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Background: Nicotinamide adenine dinucleotide (NAD+), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD+ derivatives and it's reduced forms, with 13C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD+ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD+ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD+ metabolism and the potential significance of these metabolites in various biological contexts. [ABSTRACT FROM AUTHOR]

Identification and classification of pathogenic bacterial strains is of current interest for the early treatment of diseases. In this work, protein fluorescence from eight different pathogenic bacterial strains were characterized using steady state and time resolved fluorescence spectroscopy. The spectral deconvolution method was also employed to decompose the emission contribution from different intrinsic fluorophores and extracted various key parameters, such as intensity, emission maxima, emission line width of the fluorophores, and optical redox ratio. The change in average lifetime values across different bacterial strains exhibits good statistical significance (p ≤ 0.01). The variations in the photophysical characteristics of bacterial strains are due to the different conformational states of the proteins. The stepwise multiple linear discriminate analysis of fluorescence emission spectra at 280 nm excitation across eight different bacterial strains classifies the original groups and cross validated group with 100% and 99.5% accuracy, respectively. [ABSTRACT FROM AUTHOR]

Body fluids carry several biomarkers and genetic material that enable the detection of carcinogenesis and tumor metastasis. Owing to this, liquid biopsy is gaining importance and entering clinical practice as an adjunct modality to the current standard of practice in cancer detection. Liquid biopsy involving specimens such as urine is non-invasive and has biomarkers reflecting the tumor microenvironment. We investigate the normal and altered levels of cellular metabolic by-products in liquid phantoms and demonstrate their photon-induced excited-state interaction behaviour for potential diagnostic applications. Short-span temporal analysis of metabolic by-products FAD and NADH in liquid phantoms with fiber optic fluorescence spectroscopy is carried out. The mixed liquid phantom of FAD and NADH, showed excited state interactions that manifested as simultaneous inverse variation in the fluorescence emission from FAD and NADH in varying pH environments. Linear discriminant analysis of the time-varying integrated fluorescence intensity resulted in a 100% accuracy in classifying liquid samples between high and low metabolite concentrations. An extension of application of the experiment model for analysis of body fluids such as urine is also discussed.

Background: Nicotinamide adenine dinucleotide (NAD+), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD+ derivatives and it’s reduced forms, with 13C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD+ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD+ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD+ metabolism and the potential significance of these metabolites in various biological contexts.

Adenine nucleotide translocator (ANT) is a mitochondrial protein involved in the exchange of ADP and ATP across the mitochondrial inner membrane. It plays a crucial role in cellular energy metabolism by facilitating the transport of ATP synthesized within the mitochondria to the cytoplasm. The isoform ANT1 predominately expresses in cardiac and skeletal muscles. Mutations or dysregulation in ANT1 have been implicated in various mitochondrial disorders and neuromuscular diseases. We aimed to examine whether ANT1 deletion may affect mitochondrial redox state in our established ANT1-deficient mice. Hearts and quadriceps resected from age-matched wild type (WT) and ANT1-deficient mice were snap-frozen in liquid nitrogen. The Chance redox scanner was utilized to perform 3D optical redox imaging. Each sample underwent scanning across 3–5 sections. Global averaging analysis showed no significant differences in the redox indices (NADH, flavin adenine dinucleotide containing-flavoproteins Fp, and the redox ratio Fp/(NADH+Fp) between WT and ANT1-deficient groups. However, quadriceps had higher Fp than hearts in both groups (p = 0. 0 0 0 4 and 0.01, respectively). Furthermore, the quadriceps were also more oxidized (a higher redox ratio) than hearts in WT group (p = 0. 0 0 4). NADH levels were similar in all cases. Our data suggest that under non-stressful physical condition, the ANT1-deficient muscle cells were in the same mitochondrial state as WT ones and that the significant difference in the mitochondrial redox state between quadriceps and hearts found in WT might be diminished in ANT1-deficient ones. Redox imaging of muscles under physical stress can be conducted in future. [ABSTRACT FROM AUTHOR]

Adenine nucleotide translocator (ANT) is a mitochondrial protein involved in the exchange of ADP and ATP across the mitochondrial inner membrane. It plays a crucial role in cellular energy metabolism by facilitating the transport of ATP synthesized within the mitochondria to the cytoplasm. The isoform ANT1 predominately expresses in cardiac and skeletal muscles. Mutations or dysregulation in ANT1 have been implicated in various mitochondrial disorders and neuromuscular diseases. We aimed to examine whether ANT1 deletion may affect mitochondrial redox state in our established ANT1-deficient mice. Hearts and quadriceps resected from age-matched wild type (WT) and ANT1-deficient mice were snap-frozen in liquid nitrogen. The Chance redox scanner was utilized to perform 3D optical redox imaging. Each sample underwent scanning across 3–5 sections. Global averaging analysis showed no significant differences in the redox indices (NADH, flavin adenine dinucleotide containing-flavoproteins Fp, and the redox ratio Fp/(NADH+Fp) between WT and ANT1-deficient groups. However, quadriceps had higher Fp than hearts in both groups ([Formula: see text] and 0.01, respectively). Furthermore, the quadriceps were also more oxidized (a higher redox ratio) than hearts in WT group ([Formula: see text]). NADH levels were similar in all cases. Our data suggest that under non-stressful physical condition, the ANT1-deficient muscle cells were in the same mitochondrial state as WT ones and that the significant difference in the mitochondrial redox state between quadriceps and hearts found in WT might be diminished in ANT1-deficient ones. Redox imaging of muscles under physical stress can be conducted in future.

The interplay of the redox ratio of float-glass production with the melting regime, the charge content, and the chemical composition of the glass was subjected to statistical analysis. It is shown that redox ratio influences the technical-economic performance parameters of a tank furnace and the quality of the manufactured glass. [ABSTRACT FROM AUTHOR]

Iron oxide is commonly found in natural or industrial glass compositions and can exist as ferrous (Fe2+) or ferric (Fe3+) species, with their ratios depending on glass composition, temperature, pressure and the redox reactions during the glass forming process. The iron redox ratio plays an important role on silicate glass structures and consequently various properties. This work aims to study the effect of iron oxide, and particularly the iron redox ratio, on the structures of borosilicate and boroaluminosilicate glasses using molecular dynamics simulations with newly developed iron potential parameters that are compatible with the borosilicate potentials. The results provide detailed cation coordination states of both iron species and the effect of redox ratio on boron coordination and other structural features. Particularly, competition for charge compensating modifier cations (such as Na+) among the fourfold‐coordinated cations such as B3+, Al3+, and Fe3+ is investigated by calculating the cation–cation pair distribution functions and coordination preferential ratios. The results show that the trivalent ferric ions, with a shorter Fe–O bond distance and better defined first coordiation shell with mainly four‐fold coordination, act as a glass former whereas the divalent ferrous ions mainly play the role of glass modifier. The ferrous/ferric ratio (Fe2+/Fe3+) was found to affect the glass chemistry and hence glass properties by regulating the amount of four‐coordinated boron, the fraction of non‐briding oxygen and other features. The results are compared with available experimental data to gain insights of the complex structures and charge compensation schemes of the glass system. [ABSTRACT FROM AUTHOR]

A substantial decline in nicotinamide adenine dinucleotide (NAD) has been reported in brain tissue homogenates or neurons isolated from Alzheimer's disease (AD) models. NAD, together with flavin adenine dinucleotide (FAD), critically supports energy metabolism and maintains mitochondrial redox homeostasis. Optical redox imaging (ORI) of the intrinsic fluorescence of reduced NAD (NADH) and oxidized FAD yields cellular redox and metabolic information and provides biomarkers for a variety of pathological conditions. However, its utility in AD has not been characterized at the tissue level. We performed ex vivo ORI of freshly dissected hippocampi from a well-characterized AD mouse model with five familial Alzheimer's disease mutations (5XFAD) and wild type (WT) control littermates at various ages. We found (1) a significant increase in the redox ratio with age in the hippocampi of both the WT control and the 5XFAD model, with a more prominent redox shift in the AD hippocampi; (2) a higher NADH in the 5XFAD versus WT hippocampi at the pre-symptomatic age of 2 months; and (3) a negative correlation between NADH and Aβ42 level, a positive correlation between Fp and Aβ42 level, and a positive correlation between redox ratio and Aβ42 level in the AD hippocampi. These findings suggest that the ORI can be further optimized to conveniently study the metabolism of freshly dissected brain tissues in animal models and identify early AD biomarkers. [ABSTRACT FROM AUTHOR]

Intrauterine growth restriction (IUGR) is a result of limited substrate supply to the developing fetus in utero, and can be caused by either placental, genetic or environmental factors. Babies born IUGR can have poor long‐term health outcomes, including being at higher risk of developing cardiovascular disease. Limited substrate supply in the IUGR fetus not only changes the structure of the heart but may also affect metabolism and function of the developing heart. We have utilised two imaging modalities, two‐photon microscopy and phase‐contrast MRI (PC‐MRI), to assess alterations in cardiac metabolism and function using a sheep model of IUGR. Two‐photon imaging revealed that the left ventricle of IUGR fetuses (at 140–141 d GA) had a reduced optical redox ratio, suggesting a reliance on glycolysis for ATP production. Concurrently, the use of PC‐MRI to measure foetal left ventricular cardiac output (LVCO) revealed a positive correlation between LVCO and redox ratio in IUGR, but not control fetuses. These data suggest that altered heart metabolism in IUGR fetuses is indicative of reduced cardiac output, which may contribute to poor cardiac outcomes in adulthood. [ABSTRACT FROM AUTHOR]

A substantial decline in nicotinamide adenine dinucleotide (NAD) has been reported in brain tissue homogenates or neurons isolated from Alzheimer’s disease (AD) models. NAD, together with flavin adenine dinucleotide (FAD), critically supports energy metabolism and maintains mitochondrial redox homeostasis. Optical redox imaging (ORI) of the intrinsic fluorescence of reduced NAD (NADH) and oxidized FAD yields cellular redox and metabolic information and provides biomarkers for a variety of pathological conditions. However, its utility in AD has not been characterized at the tissue level. We performed ex vivo ORI of freshly dissected hippocampi from a well-characterized AD mouse model with five familial Alzheimer’s disease mutations (5XFAD) and wild type (WT) control littermates at various ages. We found (1) a significant increase in the redox ratio with age in the hippocampi of both the WT control and the 5XFAD model, with a more prominent redox shift in the AD hippocampi; (2) a higher NADH in the 5XFAD versus WT hippocampi at the pre-symptomatic age of 2 months; and (3) a negative correlation between NADH and Aβ42 level, a positive correlation between Fp and Aβ42 level, and a positive correlation between redox ratio and Aβ42 level in the AD hippocampi. These findings suggest that the ORI can be further optimized to conveniently study the metabolism of freshly dissected brain tissues in animal models and identify early AD biomarkers.

Intrauterine growth restriction (IUGR) can result from reduced delivery of substrates, including oxygen and glucose, during pregnancy and may be caused by either placental insufficiency or maternal undernutrition. As a consequence of IUGR, there is altered programming of adipose tissue and this can be associated with metabolic diseases later in life. We have utilised two sheep models of IUGR, placental restriction and late gestation undernutrition, to determine the metabolic effects of growth restriction on foetal perirenal adipose tissue (PAT). Two‐photon microscopy was employed to obtain an optical redox ratio, which gives an indication of cell metabolism. PAT of IUGR foetuses exhibited higher metabolic activity, altered lipid droplet morphology, upregulation of cytochrome c oxidase subunit genes and decreased expression of genes involved in growth and differentiation. Our results indicate that there are adaptations in PAT of IUGR foetuses that might be protective and ensure survival in response to an IUGR insult. [ABSTRACT FROM AUTHOR]

The brain requires a large amount of energy, mostly derived from the metabolism of glucose, which decreases substantially with age and neurological diseases. While mounting evidence in model organisms illustrates the central role of brain nicotinamide adenine dinucleotide (NAD) for maintaining energy homeostasis, similar data are sparse in humans. This study explores the correlations between brain NAD, energy production and membrane phospholipid metabolism by 31-phosphorous magnetic resonance spectroscopy (31P-MRS) across 50 healthy participants including a young (mean age 27.1-year-old) and middle-aged (mean age 56.4-year-old) group. The analysis revealed that brain NAD level and NAD+/NADH redox ratio were positively associated with ATP level and the rate of energy production, respectively. Moreover, a metabolic network linking NAD with membrane phospholipid metabolism, energy production, and aging was identified. An inverted trend between age and NAD level was detected. These results pave the way for the use of 31P-MRS as a powerful non-invasive tool to support the development of new therapeutic interventions targeting NAD associated phospho-metabolic pathways in brain aging and neurological diseases.

The brain requires a large amount of energy, mostly derived from the metabolism of glucose, which decreases substantially with age and neurological diseases. While mounting evidence in model organisms illustrates the central role of brain nicotinamide adenine dinucleotide (NAD) for maintaining energy homeostasis, similar data are sparse in humans. This study explores the correlations between brain NAD, energy production and membrane phospholipid metabolism by 31-phosphorous magnetic resonance spectroscopy (31P-MRS) across 50 healthy participants including a young (mean age 27.1-year-old) and middle-aged (mean age 56.4-year-old) group. The analysis revealed that brain NAD level and NAD+/NADH redox ratio were positively associated with ATP level and the rate of energy production, respectively. Moreover, a metabolic network linking NAD with membrane phospholipid metabolism, energy production, and aging was identified. An inverted trend between age and NAD level was detected. These results pave the way for the use of 31P-MRS as a powerful non-invasive tool to support the development of new therapeutic interventions targeting NAD associated phospho-metabolic pathways in brain aging and neurological diseases. [ABSTRACT FROM AUTHOR]

Carotid body (CB) type I cells sense the blood PO2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp-NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND. [ABSTRACT FROM AUTHOR]

Purpose: Optical redox imaging (ORI) technique images cellular autofluorescence of nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp containing FAD, i.e., flavin adenine dinucleotide). ORI has found wide applications in the study of cellular energetics and metabolism and may potentially assist in disease diagnosis and prognosis. Fixed tissues have been reported to exhibit autofluorescence with similar spectral characteristics to those of NADH and Fp. However, few studies report on quantitative ORI of formalin-fixed paraffin-embedded (FFPE) unstained tissue slides for disease biomarkers. We investigate whether ORI of FFPE unstained skeletal muscle slides may provide relevant quantitative biological information.Procedures: Living mouse muscle fibers and frozen and FFPE mouse muscle slides were subjected to ORI. Living mouse muscle fibers were imaged ex vivo before and after paraformaldehyde fixation. FFPE muscle slides of three mouse groups (young, mid-age, and muscle-specific overexpression of nicotinamide phosphoribosyltransferase (Nampt) transgenic mid-age) were imaged and compared to detect age-related redox differences.Results: We observed that living muscle fiber and frozen and FFPE slides all had strong autofluorescence signals in the NADH and Fp channels. Paraformaldehyde fixation resulted in a significant increase in the redox ratio Fp/(NADH + Fp) of muscle fibers. Quantitative image analysis on FFPE unstained slides showed that mid-age gastrocnemius muscles had stronger NADH and Fp signals than young muscles. Gastrocnemius muscles from mid-age Nampt mice had lower NADH compared to age-matched controls, but had higher Fp than young controls. Soleus muscles had the same trend of change and appeared to be more oxidative than gastrocnemius muscles. Differential NADH and Fp signals were found between gastrocnemius and soleus muscles within both mid-aged control and Nampt groups.Conclusion: Aging effect on redox status quantified by ORI of FFPE unstained muscle slides was reported for the first time. Quantitative information from ORI of FFPE unstained slides may be useful for biomedical applications. [ABSTRACT FROM AUTHOR]

Purpose: Optical redox imaging (ORI), based on collecting the endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) containing a redox cofactor flavin adenine dinucleotide (FAD), provides sensitive indicators of cellular metabolism and redox status. ORI indices (such as NADH, FAD, and their ratio) have been under investigation as potential progression/prognosis biomarkers for cancer. Higher FAD redox ratio (i.e., FAD/(FAD + NADH)) has been associated with higher invasive/metastatic potential in tumor xenografts and cultured cells. This study is to examine whether ORI indices can respond to the modulation of oncogene DEK activities that change cancer cell invasive/metastatic potential.Procedures: Using lentiviral shRNA, DEK gene expression was efficiently knocked down in MDA-MB-231 breast cancer cells (DEKsh). These DEKsh cells, along with scrambled shRNA-transduced control cells (NTsh), were imaged with a fluorescence microscope. In vitro invasive potential of the DEKsh cells and NTsh cells was also measured in parallel using the transwell assay.Results: FAD and FAD redox ratios in polyclonal cells with DEKsh were significantly lower than that in NTsh control cells. Consistently, the DEKsh cells demonstrated decreased invasive potential than their non-knockdown counterparts NTsh cells.Conclusions: This study provides direct evidence that oncogene activities could mediate ORI-detected cellular redox state. [ABSTRACT FROM AUTHOR]

Purpose: Fluorescence of co-enzyme reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) provides a sensitive measure of the mitochondrial redox state and cellular metabolism. By imaging NADH and Fp, we investigated the utility of optical redox imaging (ORI) to monitor cellular metabolism and detect early metabolic response to cancer drugs.Procedures: We performed ORI of human melanoma DB-1 cells in culture and DB-1 mouse xenografts to detect the redox response to lonidamine (LND) treatment.Results: For cultured cells, LND treatment for 45 min significantly lowered NADH levels with no significant change in Fp, resulting in a significant increase in the Fp redox ratio (Fp/(NADH+Fp)); 3-h prolonged treatment led to a decrease in NADH and an increase in Fp and a more oxidized redox state compared to control. Significant decrease in the mitochondrial redox capacity of LND-treated cells was observed for the first time. For xenografts, 45-min LND treatment resulted in a significant reduction of NADH content, no significant changes in Fp content, and a trend of increase in the Fp redox ratio. Intratumor redox heterogeneity was observed in both control and LND-treated groups.Conclusion: Our results support the utility of ORI for evaluating cellular metabolism and monitoring early metabolic response to cancer drugs. [ABSTRACT FROM AUTHOR]

We created a two-channel autofluorescence test to detect oral cancer. The wavelengths 375 and 460 nm, with filters of 479 and 525 nm, were designed to excite and detect reduced-form nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Patients with oral cancer or with precancerous lesions, and a control group with healthy oral mucosae, were enrolled. The lesion in the autofluorescent image was the region of interest. The average intensity and heterogeneity of the NADH and FAD were calculated. The redox ratio [(NADH)/(NADH + FAD)] was also computed. A quadratic discriminant analysis (QDA) was used to compute boundaries based on sensitivity and specificity. We analyzed 49 oral cancer lesions, 34 precancerous lesions, and 77 healthy oral mucosae. A boundary (sensitivity: 0.974 and specificity: 0.898) between the oral cancer lesions and healthy oral mucosae was validated. Oral cancer and precancerous lesions were also differentiated from healthy oral mucosae (sensitivity: 0.919 and specificity: 0.755). The two-channel autofluorescence detection device and analyses of the intensity and heterogeneity of NADH, and of FAD, and the redox ratio combined with a QDA classifier can differentiate oral cancer and precancerous lesions from healthy oral mucosae. [ABSTRACT FROM AUTHOR]

Significance: Optical imaging using the endogenous fluorescence of metabolic cofactors has enabled nondestructive examination of dynamic changes in cell and tissue function both in vitro and in vivo. Quantifying NAD(P)H and FAD fluorescence through an optical redox ratio and fluorescence lifetime imaging (FLIM) provides sensitivity to the relative balance between oxidative phosphorylation and glucose catabolism. Since its introduction decades ago, the use of NAD(P)H imaging has expanded to include applications involving almost every major tissue type and a variety of pathologies. Recent Advances: This review focuses on the use of two-photon excited fluorescence and NAD(P)H fluorescence lifetime techniques in cancer, neuroscience, tissue engineering, and other biomedical applications over the last 5 years. In a variety of cancer models, NAD(P)H fluorescence intensity and lifetime measurements demonstrate a sensitivity to the Warburg effect, suggesting potential for early detection or high-throughput drug screening. The sensitivity to the biosynthetic demands of stem cell differentiation and tissue repair processes indicates the range of applications for this imaging technology may be broad. Critical Issues: As the number of applications for these fluorescence imaging techniques expand, identifying and characterizing additional intrinsic fluorophores and chromophores present in vivo will be vital to accurately measure and interpret metabolic outcomes. Understanding the full capabilities and limitations of FLIM will also be key to future advances. Future Directions: Future work is needed to evaluate whether a combination of different biochemical and structural outcomes using these imaging techniques can provide complementary information regarding the utilization of specific metabolic pathways. [ABSTRACT FROM AUTHOR]

Co-enzyme nicotinamide adenine dinucleotide (NAD(H)) redox plays a key role in macrophage function. Surfactant protein (SP-) A modulates the functions of alveolar macrophages (AM) and ozone (O3) exposure in the presence or absence of SP-A and reduces mouse survival in a sex-dependent manner. It is unclear whether and how NAD(H) redox status plays a role in the innate immune response in a sex-dependent manner. We investigated the NAD(H) redox status of AM from SP-A2 and SP-A knockout (KO) mice in response to O3 or filtered air (control) exposure using optical redox imaging technique. We found: (i) In SP-A2 mice, the redox alteration of AM in response to O3 showed sex-dependence with AM from males being significantly more oxidized and having a higher level of mitochondrial reactive oxygen species than females; (ii) AM from KO mice were more oxidized after O3 exposure and showed no sex differences; (iii) AM from female KO mice were more oxidized than female SP-A2 mice; and (iv) Two distinct subpopulations characterized by size and redox status were observed in a mouse AM sample. In conclusions, the NAD(H) redox balance in AM responds to O3 in a sex-dependent manner and the innate immune molecule, SP-A2, contributes to this observed sex-specific redox response.

Native fluorescence spectra are acquired from fresh normal and cancerous human prostate tissues. The fluorescence data are analyzed using an unsupervised machine learning algorithm such as non-negative matrix factorization. The nonnegative spectral components are retrieved and attributed to the native fluorophores such as collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD) in tissue. The retrieved scores of the components are used to estimate the relative concentrations of the native fluorophores such as NADH and FAD and the redox ratio. A supervised machine learning algorithm such as support vector machine (SVM) is used to classify normal and cancerous tissue samples based on either the relative concentrations of NADH and FAD or the redox ratio alone. Various statistical measures such as sensitivity, specificity, and accuracy, along with the area under receiver operating characteristic (ROC) curve are used to show the classification performance. A cross validation method such as leave-one-out is used to further evaluate the predictive performance of the SVM classifier to avoid bias due to overfitting, and the accuracy was found to be 93.3%. [ABSTRACT FROM AUTHOR]

Metabolic alterations of oral epithelial cells under oxidative stress are important signatures for early diagnosis of oral cancer. Amongst different metabolic alterations, non-invasive photo-diagnostic methods have been extensively used for determining cellular heme metabolism and accumulation of protoporphyrin IX (PpIX) under administration of suitable photosensitizer. In this study, we report these metabolic alterations by direct analysis of oral exfoliated cells obtained from individuals with prolonged smoking habit without the exogenous administration of any photosensitizer. The relative expression level of relevant biomolecules of study groups were compared with clinically diagnosed and histopathologically confirmed leukoplakia (OLPK) and oral squamous cell carcinoma (OSCC) patients. The energy imbalance and variation in ‘redox ratio’ were examined through spectroscopic studies which showed an increasing trend (p < 0.001) in smokers to OSCC groups in comparison to nonsmoker control. Gene expression of important intermediates of the heme metabolic pathway ( viz . 5-aminolevulinate synthase 1 (ALAS1), Ferrochelatase (FECH), hemeoxygenase 1 (HO-1) and ATP binding cassette subfamily G member 2 (ABCG2)) which affect production of PpIX was assessed. Relative mRNA level of ALAS1 and HO1 was upregulated whereas mRNA level of other genes ( viz . FECH and ABCG2) were found to be downregulated in smokers as well as in cancer groups. Outcome of different spectroscopic studies on exfoliated cells ( viz . fluorescence, atomic absorption and Fourier transform infrared spectroscopy) corroborated with the expression of biomarkers related to cellular endogenous metabolism related to heme cycle. This study indicates significant alterations in endogenous metabolic products, and cellular functional groups in oral epithelial cells among the study groups. Our study reports a strong possibility of diagnosis of early cancer signatures amongst habitual smokers by direct and non-invasive assessment of metabolic status of oral epithelial cells without exogenous administration of photosensitizers. The knowledge accrued from the study may guide clinicians in precise detection of precancer trend in the susceptible population through a noninvasive rapid screening method. [ABSTRACT FROM AUTHOR]

Rationale: Prediabetes can be reversed through lifestyle intervention, but its main pathologic hallmark, insulin resistance (IR), cannot be detected as conveniently as blood glucose testing. In consequence, the diagnosis of prediabetes is often delayed until patients have hyperglycemia. Therefore, developing a less invasive diagnostic method for rapid IR evaluation will contribute to the prognosis of prediabetes. Adipose tissue is an endocrine organ that plays a crucial role in the development and progression of prediabetes. Label-free visualizing the prediabetic microenvironment of adipose tissues provides a less invasive alternative for the characterization of IR and inflammatory pathology. Methods: Here, we successfully identified the differentiable features of prediabetic adipose tissues by employing the metabolic imaging of three endogenous fluorophores NAD(P)H, FAD, and lipofuscin-like pigments. Results: We discovered that 1040-nm excited lipofuscin-like autofluorescence could mark the location of macrophages. This unique feature helps separate the metabolic fluorescence signals of macrophages from those of adipocytes. In prediabetes fat tissues with IR, we found only adipocytes exhibited a low redox ratio of metabolic fluorescence and high free NAD(P)H fraction a 1 . This differential signature disappears for mice who quit the high-fat diet or high-fat-high-sucrose diet and recover from IR. When mice have diabetic hyperglycemia and inflamed fat tissues, both adipocytes and macrophages possess this kind of metabolic change. As confirmed with RNA-seq analysis and histopathology evidence, the change in adipocyte's metabolic fluorescence could be an indicator or risk factor of prediabetic IR. Conclusion: Our study provides an innovative approach to diagnosing prediabetes, which sheds light on the strategy for diabetes prevention., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Our long-term goal was to investigate the potential of incorporating redox imaging technique as a breast cancer (BC) diagnosis component to increase the positive predictive value of suspicious imaging finding and to reduce unnecessary biopsies and overdiagnosis. We previously found that precancer and cancer tissues in animal models displayed abnormal mitochondrial redox state. We also revealed abnormal mitochondrial redox state in cancerous specimens from three BC patients. Here, we extend our study to include biopsies of 16 patients. Tissue aliquots were collected from both apparently normal and cancerous tissues from the affected cancer-bearing breasts shortly after surgical resection. All specimens were snap-frozen and scanned with the Chance redox scanner, i.e., the three-dimensional cryogenic NADH/Fp (reduced nicotinamide adenine dinucleotide/oxidized flavoproteins) fluorescence imager. We found both Fp and NADH in the cancerous tissues roughly tripled that in the normal tissues (p < 0.05). The redox ratio Fp/(NADH + Fp) was ~27% higher in the cancerous tissues (p < 0.05). Additionally, Fp, or NADH, or the redox ratio alone could predict cancer with reasonable sensitivity and specificity. Our findings suggest that the optical redox imaging technique can provide parameters independent of clinical factors for discriminating cancer from noncancer breast tissues in human patients. [ABSTRACT FROM AUTHOR]

NAD+/NADH redox state has been implicated in many diseases such as cancer and diabetes as well as in the regulation of embryonic development and aging. To fluorimetrically assess the mitochondrial redox state, Dr. Chance and co-workers measured the fluorescence of NADH and oxidized flavoproteins (Fp) including flavin–adenine–dinucleotide (FAD) and demonstrated their ratio (i.e. the redox ratio) is a sensitive indicator of the mitochondrial redox states. The Chance redox scanner was built to simultaneously measure NADH and Fp in tissue at submillimeter scale in 3D using the freeze-trap protocol. This paper summarizes our recent research experience, development and new applications of the redox scanning technique in collaboration with Dr. Chance beginning in 2005. Dr. Chance initiated or actively involved in many of the projects during the last several years of his life. We advanced the redox scanning technique by measuring the nominal concentrations (in reference to the frozen solution standards) of the endogenous fluorescent analytes, i.e., [NADH] and [Fp] to quantify the redox ratios in various biological tissues. The advancement has enabled us to identify an array of the redox indices as quantitative imaging biomarkers (including [NADH], [Fp], [Fp]/([NADH]+[Fp]), [NADH]/[Fp], and their standard deviations) for studying some important biological questions on cancer and normal tissue metabolism. We found that the redox indices were associated or changed with (1) tumorigenesis (cancer versus non-cancer of human breast tissue biopsies); (2) tumor metastatic potential; (3) tumor glucose uptake; (4) tumor p53 status; (5) PI3K pathway activation in pre-malignant tissue; (6) therapeutic effects on tumors; (7) embryonic stem cell differentiation; (8) the heart under fasting. Together, our work demonstrated that the tissue redox indices obtained from the redox scanning technique may provide useful information about tissue metabolism and physiology status in normal and diseased tissues. The Chance redox scanner and other redox imaging techniques may have wide-ranging potential applications in many fields, such as cancer, diabetes, developmental process, mitochondrial diseases, neurodegenerative diseases, and aging.

There is an increasing interest in using N-acetylcysteine ( NAC) as a treatment for neurodegenerative disorders to increase glutathione ( GSH) levels and its redox status. The purpose of this study was to characterize the biosynthesis of NAC to GSH using a novel stable isotope-labeled technique, and investigate the pharmacodynamics of NAC in vivo. Female wild-type mice were given a single intravenous bolus dose of 150 mg kg−1 stable-labeled NAC. Plasma, red blood cells ( RBC), and brain tissues were collected at predesignated time points. Stable-labeled NAC and its metabolite GSH (both labeled and unlabeled forms) were quantified in blood and brain samples. Molar ratios of the reduced and oxidized forms of GSH ( GSH divided by glutathione disulfide, redox ratio) were also determined. The elimination phase half-life of NAC was approximately 34 min. Both labeled and unlabeled GSH in RBC were found to increase; however, the area under the curve above baseline ( AUCb0-280) of labeled GSH was only 1% of the unlabeled form. These data indicate that NAC is not a direct precursor of GSH. In addition, NAC has prolonged effects in brain even when the drug has been eliminated from systemic circulation. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2619-2626, 2015 [ABSTRACT FROM AUTHOR]

Hereditary Hemorrhagic Telangiectasia-1 (HHT-1) is a vascular disease caused by mutations in the endoglin (Eng)/CD105 gene. The objective of this study was to quantify the oxidative state of a rodent model of HHT-1 using an optical imaging technique. We used a cryofluorescence imaging instrument to quantitatively assess tissue metabolism in this model. Mitochondrial redox ratio (FAD/NADH), FAD RR, was used as a quantitative marker of the metabolic status and was examined in the kidneys, and eyes of wild-type and Eng +/- mice. Kidneys and eyes from wild-type P21, 6W, and 10M old mice showed, respectively, a 9% (±2), 24% (±0.4), 15% (±1), and 23% (±4), 33% (±0.6), and 30% (±2) change in the mean FAD RR compared to Eng +/- mice at the same age. Thus, endoglin haploinsufficiency is associated with less oxidative stress in various organs and mitigation of angiogenesis. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

NAD+/NADH redox state has been implicated in many diseases such as cancer and diabetes as well as in the regulation of embryonic development and aging. To fluorimetrically assess the mitochondrial redox state, Dr. Chance and co-workers measured the fluorescence of NADH and oxidized flavoproteins (Fp) including flavin-adenine-dinucleotide (FAD) and demonstrated their ratio (i.e. the redox ratio) is a sensitive indicator of the mitochondrial redox states. The Chance redox scanner was built to simultaneously measure NADH and Fp in tissue at submillimeter scale in 3D using the freeze-trap protocol. This paper summarizes our recent research experience, development and new applications of the redox scanning technique in collaboration with Dr. Chance beginning in 2005. Dr. Chance initiated or actively involved in many of the projects during the last several years of his life. We advanced the redox scanning technique by measuring the nominal concentrations (in reference to the frozen solution standards) of the endogenous fluorescent analytes, i.e., [NADH] and [Fp] to quantify the redox ratios in various biological tissues. The advancement has enabled us to identify an array of the redox indices as quantitative imaging biomarkers (including [NADH], [Fp], [Fp]/([NADH]+[Fp]), [NADH]/[Fp], and their standard deviations) for studying some important biological questions on cancer and normal tissue metabolism. We found that the redox indices were associated or changed with (1) tumorigenesis (cancer versus non-cancer of human breast tissue biopsies); (2) tumor metastatic potential; (3) tumor glucose uptake; (4) tumor p53 status; (5) PI3K pathway activation in pre-malignant tissue; (6) therapeutic effects on tumors; (7) embryonic stem cell differentiation; (8) the heart under fasting. Together, our work demonstrated that the tissue redox indices obtained from the redox scanning technique may provide useful information about tissue metabolism and physiology status in normal and diseased tissues. The Chance redox scanner and other redox imaging techniques may have wide-ranging potential applications in many fields, such as cancer, diabetes, developmental process, mitochondrial diseases, neurodegenerative diseases, and aging. [ABSTRACT FROM AUTHOR]

Highlights: [•] The reduction process of DA has not been fully examined in FSCV studies. [•] The reduction process of DA was investigated using PPV. [•] A part of DOQ could instantly desorb from the CFM surface after DA oxidation. [•] The reduction process of DA was rate-limited processing. [•] The reduction process might be one of important factors in the kinetic analysis of DA. [Copyright &y& Elsevier]