With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a c...With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.展开更多
Objectives:Monitoring of Cancer Antigen 125(CA125)during ovarian cancer(OC)maintenance treatment with poly(ADP-ribose)polymerase inhibitors(PARPis)may be insufficient when using Gynecologic Cancer Intergroup(GCIG)bioc...Objectives:Monitoring of Cancer Antigen 125(CA125)during ovarian cancer(OC)maintenance treatment with poly(ADP-ribose)polymerase inhibitors(PARPis)may be insufficient when using Gynecologic Cancer Intergroup(GCIG)biochemical progression criteria.This study aimed to evaluate the usefulness of CA125 monitoring in detecting OC recurrence during PARPis maintenance treatment.Methods:This multicenter retrospective cohort study included patients with primary OC who achieved complete or partial response after first-line platinum-based chemotherapy followed by PARPis maintenance treatment.Progressionwas defined using Response EvaluationCriteria in Solid Tumors(RECIST)and GCIG biochemical criteria.New biochemical progression definitions,based on CA125 nadir determined using receiver operating characteristic(ROC)curve analysis,were proposed.Concordance between radiological and biochemical progression was assessed.Results:Of 142 patients,progression was detected in 54(38.03%)and 29(20.42%)using RECIST and GCIG criteria,respectively.The sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)of the GCIG criteria were 53.70%[95%confidence interval(CI):39.61%–67.38%],100.00%[95%CI:95.91%–100.00%],100.00%[95%CI:88.10%–100.00%]and 77.88%[95%CI:72.54%–82.43%],respectively.A cut-off of 1.59×nadir achieved 88.90%sensitivity and 87.20%specificity[Area Under Curve(AUC):91.10%,95%CI:84.70%–97.40%]with a false positive rate(FPR)of 12.67%.Defining biochemical progression as an increase in CA125 of≥3×nadir achieved sensitivity,specificity,PPV,NPV,and FPR of 79.63%[95%CI:66.47%–89.37%],98.86%[95%CI:93.83%–99.97%],97.73%[95%CI:85.91%–99.67%],88.78%[95%CI:82.35%–93.06%],and 1.14%,respectively.Diagnostic accuracy was higher using the≥3×nadir criterion compared with GCIG definition(91.55%vs.82.39%).Conclusion:GCIG biochemical progression criteria during PARPis maintenance treatment after first-line chemotherapymissed 46.3%of progressing patients.Anewcriterion—CA125≥3×nadir—improves sensitivity and NPV,while maintaining high specificity,offering a simple and practical approach for clinical implementation.展开更多
BACKGROUND Recurrence remains the leading cause of poor prognosis in hepatocellular carcinoma(HCC),particularly among patients infected with hepatitis B virus(HBV).The telomerase reverse transcriptase(TERT)promoter is...BACKGROUND Recurrence remains the leading cause of poor prognosis in hepatocellular carcinoma(HCC),particularly among patients infected with hepatitis B virus(HBV).The telomerase reverse transcriptase(TERT)promoter is the most frequently mutated site in HBV-related HCC;however,its prognostic significance is not fully established.AIM To evaluate the prognostic impact of TERT promoter mutations and efficiency of digital polymerase chain reaction(dPCR).METHODS A total of 66 HBV-related HCC patients who underwent hepatectomy were enrolled in this study.DNA extracted from fresh tumor tissues was analyzed for TERT promoter mutations using Sanger sequencing and dPCR.The dPCR assay was optimized by adding 7-deaza-dGTP,CviQ1,and ethylenediaminetetraacetic acid to improve detection sensitivity.Concordance between methods was assessed,and nomogram survival prediction models were developed to evaluate prognostic value based on mutation status.RESULTS TERT promoter mutations were detected in 26/66(39.39%)cases by Sanger sequencing and 30/66(45.45%)by dPCR.The two methods showed high concordance(93.939%,κ=0.876),with dPCR demonstrating 100%sensitivity and 90%specificity.Patients harboring TERT promoter mutations exhibited reduced overall survival and higher recurrence risk.Nomogram models successfully distinguished mutant from non-mutant cases for both overall survival(C-index:0.7651)and disease-free survival(C-index:0.6899).CONCLUSION TERT promoter mutation predicts poor prognosis in HBV-related HCC and serves as a biomarker for risk stratification.Optimized dPCR outperforms Sanger sequencing,and nomograms with TERT status guide precision therapy.展开更多
Pamiparib is a potent and selective oral poly(adenosine diphosphate(ADP)-ribose)-polymerase(PARP)1/2inhibitor(PARPi).Pamiparib has good bioavailability and shows greater cytotoxic potency and similar DNA-trapping capa...Pamiparib is a potent and selective oral poly(adenosine diphosphate(ADP)-ribose)-polymerase(PARP)1/2inhibitor(PARPi).Pamiparib has good bioavailability and shows greater cytotoxic potency and similar DNA-trapping capacity compared to olaparib.It is not affected by adenosine triphosphate(ATP)-binding cassette transporters.展开更多
BACKGROUND Although the relationship between somatic DNA polymerase epsilon(POLE)exonuclease domain mutations(EDMs)and colorectal cancer(CRC)is well established,the role of POLE non-EDMs in CRC remains unclear.AIM To ...BACKGROUND Although the relationship between somatic DNA polymerase epsilon(POLE)exonuclease domain mutations(EDMs)and colorectal cancer(CRC)is well established,the role of POLE non-EDMs in CRC remains unclear.AIM To identify POLE non-EDMs and EDMs in CRC,and to determine their associations with accompanying mutations and microsatellite instability(MSI).METHODS In this retrospective study,next-generation sequencing was performed using a targeted colon cancer panel(Qiagen,DHS-003Z)on 356 CRC patients.Of these,191 patients were found to carry POLE mutations.For these patients,MSI status was assessed using both real-time PCR(EasyPGX^(■)Ready MSI kit)and immunohistochemistry,and accompanying somatic mutations were investigated.RESULTS POLE mutations were identified in 53.65%of the CRC patients.Among the POLE-mutant patients,87.96%were classified as pMMR(MSI-L),and 12.04%as dMMR(MSI-H).The most frequently observed POLE non-EDM variant was exon 34 c.4337_4338delTG p.V1446fs*3.The POLE EDMs were present in exon 14,with two specific variants p.Y458F(0.52%)and p.Y468N(0.52%).The most common pathogenic variants accompanying the POLE mutations were in MLH3,MSH3,KRAS,PIK3CA,and BRAF genes.POLE mutations were associated with a high mutational burden and MSI in CRC,particularly in the dMMR phenotype.This association suggests that POLE mutations may serve as important biomarkers for understanding the genetic profile of the disease and may be used in the clinical management of CRC.CONCLUSION POLE mutations,especially non-EDMs,are frequent in MSI-L CRC and often co-occur with MLH3,MSH3,KRAS,PIK3CA,and BRAF,highlighting their potential role in tumor biology and as biomarkers for personalized treatment.Functional validation and multicenter studies are needed.展开更多
OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first ...OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first predicted by network pharmacology and molecular docking,then the possible signaling pathways were systematically analyzed.Finally,the potential therapeutic targets and possible signaling pathways of AT-I on PI-IBS in Sprague-Dawley(SD)rat model were verified by experiments.RESULTS:AT-I could alleviate PI-IBS symptoms and reduce the expression of tumor necrosis factorα,interleukin-6 and Interferon-gamma in PI-IBS SD rat model and inhibit the c-Jun N-terminal kinase/inducible nitric oxide synthase(JNK/iNOS)pathway.Notably,AT-I treatment could inhibit the overexpression of polymeraseⅠand transcript release factor(PTRF).CONCLUSION:AT-I could alleviate PI-IBS symptoms through downregulation of PTRF and inhibiting the JNK/iNOS pathway.This study not only provides a scientific basis to clarify the anti-PI-IBS effect of AT-I and its mechanism but also suggests a novel promising therapeutic strategy to treat the PI-IBS.展开更多
With rapid developments of emerging technologies like synthetic biology,the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly.Thus,ration...With rapid developments of emerging technologies like synthetic biology,the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly.Thus,rational optimization of the performance of DNA polymerase is of great interest.Nuclear magnetic resonance(NMR)spectroscopy is a powerful technique used for studying protein structure and dynamics.It provides the atomic resolution information of enzymes under their functional solution environment to reveal the active sites(hot spots)of the enzyme,which could be further used for optimizing the performance of enzymes.In our previous work,we identified hot spot residues of Pyrococcus furiosus DNA polymerase(Pfu pol).We aim to employ these binding hot spots to screen for co-factors of Pfu pol,particularly targeting those molecules exhibiting weak intermolecular interactions.To validate this concept,we first demonstrated the feasibility of utilizing hot spot residues as screening probes for auxiliary factors by employing the well-characterized Tween-20 as a model system.Employing these hot spots as probes,two new co-factors,the heat shock protein TkHSP20 from Thermococcus Kodakaraensis and the chemical chaperone L-arginine,are identified to interact with Pfu pol to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu pol.This NMR-based approach requires no prior assignment information of target enzymes,guiding the rational exploration of novel cofactors for Pfu pol.Moreover,our approach is not dependent on structural data or bioinformatics.Therefore,it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.展开更多
DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbit...DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.展开更多
Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Metho...Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.展开更多
AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing techn...AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.展开更多
In non-small cell lung cancer(NSCLC),poly(ADP-ribose)polymerase 1(PARP1)induces genomic instability and promotes tumor progression by impairing DNA repair pathways.Although PARP1-targeting proteolysis-targeting chimer...In non-small cell lung cancer(NSCLC),poly(ADP-ribose)polymerase 1(PARP1)induces genomic instability and promotes tumor progression by impairing DNA repair pathways.Although PARP1-targeting proteolysis-targeting chimeras(PROTACs)offer a promising strategy for selective protein degradation,their clinical application remains limited by poor water solubility and insufficient tumor selectivity.Here,we report a pHresponsive magnetic nanoparticle system co-delivering β-lapachone(β-lap)and a PARP1-targeted PROTAC(PRO)for synergistic and tumor-targeting therapy.Designed with a hydrophobic self-assembled core and a magnetic coating,the nanoparticle(NP_(β-lap+PRO))enables pHresponsive drug release and magnetic resonance imaging(MRI)monitoring.β-Lap is a bioactivated drug that relies on NAD(P)H:quinone oxidoreductase 1(NQO1),which is overexpressed in NSCLC cells.It has the potential to deliver tumor-selective DNA damage and induce cell death.The NP_(β-lap+PRO) exploits elevated NQO1 levels in NSCLC to initiate β-lap-driven oxidative stress and DNA damage,while simultaneously enhancing PROTAC-mediated PARP1 degradation within the acidic tumor microenvironment synergistically induces apoptosis.In A549 NSCLC tumor models,this system effectively induces PARP1 degradation,blocks DNA repair,and preserves NAD(P)H pools,thereby amplifying β-lapinduced reactive oxygen species production,leading to enhanced DNA double-strand breaks and apoptosis.This study presents a biomarker-driven nanotherapeutic strategy that integrates PROTAC technology with redox-targeted combination therapy,offering a promising approach for precision treatment of NSCLC.展开更多
目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设...目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。展开更多
AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carr...AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.展开更多
Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-...Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.展开更多
AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis ...AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.展开更多
AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic...AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P＜0.05) and surgical specimens with lymph node metastasis (P＜0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P＜0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P＜0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P＜0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.展开更多
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195)。
文摘With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.
文摘Objectives:Monitoring of Cancer Antigen 125(CA125)during ovarian cancer(OC)maintenance treatment with poly(ADP-ribose)polymerase inhibitors(PARPis)may be insufficient when using Gynecologic Cancer Intergroup(GCIG)biochemical progression criteria.This study aimed to evaluate the usefulness of CA125 monitoring in detecting OC recurrence during PARPis maintenance treatment.Methods:This multicenter retrospective cohort study included patients with primary OC who achieved complete or partial response after first-line platinum-based chemotherapy followed by PARPis maintenance treatment.Progressionwas defined using Response EvaluationCriteria in Solid Tumors(RECIST)and GCIG biochemical criteria.New biochemical progression definitions,based on CA125 nadir determined using receiver operating characteristic(ROC)curve analysis,were proposed.Concordance between radiological and biochemical progression was assessed.Results:Of 142 patients,progression was detected in 54(38.03%)and 29(20.42%)using RECIST and GCIG criteria,respectively.The sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)of the GCIG criteria were 53.70%[95%confidence interval(CI):39.61%–67.38%],100.00%[95%CI:95.91%–100.00%],100.00%[95%CI:88.10%–100.00%]and 77.88%[95%CI:72.54%–82.43%],respectively.A cut-off of 1.59×nadir achieved 88.90%sensitivity and 87.20%specificity[Area Under Curve(AUC):91.10%,95%CI:84.70%–97.40%]with a false positive rate(FPR)of 12.67%.Defining biochemical progression as an increase in CA125 of≥3×nadir achieved sensitivity,specificity,PPV,NPV,and FPR of 79.63%[95%CI:66.47%–89.37%],98.86%[95%CI:93.83%–99.97%],97.73%[95%CI:85.91%–99.67%],88.78%[95%CI:82.35%–93.06%],and 1.14%,respectively.Diagnostic accuracy was higher using the≥3×nadir criterion compared with GCIG definition(91.55%vs.82.39%).Conclusion:GCIG biochemical progression criteria during PARPis maintenance treatment after first-line chemotherapymissed 46.3%of progressing patients.Anewcriterion—CA125≥3×nadir—improves sensitivity and NPV,while maintaining high specificity,offering a simple and practical approach for clinical implementation.
基金Supported by National Key Research and Development Program of China,No.2023YFF0613304Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences,No.2023-I2M-2-004,No.2024-I2M-C&T-B-069,and No.2025-I2M-C&T-B-057.
文摘BACKGROUND Recurrence remains the leading cause of poor prognosis in hepatocellular carcinoma(HCC),particularly among patients infected with hepatitis B virus(HBV).The telomerase reverse transcriptase(TERT)promoter is the most frequently mutated site in HBV-related HCC;however,its prognostic significance is not fully established.AIM To evaluate the prognostic impact of TERT promoter mutations and efficiency of digital polymerase chain reaction(dPCR).METHODS A total of 66 HBV-related HCC patients who underwent hepatectomy were enrolled in this study.DNA extracted from fresh tumor tissues was analyzed for TERT promoter mutations using Sanger sequencing and dPCR.The dPCR assay was optimized by adding 7-deaza-dGTP,CviQ1,and ethylenediaminetetraacetic acid to improve detection sensitivity.Concordance between methods was assessed,and nomogram survival prediction models were developed to evaluate prognostic value based on mutation status.RESULTS TERT promoter mutations were detected in 26/66(39.39%)cases by Sanger sequencing and 30/66(45.45%)by dPCR.The two methods showed high concordance(93.939%,κ=0.876),with dPCR demonstrating 100%sensitivity and 90%specificity.Patients harboring TERT promoter mutations exhibited reduced overall survival and higher recurrence risk.Nomogram models successfully distinguished mutant from non-mutant cases for both overall survival(C-index:0.7651)and disease-free survival(C-index:0.6899).CONCLUSION TERT promoter mutation predicts poor prognosis in HBV-related HCC and serves as a biomarker for risk stratification.Optimized dPCR outperforms Sanger sequencing,and nomograms with TERT status guide precision therapy.
基金supported in part by funding from BeiGene,Ltd.,USA(Grant No.:KPR081)with additional support from the Alessandra Bono Foundation,Italy.
文摘Pamiparib is a potent and selective oral poly(adenosine diphosphate(ADP)-ribose)-polymerase(PARP)1/2inhibitor(PARPi).Pamiparib has good bioavailability and shows greater cytotoxic potency and similar DNA-trapping capacity compared to olaparib.It is not affected by adenosine triphosphate(ATP)-binding cassette transporters.
文摘BACKGROUND Although the relationship between somatic DNA polymerase epsilon(POLE)exonuclease domain mutations(EDMs)and colorectal cancer(CRC)is well established,the role of POLE non-EDMs in CRC remains unclear.AIM To identify POLE non-EDMs and EDMs in CRC,and to determine their associations with accompanying mutations and microsatellite instability(MSI).METHODS In this retrospective study,next-generation sequencing was performed using a targeted colon cancer panel(Qiagen,DHS-003Z)on 356 CRC patients.Of these,191 patients were found to carry POLE mutations.For these patients,MSI status was assessed using both real-time PCR(EasyPGX^(■)Ready MSI kit)and immunohistochemistry,and accompanying somatic mutations were investigated.RESULTS POLE mutations were identified in 53.65%of the CRC patients.Among the POLE-mutant patients,87.96%were classified as pMMR(MSI-L),and 12.04%as dMMR(MSI-H).The most frequently observed POLE non-EDM variant was exon 34 c.4337_4338delTG p.V1446fs*3.The POLE EDMs were present in exon 14,with two specific variants p.Y458F(0.52%)and p.Y468N(0.52%).The most common pathogenic variants accompanying the POLE mutations were in MLH3,MSH3,KRAS,PIK3CA,and BRAF genes.POLE mutations were associated with a high mutational burden and MSI in CRC,particularly in the dMMR phenotype.This association suggests that POLE mutations may serve as important biomarkers for understanding the genetic profile of the disease and may be used in the clinical management of CRC.CONCLUSION POLE mutations,especially non-EDMs,are frequent in MSI-L CRC and often co-occur with MLH3,MSH3,KRAS,PIK3CA,and BRAF,highlighting their potential role in tumor biology and as biomarkers for personalized treatment.Functional validation and multicenter studies are needed.
基金The University Collaborative Innovation Project of Anhui:Creation of a Combined Animal Model of Coronary Heart Disease based on the Theory of Xin'an Medicine(No.GXXT-2020-024)Start-up Funding for Doctoral Research at Wannan Medical College(WYRCQD2018009)Horizontal Project of South Anhui Medical College(H202003)。
文摘OBJECTIVE:To explore the therapeutic effect and target of atractylenolide I(AT-I)on post-infectious irritable bowel syndrome(PI-IBS)rats.METHODS:Therefore,the preliminarily mechanism of AT-I in anti-PI-IBS were first predicted by network pharmacology and molecular docking,then the possible signaling pathways were systematically analyzed.Finally,the potential therapeutic targets and possible signaling pathways of AT-I on PI-IBS in Sprague-Dawley(SD)rat model were verified by experiments.RESULTS:AT-I could alleviate PI-IBS symptoms and reduce the expression of tumor necrosis factorα,interleukin-6 and Interferon-gamma in PI-IBS SD rat model and inhibit the c-Jun N-terminal kinase/inducible nitric oxide synthase(JNK/iNOS)pathway.Notably,AT-I treatment could inhibit the overexpression of polymeraseⅠand transcript release factor(PTRF).CONCLUSION:AT-I could alleviate PI-IBS symptoms through downregulation of PTRF and inhibiting the JNK/iNOS pathway.This study not only provides a scientific basis to clarify the anti-PI-IBS effect of AT-I and its mechanism but also suggests a novel promising therapeutic strategy to treat the PI-IBS.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences XDB0540000Natural Science Foundation of China grants 22327901,22174151 and 21991080Hubei Provincial Natural Science Foundation of China 2023AFA041。
文摘With rapid developments of emerging technologies like synthetic biology,the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly.Thus,rational optimization of the performance of DNA polymerase is of great interest.Nuclear magnetic resonance(NMR)spectroscopy is a powerful technique used for studying protein structure and dynamics.It provides the atomic resolution information of enzymes under their functional solution environment to reveal the active sites(hot spots)of the enzyme,which could be further used for optimizing the performance of enzymes.In our previous work,we identified hot spot residues of Pyrococcus furiosus DNA polymerase(Pfu pol).We aim to employ these binding hot spots to screen for co-factors of Pfu pol,particularly targeting those molecules exhibiting weak intermolecular interactions.To validate this concept,we first demonstrated the feasibility of utilizing hot spot residues as screening probes for auxiliary factors by employing the well-characterized Tween-20 as a model system.Employing these hot spots as probes,two new co-factors,the heat shock protein TkHSP20 from Thermococcus Kodakaraensis and the chemical chaperone L-arginine,are identified to interact with Pfu pol to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu pol.This NMR-based approach requires no prior assignment information of target enzymes,guiding the rational exploration of novel cofactors for Pfu pol.Moreover,our approach is not dependent on structural data or bioinformatics.Therefore,it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.
文摘DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.
文摘Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.
文摘AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.
基金supported by the Key Laboratory of Advanced Interdisciplinary Studies,The First Affiliated Hospital of Guangzhou Medical University of China(No.2023A03J0355)OpenProject of State Key Laboratory of Respiratory Disease of China(No.SKIRD OP-202311)Guangdong Provincial Zhong Nanshan Medical Foundation of China(No.ZNS-XS-ZZ-202409-007).
文摘In non-small cell lung cancer(NSCLC),poly(ADP-ribose)polymerase 1(PARP1)induces genomic instability and promotes tumor progression by impairing DNA repair pathways.Although PARP1-targeting proteolysis-targeting chimeras(PROTACs)offer a promising strategy for selective protein degradation,their clinical application remains limited by poor water solubility and insufficient tumor selectivity.Here,we report a pHresponsive magnetic nanoparticle system co-delivering β-lapachone(β-lap)and a PARP1-targeted PROTAC(PRO)for synergistic and tumor-targeting therapy.Designed with a hydrophobic self-assembled core and a magnetic coating,the nanoparticle(NP_(β-lap+PRO))enables pHresponsive drug release and magnetic resonance imaging(MRI)monitoring.β-Lap is a bioactivated drug that relies on NAD(P)H:quinone oxidoreductase 1(NQO1),which is overexpressed in NSCLC cells.It has the potential to deliver tumor-selective DNA damage and induce cell death.The NP_(β-lap+PRO) exploits elevated NQO1 levels in NSCLC to initiate β-lap-driven oxidative stress and DNA damage,while simultaneously enhancing PROTAC-mediated PARP1 degradation within the acidic tumor microenvironment synergistically induces apoptosis.In A549 NSCLC tumor models,this system effectively induces PARP1 degradation,blocks DNA repair,and preserves NAD(P)H pools,thereby amplifying β-lapinduced reactive oxygen species production,leading to enhanced DNA double-strand breaks and apoptosis.This study presents a biomarker-driven nanotherapeutic strategy that integrates PROTAC technology with redox-targeted combination therapy,offering a promising approach for precision treatment of NSCLC.
基金This work is supported by the National Natural Science Foundation of China(30070958).
文摘目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。
基金Supported by Research Fund for the Control of Infectious Diseases and Research Grant Committee of Hong Kong Government
文摘AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.
文摘Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.
基金The paper was support by a grant from the Ministry Youth Research of China,No.98-1-269
文摘AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.
基金Project supported by the zhejiang Natural Scierce Fundation No.925006.
文摘AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P＜0.05) and surgical specimens with lymph node metastasis (P＜0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P＜0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P＜0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P＜0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.
