with the eukaryotes that had phagocytized them, and they eventually evolved into mitochondria.  The origin of mitochondria from prokaryotes is a widely accepted theory.  The melatonin-synthesizing activity of the prokaryotes was retained in all eukaryotic mitochondria [2]. As a result, the mitochondria of many cells of present-day eukaryotes may also produce melatonin; thus, vertebrates have two sources of melatonin. The pineal gland of vertebrates discharges melatonin into the blood and cerebrospinal fluid (the releasable pool) for circadian and circannual rhythm regulation.  Additionally, all plant and animal species synthesize melatonin in their mitochondria, which is used as an antioxidant in the cell of origin (the non-releasable pool).

Numerous publications have shown melatonin to be a multifaceted and highly efficient direct free radical scavenger and an indirect antioxidant. Melatonin quenches highly destructive reactive oxygen (ROS) and reactive nitrogen species as does many of its metabolites in what is referred to as the antioxidant cascade.  Additionally, melatonin promotes the activities of numerous antioxidant enzymes while inhibiting pro-oxidant enzyme activities. Since mitochondria are a major source of ROS generation when electrons leak from complexes I and II of the electron transport chain and chemically reduce nearby ground-state oxygen to the superoxide anion radical, melatonin synthesis in mitochondria is ideally situated to reduce free radical damage [3].  Supplemental melatonin limits elevated oxidative stress in many pathologies, such as in models of ischemia/reperfusion including stroke and heart attack, exposure of animals to heavy metals, environmental particulate matter, microplastics, hyperglycemia and atherosclerosis, excessive alcohol, conventional medications including chemotherapies, illegal drugs, ionizing radiation, neurodegenerative diseases, and those caused by dysfunctional mitochondria. These studies have been extended to humans where melatonin has similar protective actions. Some of the functions of melatonin involve transmembrane receptors which are widely distributed while other actions are receptor-independent [4].                                            

 In multicellular organisms, cells typically communicate by sending and receiving chemical signals. Chemical messengers involved in the exocytosis of neuroendocrine cells or neurons are generally assumed to only originate from the fusing of intracellular large dense core vesicles (LDCVs) or synaptic vesicles with the cellular membrane following stimulation. Accumulated evidence suggests that exosomes - one of the main extracellular vesicles (EVs) - carrying cell-dependent DNA, mRNA and proteins etc. play an essential role in cellular communication. Due to experimental limitations, it has been difficult to monitor the real-time release of individual exosomes, which impedes a comprehensive understanding of the basic molecular mechanisms and the functions of exosomes. We introduce amperometry with microelectrodes to capture the dynamic release of single exosomes from a single living cell, distinguish them from other EVs and identify between the molecules inside exosomes and those secreted from LDCVs. We show that similar to many LDCVs and synaptic vesicles, exosomes released by neuroendocrine cells also contain catecholamine transmitters. This finding reveals long-range chemical communication via exosome-encapsulated chemical messengers and a potential interconnection between the two release pathways, changing the canonical view of exocytosis of neuroendocrine cells and possibly neurons. This defines a new mechanism for chemical communication at the fundamental level and opens new avenues in the research of the molecular biology of exosomes in the neuroendocrine and central nervous systems. In addition, electrochemically renewable surface-enhanced Raman spectroscopy (SERS) microprobes enable real-time dynamic measurement of ROS released from single cells, which lays the foundation for the development of the integrated technology of electrochemistry and Raman.                                                                                                                                                                                                                                                              

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 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. 

Why does baby smell good while the elderly has a distinctive odor? According to a publication by Shiseido Research Center in 2001(1), 2-nonenal is an unsaturated aldehyde with unpleasant odor caused by oxidative degradation reactions of ω-7 unsaturated fatty acids such as palmitoleic acid and bacenic acid found on the skin surface. This means that age-related oxidative stress is a cause of 2-nonenal. In other words, an increase in oxidative stress due to aging is the cause of age-related odor. We measured the change in age-related odor by reducing oxidative stress. Antioxidant composition Twendee X (TwX) was given who has age related odor for 4 weeks. The portable odor sensor KUNKUN BODY (Konica Minolta) was used for 10 participants to measure aging odor. Seventy-four participants were also monitored questionnaires.
Several participants whose odor KUNKUN BODY scores were 80-100, which dropped scores to zero after 3 weeks. Seventy-two of the 74 cases showed a decrease in age-related odor.
Oxidative stress increases with age and is a cause of many diseases. The development of age-related odors suggests increased oxidative stress. Reduction of age-related odors with the antioxidant composition TwX is suggested not only to reduce bad odors but also to maintain health.

Oxidative stress has long been considered to contribute to brain aging and age-related neurodegenerative disorders including Parkinson’s and Alzheimer’s disease¹.  Data over the last few years suggests that one important mechanism driving neuropathology associated with these disorders is a process known as cellular senescence. In response to stressors including agents which elicit oxidative or proteotoxic stress, cells may undergo conversion to a resting state where they are not capable of replicating and forming tumors. However, maintenance of these cells over long periods of time in the aging organism can result in increased inflammatory events known as the senescent associated secretory phenotype or SASP which is damaging to neighboring tissues, including within the brain. Here, I will present data suggesting that not only does cellular senescence occur in conjunction with age-related neurodegenerative diseases, but that elimination of senescent cells may constitute a therapeutic avenue for these disorders^2-4. This is dependent however on selectivity of senescent cell removal which avoids off-target effects on other cell types. 

Background: The vocal fold is a vibratory mucosa essential for creating voice, but it is fragile after injury, and once it is scarred, voice becomes hoarse and harsh, which is difficult to recover. Our previous research indicated that oxidative stress can cause scarring of injured vocal folds, and anti-oxidant can avoid the scarring. Basic fibroblast growth factor (FGF) has been proved to have anti-oxidant effects in vascular sclerosis. The current study examined the effects of FGF for preservation of the vocal fold after injury.
Materials and Methods: Sprague Dawley rats were used as the model of vocal fold injury. Unilateral vocal fold was resected and immediately treated with FGF or saline injection (sham control). Scarring of the vocal folds were assessed at 2 months after the injury.
Results: FGF group showed better wound healing with less scarring as compared to the sham. Hyaluronic acid was significantly better preserved. Gene expression analyses indicated significantly higher expression of HAS and HGF in FGF group.
Conclusion: FGF was revealed to prevent vocal fold scarring through anti-fibrotic and possibly anti-oxidant effects.

Stroke is a major disease burden worldwide and over 85% incidences are ischemic stroke. Tissue plasminogen activator (t-PA) is the only FDA approved drug for acute ischemic stroke, but its use is limited with the restrictive time window within 4.5 hours and the complications of the blood brain barrier (BBB) disruption and hemorrhagic transformation (HT). Exploring molecular targets to reducing the BBB permeability and HT incidence is timely important to develop novel therapeutic approaches for reducing the complications and increasing outcome in ischemic stroke.

Oxidative/nitrosative stress and neuroinflammation are two crucial pathological processes in ischemic stroke. Reactive nitrogen species (RNS) and high mobility group box 1 protein (HMGB1) are important cytotoxic factors contributing to cerebral ischemia-reperfusion injury. Peroxynitrite (ONOO^-) is a representative RNS but its roles in mediating inflammation signaling in the blood-brain barrier (BBB) damage and hemorrhagic transformation (HT) in ischemic brain injury remain unclear. In basic study, our group has tested the hypothesis that ONOO^- could directly mediate HMGB1 signaling in ischemic brains with delayed t-PA treatment [1,2]. In clinical studies, we found that plasma nitrotyrosine (NT, a surrogate marker of ONOO^-) was positively correlated with HMGB1 level in acute ischemic stroke patients. Plasma levels of nitrotyrosine and HMGB1 were increased in t-PA-treated ischemic stroke patients with hemorrhagic transformation [2]. Furthermore, animal experiments revealed that FeTmPyP, a representative ONOO^- decomposition catalyst (PDC), significantly inhibited the activations of HMGB1/TLR2/MMP-9 signaling cascades, preserved collagen IV and tight junction claudin-5 in ischemic rat brains with delayed t-PA treatment. ONOO^- donor SIN-1 directly induced HMGB1/TLR2/MMP-9 signaling cascades in naive rat brains in vivo and brain microvascular endothelial b.End3 cells in vitro. Those results suggest that ONOO^- could activate HMGB1/TLR2/MMP-9 signaling, contributing to the BBB disruption and HT in ischemic brain injury [1-2]. We also demonstrate that ONOO^- mediated MMPs and NLRP3 inflammasome could aggravate the BBB damage and HT and induce poor outcome in ischemic stroke with hyperglycemia [3]. Thus, the interactions of ONOO^- and inflammation factors play crucial roles in the BBB disruption and HT. For drug discovery, we found that medicinal plant compounds, such as baicalin and glycyrrhizin, and a classic Chinese medicinal formula named Angong Niuhuang Pill regulated the RNS/Cav-1/MMP signaling cascades, decreased the mortality rate, attenuated the BBB disruption, HT, brain swelling, and improved neurological outcomes in the ischemic stroke rat model with delayed t-PA treatment [1-4]. 

In conclusion, targeting peroxynitrite-mediated inflammation signaling cascades could be a potential adjuvant therapy to prevent hemorrhagic transformation and improve outcome in ischemic stroke with delayed t-PA treatment, potentially extending the therapeutic window for thrombolysis. 

A collection of numerous casses and stories worldwide will be described on the effect of Twendee X and MTControl on the people’s life. They range from improving the quality of life of normal healthy people at various stages of their life and more importantly on people that suffer from various diseases before, during or after treatments. This collection help develop scientific placebo based studies on the effect of Twendee. The success stories are numerous and impressive.

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 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.

Aging is a universal degenerative disease. Aging is classified into physiological and pathological. The progression rate of pathological aging varies among individuals, and reactive oxygen species is deeply involved in pathological aging. One of the most serious phenomena during aging is dementia. To prevent age-related neurodegenerative disorders, treatment with antioxidants is effective. Twendee Mt Control which is a supplement contains 15 substances and anyone can buy it. However, the detailed scientific evidence is not enough. In the present study, we treated combination supplement to aged mice, and measured their cognitive and motor functions. 

We purchased aged mice and treated them with Twendee Mt Control for one month. Cognitive and motor functions were measured by the Morris Water maze and rota-rod test [1]. Normal aged mice had significantly reduced learning ability. Treatment with Twendee Mt Control improved significantly. Twendee Mt Control significantly improved time-to-fall scores on the rota-rod test. Finally, treatment with Twendee Mt Control showed a high training effect in the treadmill test. These results indicated that treatment with Twendee Mt Control has a potential as a powerful antioxidant supplement through protection against age-related neuronal and motor dysfunctions.

Many different types of dementia exist, with Alzheimer's disease (AD) accounting for the majority of cases.
The greatest risk factor for AD is aging, which is the result of oxidative stress that has accumulated over the years. In addition, amyloid-β accumulated in the AD brain generates free radicals that cause inflammation in the surrounding neurons, leading to mitochondrial dysfunction and eventual neurodegeneration. In other words, the development of AD is the result of a vicious cycle of oxidative stress, inflammation, and mitochondrial dysfunction established over a long period of time, and the progression of AD is no exception.
The pathogenesis of AD is said to occur 20 years before clinical symptoms appear. Therefore, starting treatment after the onset of symptoms could be too late to be effective.
Twendee X (TwX, Oxicut®︎) is an antioxidant formula shown to have a preventive effect for human mild cognitive impairment (MCI) in a multicenter, randomized, double-blind, placebo-controlled, prospective intervention trial. Previous studies have shown that in diabetes, which hastens cognitive decline, TwX intake not only lowers blood glucose levels but also suppresses autophagy dysfunction. TwX has also been shown to positively affect neurogenesis in the hippocampal dentate gyrus, telomere elongation, and mitochondrial function. This suggests that TwX may prevent or halt the progression of dementia from all angles.
This study suggests that TwX may not only prevent dementia, but may also aid in its treatment.