SARS-CoV-2 main protease(M^(pro))is one of the most extensively exploited drug targets for COVID-19.Structurally disparate compounds have been reported as M^(pro) inhibitors,raising the question of their target specif...SARS-CoV-2 main protease(M^(pro))is one of the most extensively exploited drug targets for COVID-19.Structurally disparate compounds have been reported as M^(pro) inhibitors,raising the question of their target specificity.To elucidate the target specificity and the cellular target engagement of the claimed M^(pro) inhibitors,we systematically characterize their mechanism of action using the cell-free FRET assay,the thermal shift-binding assay,the cell lysate Protease-Glo luciferase assay,and the cell-based FlipGFP assay.Collectively,our results have shown that majority of the M^(pro) inhibitors identified from drug repurposing including ebselen,carmofur,disulfiram,and shikonin are promiscuous cysteine inhibitors that are not specific to M^(pro),while chloroquine,oxytetracycline,montelukast,candesartan,and dipyridamole do not inhibit M^(pro) in any of the assays tested.Overall,our study highlights the need of stringent hit validation at the early stage of drug discovery.展开更多
While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneratio...While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell（SC） remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA（mi RNA） in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.展开更多
AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of t...AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of the HCV replicon system OR6.RVT is a strong antioxidant and a kind of polyphenol that inhibits replication of various viruses.AXN is also a strong antioxidant.The replication of HCV RNA was assessed by the luciferase reporter assay.An additive effect of antioxidants on antiviral effects of interferon(IFN)and ribavirin(RBV) was investigated.RESULTS:This is the first report to investigate the effect of RVT and AXN on HCV replication.In contrast to other reported viruses,RVT significantly enhanced HCV RNA replication.Vitamin E also enhanced HCV RNA replication as reported previously,although AXN didnot affect replication.IFN and RBV significantly reduced HCV RNA replication,but these effects were dose-dependently hampered and attenuated by the addition of RVT.AXN didnot affect antiviral effects of IFN or RBV. CONCLUSION:These results suggested that RVT is not suitable as an antioxidant therapy for chronic hepatitis C.展开更多
AIM： To investigate the therapeutic potential of an RNA ligand （aptamer） specific for the catalytic ricin A-chain （RTA）, the protective effects of a 31-nucleo- tide RNA aptamer （31RA）, which formed a high affin...AIM： To investigate the therapeutic potential of an RNA ligand （aptamer） specific for the catalytic ricin A-chain （RTA）, the protective effects of a 31-nucleo- tide RNA aptamer （31RA）, which formed a high affinity complex with RTA, against ricin-induced toxicity in cell- based luciferase translation and cell cytotoxicity assays were evaluated. METHODS： To test the therapeutic potential of anti- RTA aptamers in Chinese hamster ovary （CliO） AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured us- ing luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tet- razolium compound [3-（4,5-dimethylthiazol-2-yl）- 5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H- tetrazolium]. RESULTS： Inhibition of protein synthesis by ricin in CliO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against degly- cosylated RTA （dgA） neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CliO AA8 cells. CONCLUSION： We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell- based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.展开更多
Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (access...Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.展开更多
F-type lectins(Fucolectins)are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion.In our previous research,we found that two homologous Fucolectin g...F-type lectins(Fucolectins)are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion.In our previous research,we found that two homologous Fucolectin genes,AjFTL-1 and AjFTL-2,exhibited different expression profiles after lipopolysaccharides(LPS)challenge in Apostichopus japonicus.However,the transcriptional regulation mechanism of these two genes remains largely unknown.In this study,the 5'flanking regions of Aj FTL-1 and Aj FTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid(EPC)cell system.First,in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB,CREB,and CREBP1,and both contain a TATA box.Additionally,luciferase assay and progressive 5'truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells.Moreover,the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan(PGN)and mannan(MAN),while AjFTL-2 promoter was prominently regulated by LPS and MAN,indicating Aj FTL-1 and Aj FTL-2 genes showed different transcriptional regulation pattern under different immune stimulation.More importantly,analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites(-769 bp to-761 bp,-185 bp to-172 bp)in Aj FTL-1 and one potential binding site(-530 to-517 bp)in Aj FTL-2.Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity,but not AjFTL-1 promoter activity.These findings indicated that in marine invertebrates,different Fucolectin members differ in transcription regulations and expression patterns,and might play different roles in immune defenses during pathogen infection.展开更多
AIM： To identify the variants in U rase 1 （UGT1A1） gene in Gilbert＇s syndrome （GS） and to estimate the association between homozygosity for TA insertion and GS in India, as well as the frequency of TA insertion ...AIM： To identify the variants in U rase 1 （UGT1A1） gene in Gilbert＇s syndrome （GS） and to estimate the association between homozygosity for TA insertion and GS in India, as well as the frequency of TA insertion and its impact among normal controls in India. METHODS： Ninety-five GS cases and 95 normal controls were selected. Liver function and other tests were done. The promoter and all 5 exons of UGT1A1 gene were resequenced. Functional assessment of a novel trinucleotide insertion was done by in silico analysis and by estimating UGT1A1 promoter activity carried out by ludferase reporter assay of appropriate constructs in Hep G2 cell line. RESULTS： Among the GS patients, 80% were homozygous for the TA insertion, which was several-fold higher than reports from other ethnic groups. The mean UCB level was elevated among individuals with only one copy of this insertion, which was not significantly different from those with two copies. Many new DNA variants in UGT1A1 gene were discovered, including a trinucleotide （CAT） insertion in the promoter found in a subset （10%） of GS patients, but not among normal controls. In-silico analysis showed marked changes in the DNA-folding of the promoter and functional analysis showed a 20-fold reduction in transcription efficiency of UGT1A1 gene resulting from this insertion, thereby significantly elevating the UCB level. CONCLUSION： The genetic epidemiology of GS is variable across ethnic interactions among UGT1A1 groups and the epistatic promoter variants modulate bilirubin glucuronidation.展开更多
Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL...Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.展开更多
The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for ...The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness.Here,we evaluated various novel luciferase immunosorbent assays(LISA)based on nucleocapsid protein(NP)as well as fragments derived from spike protein(S)including subunit 1(S1),N terminal domain(NTD),receptorbinding domain(RBD)and subunit 2(S2)of S for the detection of MERS-CoV-specific IgG.Fusion proteins,including nanoluciferase(NLuc)and various fragments derived from the NP or S protein of MERS-CoV,were expressed in human embryonic kidney 293 T cells.LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins.Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR,commercial S1-based ELISA,and pseudovirus particle neutralization test(ppNT).Our results showed that the S1-,RBD-,and NP-LISAs were more sensitive than the NTD-and S2-LISAs for the detection of anti-MERS-CoV IgG.Furthermore,the S1-,RBD-,and NP-LISAs were more sensitive(by at least 16-fold)than the commercially available S1-ELISA.Moreover,the S1-,RBD-,and NPLISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV(OC43,229E,HKU1,NL63)-infected patients.More importantly,these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels,monkeys,and mice,among which the RBD-LISA exhibited excellent performance.The results of this study suggest that the novel MERS-CoV RBD-and S1-LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples.These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.展开更多
Leonurus japonicus Houtt.is used in TCM to treat the metabolic syndrome.However,up to now,no active constituents could be identified.Here we describe the isolation of 17 dominant constituents of L.japonicus and the re...Leonurus japonicus Houtt.is used in TCM to treat the metabolic syndrome.However,up to now,no active constituents could be identified.Here we describe the isolation of 17 dominant constituents of L.japonicus and the related European herb Leonurus cardiaca L.-namely7R-chloro-6-desoxy-harpagide,ajugol,campneoside II,chicoric acid,ferulic acid,harpagide,isoacteoside,展开更多
基金This research was supported by the National Institute of Allergy and Infectious Diseasess at the National Instiute of Health(NIH,USA,grants AI147325,AI157046,and AI158775)the Arizona Biomedical Research Centre Young Investigator grant(ADHS18-198859,USA)to Jun Wang.
文摘SARS-CoV-2 main protease(M^(pro))is one of the most extensively exploited drug targets for COVID-19.Structurally disparate compounds have been reported as M^(pro) inhibitors,raising the question of their target specificity.To elucidate the target specificity and the cellular target engagement of the claimed M^(pro) inhibitors,we systematically characterize their mechanism of action using the cell-free FRET assay,the thermal shift-binding assay,the cell lysate Protease-Glo luciferase assay,and the cell-based FlipGFP assay.Collectively,our results have shown that majority of the M^(pro) inhibitors identified from drug repurposing including ebselen,carmofur,disulfiram,and shikonin are promiscuous cysteine inhibitors that are not specific to M^(pro),while chloroquine,oxytetracycline,montelukast,candesartan,and dipyridamole do not inhibit M^(pro) in any of the assays tested.Overall,our study highlights the need of stringent hit validation at the early stage of drug discovery.
文摘While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell（SC） remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA（mi RNA） in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.
基金Supported by The grant from the Japanese Ministry of Educationand Science
文摘AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of the HCV replicon system OR6.RVT is a strong antioxidant and a kind of polyphenol that inhibits replication of various viruses.AXN is also a strong antioxidant.The replication of HCV RNA was assessed by the luciferase reporter assay.An additive effect of antioxidants on antiviral effects of interferon(IFN)and ribavirin(RBV) was investigated.RESULTS:This is the first report to investigate the effect of RVT and AXN on HCV replication.In contrast to other reported viruses,RVT significantly enhanced HCV RNA replication.Vitamin E also enhanced HCV RNA replication as reported previously,although AXN didnot affect replication.IFN and RBV significantly reduced HCV RNA replication,but these effects were dose-dependently hampered and attenuated by the addition of RVT.AXN didnot affect antiviral effects of IFN or RBV. CONCLUSION:These results suggested that RVT is not suitable as an antioxidant therapy for chronic hepatitis C.
基金Supported by Grant from the National Institutes of Health (Tchou-Wong), No. ES-000260 and No. AI-059476
文摘AIM： To investigate the therapeutic potential of an RNA ligand （aptamer） specific for the catalytic ricin A-chain （RTA）, the protective effects of a 31-nucleo- tide RNA aptamer （31RA）, which formed a high affinity complex with RTA, against ricin-induced toxicity in cell- based luciferase translation and cell cytotoxicity assays were evaluated. METHODS： To test the therapeutic potential of anti- RTA aptamers in Chinese hamster ovary （CliO） AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured us- ing luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tet- razolium compound [3-（4,5-dimethylthiazol-2-yl）- 5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H- tetrazolium]. RESULTS： Inhibition of protein synthesis by ricin in CliO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against degly- cosylated RTA （dgA） neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CliO AA8 cells. CONCLUSION： We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell- based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.
文摘Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.
基金financially supported by the National Natural Science Foundation of China(No.31802331)the Natural Science Foundation of Zhejiang Province(Nos.LZ19C190001,LY20C190002)+1 种基金the Natural Science Foundation of Ningbo(No.2018A610340)the K.C.Wong Magna Fund in Ningbo University。
文摘F-type lectins(Fucolectins)are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion.In our previous research,we found that two homologous Fucolectin genes,AjFTL-1 and AjFTL-2,exhibited different expression profiles after lipopolysaccharides(LPS)challenge in Apostichopus japonicus.However,the transcriptional regulation mechanism of these two genes remains largely unknown.In this study,the 5'flanking regions of Aj FTL-1 and Aj FTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid(EPC)cell system.First,in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB,CREB,and CREBP1,and both contain a TATA box.Additionally,luciferase assay and progressive 5'truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells.Moreover,the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan(PGN)and mannan(MAN),while AjFTL-2 promoter was prominently regulated by LPS and MAN,indicating Aj FTL-1 and Aj FTL-2 genes showed different transcriptional regulation pattern under different immune stimulation.More importantly,analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites(-769 bp to-761 bp,-185 bp to-172 bp)in Aj FTL-1 and one potential binding site(-530 to-517 bp)in Aj FTL-2.Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity,but not AjFTL-1 promoter activity.These findings indicated that in marine invertebrates,different Fucolectin members differ in transcription regulations and expression patterns,and might play different roles in immune defenses during pathogen infection.
基金Supported by grants from the Department of Biotechnology, Government of India (to PPM) and the Department of Science & Technology, Government of West Bengal (to AC)
文摘AIM： To identify the variants in U rase 1 （UGT1A1） gene in Gilbert＇s syndrome （GS） and to estimate the association between homozygosity for TA insertion and GS in India, as well as the frequency of TA insertion and its impact among normal controls in India. METHODS： Ninety-five GS cases and 95 normal controls were selected. Liver function and other tests were done. The promoter and all 5 exons of UGT1A1 gene were resequenced. Functional assessment of a novel trinucleotide insertion was done by in silico analysis and by estimating UGT1A1 promoter activity carried out by ludferase reporter assay of appropriate constructs in Hep G2 cell line. RESULTS： Among the GS patients, 80% were homozygous for the TA insertion, which was several-fold higher than reports from other ethnic groups. The mean UCB level was elevated among individuals with only one copy of this insertion, which was not significantly different from those with two copies. Many new DNA variants in UGT1A1 gene were discovered, including a trinucleotide （CAT） insertion in the promoter found in a subset （10%） of GS patients, but not among normal controls. In-silico analysis showed marked changes in the DNA-folding of the promoter and functional analysis showed a 20-fold reduction in transcription efficiency of UGT1A1 gene resulting from this insertion, thereby significantly elevating the UCB level. CONCLUSION： The genetic epidemiology of GS is variable across ethnic interactions among UGT1A1 groups and the epistatic promoter variants modulate bilirubin glucuronidation.
基金Supported by the Major Special Projects of New Product Training of Transgenic Organisms(zx080072008-2008)
文摘Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.
基金This work was supported by the following grants:the National Major Project for Control and Prevention of Infectious Disease in China(No.2018ZX10101002 and 2018ZX10732401)the National Key Research and Development Program of China(No.2016YFD0500301 and 2017YFC1200503)。
文摘The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness.Here,we evaluated various novel luciferase immunosorbent assays(LISA)based on nucleocapsid protein(NP)as well as fragments derived from spike protein(S)including subunit 1(S1),N terminal domain(NTD),receptorbinding domain(RBD)and subunit 2(S2)of S for the detection of MERS-CoV-specific IgG.Fusion proteins,including nanoluciferase(NLuc)and various fragments derived from the NP or S protein of MERS-CoV,were expressed in human embryonic kidney 293 T cells.LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins.Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR,commercial S1-based ELISA,and pseudovirus particle neutralization test(ppNT).Our results showed that the S1-,RBD-,and NP-LISAs were more sensitive than the NTD-and S2-LISAs for the detection of anti-MERS-CoV IgG.Furthermore,the S1-,RBD-,and NP-LISAs were more sensitive(by at least 16-fold)than the commercially available S1-ELISA.Moreover,the S1-,RBD-,and NPLISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV(OC43,229E,HKU1,NL63)-infected patients.More importantly,these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels,monkeys,and mice,among which the RBD-LISA exhibited excellent performance.The results of this study suggest that the novel MERS-CoV RBD-and S1-LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples.These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.
文摘Leonurus japonicus Houtt.is used in TCM to treat the metabolic syndrome.However,up to now,no active constituents could be identified.Here we describe the isolation of 17 dominant constituents of L.japonicus and the related European herb Leonurus cardiaca L.-namely7R-chloro-6-desoxy-harpagide,ajugol,campneoside II,chicoric acid,ferulic acid,harpagide,isoacteoside,
