Real-time quaking-induced conversion(RT-QuIC)assay is a newly established PrPSc-detecting method.The development of RT-QuIC improves the diagnosis of sporadic Creutzfeldt–Jakob disease(sCJD),showing good sensitivity ...Real-time quaking-induced conversion(RT-QuIC)assay is a newly established PrPSc-detecting method.The development of RT-QuIC improves the diagnosis of sporadic Creutzfeldt–Jakob disease(sCJD),showing good sensitivity and specificity in many countries when the method was used in cerebrospinal fluid(CSF)samples.However,in China,the sensitivity and specificity of RT-QuIC has yet to be determined due to the lack of definitive diagnosis samples.展开更多
Objective The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrPSc in the brain tissues of suspected cases.Thus,developing methods ...Objective The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrPSc in the brain tissues of suspected cases.Thus,developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.Methods We prepared a PrP-specific polyclonal antibody(pAb P54)in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23–231.Thereafter,we verified that pAb in Western blot,immunohistochemistry(IHC),and immunofluorescent(IFA)assays.Results Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein,normal brain PrPC from healthy rodents and humans,and pathological PrPSc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases.The electrophoretic patterns of brain PrPC and PrPSc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies.Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54.IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139 A-infected mice and 263 K-infected hamsters.IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.Conclusion The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and,thus,may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.展开更多
Objective To describe the global profiles of acetylated proteins in the brains of scrapie agents 139Aand ME7-infected mice collected at mid-early,mid-late,and terminal stages.Methods The acetylated proteins from the c...Objective To describe the global profiles of acetylated proteins in the brains of scrapie agents 139Aand ME7-infected mice collected at mid-early,mid-late,and terminal stages.Methods The acetylated proteins from the cortex regions of scrapie agent(139A-and ME7)-infected mice collected at mid-early(80 days postinfection,dpi),mid-late(120 dpi),and terminal(180 dpi) stages were extracted,and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry.The proteins in the infected mice showing 1.5-fold higher or lower levels than that of agematched normal controls were considered as differentially expressed acetylated peptides(DEAPs).Results A total of 118,42,and 51 DEAPs were found in the brains of 139A-80,139A-120,and 139A-180dpi mice,respectively.Meanwhile,390,227,and 75 DEAPs were detected in the brains of ME7-80,ME7-120,and ME7-180 dpi mice,respectively.The overwhelming majority of DEAPs in the mid-early stage were down-regulated,and more portions of DEAPs in the mid-late and late stages were up-regulated.Approximately 22.1%(328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated.Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39(80dpi),13(120 dpi),and 10(180 dpi) significantly changed pathways in 139A-infected mice.Meanwhile,55,25,and 18 significantly changed pathways were observed in the 80,120,and 180 dpi samples of139A-and ME7-infected mice(P < 0.05),respectively.Six pathways were commonly involved in all tested samples.Moreover,many steps in the citrate cycle(tricarboxylic acid cycle) were affected,represented by down-regulated acetylation for relevant enzymes in the mid-early stage and upregulated acetylation in the mid-late and late stages.Conclusion Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection,showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.展开更多
基金Supported by Chinese National Natural Science Foundation Grants[81630062,81572048]National Key Research and Development Project[2016YFC1202700,2017YFC1200500]the State Key Laboratory of Infectious Disease Prevention and Control,China CDC[2019SKLID501 and 2019SKLID603].
文摘Real-time quaking-induced conversion(RT-QuIC)assay is a newly established PrPSc-detecting method.The development of RT-QuIC improves the diagnosis of sporadic Creutzfeldt–Jakob disease(sCJD),showing good sensitivity and specificity in many countries when the method was used in cerebrospinal fluid(CSF)samples.However,in China,the sensitivity and specificity of RT-QuIC has yet to be determined due to the lack of definitive diagnosis samples.
基金National Natural Science Foundation[Grant Nos.81572048 and 81630062]the National Key R&D Program of China[Grant Nos.2018ZX10711001,2016YFC1202700,and 2017YFC1200500]the State Key Laboratory for Infectious Disease Prevention and Control,China CDC[Grant Nos.2019SKLID501,2019SKLID603,and 2019SKLID307]。
文摘Objective The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrPSc in the brain tissues of suspected cases.Thus,developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.Methods We prepared a PrP-specific polyclonal antibody(pAb P54)in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23–231.Thereafter,we verified that pAb in Western blot,immunohistochemistry(IHC),and immunofluorescent(IFA)assays.Results Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein,normal brain PrPC from healthy rodents and humans,and pathological PrPSc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases.The electrophoretic patterns of brain PrPC and PrPSc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies.Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54.IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139 A-infected mice and 263 K-infected hamsters.IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.Conclusion The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and,thus,may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.
基金supported by National Key R&D Program of China [2020YFE0205700]Chinese National Natural Science Foundation Grants [81630062]grants from the State Key Laboratory for Infectious Disease Prevention and Control(China CDC)[Grant Nos.2019SKLID501,2019SKLID603,and 2019SKLID307]
文摘Objective To describe the global profiles of acetylated proteins in the brains of scrapie agents 139Aand ME7-infected mice collected at mid-early,mid-late,and terminal stages.Methods The acetylated proteins from the cortex regions of scrapie agent(139A-and ME7)-infected mice collected at mid-early(80 days postinfection,dpi),mid-late(120 dpi),and terminal(180 dpi) stages were extracted,and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry.The proteins in the infected mice showing 1.5-fold higher or lower levels than that of agematched normal controls were considered as differentially expressed acetylated peptides(DEAPs).Results A total of 118,42,and 51 DEAPs were found in the brains of 139A-80,139A-120,and 139A-180dpi mice,respectively.Meanwhile,390,227,and 75 DEAPs were detected in the brains of ME7-80,ME7-120,and ME7-180 dpi mice,respectively.The overwhelming majority of DEAPs in the mid-early stage were down-regulated,and more portions of DEAPs in the mid-late and late stages were up-regulated.Approximately 22.1%(328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated.Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39(80dpi),13(120 dpi),and 10(180 dpi) significantly changed pathways in 139A-infected mice.Meanwhile,55,25,and 18 significantly changed pathways were observed in the 80,120,and 180 dpi samples of139A-and ME7-infected mice(P < 0.05),respectively.Six pathways were commonly involved in all tested samples.Moreover,many steps in the citrate cycle(tricarboxylic acid cycle) were affected,represented by down-regulated acetylation for relevant enzymes in the mid-early stage and upregulated acetylation in the mid-late and late stages.Conclusion Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection,showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.