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Background: Myceliophthora thermophila has been engineered as a significant cell factory for malic acid production, yet strategies to further enhance production remain unclear and lack rational guidance. 13C-MFA (13C metabolic flux analysis) offers a means to analyze cellular metabolic mechanisms and pinpoint critical nodes for improving product synthesis. Here, we employed 13C-MFA to investigate the metabolic flux distribution of a high-malic acid-producing strain of M. thermophila and attempted to decipher the crucial bottlenecks in the metabolic pathways. Results: Compared with the wild-type strain, the high-Malic acid-producing strain M. thermophila JG207 exhibited greater glucose uptake and carbon dioxide evolution rates but lower oxygen uptake rates and biomass yields. Consistent with these phenotypes, the 13C-MFA results showed that JG207 displayed elevated flux through the EMP pathway and downstream TCA cycle, along with reduced oxidative phosphorylation flux, thereby providing more precursors and NADH for malic acid synthesis. Furthermore, based on the 13C-MFA results, we conducted oxygen-limited culture and nicotinamide nucleotide transhydrogenase (NNT) gene knockout experiments to increase the cytoplasmic NADH level, both of which were shown to be beneficial for malic acid accumulation. Conclusions: This work elucidates and validates the key node for achieving high malic acid production in M. thermophila. We propose effective fermentation strategies and genetic modifications for enhancing malic acid production. These findings offer valuable guidance for the rational design of future cell factories aimed at improving malic acid yields. [ABSTRACT FROM AUTHOR]

Introduction: Spaghetti meat (SM) and wooden breast (WB) are emerging myopathies in the breast meat of fast-growing broiler chickens. The purpose of the study was to investigate the metabolomic differences between normal (N), SM, and WB fillets 24 h postmortem. Materials and methods: Eight chicken breasts for each experimental group were collected from a commercial processing plant. Supernatant from tissue homogenates were subjected to ultra-performance liquid chromatographytandem mass spectrometry (UPLC-MS) analysis. Results and methods: A total of 3,090 metabolites were identified in the chicken breast meat. The comparison of WB and N showed 850 differential metabolites (P < 0.05), and the comparison of SM and N displayed 617 differential metabolites. The comparison of WB and SM showed 568 differential metabolites. The principal component analysis (PCA) plots showed a distinct separation between SM and N and between WB and N except for one sample, but SM and WB were not distinctly separated. Compared to N, 15-Hydroxyeicosatetraenoic acid (15-HETE) increased, and D-inositol-4-phosphate decreased in both SM and WB, indicating that cellular homeostasis and lipid metabolism can be affected in SM and WB. The abundance of nicotinamide adenine dinucleotide (NAD) + hydrogen (H) (NADH) was exclusively decreased between SM and N (P < 0.05). Purine metabolism was upregulated in SM and WB compared to N with a greater degree of upregulation in WB than SM. Folic acid levels decreased in SM and WB compared to N (P < 0.05). Steroid hormone biosynthesis was downregulated in SM compared to N (P < 0.05). Carbon metabolism was downregulated in SM and WB compared to N with greater degree of downregulation in WB than SM (P < 0.05). These data suggest both shared and unique metabolic alterations in SM and WB, indicating commonalities and differences in their underlying etiologies and meat quality traits. Dietary supplementation of deficient nutrients, such as NADH, folic acids, etc. and modulation of altered pathways in SM and WB would be strategies to reduce the incidence and severity of SM and WB. [ABSTRACT FROM AUTHOR]

The fluorescence intensities of the cellular respiratory cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD++ (oxidized flavin adenine dinucleotide) reflect energy metabolism in skin and other tissues and can be quantified in vivo by fluorescence spectroscopy (FS). However, the variability of physiological parameters largely determines the reproducibility of measurement results and the reliability of the diagnostic test. In this prospective study, we evaluated the interday reproducibility of NADH and FAD++ fluorescence intensity measurements in the skin of 51 healthy volunteers assessed by the FS at baseline, after local cooling (10°C) and heating of the skin (35°C). Results showed that the fluorescence amplitude of NADH (AFNADH) in forearm skin was the most reproducible of the FS parameters studied. Assessment of AFNADH in the dorsal forearm in combination with a thermal functional test is the most promising method for clinical use for assessing energy metabolism in the skin. [ABSTRACT FROM AUTHOR]

Mitochondrial transporters facilitate the translocation of metabolites between the cytoplasm and mitochondria and are critical for mitochondrial functional integrity. Although many mitochondrial transporters are associated with metabolic diseases, how they regulate mitochondrial function and their metabolic contributions at the cellular level are largely unknown. Here, we show that mitochondrial thiamine pyrophosphate (TPP) transporter SLC25A19 is required for mitochondrial respiration. SLC25A19 deficiency leads to reduced cell viability, increased integrated stress response (ISR), enhanced glycolysis and elevated cell sensitivity to 2-deoxyglucose (2-DG) treatment. Through a series of biochemical assays, we found that the depletion of mitochondrial NADH is the primary cause of the impaired mitochondrial respiration in SLC25A19 deficient cells. We also showed involvement of SLC25A19 in regulating the enzymatic activities of complexes I and III, the tricarboxylic acid (TCA) cycle, malate-aspartate shuttle and amino acid metabolism. Consistently, addition of idebenone, an analog of coenzyme Q10, restores mitochondrial respiration and cell viability in SLC25A19 deficient cells. Together, our findings provide new insight into the functions of SLC25A19 in mitochondrial and cellular physiology, and suggest that restoring mitochondrial respiration could be a novel strategy for treating SLC25A19-associated disorders. [Display omitted] • Loss of SLC25A19 leads to mitochondrial NADH depletion, thereby inhibiting mitochondrial respiration. • SLC25A19 is required for complex I and complex III enzyme activities. • Loss of SLC25A19 causes mitochondrial integrated stress response. • Idebenone rescues mitochondrial respiration in SLC25A19 deficient cells. [ABSTRACT FROM AUTHOR]

Abstract Background Myceliophthora thermophila has been engineered as a significant cell factory for malic acid production, yet strategies to further enhance production remain unclear and lack rational guidance. 13C-MFA (13C metabolic flux analysis) offers a means to analyze cellular metabolic mechanisms and pinpoint critical nodes for improving product synthesis. Here, we employed 13C-MFA to investigate the metabolic flux distribution of a high-malic acid-producing strain of M. thermophila and attempted to decipher the crucial bottlenecks in the metabolic pathways. Results Compared with the wild-type strain, the high-Malic acid-producing strain M. thermophila JG207 exhibited greater glucose uptake and carbon dioxide evolution rates but lower oxygen uptake rates and biomass yields. Consistent with these phenotypes, the 13C-MFA results showed that JG207 displayed elevated flux through the EMP pathway and downstream TCA cycle, along with reduced oxidative phosphorylation flux, thereby providing more precursors and NADH for malic acid synthesis. Furthermore, based on the 13C-MFA results, we conducted oxygen-limited culture and nicotinamide nucleotide transhydrogenase (NNT) gene knockout experiments to increase the cytoplasmic NADH level, both of which were shown to be beneficial for malic acid accumulation. Conclusions This work elucidates and validates the key node for achieving high malic acid production in M. thermophila. We propose effective fermentation strategies and genetic modifications for enhancing malic acid production. These findings offer valuable guidance for the rational design of future cell factories aimed at improving malic acid yields.

Brevibacillus agri C15 and Brevibacillus agri C15 CdR have been reported as a tool for do uptake of cadmium (Cd). Nevertheless, it is unclear whether these strains contrast in their adsorption behaviors and surface complexation could be one of the possible mechanisms that occurred. Therefore, this research aimed to investigate the mechanisms of cadmium uptake by B. agri C15 and B.agri C15 CdR in batch cultures and under TEM detection based on the maximum ATP or NADH production to energy metabolism using a resting cell experiment. In addition, surface complexation of cadmium in strains was studied. The results showed that the accumulation of Cd under TEM detection predicts that Cd bioaccumulation is the main mechanism in B. agri C15 and that adsorption is the main mechanism in B.agri C15 CdR. The observation of vesicles and granules, mainly poly-β-hydroxybutyrate under Cd-sublethal concentration may contribute to the adsorption and intercellular sequestration of Cd. There were differences in the cell compartments through the uptake process of Cd by the bacterial cells and the examinations of the contribution of surface complexation mechanisms in the uptake of Cd were obtained. Therefore, determining the thermodynamic stability constants of both strains from the available obtained parameters from this study was necessary.

Abstract Bivalves are an extraordinary class of animals in which species with a doubly uniparental inheritance (DUI) of mitochondrial DNA have been described. DUI is characterized as a mitochondrial homoplasmy of females and heteroplasmy of male individuals where F-type mitogenomes are passed to the progeny with mother egg cells and divergent M-type mitogenomes are inherited with fathers sperm cells. However, in most cases only male individuals retain divergent mitogenome inherited with spermatozoa. Additionally, in many of bivalves, unique mitochondrial features, like additional genes, gene duplication, gene extensions, mitochondrial introns, and recombination, were observed. In this study, we sequenced and assembled male-type mitogenomes of three Donax species. Comparative analysis of mitochondrial sequences revealed a lack of all seven NADH dehydrogenase subunits as well as the presence of three long additional open reading frames lacking identifiable homology to any of the existing genes.

Abstract Background Rosmarinic acid (RA) has a wide range of beneficial effects on human health. On the other hand, RA has been reported to induce metal-mediated reactive oxygen species (ROS) generation and DNA damage. However, its mechanism remains unknown. In this study, to clarify the underlying mechanism, we analyzed metal-mediated DNA damage in isolated DNA treated with RA and its analog isorinic acid. Results RA plus Cu(II), but not Fe(III), significantly increased 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage, in calf thymus DNA. Furthermore, a comparison of the 8-oxodG formation induced by RA and its analog isorinic acid suggested that the catechol groups in RA could be associated with their abilities to form 8-oxodG. Interestingly, the 8-oxodG formation induced by RA and isorinic acid plus Cu(II) was markedly enhanced by the addition of NADH, an endogenous reductant. To elucidate the mechanism of RA plus Cu(II)-induced oxidative DNA damage, we examined DNA damage in 32P-labeled DNA treated with RA in the presence of Cu(II). RA plus Cu(II) caused DNA cleavage, which was enhanced by piperidine treatment, suggesting that RA causes not only DNA strand breakage but also base modification. RA plus Cu(II)-induced DNA damage was inhibited by catalase (H2O2 scavenger), bathocuproine (Cu(I) chelator), and methional (scavenger of a variety of ROS other than •OH) but not by typical •OH scavengers and SOD, indicating the involvement of H2O2, Cu(I), and ROS other than •OH. DNA cleavage site analysis showing RA-induced site-specific DNA damage (frequently at thymine and some cytosine residues) supports the involvement of ROS other than •OH, because •OH causes DNA cleavage without site specificity. Based on these results, Cu(I) and H2O2 generation with concomitant RA autoxidation could lead to the production of Cu(I)-hydroperoxide, which induces oxidative DNA damage. o-Quinone and o-semiquinone radicals are likely to be again reduced to RA by NADH, which dramatically increases oxidative DNA damage, particularly at low concentrations of RA. Conclusions In this study, physiologically relevant concentrations of RA effectively induced oxidative DNA damage in isolated DNA through redox cycle reactions with copper and NADH.

SUMMARY: Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real‐time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real‐time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies. Significant Statement: Monitoring dynamic changes in pyridine nucleotides (e.g., NADPH and NADH/NAD+ ratio) in plants at the subcellular level is a major obstacle in plant bioenergetics research. Genetically encoded fluorescent biosensors are considered to be the best option so far because they provide fast, accurate, and real‐time readouts. Here, we present a detailed study on the application of pyridine nucleotide biosensors, and we anticipate that these transgenic lines will be useful in plant redox biology studies. [ABSTRACT FROM AUTHOR]

Mitochondrial disease is a devastating genetic disorder, with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke‐like episodes (MELAS) and m.3243A>G being the most common phenotype and genotype, respectively. The treatment for MELAS patients is still less effective. Here, we performed transcriptomic and proteomic analysis in muscle tissue of MELAS patients, and discovered that the expression of molecules involved in serine catabolism were significantly upregulated, and serine hydroxymethyltransferase 2 (SHMT2) increased significantly in both the mRNA and protein levels. The SHMT2 protein level was also increased in myoblasts with m.3243A>G mutation, which was transdifferentiated from patients derived fibroblasts, accompanying with the decreased nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) ratio and cell viability. After treating with SHMT2 inhibitor (SHIN1), the NAD+/NADH ratio and cell viability in MELAS myoblasts increased significantly. Taken together, our study indicates that enhanced serine catabolism plays an important role in the pathogenesis of MELAS and that SHIN1 can be a potential small molecule for the treatment of this disease. [ABSTRACT FROM AUTHOR]

Bivalves are an extraordinary class of animals in which species with a doubly uniparental inheritance (DUI) of mitochondrial DNA have been described. DUI is characterized as a mitochondrial homoplasmy of females and heteroplasmy of male individuals where F-type mitogenomes are passed to the progeny with mother egg cells and divergent M-type mitogenomes are inherited with fathers sperm cells. However, in most cases only male individuals retain divergent mitogenome inherited with spermatozoa. Additionally, in many of bivalves, unique mitochondrial features, like additional genes, gene duplication, gene extensions, mitochondrial introns, and recombination, were observed. In this study, we sequenced and assembled male-type mitogenomes of three Donax species. Comparative analysis of mitochondrial sequences revealed a lack of all seven NADH dehydrogenase subunits as well as the presence of three long additional open reading frames lacking identifiable homology to any of the existing genes. [ABSTRACT FROM AUTHOR]

Summary: Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood.In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase.OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild‐type (WT) plants under As treatment. Additionally, loss‐of‐function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain.Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice. [ABSTRACT FROM AUTHOR]

Background: Rosmarinic acid (RA) has a wide range of beneficial effects on human health. On the other hand, RA has been reported to induce metal-mediated reactive oxygen species (ROS) generation and DNA damage. However, its mechanism remains unknown. In this study, to clarify the underlying mechanism, we analyzed metal-mediated DNA damage in isolated DNA treated with RA and its analog isorinic acid. Results: RA plus Cu(II), but not Fe(III), significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage, in calf thymus DNA. Furthermore, a comparison of the 8-oxodG formation induced by RA and its analog isorinic acid suggested that the catechol groups in RA could be associated with their abilities to form 8-oxodG. Interestingly, the 8-oxodG formation induced by RA and isorinic acid plus Cu(II) was markedly enhanced by the addition of NADH, an endogenous reductant. To elucidate the mechanism of RA plus Cu(II)-induced oxidative DNA damage, we examined DNA damage in 32P-labeled DNA treated with RA in the presence of Cu(II). RA plus Cu(II) caused DNA cleavage, which was enhanced by piperidine treatment, suggesting that RA causes not only DNA strand breakage but also base modification. RA plus Cu(II)-induced DNA damage was inhibited by catalase (H2O2 scavenger), bathocuproine (Cu(I) chelator), and methional (scavenger of a variety of ROS other than •OH) but not by typical •OH scavengers and SOD, indicating the involvement of H2O2, Cu(I), and ROS other than •OH. DNA cleavage site analysis showing RA-induced site-specific DNA damage (frequently at thymine and some cytosine residues) supports the involvement of ROS other than •OH, because •OH causes DNA cleavage without site specificity. Based on these results, Cu(I) and H2O2 generation with concomitant RA autoxidation could lead to the production of Cu(I)-hydroperoxide, which induces oxidative DNA damage. o-Quinone and o-semiquinone radicals are likely to be again reduced to RA by NADH, which dramatically increases oxidative DNA damage, particularly at low concentrations of RA. Conclusions: In this study, physiologically relevant concentrations of RA effectively induced oxidative DNA damage in isolated DNA through redox cycle reactions with copper and NADH. [ABSTRACT FROM AUTHOR]

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Ageing is a nature process of microplastics that occurrs daily, and human beings are inevitably exposed to aged microplastics. However, a systematic understanding of ageing status and its toxic effect is currently still lacking. In this study, plastic cup lids-originated polypropylene (PP) microplastics were UV-photoaged until the carbonyl index (CI), a canonical indicator for plastic ageing, achieved 0.08, 0.17, 0.22 and 0.28. The adverse hepatic effect of these aged PPs (aPPs) was evaluated in Balb/c mice (75 ng/mL water, about 200 particles/day) and human-originated liver organoids (LOs, 50 particles/mL, ranged from 5.94 to 13.15 ng/mL) at low-dose equivalent to human exposure level. Low-dose of aged PP could induce hepatic reductive stress both in vitro and in vivo, by elevating the NADH/NAD+ratio in a CI-dependent manner, together with hepatoxicity (indicated by increased AST secretion and cytotoxicity), and disrupted the genes encoding the nutrients transporters and NADH subunits accompanied by the restricted ATP supply, declined mitochondrial membrane potential and mitochondrial complexI/IV activities, without significant increase in MDA levels in the liver. These changes in the liver disrupted systematic metabolism, representing a circulatory panel of increases in the lactate, triglyceride, Fgf21 levels, and decreases in the pyruvate level, linked the reductive stress to the declined body weight gain but elevated hepatic NADH contents following aPPs exposure. Additionally, assessing by the LOs, it was found that digestion drastically accelerated the ageing of aPPs and worsen the energy supply upon mitochondria, representing a “scattergun effect” induced by the formation of micro- and nano-plastics mixture toward NADH/NAD+imbalance.

Objective: The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. Methods: The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. Results: OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. Conclusions: Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.

The global surge in multidrug-resistant bacteria owing to antibiotic misuse and overuse poses considerable risks to human and animal health. With existing antibiotics losing their effectiveness and the protracted process of developing new antibiotics, urgent alternatives are imperative to curb disease spread. Notably, improving the bactericidal effect of antibiotics by using non-antibiotic substances has emerged as a viable strategy. Although reduced nicotinamide adenine dinucleotide (NADH) may play a crucial role in regulating bacterial resistance, studies examining how the change of metabolic profile and bacterial resistance following by exogenous administration are scarce. Therefore, this study aimed to elucidate the metabolic changes that occur in Edwardsiella tarda (E. tarda), which exhibits resistance to various antibiotics, following the exogenous addition of NADH using metabolomics. The effects of these alterations on the bactericidal activity of neomycin were investigated. NADH enhanced the effectiveness of aminoglycoside antibiotics against E. tarda ATCC15947, achieving bacterial eradication at low doses. Metabolomic analysis revealed that NADH reprogrammed the ATCC15947 metabolic profile by promoting purine metabolism and energy metabolism, yielding increased adenosine triphosphate (ATP) levels. Increased ATP levels played a crucial role in enhancing the bactericidal effects of neomycin. Moreover, exogenous NADH promoted the bactericidal efficacy of tetracyclines and chloramphenicols. NADH in combination with neomycin was effective against other clinically resistant bacteria, including Aeromonas hydrophila, Vibrio parahaemolyticus, methicillin-resistant Staphylococcus aureus, and Listeria monocytogenes. These results may facilitate the development of effective approaches for preventing and managing E. tarda–induced infections and multidrug resistance in aquaculture and clinical settings.

Cigarette smoke is one of the main factors in Chronic Obstructive Pulmonary Disease (COPD), a respiratory syndrome marked by persistent respiratory symptoms and increasing airway obstruction. Perturbed NAD+/NADH levels may play a role in various diseases, including lung disorders like COPD. In our study, we investigated the preventive effect of NADH supplementation in an experimental model of COPD induced by cigarette smoke extract (CSE). N = 64 mice randomly distributed in eight groups were injected with NADH (two doses of 100 mg/kg or 200 mg/kg) or dexamethasone (2 mg/kg) before being exposed to CSE for up to 9 weeks. Additionally, NADH supplementation preserved lung antioxidant defenses by preventing the functional loss of key enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase, and the expression levels of glutathione (GSH) (n = 4, p < 0.001). It also reduced oxidative damage markers, such as malondialdehyde (MDA) and nitrites (n = 4, p < 0.001). A marked increase in tissue myeloperoxidase activity was assessed (MPO), confirming neutrophils implication in the inflammatory process. The latter was significantly ameliorated in the NADH-treated groups (p < 0.001). Finally, NADH prevented the CSE-induced secretion of cytokines such as Tumor Necrosis Factor alpha (TNF-α), IL-17, and IFN-y (n = 4, p < 0.001). Our study shows, for the first time, the clinical potential of NADH supplementation in preventing key features of COPD via its unique anti-inflammatory and antioxidant properties. [ABSTRACT FROM AUTHOR]

The objective of the current review is to summarize the current state of optical methods in redox biology. It consists of two parts, the first is dedicated to genetically encoded fluorescent indicators and the second to Raman spectroscopy. In the first part, we provide a detailed classification of the currently available redox biosensors based on their target analytes. We thoroughly discuss the main architecture types of these proteins, the underlying engineering strategies for their development, the biochemical properties of existing tools and their advantages and disadvantages from a practical point of view. Particular attention is paid to fluorescence lifetime imaging microscopy as a possible readout technique, since it is less prone to certain artifacts than traditional intensiometric measurements. In the second part, the characteristic Raman peaks of the most important redox intermediates are listed, and examples of how this knowledge can be implemented in biological studies are given. This part covers such fields as estimation of the redox states and concentrations of Fe–S clusters, cytochromes, other heme-containing proteins, oxidative derivatives of thiols, lipids, and nucleotides. Finally, we touch on the issue of multiparameter imaging, in which biosensors are combined with other visualization methods for simultaneous assessment of several cellular parameters. [Display omitted] • Detailed classification of genetically encoded fluorescent redox biosensors. • Redox biosensors and fluorescence lifetime imaging microscopy (FLIM). • Raman spectroscopy for redox biology. [ABSTRACT FROM AUTHOR]

Although it is well known that coenzyme NAD(P)H is involved in anabolic and catabolic reactions in the living organism, there is still significant controversy over the reaction mechanism involved in this biochemical transformation. Thus, 1‐benzyl‐1,4‐dihydronicotinamide was used as a NAD(P)H model in the reduction reaction of 1,4‐benzoquinone (Q), 2,3,5,6‐tetrachloro‐1,4‐benzoquinone, and 2,3‐dicyano‐1,4‐benzoquinone in acetonitrile medium. The kinetic calculations support that formal hydride transfer is the main mechanism promoting Q reduction, while the two‐step process dominates 2,3‐dicyano‐1,4‐benzoquinone reduction. Interestingly, only the single‐electron transfer mechanism takes place when 2,3,5,6‐tetrachloro‐1,4‐benzoquinone is used, affording the corresponding semiquinone derivative as the main product. This mechanistic behavior is related to the presence or absence of electron‐withdrawing groups in the quinones used. Furthermore, the kinetic study results showed that calculated reaction rate constants are in close agreement with experimental results. The results support that formal hydride transfer on the reduction reaction of Q by 1‐benzyl‐1,4‐dihydronicotinamide in acetonitrile proceeds through a hydrogen coupled electron transfer mechanism. This theoretical analysis provides valuable knowledge that can be extrapolated to study the reduction of quinones performed by NADH and NADPH in physiological media. [ABSTRACT FROM AUTHOR]

Neurons exhibit a high energetic need, and the question arises as how they metabolically adapt to changing activity states. This is relevant for interpreting functional neuroimaging in different brain areas. Particularly, neurons with a broad firing range might exhibit metabolic adaptations. Therefore, we studied MNTB (medial nucleus of the trapezoid body) principal neurons, which generate action potentials (APs) at frequencies up to several hundred hertz. We performed the experiments in acute brainstem slices of the Mongolian gerbil (Meriones unguiculatus) at 22.5–24.5°C. Upon electrical stimulation of afferent MNTB fibres with 400 stimuli at varying frequencies, we monitored autofluorescence levels of NAD(P)H and FAD and determined the extremum amplitudes of their biphasic response. Additionally, we compared these data with alterations in O2 concentrations measured with an electrochemical sensor. These O2 changes are prominent since MNTB neurons rely on oxidative phosphorylation as shown by our pharmacological experiments. We calculated the O2 consumption rate as change in O2 concentration divided by stimulus durations, because these periods varied inversely with stimulus frequency as a result of the constant number of 400 stimuli applied. The O2 consumption rate increased with stimulation frequency up to a constant value at 600 Hz; that is, energy demand depends on temporal characteristics of activity despite the same number of stimuli. The rates showed no correlation with peak amplitudes of NAD(P)H or FAD, whilst the values of the two molecules were linearly correlated. This points at the complexity of analysing autofluorescence imaging for quantitative metabolic studies, because these values report only relative net changes of many superimposed oxidative and reductive processes. Monitoring O2 concentration rates is, thus, an important tool to improve the interpretation of NAD(P)H/FAD autofluorescence data, as they do not under all conditions and in all systems appropriately reflect the metabolic activity or energy demand. [ABSTRACT FROM AUTHOR]

Simple Summary: Use of cancer biomarkers for tumor aggressiveness is an unmet clinical need. Differentiation of high-risk versus low-risk tumors may guide physicians to select appropriate treatment strategies tailored to the risk level of individual patients. This study aimed to evaluate the value of an optical redox imaging technique for differentiating a human melanoma mouse xenograft model with a high risk of metastasis from a low-risk mouse model. Several imaging indices were found to be significantly different between the two models. The high-risk model was found to be in a more oxidized status and to have a higher intratumor redox heterogeneity. These findings may inform further research development of the optical redox imaging approach into translatable cancer biomarkers in the future. To develop imaging biomarkers for tumors aggressiveness, our previous optical redox imaging (ORI) studies of the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp, containing flavin adenine dinucleotide, i.e., FAD) in tumor xenografts of human melanoma associated the high optical redox ratio (ORR = Fp/(Fp + NADH)) and its heterogeneity to the high invasive/metastatic potential, without having reported quantitative results for NADH and Fp. Here, we implemented a calibration procedure to facilitate imaging the nominal concentrations of tissue NADH and Fp in the mouse xenografts of two human melanoma lines, an indolent less metastatic A375P and a more metastatic C8161. Images of the redox indices (NADH, Fp, ORR) revealed the existence of more oxidized areas (OAs) and more reduced areas (RAs) within individual tumors. ORR was found to be higher and NADH lower in C8161 compared to that of A375P xenografts, both globally for the whole tumors and locally in OAs. The ORR in the OA can differentiate xenografts with a higher statistical significance than the global averaged ORR. H&E staining of the tumors indicated that the redox differences we identified were more likely due to intrinsically different cell metabolism, rather than variations in cell density. [ABSTRACT FROM AUTHOR]

In this study, the reaction equilibrium was broken using isopropanol as a co-substrate, which was different from previous glucose as a co-substrate for furfural reduction, solving the problems of many by-products, low selectivity, and the addition of sodium phosphate buffers. The volatility of isopropanol and acetone prevented the production of by-products to the reaction system, improving the conversion and selectivity. 10 g/L furfural could be converted into furfuryl alcohol in pure water, and the furfural conversion was 90%, furfuryl alcohol selectivity was 100%, and the concentration of furfuryl alcohol could reach more than 20 g/L through accumulation reaction. No by-products were produced in this reaction system, and a single furfuryl alcohol product was obtained in the water catalytic system, which was convenient for downstream separation and purification. At the same time, based on the theory of the NADH coenzyme cycle system, this catalytic method could complete the conversion of various aldehydes into the corresponding alcohols, and it could be expanded to various biological hydrogenation projects, which has important industrial development and production prospects. [Display omitted] • Realized the biological hydrogenation of furfural in water. • The volatility of isopropanol and acetone disrupts the reaction equilibrium. • There are no by-products remaining in the system after the reaction. • Substrate spectrum can be expanded to enable biohydrogenation engineering. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide cofactor that is present in cells and in several important biological processes, including oxidative phosphorylation and production of adenosine triphosphate, DNA repair, calcium-dependent secondary messenger and gene expression. The purpose of this systematic review is to examine whether the coenzyme formulae NADþ and NADH are safe and effective when acting as a supplement to humans. This systematic review of randomized clinical trials performed a search in six electronic databases: PubMed, MEDLINE (ovid), Embase, Cochrane CENTRAL (clinical trials), Web of Science, and Scopus. Secondary search included the databases (e.g., Clinical trials.gov, Rebec, Google Scholar - advance). Two reviewers assessed and extracted the studies independently. The risk of bias in studies was performed using version 2 of the Cochrane risk of bias tool for randomized trials. This review includes 10 studies, with a total of 489 participants. The studies included different clinical conditions, such as chronic fatigue syndrome (CFS), older adults, Parkinson's disease, overweight, postmenopausal prediabetes, and Alzheimer's disease. Based on studies, the supplementation with NADH and precursors was well tolerated and observed clinical results such as, a decrease in anxiety conditions and maximum heart rate was observed after a stress test, increased muscle insulin sensitivity, insulin signaling. Quality of life, fatigue intensity, and sleep quality among others were evaluated on patients with CFS. All studies showed some side effects, thus, the most common associated with NADs use are muscle pain, nervous disorders, fatigue, sleep disturbance, and headaches. All adverse events cataloged by the studies did not present a serious risk to the health of the participants. Overall, these findings support that the oral administration of NADH can be associated to an increase in general quality of life and improvement on health parameters (e.g., a decrease in anxiety, maximum heart rate, inflammatory cytokines in serum, and cerebrospinal fluid). NADH supplementation is safe and has a low incidence of side effects. Future investigations are needed to evidence the clinical benefits regarding specific diseases and doses administered. [ABSTRACT FROM AUTHOR]

In steatotic liver diseases (SLDs), including metabolic dysfunction-associated SLD and alcohol-associated liver disease, the excessive hepatic metabolism of lipids and alcohol leads to an increase in hepatic mitochondrial reductive stress, which in turn contributes to the development of SLD. Recent genetic studies conducted on both humans and mice have provided further evidence highlighting the key role played by hepatic mitochondrial reductive stress in the pathogenesis of SLD. Several pharmaceutical agents currently in development for the treatment of SLD, such as peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, thyroid hormone receptor agonists, acetyl-CoA carboxylase (ACC) inhibitors, and mitochondrial uncouplers, share an under-recognized common characteristic in that they decrease hepatic mitochondrial reductive stress. A more comprehensive understanding of hepatic mitochondrial reductive stress could help elucidate disease pathogenesis and facilitate the identification of new therapeutic approaches. Steatotic liver diseases (SLDs) affect one-third of the population, but the pathogenesis underlying these diseases is not well understood, limiting the available treatments. A common factor in SLDs is increased hepatic mitochondrial reductive stress, which occurs as a result of excessive lipid and alcohol metabolism. Recent research has also shown that genetic risk factors contribute to this stress. This review aims to explore how these risk factors increase hepatic mitochondrial reductive stress and how it disrupts hepatic metabolism, leading to SLDs. Additionally, the review will discuss the latest clinical studies on pharmaceutical treatments for SLDs, specifically peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, thyroid hormone receptor (THR) agonists, acetyl-CoA carboxylase (ACC) inhibitors, and mitochondrial uncouplers. These treatments have a common effect of decreasing hepatic mitochondrial reductive stress, which has been largely overlooked. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a chronic metabolic disease characterized by persistent hyperglycemia, revealing a decrease in insulin efficiency. The sustained glucotoxic pancreatic microenvironment increases reactive oxygen species generation, resulting in chronic oxidative stress responsible for massive DNA damage. This triggers PARP-1 activation with both NAD+ and ATP depletion, affecting drastically pancreatic beta cells' energy storage and leading to their dysfunction and death. The aim of the present study is to highlight the main histological changes observed in pancreatic islets pre-treated with a unique NADH intraperitoneal injection in a streptozotocin-(STZ)-induced diabetes model. In order to adjust NADH doses, a preliminary study with three different doses, 500 mg/kg, 300 mg/kg, and 150 mg/kg, respectively, was conducted. Subsequently, and on the basis of the results of the aforementioned study, Wistar rats were randomly divided into four groups: non-diabetic control group, diabetics (STZ 45 mg/kg), NADH-treated group (150 mg/kg) 15 min before STZ administration, and NADH-treated group (150 mg/kg) 15 min after STZ administration. The effect of NADH was assessed by blood glucose level, TUNEL staining, histo-morphological analysis, and immunohistochemistry. The optimum protective dose of NADH was 150 mg/kg. NADH effectively decreased hyperglycemia and reduced diabetes induced by STZ. Histologically, NADH pre-treatment revealed a decrease in beta cell death favoring apoptosis over necrosis and therefore preventing inflammation with further beta cell destruction. Our data clearly demonstrate that NADH prior or post-treatment could effectively prevent the deleterious loss of beta cell mass in STZ-induced diabetes in rats and preserve the normal pancreatic islet's function. [ABSTRACT FROM AUTHOR]

The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN‐oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5–20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc‐Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity. [ABSTRACT FROM AUTHOR]

Ferroptosis is a recently discovered form of regulated cell death characterized by its distinct dependence on iron and the peroxidation of lipids within cellular membranes. Ferroptosis plays a crucial role in physiological and pathological situations and has attracted the attention of numerous scientists. Ferroptosis suppressive protein 1 (FSP1) is one of the main regulators that negatively regulates ferroptosis through the GPX4‐independent FSP1–CoQ10–NAD(P)H axis and is a potential therapeutic target for ferroptosis‐related diseases. However, the crystal structure of FSP1 has not been resolved, which hinders the development of therapeutic strategies targeting FSP1. To unravel this puzzle, we purified the human FSP1 (hFSP1) protein using the baculovirus eukaryotic cell expression system and solved its crystal structure at a resolution of 1.75 Å. Furthermore, we evaluated the oxidoreductase activity of hFSP1 with NADH as the substrate and identified E156 as the key amino acid in maintaining hFSP1 activity. Interestingly, our results indicated that hFSP1 exists and functions in a monomeric state. Mutagenesis analysis revealed the critical role of the C‐terminal domain in the binding of substrate. These findings significantly enhance our understanding of the functional mechanism of FSP1 and provide a precise model for further drug development. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD+ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD+ and NADH, have been limited by the compound's properties. We achieved highly sensitive simultaneous measurement of NAD+ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope 13C5-NAD+ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Background and Aims: Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important protective factor against hepatic lipotoxicity induced by excessive lipolysis, but its role and molecular mechanism in alcoholic liver disease (ALD) are not fully elucidated. Methods: The optimized National Institute on Alcohol Abuse and Alcoholism model was used to construct ALD model mice. Automatic biochemical analyser was used for Biochemical Parameters. The primary hepatocytes and Plin5-overexpressed HepG2 cells (including full-length Plin5 and Plin5 deleting 444--464 aa) were used for in vitro experiment. Haematoxylin and Eosin staining, Oil Red O staining, Bodipy 493/503 staining, Periodic Acid-Schiff staining, immunohistochemistry and JC-1 staining were used to evaluate cell morphology, lipids, glycogen, inflammation and membrane potential. Commercially kits are used to detect glycolipid metabolites, such as triglycerides, glycogen, glucose, reactive oxygen species, lactic acids, ketone bodies. Fluorescently labelled deoxyglucose, NBDG, was used for glucose intake. An XF96 extracellular flux analyser was used to determinate oxygen consumption rate in hepatocytes. The morphological and structural damage of mitochondria was evaluated by electron microscopy. Classical ultracentrifugation is used to separate the subcellular organelles of tissues and cells. Immunoblotting and qPCR were used to detect changes in mRNA and protein levels of related genes. Results: Our results showed that the expression of Plin5 in mouse livers was enhanced by alcohol intake, and Plin5 deficiency aggravated the alcohol-induced liver injury. To clarify the mechanism, we found that Plin5 deficiency significantly elevated the hepatic NADH levels and ketone body production in the alcohol-treated mice. As NADH elevation could promote the reduction of pyruvate into lactate and then inhibit the gluconeogenesis, alcohol-treated Plin5-deficient mice exhibited more lactate production and severer hypoglycemia. These results implied that Plin5 deficiency impaired the mitochondrial oxidative functions in the presence of alcohol. In addition, we demonstrated that Plin5 could be recruited onto mitochondria by alcohol, while Plin5 without mitochondrial targeting sequences lost its mitochondrial protection functions. Conclusion: Collectively, this study demonstrated that the mitochondrial Plin5 could protect the alcohol-induced mitochondrial injury, which provides an important new insight on the roles of Plin5 in highly oxidative tissues. [ABSTRACT FROM AUTHOR]

It has been shown that nicotinamide coenzymes (NADPH, NADH, NADP+, and NAD+) are capable of generating superoxide anions () in an alkaline environment. The superoxide-generating activity of the coenzymes is associated with high pH values and is sensitive to SOD. However, nicotinamide itself, being a functional part of coenzyme molecules, does not have this property. Polarographic studies showed that in the presence of coenzymes, molecular oxygen is consumed from the buffer, namely oxygen activation occurs due to the formation of . Based on the obtained results and in accordance with the literature, our observations suggest that the formation of adducts of nicotinamide, which is part of the coenzyme molecule, and hydroxyl anions (OH–) may lead to the formation of . Under mild conditions in the organism, the studied coenzymes, while performing their main functions, are expected to generate superoxide, meaning that they can be signaling molecules. [ABSTRACT FROM AUTHOR]

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Calcium (Ca 2 + ) signals have a crucial role in regulating various processes of almost every cell to maintain its structure and function. Calcium dynamics has been studied in various cells including hepatocytes by many researchers, but the mechanisms of calcium signals involved in regulation and dysregulation of various processes like ATP degradation rate, IP 3 and NADH production rate respectively in normal and obese cells are still poorly understood. In this paper, a reaction diffusion equation of calcium is employed to propose a model of calcium dynamics by coupling ATP degradation rate, IP 3 and NADH production rate in hepatocyte cells under normal and obese conditions. The processes like source influx, buffer, endoplasmic reticulum (ER), mitochondrial calcium uniporters (MCU) and Na + /Ca 2 + exchanger (NCX) have been incorporated in the model. Linear finite element method is used along spatial dimension, and Crank-Nicolson method is used along temporal dimension for numerical simulation. The results have been obtained for the normal hepatocyte cells and for cells due to obesity. The comparative study of these results reveal significant difference caused due to obesity in Ca 2 + dynamics as well as in ATP degradation rate, IP 3 and NADH production rate. [ABSTRACT FROM AUTHOR]

Abstract Ferroptosis is a recently discovered form of regulated cell death characterized by its distinct dependence on iron and the peroxidation of lipids within cellular membranes. Ferroptosis plays a crucial role in physiological and pathological situations and has attracted the attention of numerous scientists. Ferroptosis suppressive protein 1 (FSP1) is one of the main regulators that negatively regulates ferroptosis through the GPX4‐independent FSP1–CoQ10–NAD(P)H axis and is a potential therapeutic target for ferroptosis‐related diseases. However, the crystal structure of FSP1 has not been resolved, which hinders the development of therapeutic strategies targeting FSP1. To unravel this puzzle, we purified the human FSP1 (hFSP1) protein using the baculovirus eukaryotic cell expression system and solved its crystal structure at a resolution of 1.75 Å. Furthermore, we evaluated the oxidoreductase activity of hFSP1 with NADH as the substrate and identified E156 as the key amino acid in maintaining hFSP1 activity. Interestingly, our results indicated that hFSP1 exists and functions in a monomeric state. Mutagenesis analysis revealed the critical role of the C‐terminal domain in the binding of substrate. These findings significantly enhance our understanding of the functional mechanism of FSP1 and provide a precise model for further drug development.

Abstract Background Macrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates. Methods Here, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation. Results Based on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population. Conclusions Overall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation.

Background Cellular senescence is defined as an irreversible cell cycle arrest caused by various internal and external insults. While the metabolic dysfunction of senescent cells in normal tissue is relatively well-established, there is a lack of information regarding the metabolic features of senescent tumor cells. Methods Publicly available single-cell RNA-sequencing data from the GSE166555 and GSE178341 datasets were utilized to investigate the metabolic features of senescent tumor cells. To validate the single-cell RNA-sequencing data, we performed senescence-associated β-galactosidase (SA-β-Gal) staining to identify senescent tumor cells in fresh frozen colorectal cancer tissue. We also evaluated nicotinamide adenine dinucleotide dehydrogenase–tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH) activity using enzyme histochemical methods and compared the staining with SA-β-Gal staining. MTT assay was performed to reveal the complex 1 activity of the respiratory chain in in-vitro senescence model. Results Single-cell RNA-sequencing data revealed an upregulation in the activity of complexes 1 and 2 in oxidative phosphorylation, despite overall mitochondrial dysfunction in senescent tumor cells. Both SA-β-Gal and enzyme histochemical staining using fresh frozen colorectal cancer tissues indicated a high correlation between SA-β-Gal positivity and NADH-TR/SDH staining positivity. MTT assay showed that senescent colorectal cancer cells exhibit higher absorbance in 600 nm wavelength. Conclusions Senescent tumor cells exhibit distinct metabolic features, characterized by upregulation of complexes 1 and 2 in the oxidative phosphorylation pathway. NADH-TR and SDH staining represent efficient methods for detecting senescent tumor cells in colorectal cancer.

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.

Background: Macrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates. Methods: Here, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation. Results: Based on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population. Conclusions: Overall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation. [ABSTRACT FROM AUTHOR]

The determination of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H2O2) play critical roles in chemical, environmental, and biological processes. In this study, non-enzymatic dual sensors for the simultaneous amperometric determination of NADH and H2O2 were prepared based on a dual screen-printed carbon electrode (DSPCE) modified with reduced graphene oxide (RGO), polyneutral red (PNR), and gold nanoparticles (AuNP). The DSPCE/RGO/PNR/AuNP electrode was characterized by cyclic voltammetry and scanning electron microscopy. RGO/PNR/AuNP materials showed an electrocatalytic effect on the electrooxidation of NADH and the electroreduction of H2O2. Simultaneous amperometric determination of NADH and H2O2 was performed at + 0.15 V and − 0.45 V, respectively. The developed dual sensor allowed the simultaneous determination of both analytes in fast, high repeatability and sensitivity (2.28 µA mM−1 for NADH and 2.70 µA mM−1 for H2O2) and a low limit of detection (5.53 µA for NADH and 8.35 µA for H2O2). The application stability of the dual sensor was tested for 45 days. The simultaneous determination of NADH and H2O2 in blood serum was performed using the fabricated non-enzymatic dual sensor, and high recoveries were obtained. The developed disposable dual sensors can be used in point-of-care tests for simultaneous amperometric determination of NADH and H2O2. A disposable non-enzymatic dual sensor for simultaneous amperometric determination of NADH and H2O2 [ABSTRACT FROM AUTHOR]

Meat is an important source of high-value protein providing sustainable nutrition for human health. The discolouration of meat results in significant waste, which threatens the sustainability of meat production in terms of availability, affordability, and utilisation. Advancing the knowledge of factors and underlying mechanisms for meat discolouration supports the sustainability transformation of meat production practices. Previous studies found that colour stability may be associated with signature changes in certain metabolites, including NADH, glutamate, methionine, and testosterone. This study aimed to confirm the effect of these metabolites and sex, sire, and muscle type on lamb meat colour. NADH and glutamate improved colour stability as evidenced by the increased metmyoglobin reductase activity, while methionine and testosterone had detrimental effects. Overall, lamb meat was discoloured with retail display for up to 10 days at 4 °C. The semitendinosus muscle had higher L*, b*, and hue angle and lower a* (p < 0.05) than other muscles, especially in ewes. Lamb meat from rams had a higher L* and hue angle and lower a* than the ewes (p < 0.05), especially in the colour-labile group, suggesting an interaction between sex and sire. The outcomes of this study will help make the production of meat more sustainable by assisting the meat industry in improving the selection of animals for meat production and processing practices to reduce meat waste due to discolouration. [ABSTRACT FROM AUTHOR]

A highly sensitive electrochemical biosensor for ethanol based on a screen-printed electrode modified with gold nanoparticles—electrochemically reduced graphene oxide—poly (allylamine hydrochloride) nanocomposite (AuNPs-ERGO-PAH) is reported in this work. Ethanol was oxidized in the presence of the oxidized form of the nicotinamide adenine dinucleotide (NAD+) in a reaction catalyzed by alcohol dehydrogenase (ADH) immobilized in sol-gel. The AuNPs-ERGO-PAH nanocomposite was used as a transducer for the electrocatalytic oxidation of the reduced form the nicotinamide adenine dinucleotide (NADH) produced in the enzyme reaction. Under the optimal conditions, the ethanol biosensor exhibits a wide dynamic range from 0.05 to 5 mM with a low detection limit of 10 µM (S/N = 3) and a high sensitivity of 44.6 ± 0.07 µA/mM·cm2 for the linear range between 0.05 and 0.2 mM. The biosensor response was stable for up to 6 weeks. Furthermore, the developed biosensor has been used to detect ethanol in alcoholic beverages with good results, suggesting its potential application in various fields, including fermentation processes and food quality control. [ABSTRACT FROM AUTHOR]

The determination of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H2O2) play critical roles in chemical, environmental, and biological processes. In this study, non-enzymatic dual sensors for the simultaneous amperometric determination of NADH and H2O2 were prepared based on a dual screen-printed carbon electrode (DSPCE) modified with reduced graphene oxide (RGO), polyneutral red (PNR), and gold nanoparticles (AuNP). The DSPCE/RGO/PNR/AuNP electrode was characterized by cyclic voltammetry and scanning electron microscopy. RGO/PNR/AuNP materials showed an electrocatalytic effect on the electrooxidation of NADH and the electroreduction of H2O2. Simultaneous amperometric determination of NADH and H2O2 was performed at + 0.15 V and − 0.45 V, respectively. The developed dual sensor allowed the simultaneous determination of both analytes in fast, high repeatability and sensitivity (2.28 µA mM−1 for NADH and 2.70 µA mM−1 for H2O2) and a low limit of detection (5.53 µA for NADH and 8.35 µA for H2O2). The application stability of the dual sensor was tested for 45 days. The simultaneous determination of NADH and H2O2 in blood serum was performed using the fabricated non-enzymatic dual sensor, and high recoveries were obtained. The developed disposable dual sensors can be used in point-of-care tests for simultaneous amperometric determination of NADH and H2O2. A disposable non-enzymatic dual sensor for simultaneous amperometric determination of NADH and H2O2 [ABSTRACT FROM AUTHOR]

The capability of certain microbial strains to uptake electrons and fix CO2 can be exploited to capture greenhouse gas and convert it into products of interest through a process called microbial bioelectrosynthesis. This study evaluated the capability of C. saccharoperbutylacetonicum N1-4 to utilize bicarbonate for growth, by both supplying electrons in a single-cell reactor and using a graphite-felt assembly as electrodes. The medium was supplemented with 4 g/L bicarbonate and 200 µM NADH. An open circuit experiment was carried out in a medium with bicarbonate and no complex nitrogen sources. The applied potential was -600 mVAg/AgCl. The impedance spectroscopy and cyclic voltammetry techniques were used to characterize and monitor the reduction and oxidation peaks. The conditions that promote the highest observed specific growth rate (0.87±0.18 h-1), were -600 mV, 4 g/L HCO3 -, and NADH. The growth rates of 0.57±0.01 h-1 were observed at 4 g/L HCO3 - without any potential input, and 0.51±0.10 h-1 at a potential of -600 mV in the presence of NADH. The results showed that an environment that provides exogenous electrons and an externally applied potential promotes the capability of C. saccharoperbutylacetonicum N1-4 to reduce CO2, as evidenced when biomass concentration and specific growth rate were increased. [ABSTRACT FROM AUTHOR]