Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorio...Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.展开更多
Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunopreci...Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.展开更多
Objective:To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation.Methods Two volumes(125μL for Group C and 100μL for Group T)of elution buffer were used to explore whethe...Objective:To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation.Methods Two volumes(125μL for Group C and 100μL for Group T)of elution buffer were used to explore whether smaller volume could save testing time.Result:Time consumption of elution in Group T was significantly shorter than that in Group C,while the efficiency of eluted m6A-containing fragments and the performance of m^(6)A-Seq as indicated by m6A peak distributions showed no difference between the two groups.Conclusion:A smaller volume of elution buffer was an economical way to reduce time consumption in immunoprecipitation.展开更多
The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machiner...The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery.To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100,we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection(ChIP-GLAS).From this assay,we determined that a set of promoter fragments,including several factors in the transforming growth factor beta(TGF-β)signaling pathway,exhibited interaction with p100.The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines.展开更多
Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms ...Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy.In this study,a new luciferase immunoprecipitation system(LIPS)assay based on p REN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase(Rluc),was established and used to investigate the levels of antibody responses to SFTSV.Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019.The results showed that 82 of the 464 patients(17.7%)had antibody response to SFTSV,which were further supported by immunofluorescence assays(IFAs).Further,q RT-PCR and microneutralization tests showed that among the 82 positive cases,15 patients had viremia,10 patients had neutralizing antibody,and one had both(totally 26 patient).However,none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations.All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases.The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019,and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.展开更多
Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery m...Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.展开更多
Objective To identify ubiquitinated proteins from complex human multiple myeloma (MM) U266 cells,a malignant disorder of differentiated human B cells.Methods Employing a globally proteomic strategy combining of immu...Objective To identify ubiquitinated proteins from complex human multiple myeloma (MM) U266 cells,a malignant disorder of differentiated human B cells.Methods Employing a globally proteomic strategy combining of immunoprecipitation,LC-MS/MS and SCX-LC-MS analysis to identified ubiquitination sites,which were identified by detecting signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da).Results In total,52 ubiquitinated proteins containing 73 ubiquitination sites of which 14 and 59 sites contained LRGG-tag and GG-tag were identified,respectively.Conclusion Classification analysis by of the proteins identified in the study based on the PANTHER showed that they were associated with multiple functional groups.This suggested the involvement of many endogenous proteins in the ubiquitination in MM.展开更多
Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie acid.Methods: A new strategy for high—throughpu...Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie acid.Methods: A new strategy for high—throughput identification of direct target genes was established by combining chromatin immunoprecipitation(ChIP) with in vitro selection.Then,106 potential CKBPB binding fragments from the genome of the all-trans retinoie acid(ATRA)-treated NB4 cells were identified.Results:Of them,82 were mapped in proximity to known or previously predicted genes;7 were randomly picked up for further confirmation by ChlP-PCR and 3 genes (CALM,1TPR2 and 0RM2) were found to be specificaUy up-regulated in the ATRA-treated NB4 cells,indicating that they might lie the down-stream target genes of ATKA.Conclusions:Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.展开更多
Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological ...Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ~ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P<0.0001,P<0.0001 andP<0.0001). The mean serum M-CSFsR level of 51 idiopathic thrombocytopenic purpura (ITP) patients was significantly higher than that of normal subjects (2.05 ng/ml±2.75 ng/ml,P<0.0001). Conclusion: The serum M-CSFsR levels of patients with ALL, AML, MDS and AA were significantly lower, while the level of patients with ITP was significantly higher than that of normal individuals. Patients with severe ITP (platelet count<30×l09/L) had the highest M-CSFsR level. It suggested that the abnormal levels of serum M-CSFsR may associate with some hematological diseases and may contribute to the pathological process.展开更多
Amyloids have traditionally been considered pathologic protein aggregates which contribute to neurodegeneration.New evidence however increasingly suggests that non-pathological amyloids are formed in animals during no...Amyloids have traditionally been considered pathologic protein aggregates which contribute to neurodegeneration.New evidence however increasingly suggests that non-pathological amyloids are formed in animals during normal development.Amyloid-like aggregate formation was originally thought to be a conserved feature of animal gametogenesis.This hypothesis was based on findings which suggest that regulated amyloid formations govern yeast meiosis by way of meiosis-specific RNA binding proteins.Additional support came from studies which demonstrate that DAZL,a mammalian gametogenesis-specific RNA binding protein,also forms SDS-resistant aggregates in vivo.Here,we report evidence of aggregated BOULE formations,another DAZ family protein,during sperm development.Data suggest that in mouse testis,BOULE forms SDS-resistant amyloid-like aggregates.BOULE aggregate formation correlates with dynamic developmental expression during spermatogenesis but disappeared in Boule knockout testis.We also mapped essential small region in vitro BOULE aggregations,immediately downstream DAZ repeats,and found that aggregations positively correlated with temperature.We also performed enhanced UV cross-linking immunoprecipitation on BOULE aggregates from mouse testes and found that aggregates bind with a large number of spermatogenesis-related mRNAs.These findings provide insight into the amyloidogenic properties of gametogenesis-specific RNA binding proteins as a conserved feature in mammalian reproduction.Further investigation is warranted to understand the functional significance of BOULE amyloid-like formation during mouse spermatogenesis.展开更多
Affinity-mediated protein separation is an integral part of proteomics, the most outstanding of which is immunoproteomics. However, in the immunoprecipitate system overwhelming Ab and Ag conceal Ag interacting protein...Affinity-mediated protein separation is an integral part of proteomics, the most outstanding of which is immunoproteomics. However, in the immunoprecipitate system overwhelming Ab and Ag conceal Ag interacting proteins as the research targets, which is the rate-limiting step in the progress of comparative proteomic analyses. We presented a convenient and accurate method to tackle this problem. 1 mol/L NaCl elution buffer was applied to the complex Rubisco immunoprecipitate of Arabidopsis, the weakest force involved in the system was selectively broken up, resulting in the enrichment of Rubisco interacting proteins accessible for further comparative protein gel profile. The easy-to-use method sheds light on a narrow-down strategy supplement for comparative immunoproteomics.展开更多
As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controv...As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controversial.Until now,the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2(N.gregoryi sp2)have not been characterized.We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody.We also cultured the N.gregoryi sp2 strain and performed immunoprecipitation,chromatin immunoprecipitation(ChIP),and RNA immunoprecipitation(RIP)assays.The nucleic acids that endogenously bound NgAgo in N.gregoryi sp2 cells were sequenced and analyzed.The results showed that NgAgo endogenously bound RNA rather than DNA.NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA,transcriptional regulators,RNA polymerases,and RNA-binding proteins.NgAgo mainly binds to the transcripts inside genes or in their upstream sequences.Interestingly,the top enriched motif of peaks was the same as that of miR-1289,suggesting that NgAgo may regulate gene expression post-transcriptionally.GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes.These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.展开更多
In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein i...In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.展开更多
Objective To study the biochemecal and immunological characterization of the 200 kD schistosomulum surface antigen Method and results A very high molecular weight schistosomulum surface antigen of Mr】200kD was identi...Objective To study the biochemecal and immunological characterization of the 200 kD schistosomulum surface antigen Method and results A very high molecular weight schistosomulum surface antigen of Mr】200kD was identified and characterized using monoclonal antibodies. Carbohydrate modification experiments followed by radioimmunobinding assays demonstrated that the epitope recognised by the mAbs on this antigen was carbohydrate in nature, while protein digestion experiments followed by SDS-PAGE indicated that this antigen also contained protein. Immunoprecipitation of <sup>125</sup>I-labelled cercarial, schistosomulum, adult worm and miracidial surface antigens followed by gel analysis showed the carbohydrate epitope to be present on 5 cercarial, 2 schistosomulum and 5 miracidial surface molecules, and suggested a possible ecological function involved in adapting the parasite to the aquatic free-living stages of its life cycle and possibly also in protecting the early schistosomula from host immune damage. The 5 cercarial surfacs antigens proved to be associated with the CHR, since all the mAbs which recognised those antigens could induce a strong CHR. A kinetic investigation of the carbohydrate epitope on schistosomula of different ages demonstrated a gradual and possibly irreversible loss during the culture period. The epitope completely disappeared from the surface of adult worms. Conclusion To demonstrate an epitope common to a number of surface molecules of various developmental stages of schistosome and therefore explains the immunological cross-reactivity among different stages at the molecular level.展开更多
Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclophilin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here,...Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclophilin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here, we show that AtCYP71 physically interacts with FAS1 and LHP1, respectively, to modulate their distribution on chromatin. The Ihpl cyp71 double mutant showed more severe phenotypes than the single mutants, suggesting that AtCYP71 and LHP1 synergistically control plant development. Such synergism was in part illustrated by the observation that LHP1 association with its specific target loci requires AtCYP71 function. We also demonstrate that AtCYP71 physically interacts with FAS1 and is indispensable for FAS1 targeting to the KNAT1 locus. Together, our data suggest that AtCYP71 is involved in fundamental processes of chromatin assembly and histone modification in plants.展开更多
Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and developmen...Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and development, but the binding sites and the target genes are not clearly defined in the whole human genome. In this study, we performed a ChIP-Seq experiment to identify c-Jun binding site in the human genome. Forty-eight binding sites were selected to process further evaluation by dsDNA microarray assay. We identified 283 c-Jun binding sites in K562 cells. Data analysis showed that 48.8% binding sites located within 100 kb of the upstream of the annotated genes, 28.6% binding sites comprised consensus TRE/CRE motif (5′-TGAC/GTCA-3′, 5′-TGACGTCA-3′) and variant sequences. Forty-two out of the selected 48 binding sites were found to bind the c-Jun homodimer in dsDNA microarray analysis. Data analysis also showed that 1569 genes are located in the neighborhood of the 283 binding sites and 191 genes in the neighborhood of the 42 binding sites validated by dsDNA microarray. We consulted 38 c-Jun target genes in previous studies and 16 among these 38 genes were also detected in this study. The identification of c-Jun binding sites and potential target genes in the genome scale may improve our fundamental understanding in the molecular mechanisms underlying the transcription regulation related to c-Jun.展开更多
Background Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Prev...Background Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17β-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER ^+) breast cancer cell line MCF-7. Methods After the treatment of MCF-7 cells with E2 at different concentrations ( 10^-11 mol/L, 10.9 mol/L, 10.7 mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0. 7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10^-7 mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half- site ( 1/2 ERE) element and Spl sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (CHIP) assay was also used to confirm the binding of estrogen receptor a (ERα) on BMP-6 promoter in the presence of E2. Results E2 dose dependently increased BMP-6 mRNA expression in human ER ^+ breast cancer cell line MCF-7. At a dose of 10^-7 mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P 〈 0. 05 ). Both the 1/2 ERE response element mutant form and the Spl site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter' s response to E2. Through ChIP assay, the binding of ERot on 1/2 ERE response element in BMP-6 promoter was further validated. Conclusion Estrogen induces BMP-6 expression in human ER^+ breast cancer cell line MCF-7 receptor ERα binding on 1/2 ERE element in the BMP-6 promoter. through its展开更多
Congenital heart disease(CHD)is the most common birth defect worldwide.Long non-coding RNAs(lncRNAs)have been implicated in many diseases.However,their involvement in CHD is not well understood.This study aimed to inv...Congenital heart disease(CHD)is the most common birth defect worldwide.Long non-coding RNAs(lncRNAs)have been implicated in many diseases.However,their involvement in CHD is not well understood.This study aimed to investigate the role of dysregulated lncRNAs in CHD.We used Gene Expression Omnibus data mining,bioinformatics analysis,and analysis of clinical tissue samples and observed that the novel lncRNA SAP30-2:1 with unknown function was significantly downregulated in damaged cardiac tissues from patients with CHD.Knockdown of lncRNA SAP30-2:1 inhibited the proliferation of human embryonic kidney and AC16 cells and decreased the expression of heart and neural crest derivatives expressed 2(HAND2).Moreover,lncRNA SAP30-2:1 was associated with HAND2 by RNA immunoprecipitation.Overall,these results suggest that lncRNA SAP30-2:1 may be involved in heart development through affecting cell proliferation via targeting HAND2 and may thus represent a novel therapeutic target for CHD.展开更多
Tension wood(TW)is a specialized xylem tissue formed in angiosperm trees under gravitational stimulus or mechanical stresses(e.g.,bending).The genetic regulation that underlies this important mechanism remains poorly ...Tension wood(TW)is a specialized xylem tissue formed in angiosperm trees under gravitational stimulus or mechanical stresses(e.g.,bending).The genetic regulation that underlies this important mechanism remains poorly understood.Here,we used laser capture microdissection of stem xylem cells coupled with full transcriptome RNA-sequencing to analyze TW formation in Populus trichocarpa.After tree bending,PtrLBD39 was the most significantly induced transcription factor gene;it has a phylogenetically paired homolog,PtrLBD22.CRISPR-based knockout of PtrLBD39/22 severely inhibited TW formation,reducing cellulose and increasing lignin content.Transcriptomic analyses of CRISPR-based PtrLBD39/22 double mutants showed that these two genes regulate a set of TW-related genes.Chromatin immunoprecipitation sequencing(ChIP-seq)was used to identify direct targets of PtrLBD39.We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network(TRN)mediated by PtrLBD39.In this TRN,PtrLBD39 directly regulates 26 novel TW-responsive transcription factor genes.Our work suggests that PtrLBD39 and PtrLBD22 specifically control TW formation by mediating a TW-specific TRN in Populus.展开更多
文摘Over the past few years, many researchers have attempted to develop non-invasive prenatal testing methods in order to investigate the genetic status of the fetus. The aim is to avoid invasive procedures such as chorionic villus and amniotic fluid sampling, which result in a significant risk for pregnancy loss. The discovery of cell free fetal DNA circulating in the maternal blood has great potential for the development of non-invasive prenatal testing(NIPT) methodologies. Such strategies have been successfully applied for the determination of the fetal rhesus status and inherited monogenic disease but the field of fetal aneuploidy investigation seems to be more challenging. The main reason for this is that the maternal cell free DNA in the mother's plasma is far more abundant, and because it is identical to half of the corresponding fetal DNA. Approaches developed are mainly based on next generation sequencing(NGS) technologies and epigenetic genetic modifications, such as fetal-maternal DNA differential methylation. At present, genetic services for non-invasive fetal aneuploidy detection are offered using NGS-based approaches but, for reasons that are presented herein, they still serve as screening tests which are not readily accessed by the majority of couples. Here we discuss the limitations of both strategies for NIPT and the future potential of the methods developed.
文摘Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
文摘Objective:To develop a cost-effective method to reduce the time consumption of elution in immunoprecipitation.Methods Two volumes(125μL for Group C and 100μL for Group T)of elution buffer were used to explore whether smaller volume could save testing time.Result:Time consumption of elution in Group T was significantly shorter than that in Group C,while the efficiency of eluted m6A-containing fragments and the performance of m^(6)A-Seq as indicated by m6A peak distributions showed no difference between the two groups.Conclusion:A smaller volume of elution buffer was an economical way to reduce time consumption in immunoprecipitation.
基金This work was supported by grants from the National Basic Research Program(973 Program,2009CB918903)863 Project of the Ministry of Science and Technology of China(2007AA02Z115)+3 种基金NSFC(90919032,30970562,30670441,30811130394 and 30870562)Tianjin Municipal Science and Technology Commission(08ZCGHHZ01900 and 08JCYBJC07700)Specialized Fund for the Doctoral Program of Higher Education(20091202110001)Tianjin Educational Committee Foundation(2008ZD01).
文摘The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery.To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100,we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection(ChIP-GLAS).From this assay,we determined that a set of promoter fragments,including several factors in the transforming growth factor beta(TGF-β)signaling pathway,exhibited interaction with p100.The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines.
基金supported by the National Program on Key Research Project of China(2018YFE0200400,2019YFC1200700)the National Natural Science Foundation of China(U20A20135)+1 种基金the Strategic Biological Resources Capacity Building Project of Chinese Academy of Sciences(KFJ-BRP-017-06)the Key deployment projects of Chinese Academy of Sciences(KJZD-SW-L11)
文摘Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy.In this study,a new luciferase immunoprecipitation system(LIPS)assay based on p REN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase(Rluc),was established and used to investigate the levels of antibody responses to SFTSV.Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019.The results showed that 82 of the 464 patients(17.7%)had antibody response to SFTSV,which were further supported by immunofluorescence assays(IFAs).Further,q RT-PCR and microneutralization tests showed that among the 82 positive cases,15 patients had viremia,10 patients had neutralizing antibody,and one had both(totally 26 patient).However,none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations.All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases.The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019,and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.
基金supported by National Key Research and Development Program of China(2018YFD1000703,2018YFD1000700)Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11020400)+3 种基金National Program on Research and Development of Transgenic Plants(2016ZX08009-003-001)Startup Fund for Advanced Talents of Lushan Botanical GardenChinese Academy of Science(2020ZWZX03 and 2020ZWZX05)the“Double Hundred and Double Thousand”Talent Project of Jiujiang City(jjsbsq2020026)。
文摘Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.
基金supported by the 2007 Chang-Jiang Scholars Program, National Natural Science Foundation of China (30973393 & 30400071)"211" Projects grant (Biotechnology & Bioengineering Medicine and Biomaterial & Tissue Engineering)
文摘Objective To identify ubiquitinated proteins from complex human multiple myeloma (MM) U266 cells,a malignant disorder of differentiated human B cells.Methods Employing a globally proteomic strategy combining of immunoprecipitation,LC-MS/MS and SCX-LC-MS analysis to identified ubiquitination sites,which were identified by detecting signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da).Results In total,52 ubiquitinated proteins containing 73 ubiquitination sites of which 14 and 59 sites contained LRGG-tag and GG-tag were identified,respectively.Conclusion Classification analysis by of the proteins identified in the study based on the PANTHER showed that they were associated with multiple functional groups.This suggested the involvement of many endogenous proteins in the ubiquitination in MM.
文摘Objective:To indentify target genes of transcription factor CCA AT enhancer-binding protein P(CEBPB) in acute proinyelocytie leukemia cells induced by all-tram retinoie acid.Methods: A new strategy for high—throughput identification of direct target genes was established by combining chromatin immunoprecipitation(ChIP) with in vitro selection.Then,106 potential CKBPB binding fragments from the genome of the all-trans retinoie acid(ATRA)-treated NB4 cells were identified.Results:Of them,82 were mapped in proximity to known or previously predicted genes;7 were randomly picked up for further confirmation by ChlP-PCR and 3 genes (CALM,1TPR2 and 0RM2) were found to be specificaUy up-regulated in the ATRA-treated NB4 cells,indicating that they might lie the down-stream target genes of ATKA.Conclusions:Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.
基金National "863" High Technology Program of China ( 102-11-01-03).
文摘Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ~ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P<0.0001,P<0.0001 andP<0.0001). The mean serum M-CSFsR level of 51 idiopathic thrombocytopenic purpura (ITP) patients was significantly higher than that of normal subjects (2.05 ng/ml±2.75 ng/ml,P<0.0001). Conclusion: The serum M-CSFsR levels of patients with ALL, AML, MDS and AA were significantly lower, while the level of patients with ITP was significantly higher than that of normal individuals. Patients with severe ITP (platelet count<30×l09/L) had the highest M-CSFsR level. It suggested that the abnormal levels of serum M-CSFsR may associate with some hematological diseases and may contribute to the pathological process.
基金supported by the National Natural Science Foundation of China(No.3197060323)SKLRM grant(SKLRM-2019B2)the Jiangsu ShuangChuang Talent Program,as well as the Jiangsu graduate student innovation fellowship to Y.S.
文摘Amyloids have traditionally been considered pathologic protein aggregates which contribute to neurodegeneration.New evidence however increasingly suggests that non-pathological amyloids are formed in animals during normal development.Amyloid-like aggregate formation was originally thought to be a conserved feature of animal gametogenesis.This hypothesis was based on findings which suggest that regulated amyloid formations govern yeast meiosis by way of meiosis-specific RNA binding proteins.Additional support came from studies which demonstrate that DAZL,a mammalian gametogenesis-specific RNA binding protein,also forms SDS-resistant aggregates in vivo.Here,we report evidence of aggregated BOULE formations,another DAZ family protein,during sperm development.Data suggest that in mouse testis,BOULE forms SDS-resistant amyloid-like aggregates.BOULE aggregate formation correlates with dynamic developmental expression during spermatogenesis but disappeared in Boule knockout testis.We also mapped essential small region in vitro BOULE aggregations,immediately downstream DAZ repeats,and found that aggregations positively correlated with temperature.We also performed enhanced UV cross-linking immunoprecipitation on BOULE aggregates from mouse testes and found that aggregates bind with a large number of spermatogenesis-related mRNAs.These findings provide insight into the amyloidogenic properties of gametogenesis-specific RNA binding proteins as a conserved feature in mammalian reproduction.Further investigation is warranted to understand the functional significance of BOULE amyloid-like formation during mouse spermatogenesis.
基金Supported by the National Natural Science Foundation of China(Nos.30470159 and C01020304)
文摘Affinity-mediated protein separation is an integral part of proteomics, the most outstanding of which is immunoproteomics. However, in the immunoprecipitate system overwhelming Ab and Ag conceal Ag interacting proteins as the research targets, which is the rate-limiting step in the progress of comparative proteomic analyses. We presented a convenient and accurate method to tackle this problem. 1 mol/L NaCl elution buffer was applied to the complex Rubisco immunoprecipitate of Arabidopsis, the weakest force involved in the system was selectively broken up, resulting in the enrichment of Rubisco interacting proteins accessible for further comparative protein gel profile. The easy-to-use method sheds light on a narrow-down strategy supplement for comparative immunoproteomics.
基金The datasets generated during the current study are available in the Sequence Read Archive(SRA)repository under the accession number PRJNA720376(BioProject)GenBank under the accession number PKKI00000000.
文摘As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controversial.Until now,the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2(N.gregoryi sp2)have not been characterized.We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody.We also cultured the N.gregoryi sp2 strain and performed immunoprecipitation,chromatin immunoprecipitation(ChIP),and RNA immunoprecipitation(RIP)assays.The nucleic acids that endogenously bound NgAgo in N.gregoryi sp2 cells were sequenced and analyzed.The results showed that NgAgo endogenously bound RNA rather than DNA.NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA,transcriptional regulators,RNA polymerases,and RNA-binding proteins.NgAgo mainly binds to the transcripts inside genes or in their upstream sequences.Interestingly,the top enriched motif of peaks was the same as that of miR-1289,suggesting that NgAgo may regulate gene expression post-transcriptionally.GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes.These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.
基金supported by the Natural Science Foundation of Guangdong Province,China,No.8151051501000004
文摘In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.
文摘Objective To study the biochemecal and immunological characterization of the 200 kD schistosomulum surface antigen Method and results A very high molecular weight schistosomulum surface antigen of Mr】200kD was identified and characterized using monoclonal antibodies. Carbohydrate modification experiments followed by radioimmunobinding assays demonstrated that the epitope recognised by the mAbs on this antigen was carbohydrate in nature, while protein digestion experiments followed by SDS-PAGE indicated that this antigen also contained protein. Immunoprecipitation of <sup>125</sup>I-labelled cercarial, schistosomulum, adult worm and miracidial surface antigens followed by gel analysis showed the carbohydrate epitope to be present on 5 cercarial, 2 schistosomulum and 5 miracidial surface molecules, and suggested a possible ecological function involved in adapting the parasite to the aquatic free-living stages of its life cycle and possibly also in protecting the early schistosomula from host immune damage. The 5 cercarial surfacs antigens proved to be associated with the CHR, since all the mAbs which recognised those antigens could induce a strong CHR. A kinetic investigation of the carbohydrate epitope on schistosomula of different ages demonstrated a gradual and possibly irreversible loss during the culture period. The epitope completely disappeared from the surface of adult worms. Conclusion To demonstrate an epitope common to a number of surface molecules of various developmental stages of schistosome and therefore explains the immunological cross-reactivity among different stages at the molecular level.
文摘Chromatin is the primary carrier of epigenetic information in higher eukaryotes. AtCYP71 contains both cyclophilin domain and WD40 repeats. Loss of AtCYP71 function causes drastic pleiotropic phenotypic defects. Here, we show that AtCYP71 physically interacts with FAS1 and LHP1, respectively, to modulate their distribution on chromatin. The Ihpl cyp71 double mutant showed more severe phenotypes than the single mutants, suggesting that AtCYP71 and LHP1 synergistically control plant development. Such synergism was in part illustrated by the observation that LHP1 association with its specific target loci requires AtCYP71 function. We also demonstrate that AtCYP71 physically interacts with FAS1 and is indispensable for FAS1 targeting to the KNAT1 locus. Together, our data suggest that AtCYP71 is involved in fundamental processes of chromatin assembly and histone modification in plants.
基金supported by the National Natural Science Foundation of China(Nos.30973375 and 30600152)
文摘Determining the binding sites of the transcription factor is important for understanding of transcriptional regulation. Transcription factor c-Jun plays an important role in cell growth, differentiation and development, but the binding sites and the target genes are not clearly defined in the whole human genome. In this study, we performed a ChIP-Seq experiment to identify c-Jun binding site in the human genome. Forty-eight binding sites were selected to process further evaluation by dsDNA microarray assay. We identified 283 c-Jun binding sites in K562 cells. Data analysis showed that 48.8% binding sites located within 100 kb of the upstream of the annotated genes, 28.6% binding sites comprised consensus TRE/CRE motif (5′-TGAC/GTCA-3′, 5′-TGACGTCA-3′) and variant sequences. Forty-two out of the selected 48 binding sites were found to bind the c-Jun homodimer in dsDNA microarray analysis. Data analysis also showed that 1569 genes are located in the neighborhood of the 283 binding sites and 191 genes in the neighborhood of the 42 binding sites validated by dsDNA microarray. We consulted 38 c-Jun target genes in previous studies and 16 among these 38 genes were also detected in this study. The identification of c-Jun binding sites and potential target genes in the genome scale may improve our fundamental understanding in the molecular mechanisms underlying the transcription regulation related to c-Jun.
文摘Background Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17β-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER ^+) breast cancer cell line MCF-7. Methods After the treatment of MCF-7 cells with E2 at different concentrations ( 10^-11 mol/L, 10.9 mol/L, 10.7 mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0. 7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10^-7 mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half- site ( 1/2 ERE) element and Spl sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (CHIP) assay was also used to confirm the binding of estrogen receptor a (ERα) on BMP-6 promoter in the presence of E2. Results E2 dose dependently increased BMP-6 mRNA expression in human ER ^+ breast cancer cell line MCF-7. At a dose of 10^-7 mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P 〈 0. 05 ). Both the 1/2 ERE response element mutant form and the Spl site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter' s response to E2. Through ChIP assay, the binding of ERot on 1/2 ERE response element in BMP-6 promoter was further validated. Conclusion Estrogen induces BMP-6 expression in human ER^+ breast cancer cell line MCF-7 receptor ERα binding on 1/2 ERE element in the BMP-6 promoter. through its
基金supported by grants from the National Key Research and Development Program of China(No.2016YFC1000500)the National Science Foundation for Young Scientists(No.81801501)the Postdo ctoral Science Foundation of China(No.2018M632026).
文摘Congenital heart disease(CHD)is the most common birth defect worldwide.Long non-coding RNAs(lncRNAs)have been implicated in many diseases.However,their involvement in CHD is not well understood.This study aimed to investigate the role of dysregulated lncRNAs in CHD.We used Gene Expression Omnibus data mining,bioinformatics analysis,and analysis of clinical tissue samples and observed that the novel lncRNA SAP30-2:1 with unknown function was significantly downregulated in damaged cardiac tissues from patients with CHD.Knockdown of lncRNA SAP30-2:1 inhibited the proliferation of human embryonic kidney and AC16 cells and decreased the expression of heart and neural crest derivatives expressed 2(HAND2).Moreover,lncRNA SAP30-2:1 was associated with HAND2 by RNA immunoprecipitation.Overall,these results suggest that lncRNA SAP30-2:1 may be involved in heart development through affecting cell proliferation via targeting HAND2 and may thus represent a novel therapeutic target for CHD.
基金This work was supported by the National Key Research and Development Program of China(no.2016YFD0600106)We also acknowledge financial support from the National Natural Science Foundation of China(grant nos.32001332 and 32001331)+1 种基金the Fundamental Research Funds for the Central Universities of China(grant nos.2572018CL01 and 2572018CL02)the Heilongjiang Touyan Innovation Team Program(Tree Genetics and Breeding Innovation Team).
文摘Tension wood(TW)is a specialized xylem tissue formed in angiosperm trees under gravitational stimulus or mechanical stresses(e.g.,bending).The genetic regulation that underlies this important mechanism remains poorly understood.Here,we used laser capture microdissection of stem xylem cells coupled with full transcriptome RNA-sequencing to analyze TW formation in Populus trichocarpa.After tree bending,PtrLBD39 was the most significantly induced transcription factor gene;it has a phylogenetically paired homolog,PtrLBD22.CRISPR-based knockout of PtrLBD39/22 severely inhibited TW formation,reducing cellulose and increasing lignin content.Transcriptomic analyses of CRISPR-based PtrLBD39/22 double mutants showed that these two genes regulate a set of TW-related genes.Chromatin immunoprecipitation sequencing(ChIP-seq)was used to identify direct targets of PtrLBD39.We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network(TRN)mediated by PtrLBD39.In this TRN,PtrLBD39 directly regulates 26 novel TW-responsive transcription factor genes.Our work suggests that PtrLBD39 and PtrLBD22 specifically control TW formation by mediating a TW-specific TRN in Populus.