Search Results (116)

Nicotinamide adenine dinucleotide (NAD⁺) serves as a critical cofactor in redox reactions and metabolic transformations catalyzed by NAD-dependent enzymes and is essential for bacterial survival and virulence. The biosynthesis of NAD⁺ in the cariogenic pathogen Streptococcus Sobrinus (S. sobrinus), a pivotal participant in oral cavities of children and adolescents with a history of caries, has yet to be explored. Bioinformatics, genetics, and biochemical techniques were used to identify NAD⁺ biosynthesis pathways and corresponding regulator in S. Sobrinus. S. sobrinus lacks de novo NAD⁺ synthesis pathway but comprises NA and Nam salvage pathway I (PncA-PncB-NadD-NadE) and PnuC-NadR salvage pathway III. NiaY and PnuC were involved in the salvage pathways. N-terminal domain of SsNrtR regulator was identified as DNA-binding domain binding to the pnuC and pncB probe, and addition of ADP-ribose reversed the binding of SsNrtR to the target promoters to regulate NAD⁺ salvage pathways. C-terminal domain of SsNrtR was non-catalytic, consistent with loss of Nudix motif conservation. Furthermore, the abrogation of niaR compromised multiple pathogenic traits, including cellular proliferation, acidogenesis, and the architecture/mechanical integrity of biofilms. Consequently, this mutant exhibited attenuated virulence in a rat caries model. Our findings conclusively demonstrate that SsNrtR-mediated regulation of NAD⁺ homeostasis is a critical determinant of the cariogenic potential of S. sobrinus. This study identifies SsNrtR as a previously uncharacterized NAD⁺-responsive regulator that integrates metabolic homeostasis with the control of virulence in Streptococcus sobrinus. These findings elucidate a novel metabolic–virulence regulatory axis in this species and position SsNrtR as a promising target for the development of anti-caries interventions. Full article

Inhibition of respiratory chain complex I (NADH dehydrogenase) is a widely encountered biochemical consequence of drug intoxication and a primary consequence of mtDNA mutations and other mitochondrial defects. In an organ-selective form, it is also deployed as antidiabetic pharmacological treatment. Complex I inhibition evokes a pronounced metabolic reprogramming of uncertain purposefulness, as in several cases, anabolism appears to be fostered in a state of bioenergetic shortage. A hallmark of complex I inhibition is the enhanced biosynthesis of serine, usually accompanied by an induction of folate-converting enzymes. Here, we have revisited the differential transcriptional induction of these metabolic pathways in three published models of selective complex I inhibition: MPP-treated neuronal cells, methionine-restricted rats, and patient fibroblasts harboring an NDUFS2 mutation. We find that in a coupled fashion, serinogenesis and circular folate cycling provide an unrecognized alternative pathway of complete glucose oxidation that is mostly dependent on NADP instead of the canonic NAD cofactor (NADP:NAD ≈ 2:1) and thus evades the shortage of oxidized NAD produced by complex I inhibition. In contrast, serine utilization for anabolic purposes and C₁-folate provision for S-adenosyl-methionine production and transsulfuration cannot explain the observed transcriptional patterns, while C₁-folate provision for purine biosynthesis did occur in some models, albeit not universally. We conclude that catabolic glucose oxidation to CO₂, linked with NADPH production for indirect downstream respiration through fatty acid cycling, is the general purpose of the remarkably strong induction of serinogenesis after complex I inhibition. Full article

Azotobacter vinelandii OP is a bacterium that can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HBV), a biodegradable and biocompatible polymer with applications in the biomedical field. This study aimed to evaluate P3HBV production and its 3-hydroxyvalerate (3HV) fraction under different agitation rates and oxygen uptake rates (q_O2) in chemostat cultures of A. vinelandii OP. Steady-state conditions with either oxygen or carbon limitation were established by modulating the agitation rates. Under oxygen-limited conditions (low q_O2 values) biomass and P3HBV concentrations increased to 3.3 g L⁻¹ and 2.1 g L⁻¹, respectively. At higher q_O2 values, the chemostat cultures were limited by carbon, and P3HBV content decreased from 62% to 33% (w w⁻¹). The highest 3HV molar fractions, 33.7 and 36.4 mol %, were observed at both the lowest and highest q_O2 levels, possibly linked to comparable valeric acid consumption rates. An elevated NAD(P)H/NAD(P)⁺ ratio was also observed under oxygen limitation, favoring polymer accumulation by indicating a more favorable intracellular redox state. These findings highlight the impact of nutrient limitation and respiratory activity on the biosynthesis of P3HBV and the 3HV composition by Azotobacter vinelandii OP. Such insights can support the development of tailored bioprocesses to modulate polymer characteristics, enabling a broader range of potential biomedical applications for P3HBV. Full article

Lactiplantibacillus plantarum is a widely studied probiotic species with significant strain-specific functional diversity, yet the molecular mechanisms underlying these variations remain largely unexplored. In this study, whole genome sequencing (WGS) and untargeted metabolomics were employed to comprehensively characterize the genetic architecture and extracellular metabolic profile of Lp. plantarum FRT4 (CGMCC 17955), a probiotic strain previously studied for its metabolic effects in animal models. WGS revealed a circular chromosome and five plasmids, encoding 3301 protein-coding genes enriched in amino acid biosynthesis, carbohydrate metabolism, and environmental response pathways. Carbohydrate-active enzymes (CAZy) annotation revealed 135 carbohydrate-active enzyme genes, dominated by glycoside hydrolases and glycosyl transferases. Untargeted metabolomic analysis comparing the fermentation supernatant of FRT4 with non-inoculated MRS medium revealed significant alterations in metabolite composition, including elevated levels of acetylcholine, nicotinamide adenine dinucleotide (NAD), and trans-3-coumarate, and reduced levels of uridine, inosine, and fructose-1-phosphate, indicating active modulation of neurotransmission, redox balance, and purine metabolism. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment showed significant regulation of pathways related to amino acid metabolism, carbon metabolism, and cofactor biosynthesis. These findings highlight the metabolic versatility and functional potential of FRT4, offering mechanistic insights into its probiotic effects and providing a basis for its potential application in fermentation-based formulations. Full article

Nicotinamide mononucleotide, known as NMN, is an important nicotinamide adenine dinucleotide (NAD⁺) precursor. It is integral in cellular metabolism, energy generation, and processes associated with aging. Since NMN provides healthy value, it becomes a major focus for the biotechnological industry. This study presents a new biosynthetic pathway for producing NMN without limits on intracellular PRPP (5′-phosphoribosyl pyrophosphate) metabolic flux. The route started by converting cytidine into 1-phosphoribose via pyrimidine-nucleoside phosphorylase (PyNP), after transforming into nicotinamide riboside (NR) through either purine-nucleoside phosphorylase (XapA) or nicotinate riboside kinase (NRK). NR was phosphorylated by NRK in the presence of nicotinamide (NAM) to produce NMN. We established an in vitro enzyme activity verification system for the feasibility check. The optimization of multienzyme cascade reactions was figured out for the NMN biosynthesis. Finally, the enzymes of PyNP and NRK were expressed in the cytidine-producing strain; we established a de novo biosynthesis pathway from glucose to NMN, achieving a production titer of 33.71 mg/L at a shake-flask scale. Full article

Isocitrate dehydrogenase (IDH) catalyzes the conversion of NAD(P)⁺ and isocitrate to NAD(P)H and α-ketoglutarate (αKG). The cytoplasmic enzyme IDH1 is important for producing NADPH for biosynthesis and for protecting against oxidative stress. IDH1 mutants, such as R132H found in glioblastomas and other types of human cancers, have a neomorphic activity that uses NADPH to reduce αKG to 2-hydroxyglutarate (2HG). 2HG interferes with the activity of important enzymes such as histone demethylases and TET demethylases. We hypothesized that Caenorhabditis elegans could be a good model system for studying oncogenic properties of mutant IDH1. To test this, we purified C. elegans cytoplasmic IDH-1 and two mutants, G98N and R133H, which correspond to human IDH1 mutants G97N and R132H, respectively. We found that the wild-type IDH-1 had similar kinetic properties to human IDH1, and it could produce small amounts of 2HG. We also found that the R133H mutant had a lower K_M for αKG than human R132H in steady-state enzyme kinetic experiments, and it produced almost exclusively 2HG in the presence of NADPH and αKG. The G98N mutant had a higher k_cat in the forward direction than the comparable human G97N mutant, and the G98N mutant produced a smaller amount of 2HG compared to the R133H mutant. These results suggest that C. elegans strains with IDH-1 mutations could be a good model system for studying the effects of 2HG in eukaryotic organisms. Full article

Background/Objectives: Pancreatic cancer (PC) is the seventh leading cause of cancer-related deaths worldwide, with its incidence rising each year. Despite its relatively low incidence, the aggressiveness of pancreatic cancer results in high mortality, with only 12% of patients surviving five years post-diagnosis. Surgical resection remains the only potentially curative treatment, but the tumor is often diagnosed at an advanced stage. The goal of this work is to identify vulnerabilities that can affect the efficacy of treatments and improve the efficacy of therapy. Methods: MAP17 overexpression in pancreatic cancer cell lines, RT-qPCR analysis, xenografts, in vitro and in vivo treatments, analysis of data from pancreatic tumors in transcriptomic patient databases. Results: We studied the prognostic and predictive value of MAP17 (PDZK1IP1) expression in pancreatic cancer, and we found that high MAP17 mRNA expression was associated with poor prognosis. In addition, single-cell analysis revealed that high MAP17 expression was present only in tumor cells. We investigated whether the response to various antitumor agents depended on MAP17 expression. In 2D culture, MAP17-expressing pancreatic cancer cells responded better to gemcitabine and 5-fluorouracil. However, in vivo xenograft tumors with MAP17 expression showed resistance to all treatments. Additionally, MAP17-expressing cells had a high NAD pool, which seems to be effectively depleted in vivo by NAMPT inhibitors, the primary enzyme for NAD biosynthesis. Conclusions: Our findings suggest that MAP17 expression could enhance the prognostic stratification of pancreatic cancer patients. Moreover, the coadministration of NAMPT inhibitors with current treatments may sensitize tumors with high MAP17 expression to chemotherapy and improve the efficacy of chemotherapy. Full article

Chilling injury can limit the productivity of tomato (Solanum lycopersicum L.), especially in over-wintering greenhouse. We here explored the effect of the pre-application of chlorogenic acid (CGA) in mitigating the impact of chilling on tomato. Flowering plants subjected to either chilling (15 °C/5 °C, day/night) or pre-treatment with CGA followed by chilling for 6 days and then by a two-day control recovery period were compared to plants maintained at control conditions (25 °C/18 °C, day/night). Chilling significantly affected the expression of PSII CP43 Chlorophyll Apoprotein, NAD (P) H-Quinone Oxidoreductase Subunit 5 and ATP Synthase CF1 Beta Subunit, reduced leaf Fv/Fm and increased malondialdehyde (MDA) levels, suggesting elevated oxidative stress. These correlated with reduced shoot biomass. All these aspects were mitigated by pretreatment with CGA. Transcriptomic and metabolomic co-analysis indicated that CGA also suppressed the shikimate pathway, phenylpropanoid biosynthesis and phenylalanine accumulation but enhanced cinnamic acid and indole acetate synthesis. Hence, the pre-chilling CGA protected the tomato plant from chilling injury by maintaining light energy utilization and reprograming secondary metabolism. This study describes the mechanism through which CGA pre-treatment can be used to maintain tomato productivity under chilling conditions. Full article

Background and Objectives: Obesity is linked to liver cancer through metabolic mechanisms and can promote tumor growth through metabolic impairment, decreased lipid metabolism, and interference of the energy balance in the liver. NAMPT is an enzyme expressed in the liver and is involved in the progression of tumors in obesogenic environments, while iNAMPT is known to be the rate-limiting enzyme in the synthesis of NAD, an essential coenzyme involved in ATP synthesis which promotes a pro-growth environment in the context of obesity. Because iNAMPT and cellular energetics, a hallmark of cancer, play an important role in liver cancer progression, it has become a target for cancer therapies focused on inhibiting its functions. The objective of this study was to determine the contribution of NAD biosynthesis in obesity-associated liver cancer progression. Methods: Cell culture studies were conducted with serum from male mice randomized to diet-induced obesity (OB) or control (CR) ± FK866 (iNAMPT inhibitor) in SNU, HepG2 human liver cancer cells, and Hepa 1-6 liver murine cells. Protein analysis of pAkt and pErk was performed via immunoblot. Cytotoxicity, reactive oxygen species (ROS), cell viability, and invasion were also measured in the cells. For the mouse model, the C57BL/6J male mice were randomized to the DIO or CR group. At 21 weeks of age, the mice were injected subcutaneously with Hepa 1-6 liver cancer cells. At 23 weeks, the mice received an I.P. injection of FK866 (30 mg/kg) for 2 weeks. The tumor and mouse weights were measured. Results: The cells exposed to OB sera showed increased proliferation, lactate dehydrogenase (LDH) secretion, ROS, and invasion. FK866 decreased proliferation, LDH secretion, ROS, and invasion for all liver cancer cells. The cells exposed to CR sera and OB + FK866 resulted in more LDH, suggesting increased apoptosis compared with OB sera. The OB sera increased phosphorylation of Akt, which was suppressed by FK866 compared with the OB group. In liver cancer cells, physiological and cellular signaling is affected differently when inhibiting NAD biosynthesis in an in vitro model of obesity and liver cancer. In vivo, the diet-induced obese (DIO) mice weighed significantly more than the mice fed a control diet. In addition, 70% of the DIO mice developed tumors, compared with 20% of the CR mice, and had tumors with greater volumes and weights. NAD inhibition blocked obesity-induced tumor growth. Conclusions: In this study, we demonstrate that inhibition of iNAMPT resulted in suppression of tumor growth in the context of obesity. Identifying pre-clinical strategies to reverse the impact of obesity on liver cancer progression is important due to the strong increased risk of liver cancer and its poor prognosis. Future translational research studies can be built from this pre-clinical foundational research. Full article

The Atacama Desert is emerging as an unexpected source of microbial life and, thus, a source of bioactive compounds and novel enzymes. Baeyer–Villiger monooxygenases (BVMOs), a subclass of flavin-dependent monooxygenases (FPMOs), have gained attention as promising biocatalysts for the biosynthesis of industrially relevant molecules for a wide range of applications, such as pharmaceuticals and polymers, among others. BVMOs catalyze the oxidation of ketones and cyclic ketones to esters and lactones, respectively, by using molecular oxygen and NAD(P)H. BVMOs may also catalyze heteroatoms oxidation including sulfoxidations and N-oxidations. This work aims to search for novel BVMOs in the genomes of new bacterial strains isolated from the Atacama Desert. Bioinformatic analysis led to the identification of 10 putative BVMOs, where the monooxygenase named MO-G35A was selected. Genome context showed, downstream of the MO-G35A, a gene encoding for an enzyme from the short-chain dehydrogenase/reductase family, suggesting a closer redox loop between both enzymes. MO-G35A was successfully expressed in three Escherichia coli expression systems, where higher yields were achieved using the E. coli Shuffle T7 as host, suggesting that correct disulfide bond formation is necessary for correct folding. Enzyme characterization showed that it operates optimally at 35–38 °C, exhibiting a Km of 0.06 mM and a kcat of 0.15 s⁻¹ for bicyclo [3.2.0] hept-2-en-6-one (BHC). Furthermore, the study revealed high stability in the presence of organic solvents, making it suitable for applications in various industrial processes, especially when the substrates have poor solubility in aqueous solutions. These results highlight the robustness and adaptability of enzymes in extreme environments, making them valuable candidates for biotechnological applications. Full article

Understanding the molecular regulation of citric acid accumulation in citrus fruits is crucial, as acidity directly influences fruit flavor, consumer preference, and commercial value. Citric acid is the predominant organic acid in citrus, and its levels are shaped by several factors, including genetic and developmental factors. ‘Jinyan’ Bingtang orange (Citrus sinensis cv. Jinyan) is a novel mutant derived from ‘Jinhong’ Bingtang orange (C. sinensis cv. Jinhong) that has a noticeably sour taste. However, the molecular basis of the increased citrate content in ‘Jinyan’ fruits remains unclear. This study compared the organic acid profiles and expression of citric acid metabolism-related genes between ‘Jinyan’ and ‘Jinhong’ fruit juice sacs throughout fruit development. The trend of citric acid content in both cultivars was similar; however, ‘Jinyan’ consistently presented significantly higher levels than ‘Jinhong’ did from 95 to 215 days after flowering (DAF). After 155 DAF, the transcript levels of citrate biosynthesis-related genes (PEPC1, PEPC2, PEPC3, CS1, and CS2) and citrate transport-related genes (V₁-E1, V₁-E2, V₀-a2, V₀-d, VHP1, VHP2, and CsPH8) were significantly greater in ‘Jinyan’ than in ‘Jinhong’. In contrast, citrate degradation-related genes (NAD-IDH2 and NAD-IDH3) were expressed at lower levels than in ‘Jinhong’. Notably, the expression patterns of V₁-E2 and CsPH8 closely matched the changes in citrate content in both cultivars. These results indicate that, compared with ‘Jinhong’, high citric acid accumulation in the juice sacs of ‘Jinyan’ fruit is likely due to increased citrate synthesis (via upregulated PEPCs and CSs) and increased vacuolar citrate sequestration (via upregulated proton pumps and transporters), coupled with reduced citrate degradation (lower NAD-IDH2/3). Full article

Background: Grain protein (GPC) and grain starch (GSC) content in common wheat determines suitability for further end-use processing and is an important quality factor. The level of free asparagine in grains (GFAC) significantly affects suitability for thermal processing. The aim of this genome-wide association study (GWAS) was to identify markers associated (MTA) with the levels of GPC, GSC and GFAC in elite winter wheat breeding lines, and to identify candidate genes. Methods: In total, 344 winter wheat lines were phenotyped and genotyped with DArTseq markers. Results: This GWAS revealed 14 MTAs for GPC, 40 for GSC and 43 for GFAC. The new markers were identified and explained from 6.3% to 12.2% of phenotypic variation. For GPC, the region adjacent to marker 4990459 (QGpc.rut.2D) explained 10.2% of the variation and was stable between two years. The novel gene TraesCS7A03G037500, encoding sucrose synthase involved in starch biosynthesis, was identified in the proximity of QGsc.rut.7A.2. The TraesCS1B03G0736700 gene, coding NAD(P)H dehydrogenase subunit H involved in the mitochondrial electron transport chain, was found in the proximity of QGfac.rut.1B.1. Conclusions: These findings provide valuable insights for elucidating inheritance of GCS, and the identified MTAs provide molecular markers for the reduction of free asparagine and increase of protein content in wheat grains. Full article

Heart failure represents the terminal stage in the development of many cardiovascular diseases, and its pathological mechanisms are closely related to disturbances in energy metabolism and mitochondrial dysfunction in cardiomyocytes. In recent years, nicotinamide adenine dinucleotide (NAD⁺), a core coenzyme involved in cellular energy metabolism and redox homeostasis, has been shown to potentially ameliorate heart failure through the regulation of mitochondrial function. This review systematically investigates four core mechanisms of mitochondrial dysfunction in heart failure: imbalance of mitochondrial dynamics, excessive accumulation of reactive oxygen species (ROS) leading to oxidative stress injury, dysfunction of mitochondrial autophagy, and disturbance of Ca²⁺ homeostasis. These abnormalities collectively exacerbate the progression of heart failure by disrupting ATP production and inducing apoptosis and myocardial fibrosis. NAD⁺ has been shown to regulate mitochondrial biosynthesis and antioxidant defences through the activation of the deacetylase family (e.g., silent information regulator 2 homolog 1 (SIRT1) and SIRT3) and to increase mitochondrial autophagy to remove damaged mitochondria, thus restoring energy metabolism and redox balance in cardiomyocytes. In addition, the inhibition of NAD⁺-degrading enzymes (e.g., poly ADP-ribose polymerase (PARP), cluster of differentiation 38 (CD38), and selective androgen receptor modulators (SARMs)) increases the tissue intracellular NAD⁺ content, and supplementation with NAD⁺ precursors (e.g., β-nicotinamide mononucleotide (NMN), nicotinamide riboside, etc.) also significantly elevates myocardial NAD⁺ levels to ameliorate heart failure. This study provides a theoretical basis for understanding the central role of NAD⁺ in mitochondrial homeostasis and for the development of targeted therapies for heart failure. Full article

Background/Objectives: Athletic performance matters for athletes and fitness enthusiasts. Scientific dietary intervention may boost athletic performance alongside training. Intermittent fasting, like time-restricted fasting (TF), may enhance metabolic health. NAD⁺ supplement nicotinamide mononucleotide (NMN) improves mitochondrial activity. Both potentially boost athletic performance. However, whether TF combined with NMN treatment can further enhance athletic ability is unclear. Methods: Healthy Kunming mice were utilized to test the effects of NMN and TF on the athletic performance of mice. To simulate the in vivo state and further verify the role of TF and NMN, low glucose combined with NMN was used to intervene in C2C12 cells. The exercise capacity of mice was evaluated through motor behavior experiments. At the same time, blood gas analysis and kit tests were used to assess oxygen uptake capacity and post-exercise oxidative stress levels. Muscle development and mitochondrial function were examined through gene expression, protein analysis, and enzyme activity tests, and the distribution of intestinal microbiota and short-chain fatty acid content were also analyzed. Results: The results show that TF combined with NMN improved mitochondrial dynamics and biosynthesis, mitochondrial respiratory function, and oxidative metabolism. Then, the intervention enhanced mice’s endurance, limb strength, motor coordination, and balance and reduced oxidative damage after exercise. Moreover, TF combined with NMN significantly increased the gut microbiota diversity and upregulated Ruminococcus, Roseburia, and Akkermansia in intestinal bacteria and short-chain fatty acids, which are associated with athletic performance. Conclusion: TF combined with NMN enhanced mitochondrial function, improved energy metabolism, modulated the gut microbiota and short-chain fatty acids, and affected muscle fiber transformation, ultimately leading to an overall improvement in exercise performance. These findings provide a theoretical framework for expanding the application of NMN and TF in kinesiology. Full article

Exercise is one of the main challenges to the body’s homeostasis since it needs an immediate, substantial rise in ATP re-synthesis, which leads to the prevention of response capacity and performance of players. Therefore, it is vital to monitor sweat metabolites in soccer players during vigorous exercise to comprehend their functional variations. This flagged the requirement metabonomic approaches for the determination of the distinct metabolic pathways and signature metabolites that are involved in soccer players pre- and post-exercise. In this study, metabolomics and chemometrics approaches were integrated to accelerate and unravel signature-altered metabolites involved pre- and post-exercise. Metabolites profiling revealed a total of 57 signatures and the identified signature altered metabolites belonging to carboxylic acids, ketone, alcohols, aldehydes, aromatics, alkenes, hexoses, hydroxy fatty acids, tetracyclic N-heterocycles, aldopentose, benzenes, alkanes, phenols, and heterocyclic. Niacin is the most downregulated and abundant pre-induced exercise, which can employ its effects through energy metabolism as a precursor for nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Significant alterations were also specifically observed in the Alanine, aspartate and glutamate, Valine, leucine and isoleucine, Pantothenate and CoA biosynthesis, and Galactose metabolisms following exercise. Full article