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Tardigrades are captivating organisms known for their resilience in extreme environments, including ultra-high-dose radiation, but the underlying mechanisms of this resilience remain largely unknown. Using genome, transcriptome, and proteome analysis of Hypsibius henanensis sp. nov., we explored the molecular basis contributing to radiotolerance in this organism. A putatively horizontally transferred gene, DOPA dioxygenase 1 (DODA1), responds to radiation and confers radiotolerance by synthesizing betalains--a type of plant pigment with free radical-scavenging properties. A tardigrade-specific radiation-induced disordered protein, TRID1, facilitates DNA damage repair through a mechanism involving phase separation. Two mitochondrial respiratory chain complex assembly proteins, BCS1 and NDUFB8, accumulate to accelerate nicotinamide adenine dinucleotide (NAD+) regeneration for poly (adenosine diphosphate-ribosyl)ation (PARylation) and subsequent poly(adenosine diphosphate-ribose) polymerase 1 (PARP1)-mediated DNA damage repair. These three observations expand our understanding of mechanisms of tardigrade radiotolerance. [ABSTRACT FROM AUTHOR]

Herein we introduce a novel water-based graphite ink modified with multiwalled carbon nanotubes, designed for the development of the first wearable self-powered biosensor enabling alcohol abuse detection through sweat analysis. The stencil-printed graphite (SPG) electrodes, printed onto a flexible substrate, were modified by casting multiwalled carbon nanotubes (MWCNTs), electrodepositing polymethylene blue (pMB) at the anode to serve as a catalyst for nicotinamide adenine dinucleotide (NADH) oxidation, and hemin at the cathode as a selective catalyst for H2O2 reduction. Notably, alcohol dehydrogenase (ADH) was additionally physisorbed onto the anodic electrode, and alcohol oxidase (AOx) onto the cathodic electrode. The self-powered biosensor was assembled using the ADH/pMB-MWCNTs/SPG||AOx/Hemin-MWCNTs/SPG configuration, enabling the detection of ethanol as an analytical target, both at the anodic and cathodic electrodes. Its performance was assessed by measuring polarization curves with gradually increasing ethanol concentrations ranging from 0 to 50 mM. The biosensor demonstrated a linear detection range from 0.01 to 0.3 mM, with a detection limit (LOD) of 3 ± 1 µM and a sensitivity of 64 ± 2 μW mM−1, with a correlation coefficient of 0.98 (RSD 8.1%, n = 10 electrode pairs). It exhibited robust operational stability (over 2800 s with continuous ethanol turnover) and excellent storage stability (approximately 93% of initial signal retained after 90 days). Finally, the biosensor array was integrated into a wristband and successfully evaluated for continuous alcohol abuse monitoring. This proposed system displays promising attributes for use as a flexible and wearable biosensor employing biocompatible water-based inks, offering potential applications in forensic contexts. A novel water-based graphite ink modified with multiwalled carbon nanotubes designed for the development of a wearable self-powered biosensor enabling alcohol abuse detection through sweat analysis. [ABSTRACT FROM AUTHOR]

NADH and NAD+ act as electron donors and acceptors and NAD+ was shown to stimulate mitochondrial biogenesis and metabolic health. We here develop a non-invasive Phosphorous Magnetic Resonance Spectroscopy (31P-MRS) method to quantify these metabolites in human skeletal muscle on a clinical 3 T MRI scanner. This new MR-sequence enables NADH and NAD+ quantification by suppressing α-ATP signal, normally overlapping with NADH and NAD+. The sequence is based on a double spin echo in combination with a modified z-Filter achieving strong α-ATP suppression with little effect on NAD+ and NADH. Here we test and validate it in phantoms and in humans by measuring reproducibility and detecting a physiological decrease in NAD+ and increase in NADH induced by ischemia. Furthermore, the 31P-MRS outcomes are compared to analysis in biopsies. Additionally, we show higher NAD+ and lower NADH content in physically active older adults compared to sedentary individuals, reflecting increased metabolic health. NADH and NAD+ act as electron donors and acceptors and NAD+ was shown to stimulate mitochondrial biogenesis and metabolic health. Here, the researchers developed and validated a non-invasive Phosphorous Magnetic Resonance Spectroscopy method to non-invasively quantify NAD+ and NADH in muscle on a clinical 3 T MRI scanner. [ABSTRACT FROM AUTHOR]

The electrochemical regeneration of reduced nicotinamide adenine dinucleotide (NADH) using [Rh(Cp*)(bpy)Cl]+ holds significant promise for the industrial synthesis of chiral chemicals. However, challenges persist due to the high consumption of NADH and the limited efficiency of its cyclic regeneration, which currently hinder widespread application. To address these obstacles, based on in‐situ growth of 3D ordered metal‐organic framework (NU‐1000) on the surface of graphite felt, [Rh(Cp*)(bpy)Cl]+ were immobilized on the Zr6 nodes of NU‐1000 by solvent‐assisted ligand incorporation (SALI), and applied in a flow bioreactor. Moreover, we employ a gas diffusion electrode (GDE) to oxidize H2, providing a clean proton source for the electrochemical regeneration of NADH. Consequently, highly efficient enzymatic electrocatalytic synthesis of L‐lactate was achieved when coupled with L‐lactate dehydrogenases (LDH) as a model reaction, and the total turnover number (TTN) reached 19600 and 1750 for [Rh(Cp*)(bpy)Cl]+ and NAD+ after 48 h, corresponding to a high turnover frequency (TOF) of 2350 h−1 and 210 h−1 for [Rh(Cp*)(bpy)Cl]+ and NAD+, respectively. This work provides new insights for the construction of efficient enzymatic electrosynthesis systems in industrial production. [ABSTRACT FROM AUTHOR]

Bioactive NAD+ mediated multiple biocatalytic pathways in metabolic networks. Refining the structure of NADH oxidase-like (NOX) mimics to efficiently replenish NAD+ has been promising but challenging in NAD+-dependent dehydrogenase electrochemical cascade biosensing. Herein, we discovered that PtOx structures, formed via lattice oxygen translocation from WO3 to Pt NPs at the interface, potentially activate and modulate the NOX-like functionality in Pt@WO3 nanosheets. Incorporating PtOx leads to a more positive valence of Pt species within Pt/PtOx@WO3−x, where the PtO2 species serve as preeminent reaction sites for NADH coordination, activation, and dehydrogenation. Consequently, such nanozymes display enhanced NOX-like activity towards NADH oxidation in comparison to Pt@WO3. Ultimately, the 650-Pt/PtOx@WO3−x nanozyme is employed in an electrochemical cascade biosensor for β-hydroxybutyrate (HB) detection, achieving a calculated detection limit of 25 μM. This study offers insights into PtOx activation in Pt-based NOX mimics and supports the future development of NAD+/NADH-dependent electrochemical biosensors. [ABSTRACT FROM AUTHOR]

As the sole producers of insulin under physiological conditions, the normal functioning of pancreatic β cells is crucial for maintaining glucose homeostasis in the body. Due to the high oxygen and energy demands required for insulin secretion, hypoxia has been shown to play a critical role in pancreatic β-cell dysfunction. Lipid metabolism abnormalities, a common metabolic feature in type 2 diabetic patients, are often accompanied by tissue hypoxia caused by metabolic overload and lead to increased free fatty acid (FFA) levels. However, the specific mechanisms underlying FFA-induced β-cell dysfunction remain unclear. Nicotinamide mononucleotide (NMN), a naturally occurring bioactive nucleotide, has garnered significant attention in recent years for its effectiveness in replenishing NAD+ and alleviating various diseases. Nevertheless, studies exploring the mechanisms through which NMN influences β-cell dysfunction remain scarce. In this study, we established an in vitro β-cell dysfunction model by treating INS-1 cells with palmitate (PA), including control, PA-treated, and PA combined with NMN or activator/inhibitor groups. Compared to the control group, cells treated with PA alone showed significantly reduced insulin secretion capacity and decreased expression of proteins related to the NAD+/AMPK/SIRT1/HIF-1α pathway. In contrast, NMN supplementation significantly restored the expression of pathway-related proteins by activating NAD+ and effectively improved insulin secretion. Results obtained using HIF-1α and AMPK inhibitors/activators further supported these findings. In conclusion, our study demonstrates that NMN reversed the PA-induced downregulation of the NAD+/AMPK/SIRT1/HIF-1α pathway, thereby alleviating β-cell dysfunction. Our study investigated the mechanisms underlying PA-induced β-cell dysfunction, examined how NMN mitigates this dysfunction and offered new insights into the therapeutic potential of NMN for treating β-cell dysfunction and T2DM. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) depletion has been postulated as a contributor to the severity of COVID‐19; however, no study has prospectively characterized NAD+ and its metabolites in relation to disease severity in patients with COVID‐19. We measured NAD+ and its metabolites in 56 hospitalized patients with COVID‐19 and in two control groups without COVID‐19: (1) 31 age‐ and sex‐matched adults with comorbidities, and (2) 30 adults without comorbidities. Blood NAD+ concentrations in COVID‐19 group were only slightly lower than in the control groups (p < 0.05); however, plasma 1‐methylnicotinamide concentrations were significantly higher in patients with COVID‐19 (439.7 ng/mL, 95% CI: 234.0, 645.4 ng/mL) than in age‐ and sex‐matched controls (44.5 ng/mL, 95% CI: 15.6, 73.4) and in healthy controls (18.1 ng/mL, 95% CI 15.4, 20.8; p < 0.001 for each comparison). Plasma nicotinamide concentrations were also higher in COVID‐19 group and in controls with comorbidities than in healthy control group. Plasma concentrations of 2‐methyl‐2‐pyridone‐5‐carboxamide (2‐PY), but not NAD+, were significantly associated with increased risk of death (HR = 3.65; 95% CI 1.09, 12.2; p = 0.036) and escalation in level of care (HR = 2.90, 95% CI 1.01, 8.38, p = 0.049). RNAseq and RTqPCR analyses of PBMC mRNA found upregulation of multiple genes involved in NAD+ synthesis as well as degradation, and dysregulation of NAD+‐dependent processes including immune response, DNA repair, metabolism, apoptosis/autophagy, redox reactions, and mitochondrial function. Blood NAD+ concentrations are modestly reduced in COVID‐19; however, NAD+ turnover is substantially increased with upregulation of genes involved in both NAD+ biosynthesis and degradation, supporting the rationale for NAD+ augmentation to attenuate disease severity. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide provides the critical redox pair, NAD+ and NADH, for cellular energy metabolism. In addition, NAD+ is the precursor for de novo NADP+ synthesis as well as the co-substrates for CD38, poly(ADP-ribose) polymerase and sirtuins, thus, playing a central role in the regulation of oxidative stress and cell signaling. Declines of the NAD+ level and altered NAD+/NADH redox states have been observed in cardiometabolic diseases of various etiologies. NAD based therapies have emerged as a promising strategy to treat cardiovascular disease. Strategies that reduce NAD+ consumption or promote NAD+ production have repleted intracellular NAD+ or normalized NAD+/NADH redox in preclinical studies. These interventions have shown cardioprotective effects in multiple models suggesting a great promise of the NAD+ elevating therapy. Mechanisms for the benefit of boosting NAD+ level, however, remain incompletely understood. Moreover, despite the robust pre-clinical studies there are still challenges to translate the therapy to clinic. Here, we review the most up to date literature on mechanisms underlying the NAD+ elevating interventions and discuss the progress of human studies. We also aim to provide a better understanding of how NAD metabolism is changed in failing hearts with a particular emphasis on types of strategies employed and methods to target these pathways. Finally, we conclude with a comprehensive assessment of the challenges in developing NAD-based therapies for heart diseases, and to provide a perspective on the future of the targeting strategies. • Intracellular NAD+ level and NAD+/NADH ratio are critical regulators of cell signaling, oxidative stress and metabolic flux. • Boosting NAD+ levels has shown benefit in preclinical studies of heart failure but mechanisms are incompletely understood. • Reliable biomarkers of tissue NAD(H) levels in patients will facilitate the translation of the therapy to the clinic. [ABSTRACT FROM AUTHOR]

This work examines the electrochemically enhanced production of succinic acid using the bacterium Actinobacillus succinogenes. The principal objective is to enhance the metabolic potential of glucose and CO2 utilization via the C4 pathway in order to synthesize succinic acid. We report on the development of an electro-bioreactor system to increase succinic acid production in a power-2-X approach. The use of activated carbon fibers as electrode surfaces and contact areas allows A. succinogenes to self-initiate biofilm formation. The integration of an electrical potential into the system shifts the redox balance from NAD+ to NADH, increasing the efficiency of metabolic processes. Mediators such as neutral red facilitate electron transfer within the system and optimize the redox reactions that are crucial for increased succinic acid production. Furthermore, the role of carbon nanotubes (CNTs) in electron transfer was investigated. The electro-bioreactor system developed here was operated in batch mode for 48 h and showed improvements in succinic acid yield and concentration. In particular, a run with 100 µM neutral red and a voltage of −600 mV achieved a yield of 0.7 gsuccinate·gglucose−1. In the absence of neutral red, a higher yield of 0.72 gsuccinate·gglucose−1 was achieved, which represents an increase of 14% compared to the control. When a potential of −600 mV was used in conjunction with 500 µg∙L−1 CNTs, a 21% increase in succinate concentration was observed after 48 h. An increase of 33% was achieved in the same batch by increasing the stirring speed. These results underscore the potential of the electro-bioreactor system to markedly enhance succinic acid production. [ABSTRACT FROM AUTHOR]

In this paper, we present the synthesis of four new complexes: the dimeric precursor [Ir(dmppz)2(μ-Cl)]2 (1) (Hdmppz -- 3,5-dimethyl-1-phenyl-1H-pyrazole) and heteroleptic bis-cyclometalated complexes: [Ir(dmppz)2(Py2CO)]PF6•½CH2Cl2 (2), [Ir(dmppz)2(H2biim)]PF6•H2O (3), and [Ir(dmppz)2(PyBIm)]PF6 (4), with auxiliary N,N-donor ligands: 2-di(pyridyl)ketone (Py2CO), 2,2'-biimidazole (H2biim) and 2-(2'-pyridyl)benzimidazole (PyBIm). In the obtained complexes, SC-X-ray analysis revealed that Ir(III) has an octahedral coordination sphere with chromophores of the type {IrN2C2Cl2} (1) or {IrN4C2} (2-4). The complexes obtained, which have been fully characterised by physicochemical methods (CHN, TG, FTIR, UV-Vis, PL and ¹H, 13C, 15N NMR), were used to continue our studies on the factors influencing the cytotoxic properties of potential chemotherapeutic agents (in vitro). To this end, the following studies are presented: (i) comparative analysis of the effects on the biological properties of N,N-donor ligands and C,N-donor ligands in the studied complexes, (ii) studies of the interactions of the compounds with the selected molecular target: DNA and BSA (UV-Vis, CD and PL methods), (iii) and the reactivity towards redox molecules: GSH, NADH (UV-Vis and/or ESI-MS methods), (iv) cytotoxic activity (IC50) of potential chemotherapeutics against MCF-7, K-562 and CCRF-CEM cell lines. [ABSTRACT FROM AUTHOR]

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, detailed metabolomic characterizations of the expanding and differentiating organoids are performed. It is found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α‐ketoglutarate (α‐KG) and nicotinamide adenine dinucleotide (NAD+). Reduced expression of NAD+ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α‐KG and NAD+ promotes hair cell reprogramming both in vitro and in vivo. These findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer. [ABSTRACT FROM AUTHOR]

Barrett's esophagus (BE) is a common precancerous lesion that can progress to esophageal adenocarcinoma (EAC). There are significant alterations in the esophageal microbiome in the progression from healthy esophagus to BE to EAC, including an increased abundance of a variety of lactate-producing bacteria and an increase of lactate in the tumor microenvironment, as predicted by metabolic modeling. The role of bacterial lactate in EAC is unknown. Here, we utilize patient-derived organoid (PDO) models of EAC and demonstrate that lactate inhibits the growth and proliferation of EAC PDOs through alterations in the tumor NADH/NAD+ redox state. Further RNA sequencing of EAC PDOs identifies ID1 and RSAD2 as potential regulatory molecules crucial in mediating lactate's ability to suppress glycolysis and proliferation. Gene ontology analysis also identifies the activation of inflammatory and immunological pathways in addition to alterations in the metabolic pathways in EAC PDOs exposed to lactate, suggesting a multi-faceted role for lactate in the pathogenesis of EAC. [ABSTRACT FROM AUTHOR]

Simple Summary: In tackling the daunting challenge of glioblastoma, a severe form of brain tumor, researchers have been exploring ways to disrupt its abnormal energy production, focusing on a molecule called NAD. The drug FK866, known to deplete NAD, shows potential in curbing tumor growth but faces limitations when used alone. This study introduces a novel approach using fluorinated versions of NAD precursors, specifically a compound named F-NR, which when, combined with FK866, significantly boosts its effectiveness against glioblastoma cells. F-NR works by competing with the endogenous metabolism of NR, leading to reduced NAD levels and enhancing FK866's ability to kill cancer cells. Another key finding is the role of SARM1, activated by one of F-NR's metabolites, which contributes to the enhanced cell-killing effect. The sequence of events—NAD depletion followed by energy loss and ultimately, widespread cell death—highlights the potential of this strategy to improve FK866's therapeutic impact. This research not only deepens our understanding of NAD metabolism in glioblastoma but also provides a potential strategy for treating the brain cancer. Glioblastoma, a formidable brain tumor characterized by dysregulated NAD metabolism, poses a significant therapeutic challenge. The NAMPT inhibitor FK866, which induces NAD depletion, has shown promise in controlling tumor proliferation and modifying the tumor microenvironment. However, the clinical efficacy of FK866 as a single drug therapy for glioma is limited. In this study, we aim to disrupt NAD metabolism using fluorinated NAD precursors and explore their synergistic effect with FK866 in inducing cytotoxicity in glioblastoma cells. The synthesized analogue of nicotinamide riboside (NR), ara-F nicotinamide riboside (F-NR), inhibits nicotinamide ribose kinase (NRK) activity in vitro, reduces cellular NAD levels, and enhances FK866's cytotoxicity in U251 glioblastoma cells, indicating a collaborative impact on cell death. Metabolic analyses reveal that F-NR undergoes conversion to fluorinated nicotinamide mononucleotide (F-NMN) and other metabolites, highlighting the intact NAD metabolic pathway in glioma cells. The activation of SARM1 by F-NMN, a potent NAD-consuming enzyme, is supported by the synergistic effect of CZ-48, a cell-permeable SARM1 activator. Temporal analysis underscores the sequential nature of events, establishing NAD depletion as a precursor to ATP depletion and eventual massive cell death. This study not only elucidates the molecular intricacies of glioblastoma cell death but also proposes a promising strategy to enhance FK866 efficacy through fluorinated NAD precursors, offering potential avenues for innovative therapeutic interventions in the challenging landscape of glioblastoma treatment. [ABSTRACT FROM AUTHOR]

Aims: Increasing nicotinamide adenine dinucleotide (NAD+) availability has been proposed as a therapeutic approach to prevent neurodegeneration in amyotrophic lateral sclerosis (ALS). Accordingly, NAD+ precursor supplementation appears to exert neuroprotective effects in ALS patients and mouse models. The mechanisms mediating neuroprotection remain uncertain but could involve changes in multiple cell types. We investigated a potential direct effect of the NAD+ precursor nicotinamide mononucleotide (NMN) on the health of cultured induced pluripotent stem cell (iPSC)-derived human motor neurons and in motor neurons isolated from two ALS mouse models, that is, mice overexpressing wild-type transactive response DNA binding protein-43 (TDP-43) or the ALS-linked human superoxide dismutase 1 with the G93A mutation (hSOD1G93A). Results: NMN treatment increased the complexity of neuronal processes in motor neurons isolated from both mouse models and in iPSC-derived human motor neurons. In addition, NMN prevented neuronal death induced by trophic factor deprivation. In mouse and human motor neurons expressing ALS-linked mutant superoxide dismutase 1, NMN induced an increase in glutathione levels, but this effect was not observed in nontransgenic or TDP-43 overexpressing motor neurons. In contrast, NMN treatment normalized the TDP-43 cytoplasmic mislocalization induced by its overexpression. Innovation: NMN can directly act on motor neurons to increase the growth and complexity of neuronal processes and prevent the death induced by trophic factor deprivation. Conclusion: Our results support a direct beneficial effect of NAD+ precursor supplementation on the maintenance of the neuritic arbor in motor neurons. Importantly, this was observed in motor neurons isolated from two different ALS models, with and without involvement of TDP-43 pathology, supporting its therapeutic potential in sporadic and familial ALS. Antioxid. Redox Signal. 41, 573–589. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite that plays a crucial role in diverse biological processes, including energy metabolism, gene expression, DNA repair, and mitochondrial function. An aberrant NAD+ level mediates the development of cardiovascular dysfunction and diseases. Both in vivo and in vitro studies have demonstrated that nicotinamide mononucleotide (NMN), as a NAD+ precursor, alleviates the development of cardiovascular diseases such as heart failure, atherosclerosis, and myocardial ischemia/reperfusion injury. Importantly, NMN has suggested pharmacological activities mostly through its involvement in NAD+ biosynthesis. Several clinical studies have been conducted to investigate the efficacy and safety of NMN supplementation, indicating its potential role in cardiovascular protection without significant adverse effects. In this review, we systematically summarize the impact of NMN as a nutraceutical and potential therapeutic drug on cardiovascular diseases and emphasize the correlation between NMN supplementation and cardiovascular protection. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons. While there have been many potential factors implicated for ALS development, such as oxidative stress and mitochondrial dysfunction, no exact mechanism has been determined at this time. Nicotinamide adenine dinucleotide (NAD+) is one of the most abundant metabolites in mammalian cells and is crucial for a broad range of cellular functions from DNA repair to energy homeostasis. NAD+ can be synthesized from three different intracellular pathways, but it is the NAD+ salvage pathway that generates the largest proportion of NAD+. Impaired NAD+ homeostasis has been connected to aging and neurodegenerative disease-related dysfunctions. In ALS mice, NAD+ homeostasis is potentially disrupted prior to the appearance of physical symptoms and is significantly reduced in the nervous system at the end stage. Treatments targeting NAD+ metabolism, either by administering NAD+ precursor metabolites or small molecules that alter NAD+-dependent enzyme activity, have shown strong beneficial effects in ALS disease models. Here, we review the therapeutic interventions targeting NAD+ metabolism for ALS and their effects on the most prominent pathological aspects of ALS in animal and cell models. [ABSTRACT FROM AUTHOR]

The first cell differentiation event that occurs in the embryo determines the inner cell mass (ICM) and the trophectoderm (TE). In the mouse, glucose (GLC) is essential for this process, while oxygen tension (O 2) also interferes with TE formation. The roles of GLC and O 2 in this event in bovine embryos are not completely elucidated. We hypothesized that the absence of glucose and a higher O 2 tension negatively impact ICM and TE cell allocation in the bovine embryo. The objective of this study was to evaluate the effect of GLC within different O 2 levels on the formation of the TE. In vitro-produced embryos were cultured in serum-free KSOM medium and randomly submitted to treatments on the day of IVC, according to a 2x2 factorial model, in which GLC (present [+GLC] or absent [-GLC]) and O 2 (low [5%O 2 ] or high [20%O 2 ]) were the independent variables. Cleavage and blastocyst rates were obtained at D4 and D8, respectively. Embryos at D8 were subjected to autofluorescence analysis to quantitate NADH and FAD + or fixed for GATA3 and YAP1 immunostaining using a laser scanning confocal microscope. Total, TE, and ICM cell counts were obtained. Embryos were also harvested for gene expression quantification of GATA3, YAP1, SOX2, CDX2, TFAP2C and OCT4. Results indicate that there was an effect of O 2 (p = 0.018) on cleavage rates, although no differences were observed in blastocyst rates. NADH was higher in -GLC compared to + GLC (p = 0.014) and no differences in FAD+ were observed. Total cell count data were not different between variables. There was an increase in the ICM cell count in the +GLC 5%O 2 condition compared to the other three conditions. No effects of GLC, O 2 , or their interactions were observed on TE cell count or the TE/total cell ratio. CDX2 (p = 0.007) and TFAP2C (p = 0.038) were increased in -GLC 20%O 2 compared to + GLC 20%O 2. SOX2 was decreased in +GLC 20%O 2 compared to + GLC 5%O 2 (p = 0.027) or compared to -GLC 20%O 2 (p = 0.005). GATA3, YAP1, and OCT4 genes did not present differences among conditions. In conclusion, both GLC and high oxygen tension did not impair TE formation and TE cell number, although a +GLC-low oxygen environment led to a higher number of ICM cells. Interestingly, the expression of TE-related gene CDX2 was increased in the absence of glucose within higher O 2 tension. Our results implicate that according to the oxygen tension used in IVC, glucose can exert different effects on blastocyst cell allocation or gene expression. • Differentiation of the TE occurred in the absence of glucose. • Glucose plus 5 % oxygen tension increased the number of ICM cells. • Gene expression was influenced by glucose within a 20 % oxygen tension. [ABSTRACT FROM AUTHOR]

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Background: Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 regulates both metabolism and immune functions. This study investigated if SIRT1 inhibitory property of herbal formula Qing-Luo-Yin (QLY) contributed to its anti-rheumatic effects. Methods: Adjuvant-induced arthritis (AIA) rats were treated by QLY and nicotinamide mononucleotide (NMN, a biosynthesis precursor of NAD) for 38 days. After sacrifice, blood, paws, liver and white adipose tissues (WAT) were collected. Pre-adipocytes were cultured by the rats' serum. The medium was used for monocytes culture. Some pre-adipocytes were treated by QLY-derived SIRT1 inhibitors. SIRT1 was silenced or overexpressed beforehand. The samples were subjected to kits-based quantification, polymerase-chain reaction, western-blot, immunofluorescence, and histology experiments. Results: AIA rats experienced significant fat loss in liver and WAT. Expression of many SIRT1-related signals like PPARγ, PGC-1α, HSL, ATGL and CPT-1A were altered. QLY attenuated all these abnormalities and joint injuries. By pan-acetylation up-regulation, visfatin was obviously reduced in QLY-treated AIA rats' blood (from 191.8 to 127.0 pg/mL). NMN sustained SIRT1 activation by replenishing NAD, and weakened these effects. QLY-containing serum and the related compounds showed similar impacts on pre-adipocytes, resembling the changes in QLY-treated AIA rats' WAT. These treatments suppressed AIA serum-induced visfatin secretion (from 49.3 to 36.1 and 30.7 pg/mL). This effect was impaired by SIRT1 overexpression. The medium from the compounds-treated pre-adipocytes impaired NF-κB activation in AIA serum-cultured monocytes. Conclusion: Besides fat depletion, SIRT1 up-regulation in rheumatic subjects' WAT promotes visfatin production, and exacerbates inflammation. SIRT1 inhibition in WAT is an anti-rheumatic way of QLY independent of immune regulation. [ABSTRACT FROM AUTHOR]

The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni+2 agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 °C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations. [ABSTRACT FROM AUTHOR]

Many people convicted for drunken driving suffer from an alcohol use disorder and some traffic offenders consume denatured alcohol for intoxication purposes. Venous blood samples from people arrested for driving under the influence of alcohol were analyzed in triplicate by headspace gas chromatography (HS-GC) using three different stationary phases. The gas chromatograms from this analysis sometimes showed peaks with retention times corresponding to acetone, ethyl methyl ketone (2-butanone), 2-propanol, and 2-butanol in addition to ethanol and the internal standard (1-propanol). Further investigations showed that these drink-driving suspects had consumed an industrial alcohol (T-Red) for intoxication purposes, which contained > 90% w/v ethanol, acetone (~ 2% w/v), 2-butanone (~ 5% w/v) as well as Bitrex to impart a bitter taste. In n = 75 blood samples from drinkers of T-Red, median concentrations of ethanol, acetone, 2-butanone, 2-propanol and 2-butanol were 2050 mg/L (2.05 g/L), 97 mg/L, 48 mg/L, 26 mg/L and 20 mg/L, respectively. In a separate GC analysis, 2,3-butanediol (median concentration 87 mg/L) was identified in blood samples containing 2-butanone. When the redox state of the liver is shifted to a more reduced potential (excess NADH), which occurs during metabolism of ethanol, this favors the reduction of low molecular ketones into secondary alcohols via the alcohol dehydrogenase (ADH) pathway. Routine toxicological analysis of blood samples from apprehended drivers gave the opportunity to study metabolism of acetone and 2-butanone without having to administer these substances to human volunteers. [ABSTRACT FROM AUTHOR]

Pyruvate is a three-carbon ketoacid that occurs naturally in cells. It is produced through enzymatic reactions in the glycolytic pathway and plays a crucial role in energy metabolism. Despite promising early results, later well-controlled studies of physically active people have shown that pyruvate supplementation lasting more than 1 week has no ergogenic effects. However, some data suggest that ingested pyruvate may be preferentially metabolized without accumulation in the bloodstream. Pyruvate exhibits antioxidant activity and can affect the cellular redox state, and exogenous pyruvate can influence metabolism by affecting the acid-base balance of the blood. This brief review focuses on the potential effects of pyruvate as a supplement for active people. The current state of understanding suggests that studies of the effects of pyruvate supplementation should prioritize investigating the timing of pyruvate intake. [ABSTRACT FROM AUTHOR]

Enzymes play a fundamental role in cellular metabolism. A wide range of enzymes require the presence of complementary coenzymes and cofactors to function properly. While coenzymes are believed to have been part of the last universal ancestor (LUCA) or have been present even earlier, the syntheses of crucial coenzymes like the redox‐active coenzymes flavin adenine dinucleotide (FAD) or nicotinamide adenine dinucleotide (NAD+) remain challenging. Here, we present a pathway to NAD+ under prebiotic conditions starting with ammonia, cyanoacetaldehyde, prop‐2‐ynal and sugar‐forming precursors, yielding in situ the nicotinamide riboside. Regioselective phosphorylation and water stable light activated adenosine monophosphate derivatives allow for topographically and irradiation‐controlled formation of NAD+. Our findings indicate that NAD+, a coenzyme vital to life, can be formed non‐enzymatically from simple organic feedstock molecules via photocatalytic activation under prebiotically plausible early Earth conditions in a continuous process under aqueous conditions. [ABSTRACT FROM AUTHOR]

Cuproptosis is characterized by lipoylated protein aggregation and loss of iron–sulfur (Fe–S) proteins, which are crucial for a wide range of important cellular functions, including DNA replication and damage repair. Sirt2 and sirt4 are lipoamidases that remove the lipoyl moiety from lipoylated proteins using nicotinamide adenine dinucleotide (NAD+) as a cofactor. However, to date, it is not clear whether nicotinamide mononucleotide (NMN), a precursor of NAD+, affects cellular sensitivity to cuproptosis. Therefore, in the current study, cuproptosis was induced by the copper (Cu) ionophore elesclomol (Es) in HeLa cells. It was also found that Es/Cu treatment increased cellular DNA damage level. On the other hand, NMN treatment partially rescued cuproptosis in a dose-dependent manner, as well as reduced cellular DNA damage level. In addition, NMN upregulated the expression of Fe-S protein POLD1, without affecting the aggregation of lipoylated proteins. Mechanistic study revealed that NMN increased the expression of sirt2 and cellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) level. Overexpression of sirt2 and sirt4 did not change the aggregation of lipoylated proteins, however, sirt2, but not sirt4, increased cellular NADPH levels and partially rescued cuproptosis. Inhibition of NAD+ kinase (NADK), which is responsible for generating NADPH, abolished the rescuing function of NMN and sirt2 for Es/Cu induced cell death. Taken together, our results suggested that DNA damage is a characteristic feature of cuproptosis. NMN can partially rescue cuproptosis by upregulating sirt2, increase intracellular NADPH content and maintain the level of Fe-S proteins, independent of the lipoamidase activity of sirt2. [ABSTRACT FROM AUTHOR]

The article investigates how the lncRNA Sera and the Pkm2 pathway in the medial prefrontal cortex (mPFC) affect social competition and rank by remodeling neural ensembles in mice. Topics discussed include the role of the Sera/Pkm2 pathway in competitive success, the modulation of excitatory neurons in the prelimbic cortex, and the phosphorylation of AMPA receptors influencing social rank.

Nicotinamide Adenine Dinucleotide Phosphate (NAD(P)H) plays an important role in numerous biologically significant redox reactions. The photochemical restoration of its oxidized form (NAD(P)+) under physiological conditions is intriguing in the context of integrated photo and catalysis. Herein, we report the functionalized graphitic carbon‐based solar light active photocatalyst by doping boron and fluorine in the native graphitic carbon nitride (GCN) (nonfunctionalized) for the regeneration of enzymatically visible light active coenzyme and in photo‐acetalization reactions. The metal‐free functionalized photocatalyst systems such as BFGCN‐x leads to higher yield NADH and NADPH regeneration. They are also capable of catalyzing acetal reactions in the absence of any Lewis and Bronsted acids. The current research endeavor provides the advancement and the application of functionalized GCN‐based photocatalysts for NADH (61.89%), NADPH (59.84%) regeneration, and photo‐acetalization reactions. [ABSTRACT FROM AUTHOR]

Liver fibrosis progressing to cirrhosis is a major risk factor for liver cancer, impacting surgical treatment and survival. Our study focuses on the role of extracellular nicotinamide adenine dinucleotide (eNAD+) in liver fibrosis, analyzing liver disease patients undergoing surgery. Additionally, we explore NAD+'s therapeutic potential in a mouse model of extended liver resection and in vitro using 3D hepatocyte spheroids. eNAD+ correlated with aspartate transaminase (AST) and bilirubin after liver resection (AST: r = 0.2828, p = 0.0087; Bilirubin: r = 0.2584, p = 0.0176). Concordantly, post-hepatectomy liver failure (PHLF) was associated with higher eNAD+ peaks (n = 10; p = 0.0063). Post-operative eNAD+ levels decreased significantly (p < 0.05), but in advanced stages of liver fibrosis or cirrhosis, this decline not only diminished but actually showed a trend towards an increase. The expression of NAD+ biosynthesis rate-limiting enzymes, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), were upregulated significantly in the liver tissue of patients with higher liver fibrosis stages (p < 0.0001). Finally, the administration of NAD+ in a 3D hepatocyte spheroid model rescued hepatocytes from TNFalpha-induced cell death and improved viability (p < 0.0001). In a mouse model of extended liver resection, NAD+ treatment significantly improved survival (p = 0.0158) and liver regeneration (p = 0.0186). Our findings reveal that eNAD+ was upregulated in PHLF, and rate-limiting enzymes of NAD+ biosynthesis demonstrated higher expressions under liver fibrosis. Further, eNAD+ administration improved survival after extended liver resection in mice and enhanced hepatocyte viability in vitro. These insights may offer a potential target for future therapies. An analysis on liver disease patients suggests that extracellular Nicotinamide adenine dinucleotide (eNAD + ) correlates with liver fibrosis and post-hepatectomy liver failure, with NAD+ administration improving survival and liver regeneration in mice and hepatocyte viability in vitro. [ABSTRACT FROM AUTHOR]

Nicotinamide is an important functional compound and, in the form of nicotinamide adenine dinucleotide (NAD), is used as a co-factor by protein-based enzymes to catalyze redox reactions. In the context of the RNA world hypothesis, it is therefore reasonable to assume that ancestral ribozymes could have used co-factors such as NAD or its simpler analog nicotinamide riboside (NAR) to catalyze redox reactions. The only described example of such an engineered ribozyme uses a nicotinamide moiety bound to the ribozyme through non-covalent interactions. Covalent attachment of NAR to RNA could be advantageous, but the demonstration of such scenarios to date has suffered from the chemical instability of both NAR and its reduced form, NARH, making their use in oligonucleotide synthesis less straightforward. Here, we review the literature describing the chemical properties of the oxidized and reduced species of NAR, their synthesis, and previous attempts to incorporate either species into RNA. We discuss how to overcome the stability problem and succeed in generating RNA structures incorporating NAR. [ABSTRACT FROM AUTHOR]

Central metabolism is organised through high‐flux, Nicotinamide Adenine Dinucleotide (NAD+/NADH) and NADP+/NADPH systems operating at near equilibrium. As oxygen is indispensable for aerobic organisms, these systems are also linked to the levels of reactive oxygen species, such as H2O2, and through H2O2 to the regulation of macromolecular structures and activities, via kinetically controlled sulphur switches in the redox proteome. Dynamic changes in H2O2 production, scavenging and transport, associated with development, growth and responses to the environment are, therefore, linked to the redox state of the cell and regulate cellular function. These basic principles form the 'redox code' of cells and were first defined by D. P. Jones and H. Sies in 2015. Here, we apply these principles to plants in which recent studies have shown that they can also explain cell‐to‐cell and even plant‐to‐plant signalling processes. The redox code is, therefore, an integral part of biological systems and can be used to explain multiple processes in plants at the subcellular, cellular, tissue, whole organism and perhaps even community and ecosystem levels. As the environmental conditions on our planet are worsening due to global warming, climate change and increased pollution levels, new studies are needed applying the redox code of plants to these changes. Summary statement: The H2O2 levels and redox state of cells are tightly linked and together regulate metabolism, development, growth and stress responses, as well as coordinate these responses between different organelles, cells, tissues, the entire organism and even different organisms living in a community. [ABSTRACT FROM AUTHOR]

Recombinant protein production in Komagataella phaffi (K. phaffi), a widely utilized host organism, can be optimized by enhancing the metabolic flux in the central carbon metabolism pathways. The methanol utilization pathway (MUT) during methanol-based growth plays a crucial role in providing precursors and energy for cell growth and development. This study investigated the impact of boosting the methanol dissimilation pathway, a branch of MUT that plays a vital role in detoxifying formaldehyde and providing energy in the form of NADH, in K. phaffi. This was achieved by integrating two orthologous genes from Hansenula polymorpha into the K. phaffi genome: formaldehyde dehydrogenase (HpFLD) and formate dehydrogenase (HpFMDH). The HpFLD and HpFMDH genes were isolated from the Hansenula polymorpha genome and inserted under the regulation of the pAOX1 promoter in the genome of recombinant K. phaffi that already contained a single copy of model protein genes (eGFP or EGII). The expression levels of these model proteins were assessed through protein activity assays and gene expression analysis. The findings revealed that while both orthologous genes positively influenced model protein production, HpFMDH exhibited a more pronounced upregulation in expression compared to HpFLD. Co-expression of both orthologous genes demonstrated synergistic effects, resulting in approximately a twofold increase in the levels of the model proteins detected. This study provides valuable insights into enhancing the production capacity of recombinant proteins in K. phaffi. [ABSTRACT FROM AUTHOR]

This article provides an analysis of Oomycete Nudix effectors, which are proteins secreted by phytopathogens to aid in the colonization of host plants. The researchers conducted a phylogenetic analysis and examined the crystal structure of the P. sojae effector Avr3b. They found that oomycete Nudix effectors have distinct structural features and functions compared to fungal and bacterial Nudix effectors. The study also revealed that Avr3b and other oomycete Nudix effectors have mRNA decapping activity, suggesting their involvement in modulating host immune responses. Further research is needed to fully understand the role of these effectors in promoting pathogen infection and their impact on host immunity. [Extracted from the article]

The synthesis of carbocyclic-ddA, a potent antiviral agent against hepatitis B, relies significantly on (1 R ,3 R)-3-hydroxycyclopentanemethanol as a key intermediate. To effectively produce this intermediate, our study employed a chemoenzymatic approach. The selection of appropriate biocatalysts was based on substrate similarity, leading us to adopt the CrS enoate reductase derived from Thermus scotoductus SA-01. Additionally, we developed an enzymatic system for NADH regeneration, utilising formate dehydrogenase from Candida boidinii. This system facilitated the efficient catalysis of (S)-4-(hydroxymethyl)cyclopent-2-enone, resulting in the formation of (3 R)-3-(hydroxymethyl) cyclopentanone. Furthermore, we successfully cloned, expressed, purified, and characterized the CrS enzyme in Escherichia coli. Optimal reaction conditions were determined, revealing that the highest activity occurred at 45 °C and pH 8.0. By employing 5 mM (S)-4-(hydroxymethyl)cyclopent-2-enone, 0.05 mM FMN, 0.2 mM NADH, 10 μM CrS, 40 μM formic acid dehydrogenase, and 40 mM sodium formate, complete conversion was achieved within 45 min at 35 °C and pH 7.0. Subsequently, (1 R ,3 R)-3-hydroxycyclopentanemethanol was obtained through a simple three-step chemical conversion process. This study not only presents an effective method for synthesizing the crucial intermediate but also highlights the importance of biocatalysts and enzymatic systems in chemoenzymatic synthesis approaches. [Display omitted] • New chemoenzymatic route for the synthesis of anti-hepatitis B drug intermediates. • Novel application of CrS enoate reductase from Thermus scotoductus SA-01. • The further application of (S)-4-(hydroxymethyl)cyclopent-2-enone. [ABSTRACT FROM AUTHOR]

The direct electrochemical reduction of nicotinamide adenine dinucleotide (NAD+) results in various products, complicating the regeneration of the crucial 1,4‐NADH cofactor for enzymatic reactions. Previous research primarily focused on steady–state polarization to examine potential impacts on product selectivity. However, this study explores the influence of dynamic conditions on the selectivity of NAD+ reduction products by comparing two dynamic profiles with steady‐state conditions. Our findings reveal that the main products, including 1,4‐NADH, several dimers, and ADP‐ribose, remained consistent across all conditions. A minor by–product, 1,6‐NADH, was also identified. The product distribution varied depending on the experimental conditions (steady state vs. dynamic) and the concentration of NAD+, with higher concentrations and overpotentials promoting dimerization. The optimal yield of 1,4‐NADH was achieved under steady–state conditions with low overpotential and NAD+ concentrations. While dynamic conditions enhanced the 1,4‐NADH yield at shorter reaction times, they also resulted in a significant amount of unidentified products. Furthermore, this study assessed the potential of using pulsed electrochemical regeneration of 1,4‐NADH with enoate reductase (XenB) for cyclohexenone reduction. [ABSTRACT FROM AUTHOR]

Niacinamide is a versatile compound widely used in the personal care industry for its ample skin benefits. As a precursor to nicotinamide adenine dinucleotide (NAD+), essential for ATP production and a substrate for poly-ADP-ribose polymerase-1 (PARP-1), studies have highlighted its roles in DNA repair, cellular stress mechanisms, and anti-aging benefits. Niacinamide was also studied for its antimicrobial activity, particularly in the context of host-infection via host immune response, yet its direct antimicrobial activity and the mechanisms of action remain unclear. Its multifunctionality makes it an appealing bioactive molecule for skincare products as well as a potential preservative solution. This study explores niacinamide's antimicrobial mode of action against four common cosmetic pathogens. Our findings indicate that niacinamide is causing microbial cell cycle arrest; while cells were found to increase their volume and length under treatment to prepare for cell division, complete separation into two daughter cells was prevented. Fluorescence microscopy revealed expanded chromatin, alongside a decreased RNA expression of the DNA-binding protein gene, dps. Finally, niacinamide was found to directly interact with DNA, hindering successful amplification. These unprecedented findings allowed us to add a newly rationalized preservative facete to the wide range of niacinamide multi-functionality. [ABSTRACT FROM AUTHOR]

In this work we disclose a new family of biscyclometallated Ir(III) complexes of the general formula [Ir(C^N)2(N^N)]Cl (IrL1–IrL5), where HC^N is 1-phenyl-β-carboline and N^N ligands (L1–L5) are different diimine ligands that differ from each other in the number of aromatic rings fused to the bipyridine scaffold. The photophysical properties of IrL1–IrL5 were thoroughly studied, and theoretical calculations were performed for a deeper comprehension of the respective variations along the series. All complexes exhibited high photostability under blue light irradiation. An increase in the number of aromatic rings led to a reduction in the HOMO–LUMO band gap causing a red-shift in the absorbance bands. Although all the complexes generated singlet oxygen (1O2) in aerated aqueous solutions through a photocatalytic process, IrL5 was by far the most efficient photosensitizer. Consequently, IrL5 was highly active in the photocatalytic oxidation of NADH. The formation of aggregates in DMSO at a high concentration (25 mM) was confirmed using different techniques, but was proved to be negligible in the concentration range of biological experiments. Moreover, ICP-MS studies proved that the cellular uptake of IrL2 and IrL3 is much better relative to that of IrL1, IrL4 and IrL5. The antiproliferative activity of IrL1–IrL5 was investigated in the dark and under blue light irradiation against different cancer cell lines. Complexes IrL1–IrL4 were found to be cytotoxic under dark conditions, while IrL5 turned out to be weakly cytotoxic. Despite the low cellular uptake of IrL5, this derivative exhibited a high increase of cytotoxicity upon blue light irradiation resulting in photocytotoxicity indexes (PI) up to 38. IrL1–IrL4 showed lower photocytotoxicity indexes ranging from 1.3 to 17.0. Haemolytic experiments corroborated the compatibility of our complexes with red blood cells. Confocal microscopy studies proved their accumulation in mitochondria, leading to mitochondrial membrane depolarization, and ruled out their localization in lysosomes. Overall, the mitochondria-targeted activity of IrL5, which inhibits considerably the viability of cancer cells upon blue light irradiation, allows us to outline this PS as a new alternative to traditional chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD +) plays a pivotal role in numerous cellular functions. Reduced NAD + levels are postulated to be associated with cancer. As interest in understanding NAD + dynamics in cancer patients with therapeutic applications in mind grows, there remains a shortage of comprehensive data. This study delves into NAD + dynamics in patients undergoing surgery for different digestive system cancers. This prospective study enrolled 99 patients with eight different cancers. Fasting blood samples were obtained during the perioperative period. The concentrations of NAD + , nicotinamide mononucleotide (NMN), and nicotinamide riboside were analyzed using tandem mass spectrometry. After erythrocyte volume adjustment, NAD + remained relatively stable after surgery. Meanwhile, NMN decreased the day after surgery and displayed a recovery trend. Interestingly, liver and pancreatic cancer patients exhibited poor postoperative NMN recovery, suggesting a potential cancer type-specific influence on NAD + metabolism. This study illuminated the behavior of NAD + in surgically treated cancer patients. We identified which cancer types have particularly low levels and at what point depletion occurs during the perioperative period. These insights suggest the need for personalized NAD + supplementation strategies, calibrated to individual patient needs and treatment timelines. Clinical trial registration jRCT1020210066. [ABSTRACT FROM AUTHOR]

Background: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase—an essential enzyme in the dissimilation pathway—displayed growth defects when exposed to a methanol-containing medium. Results: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. Conclusions: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii. [ABSTRACT FROM AUTHOR]

Coenzyme Q10 (CoQ10) is an essential medicinal ingredient. In this study, we obtained a high-yielding mutant strain of CoQ10, VK-2-3, by subjecting R. sphaeroides V-0 (V-0) to a 12C6+ heavy ion beam and high-voltage prick electric field treatment. To investigate the mutation mechanism, the complete genomes of VK-2-3 and V-0 were sequenced. Collinearity analysis revealed that the nicotinamide adenine dinucleotide-dependent dehydrogenase (NAD) gene underwent rearrangement in the VK-2-3 genome. The NAD gene was overexpressed and silenced in V-0, and this construct was named RS.NAD and RS.ΔNAD. The results showed that the titers of CoQ10 in the RS.NAD and RS.ΔNAD increased and decreased by 16.00 and 33.92%, respectively, compared to those in V-0, and these differences were significant. Our results revealed the mechanism by which the VK-2-3 CoQ10 yield increases through reverse metabolic engineering, providing insights for genetic breeding and mechanistic analysis. [ABSTRACT FROM AUTHOR]

NAD-dependent deacetylase Sirt2 is involved in mammalian metabolic activities, matching energy demand with energy production and expenditure, and is relevant to a variety of metabolic diseases. Here, we constructed Sirt2 knockout and adeno-associated virus overexpression mice and found that deletion of hepatic Sirt2 accelerated primary obesity and insulin resistance in mice with concomitant hepatic metabolic dysfunction. However, the key targets of Sirt2 are unknown. We identified the M2 isoform of pyruvate kinase (PKM2) as a key Sirt2 target involved in glycolysis in metabolic stress. Through yeast two-hybrid and mass spectrometry combined with multi-omics analysis, we identified candidate acetylation modification targets of Sirt2 on PKM2 lysine 135 (K135). The Sirt2-mediated deacetylation-ubiquitination switch of PKM2 regulated the development of glycolysis. Here, we found that Sirt2 deficiency led to impaired glucose tolerance and insulin resistance and induced primary obesity. Sirt2 severely disrupted liver function in mice under metabolic stress, exacerbated the metabolic burden on the liver, and affected glucose metabolism. Sirt2 underwent acetylation modification of lysine 135 of PKM2 through a histidine 187 enzyme active site-dependent effect and reduced ubiquitination of the K48 ubiquitin chain of PKM2. Our findings reveal that the hepatic glucose metabolism links nutrient state to whole-body energetics through the rhythmic regulation of Sirt2. [ABSTRACT FROM AUTHOR]

AbstractThe expression of both lactate dehydrogenase A (LDH-A) and glucose transporter type 1 (GLUT1) is high in pancreatic, thoracic and many other types of cancer. GLUT1 is also highly expressed in endothelial cells (EC), that play an important role in tumor metastasis. We investigated the effect of inhibition of LDH-A by NHI-2 and GLUT1 by PGL14 on cellular migration, a hallmark of metastasis, in relation to changes in intracellular purine nucleotide and nicotinamide adenine dinucleotide pools in a human microvascular endothelial cell line (HMEC-1). HMEC-1 were treated with NHI-2 and PGL14 alone or in combination. Cell migration was tested by the wound healing assay. The intracellular purine nucleotides and NAD+/NADH concentrations were measured using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). Both NHI-2 at 15 µM and 45 µM and PGL14 at 10 µM and 30 µM inhibited migration by 5 to 28% while the combination led to 46% inhibition. The drugs also decreased intracellular nucleotide pools, but only 45 µM NHI-2 altered energy charge and redox status in HMEC-1 cells. Inhibitors of glycolysis attenuated migration and the energy charge of EC and support further development of LDH-A and GLUT1 inhibitors to target cancer aggressiveness and metastasis. [ABSTRACT FROM AUTHOR]

The kidney undergoes structural and physiological changes with age, predominantly studied in glomeruli and proximal tubules. However, limited knowledge is available about the impact of aging and anti‐aging interventions on distal tubules. In this study, we investigated the effects of cytochrome b5 reductase 3 (CYB5R3) overexpression and/or dietary nicotinamide riboside (NR) supplementation on distal tubule mitochondria. Initially, transcriptomic data were analyzed to evaluate key genes related with distal tubules, CYB5R3, and NAD+ metabolism, showing significant differences between males and females in adult and old mice. Subsequently, our emphasis focused on assessing how these interventions, that have demonstrated the anti‐aging potential, influenced structural parameters of distal tubule mitochondria, such as morphology and mass, as well as abundance, distance, and length of mitochondria‐endoplasmic reticulum contact sites, employing an electron microscopy approach. Our findings indicate that both interventions have differential effects depending on the age and sex of the mice. Aging resulted in an increase in mitochondrial size and a decrease in mitochondrial abundance in males, while a reduction in abundance, size, and mitochondrial mass was observed in old females when compared with their adult counterparts. Combining both the interventions, CYB5R3 overexpression and dietary NR supplementation mitigated age‐related changes; however, these effects were mainly accounted by NR in males and by transgenesis in females. In conclusion, the influence of CYB5R3 overexpression and dietary NR supplementation on distal tubule mitochondria depends on sex, genotype, and diet. This underscores the importance of incorporating these variables in subsequent studies to comprehensively address the multifaceted aspects of aging. [ABSTRACT FROM AUTHOR]

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2′-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. Key points: •Zn-containing BaDH has activity with either NAD+or NADP+at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol. [ABSTRACT FROM AUTHOR]

Here we demonstrate the preparation of enzyme‐metal biohybrids of NAD+ reductase with biocatalytically‐synthesised small gold nanoparticles (NPs, <10 nm) and core‐shell gold‐platinum NPs for tandem catalysis. Despite the variety of methods available for NP synthesis, there remains a need for more sustainable strategies which also give precise control over the shape and size of the metal NPs for applications in catalysis, biomedical devices, and electronics. We demonstrate facile biosynthesis of spherical, highly uniform, gold NPs under mild conditions using an isolated enzyme moiety, an NAD+ reductase, to reduce metal salts while oxidising a nicotinamide‐containing cofactor. By subsequently introducing platinum salts, we show that core‐shell Au@Pt NPs can then be formed. Catalytic function of these enzyme‐Au@Pt NP hybrids was demonstrated for H2‐driven NADH recycling to support enantioselective ketone reduction by an NADH‐dependent alcohol dehydrogenase. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is a universal coenzyme regulating cellular energy metabolism in many cell types. Recent studies have demonstrated the close relationships between defective NAD+ metabolism and aging and age-associated metabolic diseases. The major purpose of the present study was to test the hypothesis that NAD+ biosynthesis, mediated by a rate-limiting NAD+ biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT), is essential for maintaining normal adipose tissue function and whole body metabolic health during the aging process. To this end, we provided in-depth and comprehensive metabolic assessments for female adipocyte-specific Nampt knockout (ANKO) mice during aging. We first evaluated body fat mass in young (<4-mo-old), middle aged (10-14-mo-old), and old (≥18-mo-old) mice. Intriguingly, adipocyte-specific Nampt deletion protected against age-induced obesity without changing energy balance. However, data obtained from the hyperinsulinemic-euglycemic clamp procedure (HECP) demonstrated that, despite the lean phenotype, old ANKO mice had severe insulin resistance in skeletal muscle, heart, and white adipose tissue (WAT). Old ANKO mice also exhibited hyperinsulinemia and hypoadiponectinemia. Mechanistically, loss of Nampt caused marked decreases in WAT gene expression of lipogenic targets of peroxisome proliferator-activated receptor gamma (PPAR-γ) in an age-dependent manner. In addition, administration of a PPAR-γ agonist rosiglitazone restored fat mass and improved metabolic abnormalities in old ANKO mice. In conclusion, these findings highlight the importance of the NAMPT-NAD+-PPAR-γ axis in maintaining functional integrity and quantity of adipose tissue, and whole body metabolic function in female mice during aging. NEW & NOTEWORTHY Defective NAD+ metabolism is associated with aging and age-associated metabolic diseases. In the present study, we provided in-depth metabolic assessments in female mice with adipocyte-specific inactivation of a key NAD+ biosynthetic enzyme NAMPT and revealed an unexpected role of adipose tissue NAMPT-NAD+-PPAR-γ axis in maintaining functional integrity and quantity of adipose tissue and whole body metabolic health during the aging process. [ABSTRACT FROM AUTHOR]

Thioredoxins (TRXs) are central to redox regulation, modulating enzyme activities to adapt metabolism to environmental changes. Previous research emphasized mitochondrial and microsomal TRX o1 and h2 influence on mitochondrial metabolism, including photorespiration and the tricarboxylic acid (TCA) cycle. Our study aimed to compare TRX‐based regulation circuits towards environmental cues mainly affecting photorespiration. Metabolite snapshots, phenotypes and CO2 assimilation were compared among single and multiple TRX mutants in the wild‐type and the glycine decarboxylase T‐protein knockdown (gldt1) background. Our analyses provided evidence for additive negative effects of combined TRX o1 and h2 deficiency on growth and photosynthesis. Especially metabolite accumulation patterns suggest a shared regulation mechanism mainly on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1)‐dependent pathways. Quantification of pyridine nucleotides, in conjunction with 13C‐labelling approaches, and biochemical analysis of recombinant mtLPD1 supported this. It also revealed mtLPD1 inhibition by NADH, pointing at an additional measure to fine‐tune it's activity. Collectively, we propose that lack of TRX o1 and h2 perturbs the mitochondrial redox state, which impacts on other pathways through shifts in the NADH/NAD+ ratio via mtLPD1. This regulation module might represent a node for simultaneous adjustments of photorespiration, the TCA cycle and branched chain amino acid degradation under fluctuating environmental conditions. Summary statement: Thioredoxins o1 and h2 concertedly adapt the performance of mtLPD1‐dependent pathways towards short‐ and long‐term environmental changes. Simultaneous adjustments of mitochondrial pathway fluxes are achieved through fine tuning of mtLPD1 activity via redox regulation and subcellular NADH/NAD+ ratios. [ABSTRACT FROM AUTHOR]

Key message: The major QTL Sdp1.1+ controlling seed dormancy in cowpea was finely mapped, and two CCoAOMT1 genes were identified as candidate genes for the dormancy. Seed dormancy in wild cowpea may be useful in breeding cultivated cowpea with pre-harvest sprouting resistance. A previous study identified a major quantitative trait locus (QTL) for seed dormancy, Sdp1.1+ , using the population of the cross between cultivated cowpea 'JP81610' and wild cowpea 'JP89083.' However, the molecular basis of seed dormancy in cowpea is not yet known. In this study, we aimed to finely map the locus Sdp1.1+ and identify candidate gene(s) for it. Germination tests demonstrated that the seed coat is the major factor controlling seed dormancy in the wild cowpea JP89083. Microscopic observations revealed that wild cowpea seeds, unlike cultivated cowpea seeds, possessed a palisade cuticle layer. Fine mapping using a large F2 population of the cross JP81610 × JP89083 grown in Thailand revealed a single QTL, Sdp1.1+ , controlling seed dormancy. The Sdp1.1+ was confirmed using a small F2 population of the same cross grown in Japan. The Sdp1.1+ was mapped to a 37.34-Kb region containing three genes. Two closely linked genes, Vigun03g278900 (VuCCoAOMT1a) and Vigun03g290000 (VuCCoAOMT1b), located 4.844 Kb apart were considered as candidate genes for seed dormancy. The two genes encoded caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1). DNA sequencing and alignment of VuCCoAOMT1a and VuCCoAOMT1b between JP89083 and JP81610 revealed a single nucleotide polymorphism (SNP) causing an amino acid change in VuCCoAOMT1a and several SNPs leading to six amino acid changes in VuCCoAOMT1b. Altogether, these results indicate that VuCCoAOMT1a and VuCCoAOMT1b are candidate genes controlling physical seed dormancy in the wild cowpea JP89083. [ABSTRACT FROM AUTHOR]

Free cyanide (fCN) consisting of HCN and CN- is highly hazardous. Today, removal of cyanide from industrial (mining, plating, coke-plant) wastewaters largely relies on physicochemical processes followed by microbial degradation. Enzymatic processes are gaining ground but are still at a low technological stage. The cyanide-converting enzymes of interest are primarily cyanide dihydratases (CynDs; EC 3.5.5.1.), which hydrolyze HCN to formic acid and ammonia, and cyanide hydratases (CynHs; EC 4.2.1.66), which hydrate HCN to formamide. CynHs usually have much higher specific activities and a broader pH profile especially in the alkaline region compared to CynDs. However, the product of CynH, formamide, although much less toxic than fCN, still poses a significant health risk. Therefore, it is attractive to combine the CynH with an amidase that converts formamide to formic acid and ammonia. Here we demonstrate on a laboratory scale a two-step "one-pot" detoxification of fCN (5 mM) to formic acid using recombinantly produced purified enzymes − CynH from Exidia glandulosa and formamidase (EC 3.5.1.49) from Bacillus cereus. The reaction proceeds at pH 9.0–10.0, which reduces the risk of HCN escape. We also hypothesize that the cascade can be used for fCN determination after coupling an NAD-dependent formate dehydrogenase. [Display omitted] • Cyanide hydratase degrades hydrogen cyanide to formamide which is still toxic. • We combined cyanide hydratase with formamidase to detoxify formamide. • The recombinantly expressed, purified enzymes were used in a one-pot process. • The enzymes were active under alkaline conditions suitable for handling cyanide. • HCN was converted to formate with no significant amount of formamide. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to investigate the protective effects of dexmedetomidine (DEX) on renal injury caused by acute stress in rats and explore the protective pathways of DEX on rat kidneys in terms of oxidative stress. Methods: An acute restraint stress model was utilized, where rats were restrained for 3 hours after a 15-minute swim. Biochemical tests and histopathological sections were conducted to evaluate renal function, along with the measurement of oxidative stress and related pathway proteins. Key findings: The open-field experiments validated the successful establishment of the acute stress model. Acute stress-induced renal injury led to increased NADPH oxidase 4 (NOX4) protein expression and decreased expression levels of nuclear transcription factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1). Following DEX treatment, there was a significant reduction in renal NOX4 expression. The DEX-treated group exhibited normalized renal biochemical results and less damage observed in pathological sections compared to the acute stress group. Conclusions: The findings suggest that DEX treatment during acute stress can impact the NOX4/Nrf2/HO-1/NQO1 signaling pathway and inhibit oxidative stress, thereby preventing acute stress-induced kidney injury. Additionally, DEX shows promise for clinical applications in stress syndromes. [ABSTRACT FROM AUTHOR]

Our previous clinical metabolomics study illustrated that energy metabolism disorder is an underlying pathogenesis mechanism for the development of alcoholic liver disease (ALD). Supplementation of nicotinamide (NAM), the precursor of nicotinamide adenine dinucleotide (NAD+), may restore the energy metabolism homeostasis of ALD and thus serves as potential therapeutics to treat ALD. In this bedside‐to‐bench study, the protective effect of NAM against ALD was investigated by using the NIAAA mice model (chronic‐plus‐binge ethanol), and the liver regeneration boosting capability of NAM was evaluated by the partial hepatectomy mice model. Our results showed that NAM supplements not only protected the liver from alcohol‐induced injury and improved alcohol‐induced mitochondrial structure and function change, but also boosted liver regeneration in postpartial hepatectomy mice by increasing liver NAD+ content. These findings suggested that NAM, a water‐soluble form of vitamin B3, can promote liver regeneration and improves liver function by alleviating alcohol‐induced energy metabolism disorder. [ABSTRACT FROM AUTHOR]