CPSF蛋白相互作用的体外研究

目的 :研究在前体mRNA的切割与多聚腺嘌呤化中 4种CPSF蛋白间的相互作用及CPSF 73k、10 0k、16 0k与PAP的相互作用。方法 :将GST -CPSF 30k和GST -PAP结合于谷胱甘肽Sepharose基质上 ,与经网织红细胞体外翻译的CPSF 73k、10 0k、16 0k蛋白进行GST -pulldown试验。结果与讨论 :蛋白质电泳结果表明 ,4种CPSF蛋白可相互作用 ,以四聚体的形式与参与前体mR NA 3’端加工。此外 ,CPSF 73k、10 0k、16 0k也可分别与PAP相互作用 ,共同完成前体mRNA的切割与多聚腺嘌呤化。

Deregulated expression and subcellular localization of CPSF6,a circRNA-binding protein,promote malignant development of esophageal squamous cell carcinoma

Objective:Cleavage and polyadenylation specific factor 6(CPSF6)has been documented as an oncoprotein in different types of cancer.However,functions of CPSF6 have not been investigated yet in esophageal squamous cell carcinoma(ESCC).Here,we aimed to investigate the potential clinical values and biological functions of CPSF6 in ESCC.Methods:For determining the expression level of CPSF6 in ESCC patients,we analyzed published data,performed quantitative real-time polymerase chain reaction(RT-qPCR)and immunohistochemistry assays.Kaplan-Meier curves and log-rank tests were used for survival analyses.GO and KEGG analyses were done for CPSF6-related genes.Cell proliferation,colony formation and xenograft assays were conducted to verify the effects of CPSF6 on ESCC.In addition,cell cycle and apoptosis assays were also performed to manifest the functions of CPSF6 and circCPSF6.RNA pulldown and radioimmunoprecipitation(RIP)assays were used for confirming the interaction between circCPSF6(hsa_circ_0000417)and CPSF6 protein.The regulatory relationship between CPSF6 protein and circCPSF6 was determined by RT-qPCR.Results:We found that CPSF6 was upregulated in ESCC tissues and overexpression of cytoplasmic CPSF6 was associated with poor prognosis.GO and KEGG analyses suggested that CPSF6 could mainly affect cell division in ESCC.Further experiments manifested that CPSF6 promoted cell proliferation and colony formation in vitro.Xenograft assay showed that knockdown of CPSF6 significantly decreased tumor growth rate in vivo.Subsequently,we verified that depletion of CPSF6 led to cell cycle arrest and apoptosis.Finally,we validated that CPSF6,as a circRNA-binding protein,interacted with and regulated its circular isoform circCPSF6(hsa_circ_0000417),of which depletion also resulted in cell cycle arrest and cell apoptosis in ESCC.Conclusions:These findings gave us insight that overexpression of cytoplasmic CPSF6 protein is associated with poor prognosis in ESCC and CPSF6 may function as an oncoprotein,at least in part,through regulating circCPSF6 expression.

CPSF在真核表达载体上的克隆与瞬时表达

目的 :研究CPSF在真核表达载体上的克隆与瞬时表达。方法 :以质粒pBS176 1为模板扩增TAP -tag片段 ,PCR产物经纯化后克隆在真核表达载体pTRE2 -hyg上。再以pUK -CPSF 30k、73k、10 0k为模板扩增CPSF基因片段 ,将其克隆在质粒pTRE2 -hyg-TAP -tag中TAP -tag片段的下游 ,并将重组质粒转化入细胞株HelaTet -offS3细胞内。结果 :细胞抽提液经SDSPAGE电泳后进行蛋白质印迹杂交 ,胶片上出现野生型的CPSF条带和分子量较大的滞后条带。后者经分子量与分子标记对照 ,确系重组体TAP -tag-CPSF所表达的蛋白条带。结论 :重组质粒pTRE2hyg -TAP -tag-CPSF 30k ,73k ,10 0k在HelaTet -offS3内完好表达。

circRNA CPSF6核内转移促进同型半胱氨酸诱导的滋养细胞凋亡

目的探讨环状RNA剪切及多聚腺苷酸化特异因子6(circRNA CPSF6)在同型半胱氨酸(Hcy)致滋养细胞凋亡中的作用及机制。方法体外培养HTR-8/SVneo人绒毛膜滋养层细胞,分为对照组(0 mmol/L Hcy处理)与1 mmol/L Hcy处理组。采用免疫荧光细胞化学染色检测滋养细胞胱天蛋白酶3(caspase-3)的表达,Western blot法检测caspase-3蛋白水平;实时定量PCR检测circRNA CPSF6的mRNA表达水平;同时,运用小干涉RNA(siRNA)技术敲低滋养细胞circRNA CPSF6表达,Western blot法检测细胞caspase-3、caspase-9、B细胞淋巴瘤分子2(Bcl2)、Bcl2相关X蛋白(BAX)表达;实时定量PCR检测circRNA CPSF6在Hcy处理前后滋养细胞胞质/胞核中的表达水平。结果与对照组相比,Hcy处理组caspase-3表达明显增加,circRNA CPSF6 mRNA表达上调;敲低circRNA CPSF6后,caspase-3表达降低,线粒体凋亡途径被抑制;正常培养的滋养细胞中circRNA CPSF6在胞质大量表达,而给予Hcy处理后,circRNA CPSF6主要在胞核表达。结论通过circRNA CPSF6核内转移激活线粒体凋亡途径促进Hcy诱导的滋养细胞凋亡。

Screening of Potent Inhibitor of H1N1 Influenza NS1 CPSF30 Binding Pocket by Molecular Docking

The swine flu, H1N1 virus was outbroken in Mexico and the United States in April 2009 and then rapidly spread worldwide. The World Health Organization declared that the outbreak of influenza is caused by a new subtype of influenza H1N1 influenza virus. And researchers have isolated some oseltamivir resistance strains in 2009 swine flu which makes the imminency of research and development of new anti influenza drug. The CPSF30 binding pocket of effector domain in NS1 protein is very important in the replication of influanza A virus and is a new attractive anti flu drug target. But up to now there is no antiviral drug target this pocket. Here we employ molecular docking to screening of about 200,000 compounds. We find four novel compounds with high binding energy. Binding comformation analysis revealed that these small molecules can interact with the binding pocket by some strong hydrophobic interaction. This study find some novel small molecules can be used as lead compounds in the development of new antiinfluenza drug based on CPSF30 pocket.

SIZ1-mediated SUMOylation of CPSF100 promotes plant thermomorphogenesis by controlling alternative polyadenylation

Under warm temperatures,plants adjust their morphologies for environmental adaption via precise gene expression regulation.However,the function and regulation of alternative polyadenylation(APA),an important fine-tuning of gene expression,remains unknown in plant thermomorphogenesis.In this study,we found that SUMOylation,a critical post-translational modification,is induced by a long-term treat-ment at warm temperatures via a SUMO ligase SIZ1 in Arabidopsis.Disruption of SIZ1 altered the global usage of polyadenylation signals and affected the APA dynamic of thermomorphogenesis-related genes.CPSF100,a key subunit of the CPSF complex for polyadenylation regulation,is SUMOylated by SIZ1.Importantly,we demonstrated that SUMOylation is essential for the function of CPSF1oo in genome-wide polyadenylation site choice during thermomorphogenesis.Further analyses revealed that the SUMO conjugation on CPSF100 attenuates its interaction with two isoforms of its partner CPSF30,increasing the nuclear accumulation of CPsF1oo for polyadenylation regulation.In summary,our study uncovers a regulatory mechanism of APA via SiZ1-mediated SUMOylation in plant thermomorpho-genesis.

甲型H1N1流感病毒非结构蛋白NS1与人CPSF30结合拮抗剂筛选模型的建立及药物筛选

利用酵母双杂交技术研究甲型H1N1流感病毒非结构蛋白NS1和人切割与多聚腺苷酸化特异因子30kDa亚基(CPSF30)的相互作用,构建了一个酵母模型用于筛选NS1和CPSF30相互作用的拮抗剂,从而为筛选抗甲型H1N1流感病毒药物奠定基础。采用连续重叠PCR技术克隆得到H1N1流感病毒NS1基因。提取人HeLa细胞RNA,通过RT-PCR克隆得到人CPSF30基因。将NS1基因克隆到表达载体pGBKT7中获得诱饵载体pGBKNS1,将CPSF30基因克隆到载体pGADT7中获得捕获载体pGADCPSF;将pGBKNS1和pGADCPSF共转入酿酒酵母AH109,获得重组酿酒酵母AH109[pGADCPSF+pGBKNS1]。利用该模型筛选了30余种中成药,发现双黄连口服液等4种中成药能抑制NS1和CPSF30之间的相互作用。

中外企业博士后基金资助模式及特点的比较研究——以加拿大IRDF和中国CPSF为例

作为资助企业博士后的国家政府基金,加拿大IRDF基金与我国CPSF基金体现了鲜明的差异。IRDF强调市场导向,贴近学科前沿,注重质量优势;CPSF体现国家意志,面向传统领域,注重规模优势。设立单独面向企业博士后的基金,完善过程中管理,调整考核导向,是我国企业博士后基金未来的发展趋势。

AAUAA的分子生物学识别机制探究

mRNA3’端加poly A是很重要的生物学过程,加ployA需要识别三个信号,分别是UGUA、AAUAAA、UGUGU。以往的结构生物学已经发现了识别UGUA和UGUGU的分子生物学机制,而对于最为保守的AAUAAA的识别机制至今仍旧没有给出很清楚的解释,到目前为止,仅知道WDR33和CPSF30参与该识别过程。在本文中对参与识别的两个蛋白分别进行研究,期望找到识别最保守信号AAUAAA的结构生物学机制。通过克隆并且体外表达了这两个蛋白,借助荧光偏振实验研究了CPSF30和AAUAAA的相互作用,发现CPSF30对AAUAAA的识别是非特异性的,WDR33在大肠杆菌系统无法正确折叠。目前已经利用昆虫表达系统成功表达了蛋白,为后续的功能实验打下了坚实的基础。

CPSF30-L-mediated recognition of mRNA m^(6)A modification controls alternative polyadenylation of nitrate signaling-related gene transcripts in Arabidopsis

N6-methyladenosine(m^(6)A),a ubiquitous internal modification of eukaryotic mRNAs,plays a vital role in almost every aspect of mRNA metabolism.However,there is little evidence documenting the role of m^(6)A in regulating alternative polyadenylation(APA)in plants.APA is controlled by a large protein-RNA complex with many components,including CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30(CPSF30).In Arabidopsis,CPSF30 has two isoforms and the longer isoform(CPSF30-L)contains a YT512-B Homology(YTH)domain,which is unique to plants.In this study,we showed that CPSF30-L YTH domain binds to m^(6)A in v itro.In the cpsf30-2 mutant,the transcripts of many genes including several important nitrate signaling-related genes had shifts in polyadenylation sites that were correlated with m^(6)A peaks,indicating that these gene transcripts carrying m^(6)A tend to be regulated by APA.Wild-type CPSF30-L could rescue the defects in APA and nitrate metabolism in cpsf30-2,but m^(6)A-binding-defective mutants of CPSF30-L could not.Taken together,our results demonstrated that m^(6)A modification regulates APA in Arabidops is and revealed that the m^(6)A reader CPSF30-L affects nitrate signaling by controlling APA,shedding new light on the roles of the m^(6)A modification during RNA 3-end processing in nitrate metabolism.

Arab/c/opste N^(6)-methyladenosine reader CPSF30-L recognizes FUE signals to control polyadenylation site choice in liquid-like nuclear bodies

The biological functions of the epitranscriptomic modification N^(6)-methyladenosine(m^(6)A)in plants are not fully understood.CPSF30-L is a predominant isoform of the polyadenylation factor CPSF30 and consists of CPSF30-S and an m^(6)A-binding YTH domain.Little is known about the biological roles of CPSF30-L and the molecular mechanism underlying its m^(6)A-binding function in alternative polyadenylation.Here,we charac-terized CPSF30-L as an Arabidopsis m^(6)A reader whose m^(6)A-binding function is required for the floral tran-sition and abscisic acid(ABA)response.We found that the m^(6)A-binding activity of CPSF30-L enhances the formation of liquid-like nuclear bodies,where CPSF30-L mainly recognizes m*A-modified far-upstream elements to control polyadenylation site choice.Deficiency of CPSF30-L lengthens the 3'untranslated region of three phenotypes-related transcripts,thereby accelerating their mRNA degradation and leading to late flowering and ABA hypersensitivity.Collectively,this study uncovers a new molecular mechanism for m^(6)A-driven phase separation and polyadenylation in plants.

儿童非囊肿型先天性梨状窝瘘的诊断及内镜微创治疗效果

目的探讨儿童非囊肿型先天性梨状窝瘘(CPSF)的诊断和内镜下微创手术治疗效果。方法回顾性分析2017年10月-2022年5月该院收治的76例非囊肿型CPSF患儿的临床资料,行喉镜、颈部彩超、食道钡餐、颈部CT和磁共振成像(MRI)检查,于全身麻醉支撑喉镜引导下,行低温等离子射频消融术。结果喉镜下可见梨状窝处内瘘口,影像学检查可见瘘管与咽部相通;术后发生暂时性声者嘶哑3例,乳牙脱落3例;术后随访12~40个月,46例初治患儿复发1例,30例非初治患儿复发1例,再次行内镜下低温等离子烧灼,术后随访均无复发。结论CPSF症状无特异性,内镜下发现内瘘口是诊断的金标准,超声可作为初筛手段,食道钡餐联合CT检查可提高诊断率,MRI可作为辅助诊断;支撑喉镜下低温等离子射频消融术具有安全、微创、美观、便捷和可重复操作等优点,可作为儿童非囊肿型CPSF治疗的首选方法。

CPSF30-L: A direct connection between mRNA polyadenylation and m^(6)A RNA modification in plants

Alternative polyadenylation-the series of events that leads to the production of different mRNA isoforms from a primary transcript via usage of different polyadenylation sites within the transcript-is an important facet of gene expression in plants.Among the proteins that control alternative polyadenylation in plants is CPSF30,a core subunit of the polyadenylation complex(Chakrabarti and Hunt,2015).In plants,CPSF30 integrates cellular signaling with numerous growth and developmental processes through an interaction with calmodulin(Chakrabarti and Flunt,2015).

FRP纤维增强珊瑚混凝土基本性能研究

针对珊瑚混凝土强度低,脆性较大等缺陷,研究了不同掺量下的两种高强度FRP纤维增强材料(碳纤维CF、镀铜钢纤维CPSF)对珊瑚混凝土基本力学性能的影响,并对破坏形态进行分析。结果表明,CF和CPSF的体积掺量为0.5%、1%、1.5%时,珊瑚混凝土的性能均有一定提升;相比CF,CPSF对珊瑚混凝土的提升效果更为明显,最佳掺量为1.5%;过量的CF掺入会导致轴心抗压强度降低,但是CF对抑制裂缝的开展效果显著。

德国洪堡基金与中国博士后科学基金资助产出比较——基于文献计量分析

本文介绍了德国洪堡基金和中国博士后科学基金的资助现状,基于Web of Science数据库,对2008-2012年受洪堡基金和中国博士后科学基金资助的论文产出进行文献计量分析。通过比较洪堡基金和对中国博士后科学基金的资助绩效,发现两个基金的资助对学术成果的形成的影响,并提出中国博士后科学基金应加强国际化、提升论文影响力、增加对企业博士后的资助等政策建议。

中国博士后科学基金发展及资助政策改革设想

中国博士后科学基金对博士后成长与国家科技进步发挥着重要作用。为更好地发挥基金资助效果,采用文献法阐述了该基金会的发展过程,历年资助金的规模变化和资助博士后数量发展,资助金的资助效益,分析该基金存在着资助面较低且不稳定,资助类别设置不合理的问题,提出了"固定政府资助比例","调整资助类别与等级"和"改进评审制度"的资助政策改革措施。

The capsid revolution

Lenacapavir,targeting the human immunodeficiency virus type-1(HIV-1)capsid,is the first-in-class antiretroviral drug recently approved for clinical use.The development of Lenacapavir is attributed to the remarkable progress in our understanding of the capsid protein made during the last few years.Considered little more than a component of the virus shell to be shed early during infection,the capsid has been found to be a key player in the HIV-1 life cycle by interacting with multiple host factors,entering the nucleus,and directing integration.Here,we describe the key advances that led to this‘capsid revolution’.

罗非鱼源无乳链球菌双重PCR快速检测方法的建立

根据GenBank中罗非鱼源无乳链球菌荚膜多糖的cpsF基因和CAMP因子的cfb基因序列,设计2对特异性引物,通过对反应体系和反应条件优化,并进行特异性试验、敏感性试验及人工感染罗非鱼组织样品检测,建立了快速检测无乳链球菌的双重PCR方法。结果显示,仅无乳链球菌能扩增出cpsF和cfb两条特异性条带,而海豚链球菌、金黄色葡萄球菌、鮰爱德华氏菌、嗜水气单胞菌、杀鲑气单胞菌、豚鼠气单胞菌、嗜麦芽寡养单胞菌无条带检出;经敏感性试验,最小模板检出量为7.632×10-2ng/μL;同时对30尾人工感染无乳链球菌的罗非鱼肝和肾组织中细菌DNA进行双重PCR检测和细菌分离鉴定,检出的阳性结果一致。证实,所建立的方法具有快速、特异、灵敏的优点,适用于对罗非鱼等无乳链球菌的感染病例进行流行病学监测。

碳纤维复合材料磨损颗粒的生物学反应观察

目的 观察聚砜(PSF)及其碳纤维复合材料(CPSF)的生物相容性。方法 本实验用聚砜及其碳纤维复合材料磨损颗粒在体外对巨噬细胞株ANA- 1 吞噬功能和释放肿瘤坏死因子(TNF)、白细胞介素6(IL- 6)功能的影响进行研究,同时观察其关节内注射后对滑膜细胞的影响,并与传统的7 种人工关节材料比较。结果 颗粒被吞噬的情况与其大小关系密切,< 10 μm 的颗粒易被吞噬,> 20 μm 则多发生细胞包绕颗粒的情况。各实验组TNF和IL-6 的释放都有增高,但实验组之间差异无显著性意义。颗粒注入关节后发现滑膜细胞的吞噬功能增强,A 型细胞多见。结论 聚砜及其碳纤维复合材料磨损颗粒的生物相容性良好,力学性能优良。

基于循证设计的中国博士后科学基金整体资助绩效评估

中国博士后科学基金是政府为博士后研究人员申请科研资助而开辟的专属通道,择优资助具有创新能力和发展潜能的优秀博士后从事创新性研究。以中国博士后科学基金为对象,运用循证评估设计方法,建立基于"评估议题—关键问题—证据"的整体资助绩效评估框架,通过文本分析、数理统计、文献计量和问卷调查等评估方法进行定性与定量研究,系统总结基金资助取得的成效、存在的问题、面临的挑战,并提出相关的资助政策与管理建议。