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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]

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.

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]

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]

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]

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]

Elderly patients (age > 75) sustain larger infarcts with greater mortality from ST elevation myocardial infarcts (STEMI) despite successful reperfusion treatment. Elderly age remains an independent risk despite correction for clinical and angiographic variables. The elderly represent a high-risk population and may benefit from treatment in addition to reperfusion alone. We hypothesized that modulation of cardiac signaling and metabolism with acute, high dose metformin given at reperfusion would exhibit additional cardioprotection. Using a translational aging murine model (22-24-month C57BL/6J mice) of in vivo STEMI (45 min artery occlusion with reperfusion for 24 hours); treatment acutely at reperfusion by high dose metformin decreased infarct size and enhanced contractile recovery, demonstrating cardioprotection in the high-risk aging heart. [ABSTRACT FROM AUTHOR]

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.

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.

Introduction: Mitochondrial disease is a spectrum of debilitating disorders caused by mutations in the mitochondrial DNA (mtDNA) or nuclear DNA that compromises the respiratory chain. Mitochondrial 3243A>G (m.3243 A>G) is the most common mutation showing great heterogeneity in phenotype. Previous studies have indicated that NADH: ubiquinone oxidoreductase (complex I) deficiency accompanied by a decreased nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) ratio may play a pivotal role in the pathogenesis of m.3243A>G mutation. Methods: To evaluate the potential effects of strategies targeting the imbalanced NAD+/NADH ratio in m.3243A>G mutation, we treated fibroblasts derived from patients with the m.3243 A>G mutation using nicotinamide riboside (NR) or mitochondria-targeted H2O-forming NADH oxidase (mitoLbNOX). Results: M.3243 A>G fibroblasts showed a significant reduction in complex I core subunit 6, complex I enzymatic activity, complex I-dependent oxygen consumption rate (OCR), and adenosine triphosphate (ATP) production compared to the controls. The NAD+/NADH ratio was also significantly reduced in m.3243 A>G fibroblasts, and, using fluorescence lifetime imaging microscopy, we also found that the NADH level was elevated in m.3243 A>G fibroblasts. After NR treatment, the NAD+/NADH ratio, complex I-dependent OCR, and ATP levels increased, whereas NADH levels remained unchanged. More excitingly, after treatment with mitoLbNOX, the NAD+/NADH ratio, complex I-independent OCR, and ATP levels increased more pronouncedly compared with the NR treatment group, accompanied by significantly reduced NADH levels. Discussion: The present study suggests that compared with repletion of NAD+ alone, the combination of this therapeutic modality with alleviation of NADH overload may amplify the treatment effect of restoring NAD+/NADH balance in m.3243A>G fibroblasts. [ABSTRACT FROM AUTHOR]

The routine monitoring of bacterial populations is crucial for ensuring water quality and safeguarding public health. Thus, an electrochemical sensor based on a 1,10-phenanthroline-5,6-dione-modified electrode was developed and explored for the detection of E. coli. The modified electrode exhibited enhanced NADH oxidation ability at a low potential of 0.1 V, which effectively eliminated the interference from other redox compounds in bacteria. The sensitivity for NADH was 0.222 μA/μM, and the limit of detection was 0.0357 μM. Upon cell lysis, the intracellular NADH was released, and the concentration of E. coli was determined through establishing the relationship between the oxidation current signal and NADH concentration. The performance of the electrochemical sensor in the detection of NADH and E. coli suspensions was validated using the WST-8 colorimetric method. The blank recovery experiment in real water samples exhibited good accuracy, with recovery rates ranging from 89.12% to 93.26% and relative standard deviations of less than 10%. The proposed electrochemical sensor realized the detection of E. coli without the usage of biomarkers, which provides a promising approach for the broad-spectrum detection of microbial contents in complex water environments. [ABSTRACT FROM AUTHOR]

Introduction: Antibiotic persistence (subpopulation tolerance) occurs when a subpopulation of antibiotic sensitive cells survives prolonged exposure to a bactericidal concentration of an antibiotic, and is capable of regrowth once the antibiotic is removed. This phenomenon has been shown to contribute to prolonged treatment duration, infection recurrence, and accelerated development of genetic resistance. Currently, there are no biomarkers which would allow for segregation of these antibiotic-tolerant cells from the bulk population prior to antibiotic exposure, limiting research on this phenomenon to retrograde analyses. However, it has been previously shown that persisters often have a dysregulated intracellular redox homeostasis, warranting its investigation as a potential marker for antibiotic tolerance. Furthermore, it is currently unknown whether another antibiotic tolerant subpopulation - viable but non-culturable cells (VBNCs), are simply persisters with extreme lag phase, or are formed through separate pathways. VBNCs similarly to persisters remain viable following antibiotic exposure, however, are not capable of regrowth in standard conditions. Methods: In this article we employed an NADH:NAD+ biosensor (Peredox) to investigate NADH homeostasis of ciprofloxacin-tolerant E. coli cells on a singlecell level. [NADH:NAD+] was used as a proxy for measuring intracellular redox homeostasis and respiration rate. Results and Discussion: First, we demonstrated that ciprofloxacin exposure results in a high number of VBNCs, several orders of magnitude higher than persisters. However, we found no correlation in the frequencies of persister and VBNC subpopulations. Ciprofloxacin-tolerant cells (persisters & VBNCs) were actively undergoing respiration, although at a significantly lower rate on average when compared to the bulk population. We also noted significant heterogeneity on a single-cell level within the subpopulations, however were unable to segregate persisters from VBNCs based on these observations alone. Finally, we showed that in the highly-persistent strain of E. coli, E. coli HipQ, ciprofloxacin-tolerant cells have a significantly lower [NADH:NAD+] ratio than tolerant cells of its parental strain, providing further link between disturbed NADH homeostasis and antibiotic tolerance. [ABSTRACT FROM AUTHOR]

Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, phasor autofluorescence lifetime imaging, which does not involve any staining, can be used to understand and evaluate the severity of kidney disease.

NADH provides electrons for aerobic ATP production. In cells deprived of oxygen or with impaired electron transport chain activity, NADH accumulation can be toxic. To minimize such toxicity, elevated NADH inhibits the classical NADH-producing pathways: glucose, glutamine, and fat oxidation. Here, through deuterium-tracing studies in cultured cells and mice, we show that folate-dependent serine catabolism also produces substantial NADH. Strikingly, when respiration is impaired, serine catabolism through methylene tetrahydrofolate dehydrogenase (MTHFD2) becomes a major NADH source. In cells whose respiration is slowed by hypoxia, metformin, or genetic lesions, mitochondrial serine catabolism inhibition partially normalizes NADH levels and facilitates cell growth. In mice with engineered mitochondrial complex I deficiency (NDUSF4-/-), serine's contribution to NADH is elevated, and progression of spasticity is modestly slowed by pharmacological blockade of serine degradation. Thus, when respiration is impaired, serine catabolism contributes to toxic NADH accumulation.

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or β-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance.

In recent years, clean energy technologies that meet ever-increasing energy demands without the risk of environmental contamination has been a major interest. One approach is the utilization of plant leaves, which release redox-active NADPH as a result of photosynthesis, to generate photocurrent. In this work, we show for the first time that photocurrent can be harvested directly from the fruit of a cherry tree when associated with a bio-electrochemical cell. Furthermore, we apply electrochemical and spectroscopic methods to show that NADH in the fruit plays a major role in electric current production.

Abstract Background Saccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis. Results Consistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD+-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose–sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high Km for CO2 of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO2. Prolonged chemostat cultivation on glucose–sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway. Conclusions Combination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.

In recent years, clean energy technologies that meet ever-increasing energy demands without the risk of environmental contamination has been a major interest. One approach is the utilization of plant leaves, which release redox-active NADPH as a result of photosynthesis, to generate photocurrent. In this work, we show for the first time that photocurrent can be harvested directly from the fruit of a cherry tree when associated with a bio-electrochemical cell. Furthermore, we apply electrochemical and spectroscopic methods to show that NADH in the fruit plays a major role in electric current production. [ABSTRACT FROM AUTHOR]

IntroductionAntibiotic persistence (subpopulation tolerance) occurs when a subpopulation of antibiotic sensitive cells survives prolonged exposure to a bactericidal concentration of an antibiotic, and is capable of regrowth once the antibiotic is removed. This phenomenon has been shown to contribute to prolonged treatment duration, infection recurrence, and accelerated development of genetic resistance. Currently, there are no biomarkers which would allow for segregation of these antibiotic-tolerant cells from the bulk population prior to antibiotic exposure, limiting research on this phenomenon to retrograde analyses. However, it has been previously shown that persisters often have a dysregulated intracellular redox homeostasis, warranting its investigation as a potential marker for antibiotic tolerance. Furthermore, it is currently unknown whether another antibiotic tolerant subpopulation - viable but non-culturable cells (VBNCs), are simply persisters with extreme lag phase, or are formed through separate pathways. VBNCs similarly to persisters remain viable following antibiotic exposure, however, are not capable of regrowth in standard conditions.MethodsIn this article we employed an NADH:NAD+ biosensor (Peredox) to investigate NADH homeostasis of ciprofloxacin-tolerant E. coli cells on a single-cell level. [NADH:NAD+] was used as a proxy for measuring intracellular redox homeostasis and respiration rate.Results and DiscussionFirst, we demonstrated that ciprofloxacin exposure results in a high number of VBNCs, several orders of magnitude higher than persisters. However, we found no correlation in the frequencies of persister and VBNC subpopulations. Ciprofloxacin-tolerant cells (persisters & VBNCs) were actively undergoing respiration, although at a significantly lower rate on average when compared to the bulk population. We also noted significant heterogeneity on a single-cell level within the subpopulations, however were unable to segregate persisters from VBNCs based on these observations alone. Finally, we showed that in the highly-persistent strain of E. coli, E. coli HipQ, ciprofloxacin-tolerant cells have a significantly lower [NADH:NAD+] ratio than tolerant cells of its parental strain, providing further link between disturbed NADH homeostasis and antibiotic tolerance.

The transcription factor Nrf2 and its repressor Keap1 mediate cell stress adaptation by inducing expression of genes regulating cellular detoxification, antioxidant defence and energy metabolism. Energy production and antioxidant defence employ NADH and NADPH respectively as essential metabolic cofactors; both are generated in distinct pathways of glucose metabolism, and both pathways are enhanced by Nrf2 activation. Here, we examined the role of Nrf2 on glucose distribution and the interrelation between NADH production in energy metabolism and NADPH homeostasis using glio-neuronal cultures isolated from wild-type, Nrf2-knockout and Keap1-knockdown mice. Employing advanced microscopy imaging of single live cells, including multiphoton fluorescence lifetime imaging microscopy (FLIM) to discriminate between NADH and NADPH, we found that Nrf2 activation increases glucose uptake into neurons and astrocytes. Glucose consumption is prioritized in brain cells for mitochondrial NADH and energy production, with a smaller contribution to NADPH synthesis in the pentose phosphate pathway for redox reactions. As Nrf2 is suppressed during neuronal development, this strategy leaves neurons reliant on astrocytic Nrf2 to maintain redox balance and energy homeostasis.

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.

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However, in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is a subject of debate. The fluorescence lifetime of NADH increases when bound to an enzyme, in contrast to the short multiexponential lifetime displayed by NADH in solution. The extent of fluorescence lifetime increase depends on the enzyme to which NADH is bound. With proper preparation of lactate dehydrogenase (LDH) using oxalic acid (OA) as an allosteric factor, bound NADH to LDH has a lifetime of 3.4 ns and is positioned on the universal semicircle of the phasor plot, inferring a monoexponential lifetime for this species. Surprisingly, measurements in the cellular environments with different metabolic states show a linear trajectory between free NADH at about 0.37 ns and bound NADH at 3.4 ns. These observations support that in a cellular environment, a 3.4 ns value could be used for bound NADH lifetime. The phasor analysis of many cell types shows a linear combination of fractional contributions of free and bound species NADH.

Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer's disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 to 41% more reducing NADH redox state than cytoplasm for each age, genotype, and sex. Aging diminished the mitochondrial free NADH concentration in NTg neurons by 43% and in 3xTg-AD by 50%. The lower free NADH with age suggests a decline in capacity to regenerate free NADH for energetic supply to power oxidative phosphorylation which further worsens in AD. Applying this non-invasive approach, we showed the most explicit measures yet of bioenergetic deficits in free NADH with aging at the subcellular level in live neurons from in-bred mice and an AD model.

Abstract The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.