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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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

Elevated global pollution level is the prime reason that contributes to the onset of various harmful health diseases. The products of Biginelli reaction are enormously used in the pharmaceutical industry as they have antiviral, antibacterial, and calcium channel modulation abilities. This work reports a novel eosin Y sensitized boron graphitic carbon nitride (EY‐Ben‐g‐C3N4) as a photocatalyst that efficiently produced 3,4‐dihydropyrimidine‐2‐(1H)‐one by the Biginelli reaction of benzaldehyde, urea, and methyl acetoacetate. The photocatalyst EY‐Ben‐g‐C3N4 showed a successful generation of 3,4‐dihydropyrimidine‐2‐(1H)‐one (Biginelli product) in good yield via photocatalysis which is an eco‐friendly method and has facile operational process. In addition to the production of Biginelli products, the photocatalyst also showed a remarkable NADH regeneration of 81.18%. The incorporation of g‐C3N4 with boron helps increase the surface area and the incorporation of eosin Y which is an inexpensive and non‐toxic dye, and in Ben‐g‐C3N4, enhanced the light‐harvesting capacity of the photocatalyst. The production of 3,4‐dihydropyrimidine‐2‐(1H)‐one and NADH by the EY‐Ben‐g‐C3N4 photocatalyst is attributed to the requisite band gap, high molar absorbance, low rate of charge recombination, and increased capacity of the photocatalyst to harvest solar light energy. [ABSTRACT FROM AUTHOR]

l‐Pipecolic Acid (l‐PA) is a valuable building block for the synthesis of pharmaceuticals such as anesthetics and immunosuppressants. Thus, more efficient and greener strategies are desired for its production. Herein, we have applied a previously engineered variant of the Lysine Cyclodeaminase from Streptomyces pristinaespiralis (e‐SpLCD) for the bioconversion of l‐Lysine into l‐PA. The reaction can be performed by the free e‐SpLCD reaching full conversion to 50 mM l‐PA. From a biotechnological perspective, the process scale‐up has been trialed in a SpinChem® reactor, albeit with lower conversion yields. To further enhance the biocatalyst stability, we present a detailed study of the e‐SpLCD immobilization on microparticles. This enabled the integration of the immobilized biocatalyst into a packed‐bed reactor for the continuous flow synthesis of l‐PA. The full conversion was achieved in 90 min, maintaining also high operational stability. Remarkably, the addition of exogenous cofactor was not needed for the flow reaction, although the long‐term operational stability was improved by the addition of NAD+. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3‐(aminocarbonyl)‐1‐(2,3‐dihydroxypropyl)pyridinium, 1‐methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules. [ABSTRACT FROM AUTHOR]

Pancreatic adenocarcinoma is the fourth leading cause of malignancy-related deaths, with rapid development of drug resistance driven by pancreatic cancer stem cells. However, the mechanisms sustaining stemness and chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Here, we demonstrate that Bicaudal C homolog 1 (BICC1), an RNA binding protein regulating numerous cytoplasmic mRNAs, facilitates chemoresistance and stemness in PDAC. Mechanistically, BICC1 activated tryptophan catabolism in PDAC by up-regulating indoleamine 2,3-dioxygenase-1 (IDO1) expression, a tryptophan-catabolizing enzyme. Increased levels of tryptophan metabolites contribute to NAD+ synthesis and oxidative phosphorylation, leading to a stem cell-like phenotype. Blocking BICC1/IDO1/tryptophan metabolism signaling greatly improves the gemcitabine (GEM) efficacy in several PDAC models with high BICC1 level. These findings indicate that BICC1 is a critical tryptophan metabolism regulator that drives the stemness and chemoresistance of PDAC and thus a potential target for combinatorial therapeutic strategy against chemoresistance. [ABSTRACT FROM AUTHOR]

Profound changes in the metabolism of cancer cells have been known for almost 100 years, and many aspects of these changes have continued to be actively studied and discussed. Differences in the results of various studies can be explained by the diversity of tumours, which have differing processes of energy metabolism, and by limitations in the methods used. Here, using fluorescence lifetime needle optical biopsy in a hepatocellular carcinoma (HCC) mouse model and patients with HCC, we measured reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in control liver, and in HCC tumours and their adjacent regions. We found that NADH level (mostly responsible for energy metabolism) is increased in tumours but also in adjacent regions of the same liver. NADPH level is significantly decreased in the tumours of patients but increased in the HCC mouse model. However, in the ex vivo tumour slices of mouse HCC, reactive oxygen species production and glutathione level (both dependent on NADPH) were significantly suppressed. Thus, glucose‐dependent NADH and NADPH production in tumours changed but with a more pronounced shift to energy production (NADH), rather than NADPH synthesis for redox balance. [ABSTRACT FROM AUTHOR]

The malignancy of breast cancer poses a global challenge, with existing treatments often falling short of desired efficacy. Extensive research has underscored the effectiveness of targeting the metabolism of nicotinamide adenine dinucleotide (NAD), a pivotal molecule crucial for cancer cell survival and growth, as a promising anticancer strategy. Within mammalian cells, sustaining optimal NAD concentrations relies on two key enzymes, namely nicotinamide phosphoribosyltransferase (NAMPT) and poly(ADP-ribose) polymer 1 (PARP1). Recent studies have accentuated the potential benefits of combining NAMPT inhibitors and PARP1 inhibitors to enhance therapeutic outcomes, particularly in breast cancer. In this study, we designed and synthesized eleven novel NAMPT/PARP1 dual-target inhibitors. Among them, compound DDY02 exhibited acceptable inhibitory activities against both NAMPT and PARP1, with IC50 values of 0.01 and 0.05 µM, respectively. Moreover, in vitro evaluations revealed that treatment with DDY02 resulted in proliferation inhibition, NAD depletion, DNA damage, apoptosis, and migration inhibition in MDA-MB-468 cells. These results posit DDY02, by targeting NAD metabolism through inhibiting both NAMPT and PARP1, as a promising lead compound for the development of breast cancer therapy. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) is involved in renal physiology and is synthesized by nicotinamide mononucleotide adenylyltransferase (NMNAT). NMNAT exists as three isoforms, namely, NMNAT1, NMNAT2, and NMNAT3, encoded by Nmnat1, Nmnat2, and Nmnat3, respectively. In diabetic nephropathy (DN), NAD levels decrease, aggravating renal fibrosis. Conversely, sodium–glucose cotransporter-2 inhibitors increase NAD levels, mitigating renal fibrosis. In this regard, renal NAD synthesis has recently gained attention. However, the renal role of Nmnat in DN remains uncertain. Therefore, we investigated the role of Nmnat by establishing genetically engineered mice. Among the three isoforms, NMNAT1 levels were markedly reduced in the proximal tubules (PTs) of db/db mice. We examined the phenotypic changes in PT-specific Nmnat1 conditional knockout (CKO) mice. In CKO mice, Nmnat1 expression in PTs was downregulated when the tubules exhibited albuminuria, peritubular type IV collagen deposition, and mitochondrial ribosome (mitoribosome) excess. In CKO mice, Nmnat1 deficiency-induced mitoribosome excess hindered mitoribosomal translation of mitochondrial inner membrane-associated oxidative phosphorylation complex I (CI), CIII, CIV, and CV proteins and mitoribosomal dysfunction. Furthermore, the expression of hypermethylated in cancer 1, a transcription repressor, was downregulated in CKO mice, causing mitoribosome excess. Nmnat1 overexpression preserved mitoribosomal function, suggesting its protective role in DN. [ABSTRACT FROM AUTHOR]

We present a compact laser system for quantitative two-photon excitation spectra measurements and ratiometric two-photon imaging of fluorescent protein indicators. The fundamental of the system is a short segment of photonic crystal fiber (PCF), which supports a nonlinear transformation of low-power ultrashort pulses by preserving temporal coherence, and this generates an ultrafast almost octave-spanning supercontinuum (SC). Accurate sculpting of the SC by its amplitude and phase modulation provides implementation of the spectroscopic and microscopic modalities. The spectroscopic one was exhibited by two-photon action cross section spectra measuring for the genetically encoded fluorescent sensing proteins of the vital biochemical parameters: acidity (SypHer3s), concentration of hydrogen peroxide (HyPer3 and HyPer7), redox status of NADH and glutathione (RexYFP and Grx1-roGFP2), hypohalous acids and their derivatives (Hypocrates). For the microscopy, we investigated and optimized the intensity pump pulse profiles under the high numerical objective by dispersion scan technique. We conducted real-time monitoring of the dynamics of hydrogen peroxide in HeLa cells with subcellular spatial resolution by means of ratiometric two-photon imaging of Hyper7 sensors. The presented hybrid laser system provides an ideal optical toolbox in order to develop ratiometric fluorescent sensors, which can be visualized in vivo using two-photon microscopy. [ABSTRACT FROM AUTHOR]

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

Fructosamine-3-kinases (FN3Ks) are a conserved family of repair enzymes that phosphorylate reactive sugars attached to lysine residues in peptides and proteins. Although FN3Ks are present across the Tree of Life and share detectable sequence similarity to eukaryotic protein kinases, the biological processes regulated by these kinases are largely unknown. To address this knowledge gap, we leveraged the FN3K CRISPR Knock-Out (KO) HepG2 cell line alongside an integrative multi-omics study combining transcriptomics, metabolomics, and interactomics to place these enzymes in a pathway context. The integrative analyses revealed the enrichment of pathways related to oxidative stress response, lipid biosynthesis (cholesterol and fatty acids), and carbon and co-factor metabolism. Moreover, enrichment of nicotinamide adenine dinucleotide (NAD) binding proteins and localization of human FN3K (HsFN3K) to mitochondria suggests potential links between FN3K and NAD-mediated energy metabolism and redox balance. We report specific binding of HsFN3K to NAD compounds in a metal and concentration-dependent manner and provide insight into their binding mode using modeling and experimental site-directed mutagenesis. Our studies provide a framework for targeting these understudied kinases in diabetic complications and metabolic disorders where redox balance and NAD-dependent metabolic processes are altered. [ABSTRACT FROM AUTHOR]

Simple Summary: Marine biofouling is the undesirable accumulation of marine organisms onto man-made surfaces, which causes severe economical and environmental consequences. In recent years, using natural products as environmentally friendly antifouling agents to combat foulers has been a promising approach. However, the search for effective antifouling agents is hampered by the lack of well-defined conserved molecular targets responsible for regulating the larval settlement in fouling organisms. In this study, an in silico approach was used to screen the natural compounds' libraries against silk gland factor 1 (SGF1), which is responsible for the larval settlement of the fouling mussel Mytilopsis sallei. It was found that the targeted binding compounds against SGF1 could significantly affect the larval settlement, foot proteins' gene expression, and byssus secretion of adults in M. sallei. The laboratory bioassay results are promising, even though field trials are necessary to confirm the in silico results. Hence, this study paves the way for new experimental studies to be carried out for finding environmentally friendly antifouling agents by using SGF1-targeted compounds. Most fouling organisms have planktonic larval and benthic adult stages. Larval settlement, the planktonic–benthic transition, is the critical point when biofouling begins. However, our understanding of the molecular mechanisms of larval settlement is limited. In our previous studies, we identified that the AMP-activated protein kinase-silk gland factor 1 (AMPK-SGF1) pathway was involved in triggering the larval settlement in the fouling mussel M. sallei. In this study, to further confirm the pivotal role of SGF1, multiple targeted binding compounds of SGF1 were obtained using high-throughput virtual screening. It was found that the targeted binding compounds, such as NAD+ and atorvastatin, could significantly induce and inhibit the larval settlement, respectively. Furthermore, the qRT-PCR showed that the expression of the foot proteins' genes was significantly increased after the exposure to 10 μM NAD+, while the gene expression was significantly suppressed after the exposure to 10 μM atorvastatin. Additionally, the production of the byssus threads of the adults was significantly increased after the exposure to 10–20 μM of NAD+, while the production of the byssus threads was significantly decreased after the exposure to 10–50 μM of atorvastatin. This work will deepen our understanding of SGF1 in triggering the larval settlement in mussels and will provide insights into the potential targets for developing novel antifouling agents. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1–4, containing a methyl group at the ribose 2′-C and 3′-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1–4) have distinct conformational preferences. Biological evaluation of dinucleotides 1–4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2′-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization. [ABSTRACT FROM AUTHOR]

Cytochrome P450s (P450s), a superfamily of heme-containing enzymes, existed in animals, plants, and microorganisms. P450s can catalyze various regional and stereoselective oxidation reactions, which are widely used in natural product biosynthesis, drug metabolism, and biotechnology. In a typical catalytic cycle, P450s use redox proteins or domains to mediate electron transfer from NAD(P)H to heme iron. Therefore, the main factors determining the catalytic efficiency of P450s include not only the P450s themselves but also their redox-partners and electron transfer pathways. In this review, the electron transfer pathway engineering strategies of the P450s catalytic system are reviewed from four aspects: cofactor regeneration, selection of redox-partners, P450s and redox-partner engineering, and electrochemically or photochemically driven electron transfer. [ABSTRACT FROM AUTHOR]

Objectives: Gemcitabine, a first-line chemotherapeutic nucleoside analog drug (NAD) for pancreatic cancer, faces limitations due to drug resistance. Characterizing pancreatic cancer cells' transport characteristics may help identify the mechanisms behind drug resistance, and develop more effective therapeutic strategies. Therefore, in this study, we aimed to determine the nucleoside transport properties of Panc-1 cells, one of the commonly used pancreatic adenocarcinoma cell lines. Materials and Methods: To assess the presence of equilibrative nucleoside transporter-1 (ENT-1) in Panc-1 cells, we performed immunofluorescence staining, western blot analysis, and S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding assays. We also conducted standard uptake assays to measure the sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine. In addition, we determined the half-maximal inhibitory concentration (IC50) of gemcitabine. Statistical analyses were performed using GraphPad Prism version 8.0 for Windows. Results: The sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine was measured using standard uptake assays, and the transport rates were determined as 111.1 ± 3.4 pmol/mg protein/10 s, 62.5 ± 4.8 pmol/mg protein/10 s, and 101.3 ± 2.5 pmol/mg protein/10 s, respectively. Furthermore, the presence of ENT-1 protein was confirmed using NBTI binding assays (Bmax 1.52 ± 0.1 pmol/mg protein; equilibrium dissociation constant 0.42 ± 0.1 nM). Immunofluorescence assays and western blot analysis also revealed ENT-1 in Panc-1 cells. The determined IC50 of gemcitabine in Panc-1 cells was 2 µM, indicating moderate sensitivity. Conclusion: These results suggest that Panc-1 is a suitable preclinical cellular model for studying NAD transport properties and potential therapies in pancreatic cancer and pharmaceutical research. [ABSTRACT FROM AUTHOR]

Osteoporosis poses a substantial public health challenge due to an ageing population and the lack of adequate treatment options. The condition is marked by a reduction in bone mineral density, resulting in an elevated risk of fractures. The reduction in bone density and strength, as well as musculoskeletal issues that come with aging, present a significant challenge for individuals impacted by these conditions, as well as the healthcare system worldwide. Literature survey was conducted until May 2023 using databases such as Web of Science, PubMed, Scopus, and Google Scholar. Sirtuins 1-7 (SIRT1-SIRT7), which are a group of Nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, possess remarkable capabilities to increase lifespan and combat diseases related to aging. Research has demonstrated that these proteins play an important role in regular skeletal development and maintenance by directly impacting bone cells. Their dysfunction could be a factor in various bone conditions. Studies conducted on animals before clinical trials have shown that administering Sirtuins agonists to mice provides a safeguard against osteoporosis resulting from aging, menopause, and immobilization. These findings imply that Sirtuins may be a viable target for addressing the irregularity in bone remodeling and treating osteoporosis and other skeletal ailments. The purpose of this review was to present a thorough and current evaluation of the existing knowledge on Sirtuins biology, with a particular emphasis on their involvement in maintaining bone homeostasis and contributing to osteoporosis. Additionally, the review examines potential pharmacological interventions targeting Sirtuins for the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Mimicking the enzymatic oxidation of NADH and obtaining mechanistic insight into the electron transfer reaction pertinent to the redox inter-conversion of NADH/NAD+ couple is a long-term challenge in biological and energy-related chemistry. The kinetics and mechanism of the oxidation of NADH by [RuIII(pic)3] (pic– = picolinate anion) has been studied spectrophotometrically and kinetically in aqueous solution at pH 7.0 (MES buffer). The reaction was followed over time at 466 nm (metal to ligand charge transfer band of [RuII(pic)3]–). The rate of NADH oxidation was found to be first-order with respect to Ru(III)-complex concentration. The values of the observed rate constant (kobs) increased linearly with increase in the concentration of NADH. The rate of the reaction was independent of the ionic strength of the reacting solution. Activation parameters (ΔH≠ = 56 ± 1 kJ mol−1 and ΔS≠ = −65 ± 3 J.deg−1 mol−1) were determined from the temperature dependence studies (15-35 °C) of the reaction. Kinetic data and activation parameters are interpreted in terms of a mechanism involving outer-sphere electron transfer. [RuIII(pic)3] (pic− = picolinate anion) effectively oxidizes NADH in an outer-sphere electron transfer pathway. [ABSTRACT FROM AUTHOR]

Pierisin-1 was serendipitously discovered as a strong cytotoxic and apoptosis-inducing protein from pupae of the cabbage butterfly Pieris rapae against cancer cell lines. This 98-kDa protein consists of the N-terminal region (27 kDa) and C-terminal region (71 kDa), and analysis of their biological function revealed that pierisin-1 binds to cell surface glycosphingolipids on the C-terminal side, is taken up into the cell, and is cleaved to N- and C-terminal portions, where the N-terminal portion mono-ADP-ribosylates the guanine base of DNA in the presence of NAD to induce cellular genetic mutation and apoptosis. Unlike other ADP-ribosyltransferases, pieisin-1 was first found to exhibit DNA mono-ADP-ribosylating activity and show anti-cancer activity in vitro and in vivo against various cancer cell lines. Pierisin-1 was most abundantly produced during the transition from the final larval stage to the pupal stage of the cabbage butterfly, and this production was regulated by ecdysteroid hormones. This suggests that pierisn-1 might play a pivotal role in the process of metamorphosis. Moreover, pierisin-1 could contribute as a defense factor against parasitization and microbial infections in the cabbage butterfly. Pierisin-like proteins in butterflies were shown to be present not only among the subtribe Pierina but also among the subtribes Aporiina and Appiadina, and pierisin-2, -3, and -4 were identified in these butterflies. Furthermore, DNA ADP-ribosylating activities were found in six different edible clams. Understanding of the biological nature of pierisin-1 with DNA mono-ADP-ribosylating activity could open up exciting avenues for research and potential therapeutic applications, making it a subject of great interest in the field of molecular biology and biotechnology. [ABSTRACT FROM AUTHOR]

Photoprotective properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH)2D3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH)2D3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200–400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH)2D3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH)2D3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH)2D3 in skin to reduce UV-induced DNA damage. [ABSTRACT FROM AUTHOR]

Background: Sirtuins (SIRTs) are NAD+-dependent deacetylases that play various roles in numerous pathophysiological processes, holding promise as therapeutic targets worthy of further investigation. Among them, the SIRT2 subtype is closely associated with tumorigenesis and malignancies. Dysregulation of SIRT2 activation can regulate the expression levels of related genes in cancer cells, leading to tumor occurrence and metastasis. Methods: In this study, we used computer simulations to screen for novel SIRT2 inhibitors from the FDA database, based on which 10 compounds with high docking scores and good interactions were selected for in vitro anti-pancreatic cancer metastasis testing and enzyme binding inhibition experiments. The results showed that fluvastatin sodium may possess inhibitory activity against SIRT2. Subsequently, fluvastatin sodium was subjected to molecular docking experiments with various SIRT isoforms, and the combined results from Western blotting experiments indicated its potential as a SIRT2 inhibitor. Next, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were performed, revealing the binding mode of fluvastatin sodium at the SIRT2 active site, further validating the stability and interaction of the ligand–protein complex under physiological conditions. Results: Overall, this study provides a systematic virtual screening workflow for the discovery of SIRT2 activity inhibitors, identifies the potential inhibitory effect of fluvastatin sodium as a lead compound on SIRT2, and opens up a new direction for developing highly active and selectively targeted SIRT2 inhibitors. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can fluorescence lifetime imaging microscopy (FLIM) detect associations between the metabolic state of cumulus cell (CC) samples and the clinical outcome of the corresponding embryos? SUMMARY ANSWER FLIM can detect significant variations in the metabolism of CC associated with the corresponding embryos that resulted in a clinical pregnancy versus those that did not. WHAT IS KNOWN ALREADY CC and oocyte metabolic cooperativity are known to be necessary for the acquisition of developmental competence. However, reliable CC biomarkers that reflect oocyte viability and embryo developmental competency have yet to be established. Quantitative measures of CC metabolism could be used to aid in the evaluation of oocyte and embryo quality in ART. STUDY DESIGN, SIZE, DURATION A prospective observational study was carried out. In total, 223 patients undergoing IVF with either conventional insemination or ICSI at a tertiary care center from February 2018 to May 2020 were included, with no exclusion criteria applied. PARTICIPANTS/MATERIALS, SETTING, METHODS This cohort had a mean maternal age of 36.5 ± 4.4 years and an average oocyte yield of 16.9 (range 1–50). One to four CC clusters from each patient were collected after oocyte retrieval and vitrified. CC metabolic state was assessed using FLIM to measure the autofluorescence of the molecules NAD(P)H and FAD+, which are essential for multiple metabolic pathways. CC clusters were tracked with their corresponding oocytes and associated embryos. Patient age, Day 3 and Day 5/6 embryo morphological grades, and clinical outcomes of embryos with traceable fate were recorded. Nine FLIM quantitative parameters were obtained for each CC cluster. We investigated associations between the FLIM parameters and patient maternal age, embryo morphological rank, ploidy, and clinical outcome, where false discovery rate P -values of <0.05 were considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE A total of 851 CC clusters from 851 cumulus–oocyte complexes from 223 patients were collected. Of these CC clusters, 623 were imaged using FLIM. None of the measured CC FLIM parameters were correlated with Day 3 morphological rank or ploidy of the corresponding embryos, but FAD+ FLIM parameters were significantly associated with morphological rank of blastocysts. There were significant differences for FAD+ FLIM parameters (FAD+ fraction engaged and short lifetime) from CC clusters linked with embryos resulting in a clinical pregnancy compared with those that did not, as well as for CC clusters associated with embryos that resulted in a live birth compared those that did not. LIMITATIONS, REASONS FOR CAUTION Our data are based on a relatively low number of traceable embryos from an older patient population. Additionally, we only assessed CCs from 1 to 4 oocytes from each patient. Future work in a younger patient population with a larger number of traceable embryos, as well as measuring the metabolic state of CCs from all oocytes from each patient, would provide a better understanding of the potential utility of this technology for oocyte/embryo selection. WIDER IMPLICATIONS OF THE FINDINGS Metabolic imaging via FLIM is able to detect CC metabolic associations with maternal age and detects variations in the metabolism of CCs associated with oocytes leading to embryos that result in a clinical pregnancy and a live birth versus those that do not. Our findings suggest that FLIM of CCs may be used as a new approach to aid in the assessment of oocyte and embryo developmental competence in clinical ART. STUDY FUNDING/COMPETING INTEREST(S) National Institutes of Health grant NIH R01HD092550-03 (to C.R. and D.J.N.). Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. and C.R. are inventors on patent US20170039415A1. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

OMT-28 is a metabolically robust small molecule developed to mimic the structure and function of omega-3 epoxyeicosanoids. However, it remained unknown to what extent OMT-28 also shares the cardioprotective and antiinflammatory properties of its natural counterparts. To address this question, we analyzed the ability of OMT-28 to ameliorate hypoxia/reoxygenation (HR)-injury and lipopolysaccharide (LPS)-induced endotoxemia in cultured cardiomyocytes. Moreover, we investigated the potential of OMT-28 to limit functional damage and inflammasome activation in isolated perfused mouse hearts subjected to ischemia/reperfusion (IR) injury. In the HR model, OMT-28 (1 μM) treatment largely preserved cell viability (about 75 versus 40% with the vehicle) and mitochondrial function as indicated by the maintenance of NAD+/NADH-, ADP/ATP-, and respiratory control ratios. Moreover, OMT-28 blocked the HR-induced production of mitochondrial reactive oxygen species. Pharmacological inhibition experiments suggested that Gai, PI3K, PPARa, and Sirt1 are essential components of the OMT-28-mediated pro-survival pathway. Counteracting inflammatory injury of cardiomyocytes, OMT-28 (1 mM) reduced LPS-induced increases in TNFa protein (by about 85% versus vehicle) and NF-kB DNA binding (by about 70% versus vehicle). In the ex vivo model, OMT-28 improved post-IR myocardial function recovery to reach about 40% of the baseline value compared to less than 20% with the vehicle. Furthermore, OMT-28 (1 μM) limited IR-induced NLRP3 inflammasome activation similarly to a direct NLRP3 inhibitor (MCC950). Overall, this study demonstrates that OMT-28 possesses potent cardio-protective and anti-inflammatory properties supporting the hypothesis that extending the bioavailability of omega-3 epoxyeicosanoids may improve their prospects as therapeutic agents. [ABSTRACT FROM AUTHOR]