Coenzyme Nicotinamide Adenine Dinucleotide Research Articles

Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice

HIV-1-associated immune activation drives CD4+ T cell depletion and the development of acquired immunodeficiency syndrome. We aimed to determine the role of nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD) co-enzyme, in CD4+ T cell modulation during HIV-1 infection. We examined HIV-1 integrated DNA or transcribed RNA, intracellular p24 protein, and T cell activation markers in CD4+ T cells including invitro HIV-1-infected cells, reactivated patient-derived cells, and in HIV-1-infected humanized mice, under NMN treatment. RNA-seq and CyTOF analyses were used for investigating the effect of NMN on CD4+ T cells. We found that NMN increased the intracellular NAD amount, resulting in suppressed HIV-1 p24 production and proliferation in infected CD4+ T cells, especially in activated CD25+CD4+ T cells. NMN also inhibited CD25 expression on reactivated resting CD4+ T cells derived from cART-treated people living with HIV-1 (PLWH). In HIV-1-infected humanized mice, the frequency of CD4+ T cells was reconstituted significantly by combined cART and NMN treatment as compared with cART or NMN alone, which correlated with suppressed hyperactivation of CD4+ T cells. Our results highlight the suppressive role of NMN in CD4+ T cell activation during HIV-1 infection. It warrants future clinical investigation of NMN as a potential treatment in combination with cART in PLWH. This work was supported by the Hong Kong Research Grants Council Theme-Based Research Scheme (T11-706/18-N), University Research Committee of The University of Hong Kong, the Collaborative Research with GeneHarbor (Hong Kong) Biotechnologies Limited and National Key R&D Program of China (Grant2021YFC2301900).

Coenzyme-binding pathway on glutamate dehydrogenase suggested from multiple-binding sites visualized by cryo-electron microscopy.

The structure of hexameric glutamate dehydrogenase (GDH) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) was visualized using cryogenic transmission electron microscopy to investigate the ligand-binding pathways to the active site of the enzyme. Each subunit of GDH comprises one hexamer-forming core domain and one nucleotide-binding domain (NAD domain), which spontaneously opens and closes the active-site cleft situated between the two domains. In the presence of NADP, the potential map of GDH hexamer, assuming D3 symmetry, was determined at a resolution of 2.4 Å, but the NAD domain was blurred due to the conformational variety. After focused classification with respect to the NAD domain, the potential maps interpreted as NADP molecules appeared at five different sites in the active-site cleft. The subunits associated with NADP molecules were close to one of the four metastable conformations in the unliganded state. Three of the five binding sites suggested a pathway of NADP molecules to approach the active-site cleft for initiating the enzymatic reaction. The other two binding modes may rarely appear in the presence of glutamate, as demonstrated by the reaction kinetics. Based on the visualized structures and the results from the enzymatic kinetics, we discussed the binding modes of NADP to GDH in the absence and presence of glutamate.

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection.

Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the infested human or animal in the long term and, thus, impact agricultural and socio-economic outcomes. However, parasite-driven immune-regulatory effects have been described, with potential therapeutic relevance for autoimmune diseases. While the metabolism in both the host and parasites contributes to their defense and is the basis for nematode survival in the intestine, it has remained largely understudied due to a lack of adequate technologies. We have developed and applied NAD(P)H fluorescence lifetime imaging to explanted murine intestinal tissue during infection with the natural nematode Heligmosomoides polygyrus to study the metabolic processes in both the host and parasites in a spatially resolved manner. The exploitation of the fluorescence lifetime of the co-enzymes nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, which are preserved across species, depends on their binding status and the binding site on the enzymes catalyzing metabolic processes. Focusing on the most abundantly expressed NAD(P)H-dependent enzymes, the metabolic pathways associated with anaerobic glycolysis, oxidative phosphorylation/aerobic glycolysis, and NOX-based oxidative burst, as a major defense mechanism, were distinguished, and the metabolic crosstalk between the host and parasite during infection was characterized.

Light‐Driven NADPH Cofactor Recycling by Photosystem I for Biocatalytic Reactions

AbstractBiocatalytic asymmetric reduction of C=C and C=O bonds is highly attractive to produce valuable (chiral) chemicals for the fine and pharmaceutical industry, yet occurs at the expense of reduced nicotinamide adenine dinucleotide coenzyme NADPH that requires recycling. Established methods each have their challenges. Here we developed a light‐driven approach based on photosystem I (PSI) by mimicking the natural electron transfer from PSI via ferredoxin (Fd) towards ferredoxin NADP+ reductase (FNR) in vitro. Illumination with red light led to reduction of NADP+ to NADPH with a turnover frequency of 2.55 s−1 (>9000 h−1) at pH 7.5. Light‐driven NADPH regeneration by PSI‐Fd‐FNR was coupled with three oxidoreductases for asymmetric reduction of C=C and C=O bonds, reaching up to 99 % conversion with a turnover number of 3035, and retaining enantioselectivity. This study demonstrates the capacity of a PSI system to drive continuous NADPH‐dependent biocatalytic conversions with light.

Enhanced Production of β-Nicotinamide Mononucleotide with Exogenous Nicotinamide Addition in Saccharomyces boulardii-YS01.

β-Nicotinamide mononucleotide (NMN), as a key precursor of an essential coenzyme nicotinamide adenine dinucleotide (NAD+), is most recognized for its pathological treatment effects and anti-aging functions. Here, the biosynthesis of NMN from the inexpensive feedstock substrate nicotinamide (Nam) using previously isolated Saccharomyces boulardii-YS01 was investigated. Ultra-high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UPLC-ESI-QqQ-MS/MS) was established for the determination and targeted analysis of NMN, nicotinamide riboside (NR), nicotinic acid (NA), Nam, and NAD+ in YS01 cells. Satisfactory precision and accuracy values were achieved with recoveries above 70% for five analytes. A 5~100 times higher content of NMN in YS01 (0.24~103.40 mg/kg) than in some common foods (0.0~18.8 mg/kg) was found. Combined with genome sequencing and enzyme function annotation, target-acting enzymes, including nudC, ISN1, URH1, PNP, and SIR2, were identified, and the biosynthetic pathway of NMN via Nam was suggested. The initial addition of 3 g/L Nam in the culture medium effectively promoted the generation of NMN, which raised the content of NMN by 39%. This work supplements an alternative resource for NMN production and lays the theoretical foundation for the further construction of NMN transgenic synthesis hosts.

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array.

Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a to acquisition speed advantage for the light sheet compared to the laser scanning geometry. FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella.

Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD+ ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (FdhSNO ) from the soil bacterium Starkeya novella strictly specific for NAD+ . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of FdhSNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+ ) with better catalytic efficiency than NAD+ . The single mutation D221Q enabled the reduction of NADP+ with a catalytic efficiency kCAT /KM of 0.4 s-1 ·mm-1 at 200 mm formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a fivefold increase in catalytic efficiency for NADP+ compared with the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP+ . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of FdhSNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds.

Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase.

Methylenetetrahydrofolate reductase (MTHFR) is a key metabolic enzyme in colonization and virulence of Neisseria meningitidis, a causative agent of meningococcal diseases. Here, the biochemical and structural properties of MTHFR from a virulent strain of N. meningitidis serogroup B (NmMTHFR) were characterized. Unlike other orthologs, NmMTHFR functions as a unique homohexamer, composed of three homo-dimerization partners, as shown in our 2.7 Å resolution crystal structure. Six active sites were formed solely within monomers and located away from the oligomerization interfaces. Flavin adenine dinucleotide cofactor formed hydrogen bonds with conserved sidechains, positioning its isoalloxazine ring adjacent to the overlapping binding sites of nicotinamide adenine dinucleotide (NADH) coenzyme and CH2 -H4 folate substrate. NmMTHFR utilized NADH (Km = 44 μM) as an electron donor in the NAD(P)H-CH2 -H4 folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. In silico analysis and mutagenesis studies highlighted the significant difference in orientation of helix α7A (Phe215-Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix α7A plays a role in stabilizing the folded structure of NADH in the hydrophobic box. This supports the NADH specificity by restricting the phosphate group of NADPH that causes steric clashes with Glu26. The movement of Met221 sidechain allows the CH2 -H4 folate substrate to bind. The unique topology of its NADH and CH2 -H4 folate binding pockets makes NmMTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off-target side effects.

Ex Vivo Transdermal Delivery of Nicotinamide Mononucleotide Using Polyvinyl Alcohol Microneedles.

Nicotinamide mononucleotide (NMN), which has recently been spotlighted as an anti-aging agent, is a precursor of the coenzyme nicotinamide adenine dinucleotide that plays an important role in intracellular redox reactions. NMN capsules for oral administration currently on the market have a problem in that they are almost fully metabolized in the stomach and liver and excreted as nicotinamide. Therefore, there is a need to develop a patient-friendly delivery method that can improve the bioavailability of NMN. For this purpose, various polyvinyl alcohol (PVA)-based microneedle patches were fabricated to develop a transdermal delivery system for NMN. First, the molecular weight effect of PVA on the shape and microstructure of microneedles was studied. After selecting the optimal molecular weight PVA, the swelling of the microneedles and the ex vivo release of NMN were studied. The effect of carboxymethyl cellulose (CMC) and dimethyl sulfoxide on NMN release was also investigated. The highest NMN release of 91.94% in 18 h was obtained using a 9.5 kDa molecular weight PVA microneedle containing NMN and CMC.

Paralogous FgIDO genes with differential roles in tryptophan catabolism, fungal development and virulence in Fusarium graminearum

Indoleamine 2,3-dioxygenase (Ido) is a tryptophan-degrading enzyme that is widely distributed across species. Ido catalyzes the first step of tryptophan (TRP) degradation and drives the de novo synthesis of nicotinamide adenine dinucleotide (NAD+) coenzymes via the kynurenine (KYN) pathway. The budding yeast Saccharomyces cerevisiae possesses a single IDO gene (BNA2) that is responsible for NAD+ synthesis, whereas a number of fungal species contain multiple IDO genes. However, the biological roles of IDO paralogs in plant pathogens remain unclear. In the current study, we identified three FgIDOs from the wheat head blight fungus Fusarium graminearum. FgIDOA/B/C expression was significantly induced upon TRP treatment. Targeted disruption of FgIDOA and/or FgIDOB caused different levels of NAD+ auxotrophy, thus resulting in pleotropic phenotypic defects. Loss of FgIDOA resulted in abnormal conidial morphology, reduced mycelial growth, decreased virulence in wheat heads and reduced deoxynivalenol accumulation. Exogenous addition of KYN or various intermediates involved in the KYN pathway rescued auxotrophy of the mutants. Metabolomics analysis revealed shifts toward alternative TRP degradation pathways to melatonin and indole derivatives in mutants lacking FgIDOB. Upregulation of partner genes in auxotrophic mutants and the capacity to rescue the auxotroph by overexpressing a partner gene indicated functional complementation among FgIDOA/B/C. Taken together, the results of this study provide insights into differential roles in paralogous FgIDOs and how fungal TRP catabolism modulates fungal development and virulence.

Single cell metabolic imaging of tumor and immune cells in vivo in melanoma bearing mice.

Metabolic reprogramming of cancer and immune cells occurs during tumorigenesis and has a significant impact on cancer progression. Unfortunately, current techniques to measure tumor and immune cell metabolism require sample destruction and/or cell isolations that remove the spatial context. Two-photon fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent metabolic coenzymes nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) provides in vivo images of cell metabolism at a single cell level. Here, we report an immunocompetent mCherry reporter mouse model for immune cells that express CD4 either during differentiation or CD4 and/or CD8 in their mature state and perform in vivo imaging of immune and cancer cells within a syngeneic B78 melanoma model. We also report an algorithm for single cell segmentation of mCherry-expressing immune cells within in vivo images. We found that immune cells within B78 tumors exhibited decreased FAD mean lifetime and an increased proportion of bound FAD compared to immune cells within spleens. Tumor infiltrating immune cell size also increased compared to immune cells from spleens. These changes are consistent with a shift towards increased activation and proliferation in tumor infiltrating immune cells compared to immune cells from spleens. Tumor infiltrating immune cells exhibited increased FAD mean lifetime and increased protein-bound FAD lifetime compared to B78 tumor cells within the same tumor. Single cell metabolic heterogeneity was observed in both immune and tumor cells in vivo. This approach can be used to monitor single cell metabolic heterogeneity in tumor cells and immune cells to study promising treatments for cancer in the native in vivo context.

Flavin Monooxygenases: A Multifaceted Class of Enzymes with Abundant Biomedical Applications

Flavin-dependent monooxygenases (FMOs) catalyze oxidation of a variety of organic molecules many of which have a profound biomedical applications. Oxidation occurs through the incorporation of a single atom of dioxygen into the substrate while the other oxygen atom is reduced to water. Since 2014, FMOs are grouped into eight types listed as Groups A through H. Grouping is based on structural homology, flavin type and the strategy taken to reduce the isoalloxazine ring of the cofactor for subsequent oxygen activation and substrate monooxygenation. Groups A and B FMOs are single component enzymes that carry out their entire reaction cycle in a single protein core using a nicotinamide adenine dinucleotide coenzyme as electron source. Groups C through F are two comments FMOs. These systems contain a reductase to reduce the flavin cofactor that is then delivered to a monooxygenase component as a substrate. Reduced flavin then activates molecular oxygen in the second component to oxidize the organic metabolite of the system. Finally, Groups G and H are self- sufficient internal monooxygenases that receive electrons from one substrate for the oxidation of a second one all within a single protein unit. FMOs carry out an array of biomedically relevant reactions including the degradation of antibiotics by pathogens, the biocatalytic production of drugs in pharmaceutical industries and cholesterol anabolism in humans and fungi. Given this diversity of both reaction type and application, the field of FMO enzymology can rapidly become overwhelming. This review provides a general overview of each FMO group and is intended to guide researchers just entering the field. Canonical enzymes are detailed along with biomedically important members of each group. It is hoped the references herein will be provide a quick source to make more extensive literature reviews more accessible.

Photoelectrochemical NADH regeneration on a polymer semiconductor-based photocathode

Semiconductor pTTh shows an unprecedentedly positive onset potential of 0.6 V vs. RHE for achieving an enzymatically active product of 1,4-NADH.

Enabling Characterisation of the NAD+ Biosynthesis Pathway via in vitro CEST MRI

AbstractBackgroundThe Chemical Exchange Saturation Transfer (CEST) MRI properties of six metabolites required for synthesis of coenzyme nicotinamide adenine dinucleotide (NAD+) and to evaluate the potential for clinical imaging of altered NAD+ regulation in neurodegenerative disordersMethodCEST images of metabolites in the NAD+ biosynthesis pathway were acquired at physiological temperature with high‐field MRI (9.4T) running a modified magnetisation transfer (MTR) prepared RARE sequence over a range of saturation powers and frequency ofsets, for pH values 5.5‐7.4. CEST data were fitted using spline interpolation, the exchange rate was calculated, and frequency specific MTR maps were generated using pixelwise analysis.ResultTryptophan, nicotinamide, nicotinic acid (NA), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) and NADH were evaluated. At pH 7.4, all molecules exhibited CEST contrast between 0.1 and 0.9 ppm, originating from the OH hydroxyl proton. NADH exhibited up to 15% CEST contrast in a B1 field of 2.4µT from its fast‐exchanging amine groups at 2 and 3 ppm, and showed a positive dependence of CEST efciency on pH. Tryptophan exhibited three independent peaks at 0.7, 2.6, and 5.4 ppm corresponding to pH 6.7, 5.5 and 7.4. NMN showed high CEST contrast at 0.7ppm originating from its phosphorous OH enabling higher CEST efciency due to enhanced charge delocalisation (Fig.1).ConclusionThis in vitro feasibility study demonstrates signal detection and characterization of NAD+ biosynthesis pathway precursor molecules over precise temperature, concentration and physiological pH ranges. All molecules exhibited a peak at 0.7 from the hydroxyl group with NMN showing highest CEST efficiency due to phosphate charge delocalisation. NADH showed a peak at 2 and 3ppm from amine group protons and a dependency on temperature and pH.

ODP443 Targeting NAD+ availability modulates 11β- Hydroxysteroid dehydrogenase 1 activity in mouse and human cell lines and primary cells

Abstract Four nicotinamide adenine dinucleotide coenzymes – NAD+, NADH, NADP+, and NADPH – are the central catalysts of metabolism. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is NADPH-dependent reductase that catalyses the reduction of the inactive glucocorticoid cortisone) to active cortisol (11-dehydrocorticosterone to corticosterone in rodents). 11β-HSD1 is in the endoplasmic reticulum (ER) lumen, and the level of NADPH in the ER regulates enzyme activity. Little is known about the biochemical mechanisms controlling cytosolic/ER NAD(P)(H) crosstalk. Here we examined how perturbed cellular NAD+ availability modulates 11β-HSD1 enzyme activity in the ER across a range of mouse and human cell types. 11β-HSD1 activity was measured in a series of transformed and primary mouse and human liver (HepG2), muscle, and Human Dermal Fibroblast cells (HDFn) depleted for NAD+ using FK866 to inhibit nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. To replete NAD+ we supplemented cells with 0.5mM of NAD+ precursors nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). FK866 (100nM, 48h) was effective at depleting NAD+ by approximately 90% in C2C12 myotubes and 50% in HepG2 liver cells. In each cell line, 11β-HSD1 reductase activity was decreased by FK866 compared to untreated controls (80% in C2C12 P<0. 0001, 55% in HepG2, P=0. 03 and 65% in HDFn P=0. 002). Restoring NAD+ level with NR (0.5mM, 6h) in C2C12 and (0.5mM, 24h) in HepG2 and HDFn rescued 11β-HSD1 enzyme activity compared with no precursor treatment (60% in C2C12 P=0. 0007, 52% in HepG2 P=0. 03 and 68% in HDFn P=0. 0006). Human dermal fibroblasts treated with TNFa (10nM, 24h) boosted 11β-HSD1 expression 4-fold, however this was not able to overcome the FK866 induced drop-in reductase activity. These data show that depletion of NAD+ and impaired 11β-HSD1 reductase activity in the ER is a generalised phenomenon across cell types. This suggests a crosstalk between cytosolic and ER NAD(P)(H) pools that can respond rapidly to changes in NAD+ precursor availability. This could provide new insight into the regulation of cellular glucocorticoid metabolism and ER NAD(P)(H) homeostasis. Presentation: No date and time listed

Niacin Status Indicators and Their Relationship with Metabolic Parameters in Dairy Cows during Early Lactation.

Simple SummaryThe active forms of niacin that represent niacin status are nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP) and the NAD:NADP ratio. Previous studies have shown metabolic changes in the function of niacin form and dose, but it has not been determined whether there are changes in the function of active form of niacin that indicate the vitamin status in the body. In this study, we examined differences in NAD, NADP and NAD:NADP concentration in blood and their relationship with metabolic parameters in cows receiving and not receiving additional niacin in food. We concluded that NAD and NADP are good indicators of the ability of an additional niacin source to create functional cofactors due to their concentration changes, while the NAD:NADP ratio is a good indicator of the biological effects of additional niacin due to correlation with many metabolites.Previous experimental models on cows have examined the difference in the metabolic adaptation in cows after niacin administration, without identifying the most important mediators between niacin administration and its biological effects, namely active forms of niacin. All tissues in the body convert absorbed niacin into its main metabolically active form, the coenzyme nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The aim of this study was to determine the influence of niacin administration in periparturient period on NAD, NADP and the NAD:NADP ratio and to determine relationship between these indicators of an active form of niacin with metabolic parameters in cow blood. The study included 90 healthy cows: 45 cows receiving niacin and 45 cows were negative control. The niacin group was treated with nicotinic acid for two weeks before, as well as two weeks after parturition. Nicotinic acid was applied per os with feed. In cows receiving niacin, there was a significantly higher concentration of NAD and NADP, but the NAD:NADP ratio did not differ compared with control. All three indicators were able to separate cows who received and who did not receive additional niacin. NAD and NADP are good indicators of the availability of niacin from additional sources. The NAD:NADP ratio is a good indicator of the biological effect of applied niacin on metabolites in cows due to its correlation with a number of metabolites: positive correlation with glucose, insulin, glucose to insulin ratio and the revised quantitative insulin sensitivity check index (RQUICKIBHB) of insulin resistance, triglycerides and cholesterol, and a negative correlation with nonesterified fatty acid (NEFA), beta hydroxybutyrate (BHB), gamma-glutamyltranspherase (GGT) and urea in cows receiving niacin. The same amount of added niacin in feed can produce different concentrations of NAD, NADP and NAD:NADP in the blood, and this was not related to their concentration before the addition of niacin. The change in the concentration of the active form of niacin (NAD, NADP and NAD:NADP) further correlates with the concentration of metabolic parameters, which indicates that the intensity of the biological effect of additional niacin can be accurately determined only if we know the concentrations of its active forms in blood. Under basal conditions (without additional niacin), active forms of niacin that already exist in the blood do not show significant correlations with metabolic parameters.

Development of Biosensors for Ethanol Gas Detection

This work developed a biosensor for the measurement of ethanol gas in the air. The biosensors were synthesized by mixing signal layer materials containing SiO2 and polyimide (PI) substrates using the enzyme Alcohol Dehydrogenase (ADH) and coenzyme Nicotinamide Adenine Dinucleotide (NAD+) as a biosensor. The electrodes were coated on biosensors by DC magnetron sputtering method for test the response performance of the developed biosensors. The ADH/NAD+ was immobilized on the Ag electrode by Glutaric dialaehyde 25 wt. % cross-linking procedure. It was found that, alcohol biosensors can be exhibited sensing ethanol gas at even low concentrations from 300 ppb to very high concentrations up to 1900 ppm, response time 3 s, recovery times 1-2 minutes and good sensitivity. The SiO2 substrate has excellent, which provides significant advantages for wearable electronic device that compact, easy to use and reduce direct contact with alcoholics. The alcohol biosensors can adoption in next generation to other electronic devices, because easy to integrate, such as a module alcohol biosensor with wireless or the fabrication of the RCL circuit. Furthermore, the alcohol biosensors based on SiO2/Ag/ADH, PI/Ag/ADH is artificial intelligence strategy for stable practical wearable electronic devices.

How Photoactivation Triggers Protochlorophyllide Reduction: Computational Evidence of a Stepwise Hydride Transfer during Chlorophyll Biosynthesis.

The photochemical reaction catalyzed by enzyme protochlorophyllide oxidoreductase (POR), a rare example of a photoactivated enzyme, is a crucial step during chlorophyll biosynthesis and involves the fastest known biological hydride transfer. Structures of the enzyme with bound substrate protochlorophyllide (PChlide) and coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) have recently been published, opening up the possibility of using computational approaches to provide a comprehensive understanding of the excited state chemistry. Herein, we propose a complete mechanism for the photochemistry between PChlide and NADPH based on density functional theory (DFT) and time-dependent DFT calculations that is consistent with recent experimental data. In this multi-step mechanism, photoexcitation of PChlide leads to electron transfer from NADPH to PChlide, which in turn facilitates hydrogen atom transfer by weakening the breaking C–H bond. This work rationalizes how photoexcitation facilitates hydride transfer in POR and has more general implications for biological hydride transfer reactions.

Alpha-synuclein/MPP+ mediated activation of NLRP3 inflammasome through microtubule-driven mitochondrial perinuclear transport

1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can activate nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) inflammasome in Parkinson's disease (PD) mice, while 1-methyl-4-phenyl- 1, 2, 3, 6-tetrahydropyridinium ion (MPP+), the toxic metabolite of MPTP was not enough to achieve it in vitro. We hypothesized that the accumulation of Alpha-synuclein (α-syn) caused by MPP+ can be a priming signal of MPP+ mediated NLRP3 activation, and its mechanism was explored. This study demonstrated the α-syn can mediate NLRP3 priming in BV2 cells. It can also act on ERK-p67phox-nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) axis and induce mitochondrial damage. The co-treatment of α-syn/MPP+ can cause aberrant mitochondrial homeostasis to diminish the concentration of the coenzyme nicotinamide adenine dinucleotide (NAD+), mediate accumulation of ac-α-tubulin, and induce mitochondrial perinuclear aggregation, navigating the co-localization of NLRP3 and apoptosis-associated speck-like protein containing a CARD domain (ASC). This study suggested that α-syn/MPP+ mediated NLRP3 inflammasome activation through microtubule-driven mitochondrial perinuclear transport.

Indirect Export of Reducing Equivalents From the Chloroplast to Resupply NADP for C3 Photosynthesis-Growing Importance for Stromal NAD(H)?

Plant productivity greatly relies on a flawless concerted function of the two photosystems (PS) in the chloroplast thylakoid membrane. While damage to PSII can be rapidly resolved, PSI repair is complex and time-consuming. A major threat to PSI integrity is acceptor side limitation e.g., through a lack of stromal NADP ready to accept electrons from PSI. This situation can occur when oscillations in growth light and temperature result in a drop of CO2 fixation and concomitant NADPH consumption. Plants have evolved a plethora of pathways at the thylakoid membrane but also in the chloroplast stroma to avoid acceptor side limitation. For instance, reduced ferredoxin can be recycled in cyclic electron flow or reducing equivalents can be indirectly exported from the organelle via the malate valve, a coordinated effort of stromal malate dehydrogenases and envelope membrane transporters. For a long time, the NADP(H) was assumed to be the only nicotinamide adenine dinucleotide coenzyme to participate in diurnal chloroplast metabolism and the export of reductants via this route. However, over the last years several independent studies have indicated an underappreciated role for NAD(H) in illuminated leaf plastids. In part, it explains the existence of the light-independent NAD-specific malate dehydrogenase in the stroma. We review the history of the malate valve and discuss the potential role of stromal NAD(H) for the plant survival under adverse growth conditions as well as the option to utilize the stromal NAD(H) pool to mitigate PSI damage.