Green autofluorescence of the skin and fingernails is a novel biomarker for evaluating the risk for developing acute ischemic stroke.

The only existing approach for assessing the risk of developing acute ischemic stroke (AIS) necessitates that individuals possess a strong understanding of their health status. Our research gathered compelling evidence in favor of our hypothesis, suggesting that the likelihood of developing AIS can be assessed by analyzing the green autofluorescence (AF) of the skin and fingernails. Utilizing machine learning-based analyses of AF images, we found that the area under the curve (AUC) for distinguishing subjects with three risk factors from those with zero, one, or two risk factors was 0.79, 0.76, and 0.75, respectively. Our research has revealed that green AF serves as an innovative biomarker for assessing the risk of developing AIS. Our method is objective, non-invasive, efficient, and economic, which shows great promise to boost a technology for screening natural populations for risk of developing AIS.

Methylene blue reduces the serum levels of interleukin-6 and inhibits STAT3 activation in the brain and the skin of lipopolysaccharide-administered mice.

It is valuable to search for novel and economical agents for inhibiting STAT3 activation and blocking increases in IL-6 levels, due to the important roles of STAT3 and IL-6 in inflammation. Since Methylene Blue (MB) has shown therapeutical potential for multiple diseases, it has become increasingly important to investigate the mechanisms underlying the effects of MB on inflammation. Using a mouse model of lipopolysaccharide (LPS)-induced inflammation, we investigated the mechanisms underlying the effects of MB on inflammation, obtaining the following findings: First, MB administration attenuated the LPS-induced increases in the serum levels of IL-6; second, MB administration attenuated LPS-induced STAT3 activation of the brain; and third, MB administration attenuated LPS-induced STAT3 activation of the skin. Collectively, our study has suggested that MB administration can decrease the levels of IL-6 and STAT3 activation - two important factors in inflammation. Since MB is a clinically used and relatively economical drug, our findings have suggested therapeutic potential of MB for multiple inflammation-associated diseases due to its effects on STAT3 activation and IL-6 levels.

NAD+ administration profoundly decreases UVC-induced skin damage by attenuating oxidative stress, inflammation, DNA damage and apoptosis.

Ultraviolet (UV) radiation is a major cause of multiple major skin diseases including skin cancer. It is crucial to discover new agents that can produce profound protective effects on UV-produced skin damage. Using a mouse model, in this study we determined the effects of NAD+ on UVC-induced skin damage and investigated the mechanisms underlying the effects, obtaining the following discoveries: First, UVC-induced skin's green autofluorescence (AF) was highly correlated with the extent of UVC-indued skin's damage; second, NAD+ administration profoundly decreased UVC-induced skin damage; third, NAD+ administration significantly attenuated UVC-induced decreases in the levels of mitochondrial superoxide dismutase and catalase; fourth, NAD+ administration significantly attenuated UVC-induced increase in the level of cyclooxygenase (COX) 2 - a marker of inflammation; fifth, NAD+ administration profoundly attenuated UVC-induced increase in double-strand DNA (dsDNA) damage; and sixth, NAD+ administration profoundly attenuated UVC-induced decreases in the ratios of Bcl-2/Bax - an index of apoptosis. Collectively, our study has found that NAD+ administration can profoundly decrease UVC-induced skin damage by attenuating oxidative stress, inflammation, DNA damage, and apoptosis, suggesting great potential of NAD+ as a protective agent for UVC-induced skin damage. Moreover, our study has further indicated that the skin's green AF is a biomarker for predicting UVC-induced skin damage.

Phenomic Studies on Diseases: Potential and Challenges.

The rapid development of such research field as multi-omics and artificial intelligence (AI) has made it possible to acquire and analyze the multi-dimensional big data of human phenomes. Increasing evidence has indicated that phenomics can provide a revolutionary strategy and approach for discovering new risk factors, diagnostic biomarkers and precision therapies of diseases, which holds profound advantages over conventional approaches for realizing precision medicine: first, the big data of patients' phenomes can provide remarkably richer information than that of the genomes; second, phenomic studies on diseases may expose the correlations among cross-scale and multi-dimensional phenomic parameters as well as the mechanisms underlying the correlations; and third, phenomics-based studies are big data-driven studies, which can significantly enhance the possibility and efficiency for generating novel discoveries. However, phenomic studies on human diseases are still in early developmental stage, which are facing multiple major challenges and tasks: first, there is significant deficiency in analytical and modeling approaches for analyzing the multi-dimensional data of human phenomes; second, it is crucial to establish universal standards for acquirement and management of phenomic data of patients; third, new methods and devices for acquirement of phenomic data of patients under clinical settings should be developed; fourth, it is of significance to establish the regulatory and ethical guidelines for phenomic studies on diseases; and fifth, it is important to develop effective international cooperation. It is expected that phenomic studies on diseases would profoundly and comprehensively enhance our capacity in prevention, diagnosis and treatment of diseases.

UV-induced skin's green autofluorescence is a biomarker for both non-invasive evaluations of the dosages of UV exposures of the skin and non-invasive prediction of UV-induced skin damage.

It is crucial to discover biomarkers for non-invasive evaluations of the dosages of UV exposures to a person during post-UV exposure period, and for non-invasive prediction of UV-induced skin damage. Our current study has obtained findings: UVB exposures produced dose-dependent increases in skin's green autofluorescence (AF) intensity of mice, which were significantly associated with the UVB dosages. The UVC-induced green AF increases were dose dependent, which were highly associated with the UVC dosages. Moreover, both previous reports and our current study have collectively shown significant association between UVB/UVC dosages and UVB/UVC-induced skin damage. Collectively, our study has indicated that the UVB/UVC-induced skin's AF are first biomarkers for both non-invasive evaluations of the dosages of UV exposures to a person during post-UV exposure period and non-invasive and label-free prediction of UVB/UVC-induced skin damage.

A Comprehensive Study of De Novo Mutations on the Protein-Protein Interaction Interfaces Provides New Insights into Developmental Delay.

Mutations, especially those at the protein-protein interaction (PPI) interface, have been associated with various diseases. Meanwhile, though de novo mutations (DNMs) have been proven important in neuropsychiatric disorders, such as developmental delay (DD), the relationship between PPI interface DNMs and DD has not been well studied. Here we curated developmental delay DNM datasets from the PsyMuKB database and showed that DD patients showed a higher rate and deleteriousness in DNM missense on the PPI interface than sibling control. Next, we identified 302 DD-related PsychiPPIs, defined as PPIs harboring a statistically significant number of DNM missenses at their interface, and 42 DD candidate genes from PsychiPPI. We observed that PsychiPPIs preferentially affected the human protein interactome network hub proteins. When analyzing DD candidate genes using gene ontology and gene spatio-expression, we found that PsychiPPI genes carrying PPI interface mutations, such as FGFR3 and ALOX5, were enriched in development-related pathways and the development of the neocortex, and cerebellar cortex, suggesting their potential involvement in the etiology of DD. Our results demonstrated that DD patients carried an excess burden of PPI-truncating DNM, which could be used to efficiently search for disease-related genes and mutations in large-scale sequencing studies. In conclusion, our comprehensive study indicated the significant role of PPI interface DNMs in developmental delay pathogenicity.

Green autofluorescence of the index fingernails is a novel biomarker for noninvasive determinations on the status of tobacco smoking.

It is critical to discover novel biomarkers for tobacco smoking. Our study has indicated the green autofluorescence (AF) of Index Fingernails as a novel biomarker for rapid and noninvasive determinations on the status of tobacco smoking: The green AF intensity of the Index Fingernails of the smokers was remarkably higher than that of the nonsmokers in the natural populations. When the AF intensity of the Fingernails was used as the variable, the area under curve (AUC) for differentiating the smokers from the nonsmokers was 0.91. Similar results were obtained by analyzing the green AF of the Index Fingernails of the healthy populations and the patients of acute ischemic stroke. Collectively, our study has indicated the green AF of the Index Fingernails as a novel biomarker for tobacco smoking, based on which the first method for noninvasive, rapid and economical determinations on the status of tobacco smoking may be established.

Decreased green autofluorescence of lung parenchyma is a biomarker for lung cancer tissues.

It is highly valuable to discover novel biomarkers for differentiating noninvasively the cancerous tissues from the nonneoplastic tissues of lung cancer. In current study, we determined the green autofluorescence (AF) of the pulmonary parenchyma of lung cancer patients, indicating that decreased green AF of pulmonary parenchyma may be the biomarker of this type: First, the green AF intensity of the cancerous tissues was significantly lower than that of the nonneoplastic tissues of the lung cancer patients; second, the green AF intensity of the nonneoplastic tissues of the lung squamous cell carcinoma was significantly lower than that of the lung adenocarcinoma; and third, "decreased green AF intensity" could be used for differentiating the nonneoplastic tissues and the cancerous tissues. Collectively, our study has suggested that decreased green AF of lung parenchyma is a biomarker for differentiating the cancerous tissues from the nonneoplastic tissues of lung cancer.

Skin's green autofluorescence at dorsal centremetacarpus may become a novel biomarker for diagnosis of lung cancer.

It is critical to discover novel biomarkers of lung cancer for establishing economical technology for diagnosis of lung cancer. Our study has suggested that the autofluorescence (AF) of the skin may become a novel biomarker of this type: First, development of lung cancer led to a significant increase in the skin's green AF in a mouse model of lung cancer; second, lung cancer patients had significantly higher skin's green AF at certain positions compared with healthy volunteers and pulmonary infection patients; and third, using the skin's green AF intensity at dorsal centremetacarpus as the variable, the areas under curve (AUC) for differentiating lung cancer patients and pulmonary infection patients and for differentiating lung cancer patients and healthy volunteers was 0.871 and 0.813, respectively. Collectively, our study has indicated that the skin's green AF at dorsal centremetacarpus may become a novel biomarker for establishing a ground-breaking diagnostic strategy for lung cancer.

Malate-Aspartate Shuttle Plays an Important Role in LPS-Induced Neuroinflammation of Mice Due to its Effect on STAT3 Phosphorylation.

Neuroinflammation is a key pathological factor in numerous neurological disorders. Cumulating evidence has indicated critical roles of NAD+/NADH metabolism in multiple major diseases, while the role of malate-aspartate shuttle (MAS) - a major NADH shuttle - in inflammation has remained unclear. In this study we investigated the roles of MAS in LPS-induced neuroinflammation both in vivo and in vitro. Immunofluorescence staining, Western blot assay and Real-time PCR assays were conducted to determine the activation of Iba-1, the protein levels of iNOS and COX2 and the mRNA levels of IL-1ß, IL-6, and TNF-α in vivo, showing that both pre-treatment and post-treatment of aminooxyacetic acid (AOAA) - an MAS inhibitor - profoundly decreased the LPS-induced neuroinflammation in mice. BV2 microglia was also used as a cellular model to investigate the mechanisms of this finding, in which such assays as Western blot assay and nitrite assay. Our study further indicated that AOAA produced its effects on LPS-induced microglial activation by its effects on MAS: Pyruvate treatment reversed the effects of AOAA on the cytosolic NAD+/NADH ratio, which also restored the LPS-induced activation of the AOAA-treated microglia. Moreover, the lactate dehydrogenase (LDH) inhibitor GSK2837808A blocked the effects of pyruvate on the AOAA-produced decreases in both the cytosolic NAD+/NADH ratio and LPS-induced microglial activation. Our study has further suggested that AOAA produced inhibition of LPS-induced microglial activation at least partially by decreasing STAT3 phosphorylation. Collectively, our findings have indicated AOAA as a new and effective drug for inhibiting LPS-induced neuroinflammation. Our study has also indicated that MAS is a novel mediator of LPS-induced neuroinflammation due to its capacity to modulate LPS-induced STAT3 phosphorylation, which has further highlighted a critical role of NAD+/NADH metabolism in inflammation.

Oxidative stress induces cell death partially by decreasing both mRNA and protein levels of nicotinamide phosphoribosyltransferase in differentiated PC12 cells.

BACKGROUND: Multiple studies have indicated crucial roles of NAD+ deficiency in several neurological diseases and aging. It is critical to discover the mechanisms underlying the NAD+ deficiency. A decreased level of Nicotinamide phosphoribosyltransferase (Nampt)-an important enzyme in the salvage pathway of NAD+ synthesis-has been found under certain pathological conditions, while the mechanisms underlying the Nampt decrease are unclear. The purpose of this study is to test the hypothesis that oxidative stress can produce decreased Nampt, and to investigate the biological effects of Nampt on NAD+ synthesis and cell survival under both basal and oxidative stress conditions. METHODS: We used differentiated PC12 cells as a cellular model to investigate the effects of oxidative stress on the levels of Nampt. Multiple assays, including flow cytometry-based cell death assays and NAD+ assays were conducted. RESULTS: First, oxidative stress can decrease the levels of Nampt mRNA and Nampt protein; second, Nampt plays significant roles in NAD+ synthesis under both basal conditions and oxidative stress conditions; third, Nampt plays critical roles in cell survival under both basal conditions and oxidative stress conditions; and fourth, oxidative stress produced decreased NAD+ levels and cell survival partially by decreasing Nampt. Collectively, our study has indicated that oxidative stress is a pathological factor leading to decreased Nampt, which plays important roles in oxidative stress-produced decreases in NAD+ levels and cell survival. Our findings have indicated major roles of Nampt in maintaining NAD+ levels and cell survival under both basal and oxidative stress conditions.

Keratin 1 plays significant roles in maintaining the survival and oxidative stress state of B16-F10 melanoma cell lines.

Keratins play multiple significant biological roles in epithelium. Keratin 1 (K1)/keratin 10 (K10) heterodimer is a hallmark for keratinocyte differentiation. While keratins are absent in normal melanocyte, keratins have been found in both melanoma cell lines and human melanoma. The biological significance of the keratins in melanoma cells has remained unclear. In our current study we applied K1 siRNA to investigate the biological significance of K1 in B16-F10 melanoma cells. We found that as low as a 16% decrease in the K1 level led to significant increases in both apoptosis and necrosis of the cells. Moreover, the mild K1 decrease led to significant increases in both dichlorofluorescein (DCF) and ethidium signals - two indicators of oxidative stress - in the cells. Collectively, our findings have provided the first evidence indicating both a critical role of the K1 in maintaining the survival of melanoma cells and an important role of the K1 in modulating the oxidative stress state of the cells. These findings have exposed new functions of keratins in cancer cells, suggesting that K1 may become a novel therapeutic target for melanoma.

Testosterone in Female Depression: A Meta-Analysis and Mendelian Randomization Study.

Testosterone's role in female depression is not well understood, with studies reporting conflicting results. Here, we use meta-analytical and Mendelian randomization techniques to determine whether serum testosterone levels differ between depressed and healthy women and whether such a relationship is casual. Our meta-analysis shows a significant association between absolute serum testosterone levels and female depression, which remains true for the premenopausal group while achieving borderline significance in the postmenopausal group. The results from our Mendelian randomization analysis failed to show any causal relationship between testosterone and depression. Our results show that women with depression do indeed display significantly different serum levels of testosterone. However, the directions of the effect of this relationship are conflicting and may be due to menopausal status. Since our Mendelian randomization analysis was insignificant, the difference in testosterone levels between healthy and depressed women is most likely a manifestation of the disease itself. Further studies could be carried out to leverage this newfound insight into better diagnostic capabilities culminating in early intervention in female depression.

NAD+ administration decreases microvascular damage following cardiac ischemia/reperfusion by restoring autophagic flux.

Microvascular damage is a key pathological change in myocardial ischemia/reperfusion (I/R) injury. Using a rat model of myocardial I/R, our current study has provided the first evidence that nicotinamide adenine dinucleotide (NAD+) administration can significantly attenuate myocardial I/R-induced microvascular damage, including reduced regional blood perfusion, decreased microvessel density and integrity, and coronary microvascular endothelial cells (CMECs) injury. In studies with primary cultured CMECs under hypoxia/reoxygenation (HR) and a rat model of I/R, our results suggested that the protective effect of NAD+ on CMECs exposed to HR or I/R is at least partially mediated by the NAD+-induced restoration of autophagic flux, especially lysosomal autophagy: NAD+ treatment markedly induced transcription factor EB (TFEB) activation and attenuated lysosomal dysfunction in the I/R or HR-exposed cells. Collectively, our study has provided the first in vivo and in vitro evidence that NAD+ significantly rescued the impaired autophagic flux and cell apoptosis that was induced by I/R in rat CMECs, which is mediated in part through the action of TFEB-mediated lysosomal autophagy.

Cryptotanshinone Attenuates Inflammatory Response of Microglial Cells via the Nrf2/HO-1 Pathway.

Cryptotanshinone (CTN), a monomer compound extracted from the dried roots and rhizomes of Salvia miltiorrhiza Bge, has a variety of pharmacological effects. However, little research has been done on the mechanism of CTN in attenuating neuroinflammation. The present study aimed to investigate whether CTN can ameliorate neuroinflammation induced by lipopolysaccharide (LPS) through the Nrf2/heme-oxygenase 1 (HO-1) signaling pathway in BV-2 microglial cells. We found that CTN attenuated the upregulated expression of inducible nitric oxide synthase, cyclooxygenase 2, NOD-like receptor pyrin domains-3, and nitric oxide induced by LPS in microglial cells. In addition, it curtailed the increased release of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α in LPS-activated microglial cells. Furthermore, CTN significantly increased the levels of NF-κB, Nrf2, HO-1, and Akt proteins. We demonstrated that the anti-inflammatory action of CTN in BV-2 microglial cells was partially through the activation of the Nrf2/HO-1 pathway, which was regulated by the PI3K/Akt signaling pathway. Taken together, our results indicated that CTN downregulated the production and release of proinflammatory mediators in BV-2 microglial cells through activating the Nrf2/HO-1 pathway and subsequently protected neurons from inflammatory injury.

SIRT2, ERK and Nrf2 Mediate NAD+ Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions.

NAD+ (oxidized form of nicotinamide adenine dinucleotide) administration is highly beneficial in numerous models of diseases and aging. It is becoming increasingly important to determine if NAD+ treatment may directly increase the antioxidant capacity of cells under basal conditions. In the current study, we tested our hypothesis that NAD+ can directly enhance the antioxidant capacity of cells under basal conditions by using PC12 cells as a cellular model. We found that NAD+ treatment can increase the GSH/GSSG ratios in the cells under basal conditions. NAD+ can also increase both the mRNA and protein level of γ-glutamylcysteine ligase (γ-GCL)-a key enzyme for glutathione synthesis, which appears to be mediated by the NAD+-induced increase in Nrf2 activity. These NAD+-induced changes can be prevented by both SIRT2 siRNA and the SIRT2 inhibitor AGK2. The NAD+-induced changes can also be blocked by the ERK signaling inhibitor U0126. Moreover, the NAD+-induced ERK activation can be blocked by both SIRT2 siRNA and AGK2. Collectively, our study has provided the first evidence that NAD+ can enhance directly the antioxidant capacity of the cells under basal conditions, which is mediated by SIRT2, ERK, and Nrf2. These findings have suggested not only the great nutritional potential of NAD+, but also a novel mechanism underlying the protective effects of the NAD+ administration in the disease models: the NAD+ administration can enhance the resistance of the normal cells to oxidative insults by increasing the antioxidant capacity of the cells.

NAD+ Deficiency Is a Common Central Pathological Factor of a Number of Diseases and Aging: Mechanisms and Therapeutic Implications.

SIGNIFICANCE: Increasing evidence has indicated critical roles of nicotinamide adenine dinucleotide, oxidized form (NAD+) in various biological functions. NAD+ deficiency has been found in models of a number of diseases such as cerebral ischemia, myocardial ischemia, and diabetes, and in models of aging. Applications of NAD+ or other approaches that can restore NAD+ levels are highly protective in these models of diseases and aging. NAD+ produces its beneficial effects by targeting at multiple pathological pathways, including attenuating mitochondrial alterations, DNA damage, and oxidative stress, by modulating such enzymes as sirtuins, glyceraldehyde-3-phosphate dehydrogenase, and AP endonuclease. These findings have suggested great therapeutic and nutritional potential of NAD+ for diseases and senescence. Recent Advances: Approaches that can restore NAD+ levels are highly protective in the models of such diseases as glaucoma. The NAD+ deficiency in the diseases and aging results from not only poly(ADP-ribose) polymerase-1 (PARP-1) activation but also decreased nicotinamide phosphoribosyltransferase (Nampt) activity and increased CD38 activity. Significant biological effects of extracellular NAD+ have been found. Increasing evidence has suggested that NAD+ deficiency is a common central pathological factor in a number of diseases and aging. Critical Issues and Future Directions: Future studies are required for solidly establishing the concept that "NAD+ deficiency is a common central pathological factor in a number of disease and aging." It is also necessary to further investigate the mechanisms underlying the NAD+ deficiency in the diseases and aging. Preclinical and clinical studies should be conducted to determine the therapeutic potential of NAD+ for the diseases and aging.

Extracellular Degradation Into Adenosine and the Activities of Adenosine Kinase and AMPK Mediate Extracellular NAD+-Produced Increases in the Adenylate Pool of BV2 Microglia Under Basal Conditions.

Cumulating evidence has indicated NAD+ deficiency as a common central pathological factor of multiple diseases and aging. NAD+ supplement is highly protective in various disease and aging models, while two key questions have remained unanswered: (1) Does extracellular NAD+ also produce its effects through its degradation product adenosine? (2) Does extracellular NAD+ produce the protective effects by affecting cells under pathological insults only, or by affecting both normal cell and the cells under pathological insults? Since extracellular NAD+ can be degraded into adenosine, and endogenous adenosine levels are in the nanomolar range under physiological conditions, extracellular NAD+ may produce its effects through its degradation into adenosine. In this study we used BV2 microglia as a cellular model to test our hypothesis that NAD+ treatment can increase the intracellular adenylate pool under basal conditions through its extracellular degradation into adenosine. Our study has shown that extracellular NAD+ is degraded into adenosine extracellularly, which enters BV2 microglia through equilibrative nucleoside transporters under basal conditions. The intracellular adenosine is converted to AMP by adenosine kinase, which increases the intracellular ATP levels by both activating AMPK and increasing the intracellular adenylate pool. Collectively, our study has suggested a novel mechanism underlying the protective effects of NAD+ administration, which is mediated by extracellular NAD+ degradation into adenosine as well as the activities of adenosine kinase and AMPK. Our findings have also suggested that NAD+ administration in various disease and aging models may also produce its effects by affecting the microglia that are not under pathological insults.

Cytosolic aspartate aminotransferase mediates the mitochondrial membrane potential and cell survival by maintaining the calcium homeostasis of BV2 microglia.

Both mitochondrial aspartate aminotransferase (mAST) and cytosolic aspartate aminotransferase (cAST) are important components in the malate-aspartate shuttle - one of the two types of NADH shuttles in cells. A major goal of our current study was to determine specifically the roles of cAST in maintaining the [Ca]i, mitochondrial membrane potential and the survival of BV2 microglia by applying molecular approach to modulate the cAST levels. Our study found that decreased cAST by cAST siRNA can lead to significant increases in the [Ca]i, mitochondrial depolarization and apoptosis of BV2 microglia. The cAST siRNA-induced mitochondrial depolarization can be significantly attenuated by an inhibitor of calpain. We further found that the cAST siRNA-induced apoptosis can be prevented by the calpain inhibitor. Collectively, our study suggests that decreased cAST induces calpain activation by increasing the [Ca]i of BV2 microglia, resulting in mitochondrial depolarization and cell death. Moreover, our data suggest that decreased cAST may produce these pathological effects by malate-aspartate shuttle-independent pathways.

SIRT2 and Akt mediate NAD+-induced and NADH-induced increases in the intracellular ATP levels of BV2 microglia under basal conditions.

NAD replenishment can restore ATP levels and rescue premature aging in Cockayne syndrome mice. However, there has been no mechanistic study regarding the effects of NAD and NADH on intracellular ATP levels under basal conditions. In our current study, we used BV2 microglia to test our hypothesis that NAD and NADH can increase intracellular ATP levels under basal conditions. We found that both NAD and NADH significantly increased the intracellular ATP levels of BV2 microglia, which were attenuated by SIRT2 siRNA, the SIRT2 inhibitor AGK2, and the phosphatidylinositol 3-kinase/Akt inhibitor LY294002. Our study has also suggested that SIRT2 mediates the NAD-induced and NADH-induced increase in Akt phosphorylation in BV2 microglia. Collectively, our study has suggested that SIRT2 mediates both NAD-induced and NADH-induced increases in the intracellular ATP levels of BV2 microglia by modulating Akt phosphorylation.