Objectives:Cancer treatment relies heavily on accurate diagnosis and effective monitoring of the disease.These processes often involve invasive procedures,such as colonoscopy,to detect malignant tissues,followed by mo...Objectives:Cancer treatment relies heavily on accurate diagnosis and effective monitoring of the disease.These processes often involve invasive procedures,such as colonoscopy,to detect malignant tissues,followed by molecular analyses to determine relevant biomarkers.This study aimed to evaluate the clinical performance of droplet digital PCR(ddPCR)for detecting Kirsten Rat Sarcoma Viral Proto-Oncogene(KRAS),Neuroblastoma RAS Viral Oncogene Homolog(NRAS),and B-Raf Murine Sarcoma Viral Oncogene Homolog B(BRAF)mutations in circulating tumor DNA(ctDNA)from colorectal cancer patients using liquid biopsy.Methods:ctDNA was isolated from colorectal cancer(CRC)patients(n=110)and analyzed for KRAS,BRAF,and NRAS mutations.The ctDNA obtained through liquid biopsy was analyzed using ddPCR,and the findings were compared with sequencing data from tumor DNA archived in formalin-fixed paraffin-embedded(FFPE)blocks.Results:For KRAS mutations,ddPCR achieved a sensitivity of 72.0%and a specificity of 71.4%.However,when pooling all target mutations(KRAS,NRAS and BRAF),the overall sensitivity and specificity were lower,at 48.3%and 51.1%,respectively.Conclusion:The results of this study indicate that the ddPCR analysis of ctDNA may provide complementary information for the molecular diagnosis of CRC patients.展开更多
p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners.The hierarchical order and subcellular location of these events are still poorly understood.T...p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners.The hierarchical order and subcellular location of these events are still poorly understood.The activation of p53 during the DNA damage response(DDR)requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53.This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis.Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53.A single synonymous mutation in p53 codon 22(L22L)prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress.The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2,which requires its interaction with the ribosomal proteins RPL5 and RPL11.These results show how the ATM kinase phosphorylates the p53 protein while it is bang synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.展开更多
基金funded by the Ministry of Health of the Czech Republic—conceptual development of research organization(MMCI,00209805)Czech Science Foundation(No.25-15990S)+1 种基金the project 7D241003 EUREKA EUROSTARS35897,project SALVAGE(P JAC,reg.No.CZ.02.01.01/00/22_008/0004644)—funded by the European Unionby the State Budget of the Czech Republic,and by the LRI project BBMRI.cz(Nos.LM2023033 and CZ.02.1.01/0.0/0.0/16_013/0001674.).
文摘Objectives:Cancer treatment relies heavily on accurate diagnosis and effective monitoring of the disease.These processes often involve invasive procedures,such as colonoscopy,to detect malignant tissues,followed by molecular analyses to determine relevant biomarkers.This study aimed to evaluate the clinical performance of droplet digital PCR(ddPCR)for detecting Kirsten Rat Sarcoma Viral Proto-Oncogene(KRAS),Neuroblastoma RAS Viral Oncogene Homolog(NRAS),and B-Raf Murine Sarcoma Viral Oncogene Homolog B(BRAF)mutations in circulating tumor DNA(ctDNA)from colorectal cancer patients using liquid biopsy.Methods:ctDNA was isolated from colorectal cancer(CRC)patients(n=110)and analyzed for KRAS,BRAF,and NRAS mutations.The ctDNA obtained through liquid biopsy was analyzed using ddPCR,and the findings were compared with sequencing data from tumor DNA archived in formalin-fixed paraffin-embedded(FFPE)blocks.Results:For KRAS mutations,ddPCR achieved a sensitivity of 72.0%and a specificity of 71.4%.However,when pooling all target mutations(KRAS,NRAS and BRAF),the overall sensitivity and specificity were lower,at 48.3%and 51.1%,respectively.Conclusion:The results of this study indicate that the ddPCR analysis of ctDNA may provide complementary information for the molecular diagnosis of CRC patients.
文摘p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners.The hierarchical order and subcellular location of these events are still poorly understood.The activation of p53 during the DNA damage response(DDR)requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53.This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis.Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53.A single synonymous mutation in p53 codon 22(L22L)prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress.The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2,which requires its interaction with the ribosomal proteins RPL5 and RPL11.These results show how the ATM kinase phosphorylates the p53 protein while it is bang synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.
