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Non-invasive detection of polarized macrophages in tumors is an urgent task in terms of combined antitumor therapy. By analyzing the fluorescence lifetime of the metabolic cofactors—the reduced form of nicotinamide adenine dinucleotide (NADH) and flavins—differences in cellular metabolism of normal tissue, tumor, inflammatory and anti-inflammatory macrophages were demonstrated. In this work we studied changes in the polarization of macrophages obtained from THP-1 monocytes in response to photodynamic therapy with 5-aminolevulinic acid (ALA–PDT). Moderate ALA–PDT in vitro led to changes in M0 macrophages metabolism towards M1 polarization, wherein M1 and M2 macrophages died and were replaced by non-polarized cells. The interstitial distribution of polarized macrophages after ALA–PDT was studied in a mouse tumor model of grafted Lewis lung carcinoma. In response to ALA–PDT, there was an increase in the inflammatory macrophages fraction in the tumor node. Metabolic fluorescence-lifetime imaging microscopy (FLIM) was performed for macrophages in vitro and for tumor cryosections. It was shown that analysis of phasor diagrams for the NADH, flavins, and 5-ALA-induced protoporphyrin IX (PpIX) fluorescence lifetime helps to determine the change in metabolism in response to different modes of PDT at the cellular and tissue levels. These data can be used for post-surgery tissue inspection. [ABSTRACT FROM AUTHOR]

The stress induced protein NQO1 can participate in a wide range of biological pathways which are dependent upon the interaction of NQO1 with protein targets. Many of the protein-protein interactions involving NQO1 have been shown to be regulated by the pyridine nucleotide redox balance. NQO1 can modify its conformation as a result of redox changes in pyridine nucleotides and sites on the C-terminal and helix seven regions of NQO1 have been identified as potential areas that may be involved in redox-dependent protein-protein interactions. Since post-translational modifications can modify the functionality of proteins, we examined whether redox-dependent conformational changes induced in NQO1 would alter lysine acetylation. Recombinant NQO1 was incubated with and without NADH then acetylated non-enzymatically by acetic anhydride or S-acetylglutathione (Ac-GSH). NQO1 acetylation was determined by immunoblot and site-specific lysine acetylation was quantified by mass spectrometry (MS). NQO1 was readily acetylated by acetic anhydride and Ac-GSH. Interestingly, despite a large number of lysine residues (9%) in NQO1 only a small subset of lysines were acetylated and the majority of these were located in or near the functional C-terminal or helix seven regions. Reduction of NQO1 by NADH prior to acetylation resulted in almost complete protection of NQO1 from lysine acetylation as confirmed by immunoblot analysis and MS. Lysines located within the redox-active C-terminus and helix seven regions were readily acetylated when NQO1 was in an oxidized conformation but were protected from acetylation when NQO1 was in the reduced conformation. To investigate regulatory mechanisms of enzymatic deacetylation, NQO1 was acetylated by Ac-GSH then exposed to purified sirtuins (SIRT 1-3) or histone deacetylase 6 (HDAC6). NQO1 could be deacetylated by all sirtuin isoforms and quantitative MS analysis performed using SIRT2 revealed very robust deacetylation of NQO1, specifically at K262 and K271 in the C-terminal region. No deacetylation of NQO1 by HDAC6 was detected. These data demonstrate that the same subset of key lysine residues in the C-terminal and helix seven regions of NQO1 undergo redox dependent acetylation and are regulated by sirtuinmediated deacetylation. [ABSTRACT FROM AUTHOR]

Confinement of reaction spaces was achieved in a biomimetic manner by using liposome vesicles that are based on phospholipid bilayer membranes, similar to cellular compartments. Encapsulation of photosensitizer (PS) and substrate within the inner aqueous compartment of liposomes accelerated the photosensitized model reaction of nicotinamide adenine dinucleotide (NADH) conversion to its oxidized form (NAD+) by one order of magnitude compared to classical homogeneous reaction conditions. Furthermore, it was found that the reaction proceeds around 40 % faster when the photosensitizer is dissolved in the inner aqueous compartment instead of being embedded within the phospholipid bilayer which is attributed to the diffusion behavior of singlet oxygen which acts as oxidant in this reaction. These experimental findings will allow for reaction and molecular systems design for photochemical and catalytic conversions and will be relevant in the context of creating artificial cellular compartments such as fully artificial chloroplasts. [ABSTRACT FROM AUTHOR]

Drug memory is associated with drug-taking experience and environmental cues, which mainly contribute to addiction. Recent studies report that glycogenolysis-derived lactate from astrocyte transport to neurons is necessary for long-term potentiation and memory formation instead of its function as an energy substrate. However, the role of astrocyte-neuron lactate transfer in neuronal plasticity and methamphetamine (METH)-induced addiction memory consolidation and retrieval, especially the underlying mechanisms, are not clear. C57BL/6 J mice trained for METH-induced conditioned place preference (CPP) were stereotaxically injected with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) into the dorsal hippocampus (dHPC) 15 min before training. The CPP score was recorded, and neuronal synaptic plasticity was detected with Golgi staining. The neuronal Ca2+ levels were examined using AAV-GCaMP6 injection. Moreover, monocarboxylate transporters (MCT1, MCT2, MCT4) were inhibited with oligodeoxynucleotides in the dHPC to further prove the METH appetitive memory changes. The data showed that inhibiting lactate transport by microinjection with DAB or monocarboxylate transporter oligodeoxynucleotides in the dHPC completely destroyed METH-induced CPP, reduced Npas4 and other plasticity-associated gene expression and decreased neuronal Ca2+ levels and neuronal arborization and spine density, all of which were fully rescued by L-lactate coadministration except for MCT2-ODN administration. Furthermore, the downstream signaling molecule NADH could mimic lactate's effects and trigger METH CPP by influencing the redox state of neurons and regulating NMDA receptor activity. Collectively, these findings indicate that astrocyte-neuron lactate transfer is crucial for METH-induced memory consolidation and retrieval. [ABSTRACT FROM AUTHOR]

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. Wholegenome 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 PRKRuBisCO 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. [ABSTRACT FROM AUTHOR]

Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4′,6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes. [ABSTRACT FROM AUTHOR]

In the present review, which is aimed at researchers, teachers and students in life sciences, we try to show how the physicochemical properties of the elements and molecules define the concept of redox balance. Living organism are open systems traversed by fluxes of energy and matter. During catabolic oxidative metabolism, matter—mostly hydrogenated organic molecules—is oxidized and ultimately released as CO2. Electrons are passed over to coupling molecules, such as NAD+ and FAD, whose reduced forms serve as electrons donors in anabolic reactions. Early photosynthetic activity led to the accumulation of O2 and the transformation of the reduction to an oxidizing atmosphere, favoring the development of oxidative metabolism in living organisms. We focus on the specific properties of O2 that provide the chemical energy for the combustion reactions occurring in living cells. We explain the concepts of redox potential and redox balance in complex systems such as living cells, we present the main redox couples involved in cellular redox balance and we discuss the chemical properties underlying their cellular roles and, in particular, their antioxidant properties in the defense against reactive oxygen species (ROS). Finally, we try to provide an integrative view emphasizing the interplay between metabolism, oxidative stress and metabolic compartmentation in mammalian cells. [ABSTRACT FROM AUTHOR]

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.

Photo-enzyme coupling system are receiving widespread attention due to the unique opportunity by integrating photochemistry with nature's enzymatic machinery. Here we highlight a recent work about MOF encapsulated tandem enzymes for photoenzymatic CO2 conversion, and provide an outlook for the further development of photo-enzyme systems for green bio-manufacturing., (© 2024 Wiley‐VCH GmbH.)

The long-lasting activity of photosensitizers during photodynamic therapy (PDT) causes excessive damage and arouses great concerns about biosafety. Herein, we synthesized a pyridinium-decorated diiodo-BODIPY compound (PyBDP) and investigated its photosensitizing activity under hypoxic condition in the presence of NADH that is abundant in the mitochondria of hypoxic tumors. The unique property of PyBDP lies in the redox environment-dependent photo-response. At green light exposure, PyBDP is converted into a colorless inactive form by interacting with NADH in a two-step one-electron transfer process. Interestingly, the NADH-dependent hydrogenation of PyBDP is affected by the presence of cytochrome c (Cyt cox) that is an important component of mitochondrial electron transport chain (Mito-ETC), unless Cyt cox is exhausted. Active radical species is produced during the photocatalytic reaction, which adds the understanding of PyBDP-induced photodamage. Therefore, we applied the strategy of auto-deactivation PDT using a BODIPY photosensitizer by tethering triphenylphosphonium to PyBDP. After PDT effect in a type I pathway, the photosensitizer underwent almost entire auto-deactivation in hypoxic HeLa cells. This work paves a way for the development of reductive PDT with enhanced safety and efficacy in fighting hypoxic tumors independent on reactive oxygen species (ROS)., (© 2024 Wiley‐VCH GmbH.)

Horse Liver Alcohol Dehydrogenase (HLADH) is an extensively studied enzyme isolated from equine liver tissue, and holds a central role in numerous enzymatic processes, underscoring the need for thorough investigation. This study delves into the kinetic behavior and structural dynamics of HLADH, shedding light on complex mechanisms governing its catalytic activity and interactions with the cofactor. Notably, deviations from traditional Michaelis-Menten kinetics are observed, manifesting as a slowdown in catalytic rate under high NADH concentrations. Utilizing molecular dynamics simulations, an allosteric site is identified, clarifying how excessive cofactor levels impact protein dynamics and catalytic properties. Structural alterations induced by inhibitory NADH concentrations are revealed, indicating reduced protein flexibility and modifications in catalytic cavity size, thereby elucidating the inhibitory mechanism at high cofactor concentrations. This comprehensive investigation unveils intricate facets of HLADH's catalytic mechanisms, providing a platform for further exploration in enzymology and biocatalysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme, existing in its oxidized form (NAD + ) and reduced form (NADH). Both are essential in cellular redox reactions and are implicated in energy production and cancer. Current NADH detection methods often involve complex optical measurements. We propose a miniaturized, on-chip photoelectric sensor array using AlGaN/GaN two-dimensional electron gas (2DEG) photodetectors for NADH quantification. The device exhibits an ultralow dark current and ultrahigh UV light responsivity, enabling sensitive NADH detection. By exploiting the absorbance disparity between NADH and NAD + , our sensor achieves rapid, sensitive detection, surpassing commercial assays. It effectively detects NADH levels in 3D multicellular models, promising cancer screening and monitoring. This sensor platform offers a significant advancement in NADH quantification, with the potential for high-throughput testing and point-of-care diagnostics. Our study presents an efficient approach for NADH sensing, addressing the need for rapid and sensitive detection methods in biomedical research and clinical practice.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Choi, Shakeri, Kim, Kong, Bowker and Zhuang.)

The β-hydroxyacid dehydrogenase family exhibits diverse cofactor preferences: some enzymes favor NAD, others favor NADP, and a subset can utilize both NAD and NADPH. Glyoxylate reductase from Acetobacter aceti JCM 20276 (AacGR) exhibits a dual cofactor specificity for NADPH and NADH in its catalytic reduction of glyoxylate to glycolate. In contrast to conventional cofactor-discriminating motifs, NRX and DXX, found in NADP- and NAD-specific enzymes, respectively, AacGR has a TPS motif in the equivalent position. Here we report X-ray crystallographic analysis of AacGR in its ligand-free form, and in complexes with NADPH and NADH, revealing critical interactions: Ser41 of the TPS motif interacted with the 2'-phosphate group of NADPH, while no analogous interaction occurred with the ribose hydroxy groups of NADH. Moreover, the TPS motif resided within a characteristic β-turn-like structure adjacent to a long flexible loop. Site-directed mutagenesis and kinetic analyses suggest that Ser41 facilitates NADPH binding, while the lack of a direct interaction of the TPS motif with NADH may allow for NADH utilization. The conformational dynamics of the TPS-containing β-turn-like structure along with the flexible loop likely govern the dual cofactor specificity and catalytic turnover of AacGR., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

ABSTRACT Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv. campestris (XccSAH) and found that it metabolizes both cellular alarmones and cofactors. In vitro, XccSAH displays abilities to hydrolyze multiple nucleotides, including pppGpp, ppGpp, pGpp, pppApp, and NADPH. In vivo, X. campestris pv. campestris cells lacking sah accumulated higher levels of cellular (pp)pGpp and NADPH compared to wild-type cells upon amino acid starvation. In addition, X. campestris pv. campestris mutants lacking sah were more sensitive to killing by Pseudomonas during interbacterial competition. Interestingly, loss of sah also resulted in reduced growth in amino acid-replete medium, a condition that did not induce (pp)pGpp or pppApp accumulation. Further metabolomic characterization revealed strong depletion of NADH levels in the X. campestris pv. campestris mutant lacking sah, suggesting that NADPH/NADH regulation is an evolutionarily conserved function of both bacterial and metazoan SAHs and Mesh1. Overall, our work demonstrates a regulatory role of bacterial SAHs as tuners of stress responses and metabolism, beyond functioning as antitoxins. IMPORTANCE Small alarmone hydrolases (SAHs) comprise a widespread family of alarmone metabolizing enzymes. In metazoans, SAHs have been reported to control multiple aspects of physiology and stress resistance through alarmone and NADPH metabolisms, but their physiological functions in bacteria is mostly uncharacterized except for a few reports as antitoxins. Here, we identified an SAH functioning independently of toxins in the phytopathogen Xanthomonas campestris pv. campestris. We found that XccSAH hydrolyzed multiple alarmones and NADPH in vitro, and X. campestris pv. campestris mutants lacking sah displayed increased alarmone levels during starvation, loss of interspecies competitive fitness, growth defects, and strong reduction in NADH. Our findings reveal the importance of NADPH hydrolysis by a bacterial SAH. Our work is also the first report of significant physiological roles of bacterial SAHs beyond functioning as antitoxins and suggests that SAHs have far broader physiological roles and share similar functions across domains of life.

The stress induced protein NQO1 can participate in a wide range of biological pathways which are dependent upon the interaction of NQO1 with protein targets. Many of the protein-protein interactions involving NQO1 have been shown to be regulated by the pyridine nucleotide redox balance. NQO1 can modify its conformation as a result of redox changes in pyridine nucleotides and sites on the C-terminal and helix seven regions of NQO1 have been identified as potential areas that may be involved in redox-dependent protein-protein interactions. Since post-translational modifications can modify the functionality of proteins, we examined whether redox-dependent conformational changes induced in NQO1 would alter lysine acetylation. Recombinant NQO1 was incubated with and without NADH then acetylated non-enzymatically by acetic anhydride or S-acetylglutathione (Ac-GSH). NQO1 acetylation was determined by immunoblot and site-specific lysine acetylation was quantified by mass spectrometry (MS). NQO1 was readily acetylated by acetic anhydride and Ac-GSH. Interestingly, despite a large number of lysine residues (9%) in NQO1 only a small subset of lysines were acetylated and the majority of these were located in or near the functional C-terminal or helix seven regions. Reduction of NQO1 by NADH prior to acetylation resulted in almost complete protection of NQO1 from lysine acetylation as confirmed by immunoblot analysis and MS. Lysines located within the redox-active C-terminus and helix seven regions were readily acetylated when NQO1 was in an oxidized conformation but were protected from acetylation when NQO1 was in the reduced conformation. To investigate regulatory mechanisms of enzymatic deacetylation, NQO1 was acetylated by Ac-GSH then exposed to purified sirtuins (SIRT 1-3) or histone deacetylase 6 (HDAC6). NQO1 could be deacetylated by all sirtuin isoforms and quantitative MS analysis performed using SIRT2 revealed very robust deacetylation of NQO1, specifically at K262 and K271 in the C-terminal region. No deacetylation of NQO1 by HDAC6 was detected. These data demonstrate that the same subset of key lysine residues in the C-terminal and helix seven regions of NQO1 undergo redox dependent acetylation and are regulated by sirtuin-mediated deacetylation.

NADH is a cofactor used by a wide range of dehydrogenases. Measurement of the concentration of NADH is widely used to measure the activity of enzymes. Extensive efforts have been made to develop electrochemical sensors for NADH determination at low overpotentials to avoid interferences. The development of an enzymatic electrochemical biosensor for the detection of NADH is described. Nanoporous gold electrodes were utilised for the immobilization of diaphorase and osmium-based polymer Os(bpy)2(PVI). Nanoporous gold electrodes of different pore sizes were manufactured by varying the dealloying temperature. To optimise the enzymatic response, the concentrations of polymer and enzyme, average pore size and the operating temperature were examined with the optimal performance observed using 10 µL of Os(bpy)2(PVI) and 5 µL of diaphorase at concentrations of 6 and 10 mg/mL, respectively, on nanoporous gold electrodes with an average pore size of 5.9 nm. The biosensor showed a high sensitivity of 89.6 µA/cm2 mM, a low LOD of 0.8 µM and a linear range from 5 to 100 µM at a potential of 0.35 V vs Ag/AgCl at a temperature of 40 °C.

ABSTRACT This study aimed to investigate the antibacterial mechanism of cefiderocol (CFDC) using data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays. Numerous differentially expressed proteins related to the production of NADH, reduced cofactor flavin adenine dinucleotide (FADH2), NADPH and reactive oxygen species (ROS), iron-sulfur cluster binding, and iron ion homeostasis were found to be upregulated by CFDC. Furthermore, parallel reaction monitoring analysis validated these results. Meanwhile, we confirmed that the levels of NADH, ROS, H2O2, and iron ions were induced by CFDC, and the sensitivity of Escherichia coli to CFDC was inhibited by the antioxidant vitamin C, N-acetyl-l-cysteine, and deferoxamine. Moreover, deferoxamine also suppressed the H2O2 stress induced by CFDC. In addition, knockout of the NADH-quinone oxidoreductase genes (nuoA, nuoC, nuoE, nuoF, nuoG, nuoJ, nuoL, nuoM) in the respiratory chain attenuated the sensitivity of E. coli to CFDC far beyond the effects of cefepime and ceftazidime; in particular, the E. coli BW25113 ΔnuoJ strain produced 60-fold increases in MIC to CFDC compared to that of the wild-type E. coli BW25113 strain. The present study revealed that CFDC exerts its antibacterial effects by inducing ROS stress by elevating the levels of NADH and iron ions in E. coli. IMPORTANCE CFDC was the first FDA-approved siderophore cephalosporin antibiotic in 2019 and is known for its Trojan horse tactics and broad antimicrobial activity against Gram-negative bacteria. However, its antibacterial mechanism is not fully understood, and whether it has an impact on in vivo iron ion homeostasis remains unknown. To comprehensively reveal the antibacterial mechanisms of CFDC, data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays were performed in this study. The findings will further facilitate our understanding of the antibacterial mechanism of CFDC and may provide a theoretical foundation for controlling CFDC resistance in the future.

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.

Kidney transplantation is the only definitive therapy for end-stage kidney disease. The shortage of organs for transplantation is the main limitation of this life-saving treatment. Normothermic machine perfusion (NMP) is a novel preservation technique with the potential to increase the number of transplantable kidneys through reducing delayed graft function and organ evaluation under physiological conditions. To date, the cellular effects and possible pharmacological interventions during machine perfusion are incompletely understood. A major limitation is the technically complex, time-consuming, and small-scale replication of NMP in rodent models. To overcome this, we developed a 3D-printed, high throughput ex-vivo mouse kidney slice incubator (KSI) mimicking mouse kidney NMP by working under closely resembling conditions. KSI significantly reduced the time per experiment and increased the sample throughput (theoretical: 54 incubations with n = 500/day). The model recapitulated the cellular responses during NMP, namely increased endoplasmic reticulum stress (ER stress). Using KSI, five pharmacological interventions against ER stress taken from the literature were tested. While four were ineffective and excluded, one, β-Nicotinamide-adeninedinucleotide (NADH), ameliorated ER stress significantly during KSI. The test of NADH in mouse kidney NMP replicated the positive effects against ER stress. This suggests that testing the addition of NADH during clinical kidney NMP might be warranted. [ABSTRACT FROM AUTHOR]

Phenazine compounds have been used as simple organic catalysts for the rapid oxidation of NADH. Although positively charged phenazine compounds are highly water‐soluble, their uncharged, reduced forms are not. We report the use of a zwitterionic phenazine compound, 3‐(1‐methoxyphenazin‐5‐ium‐5‐yl)propane‐1‐sulfonate (mPPS), as an organic catalyst and electron mediator for the electrochemical oxidation of NADH and detection of glucose and lactate dehydrogenase (LDH). mPPS is highly stable in phosphate buffered saline (pH 7.4). Furthermore, the negatively charged, reduced form of mPPS is highly water‐soluble. The detection limits of glucose and LDH in artificial serum were approximately 0.02 mM and 2 ng/mL, respectively. [ABSTRACT FROM AUTHOR]

Malate dehydrogenase (MDH), which catalyzes a reversible conversion of L-malate to oxaloacetate, plays essential roles in common metabolic processes, such as the tricarboxylic acid cycle, the oxaloacetate–malate shuttle, and the glyoxylate cycle. MDH2 has lately been recognized as a promising anticancer target; however, the structural information for the human homologue with natural ligands is very limited. In this study, various complex structures of hMDH2, with its substrates and/or cofactors, were solved by X-ray crystallography, which could offer knowledge about the molecular and enzymatic mechanism of this enzyme and be utilized to design novel inhibitors. The structural comparison suggests that phosphate binds to the substrate binding site and brings the conformational change of the active loop to a closed state, which can secure the substate and cofactor to facilitate enzymatic activity. [ABSTRACT FROM AUTHOR]

Elucidating the underlying photochemical mechanisms of action (MoA) of photodynamic therapy (PDT) may allow its efficacy to be improved and could set the stage for the development of new classes of PDT photosensitizers. Here, we provide evidence that "photoredox catalysis in cells," wherein key electron transport pathways are disrupted, could constitute a general MoA associated with PDT. Taking the cellular electron donor nicotinamide adenine dinucleotide as an example, we have found that well-known photosensitizers, such as Rose Bengal, BODIPY, phenoselenazinium, phthalocyanine, and porphyrin derivatives, are able to catalyze its conversion to NAD+. This MoA stands in contrast to conventional type I and type II photoactivation mechanisms involving electron and energy transfer, respectively. A newly designed molecular targeting photocatalyst (termed CatER) was designed to test the utility of this mechanism-based approach to photosensitizer development. Photoexcitation of CatER induces cell pyroptosis via the caspase 3/GSDME pathway. Specific epidermal growth factor receptor positive cancer cell recognition, high signal-to-background ratio tumor imaging (SBRTI = 12.2), and good tumor growth inhibition (TGI = 77.1%) are all hallmarks of CatER. CatER thus constitutes an effective near-infrared pyroptotic cell death photo-inducer. We believe the present results will provide the foundation for the synthesis of yet-improved phototherapeutic agents that incorporate photocatalytic chemistry into their molecular design. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human platelet polyphosphate (polyP) is a multifunctional molecule; however, its functions are not yet fully understood. A recent study demonstrated that similar to skeletal muscle, polyP is involved in energy metabolism in platelets, which suggests that well‐trained athletes may exhibit elevated platelet polyP levels for energy storage. To test this hypothesis, we quantified platelet polyP along with NADH, a component involved in ATP production in non‐trained and well‐trained male Japanese participants of the same generation. Washed platelets were prepared from the venous blood of young, healthy, non‐athletes, and professional soccer players (pro‐athletes). NADH and polyP levels were spectrophotometrically determined using tetrazolium reduction and fluorometrically determined using 4′,6‐diamidino‐2‐phenylindole at the excitation/emission wavelengths of 425/525 nm. Body weight and impedances were measured simultaneously. Statistical analyses were performed using the Mann–Whitney U test and Spearman correlation coefficient. Although basal metabolic rate levels were significantly higher, platelet polyP levels were significantly lower in pro‐athletes than in that in non‐athletes. No significant differences were detected in other body compositions or platelet indices between the two groups. The pro‐athlete group showed a moderate, nearly significant correlation (R = 0.439; p = 0.0512) between platelet polyP and NADH levels. Taken together with the weak correlation data between polyP and body mass index, it is suggested that platelet polyP levels may be influenced by platelet and body energy metabolic activity. Further biochemical studies are needed to elucidate this mechanism. [ABSTRACT FROM AUTHOR]

Near-infrared fluorescent probe was prepared for selective detection of reduced nicotinamide adenine dinucleotide (NADH) in live cells. [Display omitted] • A novel fluorescent probe was prepared for sensitive detection of NADH in the near-infrared emission region of 740 nm. • Computation chemistry was used to predict optical properties of the probe in the absence and presence of NADH. • The probe was used to detect NADH concentration changes in HeLa cells caused by glucose treatment and under different pyruvate/lactate ratio levels. A near-infrared fluorescent probe was prepared for selective detection of reduced nicotinamide adenine dinucleotide (NADH) in live cells. The probe turns off the fluorescence with a closed spironolactone switch. However, reduction of the probe by NADH turns on fluorescence at 740 nm. Theoretical calculations suggest a more planar arrangement between the rhodamine and quinoline moieties with increased π-delocalization resulting from reduction. [ABSTRACT FROM AUTHOR]

Microbial engineering based on synthetic biology can facilitate large-scale production of target products. In this study, the introduction of lactate dehydrogenase (LDH) enabled Saccharomyces cerevisiae to acquire the capacity for L-lactic acid (LA) production and the NADH/NAD+ ratio from 0.228 to 0.156, while the subsequent modification of carbon metabolism pathway led to a rapid increase of NADH/NAD+ even up to 0.337. By testing the effectiveness of four different redox systems, we demonstrated that dynamic regulation of additional redox genes to consume excessive NADH is more beneficial for LA accumulation, alleviating the negative effects of metabolic modification on hosts, and altering the distribution of metabolic flow. We first reported expression of GLT1 which coding glutamate synthase has the strongest ability to increase LA production and reduce NADH/NAD+. Combining metabolic engineering and cofactor engineering, the LA yield reached from 0.04 g/g to 0.37 g/g in YNB medium. Subsequently, strain PK27 produced 37.94 g/L LA with production yield of 0.66 g/g in YPD medium. Finally, the results could provide a reference that the potential under poor nutrient culture conditions and the direction and intensity of regulation of intracellular NADH/NAD+ for LA accumulation. [ABSTRACT FROM AUTHOR]

The preservation of cognitive ability by increasing nicotinamide adenine dinucleotide (NAD+) levels through supplementation with NAD+ precursors has been identified as a promising treatment strategy for a number of conditions; principally, age-related cognitive decline (including Alzheimer's disease and vascular dementia), but also diabetes, stroke, and traumatic brain injury. Candidate factors have included NAD+ itself, its reduced form NADH, nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and niacin (or nicotinic acid). This review summarises the research findings for each source of cognitive impairment for which NAD+ precursor supplementation has been investigated as a therapy. The findings are mostly positive but have been made primarily in animal models, with some reports of null or adverse effects. Given the increasing popularity and availability of these factors as nutritional supplements, further properly controlled clinical research is needed to provide definitive answers regarding this strategy's likely impact on human cognitive health when used to address different sources of impairment. [ABSTRACT FROM AUTHOR]

MitoNEET is the first iron-sulfur protein found in mitochondrial outer membrane. Abnormal expression of mitoNEET in cells has been linked to several types of cancer, type II diabetes, and neurodegenerative diseases. Structurally, mitoNEET is anchored to mitochondrial outer membrane via its N-terminal single transmembrane alpha helix. The C-terminal cytosolic domain of mitoNEET binds a [2Fe–2S] cluster via three cysteine and one histidine residues. It has been shown that mitoNEET has a crucial role in energy metabolism, iron homeostasis, and free radical production in cells. However, the exact function of mitoNEET remains elusive. Previously, we reported that the C-terminal soluble domain of mitoNEET has a specific binding site for flavin mononucleotide (FMN) and can transfer electrons from FMNH 2 to oxygen or ubiquinone-2 via its [2Fe–2S] cluster. Here we have constructed a hybrid protein using the N-terminal transmembrane domain of Escherichia coli YneM and the C-terminal soluble domain of human mitoNEET and assembled the hybrid protein YneM-mitoNEET into phospholipid nanodiscs. The results show that the [2Fe–S] clusters in the nanodisc-bound YneM-mitoNEET can be rapidly reduced by FMNH 2 which is reduced by flavin reductase using NADH as the electron donor. Addition of lumichrome, a FMN analog, effectively inhibits the FMNH 2 -mediated reduction of the [2Fe–2S] clusters in the nanodisc-bound YneM-mitoNEET. The reduced [2Fe–2S] clusters in the nanodisc-bound YneM-mitoNEET are quickly oxidized by oxygen under aerobic conditions or by ubiquinone-10 in the nanodiscs under anaerobic conditions. Because NADH oxidation is required for cellular glycolytic activity, we propose that the mitochondrial outer membrane protein mitoNEET may promote glycolysis by transferring electrons from FMNH 2 to oxygen or ubiquinone-10 in mitochondria. [Display omitted] • MitoNEET is a mitochondrial outer membrane protein with a [2Fe–2S] cluster. • A hybrid protein of E. coli YneM and human mitoNEET was constructed. • YneM-mitoNEET transfers electrons from FMNH 2 to O 2 or ubiquinone. • Membrane-bound mitoNEET may act as a novel redox enzyme. [ABSTRACT FROM AUTHOR]

Iron, an essential element for most of living organisms, participates in many biological functions. Since iron is redox-active transition metal, it is known that excessive levels stimulate the formation of reactive oxygen species (ROS) and exacerbate cytotoxicity. An iron deficiency is the most common nutritional deficiency disorder in the world (about 30% of the population) and is more common than cases of iron overload. However, the effects of iron deficiency on ROS-induced cytotoxicity and the maintenance of intracellular redox homeostasis are not fully understood. The present study reports on an evaluation of the effects of iron deficiency on cytotoxicity induced by several ROS generators. In contrast to hydrogen peroxide and erastin, the cytotoxicity of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling agent that induces intracellular superoxide anion formation, was exacerbated by iron deficiency. Cytochrome b5 reductase was identified as a candidate enzyme responsible for the redox cycling of DMNQ under conditions of iron depletion. Moreover, the DMNQ-induced intracellular accumulation of ROS and a decrease in NADH/NAD+ ratios were enhanced by an iron deficiency. These negative changes were found to be ameliorated by overexpressing NAD(P)H:quinone oxidoreductase 1 (NQO1) in kidney-derived cells that originally showed a very low expression of NQO1. These results indicate that NQO1 plays a protective role against redox cycling quinone-mediated cytotoxicity under iron-depleted conditions. This is because NQO1 generates less-toxic hydroquinones via the two-electron reduction of quinones. The collective findings reported herein demonstrate that not only an iron overload but also an iron deficiency exacerbates ROS-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

Dinuclear complex [Ir 2 (μ-L1)(η5-Cp*) 2 Cl 2 ](PF 6) 2 (1) exhibits low micromolar cytotoxic activity in vitro in various human cancer cells (GI 50 = 1.7–3.0 μM) and outperformed its mononuclear analogue [Ir(η5-Cp*)Cl(L2)]PF 6 (2 ; GI 50 > 40.0 μM); Cp* = pentamethylcyclopentadienyl, L1 = 4-chloro-2,6-bis[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl]pyridine, L2 = 5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amine. Compound 1 upregulated the Keap1/Nrf2 oxidative stress-protective pathway in the treated MV4‐11 acute myeloid leukemia cells. In connection with the redox-mediated mode of action of 1 , its NADH-oxidizing activity was detected in solution (1H NMR), while NAD+ remained intact (with formate as a hydride source). Surprisingly, only negligible NADH oxidation was detected in the presence of the reduced glutathione and ascorbate. Following the results of in-solution experiments, NAD(H) concentration was assessed in 1 -treated MV4‐11 cancer cells. Besides the intracellular NADH oxidation in the presence of 1 , the induced oxidative stress also led to a decrease of NAD+, resulting in depletion of both NAD+/NADH coenzymes. The discussed findings provide new insight into the biochemical effects of catalytic anticancer compounds that induce cell death via a redox-mediated mode of action. Dinuclear Ir(III) complex 1 upregulates the Keap1/Nrf2 oxidative stress-protective pathway in cancer cells. In solution, the ability of 1 to oxidize NADH was suppressed by ascorbate, while no interaction with NAD+ was observed. In contrast, a relevant decrease of both NAD(H) coenzymes was observed in the 1 -treated cancer cells. [Display omitted] • dinuclear Ir(III) complex 1 exhibited low micromolar activity in human cancer cells. • in-solution NADH conversion is induced by 1 and reduced by ascorbate. • no NAD+ conversion was observed in solution in the presence of 1. • in cells, 1 upregulated the Keap1/Nrf2 pathway protecting against oxidative stress. • a significant decrease of both NAD(H) coenzymes was induced in cells by 1. [ABSTRACT FROM AUTHOR]

BackgroundThe leading edge of the global problem of antibiotic resistance necessitates novel therapeutic strategies. This study develops a novel systems biology driven approach for killing antibiotic resistant pathogens using benign metabolites.ResultsControlled laboratory evolutions established chloramphenicol and streptomycin resistant pathogens of Chromobacterium. These resistant pathogens showed higher growth rates and required higher lethal doses of antibiotic. Growth and viability testing identified malate, maleate, succinate, pyruvate and oxoadipate as resensitising agents for antibiotic therapy. Resistant genes were catalogued through whole genome sequencing. Intracellular metabolomic profiling identified violacein as a potential biomarker for resistance. The temporal variance of metabolites captured the linearized dynamics around the steady state and correlated to growth rate. A constraints-based flux balance model of the core metabolism was used to predict the metabolic basis of antibiotic susceptibility and resistance.ConclusionsThe model predicts electron imbalance and skewed NAD/NADH ratios as a result of antibiotics - chloramphenicol and streptomycin. The resistant pathogen rewired its metabolic networks to compensate for disruption of redox homeostasis. We foresee the utility of such scalable workflows in identifying metabolites for clinical isolates as inevitable solutions to mitigate antibiotic resistance.

The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

Kidney transplantation is the only definitive therapy for end-stage kidney disease. The shortage of organs for transplantation is the main limitation of this life-saving treatment. Normothermic machine perfusion (NMP) is a novel preservation technique with the potential to increase the number of transplantable kidneys through reducing delayed graft function and organ evaluation under physiological conditions. To date, the cellular effects and possible pharmacological interventions during machine perfusion are incompletely understood. A major limitation is the technically complex, time-consuming, and small-scale replication of NMP in rodent models. To overcome this, we developed a 3D-printed, high throughput ex-vivo mouse kidney slice incubator (KSI) mimicking mouse kidney NMP by working under closely resembling conditions. KSI significantly reduced the time per experiment and increased the sample throughput (theoretical: 54 incubations with n = 500/day). The model recapitulated the cellular responses during NMP, namely increased endoplasmic reticulum stress (ER stress). Using KSI, five pharmacological interventions against ER stress taken from the literature were tested. While four were ineffective and excluded, one, β-Nicotinamide-adenine-dinucleotide (NADH), ameliorated ER stress significantly during KSI. The test of NADH in mouse kidney NMP replicated the positive effects against ER stress. This suggests that testing the addition of NADH during clinical kidney NMP might be warranted.

Abstract Human platelet polyphosphate (polyP) is a multifunctional molecule; however, its functions are not yet fully understood. A recent study demonstrated that similar to skeletal muscle, polyP is involved in energy metabolism in platelets, which suggests that well‐trained athletes may exhibit elevated platelet polyP levels for energy storage. To test this hypothesis, we quantified platelet polyP along with NADH, a component involved in ATP production in non‐trained and well‐trained male Japanese participants of the same generation. Washed platelets were prepared from the venous blood of young, healthy, non‐athletes, and professional soccer players (pro‐athletes). NADH and polyP levels were spectrophotometrically determined using tetrazolium reduction and fluorometrically determined using 4′,6‐diamidino‐2‐phenylindole at the excitation/emission wavelengths of 425/525 nm. Body weight and impedances were measured simultaneously. Statistical analyses were performed using the Mann–Whitney U test and Spearman correlation coefficient. Although basal metabolic rate levels were significantly higher, platelet polyP levels were significantly lower in pro‐athletes than in that in non‐athletes. No significant differences were detected in other body compositions or platelet indices between the two groups. The pro‐athlete group showed a moderate, nearly significant correlation (R = 0.439; p = 0.0512) between platelet polyP and NADH levels. Taken together with the weak correlation data between polyP and body mass index, it is suggested that platelet polyP levels may be influenced by platelet and body energy metabolic activity. Further biochemical studies are needed to elucidate this mechanism.

[Display omitted] • PP-MP particles made from plastic cup lids by UV-ageing to mimic the realistic oral intake way. • The relationship between carbonyl index (CI) of aged PP and hepatotoxicity was firstly explored. • Low-dose of aPP induced reductive stress in liver organoids with disrupted mitochondrial respiratory chain function. • aPP-elevated NADH/NAD+ratio in the liver and declined bioenergy supply in CI-dependent manner. • aPP-altered the serum profile of indicators hinting reductive stress in CI-dependent manner. 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. [ABSTRACT FROM AUTHOR]

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. 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. 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. Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry. • Real-time simultaneous metabolic analysis of different cell populations. • Label-free detection of the optical redox ratio of FAD/NADH fluorescence via flow cytometry. • Correlation with Seahorse extracellular flux analysis and enzymatic NAD(P)H detection. • Applicable to human T cell lymphoma, primary mouse and human lymphocytes. • Distinction of naïve and memory B cell populations in healthy and inflamed background. [ABSTRACT FROM AUTHOR]

Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages. [Display omitted] • Fluorescent tools that monitor cellular redox metabolites in situ and in real time. • Different fluorescence recording modalities (intensity, lifetime, anisotropy). • Available biosensors to monitor cellular GSH/GSSG, hydrogen peroxide, NADH, NADPH. • Description of biosensors architecture, properties and limitations. • Novel biosensor architectures that use chemigenetic reporters: HaloTag, UnaG, FAST. [ABSTRACT FROM AUTHOR]

Hydrogen gas, H2, is generated in serpentinizing hydrothermal systems, where it has supplied electrons and energy for microbial communities since there was liquid water on Earth. In modern metabolism, H2 is converted by hydrogenases into organically bound hydrides (H–), for example, the cofactor NADH. It transfers hydrides among molecules, serving as an activated and biologically harnessed form of H2. In serpentinizing systems, minerals can also bind hydrides and could, in principle, have acted as inorganic hydride donors—possibly as a geochemical protoenzyme, a 'geozyme'— at the origin of metabolism. To test this idea, we investigated the ability of H2 to reduce NAD+ in the presence of iron (Fe), cobalt (Co) and nickel (Ni), metals that occur in serpentinizing systems. In the presence of H2, all three metals specifically reduce NAD+ to the biologically relevant form, 1,4‐NADH, with up to 100% conversion rates within a few hours under alkaline aqueous conditions at 40 °C. Using Henry's law, the partial pressure of H2 in our reactions corresponds to 3.6 mm, a concentration observed in many modern serpentinizing systems. While the reduction of NAD+ by Ni is strictly H2‐dependent, experiments in heavy water (2H2O) indicate that native Fe can reduce NAD+ both with and without H2. The results establish a mechanistic connection between abiotic and biotic hydride donors, indicating that geochemically catalysed, H2‐dependent NAD+ reduction could have preceded the hydrogenase‐dependent reaction in evolution. [ABSTRACT FROM AUTHOR]

Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. To our knowledge, this is the first correlative FLIM and EM workflow. Combining the high sensitivity of FLIM with the high spatial resolution of EM could boost the research of pathophysiological processes involving cell metabolism, such as cancer, neurodegenerative disorders, and viral infection. [ABSTRACT FROM AUTHOR]

Hydrogen gas, H2, is generated in serpentinizing hydrothermal systems, where it has supplied electrons and energy for microbial communities since there was liquid water on Earth. In modern metabolism, H2 is converted by hydrogenases into organically bound hydrides (H–), for example, the cofactor NADH. It transfers hydrides among molecules, serving as an activated and biologically harnessed form of H2. In serpentinizing systems, minerals can also bind hydrides and could, in principle, have acted as inorganic hydride donors—possibly as a geochemical protoenzyme, a 'geozyme'— at the origin of metabolism. To test this idea, we investigated the ability of H2 to reduce NAD+ in the presence of iron (Fe), cobalt (Co) and nickel (Ni), metals that occur in serpentinizing systems. In the presence of H2, all three metals specifically reduce NAD+ to the biologically relevant form, 1,4‐NADH, with up to 100% conversion rates within a few hours under alkaline aqueous conditions at 40 °C. Using Henry's law, the partial pressure of H2 in our reactions corresponds to 3.6 mm, a concentration observed in many modern serpentinizing systems. While the reduction of NAD+ by Ni is strictly H2‐dependent, experiments in heavy water (2H2O) indicate that native Fe can reduce NAD+ both with and without H2. The results establish a mechanistic connection between abiotic and biotic hydride donors, indicating that geochemically catalysed, H2‐dependent NAD+ reduction could have preceded the hydrogenase‐dependent reaction in evolution. [ABSTRACT FROM AUTHOR]

Drug‐resistant cells and anti‐inflammatory immune cells within tumor masses contribute to tumor aggression, invasion, and worse patient outcomes. These cells can be a small proportion (<10%) of the total cell population of the tumor. Due to their small quantity, the identification of rare cells is challenging with traditional assays. Single cell analysis of autofluorescence images provides a live‐cell assay to quantify cellular heterogeneity. Fluorescence intensities and lifetimes of the metabolic coenzymes reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide allow quantification of cellular metabolism and provide features for classification of cells with different metabolic phenotypes. In this study, Gaussian distribution modeling and machine learning classification algorithms are used for the identification of rare cells within simulated autofluorescence lifetime image data of a large tumor comprised of tumor cells and T cells. A Random Forest machine learning algorithm achieved an overall accuracy of 95% for the identification of cell type from the simulated optical metabolic imaging data of a heterogeneous tumor of 20,000 cells consisting of 70% drug responsive breast cancer cells, 5% drug resistant breast cancer cells, 20% quiescent T cells and 5% activated T cells. High resolution imaging methods combined with single‐cell quantitative analyses allows identification and quantification of rare populations of cells within heterogeneous cultures [ABSTRACT FROM AUTHOR]

A highly sensitive and selective modified electrode was successfully developed for the monitoring of nicotinamide adenine dinucleotide (NADH) in the presence of folic acid. In this regard, a carbon paste electrode (CPE) was functionalized by the nitrogen-doped carbon quantum dots/tin oxide (N-CQDs/SnO2) nanocomposite and 1-butyl-2,3-dimethyl imidazolium hexafluorophosphate ([C4DMIM][PF6]) ionic liquid (IL). The structure and surface morphology of the nanocomposite were characterized by various methods, including field emission scanning electron microscopy, energy dispersive spectroscopy (EDS), high-resolution transmission electron microscopy (HR-TEM), and x-ray diffraction (XRD). The modified electrode displayed powerful and long-lasting electron mediating activity, with well-separated NADH and folic acid oxidation peaks. The sensing response of the developed [C4DMIM][PF6]/N-CQDs/SnO2/CPE platform was evaluated by determining NADH via the voltammetric technique under the optimized operating conditions. The current peaks of the square wave voltammograms of NADH and folic acid increased linearly with enhancing its concentrations within the ranges of 0.003â€"275 ÎĽ M NADH and 0.4â€"380 ÎĽ M folic acid. The detection limits for NADH and folic acid were obtained at 0.8 nM and 0.1 ÎĽ M, respectively. Interference species such as glucose, urea, tryptophan, glycine, methionine, and vitamin B12 had no influence on the ability of the fabricated modified electrode to detect the target species. The low detection limit, high sensitivity, excellent selectivity, superior stability, and cost-effectiveness made it suitable for the quantification of NADH in the real biological samples with the recovery percent values in the range of 97.5%â€"103%. [ABSTRACT FROM AUTHOR]

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 μM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time. [ABSTRACT FROM AUTHOR]

A highly sensitive tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) electrogenerated chemiluminescence (ECL) sensor based on a graphene-titania-Nafion composite film has been prepared in a simple one-step manner. In the present work, a highly concentrated 0.1 M Ru(bpy)32+ solution was mixed with an as-prepared graphene-titania-Nafion composite solution (1:20, v/v), and then a small aliquot (2 µL) of the resulting mixture solution was cast on a glassy carbon electrode surface. This one-step process for the construction of an ECL sensor shortens the fabrication time and leads to reproducible ECL signals. Due to the synergistic effect of conductive graphene and mesoporous sol-gel derived titania-Nafion composite, the present ECL sensor leads to a highly sensitive detection of tripropylamine from 1.0 × 10−8 M to 2.0 × 10−3 M with a detection limit of 0.8 nM (S/N = 3), which is lower in comparison to that of the ECL sensor based on the corresponding ECL sensor based on the titania-Nafion composite containing carbon nanotube. The present ECL sensor also shows a good response for nicotinamide adenine dinucleotide hydrogen (NADH) from 1.0 × 10−6 M to 1.0 × 10−3 M with a detection limit of 0.4 µM (S/N = 3). Thus, the present ECL sensor can offer potential benefits in the development of dehydrogenase-based biosensors. [ABSTRACT FROM AUTHOR]

Major depressive disorder (MDD) is a serious mental disease with a pathophysiology that is not yet fully clarified. An increasing number of studies show an association of MDD with energy metabolism alteration and the presence of oxidative stress. We aimed to evaluate plasma levels of 3-hydroxybutyrate (3HB), NADH, myeloperoxidase, and dityrosine (di-Tyr) in adolescent and adult patients with MDD, compare them with healthy age-matched controls, and assess the effect of antidepressant treatment during hospitalisation on these levels. In our study, plasmatic levels of 3HB were elevated in both adolescents (by 55%; p = 0.0004) and adults (by 88%; p < 0.0001) with MDD compared to controls. Levels of dityrosine were increased in MDD adults (by 19%; p = 0.0092) but not adolescents. We have not found any significant effect of antidepressants on the selected parameters during the short observation period. Our study supports the findings suggesting altered energy metabolism in MDD and demonstrates its presence independently of the age of the patients. [ABSTRACT FROM AUTHOR]

A reducing system of SoxR, a regulator of redox-active molecules, was identified as rsxABCDGE gene products and RseC in Escherichia coli through genetic studies. We found that ApbE was an additional component of the reducer system. Bacterial two hybrid analysis revealed that these proteins indeed had multiple interactions among themselves. RseC and RsxB formed the core of the complex, interacting with more than five other components. RsxC, the only cytoplasmic component of the system, interacted with SoxR. It might be linked with the rest of the complex via RsxB. Membrane fractions containing the wild type complex but not the mutant complex reduced purified SoxR using NADH as an electron source. These results suggest that Rsx genes, RseC, and ApbE can form a complex using NAD(P)H to reduce SoxR. [ABSTRACT FROM AUTHOR]

Abstract Background Ascomycin is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. As a secondary metabolite, the production of ascomycin is often limited by the shortage of precursors during the late fermentation phase. Polyhydroxybutyrate is an intracellular polymer accumulated by prokaryotic microorganisms. Developing polyhydroxybutyrate as an intracellular carbon reservoir for precursor synthesis is of great significance to improve the yield of ascomycin. Results The fermentation characteristics of the parent strain S. hygroscopicus var. ascomyceticus FS35 showed that the accumulation and decomposition of polyhydroxybutyrate was respectively correlated with cell growth and ascomycin production. The co-overexpression of the exogenous polyhydroxybutyrate synthesis gene phaC and native polyhydroxybutyrate decomposition gene fkbU increased both the biomass and ascomycin yield. Comparative transcriptional analysis showed that the storage of polyhydroxybutyrate during the exponential phase accelerated biosynthesis processes by stimulating the utilization of carbon sources, while the decomposition of polyhydroxybutyrate during the stationary phase increased the biosynthesis of ascomycin precursors by enhancing the metabolic flux through primary pathways. The comparative analysis of cofactor concentrations confirmed that the biosynthesis of polyhydroxybutyrate depended on the supply of NADH. At low sugar concentrations found in the late exponential phase, the optimization of carbon source addition further strengthened the polyhydroxybutyrate metabolism by increasing the total concentration of cofactors. Finally, in the fermentation medium with 22 g/L starch and 52 g/L dextrin, the ascomycin yield of the co-overexpression strain was increased to 626.30 mg/L, which was 2.11-fold higher than that of the parent strain in the initial medium (296.29 mg/L). Conclusions Here we report for the first time that polyhydroxybutyrate metabolism is beneficial for cell growth and ascomycin production by acting as an intracellular carbon reservoir, stored as polymers when carbon sources are abundant and depolymerized into monomers for the biosynthesis of precursors when carbon sources are insufficient. The successful application of polyhydroxybutyrate in increasing the output of ascomycin provides a new strategy for improving the yields of other secondary metabolites.