Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The res...Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for addi- tional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated lambda DNA af- ter overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all REs tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed di- rectly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of REs in PCR buffer. This simplified method for RE digestion of PCR prod- ucts could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymor- phism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.展开更多
DNA exonucleases and endonucleases are key executors of the genome during many physiological processes.They generate double-stranded DNA by cleaving damaged endogenous or exogenous DNA,triggering the activation of the...DNA exonucleases and endonucleases are key executors of the genome during many physiological processes.They generate double-stranded DNA by cleaving damaged endogenous or exogenous DNA,triggering the activation of the innate immune pathways such as cGAS-STING-IFN,and enabling the body to produce antiviral or anti-tumor immune responses.This is of great significance for maintaining the stability of the genome and improving the therapeu-tic efficacy of tumors.In addition,genomic instability caused by exonuclease mutations contributes to the development of various autoimmune diseases.This review summarizes the DNA exonucleases and endonucleases which have critical functions in immunity and associated diseases.展开更多
Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patien...Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patients) Mothods This sequence section bears interest because ① it harbors several potential methylation (Cp rich) sites, and ② it represents the largest part of its internal ribosomal entry site A pre PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences Results The suspected HCV specific sequence was found in the DNA of each subject tested The pre PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena Conclusions The results provide formal proof that these HCV specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences展开更多
Aqueous nanoparticle suspension of fullerene and its derivatives are currently attracting much attention. To determine the effects of aqueous nanoparticle suspension of a mono-methanophosphonate fullerene and bis-meth...Aqueous nanoparticle suspension of fullerene and its derivatives are currently attracting much attention. To determine the effects of aqueous nanoparticle suspension of a mono-methanophosphonate fullerene and bis-methanophosphonate fullerene (denoted as n-MMPF and n-BMPF, respectively) on the activities of DNA restrictive endonucleases, plasmid pEGFP-N1 was cleaved at a single but differently restrictive site by EcoR I, BamH I, and isozymes Cfr9 I and Xma I, respectively. Both n-MMPF and n-BMPF inhibited the activity of EcoR I, while n-BMPF exhibited stronger inhibition than n-MMPF. Addition of n-BMPF into reaction mixtures inhibited the activities of all the four enzymes, and IC50 values for EcoR I, BamH I, Cfr9 I and Xma I were 4.3, >30, 11.7 and 8.3 μmol/L, respectively. When EcoR I was completely inhibited by n-BMPF, addition of excess amounts of pEGFP-N1 could not produce the product linear plasmid; however, increase of EcoR I amounts antagonized EcoR I inhibition of n-BMPF. Two scavengers of reactive oxygen species (ROS), mannitol and sodium azide at the concentrations of 2-10 mmol/L, did not reverse inhibition of n-BMPF, implying that this inhibition probably is not correlated to ROS. These results suggested that aqueous nano-fullerenes might act as inhibitors of DNA restrictive endonucleases.展开更多
Human chromosome banding was carried out with restriction endonucleases (REs)HaeⅢ, HinfⅠ, PstⅠ and the effects of some factors on it were examined. HaeⅢ induced Cand G banding patterns, HinfⅠ induced negative C b...Human chromosome banding was carried out with restriction endonucleases (REs)HaeⅢ, HinfⅠ, PstⅠ and the effects of some factors on it were examined. HaeⅢ induced Cand G banding patterns, HinfⅠ induced negative C bands but the centromeric heterochro-matin of chromosomes 3 and 4 remained selectively dark-stained. A new heteromorphismof C banded region was discovered in chromosome 4. PstⅠ induced G-like banding pattern.The duration of enzymatic digestion, the concentration of glycerol in the reaction buffer,as well as the ageing and heating of chromosome preparations all had a significant influ-ence on the banding effects of the REs.展开更多
BACKGROUND Colorectal cancer(CRC)is a common malignant tumor of the digestive tract worldwide,characterized by high incidence and mortality rates.AIM To investigate the expression of serum apurinic/apyrimidinic endonu...BACKGROUND Colorectal cancer(CRC)is a common malignant tumor of the digestive tract worldwide,characterized by high incidence and mortality rates.AIM To investigate the expression of serum apurinic/apyrimidinic endonuclease 1 autoantibodies(APE1-AAbs),peripheral pentraxin-3(PTX-3),and miR-486-3p in patients with CRC undergoing radical surgery and their relationship with postoperative recurrence and metastasis.METHODS A retrospective analysis was conducted on the clinical data of 154 CRC patients who underwent laparoscopic radical surgery in our hospital from January 2022 to January 2024.Patients were followed for one year postoperatively and divided into an occurrence group(n=28)and a non-occurrence group(n=126)based on whether they experienced recurrence or metastasis.The clinical data and the expression levels of APE1-AAbs,PTX-3,and miR-486-3p were compared between the two groups.Multivariate logistic regression analysis was performed to identify risk factors for postoperative recurrence and metastasis in CRC patients.The relationship of APE1-AAbs,PTX-3,and miR-486-3p with postoperative recurrence and metastasis was analyzed using Spearman correlation analysis.Receiver operating characteristic curves were drawn to evaluate the predictive value of serum APE1-AAbs,PTX-3,and miR-486-3p levels alone and their combination for postoperative recurrence and metastasis in CRC.RESULTS The occurrence group had significantly higher proportions of patients with an age≥60 years,lymph node metastasis,stage III disease,poor differentiation,tumor diameter>5 cm,and higher platelet count,carcinoembryonic antigen,and carbohydrate antigen 19-9 levels than the non-occurrence group(P<0.05).The expression levels of APE1-AAbs,PTX-3,and miR-486-3p in the occurrence group were significantly higher than those in the non-occurrence group(P<0.05).Multivariate logistic regression analysis showed that lymph node metastasis,stage III disease,poor differentiation,and elevated levels of APE1-AAbs,PTX-3,and miR-486-3p were risk factors for postoperative recurrence and metastasis in CRC patients(odds ratio>1,P<0.05).Spearman correlation analysis revealed that the levels of APE1-AAbs,PTX-3,and miR-486-3p were positively correlated with postoperative recurrence and metastasis in CRC patients(r=0.642,0.653,and 0.631,respectively,P<0.05).Receiver operating characteristic curve analysis showed that the area under the curve values for APE1-AAbs,PTX-3,and miR-486-3p levels alone and their combination in predicting postoperative recurrence and metastasis in CRC were 0.764,0.783,0.806,and 0.875,respectively,with the combination significantly outperforming individual markers(P<0.05).CONCLUSION Serum APE1-AAbs,PTX-3,and miR-486-3p levels are higher in CRC patients with postoperative recurrence and metastasis.These three markers are risk factors for postoperative recurrence and metastasis in CRC and can be used as predictive biomarkers.The combined detection of these markers has higher predictive value compared to individual tests.展开更多
Mesophilic Argonautes(Agos)from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics.This study characterizes a novel Ago from Pseudob...Mesophilic Argonautes(Agos)from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics.This study characterizes a novel Ago from Pseudobutyrivi-brio ruminis(PrAgo),which can cleave single-stranded DNA using guide DNA(gDNA).PrAgo,functioning as a multi-turnover enzyme,effectively cleaves DNA using 5′-phosphate gDNA,14-30 nucleotides in length,in the presence of both Mn2+and Mg2+ions.PrAgo demonstrates DNA cleavage activity over a broad pH range(pH 4-12),with optimal activity at pH 11.As a mesophilic enzyme,PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65°C,particularly at 65°C.PrAgo does not show strong preferences for the 5′-nucleotide in gDNA.It shows high tolerance for single-base mismatches,except at positions 13 and 15 of gDNA.Continuous double-nucleotide mismatches at positions 10-16 of gDNA significantly reduce cleavage activity.Furthermore,PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65°C.Additionally,molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency.These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology.展开更多
Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragm...Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.展开更多
The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal...The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.展开更多
Liver diseases are one of the leading causes of mortality in the world. The hepatic illnesses, which include inherited metabolic disorders, hemophilias and viralhepatitides, are complex and currently difficult to trea...Liver diseases are one of the leading causes of mortality in the world. The hepatic illnesses, which include inherited metabolic disorders, hemophilias and viralhepatitides, are complex and currently difficult to treat. The maturation of gene therapy has heralded new avenues for developing effective intervention for these diseases. DNA modification using gene therapy is now possible and available technology may be exploited to achieve long term therapeutic benefit. The ability to edit DNA sequences specifically is of paramount importance to advance gene therapy for application to liver diseases. Recent development of technologies that allow for this has resulted in rapid advancement of gene therapy to treat several chronic illnesses. Improvements in application of derivatives of zinc finger proteins(ZFPs), transcription activator-like effectors(TALEs), homing endonucleases(HEs) and clustered regularly interspaced palindromic repeats(CRISPR) and CRISPR associated(Cas) systems have been particularly important. These sequence-specific technologies may be used to modify genes permanently and also to alter gene transcription for therapeutic purposes. This review describes progress in development of ZFPs, TALEs, HEs and CRISPR/Cas for application to treating liver diseases.展开更多
Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutat...Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases(RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were2.6% and 2.2% in the Arabidopsis FLOWERING TIME(At FT) and TERMINAL FLOWER 1(At TFL1)loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.展开更多
Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method ...Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonu-clease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, cir-cularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.展开更多
Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(...Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(BXM),was approved in Japan and the US in 2018 and several other countries subsequently.Along with the clinical use of BXM,the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern.Herein,we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A,an analogue of BXM.The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes,including pH1N1,H3N2,H7N9 and H9N2,in MDCK cells,and the 50%effective concentration(EC_(50))was calculated to nanomole level and comparable to that of baloxavir acid(BXA),the active form of BXM.Furthermore,in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice,with reduced viral RNA loads and alleviated pulmonary damage.Importantly,serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage.Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA.Taken together,our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance,which provides critical clues for future drug development and drug resistance surveillance.展开更多
AIM:To evaluate the relationship between apurinic endonuclease 1(APE1) Asp148 Glu polymorphism and the susceptibility to gastrointestinal(GI) cancers.METHODS:We searched Pub Med, ISI Web of Knowledge, and Chinese Nati...AIM:To evaluate the relationship between apurinic endonuclease 1(APE1) Asp148 Glu polymorphism and the susceptibility to gastrointestinal(GI) cancers.METHODS:We searched Pub Med, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure(CNKI) databases updated on July 15, 2014 for relevant studies.Only case-control studies comparing APE1 Asp148 Glu polymorphism and GI cancer risk were included.We excluded studies reporting only standardized incidence ratios without control groups and those without detailed genotyping data.Meta-analysis was performed on 17 studies involving 4856 cancer patients and 6136 cancer-free controls.Review Manager version 5.1 was used to perform the meta-analysis.The pooled odds ratios(ORs) and 95% confidence intervals(CIs) were estimated under the allele contrast, homozygous, heterozygous, dominant and recessive genetic models.We also conducted subgroup analyses stratified by ethnicity and cancer type.Publication bias was evaluated using Begg's test.RESULTS:The meta-analysis showed a significant association between APE1 Asp148Glu polymorphism and GI cancer risk in three genetic models in the overall population(G vs T:OR=1.18;95%CI:1.05-1.32;TG vs TT:OR=1.28;95%CI:1.08-1.52;TG+GG vs TT:OR=1.32;95%CI:1.10-1.57).Stratified analysis by ethnicity revealed a statistically increased GI cancer risk in Asians(G vs T:OR=1.27;95%CI:1.07-1.51;GG vs TT:OR=1.58;95%CI:1.05-2.38;TG vs TT:OR=1.30;95%CI,1.01-1.67;and TG+GG vs TT:OR=1.38;95%CI:1.07-1.78),but not in Caucasians.Furthersubgroup analysis by cancer type indicated that APE1Asp148Glu polymorphism may contribute to gastric cancer risk.However,Asp148Glu has no significant association with colorectal or esophageal cancer risk in any genetic model.CONCLUSION:This meta-analysis suggests that the APE1 Asp148Glu polymorphism G allele is associated with an increased GI cancer risk,especially in gastric cancer.展开更多
Objectives: To investigate the methylation status of the CDKN2/pl6 gene 5′-CpG island, and study the relationship between the CDKN2/pl6 gene inactivation by methylation and lung cancer. Methods: Genomic DNA extracted...Objectives: To investigate the methylation status of the CDKN2/pl6 gene 5′-CpG island, and study the relationship between the CDKN2/pl6 gene inactivation by methylation and lung cancer. Methods: Genomic DNA extracted from the specimens of lung cancer and normal lung tissue was digested with methylation-sensitive endonucleases, and Southern blotting was used to analyze the methylation status of the CDKN2/pl6 gene in 89 cases of lung cancer and 10 cases of normal lung tissue. Results: In 89 cases of lung cancer studied, the CDKN2/pl6 gene was shown to be methylated in 21 cases with the total methylation rate of 23.6% (21/89), in which there were 15 cases (16.9%) methylated atSmaI sites, 12 cases (13.5%) atSmaI sites and 6 cases at bothSmaI andSacII sites. The methylation of the CDKN2/pl6 gene occurred in 17 among 42 of pl6 protein negative cases of lung cancer with a rate of 40.5% (17/42), and in 3 among 47 of pl6 protein positive cases with a rate of 6.4% (3/47). The CDKN2/pl6 gene was not methylated in 10 cases of normal lung tissue. Conclusion: The aberrant methylation of the CDKN2/pl6 gene 5′-CpG island is probably an important mechanism of the gene inactivation, it may be involved in the genesis and progress of lung cancer.展开更多
AIM:To determine hepatic expression of apurinic/apyrimidinic endonuclease 1(APE-1)and 8-hydroxydeoxyguanosine(8-OHdG)in patients with chronic hepatitis B and C.METHODS:Liver biopsies were obtained from 27 patients wit...AIM:To determine hepatic expression of apurinic/apyrimidinic endonuclease 1(APE-1)and 8-hydroxydeoxyguanosine(8-OHdG)in patients with chronic hepatitis B and C.METHODS:Liver biopsies were obtained from 27 patients with chronic hepatitis B virus(HBV),30 with chronic hepatitis C virus(HCV),6 with autoimmune hepatitis(AIH),and 6 with primary biliary cirrhosis(PBC).Normal liver tissue was obtained from surgical resection specimens of four patients.Hepatic APE-1 protein and mRNA expression were assayed by Western blot and by real-time polymerase chain reaction,respectively.Hepatocellular APE-1 and 8-OHdG expression were determined by immunohistochemistry.RESULTS:The staining intensity of hepatocellular nuclear APE-1 was lower in the HBV group than in the other groups(P < 0.05).Hepatic APE-1 protein levels were reduced in the HBV group relative to the other groups.Hepatic APE-1 mRNA levels were also lower in the HBV group.The proportion of hepatocytes with 8-OHdG-positive nuclei was increased in the HCV,AIH and PBC groups(P < 0.05),but not in the HBV group.Hepatocellular nuclear APE-1 levels were positively correlated with hepatocellular 8-OHdG levels in both the HBV and HCV groups(HBV,r = 0.34,P < 0.05;HCV,r = 0.54,P < 0.01).CONCLUSION:An imbalance between oxidative DNA damage and APE-1 expression may contribute to hepatocarcinogenesis in chronic viral hepatitis.展开更多
DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly relate...DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly related to its length. This intriguing phenomenon only appears at the interfaces of DNAfunctionalized gold nanoparticles. We systematically investigated the causes of this phenomenon. We conjectured that the DNAzyme with extended nucleotides that do not match its substrate strand is vulnerable to non-specific adsorption, electrostatic repulsion, and steric hindrance. Based on our improved understanding of this phenomenon, we have successfully developed a highly sensitive and specific amplifiable biosensor to detect human apurinic/apyrimidinic endonuclease 1.展开更多
基金This research was supported by the Fund for ICP Cup of the Northeast Forestry University and partially by the Key Project (NO. 96-20) of State Forestry Administration.
文摘Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for addi- tional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated lambda DNA af- ter overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all REs tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed di- rectly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of REs in PCR buffer. This simplified method for RE digestion of PCR prod- ucts could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymor- phism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.
基金supported by the Beijing Xisike Clinical Oncology Research Foundation (China) (No.Y-HR2020MS-0156 to T.M.)the National Natural Science Foundation of China (No.82273130 to T.M.).
文摘DNA exonucleases and endonucleases are key executors of the genome during many physiological processes.They generate double-stranded DNA by cleaving damaged endogenous or exogenous DNA,triggering the activation of the innate immune pathways such as cGAS-STING-IFN,and enabling the body to produce antiviral or anti-tumor immune responses.This is of great significance for maintaining the stability of the genome and improving the therapeu-tic efficacy of tumors.In addition,genomic instability caused by exonuclease mutations contributes to the development of various autoimmune diseases.This review summarizes the DNA exonucleases and endonucleases which have critical functions in immunity and associated diseases.
文摘Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patients) Mothods This sequence section bears interest because ① it harbors several potential methylation (Cp rich) sites, and ② it represents the largest part of its internal ribosomal entry site A pre PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences Results The suspected HCV specific sequence was found in the DNA of each subject tested The pre PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena Conclusions The results provide formal proof that these HCV specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences
基金Supported by the National Natural Science Foundation of China (Grant No. 20672012)Beijing Natural Science Foundation (Grant No. 20822020)
文摘Aqueous nanoparticle suspension of fullerene and its derivatives are currently attracting much attention. To determine the effects of aqueous nanoparticle suspension of a mono-methanophosphonate fullerene and bis-methanophosphonate fullerene (denoted as n-MMPF and n-BMPF, respectively) on the activities of DNA restrictive endonucleases, plasmid pEGFP-N1 was cleaved at a single but differently restrictive site by EcoR I, BamH I, and isozymes Cfr9 I and Xma I, respectively. Both n-MMPF and n-BMPF inhibited the activity of EcoR I, while n-BMPF exhibited stronger inhibition than n-MMPF. Addition of n-BMPF into reaction mixtures inhibited the activities of all the four enzymes, and IC50 values for EcoR I, BamH I, Cfr9 I and Xma I were 4.3, >30, 11.7 and 8.3 μmol/L, respectively. When EcoR I was completely inhibited by n-BMPF, addition of excess amounts of pEGFP-N1 could not produce the product linear plasmid; however, increase of EcoR I amounts antagonized EcoR I inhibition of n-BMPF. Two scavengers of reactive oxygen species (ROS), mannitol and sodium azide at the concentrations of 2-10 mmol/L, did not reverse inhibition of n-BMPF, implying that this inhibition probably is not correlated to ROS. These results suggested that aqueous nano-fullerenes might act as inhibitors of DNA restrictive endonucleases.
基金Project supported by National Natural Science Foundation of China.
文摘Human chromosome banding was carried out with restriction endonucleases (REs)HaeⅢ, HinfⅠ, PstⅠ and the effects of some factors on it were examined. HaeⅢ induced Cand G banding patterns, HinfⅠ induced negative C bands but the centromeric heterochro-matin of chromosomes 3 and 4 remained selectively dark-stained. A new heteromorphismof C banded region was discovered in chromosome 4. PstⅠ induced G-like banding pattern.The duration of enzymatic digestion, the concentration of glycerol in the reaction buffer,as well as the ageing and heating of chromosome preparations all had a significant influ-ence on the banding effects of the REs.
文摘BACKGROUND Colorectal cancer(CRC)is a common malignant tumor of the digestive tract worldwide,characterized by high incidence and mortality rates.AIM To investigate the expression of serum apurinic/apyrimidinic endonuclease 1 autoantibodies(APE1-AAbs),peripheral pentraxin-3(PTX-3),and miR-486-3p in patients with CRC undergoing radical surgery and their relationship with postoperative recurrence and metastasis.METHODS A retrospective analysis was conducted on the clinical data of 154 CRC patients who underwent laparoscopic radical surgery in our hospital from January 2022 to January 2024.Patients were followed for one year postoperatively and divided into an occurrence group(n=28)and a non-occurrence group(n=126)based on whether they experienced recurrence or metastasis.The clinical data and the expression levels of APE1-AAbs,PTX-3,and miR-486-3p were compared between the two groups.Multivariate logistic regression analysis was performed to identify risk factors for postoperative recurrence and metastasis in CRC patients.The relationship of APE1-AAbs,PTX-3,and miR-486-3p with postoperative recurrence and metastasis was analyzed using Spearman correlation analysis.Receiver operating characteristic curves were drawn to evaluate the predictive value of serum APE1-AAbs,PTX-3,and miR-486-3p levels alone and their combination for postoperative recurrence and metastasis in CRC.RESULTS The occurrence group had significantly higher proportions of patients with an age≥60 years,lymph node metastasis,stage III disease,poor differentiation,tumor diameter>5 cm,and higher platelet count,carcinoembryonic antigen,and carbohydrate antigen 19-9 levels than the non-occurrence group(P<0.05).The expression levels of APE1-AAbs,PTX-3,and miR-486-3p in the occurrence group were significantly higher than those in the non-occurrence group(P<0.05).Multivariate logistic regression analysis showed that lymph node metastasis,stage III disease,poor differentiation,and elevated levels of APE1-AAbs,PTX-3,and miR-486-3p were risk factors for postoperative recurrence and metastasis in CRC patients(odds ratio>1,P<0.05).Spearman correlation analysis revealed that the levels of APE1-AAbs,PTX-3,and miR-486-3p were positively correlated with postoperative recurrence and metastasis in CRC patients(r=0.642,0.653,and 0.631,respectively,P<0.05).Receiver operating characteristic curve analysis showed that the area under the curve values for APE1-AAbs,PTX-3,and miR-486-3p levels alone and their combination in predicting postoperative recurrence and metastasis in CRC were 0.764,0.783,0.806,and 0.875,respectively,with the combination significantly outperforming individual markers(P<0.05).CONCLUSION Serum APE1-AAbs,PTX-3,and miR-486-3p levels are higher in CRC patients with postoperative recurrence and metastasis.These three markers are risk factors for postoperative recurrence and metastasis in CRC and can be used as predictive biomarkers.The combined detection of these markers has higher predictive value compared to individual tests.
基金supported by the Ministry of Science and Technology(2020YFA0907700)National Natural Science Foundation of China(31770078,32270051)Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University(21TQ1400204).
文摘Mesophilic Argonautes(Agos)from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics.This study characterizes a novel Ago from Pseudobutyrivi-brio ruminis(PrAgo),which can cleave single-stranded DNA using guide DNA(gDNA).PrAgo,functioning as a multi-turnover enzyme,effectively cleaves DNA using 5′-phosphate gDNA,14-30 nucleotides in length,in the presence of both Mn2+and Mg2+ions.PrAgo demonstrates DNA cleavage activity over a broad pH range(pH 4-12),with optimal activity at pH 11.As a mesophilic enzyme,PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65°C,particularly at 65°C.PrAgo does not show strong preferences for the 5′-nucleotide in gDNA.It shows high tolerance for single-base mismatches,except at positions 13 and 15 of gDNA.Continuous double-nucleotide mismatches at positions 10-16 of gDNA significantly reduce cleavage activity.Furthermore,PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65°C.Additionally,molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency.These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology.
基金NIDA (DAll284). M. X. is a National Alliance for Research on Schizophrenia and Depression investigator and issupported by NI
文摘Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.
文摘The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.
基金The South African National Research Foundation(NRF,GUNs 81768,81692,68339,85981 and 77954)Poliomyelitis Research Foundation+1 种基金Claude Leon Foundation(SAN)The University of the Witwatersrand Research Council(BM)and Medical Research Council
文摘Liver diseases are one of the leading causes of mortality in the world. The hepatic illnesses, which include inherited metabolic disorders, hemophilias and viralhepatitides, are complex and currently difficult to treat. The maturation of gene therapy has heralded new avenues for developing effective intervention for these diseases. DNA modification using gene therapy is now possible and available technology may be exploited to achieve long term therapeutic benefit. The ability to edit DNA sequences specifically is of paramount importance to advance gene therapy for application to liver diseases. Recent development of technologies that allow for this has resulted in rapid advancement of gene therapy to treat several chronic illnesses. Improvements in application of derivatives of zinc finger proteins(ZFPs), transcription activator-like effectors(TALEs), homing endonucleases(HEs) and clustered regularly interspaced palindromic repeats(CRISPR) and CRISPR associated(Cas) systems have been particularly important. These sequence-specific technologies may be used to modify genes permanently and also to alter gene transcription for therapeutic purposes. This review describes progress in development of ZFPs, TALEs, HEs and CRISPR/Cas for application to treating liver diseases.
基金financially supported by the National Natural Science Foundation of China(No.31361140364)the National Major Project for Developing New GM Crops(No.2016ZX080009-001)the Agricultural Science and Technology Innovation Program(ASTIP)of CAAS to Chuanxiao Xie
文摘Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases(RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were2.6% and 2.2% in the Arabidopsis FLOWERING TIME(At FT) and TERMINAL FLOWER 1(At TFL1)loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.
基金Project supported by the Hi-Tech Research and Development (863) Program of China (No. 2007AA02Z151)the National Natural Science Foundation of China (No. 30872223)
文摘Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonu-clease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, cir-cularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.
基金supported by the National Science and Technology Major Project (2018ZX097110003-005-002)the Key-Area Research and Development Program of Guangdong Province (2022B1111020002)+3 种基金the National Natural Science Foundation of China (NSFC) (32170159,and 82174055)supported by the National Science Fund for Distinguished Young Scholars (81925025)the Innovative Research Group (81621005)of the NSFCthe Innovation Fund for Medical Sciences (2019-I2M-5-049)of the Chinese Academy of Medical Sciences.
文摘Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(BXM),was approved in Japan and the US in 2018 and several other countries subsequently.Along with the clinical use of BXM,the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern.Herein,we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A,an analogue of BXM.The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes,including pH1N1,H3N2,H7N9 and H9N2,in MDCK cells,and the 50%effective concentration(EC_(50))was calculated to nanomole level and comparable to that of baloxavir acid(BXA),the active form of BXM.Furthermore,in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice,with reduced viral RNA loads and alleviated pulmonary damage.Importantly,serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage.Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA.Taken together,our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance,which provides critical clues for future drug development and drug resistance surveillance.
基金Supported by National Natural Science Foundation of China,No.81471670 and No.81102711the International Cooperative Project of Shaanxi Province,China,No.2013KW-32-01the Fundamental Research Funds for the Central Universities,China and Specialized Research Fund of the Second Affiliated Hospital of Xi'an Jiaotong University,China,No.RC(GG)201203
文摘AIM:To evaluate the relationship between apurinic endonuclease 1(APE1) Asp148 Glu polymorphism and the susceptibility to gastrointestinal(GI) cancers.METHODS:We searched Pub Med, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure(CNKI) databases updated on July 15, 2014 for relevant studies.Only case-control studies comparing APE1 Asp148 Glu polymorphism and GI cancer risk were included.We excluded studies reporting only standardized incidence ratios without control groups and those without detailed genotyping data.Meta-analysis was performed on 17 studies involving 4856 cancer patients and 6136 cancer-free controls.Review Manager version 5.1 was used to perform the meta-analysis.The pooled odds ratios(ORs) and 95% confidence intervals(CIs) were estimated under the allele contrast, homozygous, heterozygous, dominant and recessive genetic models.We also conducted subgroup analyses stratified by ethnicity and cancer type.Publication bias was evaluated using Begg's test.RESULTS:The meta-analysis showed a significant association between APE1 Asp148Glu polymorphism and GI cancer risk in three genetic models in the overall population(G vs T:OR=1.18;95%CI:1.05-1.32;TG vs TT:OR=1.28;95%CI:1.08-1.52;TG+GG vs TT:OR=1.32;95%CI:1.10-1.57).Stratified analysis by ethnicity revealed a statistically increased GI cancer risk in Asians(G vs T:OR=1.27;95%CI:1.07-1.51;GG vs TT:OR=1.58;95%CI:1.05-2.38;TG vs TT:OR=1.30;95%CI,1.01-1.67;and TG+GG vs TT:OR=1.38;95%CI:1.07-1.78),but not in Caucasians.Furthersubgroup analysis by cancer type indicated that APE1Asp148Glu polymorphism may contribute to gastric cancer risk.However,Asp148Glu has no significant association with colorectal or esophageal cancer risk in any genetic model.CONCLUSION:This meta-analysis suggests that the APE1 Asp148Glu polymorphism G allele is associated with an increased GI cancer risk,especially in gastric cancer.
基金a grant from the National 9th Five-Year Plan Key Project (No. 96-906-01-18) and the National Natural Science Foundation of China
文摘Objectives: To investigate the methylation status of the CDKN2/pl6 gene 5′-CpG island, and study the relationship between the CDKN2/pl6 gene inactivation by methylation and lung cancer. Methods: Genomic DNA extracted from the specimens of lung cancer and normal lung tissue was digested with methylation-sensitive endonucleases, and Southern blotting was used to analyze the methylation status of the CDKN2/pl6 gene in 89 cases of lung cancer and 10 cases of normal lung tissue. Results: In 89 cases of lung cancer studied, the CDKN2/pl6 gene was shown to be methylated in 21 cases with the total methylation rate of 23.6% (21/89), in which there were 15 cases (16.9%) methylated atSmaI sites, 12 cases (13.5%) atSmaI sites and 6 cases at bothSmaI andSacII sites. The methylation of the CDKN2/pl6 gene occurred in 17 among 42 of pl6 protein negative cases of lung cancer with a rate of 40.5% (17/42), and in 3 among 47 of pl6 protein positive cases with a rate of 6.4% (3/47). The CDKN2/pl6 gene was not methylated in 10 cases of normal lung tissue. Conclusion: The aberrant methylation of the CDKN2/pl6 gene 5′-CpG island is probably an important mechanism of the gene inactivation, it may be involved in the genesis and progress of lung cancer.
文摘AIM:To determine hepatic expression of apurinic/apyrimidinic endonuclease 1(APE-1)and 8-hydroxydeoxyguanosine(8-OHdG)in patients with chronic hepatitis B and C.METHODS:Liver biopsies were obtained from 27 patients with chronic hepatitis B virus(HBV),30 with chronic hepatitis C virus(HCV),6 with autoimmune hepatitis(AIH),and 6 with primary biliary cirrhosis(PBC).Normal liver tissue was obtained from surgical resection specimens of four patients.Hepatic APE-1 protein and mRNA expression were assayed by Western blot and by real-time polymerase chain reaction,respectively.Hepatocellular APE-1 and 8-OHdG expression were determined by immunohistochemistry.RESULTS:The staining intensity of hepatocellular nuclear APE-1 was lower in the HBV group than in the other groups(P < 0.05).Hepatic APE-1 protein levels were reduced in the HBV group relative to the other groups.Hepatic APE-1 mRNA levels were also lower in the HBV group.The proportion of hepatocytes with 8-OHdG-positive nuclei was increased in the HCV,AIH and PBC groups(P < 0.05),but not in the HBV group.Hepatocellular nuclear APE-1 levels were positively correlated with hepatocellular 8-OHdG levels in both the HBV and HCV groups(HBV,r = 0.34,P < 0.05;HCV,r = 0.54,P < 0.01).CONCLUSION:An imbalance between oxidative DNA damage and APE-1 expression may contribute to hepatocarcinogenesis in chronic viral hepatitis.
基金the National Natural Science Foundation of China (Nos. 82172372 and 21904045)COVID-19 Pneumonia Emergency Scientific Research Special Fund of Wuhan (No. EX20D03)the Fundamental Research Funds for the Central Universities (Nos. 2019kfy XJJS169 and 2021yjs CXCY127)。
文摘DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly related to its length. This intriguing phenomenon only appears at the interfaces of DNAfunctionalized gold nanoparticles. We systematically investigated the causes of this phenomenon. We conjectured that the DNAzyme with extended nucleotides that do not match its substrate strand is vulnerable to non-specific adsorption, electrostatic repulsion, and steric hindrance. Based on our improved understanding of this phenomenon, we have successfully developed a highly sensitive and specific amplifiable biosensor to detect human apurinic/apyrimidinic endonuclease 1.
