Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide （rhBNP）. In previous study, we successfully developed a genetically modified cell line 293...Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide （rhBNP）. In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGIo-G1 was constructed by transfecting pGloSensorTM 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGIo-G1 showed high precision with intraassay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R^2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay （paired t test,p = 0.630）. All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.展开更多
Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential i...Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential in the development,registration,and quality control of rhGH pharmaceutical products[2].However,the existing in vivo bioassay procedure based on somatropin-induced weight gain in rats is complicated,and the use of a rat cell line-based approach(Nb2-11 bioassay),which measures the production of adenosine triphosphate(ATP)as a direct indicator of cell growth,has a low mechanism of action(MOA)relevance.Therefore,novel rhGH bioassays are still needed.To this end,we developed a reporter gene assay(RGA)based on the GH/insulin-like growth factor-1(IGF-1)axis.展开更多
Food,especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans(PCDD/Fs),and dioxin-like polychlorinated biphenyls(dl-PCBs)for human exposure.So,a simple,rapid and cheap bi...Food,especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans(PCDD/Fs),and dioxin-like polychlorinated biphenyls(dl-PCBs)for human exposure.So,a simple,rapid and cheap bioassay method is needed for determination of dioxins in food samples.In this study,we used a new highly sensitive reporter cell line to determine the concentration of dioxins in 33 fish and seafood samples.The samples were extracted by shaking with water/isopropanol(1:1 v/v)and hexane and cleaned-up by a multi layered silica gel column and an alumina column,then analyzed using CBG 2.8 D cell line.We compared the results obtained from the CBG 2.8 D cell assay to those obtained from conventional High-Resolution Gas Chromatography-High Resolution Mass Spectrometry(HRGC-HRMS)analysis.Good correlations were observed between these two methods(r^2=0.93).While the slope of regression line was 1.76,the bioanalytical equivalent(BEQ)values were 1.76 folds higher than WHO-TEQ values and the conversion coefficient was 0.568(the reciprocal of 1.76).In conclusion,CBG 2.8 D cell assay was an applicable method to determine dioxins levels in fish and sea food samples.展开更多
Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located...Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.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
Apolipoprotein C2 is an important member of the apolipoprotein C family, and is a potent activator of lipoprotein lipase. In the central nervous system, apolipoprotein C2 plays an important role in the catabolism of t...Apolipoprotein C2 is an important member of the apolipoprotein C family, and is a potent activator of lipoprotein lipase. In the central nervous system, apolipoprotein C2 plays an important role in the catabolism of triglyceride-rich lipoproteins. Studies into the exact regulatory mechanism of mouse apolipoprotein C2 expression have not been reported. In this study, seven luciferase expression vectors, which contained potential mouse apolipoprotein C2 gene promoters, were constructed and co-transfected with pRL-TK into HEK293T cells to investigate apolipoprotein C2 promoter activity. Luciferase assays indicated that the apolipoprotein C2 promoter region was mainly located in the ＋104 bp to ＋470 bp region. The activity of the different lengths of apolipoprotein C2 promoter region varied. This staggered negative-positive-negative arrangement indicates the complex regulation of apolipoprotein C2 expression and provides important clues for elucidating the regulatory mechanism of apolipoprotein C2 gene transcription.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
T cell immunoglobulin and ITIM domain(TIGIT)is a novel immune checkpoint that has been considered as a target in cancer immunotherapy.Current available bioassays for measuring the biological activity of therapeutic an...T cell immunoglobulin and ITIM domain(TIGIT)is a novel immune checkpoint that has been considered as a target in cancer immunotherapy.Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable.Here,we designed a reporter gene assay comprising two cell lines,namely,CHO-CD112-CD3 scFv,which stably expresses CD 112(PVRL2,nectin-2)and a membranebound anti-CD3 single-chain fragment variable(scFv)as the target cell,and Jurkat-NFAT-TIGIT,which stably expresses TIGIT as well as the nuclear factor of activated T-cells(NFAT)response element-controlled luciferase gene,as the effector cell.The anti-CD3 scFv situated on the target cells activates Jurkat-NFATTIGIT cells through binding and crosslinking CD3 molecules of the effector cell,whereas interactions between CD 112 and TIGIT prevent activation.The presence of anti-TIGIT mAbs disrupts their interaction,which in turn reverses the inactivation and luciferase expression.Optimization and validation studies have demonstrated that this assay is superior in terms of specificity,accuracy,linearity,and precision.In summary,this reliable and effective reporter gene assay may potentially be utilized in lot release control,stability assays,screening,and development of novel TIGIT-targeted therapeutic antibodies.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
While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs)...While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs),which are critical components of epigenetic regulation.lncRNAs are emerging as critical regulators of gene expression and genomic stability.However,it remains largely unknown about how lncRNAs are regulated.Here,we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion.Specifically,we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase(MODC)to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein,designated as dBiFP,and show that the dBiFP protein is highly destabilized,compared with the commonly-used eGFP protein.We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs.Therefore,our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs,synthetic regulatory RNA molecules,RNA binding proteins,and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.展开更多
Dear EditorProbing protein-protein interaction has become a routine practice in the post genomic era. Multiple in vitro or in vivo techniques have been developed to detect or report direct or indirect interactions of ...Dear EditorProbing protein-protein interaction has become a routine practice in the post genomic era. Multiple in vitro or in vivo techniques have been developed to detect or report direct or indirect interactions of functionally related proteins （Lalonde et al., 2008）. These techniques sometimes are technically challenging, however, because the readout would demand sophisticated detectors and/or complicated calculations. Besides, a common drawback of many of these techniques is they can render inherent false positives to various degrees so that an interaction often cannot be judged unambiguously.展开更多
Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carri...Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.展开更多
More than 90%of disease-and trait-associated human variants are noncoding.By systematically screening multiple large-scale studies,we compiled REVA,a manually curated database for over 11.8 million experimentally test...More than 90%of disease-and trait-associated human variants are noncoding.By systematically screening multiple large-scale studies,we compiled REVA,a manually curated database for over 11.8 million experimentally tested noncoding variants with expression-modulating potentials.We provided 2424 functional annotations that could be used to pinpoint the plausible regulatory mechanism of these variants.We further benchmarked multiple state-of-the-art computational tools and found that their limited sensitivity remains a serious challenge for effective large-scale analysis.REVA provides high-quality experimentally tested expression-modulating variants with extensive functional annotations,which will be useful for users in the noncoding variant community.REVA is freely available at http://reva.gao-lab.org.展开更多
The present study was aimed to develop and optimize isoniazid(INZ)loaded solid lipid nanoparticles(SLNs)for exploring in vitro anti-tubercular and cytotoxic activity.The INZ-SLNs were successfully prepared by high pre...The present study was aimed to develop and optimize isoniazid(INZ)loaded solid lipid nanoparticles(SLNs)for exploring in vitro anti-tubercular and cytotoxic activity.The INZ-SLNs were successfully prepared by high pressure homogenization followed by ultrasonication technique and optimized 3 using 2 full factorial designs.INZ-SLNs were characterized for particle size(PS),zeta potential(ZP),entrapment efficiency percentage(EE%)and cumulative percentage drug release(CDR%).Physicochemical properties were investigated using transmission electron microscopy(TEM),differential scanning calorimeter(DSC),X-ray diffraction and Fourier transmission infrared spectroscopy(FTIR).The average PS,ZP and EE%of the optimized formulation were found to be 167.1 nm,-32.4 mV and 73.17%respectively.The optimized formulation showed a CDR of 79.14%up to 36 h.In vitro anti-tubercular(luciferase reporter phage(LRP)assay in H37Rv viable and resistant strain)and cytotoxicity efficacy(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)assay in J774A.1 cells)of INZ-SLNs were evaluated and compared with free INZ.Results of LRP assay in H37Rv strain showed that percentage reduction in relative light unit(RLU)for INZ-SLNs and free INZ were 99.75 and 99.898%respectively,whereas in case of INZ resistant strain they were found to be 90.27 and 90.52%respectively,confirming notable antitubercular activity.MTT assay revealed that the percentage of cell viability upon exposure with INZ-SLNs was significantly higher(>90%)than free INZ(<80%),confirming its safety.Thus,INZ-SLNs could be an effective dosage form with sustained drug release profile,significant anti-tubercular activity,and reduced normal cell toxicity for achieving better therapeutic activity.展开更多
基金supported by grants from the National Science and Technology Major Project(No.2015ZX09501008-001)the Middle-aged and Young Development Research Foundation of NIFDC(No.2017B3)
文摘Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide （rhBNP）. In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGIo-G1 was constructed by transfecting pGloSensorTM 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGIo-G1 showed high precision with intraassay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R^2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay （paired t test,p = 0.630）. All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.
基金supported by the first batch of grants from the State Key Laboratory of Drug Regulatory Science,China(Grant No.:2023SKLDRS0108).
文摘Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential in the development,registration,and quality control of rhGH pharmaceutical products[2].However,the existing in vivo bioassay procedure based on somatropin-induced weight gain in rats is complicated,and the use of a rat cell line-based approach(Nb2-11 bioassay),which measures the production of adenosine triphosphate(ATP)as a direct indicator of cell growth,has a low mechanism of action(MOA)relevance.Therefore,novel rhGH bioassays are still needed.To this end,we developed a reporter gene assay(RGA)based on the GH/insulin-like growth factor-1(IGF-1)axis.
基金the National Natural Science Foundation of China(Nos.21525730 and 21527901)the National Key Research and Development Program of China(No.2018YFA0901103)。
文摘Food,especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans(PCDD/Fs),and dioxin-like polychlorinated biphenyls(dl-PCBs)for human exposure.So,a simple,rapid and cheap bioassay method is needed for determination of dioxins in food samples.In this study,we used a new highly sensitive reporter cell line to determine the concentration of dioxins in 33 fish and seafood samples.The samples were extracted by shaking with water/isopropanol(1:1 v/v)and hexane and cleaned-up by a multi layered silica gel column and an alumina column,then analyzed using CBG 2.8 D cell line.We compared the results obtained from the CBG 2.8 D cell assay to those obtained from conventional High-Resolution Gas Chromatography-High Resolution Mass Spectrometry(HRGC-HRMS)analysis.Good correlations were observed between these two methods(r^2=0.93).While the slope of regression line was 1.76,the bioanalytical equivalent(BEQ)values were 1.76 folds higher than WHO-TEQ values and the conversion coefficient was 0.568(the reciprocal of 1.76).In conclusion,CBG 2.8 D cell assay was an applicable method to determine dioxins levels in fish and sea food samples.
文摘Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.
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 grants from the National Natural Science Foundation of China, No. 30770818a grant from Education Department of Liaoning Province, No. 2009s109
文摘Apolipoprotein C2 is an important member of the apolipoprotein C family, and is a potent activator of lipoprotein lipase. In the central nervous system, apolipoprotein C2 plays an important role in the catabolism of triglyceride-rich lipoproteins. Studies into the exact regulatory mechanism of mouse apolipoprotein C2 expression have not been reported. In this study, seven luciferase expression vectors, which contained potential mouse apolipoprotein C2 gene promoters, were constructed and co-transfected with pRL-TK into HEK293T cells to investigate apolipoprotein C2 promoter activity. Luciferase assays indicated that the apolipoprotein C2 promoter region was mainly located in the ＋104 bp to ＋470 bp region. The activity of the different lengths of apolipoprotein C2 promoter region varied. This staggered negative-positive-negative arrangement indicates the complex regulation of apolipoprotein C2 expression and provides important clues for elucidating the regulatory mechanism of apolipoprotein C2 gene transcription.
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supported by the Major Scientific and Technological Special Project for“Significant New Drugs Development”(Grant No.2018ZX09736016-007,China)。
文摘T cell immunoglobulin and ITIM domain(TIGIT)is a novel immune checkpoint that has been considered as a target in cancer immunotherapy.Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable.Here,we designed a reporter gene assay comprising two cell lines,namely,CHO-CD112-CD3 scFv,which stably expresses CD 112(PVRL2,nectin-2)and a membranebound anti-CD3 single-chain fragment variable(scFv)as the target cell,and Jurkat-NFAT-TIGIT,which stably expresses TIGIT as well as the nuclear factor of activated T-cells(NFAT)response element-controlled luciferase gene,as the effector cell.The anti-CD3 scFv situated on the target cells activates Jurkat-NFATTIGIT cells through binding and crosslinking CD3 molecules of the effector cell,whereas interactions between CD 112 and TIGIT prevent activation.The presence of anti-TIGIT mAbs disrupts their interaction,which in turn reverses the inactivation and luciferase expression.Optimization and validation studies have demonstrated that this assay is superior in terms of specificity,accuracy,linearity,and precision.In summary,this reliable and effective reporter gene assay may potentially be utilized in lot release control,stability assays,screening,and development of novel TIGIT-targeted therapeutic antibodies.
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,
基金The reported work was supported in part by research grants from the National Institutes of Health(AT004418,DE020140 to TCH and RRR)the US Department of Defense(OR130096 to JMW)+5 种基金the Scoliosis Research Society(TCH and MJL)the National Key Research and Development Program of China(2016YFC1000803 and 2011CB707906 to TCH)the National Natural Science Foundation of China(#81201916 to XW)ZZ was a recipient of protectorate fellowship from China Scholarship CouncilThis project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430.
文摘While the human genome is pervasively transcribed,<2%of the human genome is transcribed into protein-coding mRNAs,leaving most of the transcripts as noncoding RNAs,such as microRNAs and long-noncoding RNAs(lncRNAs),which are critical components of epigenetic regulation.lncRNAs are emerging as critical regulators of gene expression and genomic stability.However,it remains largely unknown about how lncRNAs are regulated.Here,we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion.Specifically,we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase(MODC)to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein,designated as dBiFP,and show that the dBiFP protein is highly destabilized,compared with the commonly-used eGFP protein.We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs.Therefore,our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic transcript reporter to monitor the inhibition of lncRNAs by microRNAs,synthetic regulatory RNA molecules,RNA binding proteins,and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.
文摘Dear EditorProbing protein-protein interaction has become a routine practice in the post genomic era. Multiple in vitro or in vivo techniques have been developed to detect or report direct or indirect interactions of functionally related proteins （Lalonde et al., 2008）. These techniques sometimes are technically challenging, however, because the readout would demand sophisticated detectors and/or complicated calculations. Besides, a common drawback of many of these techniques is they can render inherent false positives to various degrees so that an interaction often cannot be judged unambiguously.
基金grantsfromtheNationalNaturalScienceFoundationofChina (No 396 70 35 2 )
文摘Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.
基金supported by funds from the National Key R&D Program of China(Grant No.2016YFC0901603)the National High Technology Research and Development Program of China(Grant No.2015AA020108)+2 种基金the State Key Laboratory of Protein and Plant Gene Research and the Beijing Advanced Innovation Center for Genomics(ICG)at Peking University,Chinasupported in part by the National Program for Support of Top-notch Young Professionalssupported by the High-performance Computing Platform of Peking University。
文摘More than 90%of disease-and trait-associated human variants are noncoding.By systematically screening multiple large-scale studies,we compiled REVA,a manually curated database for over 11.8 million experimentally tested noncoding variants with expression-modulating potentials.We provided 2424 functional annotations that could be used to pinpoint the plausible regulatory mechanism of these variants.We further benchmarked multiple state-of-the-art computational tools and found that their limited sensitivity remains a serious challenge for effective large-scale analysis.REVA provides high-quality experimentally tested expression-modulating variants with extensive functional annotations,which will be useful for users in the noncoding variant community.REVA is freely available at http://reva.gao-lab.org.
文摘The present study was aimed to develop and optimize isoniazid(INZ)loaded solid lipid nanoparticles(SLNs)for exploring in vitro anti-tubercular and cytotoxic activity.The INZ-SLNs were successfully prepared by high pressure homogenization followed by ultrasonication technique and optimized 3 using 2 full factorial designs.INZ-SLNs were characterized for particle size(PS),zeta potential(ZP),entrapment efficiency percentage(EE%)and cumulative percentage drug release(CDR%).Physicochemical properties were investigated using transmission electron microscopy(TEM),differential scanning calorimeter(DSC),X-ray diffraction and Fourier transmission infrared spectroscopy(FTIR).The average PS,ZP and EE%of the optimized formulation were found to be 167.1 nm,-32.4 mV and 73.17%respectively.The optimized formulation showed a CDR of 79.14%up to 36 h.In vitro anti-tubercular(luciferase reporter phage(LRP)assay in H37Rv viable and resistant strain)and cytotoxicity efficacy(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)assay in J774A.1 cells)of INZ-SLNs were evaluated and compared with free INZ.Results of LRP assay in H37Rv strain showed that percentage reduction in relative light unit(RLU)for INZ-SLNs and free INZ were 99.75 and 99.898%respectively,whereas in case of INZ resistant strain they were found to be 90.27 and 90.52%respectively,confirming notable antitubercular activity.MTT assay revealed that the percentage of cell viability upon exposure with INZ-SLNs was significantly higher(>90%)than free INZ(<80%),confirming its safety.Thus,INZ-SLNs could be an effective dosage form with sustained drug release profile,significant anti-tubercular activity,and reduced normal cell toxicity for achieving better therapeutic activity.
