Search Results (110)

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

Scleroglucan is the extracellular polysaccharide (EPS) produced by Sclerotium rolfsii (S. rolfsii). The low EPS titer and limited substrate utilization of S. rolfsii present significant challenges in the fermentation process, restricting industrial applications of scleroglucan. In this study, we performed a transcriptomic analysis on the mycelium of S. rolfsii fermented with different carbon sources. The key genes involved in polysaccharide biosynthesis (6-phosphofructokinase 1 (PFK1), pyruvate decarboxylase (PDC), aldehyde dehydrogenase (NAD (P)+) (ALDH3), and acetyl-CoA synthase (ACS)) were identified and their roles in the process were investigated. The supplementation of specific precursors—fructose-6-phosphate, pyruvate, aldehydes, and acetate—was shown to enhance both the polysaccharide titer and substrate utilization. By adding precursors, the titer of SEPS produced in a 5 L fermentation tank reached 48.69 ± 3.8 g/L. Notably, the addition of these precursors increased the titer of EPS fermented with sucrose (SEPS) by 65.63% and substrate utilization by 119.3%, while the titer of EPS fermented with lactose (LEPS) rose by 80.29% and substrate utilization rose by 47.08%. These findings suggest that precursor supplementation can effectively improve polysaccharide production and substrate efficiency, thereby minimizing resource waste and environmental impact. Full article

Cyst nematodes remain a major threat to global agricultural production, causing huge losses. To understand the parasitism of the cyst nematodes Heterodera trifolii (HT) and Heterodera schachtii (HS), we constructed whole-genome assemblies using short- and long-read sequencing technologies. The nematode genomes were 379 Mb and 183 Mb in size, with the integrated gene models predicting 40,186 and 18,227 genes in HT and HS, respectively. We found more than half of the genes predicted in HT (64.7%) and HS (53.2%) were collinear to their nearest neighbor H. glycines (HG). Large-scale duplication patterns in HT and segmental duplications of more than half of the orthologous genes indicate that the genome of HT is polyploid in nature. Functional analysis of the genes indicated that 65.6% of the HG genes existed within the HT genome. Most abundant genes in HT and HS were involved in gene regulation, DNA integration, and chemotaxis. Differentially expressed genes showed upregulation of cuticle structural constituent genes during egg and female stages and cytoskeletal motor activity-related genes in juvenile stage 2 (J2). Horizontal gene transfer analyses identified four new vitamin biosynthesis genes, pdxK, pdxH, pdxS, and fabG, of bacterial origin, to be first reported in HT and HS. Mitogenomes of HT, HS, and HG showed similar structure, composition, and codon usage. However, rates of substitution of bases in the gene nad4l were significantly different between HT and HS. The described genomes, transcriptomes, and mitogenomes of plant-parasitic nematodes HT and HS are potential bio-resources used to identify several strategies of control of the nematode. Full article

Background/Objectives: Cancer cells rely on metabolic reprogramming that is supported by altered mitochondrial redox status and an increased demand for NAD⁺. Over expression of Nampt, the rate-limiting enzyme of the NAD⁺ biosynthesis salvage pathway, is common in breast cancer cells, and more so in triple negative breast cancer (TNBC) cells. Targeting the salvage pathway has been pursued for cancer therapy. However, TNBC cells have heterogeneous responses to Nampt inhibition, which contributes to the diverse outcomes. There is a lack of imaging biomarkers to differentiate among TNBC cells under metabolic stress and identify which are responsive. We aimed to characterize and differentiate among a panel of TNBC cell lines under NAD-deficient stress and identify which subtypes are more dependent on the NAD salvage pathway. Methods: Optical redox imaging (ORI), a label-free live cell imaging microscopy technique was utilized to acquire intrinsic fluorescence intensities of NADH and FAD-containing flavoproteins (Fp) thus the mitochondrial redox ratio Fp/(NADH + Fp) in a panel of TNBC cell lines. Various fluorescence probes were then added to the cultures to image the mitochondrial ROS, mitochondrial membrane potential, mitochondrial mass, and cell number. Results: Various TNBC subtypes are sensitive to Nampt inhibition in a dose- and time-dependent manner, they have differential mitochondrial redox responses; furthermore, the mitochondrial redox indices linearly correlated with mitochondrial ROS induced by various doses of a Nampt inhibitor. Moreover, the changes in the redox indices correlated with growth inhibition. Additionally, the redox state was found fully recovered after removing the Nampt inhibitor. Conclusions: This study supports the utility of ORI in rapid metabolic phenotyping of TNBC cells under NAD-deficient stress to identify responsive cells and biomarkers of treatment response, facilitating combination therapy strategies. Full article

NAD+ plays a pivotal role in energy metabolism and adaptation to external stimuli and stressful conditions. A significant reduction in intracellular NAD+ levels is associated with aging and contributes to the development of chronic cardiovascular, neurodegenerative, and metabolic diseases. It is of particular importance to maintain optimal levels of NAD+ in cells with high energy consumption, particularly in the brain. Maintaining the tissue level of NAD+ with pharmacological tools has the potential to slow down the aging process, to prevent the development of age-related diseases. This review covers key aspects of NAD+ metabolism in terms of brain metabolic plasticity, including NAD+ biosynthesis and degradation in different types of brain cells, as well as its contribution to the development of neurodegeneration and aging, and highlights up-to-date approaches to modulate NAD+ levels in brain cells. Full article

Background: Nicotinamide adenine dinucleotide (NAD⁺), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD⁺ derivatives and it’s reduced forms, with ¹³C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD⁺ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD⁺ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD⁺ metabolism and the potential significance of these metabolites in various biological contexts. Full article

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli. First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli. HPLC and LC–MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli. In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli. To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host. Full article