京海黄鸡柔嫩艾美耳球虫感染后盲肠转录组分析

为了探究京海黄鸡感染柔嫩艾美尔球虫(E. tenella)后盲肠组织差异表达基因,以及球虫感染分子应答过程和免疫应答机制,试验采用RNA-seq技术对E. tenella感染和非感染组第7天的盲肠组织进行转录组测序,筛选差异表达基因,并进行差异基因的功能、通路富集分析。结果表明,在感染和非感染组中有显著差异的表达基因2 830个(P0.05),其中1 419个基因上调,1 411个基因下调。随机选取10个差异基因进行qRT-PCR验证,结果显示差异基因的表达倍数与RNA-seq检测结果显著相关(r=0.988,P0.000),决定系数达0.975。GO分析表明,有2 356个差异基因获得GO功能注释,显著富集的前30个GO terms主要涉及细胞交流、信号转导、血管生成、氧化还原酶活性等。KEGG分析发现差异基因显著富集的信号通路有黏着斑、细胞外基质-受体相互作用、过氧化物酶体增殖物激活受体等。这些通路中的差异基因有ANGPTL4、ACSL5、VEGFC、CD44和MAKP10等,提示这些基因在宿主柔嫩艾美耳球虫感染过程中发挥重要作用。     

基于高通量转录组测序的草鱼雌雄性腺差异表达基因分析

草鱼雌性个体生长优势明显高于雄性,挖掘精巢和卵巢差异表达的功能基因对草鱼生殖调控具有重要的价值及应用前景。采用Illumina HiSep 2500转录组测序技术分别对12月龄的3尾雌性草鱼和3尾雄性草鱼的性腺组织的mRNA进行测序分析,精巢和卵巢共得到8 363个差异表达基因。与卵巢相比,精巢上调基因6 446个(77%),下调基因1 917个(23%),差异明显。将上述差异基因在Nr、GO和KEGG数据库进行比对,获得48种Go功能注释分类和313条KEGG代谢通路。分析发现,Go功能分类中"绑定"功能富集差异基因的数量最多,占总数的15.8%;KEGG代谢通路中"信号转导"通路富集差异基因的数量最多,占总数的10.2%。结合转录本数据,挑选与草鱼性腺发育相关的关键差异基因Dmrt1、amh、foxl2、cyp19a1a进行分析,研究显示,与卵巢相比,Dmrt1和amh基因在草鱼精巢中极显著高表达(p0.01);与精巢相比,cyp19a1a和foxl2基因在草鱼卵巢中显著高表达(p0.05),与转录组数据分析结论一致,说明Dmrt1和amh基因与早期草鱼精巢发育调控相关,cyp19a1a和foxl2基因与早期草鱼卵巢发育调控相关。本实验为进一步了解草鱼性别基因的调控机理提供数据依据。     

小鼠正常黑色素细胞与黑色素瘤细胞(B16)mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径,主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrinβ1和Integrinβ5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

小鼠正常黑素细胞与黑色素瘤细胞（B16）mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径, 主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrin-β1和Integrin-β5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

USP1通过调控E2F1对膀胱癌细胞增殖和周期的作用研究

该文探讨了泛素特异蛋白酶1(USP1)对膀胱癌细胞增殖和周期等生物学行为的作用,并进一步探索其作用机制。通过分子克隆技术构建膀胱癌T24细胞USP1过表达细胞株; CRISPRCas9技术构建膀胱癌UMUC3细胞USP1敲除细胞株; CCK8和克隆形成实验检测细胞增殖和克隆形成能力;划痕实验检测细胞迁移; PI染色流式细胞术检测细胞周期;转录组测序检测USP1敲除后基因表达差异及其相关的功能与信号通路;双荧光素酶报告基因检测USP1对信号通路的影响,并通过免疫印迹技术进行验证。结果显示, USP1过表达可以促进膀胱癌细胞增殖,敲除后显著抑制了膀胱癌细胞的增殖和克隆形成及迁移能力,促进S期细胞阻滞。转录组测序结果显示, USP1敲除后差异表达基因共4 522个,其中上调基因2 078个,下调基因2 444个。KEGG分析结果显示,这些基因涉及多个方面,包括细胞周期调控、细胞信号转导、转录翻译、蛋白折叠降解、自噬凋亡等。Hallmark数据库分析结果显示,差异表达基因与E2F信号通路密切相关。双荧光素酶报告基因显示, USP1过表达后, E2F1信号通路明显上调并且呈剂量依赖式,免疫印迹结...     

抗肿瘤多肽9R-P201诱导下的肝癌HepG2细胞转录组测序分析

旨在探索多肽9R-P201处理肝癌HepG2细胞后基因融合、单核苷酸多态性(Single nucleotide polymorphism,SNP)突变、可变剪接等事件,并分析差异表达基因所参与的生物学进程与信号通路,以期解析多肽9R-P201在转录组水平对肝癌细胞的调控。通过转录组测序检测9R-P201处理肝癌HepG2细胞前后基因差异表达情况,tophat-fusion软件检测基因融合,SAMTOOLS软件检测SNP位点,r MATS软件鉴定可变剪接,使用基因本体(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析方法对差异表达基因进行功能富集分析。结果共检测到可变剪接事件276个、SNP位点5 557个、基因融合事件45个;同时共得到显著差异表达基因403个,其中上调269个而下调134个,基因的功能富集分析结果显示差异表达基因显著富集细胞生长、迁移等肿瘤相关生物进程,并参与多条与癌症相关的信号通路。研究表明在9R-P201诱导HepG2细胞后,导致表达差异基因显著与肿瘤生物学进程和通路相关,并发生了大量可变剪接、SNP突变、基因融合等事件,这暗示着该多肽有望作为后续肝癌介入治疗潜在药物分子。     

眼优角蚱两性成体转录组差异分析

为了解蚱类昆虫两性间基因转录水平上的整体差异,本研究以眼优角蚱Eucriotettix oculatus为研究对象,通过高通量转录组(RNA-Seq)测序和分析技术对其雌、雄成体的转录组进行了测定和分析。结果显示,在雌、雄转录组中共获得13 432条转录本,两性间差异表达基因共3 597个,其中雌性相对于雄性显著上调基因1 295个,显著下调基因2 302个。对其中9个差异基因进行实时荧光定量PCR验证,其结果与RNA-Seq分析一致。差异基因显著富集的GO条目大多是与细胞的形成有关,结合、细胞组分和细胞过程等条目相关基因显著高表达。KEGG富集显示差异基因主要是与性腺发育、遗传信息加工、能量代谢和消化等通路有关。研究结果对从分子水平进一步研究眼优角蚱两性差异提供一定的理论基础。     

镧对铜胁迫下水稻转录组模式的调控分析

探究稀土镧对铜胁迫下水稻转录组的影响,鉴定镧在调控水稻铜胁迫应答中的关键基因和功能。利用抗氧化酶活性筛选最适铜胁迫及镧处理浓度,进行转录组测序和差异表达分析,并用qRT-PCR验证差异表达水平。对测序数据进行差异表达分析发现3 222个基因显著上调,3 798个显著下调,qRT-PCR验证了4个细胞壁防御相关基因CHIT13、Laccase、Expansin和GH3.4。功能和通路分析获得95组GO条目和112条KEGG通路,富集于激酶和氧分子结合等分子功能、细胞壁及质膜等细胞组分、次级代谢和脂质代谢等生物学过程以及苯丙烷生物合成和植物激素信号转导通路。镧通过调节细胞壁形成和组分提高水稻铜胁迫耐受性。     

基于生物信息学方法识别非小细胞肺癌(NSCLC)潜在治疗靶基因

本研究运用生物信息学方法识别非吸烟女性非小细胞肺癌(NSCLC)潜在的靶基因,并从分子水平探索其潜在的发病机制。从GEO数据库下载非吸烟女性非小细胞肺癌相关基因芯片数据集,经癌症组和癌旁对照组差异表达基因识别,并利用R软件对差异基因进行层次聚类分析,DAVID进行基因本体(gene ontology)和KEGG通路富集分析,STRING和Cytoscape软件构建蛋白-蛋白交互(PPI)网络,以及运用PASTAA分析,识别NSCLC相关转录因子,构建转录因子-基因共表达网络。结果表明,185个基因在NSCLC中差异表达,其中40个上调,145个下调;通过PASTAA分析识别出5个NSCLC基因相关转录因子。差异基因与胶原分解代谢过程、炎症反应的正调控等生物过程密切相关,基因的产物主要参与蛋白质细胞外基质、胶原三聚体等细胞组分,且主要发挥调节金属内肽酶活性、肝素结合和调节受体活性等分子功能;KEGG通路富集分析表明差异基因显著富集到胞外基质-受体信号通路、粘着斑信号通路、PPAR信号通和PI3K-Akt信号通路等,与非小细胞肺癌的发生发展密切相关。通过生物信息学方法,最终筛选到4个NSCLC关键基因:IL6、MMP1、COL1A1、CD36,其可能是非吸烟女性NSCLC潜在的治疗靶点。     

茶树种子发育过程的转录组分析

该研究以‘铁观音’茶树品种的种子为试验材料,采用转录组测序技术分析种子发育的3个时期(幼果期、膨大期、成熟期)的表达差异,探究茶树种子油脂代谢的分子机制。结果表明:(1)经转录组测序、组装后共获得30 940 581个clean reads,经数据合并拼接最终得到36 951条非冗余Unigene序列,其中28 476个Unigene可得到功能注释;在转录本中能够被注释到GO分类的Unigene有11 201条(30.3%),KEGG分析发现共有17 172个基因参与了127个代谢通路。(2)经KEGG通路筛选出14条与脂肪酸代谢相关的通路,且随着茶籽的发育,大部分脂肪酸调控途径相关基因呈下调趋势,其中上调基因数最多的有α-亚麻酸代谢途径和脂肪酸降解途径(有17个基因表达量上调),下调基因数最多的是甘油磷脂代谢途径(有58个基因表达量下调);在茶籽发育幼果期α-亚麻酸代谢途径中表达量上调的基因数超过表达量下调的基因数。(3)研究发现茶籽脂肪酸合成相关的基因涉及14个脂类调控途径,共409条差异基因;随着茶树种子发育到成熟期,上调的差异表达基因数量在减少,下调的差异表达基因数量增加,其中α-亚麻酸途径中的基因PLA2G16、DAD1、pldA、FabF、FabI表达量上调显著,随后表达量下调。(4)qRT-PCR检测结果表明,7个茶树FAD和1个ACP差异表达基因的水平与转录组测序结果基本一致;随着茶籽的发育,基因CsFAD7和Δ6-CsFAD从幼果期、果实膨大期至果实成熟期都为差异下调表达,CsFAD2、CsFAD6和Δ7-CsFAD为差异上调表达,CsFAD8、Δ8-CsFAD和CsACP在幼果期至果实膨大期差异上调表达,在果实膨大期至果实成熟期差异下调表达。     

Cytotoxic T-cell abundance and virus load in human immunodeficiency virus type 1 and human T-cell leukaemia virus type 1.

The correlation between virus load and specific cytotoxic T-lymphocyte (CTL) frequency during the chronic phase in human immunodeficiency virus type 1 (HIV-1) infection has been found to be negative in cross-sectional studies. We report here that, in infection with the related retrovirus human T-cell leukaemia virus type 1 (HTLV-1), the correlation is positive in asymptomatic carriers and zero in patients with the associated inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We demonstrate that the direction of the correlation may depend on the efficacy of the CTL response using mathematical models. We conclude that the CTL response is effective in asymptomatic carriers of HTLV-1, but ineffective in patients with HAM/TSP. Virus-mediated impairment of specific CTL production in HIV-1 infection can account for the negative correlation observed.     

Galectin-1 binds to influenza virus and ameliorates influenza virus pathogenesis

Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant (K(d)) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.     

DC-SIGN interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus

Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.     

人H7N9禽流感病毒、高致病H5N1禽流感病毒及H1N1流感病毒感染小鼠特征分析

目的对比分析人高致病H5N1禽流感病毒、H7N9禽流感病毒及H1N1流感病毒分别感染BALB/c小鼠后的机体反应特征。方法分别以H7N9病毒、H5N1病毒和H1N1病毒滴鼻感染BALB/c小鼠,观察小鼠存活率、体征变化及感染后肺组织病理损伤差异,检测小鼠感染流感病毒后肺组织增殖细胞核抗原(PCNA)表达并观察小鼠感染后修复状况。结果 H7N9病毒、H5N1病毒和H1N1病毒均感染BALB/c小鼠,小鼠存活率依次为H7N9H1N1H5N1,肺组织病理损伤严重程度依次为H5N1H1N1H7N9,PCNA表达水平依次为H7N9H1N1H5N1。结论 H7N9病毒感染后宿主炎症反应较小,感染后小鼠肺组织自我修复能力较强;H5N1病毒感染BALB/c小鼠后的机体反应最为强烈,感染后恢复能力差,致死率高。     

Pf1 virus particle dynamics

The overall dynamics of the Pf1 filamentous bacteriophage particle in solution are characterized by nmr experiments. The chemical-shift anisotropy powder-pattern lineshapes from both DNA and protein backbone sites of the virus are motionally averaged in the same way, indicating that the entire particle undergoes rapid (< 10⁴ Hz) reorientation about the long axis of the filament when the virus is in solution at high pH. In contrast, the virus particles in samples at low pH are immobile on this time scale.     

An important role for syndecan-1 in herpes simplex virus type-1 induced cell-to-cell fusion and virus spread

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread.     

Human immunodeficiency virus type 1 and influenza virus exit via different membrane microdomains

Directed release of human immunodeficiency virus type 1 (HIV-1) into the cleft of the virological synapse that can form between infected and uninfected T cells, for example, in lymph nodes, is thought to contribute to the systemic spread of this virus. In contrast, influenza virus, which causes local infections, is shed into the airways of the respiratory tract from free surfaces of epithelial cells. We now demonstrate that such differential release of HIV-1 and influenza virus is paralleled, at the subcellular level, by viral assembly at different microsegments of the plasma membrane of HeLa cells. HIV-1, but not influenza virus, buds through microdomains containing the tetraspanins CD9 and CD63. Consequently, the anti-CD9 antibody K41, which redistributes its antigen and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins.     

Adeno-associated virus site-specific integration and AAVS1 disruption

Adeno-associated virus (AAV) is a single-stranded DNA virus with a unique biphasic lifestyle consisting of both a productive and a latent phase. Typically, the productive phase requires coinfection with a helper virus, for instance adenovirus, while the latent phase dominates in healthy cells. In the latent state, AAV is found integrated site specifically into the host genome at chromosome 19q13.4 qtr (AAVS1), the only animal virus known to integrate in a defined location. In this study we investigated the latent phase of serotype 2 AAV, focusing on three areas: AAV infection, rescue, and integration efficiency as a function of viral multiplicity of infection (MOI); efficiency of site-specific integration; and disruption of the AAVS1 locus. As expected, increasing the AAV MOI resulted in an increase in the percentage of cells infected, with 80% of cells infected at an MOI of 10. Additional MOI only marginally effected a further increase in percentage of infected cells. In contrast to infection, we found very low levels of integration at MOIs of less than 10. At an MOI of 10, at which 80% of cells are infected, less than 5% of clonal cell lines contained integrated AAV DNA. At an MOI of 100 or greater, however, 35 to 40% of clonal cell lines contained integrated AAV DNA. Integration and the ability to rescue viral genomes were highly correlated. Analysis of integrated AAV indicated that essentially all integrants were AAVS1 site specific. Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 ( approximately 100% of infected cells). Rep expression by itself and in the presence of a plasmid integration substrate was able to mediate this disruption of the AAVS1 site. We further characterized the disruption event and demonstrated that it resulted in amplification of the AAVS1 locus. The data are consistent with a revised model of AAV integration that includes preliminary expansion of a defined region in AAVS1.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.