BACKGROUND Sepsis is a severe illness characterized by systemic and multiorgan reactive responses and damage.However,the impact of sepsis on the bone marrow,particularly on bone marrow mesenchymal stem cells(BMSCs),is...BACKGROUND Sepsis is a severe illness characterized by systemic and multiorgan reactive responses and damage.However,the impact of sepsis on the bone marrow,particularly on bone marrow mesenchymal stem cells(BMSCs),is less reported.BMSCs are critical stromal cells in the bone marrow microenvironment that maintain bone stability and hematopoietic homeostasis;however,the impairment caused by sepsis remains unknown.AIM To investigate the effects of sepsis on BMSCs and the underlying mechanisms.METHODS BMSCs were obtained from healthy donors and patients with sepsis.We compared the self-renewal capacity,differentiation potential,and hematopoietic supportive ability in vitro.Senescence of septic BMSCs was assessed usingβ-galactosidase staining,senescence-associated secretory phenotype,intracellular reactive oxygen species levels,and the expression of P16 and P21.Finally,the changes in septic BMSCs after nicotinamide adenine dinucleotide(NAD)treatment were evaluated.RESULTS Septic BMSCs showed decreased proliferation and self-renewal,bias towards adipogenic differentiation,and weakened osteogenic differentiation.Additionally,hematopoietic supportive capacity declines in sepsis.The levels of aging markers were significantly higher in the septic BMSCs.After NAD treatment,the proliferation capacity of septic BMSCs showed a recovery trend,with increased osteogenic and hematopoietic supportive capacities.Sepsis resulted in decreased expression of sirtuin 3(SIRT3)in BMSCs,whereas NAD treatment restored SIRT3 expression,enhanced superoxide dismutase enzyme activity,reduced intracellular reactive oxygen species levels,maintained mitochondrial stability and function,and ultimately rejuvenated septic BMSCs.CONCLUSION Sepsis accelerates the aging of BMSCs,as evidenced by a decline in self-renewal and osteogenic capabilities,as well as weakened hematopoietic support functions.These deficiencies can be effectively reversed via the NAD/SIRT3/superoxide dismutase pathway.展开更多
Neonatal hypoxic-ischemic brain injury is the main cause of hypoxic-ischemic encephalopathy and cerebral palsy.Currently,there are few effective clinical treatments for neonatal hypoxic-ischemic brain injury.Here,we i...Neonatal hypoxic-ischemic brain injury is the main cause of hypoxic-ischemic encephalopathy and cerebral palsy.Currently,there are few effective clinical treatments for neonatal hypoxic-ischemic brain injury.Here,we investigated the neuroprotective and molecular mechanisms of exogenous nicotinamide adenine dinucleotide,which can protect against hypoxic injury in adulthood,in a mouse model of neonatal hypoxic-ischemic brain injury.In this study,nicotinamide adenine dinucleotide(5 mg/kg)was intraperitoneally administered 30 minutes befo re surgery and every 24 hours thereafter.The results showed that nicotinamide adenine dinucleotide treatment improved body weight,brain structure,adenosine triphosphate levels,oxidative damage,neurobehavioral test outcomes,and seizure threshold in experimental mice.Tandem mass tag proteomics revealed that numerous proteins were altered after nicotinamide adenine dinucleotide treatment in hypoxic-ischemic brain injury mice.Parallel reaction monitoring and western blotting confirmed changes in the expression levels of proteins including serine(or cysteine)peptidase inhibitor,clade A,member 3N,fibronectin 1,5'-nucleotidase,cytosolic IA,microtubule associated protein 2,and complexin 2.Proteomics analyses showed that nicotinamide adenine dinucleotide ameliorated hypoxic-ischemic injury through inflammation-related signaling pathways(e.g.,nuclear factor-kappa B,mitogen-activated protein kinase,and phosphatidylinositol 3 kinase/protein kinase B).These findings suggest that nicotinamide adenine dinucleotide treatment can improve neurobehavioral phenotypes in hypoxic-ischemic brain injury mice through inflammation-related pathways.展开更多
Background: Bioenhancers augment the bioavailability of co-administered molecules without showing any significant effect on their own. Piperine, an alkaloid from Piper nigrum, is an established natural bioenhancer. Ni...Background: Bioenhancers augment the bioavailability of co-administered molecules without showing any significant effect on their own. Piperine, an alkaloid from Piper nigrum, is an established natural bioenhancer. Nicotinamide mononucleotide (NMN), an antiaging supplement, is the precursor of coenzyme nicotinamide adenine dinucleotide (NAD) that plays an important role in intracellular redox reactions. Objective: The study compared the serum concentrations of NAD in normal healthy participants, supplemented with NMN 500 mg and NMN 500 mg + 5 mg BioPerine® (95% piperine). Methods: In a randomized, open-label, crossover study, NMN (500 mg) was compared to NMN + BioPerine® (500 mg + 5 mg) in 6 healthy adults, aged 18 - 45 years. The participants received a single oral dose of NMN or NMN + BioPerine® capsules with 240 mL water, and blood samples were collected over 8hr. After a 4-day washout period, the same procedures were repeated as per the crossover design. Total NAD (NADtotal), including oxidized NAD (the oxidized) and its reduced form NADH, was measured in human serum samples. Results: The maximum concentration (Cmax) of NAD in serum was higher with NMN + BioPerine® (282 pmol/mL) compared to NMN (246 pmol/mL) alone. In the presence of BioPerine®, the NAD concentrations reached 257 pmol/mL during the first 2 hr, whereas a comparable serum concentration (246 pmol/mL) was attained only after 6 hr in NMN alone. The AUC0-8hr was 1738 pmol/mL/hr in NMN compared to 2004 pmol/mL/hr in NMN+ BioPerine®. The time to reach peak concentration (t1/2) was similar (6hr) in both groups. No clinically relevant adverse events (AE) were observed, and safety parameters remained within normal ranges in all the participants with both formulations. Conclusion: These results reveal that BioPerine® can effectively increase the NAD concentrations in the serum following NMN supplementation in healthy volunteers. The present study was registered prospectively with the Clinical Trials Registry-India (CTRI/2023/11/059982).展开更多
Pancreatitis and pancreatic cancer(PC)stand as the most worrisome ailments affecting the pancreas.Researchers have dedicated efforts to unraveling the mechanisms underlying these diseases,yet their true nature continu...Pancreatitis and pancreatic cancer(PC)stand as the most worrisome ailments affecting the pancreas.Researchers have dedicated efforts to unraveling the mechanisms underlying these diseases,yet their true nature continues to elude their grasp.Within this realm,oxidative stress is often believed to play a causal and contributory role in the development of pancreatitis and PC.Excessive accumulation of reactive oxygen species(ROS)can cause oxidative stress,and the key enzyme responsible for inducing ROS production in cells is nicotinamide adenine dinucleotide phosphate hydrogen oxides(NOX).NOX contribute to pancreatic fibrosis and inflammation by generating ROS that injure acinar cells,activate pancreatic stellate cells,and mediate macrophage polarization.Excessive ROS production occurs during malignant transformation and pancreatic carcinogenesis,creating an oxidative microenvironment that can cause abnormal apoptosis,epithelial to mesenchymal transition and genomic instability.Therefore,understanding the role of NOX in pancreatic diseases contributes to a more in-depth exploration of the exact pathogenesis of these diseases.In this review,we aim to summarize the potential roles of NOX and its mechanism in pancreatic disorders,aiming to provide novel insights into understanding the mechanisms underlying these diseases.展开更多
Formate oxidase(FOx),which contains 8-formyl flavin adenine dinucleotide(FAD),exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-cho...Formate oxidase(FOx),which contains 8-formyl flavin adenine dinucleotide(FAD),exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline(GMC)oxidoreductase enzymes that contain only the standard FAD cofactor.The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity.However,the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood,which impedes further utilization of FOx.In this study,we employ molecular dynamics simulation,QM/MM umbrella sampling simulation,enzyme activity assay,site-directed mutagenesis,and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD.Our results reveal that a catalytic water molecule,rather than any catalytic amino acids,serves as a general base to deprotonate the C8 methyl group on FAD,thus facilitating the formation of a quinone-methide tautomer intermediate.An oxygen molecule subsequently oxidizes this intermediate,resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH−.During the oxidation of FAD to 8-formyl FAD,the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol,which corresponds to the required 14-hour transformation time observed experimentally.Further,the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues,R87 and S94,respectively.Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75%of the wild-type,while the mutation to histidine results in a complete loss of activity.Similarly,the mutant S94I also leads to the deactivation of enzyme.This dependency arises because the nucleophilic OH−group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94.These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD,thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties.展开更多
Cyclic dinucleotides(CDNs)play important physiological roles in bacteria,mammals and insects as a novel class of signaling molecules.However,the application of CDNs in agricultural pest control has not been reported y...Cyclic dinucleotides(CDNs)play important physiological roles in bacteria,mammals and insects as a novel class of signaling molecules.However,the application of CDNs in agricultural pest control has not been reported yet.To explore the potential bioactivity of CDNs on agricultural pests,we synthesized ten kinds of CDNs containing adenine and guanine bases with different internucleotide linkages(30,30;20,30;20,20).The target CDNs were used to determine the antifeedant and insecticidal activity against common Lepidoptera pests including S.frugiperda,M.separata,and H.armigera.The bioassay tests indicated that 30,30-c-di-AMP showed the highest antifeedant activity(EC50?0.59 mg/L)against M.separata larvae among all the tested CDNs.Regarding insecticidal activity,20,30-c-di-AMP showed higher insecticidal activity against M.separata larvae with LC50 of 55.4 mg/L.RNA-seq further revealed that 30,30-c-di-AMP and 20,30-c-di-AMP exhibited a significant effect on the growth and development process of insects.More importantly,the bioactivity of 30,30-c-di-AMP was also closely correlated with the stimulation of insect immune inflammation.These results indicated that cyclic dinucleotides can affect the normal physiological process of insects,providing a new direction for managing pests.展开更多
Three dinucleotides containing L-isonucleosides at 5＇-end were synthesized by an elegant phosphoramidite one-pot method. Their binding modes with HIV integrase were simulated and their anti-HIV activities in pseudoty...Three dinucleotides containing L-isonucleosides at 5＇-end were synthesized by an elegant phosphoramidite one-pot method. Their binding modes with HIV integrase were simulated and their anti-HIV activities in pseudotyped virus system were examined.展开更多
Nicotinamide adenine dinucleotide phosphate oxidase(NOX) is a multisubunit enzyme complex that utilizes nicotinamide adenine dinucleotide phosphate to produce superoxide anions and other reactive oxygen species. Under...Nicotinamide adenine dinucleotide phosphate oxidase(NOX) is a multisubunit enzyme complex that utilizes nicotinamide adenine dinucleotide phosphate to produce superoxide anions and other reactive oxygen species. Under normal circumstances, reactive oxygen species mediate a number of important cellular functions, including the facilitation of adaptive immunity. In pathogenic circumstances, however,excess reactive oxygen species generated by NOX promotes apoptotic cell death. In ischemic stroke, in particular, it has been shown that both NOX activation and derangements in glucose metabolism result in increased apoptosis. Moreover, recent studies have established that glucose, as a NOX substrate, plays a vital role in the pathogenesis of reperfusion injury. Thus, NOX inhibition has the potential to mitigate the deleterious impact of hyperglycemia on stroke. In this paper, we provide an overview of this research,coupled with a discussion of its implications for the development of NOX inhibition as a strategy for the treatment of ischemic stroke. Both inhibition using apocynin, as well as the prospect of developing more specific inhibitors based on what is now understood of the biology of NOX assembly and activation, will be highlighted in the course of our discussion.展开更多
Oxaliplatin, an anticancer drug commonly used to treat colorectal cancer and other tumors, has a number of serious side effects, most notably neuropathy and ototoxicity. To gain insights into its ototoxic profile, oxa...Oxaliplatin, an anticancer drug commonly used to treat colorectal cancer and other tumors, has a number of serious side effects, most notably neuropathy and ototoxicity. To gain insights into its ototoxic profile, oxaliplatin was applied to rat cochlear organ cultures. Consistent with it neurotoxic propensity, oxaliplatin selectively damaged nerve fibers at a very low dose 1 μM. In contrast, the dose required to damage hair cells and spiral ganglion neurons was 50 fold higher (50 μM). Oxailiplatin-induced cochlear lesions initial-ly increased with dose, but unexpectedly decreased at very high doses. This non-linear dose response could be related to depressed oxaliplatin uptake via active transport mechanisms. Previous studies have demon-strated that axonal degeneration involves biologically active processes which can be greatly attenuated by nicotinamide adenine dinucleotide (NAD+). To determine if NAD+would protect spiral ganglion axons and the hair cells from oxaliplatin damage, cochlear cultures were treated with oxaliplatin alone at doses of 10 μM or 50 μM respectively as controls or combined with 20 mM NAD+. Treatment with 10 μM oxaliplatin for 48 hours resulted in minor damage to auditory nerve fibers, but spared cochlear hair cells. However, when cochlear cultures were treated with 10 μM oxaliplatin plus 20 mM NAD+, most auditory nerve fibers were intact. 50 μM oxaliplatin destroyed most of spiral ganglion neurons and cochlear hair cells with apop-totic characteristics of cell fragmentations. However, 50 μM oxaliplatin plus 20 mM NAD+treatment great-ly reduced neuronal degenerations and hair cell missing. The results suggested that NAD+provides signifi-cant protection against oxaliplatin-induced neurotoxicity and ototoxicity, which may be due to its actions of antioxidant, antiapoptosis, and energy supply.展开更多
The inhibition of 5-a reductase type 2(SRD5A2)by finasteride is commonly used for the management of urinary obstruction resulting from benign prostatic enlargement(BPE).Certain BPE patients showing no SRD5A2 protein e...The inhibition of 5-a reductase type 2(SRD5A2)by finasteride is commonly used for the management of urinary obstruction resulting from benign prostatic enlargement(BPE).Certain BPE patients showing no SRD5A2 protein expression are resistant to finasteride therapy.Our previous work showed that methylated cytosine-phosphate-guanine(CpG)islands in the SRD5A2 gene might account for the absence or reduction of SRD5A2 protein expression.Here,we found that the expression of the SRD5A2 protein was variable and that weak expression of the SRD5A2 protein(scored 0-100)occurred in 10.0%(4/40)of benign adult prostates.We showed that the expression of SRD5A2 was negatively correlated with DNA methyltransferase 1(DNMT1)expression.In vitro SRD5A2-negative BPH-1 cells were resistant to finasteride treatment,and SRD5A2 was re-expressed in BPH-1 cells when SRD5A2 was demethylated by 5-Aza-2T-deoxycytidine(5-Aza-CdR)or N-phthalyl-L-tryptophan(RG108).Furthermore,we determined the exact methylation ratios of CpG dinucleotides in a CpG island of SRD5A2 through MassArray quantitative methylation analysis.Ten methylated CpG dinucleotides,including four CpG dinucleotides in the promoter and six CpG dinucleotides in the first exon,were found in a CpG island located from-400 bp to+600 bp in SRD5A2,which might lead to the silencing of SRD5A2 and the absence or reduction of SRD5A2 protein expression.Finasteride cannot exert a therapeutic effect on patients lacking SRD5A2,which may partially account for the resistance to finasteride observed in certain BPE patients.展开更多
The occurrence frequencies of the dinucleotides of genes of three thermophilic and three mesophilic species from both archaea and eubacteria were investigated in this study. The genes encoding water soluble proteins w...The occurrence frequencies of the dinucleotides of genes of three thermophilic and three mesophilic species from both archaea and eubacteria were investigated in this study. The genes encoding water soluble proteins were rich in the dinucleotides of purine dimers, whereas the genes encoding membrane proteins were rich in pyrimidine dimers. The dinucleotides of purine dimers are the counterparts of pyrimidine dimers in a double-stranded DNA. The purine/pyrimidine dimers were favored in the thermophiles but not in the mesophiles, based on comparisons of observed and expected frequencies. This finding is in agreement with our previous study which showed that purine/pyrimidine dimers are positive factors that increase the thermal stability of DNA. The dinucleotides AA, AG, and GA are components of the codons of charged residues of Glu, Asp, Lys, and Arg, and the dinucleotides TT, CT, and TC are components of the codons of hydrophobic residues of Leu, Ile, and Phe. This is consistent with the suitabilities of the different amino acid residues for water soluble and membrane proteins. Our analysis provides a picture of how thermophilic species produce water soluble and membrane proteins with distinctive characters: the genes encoding water soluble proteins use DNA sequences rich in purine dimers, and the genes encoding membrane proteins use DNA sequences rich in pyrimidine dimers on the opposite strand.展开更多
AIM: The molecular diagnosis of microsatellite instability (MSI) in colorectal cancer (CRC) is based on the analysis of five microsatellite markers. Among them, the two mononucleotide microsatellite repeats are consid...AIM: The molecular diagnosis of microsatellite instability (MSI) in colorectal cancer (CRC) is based on the analysis of five microsatellite markers. Among them, the two mononucleotide microsatellite repeats are considered more informative for this analysis than the three dinucleotide ones. The aim of this study is to establish the most relevant markers for MSI analysis in colorectal cancers from Asian patients. METHODS: The MSI analysis of 143 CRC cases in a routine molecular diagnostic laboratory was reviewed. Analysis by fluorescence-based PCR of the five recommended microsatellites was performed, followed by data interpretation according to internationally accepted guidelines. The results were analyzed to address (1) the rate of success in the analysis of histopathological samples not specifically preparedfor molecular analysis; (2) the relative importance ofindividual markers in the diagnosis of high-MSI (H-MSI).RESULTS: MSI analysis was unsuccessful in 34 cases (24%), but for tissues archived in recent years the unsuccessful rate was 5%. We found the D2S123 marker the mostvulnerable to inadequate tissue preservation, failing to amplify in 58 instances. Approximately 30% (32/109) of the cases were H-MSI, while 7/109 (6%) were low-MSI.A detailed analysis of the H-MSI cases revealed that the dinucleotide repeats (and D5S346 in particular) were more relevant than the mononucleotide repeats in assigning the correct MSI status. CONCLUSION: The analysis of dinucleotide repeats isessential for the establishment of MSI status in Asian CRC patients.展开更多
Dinucleotide (TpAZT) phosphoramidates were synthesized by Todd reaction of dinucleoside H-phosphonates and amino acid methyl esters, and their diastereomers (Rp and Sp) were separated by crystallization, and the resul...Dinucleotide (TpAZT) phosphoramidates were synthesized by Todd reaction of dinucleoside H-phosphonates and amino acid methyl esters, and their diastereomers (Rp and Sp) were separated by crystallization, and the results showed that natural and cheap methyl esters of alanine and phenylalanine can be used for large-scale synthesis of dinucleotide analogs.展开更多
Recently biospecific affinity chromatography has been widely used for the separation and purification of various enzymes and nucleic acids. In this paper, a series of synthetic reactions of solid-liquid phase were car...Recently biospecific affinity chromatography has been widely used for the separation and purification of various enzymes and nucleic acids. In this paper, a series of synthetic reactions of solid-liquid phase were carried out on silica surface, using a macroporous(30 mu m), microspherical silica (8 mu m) as the matrix and gamma-aminopropyltriethoxysilane as the activating agent, the nicotinamide adenine dinucleotide(NAD) was bonded through its amino groups to the carboxylic groups of linked phospholipid which was bonded covalently on aminated support. The bonded stationary phase has high thermal stability, and could be used to separate of nucleotides with good resolution.展开更多
Deformability of DNA is important for its superhelical folding in the nucleosome and has long been thought to be facilitated by periodic occurrences of certain dinucleotides along the sequences, with the period close ...Deformability of DNA is important for its superhelical folding in the nucleosome and has long been thought to be facilitated by periodic occurrences of certain dinucleotides along the sequences, with the period close to 10.5 bases. This study statistically examines the conformational properties of dinucleotides containing the 10.5 - base periodicity and those without that periodicity through scanning all nucleosome structures provided in PDB. By categorizing performances on the distribution of step parameter values, averaged net values, standard deviations and deformability based on step conformational energies, we give a detailed description as to the deformation preferences correlated with the periodicity for the 10 unique types of dinucleotides and summarize the possible roles of various steps in how they facilitate DNA bending. The results show that the structural properties of dinucleotide steps are influenced to various extents by the periodicity in nucleosomes and some periodic steps have shown a clear tendency to take specific bending or shearing patterns.展开更多
Nicotinamide adenine dinucleotide (NAD) oscillation was observed when the isolated mitochondria were immersed in a pyruvate solution. In addition, when an adenosine diphosphate (ADP) was added to the mitochondrial sus...Nicotinamide adenine dinucleotide (NAD) oscillation was observed when the isolated mitochondria were immersed in a pyruvate solution. In addition, when an adenosine diphosphate (ADP) was added to the mitochondrial suspension containing pyruvate, adenosine triphosphate (ATP) oscillation was observed as well as NADH oscillation. At this time, the pH within mitochondria also oscillated. It was found that the oscillatory reaction of NADH caused by the membrane permeation of pyruvate continues, causing the oscillation of NADH and H+ in the subsequent reactions. The pH oscillation led to the ATP oscillation. It is considered that the oscillatory reaction caused by the gradual entry of pyruvate into mitochondria was thought to be carried over to both the citric acid cycle and the respiratory chain, ultimately leading to the ATP oscillation in oxidative phosphorylation. Similarly, it was found that membrane permeation of malate causes the gradual occurrence of NADH, at which point NADH oscillates, followed by an oscillatory reaction of the respiratory chain, and finally ATP oscillation. It was found that the oscillations of NADH and ATP occur without going through the citric acid cycle. Oscillations of NADH and other intermediates in both the citric acid cycle and respiratory chain were also confirmed by experiments using semipermeable membranes. These results support our hypothesis that the gradual entry of the substrate by membrane permeation triggers an oscillatory reaction of the enzyme, which is also carried over to subsequent reactions.展开更多
Chain elongation via dinucleotide(dimer)block coupling was considered as an improved chemical technique capable of synthesizing high-quality longer oligonucleotide for de novo DNA synthesis in synthetic biology.Howeve...Chain elongation via dinucleotide(dimer)block coupling was considered as an improved chemical technique capable of synthesizing high-quality longer oligonucleotide for de novo DNA synthesis in synthetic biology.However,this dimer block-wise approach was constrained by readily available dimer phosphoramidite with sufficient quality.Herein,through the usage of a one-pot coupling-oxidation-deprotection cascade process for preparing the key precursors 3'-hydroxyl dimers,then condensation with phosphorodiamidite,purification by flash column chromatography and precipation in methyl tert-butyl ether,a rationally designed dimer phosphoramidite bearing an internucleotide allyl phosphate and aβ-cyanoethyl phosphoramidite at the 3’-hydroxyl was synthesized.All sixteen allylic dimer phosphoramidites 2a-p were smoothly prepared with overall yields exceeding 50%and HPLC purities ranging from 97.40%to 99.69%.With these allylic reagents,oligonucleotides were successfully synthesized using a modified solid-phase phosphoramidite method and were completely deprotected under normal ammonialysis condition.Our results indicated that these dimer block-wise synthesized oligonucleotides were of sufficient quality for gene assembly and protein expression,thus,the allylic phosphate linked dimer phosphoramidite can serve as a promising dimer reagent that will enable the applications of long oligonucleotides.展开更多
Cyclic dinucleotides(CDNs)are natural agonists of the stimulator of interferon genes(STING),which is an attractive immunotherapy target.Currently,CDNs and their derivatives are being investigated clinically.However,th...Cyclic dinucleotides(CDNs)are natural agonists of the stimulator of interferon genes(STING),which is an attractive immunotherapy target.Currently,CDNs and their derivatives are being investigated clinically.However,the poor bioavailability of exogenous CDNs has limited their application in immunotherapy.Although nanocarriers are widely used for cytosolic delivery of CDNs,their loading capacity is insufficient,and their complicated composition and purification process raises bio-compatibility concerns.Herein,we report a super-simplified CDN self-assembly strategy for carrier-free delivery of CDNs.In the presence of excess K^(+),CDNs form oligomers which further self-assemble with divalent metal ions(such as Mn^(2+))to form nanoparticles(NPs)in aqueous solution.We demonstrate that the self-assembled CDN NPs promote cellular uptake of CDNs and enhance tumor immunogenicity by remodeling the tumor microenvironment,inducing immunogenic tumor cell death and increasing tumorinfiltrating lymphocytes,which is conducive to the generation of tumor neoantigen-specific T-cell responses.We also demonstrate that the use of CDN NPs alone or in combination with immune checkpoint blockades inhibits tumor growth,highlighting the fact that CDN NPs are a potent platform for cancer immunotherapy.展开更多
Mitochondria are central organelles in cellular metabolism,orchestrating energy production,biosynthetic pathways,and signaling networks.Nicotinamide adenine dinucleotide(NAD+)and its reduced form(NADH)are essential fo...Mitochondria are central organelles in cellular metabolism,orchestrating energy production,biosynthetic pathways,and signaling networks.Nicotinamide adenine dinucleotide(NAD+)and its reduced form(NADH)are essential for mitochondrial metabolism,functioning both as redox coenzymes and as signaling agents that help regulate cellular balance.Thus,while its major role is in energy production,NAD+is widely recognized as a metabolic cofactor and also serves as a substrate for various enzymes involved in cellular signaling,like sirtuins(SIRTs),poly(ADP-ribosyl)polymerases(PARPs),mono(ADP-ribosyl)transferases,and CD38.Sirtuins,a family of NAD+-dependent deacetylases,are critical in this regulatory network.SIRT3 removes acetyl groups from and enhances the activity of key enzymes that participate in fatty acid breakdown,the tricarboxylic acid(TCA)cycle,and the electron transport chain(etc),thereby enhancing mitochondrial efficiency and energy production.Mitochondrial NAD+biosynthesis involves multiple pathways,including the de novo synthesis from tryptophan via the kynurenine and the salvage pathway,which recycles nicotinamide back to NAD+.Moreover,NAD+concentrations influence mitochondrial dynamics such as fusion,fission,and mitophagy,which are essential for preserving mitochondrial integrity and function.NAD+alsomodulates the balance between glycolysis and oxidative phosphorylation,influencing the metabolic flexibility of cells.During NAD+depletion,mainly in metabolic disorders,cells often shift towards anaerobic glycolysis,reducing ATP production efficiency and increasing lactate production.This metabolic shift is associated with various pathophysiological conditions,including insulin resistance,neurodegeneration,and muscle wasting.This reviewexplores themultifaceted functions of NAD+in regulating mitochondrialmetabolism.It highlights the underlying causes and pathological outcomes of disrupted NAD+metabolism while exploring potential therapeutic targets and treatment strategies.展开更多
The study aimed to explore the efficacy and potential mechanisms of a naturally aromatic cyanogenic compound amygdalin(AMY)in treating glucocorticoid(GC)-associated necrosis of the femoral head(GANFH).We demonstrated ...The study aimed to explore the efficacy and potential mechanisms of a naturally aromatic cyanogenic compound amygdalin(AMY)in treating glucocorticoid(GC)-associated necrosis of the femoral head(GANFH).We demonstrated that GC exacerbates the oxidative stress(OS)microenvironment via promoting nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)expression in human,rat,and mesenchymal stem cells(MSCs)samples,thus generating excessive reactive oxygen species(ROS),leading to increased apoptosis and unbalanced osteolipogenic differentiation.Furthermore,computational docking results revealed that AMY could bind specifically to the predicted binding sites of NOX4.Additionally,AMY ameliorated the OS microenvironment of MSCs via decreasing NOX4 expression and inhibiting NOX4/ROS/p38MAPK signaling,thereby reversing the GC-induced apoptosis and imbalanced osteolipogenic differentiation,and ultimately alleviating GANFH.In summary,we demonstrated for the first time that AMY attenuated apoptosis and maintained osteolipogenic differentiation balance in MSCs via specifically targeting NOX4,inhibiting NOX4/ROS/p38MAPK signaling,thereby treating GANFH.展开更多
基金Shanghai Natural Science Foundation,No.21ZR1452300the Clinical Research Innovation Plan of Shanghai General Hospital,No.CCTR-2022B04.
文摘BACKGROUND Sepsis is a severe illness characterized by systemic and multiorgan reactive responses and damage.However,the impact of sepsis on the bone marrow,particularly on bone marrow mesenchymal stem cells(BMSCs),is less reported.BMSCs are critical stromal cells in the bone marrow microenvironment that maintain bone stability and hematopoietic homeostasis;however,the impairment caused by sepsis remains unknown.AIM To investigate the effects of sepsis on BMSCs and the underlying mechanisms.METHODS BMSCs were obtained from healthy donors and patients with sepsis.We compared the self-renewal capacity,differentiation potential,and hematopoietic supportive ability in vitro.Senescence of septic BMSCs was assessed usingβ-galactosidase staining,senescence-associated secretory phenotype,intracellular reactive oxygen species levels,and the expression of P16 and P21.Finally,the changes in septic BMSCs after nicotinamide adenine dinucleotide(NAD)treatment were evaluated.RESULTS Septic BMSCs showed decreased proliferation and self-renewal,bias towards adipogenic differentiation,and weakened osteogenic differentiation.Additionally,hematopoietic supportive capacity declines in sepsis.The levels of aging markers were significantly higher in the septic BMSCs.After NAD treatment,the proliferation capacity of septic BMSCs showed a recovery trend,with increased osteogenic and hematopoietic supportive capacities.Sepsis resulted in decreased expression of sirtuin 3(SIRT3)in BMSCs,whereas NAD treatment restored SIRT3 expression,enhanced superoxide dismutase enzyme activity,reduced intracellular reactive oxygen species levels,maintained mitochondrial stability and function,and ultimately rejuvenated septic BMSCs.CONCLUSION Sepsis accelerates the aging of BMSCs,as evidenced by a decline in self-renewal and osteogenic capabilities,as well as weakened hematopoietic support functions.These deficiencies can be effectively reversed via the NAD/SIRT3/superoxide dismutase pathway.
基金supported by the National Natural Science Foundation of China,Nos.81871024 (to HN),82301957 (to XW),82001382 (to LL),62127810 (to HN)the Natural Science Foundation of Jiangsu Province of China,No.SBK2020040785 (to LL)。
文摘Neonatal hypoxic-ischemic brain injury is the main cause of hypoxic-ischemic encephalopathy and cerebral palsy.Currently,there are few effective clinical treatments for neonatal hypoxic-ischemic brain injury.Here,we investigated the neuroprotective and molecular mechanisms of exogenous nicotinamide adenine dinucleotide,which can protect against hypoxic injury in adulthood,in a mouse model of neonatal hypoxic-ischemic brain injury.In this study,nicotinamide adenine dinucleotide(5 mg/kg)was intraperitoneally administered 30 minutes befo re surgery and every 24 hours thereafter.The results showed that nicotinamide adenine dinucleotide treatment improved body weight,brain structure,adenosine triphosphate levels,oxidative damage,neurobehavioral test outcomes,and seizure threshold in experimental mice.Tandem mass tag proteomics revealed that numerous proteins were altered after nicotinamide adenine dinucleotide treatment in hypoxic-ischemic brain injury mice.Parallel reaction monitoring and western blotting confirmed changes in the expression levels of proteins including serine(or cysteine)peptidase inhibitor,clade A,member 3N,fibronectin 1,5'-nucleotidase,cytosolic IA,microtubule associated protein 2,and complexin 2.Proteomics analyses showed that nicotinamide adenine dinucleotide ameliorated hypoxic-ischemic injury through inflammation-related signaling pathways(e.g.,nuclear factor-kappa B,mitogen-activated protein kinase,and phosphatidylinositol 3 kinase/protein kinase B).These findings suggest that nicotinamide adenine dinucleotide treatment can improve neurobehavioral phenotypes in hypoxic-ischemic brain injury mice through inflammation-related pathways.
文摘Background: Bioenhancers augment the bioavailability of co-administered molecules without showing any significant effect on their own. Piperine, an alkaloid from Piper nigrum, is an established natural bioenhancer. Nicotinamide mononucleotide (NMN), an antiaging supplement, is the precursor of coenzyme nicotinamide adenine dinucleotide (NAD) that plays an important role in intracellular redox reactions. Objective: The study compared the serum concentrations of NAD in normal healthy participants, supplemented with NMN 500 mg and NMN 500 mg + 5 mg BioPerine® (95% piperine). Methods: In a randomized, open-label, crossover study, NMN (500 mg) was compared to NMN + BioPerine® (500 mg + 5 mg) in 6 healthy adults, aged 18 - 45 years. The participants received a single oral dose of NMN or NMN + BioPerine® capsules with 240 mL water, and blood samples were collected over 8hr. After a 4-day washout period, the same procedures were repeated as per the crossover design. Total NAD (NADtotal), including oxidized NAD (the oxidized) and its reduced form NADH, was measured in human serum samples. Results: The maximum concentration (Cmax) of NAD in serum was higher with NMN + BioPerine® (282 pmol/mL) compared to NMN (246 pmol/mL) alone. In the presence of BioPerine®, the NAD concentrations reached 257 pmol/mL during the first 2 hr, whereas a comparable serum concentration (246 pmol/mL) was attained only after 6 hr in NMN alone. The AUC0-8hr was 1738 pmol/mL/hr in NMN compared to 2004 pmol/mL/hr in NMN+ BioPerine®. The time to reach peak concentration (t1/2) was similar (6hr) in both groups. No clinically relevant adverse events (AE) were observed, and safety parameters remained within normal ranges in all the participants with both formulations. Conclusion: These results reveal that BioPerine® can effectively increase the NAD concentrations in the serum following NMN supplementation in healthy volunteers. The present study was registered prospectively with the Clinical Trials Registry-India (CTRI/2023/11/059982).
基金Supported by Youth Independent Innovation Science Fund Project from Chinese PLA General Hospital,No.22QNFC075.
文摘Pancreatitis and pancreatic cancer(PC)stand as the most worrisome ailments affecting the pancreas.Researchers have dedicated efforts to unraveling the mechanisms underlying these diseases,yet their true nature continues to elude their grasp.Within this realm,oxidative stress is often believed to play a causal and contributory role in the development of pancreatitis and PC.Excessive accumulation of reactive oxygen species(ROS)can cause oxidative stress,and the key enzyme responsible for inducing ROS production in cells is nicotinamide adenine dinucleotide phosphate hydrogen oxides(NOX).NOX contribute to pancreatic fibrosis and inflammation by generating ROS that injure acinar cells,activate pancreatic stellate cells,and mediate macrophage polarization.Excessive ROS production occurs during malignant transformation and pancreatic carcinogenesis,creating an oxidative microenvironment that can cause abnormal apoptosis,epithelial to mesenchymal transition and genomic instability.Therefore,understanding the role of NOX in pancreatic diseases contributes to a more in-depth exploration of the exact pathogenesis of these diseases.In this review,we aim to summarize the potential roles of NOX and its mechanism in pancreatic disorders,aiming to provide novel insights into understanding the mechanisms underlying these diseases.
基金supported by the National Natural Science Foundation of China(32201030,32271319 and 32071267)the Science and Technology Department of Jilin Province(20230402041GH and YDZJ202301ZYTS537)+2 种基金the Education Department of Jilin Province(JJKH20220970KJ)the Development and Reform Commission of Jilin Province(2023C015)the Fundamental Research Funds of the Central Universities in China(2024-JCXK-11).
文摘Formate oxidase(FOx),which contains 8-formyl flavin adenine dinucleotide(FAD),exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline(GMC)oxidoreductase enzymes that contain only the standard FAD cofactor.The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity.However,the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood,which impedes further utilization of FOx.In this study,we employ molecular dynamics simulation,QM/MM umbrella sampling simulation,enzyme activity assay,site-directed mutagenesis,and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD.Our results reveal that a catalytic water molecule,rather than any catalytic amino acids,serves as a general base to deprotonate the C8 methyl group on FAD,thus facilitating the formation of a quinone-methide tautomer intermediate.An oxygen molecule subsequently oxidizes this intermediate,resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH−.During the oxidation of FAD to 8-formyl FAD,the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol,which corresponds to the required 14-hour transformation time observed experimentally.Further,the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues,R87 and S94,respectively.Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75%of the wild-type,while the mutation to histidine results in a complete loss of activity.Similarly,the mutant S94I also leads to the deactivation of enzyme.This dependency arises because the nucleophilic OH−group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94.These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD,thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties.
基金supported by National Key Research and Development Program of China(Grant Number:2023YFD1700502)Frontiers Science Center for New Organic Matter,Nankai University,(Grant Number:63181206)the Haihe Laboratory of Sustainable Chemical Transformations,China(Grant Number:YYJC202102).
文摘Cyclic dinucleotides(CDNs)play important physiological roles in bacteria,mammals and insects as a novel class of signaling molecules.However,the application of CDNs in agricultural pest control has not been reported yet.To explore the potential bioactivity of CDNs on agricultural pests,we synthesized ten kinds of CDNs containing adenine and guanine bases with different internucleotide linkages(30,30;20,30;20,20).The target CDNs were used to determine the antifeedant and insecticidal activity against common Lepidoptera pests including S.frugiperda,M.separata,and H.armigera.The bioassay tests indicated that 30,30-c-di-AMP showed the highest antifeedant activity(EC50?0.59 mg/L)against M.separata larvae among all the tested CDNs.Regarding insecticidal activity,20,30-c-di-AMP showed higher insecticidal activity against M.separata larvae with LC50 of 55.4 mg/L.RNA-seq further revealed that 30,30-c-di-AMP and 20,30-c-di-AMP exhibited a significant effect on the growth and development process of insects.More importantly,the bioactivity of 30,30-c-di-AMP was also closely correlated with the stimulation of insect immune inflammation.These results indicated that cyclic dinucleotides can affect the normal physiological process of insects,providing a new direction for managing pests.
基金National Natural Science Foundation of China (Grant No. 20832008 and 21002004)
文摘Three dinucleotides containing L-isonucleosides at 5＇-end were synthesized by an elegant phosphoramidite one-pot method. Their binding modes with HIV integrase were simulated and their anti-HIV activities in pseudotyped virus system were examined.
基金partially supported by Merit Review Award(I01RX-001964-01)from the US Department of Veterans Affairs Rehabilitation Research and Development Service(to YD)the National Natural Science Foundation of China(81501141)+1 种基金Beijing New Star of Science and Technology Program of China(xx2016061)Beijing Tongzhou District Financial Fund,and Scientific Research Common Program of Beijing Municipal Commission of Education,China(KM201610025028)(to XG)
文摘Nicotinamide adenine dinucleotide phosphate oxidase(NOX) is a multisubunit enzyme complex that utilizes nicotinamide adenine dinucleotide phosphate to produce superoxide anions and other reactive oxygen species. Under normal circumstances, reactive oxygen species mediate a number of important cellular functions, including the facilitation of adaptive immunity. In pathogenic circumstances, however,excess reactive oxygen species generated by NOX promotes apoptotic cell death. In ischemic stroke, in particular, it has been shown that both NOX activation and derangements in glucose metabolism result in increased apoptosis. Moreover, recent studies have established that glucose, as a NOX substrate, plays a vital role in the pathogenesis of reperfusion injury. Thus, NOX inhibition has the potential to mitigate the deleterious impact of hyperglycemia on stroke. In this paper, we provide an overview of this research,coupled with a discussion of its implications for the development of NOX inhibition as a strategy for the treatment of ischemic stroke. Both inhibition using apocynin, as well as the prospect of developing more specific inhibitors based on what is now understood of the biology of NOX assembly and activation, will be highlighted in the course of our discussion.
文摘Oxaliplatin, an anticancer drug commonly used to treat colorectal cancer and other tumors, has a number of serious side effects, most notably neuropathy and ototoxicity. To gain insights into its ototoxic profile, oxaliplatin was applied to rat cochlear organ cultures. Consistent with it neurotoxic propensity, oxaliplatin selectively damaged nerve fibers at a very low dose 1 μM. In contrast, the dose required to damage hair cells and spiral ganglion neurons was 50 fold higher (50 μM). Oxailiplatin-induced cochlear lesions initial-ly increased with dose, but unexpectedly decreased at very high doses. This non-linear dose response could be related to depressed oxaliplatin uptake via active transport mechanisms. Previous studies have demon-strated that axonal degeneration involves biologically active processes which can be greatly attenuated by nicotinamide adenine dinucleotide (NAD+). To determine if NAD+would protect spiral ganglion axons and the hair cells from oxaliplatin damage, cochlear cultures were treated with oxaliplatin alone at doses of 10 μM or 50 μM respectively as controls or combined with 20 mM NAD+. Treatment with 10 μM oxaliplatin for 48 hours resulted in minor damage to auditory nerve fibers, but spared cochlear hair cells. However, when cochlear cultures were treated with 10 μM oxaliplatin plus 20 mM NAD+, most auditory nerve fibers were intact. 50 μM oxaliplatin destroyed most of spiral ganglion neurons and cochlear hair cells with apop-totic characteristics of cell fragmentations. However, 50 μM oxaliplatin plus 20 mM NAD+treatment great-ly reduced neuronal degenerations and hair cell missing. The results suggested that NAD+provides signifi-cant protection against oxaliplatin-induced neurotoxicity and ototoxicity, which may be due to its actions of antioxidant, antiapoptosis, and energy supply.
基金the National Natural Science Foundation of China(No.81770754)the Beiing Natural Science Foundation(No.7172081)at Beijing Chaoyang Hospital,Capital Medical University,through funding provided to YNN.
文摘The inhibition of 5-a reductase type 2(SRD5A2)by finasteride is commonly used for the management of urinary obstruction resulting from benign prostatic enlargement(BPE).Certain BPE patients showing no SRD5A2 protein expression are resistant to finasteride therapy.Our previous work showed that methylated cytosine-phosphate-guanine(CpG)islands in the SRD5A2 gene might account for the absence or reduction of SRD5A2 protein expression.Here,we found that the expression of the SRD5A2 protein was variable and that weak expression of the SRD5A2 protein(scored 0-100)occurred in 10.0%(4/40)of benign adult prostates.We showed that the expression of SRD5A2 was negatively correlated with DNA methyltransferase 1(DNMT1)expression.In vitro SRD5A2-negative BPH-1 cells were resistant to finasteride treatment,and SRD5A2 was re-expressed in BPH-1 cells when SRD5A2 was demethylated by 5-Aza-2T-deoxycytidine(5-Aza-CdR)or N-phthalyl-L-tryptophan(RG108).Furthermore,we determined the exact methylation ratios of CpG dinucleotides in a CpG island of SRD5A2 through MassArray quantitative methylation analysis.Ten methylated CpG dinucleotides,including four CpG dinucleotides in the promoter and six CpG dinucleotides in the first exon,were found in a CpG island located from-400 bp to+600 bp in SRD5A2,which might lead to the silencing of SRD5A2 and the absence or reduction of SRD5A2 protein expression.Finasteride cannot exert a therapeutic effect on patients lacking SRD5A2,which may partially account for the resistance to finasteride observed in certain BPE patients.
文摘The occurrence frequencies of the dinucleotides of genes of three thermophilic and three mesophilic species from both archaea and eubacteria were investigated in this study. The genes encoding water soluble proteins were rich in the dinucleotides of purine dimers, whereas the genes encoding membrane proteins were rich in pyrimidine dimers. The dinucleotides of purine dimers are the counterparts of pyrimidine dimers in a double-stranded DNA. The purine/pyrimidine dimers were favored in the thermophiles but not in the mesophiles, based on comparisons of observed and expected frequencies. This finding is in agreement with our previous study which showed that purine/pyrimidine dimers are positive factors that increase the thermal stability of DNA. The dinucleotides AA, AG, and GA are components of the codons of charged residues of Glu, Asp, Lys, and Arg, and the dinucleotides TT, CT, and TC are components of the codons of hydrophobic residues of Leu, Ile, and Phe. This is consistent with the suitabilities of the different amino acid residues for water soluble and membrane proteins. Our analysis provides a picture of how thermophilic species produce water soluble and membrane proteins with distinctive characters: the genes encoding water soluble proteins use DNA sequences rich in purine dimers, and the genes encoding membrane proteins use DNA sequences rich in pyrimidine dimers on the opposite strand.
基金Supported by the Health Services Development Program, Ministry of Health, Singapore (Project Reference No.: HSDP01N02). The study was carried out following the ethical approval of our Institutional Review Board (IRB Reference Code 03.104)
文摘AIM: The molecular diagnosis of microsatellite instability (MSI) in colorectal cancer (CRC) is based on the analysis of five microsatellite markers. Among them, the two mononucleotide microsatellite repeats are considered more informative for this analysis than the three dinucleotide ones. The aim of this study is to establish the most relevant markers for MSI analysis in colorectal cancers from Asian patients. METHODS: The MSI analysis of 143 CRC cases in a routine molecular diagnostic laboratory was reviewed. Analysis by fluorescence-based PCR of the five recommended microsatellites was performed, followed by data interpretation according to internationally accepted guidelines. The results were analyzed to address (1) the rate of success in the analysis of histopathological samples not specifically preparedfor molecular analysis; (2) the relative importance ofindividual markers in the diagnosis of high-MSI (H-MSI).RESULTS: MSI analysis was unsuccessful in 34 cases (24%), but for tissues archived in recent years the unsuccessful rate was 5%. We found the D2S123 marker the mostvulnerable to inadequate tissue preservation, failing to amplify in 58 instances. Approximately 30% (32/109) of the cases were H-MSI, while 7/109 (6%) were low-MSI.A detailed analysis of the H-MSI cases revealed that the dinucleotide repeats (and D5S346 in particular) were more relevant than the mononucleotide repeats in assigning the correct MSI status. CONCLUSION: The analysis of dinucleotide repeats isessential for the establishment of MSI status in Asian CRC patients.
基金The authors would like to thank the financial supports from the National Natural Science Foundation of China(No.29902003,20132020 and 20175026),the Ministry of Science and Technology,the Chinese Ministry of Education and Tsinghua University.
文摘Dinucleotide (TpAZT) phosphoramidates were synthesized by Todd reaction of dinucleoside H-phosphonates and amino acid methyl esters, and their diastereomers (Rp and Sp) were separated by crystallization, and the results showed that natural and cheap methyl esters of alanine and phenylalanine can be used for large-scale synthesis of dinucleotide analogs.
文摘Recently biospecific affinity chromatography has been widely used for the separation and purification of various enzymes and nucleic acids. In this paper, a series of synthetic reactions of solid-liquid phase were carried out on silica surface, using a macroporous(30 mu m), microspherical silica (8 mu m) as the matrix and gamma-aminopropyltriethoxysilane as the activating agent, the nicotinamide adenine dinucleotide(NAD) was bonded through its amino groups to the carboxylic groups of linked phospholipid which was bonded covalently on aminated support. The bonded stationary phase has high thermal stability, and could be used to separate of nucleotides with good resolution.
文摘Deformability of DNA is important for its superhelical folding in the nucleosome and has long been thought to be facilitated by periodic occurrences of certain dinucleotides along the sequences, with the period close to 10.5 bases. This study statistically examines the conformational properties of dinucleotides containing the 10.5 - base periodicity and those without that periodicity through scanning all nucleosome structures provided in PDB. By categorizing performances on the distribution of step parameter values, averaged net values, standard deviations and deformability based on step conformational energies, we give a detailed description as to the deformation preferences correlated with the periodicity for the 10 unique types of dinucleotides and summarize the possible roles of various steps in how they facilitate DNA bending. The results show that the structural properties of dinucleotide steps are influenced to various extents by the periodicity in nucleosomes and some periodic steps have shown a clear tendency to take specific bending or shearing patterns.
文摘Nicotinamide adenine dinucleotide (NAD) oscillation was observed when the isolated mitochondria were immersed in a pyruvate solution. In addition, when an adenosine diphosphate (ADP) was added to the mitochondrial suspension containing pyruvate, adenosine triphosphate (ATP) oscillation was observed as well as NADH oscillation. At this time, the pH within mitochondria also oscillated. It was found that the oscillatory reaction of NADH caused by the membrane permeation of pyruvate continues, causing the oscillation of NADH and H+ in the subsequent reactions. The pH oscillation led to the ATP oscillation. It is considered that the oscillatory reaction caused by the gradual entry of pyruvate into mitochondria was thought to be carried over to both the citric acid cycle and the respiratory chain, ultimately leading to the ATP oscillation in oxidative phosphorylation. Similarly, it was found that membrane permeation of malate causes the gradual occurrence of NADH, at which point NADH oscillates, followed by an oscillatory reaction of the respiratory chain, and finally ATP oscillation. It was found that the oscillations of NADH and ATP occur without going through the citric acid cycle. Oscillations of NADH and other intermediates in both the citric acid cycle and respiratory chain were also confirmed by experiments using semipermeable membranes. These results support our hypothesis that the gradual entry of the substrate by membrane permeation triggers an oscillatory reaction of the enzyme, which is also carried over to subsequent reactions.
基金supported by the National Key Research and Development Program of China(No.2018YFA0902300)。
文摘Chain elongation via dinucleotide(dimer)block coupling was considered as an improved chemical technique capable of synthesizing high-quality longer oligonucleotide for de novo DNA synthesis in synthetic biology.However,this dimer block-wise approach was constrained by readily available dimer phosphoramidite with sufficient quality.Herein,through the usage of a one-pot coupling-oxidation-deprotection cascade process for preparing the key precursors 3'-hydroxyl dimers,then condensation with phosphorodiamidite,purification by flash column chromatography and precipation in methyl tert-butyl ether,a rationally designed dimer phosphoramidite bearing an internucleotide allyl phosphate and aβ-cyanoethyl phosphoramidite at the 3’-hydroxyl was synthesized.All sixteen allylic dimer phosphoramidites 2a-p were smoothly prepared with overall yields exceeding 50%and HPLC purities ranging from 97.40%to 99.69%.With these allylic reagents,oligonucleotides were successfully synthesized using a modified solid-phase phosphoramidite method and were completely deprotected under normal ammonialysis condition.Our results indicated that these dimer block-wise synthesized oligonucleotides were of sufficient quality for gene assembly and protein expression,thus,the allylic phosphate linked dimer phosphoramidite can serve as a promising dimer reagent that will enable the applications of long oligonucleotides.
基金the National Key R&D Program of China(grant nos.2019YFA0904200 and 2018YFA0507600)the National Natural Science Foundation of China(grant nos.22237003 and 92053108)the Tsinghua University Spring Breeze Fund(grant no.2020Z99CFY042).
文摘Cyclic dinucleotides(CDNs)are natural agonists of the stimulator of interferon genes(STING),which is an attractive immunotherapy target.Currently,CDNs and their derivatives are being investigated clinically.However,the poor bioavailability of exogenous CDNs has limited their application in immunotherapy.Although nanocarriers are widely used for cytosolic delivery of CDNs,their loading capacity is insufficient,and their complicated composition and purification process raises bio-compatibility concerns.Herein,we report a super-simplified CDN self-assembly strategy for carrier-free delivery of CDNs.In the presence of excess K^(+),CDNs form oligomers which further self-assemble with divalent metal ions(such as Mn^(2+))to form nanoparticles(NPs)in aqueous solution.We demonstrate that the self-assembled CDN NPs promote cellular uptake of CDNs and enhance tumor immunogenicity by remodeling the tumor microenvironment,inducing immunogenic tumor cell death and increasing tumorinfiltrating lymphocytes,which is conducive to the generation of tumor neoantigen-specific T-cell responses.We also demonstrate that the use of CDN NPs alone or in combination with immune checkpoint blockades inhibits tumor growth,highlighting the fact that CDN NPs are a potent platform for cancer immunotherapy.
基金A fellowship support from the Golda Meir Fellowship Fund,The Hebrew University of Jerusalem,Israel。
文摘Mitochondria are central organelles in cellular metabolism,orchestrating energy production,biosynthetic pathways,and signaling networks.Nicotinamide adenine dinucleotide(NAD+)and its reduced form(NADH)are essential for mitochondrial metabolism,functioning both as redox coenzymes and as signaling agents that help regulate cellular balance.Thus,while its major role is in energy production,NAD+is widely recognized as a metabolic cofactor and also serves as a substrate for various enzymes involved in cellular signaling,like sirtuins(SIRTs),poly(ADP-ribosyl)polymerases(PARPs),mono(ADP-ribosyl)transferases,and CD38.Sirtuins,a family of NAD+-dependent deacetylases,are critical in this regulatory network.SIRT3 removes acetyl groups from and enhances the activity of key enzymes that participate in fatty acid breakdown,the tricarboxylic acid(TCA)cycle,and the electron transport chain(etc),thereby enhancing mitochondrial efficiency and energy production.Mitochondrial NAD+biosynthesis involves multiple pathways,including the de novo synthesis from tryptophan via the kynurenine and the salvage pathway,which recycles nicotinamide back to NAD+.Moreover,NAD+concentrations influence mitochondrial dynamics such as fusion,fission,and mitophagy,which are essential for preserving mitochondrial integrity and function.NAD+alsomodulates the balance between glycolysis and oxidative phosphorylation,influencing the metabolic flexibility of cells.During NAD+depletion,mainly in metabolic disorders,cells often shift towards anaerobic glycolysis,reducing ATP production efficiency and increasing lactate production.This metabolic shift is associated with various pathophysiological conditions,including insulin resistance,neurodegeneration,and muscle wasting.This reviewexplores themultifaceted functions of NAD+in regulating mitochondrialmetabolism.It highlights the underlying causes and pathological outcomes of disrupted NAD+metabolism while exploring potential therapeutic targets and treatment strategies.
基金supported by the Natural Science Foundation of China(81873325)the State Administration of Traditional Chinese Medicine of Zhejiang Province(2021ZZ014).
文摘The study aimed to explore the efficacy and potential mechanisms of a naturally aromatic cyanogenic compound amygdalin(AMY)in treating glucocorticoid(GC)-associated necrosis of the femoral head(GANFH).We demonstrated that GC exacerbates the oxidative stress(OS)microenvironment via promoting nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)expression in human,rat,and mesenchymal stem cells(MSCs)samples,thus generating excessive reactive oxygen species(ROS),leading to increased apoptosis and unbalanced osteolipogenic differentiation.Furthermore,computational docking results revealed that AMY could bind specifically to the predicted binding sites of NOX4.Additionally,AMY ameliorated the OS microenvironment of MSCs via decreasing NOX4 expression and inhibiting NOX4/ROS/p38MAPK signaling,thereby reversing the GC-induced apoptosis and imbalanced osteolipogenic differentiation,and ultimately alleviating GANFH.In summary,we demonstrated for the first time that AMY attenuated apoptosis and maintained osteolipogenic differentiation balance in MSCs via specifically targeting NOX4,inhibiting NOX4/ROS/p38MAPK signaling,thereby treating GANFH.
