铜坑矿排水排泥系统改造

金属矿山井下排水排泥系统是重要的安全生产设施。长期以来,铜坑矿井下排水与排泥是分开的,即先排水后,再把沉淀下来的泥浆用人工清浆方式进行清理妆车,再转运至采空区或地面废石场进行处理,采用原始的人工清浆方法,时间长,工效低。文章提出以机械化清浆来代替原始的人工清浆方式,以提高效率,减少影响。

Verification of the Interaction between SET and eEF1A1 in Human Liver Cells by Co-immunoprecipitation

[Objective] The aim was to investigate the possible interaction between SET and eEF1A1 in human liver cells. [Method] Firstly the total proteins of human L-02 liver cells were extracted under non-denaturing conditions; then,mouse anti-human SET and rabbit anti-human eEF1A1 antibodies were used to perform the co-immunoprecipitation respectively; subsequently,the immunoprecipitations was correspondingly detected with rabbit anti-human eEF1A1 and mouse anti-human SET antibodies by Western Blot. [Result] EEF1A1 was detected in protein complex from the immunoprecipitations by using anti-SET antibody,and SET also was detected in immunoprecipitations by using anti-eEF1A1 antibody. [Conclusion] The interaction between SET and eEF1A1 in human liver cells was confirmed.

Dishevelled interacts with p65 and acts as a repressor of NF-KB-mediated transcription

Dishevelled （Dvl） is a highly conserved protein family that plays an important role in mediating Wnt signal- ing from membrane to cytoplasm. Recently we reported that Dvl also functions in the nucleus by stabilizing the β-catenin/TCFs transcriptional complex. Here we describe that Dvl may function as a repressor of NF-kB. Our data show that Dvl directly binds to p65 and their interaction occurs in the nucleus. Dvl expression inhibits p65-mediated or TNF-α-stimulated activation of the NF-KB dependent reporter. This action of Dvl, however, is not dependent on Wnt or its downstream effector β-catenin. Chromatin immunoprecipitation assay shows that recruitment of p65 to the promoters of NF-KB target genes is significantly enhanced when expression of Dvl is knocked down. Consistently, the expression level of a subset of NF-KB target genes is also increased after knock-down of Dvl. Moreover, our data suggest that Dvl may relieve the anti-apoptotic effect of NF-KB, thus play a role in promoting apoptosis. Therefore, this work demonstrates a novel function of Dvl in modulating NF-KB-regulated gene transcription.

AtFKBP53 is a histone chaperone required for repression of ribosomal RNA gene expression in Arabidopsis

Chromatin structure is important for controlling gene expression, but mechanisms underlying chromatin remodel- ing are not fully understood. Here we report that an FKBP （FK506 binding protein） type immunophilin, AtFKBP53, possesses histone chaperone activity and is required for repressing ribosomal gene expression in Arabidopsis. The At- FKBP53 protein is a multidomain FKBP with a typical peptidylprolyl isomerase （PPIase） domain and several highly charged domains. Using nucleosome assembly assays, we showed that AtFKBP53 has histone chaperone activity and the charged acidic domains are sufficient for the activity. We show that AtFKBP53 interacts with histone H3 through the acidic domains, whereas the PPIase domain is dispensable for histone chaperone activity or histone binding. Ri- bosomal RNA gene （18S rDNA） is overexpressed when AtFKBP53 activity is reduced or eliminated in Arabidopsis plants. Chromatin immunoprecipitation assay showed that AtFKBP53 is associated with the 18S rDNA gene chro- matin, implicating that AtFKBP53 represses rRNA genes at the chromatin level. This study identifies a new histone chaperone in plants that functions in chromatin remodeling and regulation of transcription.

Fhyroid hormone controls the gene expression of HSV-1 .AT and ICPO in neuronal cells

Various factors/pathways including hormonal regulation have been suggested to control herpes simplex virus type 1 （HSV-1） latency and reactivation. Our computer analysis identified a DNA repeat containing thyroid hormoneresponsive elements （TRE） in the regulatory region of HSV-1 latency-associated transcript （LAT）. Thyroid hormone （triiodothyronine, T3） functions via its receptor TR （thyroid hormone receptor）, a transcription factor. Present study investigated the roles of TR and T3 in HSV-1 gene expression using cultured neuoroblastoma cell lines. We demonstrated that liganded TR activated LAT transcription, but repressed infected cell protein no. 0 （ICP0） transcription in the presence of LAT TRE. Chromatin immunoprecipitation （CHIP） assays showed that TRs were recruited to LAT TREs independently of T3 and hyperacetylated H4 was associated with the LAT promoter that was transcriptionally active. In addition, ChIP results showed that the chromatin insulator protein CCCTC-binding factor was enriched at the LAT TREs in the presence of TR and T3. In addition, the BRG1 chromatin remodeling complex is found to participate in the T3/TR-mediated LAT activation since overexpression of BRG1 enhanced the LAT transcription and the dominant-negative mutant K785R abolished the activation. This is the first report revealing that TR elicits epigenetic regulation on HSV-1 ICP0 expression in neuronal cells and could have a role in the complex processes of HSV-1 latency/reactivation.

Screening and identifkation of proteins interacting with nudeostemin

AIM： To identify the proteins interacting with nucleostemin （NS）, thereby gaining an insight into the function of NS. METHODS： Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and β-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially. RESULTS： Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B （B56） （PPP2RSA） and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A. CONCLUSION： NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

C/EBPα regulates SIRT1 expression during adipogenesis

SIRT1 plays an important role in adipogenesis, but how SIRT1 is regulated in adipogenesis is largely unknown. In this study, we show that both SIRT1 protein and mRNA levels were increased along with CCAAT/enhancer-binding protein a （C/EBPa） during adipocyte differentiation. C/EBPa, but not C/EBPap30, activated SIRT1 promoter in both HeLa cells and 3T3-L1 preadipocytes. Furthermore, C/EBPa upregulated SIRT1 mRNA and protein levels in HeLa cells and increased SIRT1 expression in a p53-independent manner in Soas2 cells. In preadipocytes, ectopic expression of C/EBPa upregulated SIRT1 protein level and knockdown of C/EBPa led to the decrease of SIRTI pro- tein level. Moreover, by promoter deletion analysis, gel shift assay and chromatin immunoprecipitation, we found that C/EBPa bound to the SIRT1 promoter at a consensus C/EBPα binding site. These data demonstrate that C/ EBPα regulates SIRT1 expression during adipogenesis by directly binding to the SIRT1 promoter.

NF-E2:a Novel Regulator of Alpha-hemoglobin Stabilizing Protein Gene Expression

Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.

Mutual regulation between microRNA-373 and methyl-CpGbinding domain protein 2 in hilar cholangiocarcinoma

AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.

Inhibition of SIRT1 Increases EZH2 Protein Level and Enhances the Repression of EZH2 on Target Gene Expression

Objective To study the regulatory roles of SIRT1 on EZH2 expression and the further ef-fects on EZH2's repression of target gene expression. Methods The stable SIRT1 RNAi and Control RNAi HeLa cells were established by in-fection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells. Results Western blot results showed that EZH2 protein level increased upon SIRT1 de-pletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter region of EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases. Conclusions Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expres-sion.

Microfluidic Immunoprecipitation for Post-Translational Modified Protein Purification

In this paper, we report an antibody functionalized microimmunopreci- pitation （IX IP） method used for enrich lowabundant post-translational modified （PT~ proteins. The device is fabricated by inert, nontoxic and disposable polydimethylsiloxane （PDMS） using a silane-based chemical modification protocol, which yield antibody- terminated PDMS surfaces. In this study, the IX IP device is specifically designed for the purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model in vitro oxidized bovine serum albumin （BSA） was first derivitized by dinitrophenylhydrazide （DNPH） and then captured by the anti-DNP immobilized on this Ix lP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment Ix IP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.

Ran binding protein 9(RanBPM) binds IFN-λR1 in the IFN-λsignaling pathway

Like the type I interferons(IFNs),the recently discovered cytokine IFN-λ displays antiviral,antiproliferative,and proapoptotic activities,mediated by a heterodimeric IFN-λ receptor complex composed of a unique IFN-λR1 chain and the IL-10R2 chain.However,the molecular mechanism of the IFN-λ-regulated pathway remains unclear.In this study,we newly identified RAN-binding protein M(RanBPM) as a binding partner of IFN-λR1.The interaction between RanBPM and IFN-λRl was identified with a glutathione S-transferase pull-down assay and coimmunoprecipitation experiments.IFN-λ1 stimulates this interaction and affects the cellular distribution of RanBPM.However,the interaction between RanBPM and IFN-λR1 does not correlate with their conserved TRAF6-binding sites.Furthermore,we also found that RanBPM is a scaffolding protein with a modulatory function that regulates the activities of IFN-stimulated response elements.Therefore,RanBPM plays a novel role in the IFN-λ-regulated signaling pathway.

Genomewide decoupling of H2AK119ub1 and H3K27me3 in early mouse development

Polycomb group(Pc G)proteins are crucial chromatin regulators during development.H2 AK119 ub1(H2 Aub)and H3 K27 me3 are catalyzed by Polycomb-repressive complex 1 and 2(PRC1/2)respectively,and they largely overlap in the genome due to mutual recruitment of the two complexes.However,it is unclear whether PRC1/H2 Aub and PRC2/H3 K27 me3 can also function independently.By developing an ultra-sensitive carrier-DNA-assisted chromatin immunoprecipitation sequencing method termed CATCH-Seq,we generated allelic H2 Aub profiles in mouse gametes and early embryos.Our results revealed an unexpected genomewide decoupling of H2 Aub and H3 K27 me3 in mouse preimplantation embryos,where H2 Aub but not H3 K27 me3 was enriched at Pc G targets while only H3 K27 me3 was deposited in the broad distal domains associated with DNA methylation-independent non-canonical imprinting.These observations suggest that H2 Aub represses future bivalent genes during early embryogenesis without H3 K27 me3,but it is not required for the maintenance of non-canonical imprinting,which is mediated by maternal H3 K27 me3.Thus,our study reveals the distinct depositions and independent functions of H2 Aub and H3 K27 me3 during early mammalian development.

Kicking down the ladder： adding cleavable features to genetically encoded photocrosslinkers

Protein-protein interactions(PPIs)play vital roles in biological processes.However,the discovery of transient and biologically relevant PPIs remain a formidable challenge using conventional strategies such as co-immunoprecipitation(CoIP),yeast two hybrids(Y2H)and Forster resonance energy transfer(FRET)[1].Genetically encoded photocrosslinkers have recently emerged as a powerful approach for probing intracellular PPIs with the capability for in situ studies,low perturbation to cells as well as the wide generality.This facile strategy also demonstrated an advantage of high spatiotemporal resolution,which offers a robust capture strategy for the discovery of transient and/or weak PPIs with lowered false-positive backgrounds[2].