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
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
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
Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitiv...Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15–38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.展开更多
The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chlo...The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB), diisopropyl fluorophosphate (DEP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3′-sulfonate (woodward's reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity ofPst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant,A and the microcosmic inhibition rate constants,k +0 andk′ +0 of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration ofPst I conformation and then influence the ability ofPst I recognizing and incising DNA specifically.展开更多
The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be inf...The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be influenced by th e sin gle alteration of the concentration of Tris·HCl, Mg 2+ or Na+ in the rea ction s ystem, but it can be altered by the reduction of any two of them. The specificit y can not be altered by the single alteration of pH or the replacement of Mg 2+ w ith Mn 2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the rea ction system results in the relaxation of the substrate-specificity of Pst Ⅰ , b ut dim ethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nuc leot ides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6 G→T). Used with the enzyme analysis of an artificially synthetic DNA segment co ntaining a special sequence, the nucleotides of No.1 and 6 have both changed (1C →A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be chan ged from CTGCA↓G to TGCA↓.展开更多
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
In this work, we designed a facile and promising strategy for fluorescence determination of S1 endonuclease activity and inhibition based on fluorescence resonance energy transfer(FRET) between positively Ag nanorods(...In this work, we designed a facile and promising strategy for fluorescence determination of S1 endonuclease activity and inhibition based on fluorescence resonance energy transfer(FRET) between positively Ag nanorods(AgNRs) and negatively-charged ROX-labeled sing-stranded DNA(ROX-ssDNA). In the absence of Sl endonuclease, the fluorescence signal of the ROX-ssDNA was efficiently quenched when the ROX-ssDNA was adsorbed on the surface of AgNRs via strong electrostatic interaction. However, upon addition of different concentration of Sl endonuclease, the fluorescence was gradually restored owing to the reduction of FRET efficiency caused by S1 endonuclease specific cleavage ROX-ssDNA into short fragments, which reduced the electrostatic interaction between AgNRs and short oligonucleotide fragment. This assay strategy exhibited a high sensitivity and excellent specificity for S1 endonuclease with a detection limit of 0.004 U/mL and a dynamic concentration range from 0.01 U/mL to 5.0 U/mL. In addition, the capabilities for screening of S1 endonuclease inhibitors and S1 endonuclease detection from complex biological matrixes were also verified.展开更多
Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their se...Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells.Recently,programmable endonucleases such as clustered regularly interspaced short palindromic repeats(CRISPR)associated protein(Cas)and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions,enabling their detection with deep next generation sequencing(NGS).However,the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage.To overcome these shortcomings,we conceived a new assay(Programmable Enzyme-Assisted Selective Exponential Amplification,PASEA)that combines the cleavage of wild type alleles with concurrent polymerase amplification.While PASEA increases the numbers of both wild type and mutant alleles,the numbers of mutant alleles increase at much greater rates,allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles.By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification,we converted samples with0.01%somatic mutant allele fractions(MAFs)to products with 70%MAFs in a single step within 20 min,enabling inexpensive,rapid genotyping with such as Sanger sequencers.Furthermore,PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes.Real-time PASEA'performance was proved equivalent to clinical amplification refractory mutation system(ARMS)-PCR and NGS when testing over hundred cancer patients'samples.This strategy has the potential to reduce the cost and time of cancer screening and genotyping,and to enable targeted therapies in resource-limited settings.展开更多
Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease dia...Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.展开更多
Objective. To clone complete EcoRII restriction endonuclease gene (ecoRIIR) and methyltransferase gene (ecoRIIM) in one vector and to analyze the coordinating expression of this whole R M system.Methods. Unidirectiona...Objective. To clone complete EcoRII restriction endonuclease gene (ecoRIIR) and methyltransferase gene (ecoRIIM) in one vector and to analyze the coordinating expression of this whole R M system.Methods. Unidirectional deletion subclones were constructed with ExoIII.ecoRIIR/M genes were preliminarily located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were determined by sequencing, and transcriptional start sites were determined by S1 mapping. Results. The DNA fragment which was cloned into pBluescript SK+contained intact ecoRIIR gene andecoRIIM gene, and two transcriptional start sites of ecoRIIR gene were determined. 132bp to 458bp from 3’end of ecoRIIR gene are indispensable to enzyme activities and deletion of 202bp from 3’end of ecoRIIM gene made enzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII.R). Deletion of the coding and flanking sequences of one gene did not affect the expression of the other gene, and the recombinants only containing ecoRIIR gene appeared to be lethal to dcm+host. Conclusion. ecoRIIM gene linking closely to ecoRIIR gene is very important for the existence of the R M system in process of evolution, but the key tocontrol EcoRII R M order may not exist in transcriptional level .展开更多
Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage cause...Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage caused by free radicals. We hypothesized that tea polyphenols repair DNA damage and inhibit neuronal apoptosis during global cerebral ischemia/reperfusion. To test this hypothesis, we employed a rat model of global cerebral ischemia/reperfusion. We demonstrated that intraperitoneal injection of tea polyphenols immediately after reperfusion significantly reduced apoptosis in the hippocampal CA1 region; this effect started 6 hours following reperfusion. Immunohistochemical staining showed that tea polyphenols could reverse the ischemia/reperfusion-induced reduction in the expression of DNA repair proteins, X-ray repair cross-complementing protein 1 and apudnic/apyrimidinic endonuclease/redox factor-1 starting at 2 hours. Both effects lasted at least 72 hours. These experimental findings suggest that tea polyphenols promote DNA damage repair and protect against apoptosis in the brain.展开更多
Amongst endonuclease, the homodimeric variety is found in many prokaryotes for processing of the introns out from pre-RNAs. But as the variety and the complexity of introns rise with evolution, do the homodimeric endo...Amongst endonuclease, the homodimeric variety is found in many prokaryotes for processing of the introns out from pre-RNAs. But as the variety and the complexity of introns rise with evolution, do the homodimeric endonuclease adapt to the changes? The correlations between evolving pre-RNAs and adapting homodimeric endonuclease in lower prokaryotes is investigated in this paper. First, we construct and observe the appearance of a long branch in the phylogeny based on homodimeric endonuclease. To appreciate the finer aspects of accelerating evolution near this long branch, we delve deeper into the pre-RNA substrates of the endonuclease. Computational evidence of an as-yet-unreported noncoding RNA gene then emerges from this study. The capabilities of homodimeric endonuclease and the complexities of its pre-RNA substrates appear to evolve in steps together.展开更多
Apoptosis in single-cell organisms like Trypanosoma or Leishmania was characterized in several studies in the last few years [1]-[4]. Cell death in these caspase lacking protozoa is still poorly understood and a concl...Apoptosis in single-cell organisms like Trypanosoma or Leishmania was characterized in several studies in the last few years [1]-[4]. Cell death in these caspase lacking protozoa is still poorly understood and a conclusive apoptotic pathway has not been identified so far. In the work presented here, we studied the effects of prostaglandin D2 and staurosporine induced cell death in blood-forms of Trypanosoma brucei in a time dependent manner and focused on the role of a nuclease similar to endonuclease G of higher eukaryotes. We found that these parasites undergo apoptotic cell death as demonstrated by the appearance of several canonical hallmarks of apoptosis in higher eukaryotes, but that different stimuli induce remarkable differences in the way these cells die. We compared the effects of prostaglandin D2 and staurosporine in trypanosomes with and without endonuclease G overexpression by flow cytometric and electron microscopic methods with the result that endonuclease G overexpression led to a significant modification of intracellular organelles and accelerated apoptotic cell death in prostaglandin D2 or staurosporine treated cells. Our results demonstrate that different stimuli induce apoptosis even in these ancient organisms in different caspase-independent ways. Whereas central processes of apoptosis like ROS formation, loss of mitochondrial membrane potential, endonuclease G release, phosphatidylserine exposure and DNA fragmentation appeared in the same chronology during treatment with either one of both drugs, other effects like cell cycle arrest or change of cell shape occurred only in the case of prostaglandin D2 or staurosporine treatment. We conclude from these results that trypanosomes react to stimuli of apoptosis with the concerted action of cellular responses but cannot control the final outcome if additional stress, as in the case of staurosporine, is superimposed.展开更多
The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detect...The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.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
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.
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.
文摘Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15–38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.
基金Supported by the Research Fund for the Doctoral Program of Higher Education ( 19990 486 0 1)
文摘The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB), diisopropyl fluorophosphate (DEP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3′-sulfonate (woodward's reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity ofPst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant,A and the microcosmic inhibition rate constants,k +0 andk′ +0 of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration ofPst I conformation and then influence the ability ofPst I recognizing and incising DNA specifically.
基金Supported by the Research Fund for the Doctoral Program of Higher Education.
文摘The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be influenced by th e sin gle alteration of the concentration of Tris·HCl, Mg 2+ or Na+ in the rea ction s ystem, but it can be altered by the reduction of any two of them. The specificit y can not be altered by the single alteration of pH or the replacement of Mg 2+ w ith Mn 2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the rea ction system results in the relaxation of the substrate-specificity of Pst Ⅰ , b ut dim ethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nuc leot ides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6 G→T). Used with the enzyme analysis of an artificially synthetic DNA segment co ntaining a special sequence, the nucleotides of No.1 and 6 have both changed (1C →A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be chan ged from CTGCA↓G to TGCA↓.
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financially supported by the Promotion Project of Basic Ability of Young and Middle-aged Teachers of Universities in Guangxi(No. 2018KY0433)the National Natural Science Foundation of China(Nos.21822407,21405163)
文摘In this work, we designed a facile and promising strategy for fluorescence determination of S1 endonuclease activity and inhibition based on fluorescence resonance energy transfer(FRET) between positively Ag nanorods(AgNRs) and negatively-charged ROX-labeled sing-stranded DNA(ROX-ssDNA). In the absence of Sl endonuclease, the fluorescence signal of the ROX-ssDNA was efficiently quenched when the ROX-ssDNA was adsorbed on the surface of AgNRs via strong electrostatic interaction. However, upon addition of different concentration of Sl endonuclease, the fluorescence was gradually restored owing to the reduction of FRET efficiency caused by S1 endonuclease specific cleavage ROX-ssDNA into short fragments, which reduced the electrostatic interaction between AgNRs and short oligonucleotide fragment. This assay strategy exhibited a high sensitivity and excellent specificity for S1 endonuclease with a detection limit of 0.004 U/mL and a dynamic concentration range from 0.01 U/mL to 5.0 U/mL. In addition, the capabilities for screening of S1 endonuclease inhibitors and S1 endonuclease detection from complex biological matrixes were also verified.
基金supported by China Scholarship CouncilNIH grant to the University of Pennsylvania(No.K011K01TW011190-01A1)+1 种基金NIH grant to the University of Pennsylvania(No.R21CA228614-01A1)Beijing Hope Run Special Fund from the Cancer Foundation of China(Nos.LC2019L04 and LC2020A36)。
文摘Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells.Recently,programmable endonucleases such as clustered regularly interspaced short palindromic repeats(CRISPR)associated protein(Cas)and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions,enabling their detection with deep next generation sequencing(NGS).However,the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage.To overcome these shortcomings,we conceived a new assay(Programmable Enzyme-Assisted Selective Exponential Amplification,PASEA)that combines the cleavage of wild type alleles with concurrent polymerase amplification.While PASEA increases the numbers of both wild type and mutant alleles,the numbers of mutant alleles increase at much greater rates,allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles.By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification,we converted samples with0.01%somatic mutant allele fractions(MAFs)to products with 70%MAFs in a single step within 20 min,enabling inexpensive,rapid genotyping with such as Sanger sequencers.Furthermore,PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes.Real-time PASEA'performance was proved equivalent to clinical amplification refractory mutation system(ARMS)-PCR and NGS when testing over hundred cancer patients'samples.This strategy has the potential to reduce the cost and time of cancer screening and genotyping,and to enable targeted therapies in resource-limited settings.
基金financially supported by the National Natural Science Foundation of China(Grant No.:21904045)the Fundamental Research Funds for the Central Universities(HUST:Grant No.:2019kfyXJJS169)Training Program of Innovation and Entrepreneurship for Undergraduates of Hubei Province(Grant No.:S202010487225).
文摘Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.
文摘Objective. To clone complete EcoRII restriction endonuclease gene (ecoRIIR) and methyltransferase gene (ecoRIIM) in one vector and to analyze the coordinating expression of this whole R M system.Methods. Unidirectional deletion subclones were constructed with ExoIII.ecoRIIR/M genes were preliminarily located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were determined by sequencing, and transcriptional start sites were determined by S1 mapping. Results. The DNA fragment which was cloned into pBluescript SK+contained intact ecoRIIR gene andecoRIIM gene, and two transcriptional start sites of ecoRIIR gene were determined. 132bp to 458bp from 3’end of ecoRIIR gene are indispensable to enzyme activities and deletion of 202bp from 3’end of ecoRIIM gene made enzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII.R). Deletion of the coding and flanking sequences of one gene did not affect the expression of the other gene, and the recombinants only containing ecoRIIR gene appeared to be lethal to dcm+host. Conclusion. ecoRIIM gene linking closely to ecoRIIR gene is very important for the existence of the R M system in process of evolution, but the key tocontrol EcoRII R M order may not exist in transcriptional level .
基金supported by the National Natural Science Foundation of China, No. 30571790
文摘Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage caused by free radicals. We hypothesized that tea polyphenols repair DNA damage and inhibit neuronal apoptosis during global cerebral ischemia/reperfusion. To test this hypothesis, we employed a rat model of global cerebral ischemia/reperfusion. We demonstrated that intraperitoneal injection of tea polyphenols immediately after reperfusion significantly reduced apoptosis in the hippocampal CA1 region; this effect started 6 hours following reperfusion. Immunohistochemical staining showed that tea polyphenols could reverse the ischemia/reperfusion-induced reduction in the expression of DNA repair proteins, X-ray repair cross-complementing protein 1 and apudnic/apyrimidinic endonuclease/redox factor-1 starting at 2 hours. Both effects lasted at least 72 hours. These experimental findings suggest that tea polyphenols promote DNA damage repair and protect against apoptosis in the brain.
文摘Amongst endonuclease, the homodimeric variety is found in many prokaryotes for processing of the introns out from pre-RNAs. But as the variety and the complexity of introns rise with evolution, do the homodimeric endonuclease adapt to the changes? The correlations between evolving pre-RNAs and adapting homodimeric endonuclease in lower prokaryotes is investigated in this paper. First, we construct and observe the appearance of a long branch in the phylogeny based on homodimeric endonuclease. To appreciate the finer aspects of accelerating evolution near this long branch, we delve deeper into the pre-RNA substrates of the endonuclease. Computational evidence of an as-yet-unreported noncoding RNA gene then emerges from this study. The capabilities of homodimeric endonuclease and the complexities of its pre-RNA substrates appear to evolve in steps together.
文摘Apoptosis in single-cell organisms like Trypanosoma or Leishmania was characterized in several studies in the last few years [1]-[4]. Cell death in these caspase lacking protozoa is still poorly understood and a conclusive apoptotic pathway has not been identified so far. In the work presented here, we studied the effects of prostaglandin D2 and staurosporine induced cell death in blood-forms of Trypanosoma brucei in a time dependent manner and focused on the role of a nuclease similar to endonuclease G of higher eukaryotes. We found that these parasites undergo apoptotic cell death as demonstrated by the appearance of several canonical hallmarks of apoptosis in higher eukaryotes, but that different stimuli induce remarkable differences in the way these cells die. We compared the effects of prostaglandin D2 and staurosporine in trypanosomes with and without endonuclease G overexpression by flow cytometric and electron microscopic methods with the result that endonuclease G overexpression led to a significant modification of intracellular organelles and accelerated apoptotic cell death in prostaglandin D2 or staurosporine treated cells. Our results demonstrate that different stimuli induce apoptosis even in these ancient organisms in different caspase-independent ways. Whereas central processes of apoptosis like ROS formation, loss of mitochondrial membrane potential, endonuclease G release, phosphatidylserine exposure and DNA fragmentation appeared in the same chronology during treatment with either one of both drugs, other effects like cell cycle arrest or change of cell shape occurred only in the case of prostaglandin D2 or staurosporine treatment. We conclude from these results that trypanosomes react to stimuli of apoptosis with the concerted action of cellular responses but cannot control the final outcome if additional stress, as in the case of staurosporine, is superimposed.
文摘The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.
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.
