Photophysical Behavior of Heteroduplexes Formed from Naphthalene Incorporated Foldamers,and Linear Donors and Pyromellitic Diimide Incorporated Foldamers

The authors synthesized the electron-rich 1,5-dioxonaphthyl group incorporated foldamers, and linear donors and electron-deficient pyromellitic diimide incorporated foldamers, which were investigated in organic solvents. There is an evident charge transfer process from the donors to the electron-deficient foldamers, due to the donor-acceptor interaction of the heteroduplexes formed from the donors and the electron-deficient foldamers in chloroform, observed by using the ultraviolet（UV） visible method. The charge transfer process of the heteroduplexes depends on the molecular structure（not only the number of 1,5-dioxonaphthyl groups and pyromellitic diimide groups in the heteroduplex, but also the intermolecular double hydrogen bond in the heteroduplex）, the intercalative π-π stacking motif, the initial concentration of heteroduplexes, and the polarity of the solvent. Fortunately, the authors found a critical bumping concentration（CBC） of the donors in acetonitrile or chloroform, which is useful in quantitative research, by using the fluorescence method. The value of CBC depends not only on intramolecular folding and the intramolecular hydrogen bond, but also on intramolecular π-π stacking and the solvent. The electron-deficient foldamers quenched the fluorescence of the donors. Fluorescence quenching depends not only on the number of pyromellitic diimide groups and 1,5-dioxonaphthyl groups in the heteroduplex, but also on the intercalative π-π stacking motif between the donor and the acceptor.

Detection of RB Germline Mutations Using Exon-by-exon Heteroduplex Analysis Compared with SSCP

Purpose: To compare heteroduplex analysis with SSCP and to develop a simple and effective method for mutational screening of RB gene.Materials and Methods: Leukocyte DNA was prepared from 12 unrelated Japanese patients with hereditary retinoblstoma. PCR combined with simultaneous nonisotopic heteroduplex and SSCP analysis was used to screen leukocyte DNA for such mutations, exon-by-exon, without the use of restriction endonu-clease digestion. PCR was conducted using 28 pairs of primers flanking all 27 ex-ons and the promoter region of the RB gene, with PCR products ranging from 159bp to 326bp. Mutations were identified by sequencing.Results: Heterozygous germline mutations were detected in 8 of 12 Japanese patients. The mutations were identified by sequencing as follows; G→C/acceptor of exon 11, T insertion/codon 389, C→T/codon 455, 33bp insertion/codon 455 (C GA), G→T/codon 533, C→T/codon 579, C deletion/codon 674, and C→T/ codon 787.Conclusion: Our results suggest that small RB gene mutations

Exon-by-exon Screening for RB Germline Mutations Using Heteroduplex-SSCP Analysis

Objective: To disclose the nature of RBI germline mutations in Chinese and to develop a practical and effective way for mutational screening. Methods: Leukocyte DNA was prepared from 8 Chinese patients with hereditary retinoblastoma. PCR combined with nonisotopic heteroduplex-SSCP analysis was used to screen leukocyte DNA for RBI germline mutations, exon-by-exon, without the use of restriction endonuclease digestion. The mutations were finally identified by sequencing. In order to testify the effectiveness of this method, the same method was used to detect other 17 samples which have been previously analyzed by other methods. Results: Heterozygous germline mutations were detected in the leukocyte DNA of 6 out of 8 Chinese patients;G del/codon 46,T del/codon 131 ,CAGAA del/condon 257 -258, GCAgta→GCAgca/donor of exon 16, C@T/codon 661, and C→T/codon 787. Heteroduplex-SSCP analysis may detect RBI germline mutations in 68% (177 25) unselected patients, which is more effective than SSCP(56%) or

Therapeutic Targeting of PKM2 Ameliorates NASH Fibrosis Progression in a Macrophage-Specific and Liver-Specific Manner

Nonalcoholic steatohepatitis(NASH)may soon become the leading cause of end-stage liver disease worldwide with limited treatment options.Liver fibrosis,which is driven by chronic inflammation and hepatic stellate cell(HSC)activation,critically determines morbidity and mortality in patients with NASH.Pyruvate kinase M2(PKM2)is involved in immune activation and inflammatory liver diseases;however,its role and therapeutic potential in NASH-related fibrosis remain largely unexplored.Bioinformatics screening and analysis of human and murine NASH livers indicated that PKM2 was upregulated in nonparenchymal cells(NPCs),especially macrophages,in the livers of patients with fibrotic NASH.Macrophage-specific PKM2 knockout(PKM2^(FL/FL)LysM-Cre)significantly ameliorated hepatic inflammation and fibrosis severity in three distinct NASH models induced by a methionine-and choline-deficient(MCD)diet,a high-fat high-cholesterol(HFHC)diet,and a western diet plus weekly carbon tetrachloride injection(WD/CCl_(4)).Single-cell transcriptomic analysis indicated that deletion of PKM2 in macrophages reduced profibrotic Ly6C^(high) macrophage infiltration.Mechanistically,PKM2-dependent glycolysis promoted NLR family pyrin domain containing 3(NLRP3)activation in proinflammatory macrophages,which induced HSC activation and fibrogenesis.A pharmacological PKM2 agonist efficiently attenuated the profibrotic crosstalk between macrophages and HSCs in vitro and in vivo.Translationally,ablation of PKM2 in NPCs by cholesterol-conjugated heteroduplex oligonucleotides,a novel oligonucleotide drug that preferentially accumulates in the liver,dose-dependently reversed NASH-related fibrosis without causing observable hepatotoxicity.The present study highlights the pivotal role of macrophage PKM2 in advancing NASH fibrogenesis.Thus,therapeutic modulation of PKM2 in a macrophage-specific or liver-specific manner may serve as a novel strategy to combat NASH-related fibrosis.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals：A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout（KO） or knockin（KI） animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA（gDNA） followed by an automated capillary electrophoresis step which reveals a heteroduplexes（HD） signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

Construction of heteroduplex DNA and in vitro model for functional analysis of mismatch repair

Functional deficiency of mismatch repair(MMR) system is one of the mechanisms of tumorigenesis.With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZa gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G-G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (routS^-) without the MMR ability on the indicator plate with x-gal and IPTG,there are three kinds of plaques, mixture plaque as the charaeteristie phenotype of heteroduplex DNA, blue and clearplaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100x (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functionai status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G-G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G-G is less than 20%.Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target,TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (mierosatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the funetion status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.

我国几种植物植原体的快速分子鉴别与鉴定的研究

选用桑萎缩病(M u lberry dw arf,MD)、枣疯病(Ju jube w itches-broom,JW B)、酸枣丛枝病(W ild ju jube w itches-broom,W JW B)、泡桐丛枝病(Pau lownia w itches-broom,PaW B)和苦楝丛枝病(Chinaberry tree w itches-broom,CW B)5种不同植物植原体和来源于3个不同地区PaW B和JW B材料进行16S rDNA和23S rDNA PCR扩增、异源双链迁移率分析(HMA)、PCR产物的RFLP分析和16S rDNA的克隆和测序等比较研究,建立了一种快速确定未知植原体种类和分类地位的分子鉴别与鉴定优化程序;并可对田间采集的各种植物植原体样品进行快速鉴定和鉴别。16S rDNA PCR产物HMA分析结果显示,JW B与CW B、MD和PaW B皆可形成明显的异源杂交双链;而CW B、MD和PaW B植原体之间未能形成异源双链。JW B和PaW B不同地区样品之间、JW B和W JW B之间也未发现异源杂交双链的形成。而23S rDNA PCR产物HMA分析则可以将MD与PaW B区分开。进一步对未知分类地位的CW B序列测定及与其它植原体16S rDNA的RFLP和同源性比较结果显示,CW B与PaW B同源性为99.5%,其中与MD的同源性高达99.7%,因而应将CW B归为翠菊黄花组16S r I-B,16S r I-B(rp-B)。

二核苷酸微卫星PCR扩增的影子带及其来源

2个绵羊二核苷酸微卫星BM143和BMS2508的PCR扩增产物在非变性(中性)聚丙烯酰胺凝胶电泳(nPAGE)中形成滞后于主带的影子带。影子带比主带小2 bp。实际上主带由两部分组成:表明微卫星正确大小的PCR特异扩增的高浓度条带(特异扩增带)和比特异扩增带小2 bp的PCR非特异扩增的低浓度条带(非特异扩增带)。本试验初步证明影子带是微卫星的一条特异扩增带和一条非特异扩增带错配形成的异源双链。本研究结果表明,影子带的出现并不改变特异扩增带在nPAGE中的正常迁移率。

异源双链泳动分析法对流行性角膜结膜炎致病腺病毒分型的研究

背景 目前对流行性角膜结膜炎致病腺病毒的分型多采用直接DNA测序法,而异源双链泳动分析（HMA）法可对腺病毒进行基因分型,且更符合成本-效益原则,HMA法对流行性角膜结膜炎致病腺病毒分型的应用尚少有研究.目的 将流行性角膜结膜炎致病腺病毒六邻体蛋白编码区内保守区的测序结果与HMA的结果进行对照,评价HMA法在流行性角膜结膜炎病原体检查中的有效性和可行性.方法 在知情同意的情况下收集2010年1月至2012年12月在上海市眼病防治中心和上海市急性出血性结膜炎防控办公室下属红眼病监测站点就诊的可疑流行性角膜结膜炎患者214例,取患者结膜囊的结膜拭子标本214份.使用QIA-amp minikit提取结膜拭子标本中的病毒DNA,然后通过PCR法扩增病毒DNA六邻体蛋白编码区内366 bp的保守序列,并对该片段进行基因测序,确定是否为腺病毒及其分型.采用HMA法同时对PCR扩增产物进行分析,将HMA结果与基因测序法进行比较.结果 使用QIA-amp minikit提取的病毒基因组DNA经质量分数1％琼脂糖凝胶电泳可见边界清楚的长约35 kb的金黄色荧光条带,无拖尾现象,DNA吸光度比值（A260/A280）约为1.7.214例患者中,经PCR检测确定为腺病毒感染者109例,经基因测序确定为腺病毒l型（AdVl）者4例,AdV2者33例,AdV3者15例,AdV4者12例,AdV8者19例,AdV19者15例,AdV37者8例.HMA法可以鉴别AdV1、AdV2、AdV3、AdV8、AdV19和AdV37,其结果与基因测序结果一致.AdV4的DNA突变点为59.6％,超过10％,不适合HMA法.结论 针对六邻体蛋白编码区内保守区域的基因测序是腺病毒分型的重要方法之一,可以快速确定流行性角膜结膜炎是否系腺病毒感染引起,并可以确定腺病毒分型.HMA的结果与基因测序相一致,可作为大规模分子流行病学调查的检测工具.

湖北汉族人群甘露糖结合凝集素基因外显子1多态性与SLE关联性的研究

目的 :分析湖北汉族人群甘露糖结合凝集素 (Mannose BindingLectin或Mannose BindingProtein ,MBL或MBP)基因外显子 1多态性 ,探讨其与系统性红斑狼疮 (SystemicLupusErythematosus ,SLE)的易感性关系。方法 :采用异源双链杂交技术对 111例健康正常人和 4 1例SLE患者的MBL基因外显子 1多态性进行分析。结果 :①共检测出两种等位基因 :野生型A和变异型B(在 5 4位密码子由GGC→GAC) ,未检出变异型C、D等位基因。②正常对照中A等位基因及B等位基因的基因频率分别为 0 910和 0 0 90 ,符合Hardy Wernberg定律 ;与此前在日本人中报道的A及B等位基因频率 0 76 7和 0 2 33相比 ,变异型B的等位基因频率明显低于后者 (P <0 0 5 )。③SLE病人组中B等位基因频率为 0 14 6 ,高于正常对照组 ,但差异无显著性意义。结论 :在湖北汉族人群中MBL基因外显子 1具有遗传多态性 ,而且与已知其他人群的分布频率有一定差异。MBL基因外显子 1的遗传多态性并不与系统性红斑狼疮相关联。

人类免疫缺陷病毒膜蛋白基因(env)F亚型毒株在中国的首例发现

１９９８年广东某戒毒所ＨＩＶ阳性感染者的血样，经ＰＣＲ扩增未培养细胞内ＨＩＶ前病毒基因并对Ｃ２－Ｖ３区进行测序和分析，发现两例感染者与巴西Ｆ亚型毒株ＢＺ１６３的基因离散率小于４％；Ｎｅｉｇｈｂｏｒ－ｊｏｉｎｉｎｇ系统树表明，它们与Ｆ亚型聚在一起；通过异源双链泳动技术分析法（ＨＭＡ）分析，这两例感染者与Ｆ亚型形成明显的异源二聚体。上述各项研究结果表明，Ｆ亚型ＨＩＶ－１已传入我国。

常染色体显性视网膜色素变性NRL基因的突变研究

目的 对中国人常染色体显性视网膜色素变性 (RP)NRL基因的突变现状进行研究。方法 抽取 12 0例RP先证者的静脉血提取DNA ,行NRL基因的引物设计合成 ,PCR扩增 ,琼脂糖电泳鉴定 ,异源双连 SSCP电泳。结果 12 0例散发型RP患者未发现NRL基因突变。

福建省HIV-1病毒基因亚型分布的研究

目的 了解福建省HIV 1各亚型的流行情况 ,帮助分析其传染来源和判断流行趋势。方法 采集福建省检出的 13例HIV 1感染者的外周血单核细胞 (PBMCs) ,提取核酸后用套式聚合酶链反应技术进行扩增HIV 1膜蛋白 (env)基因C2 V3区目的核酸片段 ,应用异源双链泳动分析法 (HMA)进行HIV 1亚型的分析。结果 13份标本PCR扩增均阳性 ,亚型分析结果表明福建省存在HIV 1B亚型和E亚型。结论 HIV 1B亚型和E亚型可能是福建省流行的主要亚型 ,主要经性途径由境外传入 ,且经性传播途径在人群中流行。异源双链泳动分析法可作为HIV

S1核酸酶突变检测法的优化

优化了S1核酸酶突变检测体系,结果表明(1)扩增法比杂交法产生异源双链DNA更节省时间;(2)PCR产物可以直接作为突变检测的底物;(3)适当降低反应温度、适当增加反应缓冲液中的NaCl浓度、适当缩短反应时间可提高突变检测效果。

定量PCR的非同源对照模板的构建

建立了HCVRNA和hTERTmRNA的竞争定量PCR系统。对照模板的构建方法是 :利用计算机辅助优选设计连接引物 ,低严紧型扩增大肠杆菌DNA ,回收并克隆预期的DNA片段。该片段与靶序列除两端引物序列完全相同外 ,无同源性 ,因此可作为非同源对照模板。这种构建非同源对照模板的方法 ,简便易行 。

变性梯度凝胶电泳技术在微生物分子生态学研究中的应用

变性梯度凝胶电泳在微生物分子生态学研究中的应用日益广泛,已成为研究微生物多样性和动态变化的有力工具。对变性梯度凝胶电泳技术中存在的问题,如基因片段的选择、共迁移、背景色和异源双链分子的形成等作了综述,以期为进一步更好地利用该技术进行微生态的研究奠定基础。

鸡Visfatin基因9 bp indel杂合突变异源双链DNA的鉴定

内脂素(Visfatin)是脂肪细胞因子家族的新成员,主要由内脏脂肪组织产生.研究表明内脂素具有类胰岛素样作用.在检测固始鸡-安卡鸡资源群体3代(亲本,F1,F2)964只鸡Visfatin基因9bp插入/缺失(9 bp 'TAACCTGTG' insertion-deletion)多态的过程中,发现其杂合子的变性和非变性聚丙烯酰胺胶上除2条同源双链DNA(282bp和273bp)外有2条未知条带(命名为A和B).A,B条带经回收、二次PCR、再次聚丙烯酰胺凝胶电泳及DNA测序表明:Visfatin基因第10内含子中9bp insertion-deletion突变杂合子的PCR产物中,本身包含2种同源双链DNA片段和2种异源双链DNA片段,不需要经过额外的变性、退火处理,其PCR产物可以直接进行突变检测,在229个杂合突变中异源双链DNA的检出率为100%.因此,通过异源双链DNA这一标示物作为基因分型时的依照或者参考,建立适当的异源双链DNA分析法可进行基因中几个核苷酸插入/缺失多态的检测.

慢性丙型肝炎患者HCV准种变异研究

目的研究慢性丙型肝炎患者体内准种的多样性及动态变化过程,探讨其与病程进展的关系及感染慢性化的原因。方法应用异源双链泳动技术(HMA)研究在干扰素治疗压力下慢性丙型肝炎患者体内准种的变化。结果慢性HCV患者无论对干扰素应答情况如何,其体内HCV准种的数量没有明显变化,但无应答者体内HCV准种的异质性发生了明显改变;治疗前占支配地位的准种株群在不同感染者体内的比例并不相同,随着抗病毒治疗的进行,不断有新的异质性程度更高的准种株出现,打破原有的平衡比例。结论慢性HCV患者对干扰素治疗有无应答,取决于变异株的异质性程度,而不是变异株数量。在干扰素治疗压力下,原来宿主与病毒之间的平衡已经被打破,耐药的准种株被选择出来,占据支配地位,是导致感染持续存在的原因之一。

异源双链泳动分析丙型肝炎病毒准种及变异研究

目的探讨慢性丙型肝炎病毒(HCV)患者体内准种的多样性及动态变化过程与病程进展的关系。方法应用异源双链泳动技术(HMA)研究在干扰素治疗压力下慢性HCV患者体内准种的变化。结果慢性HCV患者无论对干扰素应答情况如何,其体内HCV准种的数量无明显变化;治疗前占支配地位的准种株群在不同感染者体内的比例并不相同,随着抗病毒治疗的进行,不断有新的异质性程度更高的准种株出现,打破原有的平衡比例。结论慢性HCV患者对干扰素治疗有无应答,取决于变异株的异质性程度,而不是变异株数量。