Micropthalmia transcription factor a new prognostic marker

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用生物信息学挖掘玉米中的microRNAs及其靶基因

。近年来 , miRNA 的研究已经成为分子子 RNA 发展到在不同物种中大量发现 miRNA, 而随着研究的不断深入以及相关理论和实验技术的完善 , 发现在植物中存在一些序
本研究由国家自然科学基金项目 (30900901)和教育部高等学校博士学科点专项科研基金项目 (20095103120002)资助。
microRNAs (miRNA)是一大类内源性的、 19~24 碱基长度的小分子非编码 RNA, 它可以通过与靶 mRNA 分子完全或部分匹配 , 指导 mRNA 剪切或者抑止翻译等方式调控动植物的生命过程 [1], 从而在多细胞生物的基因表达调节和控制中扮演十分重要的角色 , 是以 RNA 为基础的基因表达调控系统的关键成员
用生物信息学挖掘玉米中的 microRNAs 及其靶基因
张志明
摘
宋锐
彭华
罗茂
沈亚欧
刘丽
赵茂俊
潘光堂*
四川农业大学玉米研究所 , 四川雅安 625014
要 : microRNA (miRNA)是一类内源性的、 19~24 碱基长度的小分子非编码 RNA, 通过碱基互补调控靶基因的表
达 , 在多细胞生物的基因表达调控过程中扮演着十分重要的角色。植物中的 miRNAs 具有高度的保守性 , 这为通过同源比对发现保守的 miRNAs 提供了思路和途径。本研究通过对拟南芥、水稻等植物已知的 miRNAs 与玉米 EST 和 GSS 数据库的比对 , 并设置一系列严格的筛选标准 , 共筛选到 23 条新的玉米 miRNAs; 利用 WMD 3 在线植物 miRNAs 靶基因预测软件 , 对新发现的玉米 miRNAs 进行靶基因预测 , 总共预测到 89 个靶基因 , 进一步功能分析发现 , 这些靶基因参与玉米的生长发育、信号转导、转录调节、新陈代谢及逆境胁迫响应等调控过程。关键词 : microRNA (miRNA); 生物信息学 ; 预测 ; 靶基因 ; 玉米

分子生物学复习题及答案附带模拟考卷

分子生物学复习思考题1．写出分子生物学广义的与狭义的定义，现代分子生物学研究的主要内容，以及5个分子生物学发展的主要大事纪（年代、发明者、简要内容）。

广义上:分子生物学包括对蛋白质和核酸等生物大分子结构与功能的研究、以及从分子水平上阐明生命的现象和生物学规律。

狭义概念:既将分子生物学的范畴偏重于核酸（基因）的分子生物学，主要研究基因或DNA结构与功能、复制、转录、表达和调节控制等过程。

其中也涉及到与这些过程相关的蛋白质和酶的结构与功能的研究。

现代分子生物学研究的主要内容有：基因与基因组的结构与功能，DNA的复制、转录和翻译，基因表达调控的研究，DNA重组技术，结构分子生物学等。

5个分子生物学发展的主要大事纪（年代、发明者、简要内容）:1.1944年,著名微生物学家Avery 等人在对肺炎双球菌的转化实验中证实了DNA是生物的遗传物质。

这一重大发现打破了长期以来，许多生物学家认为的只有象蛋白质那样的大分子才能作为细胞遗传物质的观点，在遗传学上树立了DNA是遗传信息载体的理论。

2. 2.1953年，是开创生命科学新时代具有里程碑意义的一年，Watson和Crick发表了“脱氧核糖核酸的结构”的著名论文，他们在Franklin和Wilkins X-射线衍射研究结果的基础上，推导出DNA双螺旋结构模型，为人类充分揭示遗传信息的传递规律奠定了坚实的理论基础。

同年，Sanger历经8年，完成了第一个蛋白质——胰岛素的氨基酸全序列分析。

3. 1954年Gamnow从理论上研究了遗传密码的编码规律, Crick在前人研究工作基础上，提出了中心法则理论，对正在兴起的分子生物学研究起了重要的推动作用。

4. 1956年Volkin和Astrachan发现了mRNA(当时尚未用此名)。

5. 1985年,Saiki等发明了聚合酶链式反应(PCR)；Sinsheimer首先提出人类基因组图谱制作计划设想；Smith等报导了DNA测序中应用荧光标记取代同位素标记的方法；Miller等发现DNA结合蛋白的锌指结构。

Translation-翻译

• Cap binding protein, eIF4E, binds to cap
• The N-terminus of eIF4G binds eIF4E and the Cterminus binds eIF4A
• Th20e21/44/40S subunit binds to eIF4G via eIF3
AAAAAA
1
A “Simple” Eukaryotic Gene
Transcription Start Site
5’ UTR
Introns
3’ UTR
5’
Exon 1 Int. 1 Exon 2 Int. 2 Exon 3
3’
Promoter/ Control Region
Exons
Transcription
• 70-80 nt long
• 3’ end has the 5’- CCA sequence to which aa are linked
• The opposite end contains the anticodon loop
• Contains modified
ba2s02e1/4s/4
7
2021/4/4
4
Model of eukaryotic ribosome
• rRNAs are believed to play a catalytic role in protein synthesis.
• After removal of 95% of the ribosomal proteins, the 60S subunit can catalyze formation of peptide bonds.

动物园非洲野犬繁殖研究初探

2017年第12期[9]马亚南.miR-27a 在WM239细胞中的表达及对其生物学特性的影响[D].济南大学,2015[10]Dong C,et al.Coat color determination by miR-137mediateddown-regulation of microphthalmia-associated transcription factorin a mouse model[J].Rna-a Publication of the Rna Society,2012,18(9):1679-86.[11]Yan B,et al.microRNA regulation of skin pigmentation in fish[J].Journal of Cell Science,2013,126(15):3401-8.[12]Kennell J A,et al.The microRNA miR-8is a positive regulatorof pigmentation and eclosion in Drosophila[J].DevelopmentalDynamics,2012,241(1):161-168.[13]Zhang J,et al.Role of microRNA508-3p in melanogenesis bytargeting microphthalmia transcription factor in melanocytes of alpaca[J].Animal,2017,11(2):236-243.[14]Shi Q,et al.Oxidative stress-induced overexpression of miR-25:the mechanism underlying the degeneration of melanocytes in vitiligo[J].Cell Death &Differentiation,2016,23(3):496-508.[15]Yang S,et al.Identification of a novel microRNA importantfor melanogenesis in alpaca (Vicugna pacos)[J].Journal of AnimalScience,2015,93(4):1622-31.非洲野犬（Lycaon pictus ）是食肉目犬科非洲野犬属的唯一物种。

斑马鱼的mophalino

Targeted Inhibition of miRNA Maturationwith Morpholinos Reveals a Role for miR-375 in Pancreatic Islet DevelopmentWigard P.Kloosterman1,Anne gendijk1,Rene´F.Ketting1*,Jon D.Moulton2,Ronald H.A.Plasterk11Hubrecht Laboratory-KNAW,Utrecht,The Netherlands,2Gene Tools,Philomath,Oregon,United States of AmericaSeveral vertebrate microRNAs(miRNAs)have been implicated in cellular processes such as muscle differentiation, synapse function,and insulin secretion.In addition,analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis.Nonetheless,only a few loss-of-function phenotypes for individual miRNAs have been described to date.Here,we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development.Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs.Morpholinos can block processing of the primary miRNA(pri-miRNA)or the pre-miRNA,and they can inhibit the activity of the mature miRNA.We used this strategy to knock down13miRNAs conserved between zebrafish and mammals.For most miRNAs,this does not result in visible defects,but knockdown of miR-375causes defects in the morphology of the pancreatic islet.Although the islet is still intact at24hours postfertilization,in later stages the islet cells become scattered.This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375precursor,excluding off-target effects as cause of the phenotype.The aberrant formation of the endocrine pancreas,caused by miR-375knockdown,is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development.The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.Citation:Kloosterman WP,Lagendijk AK,Ketting RF,Moulton JD,Plasterk RHA(2007)Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375in pancreatic islet development.PLoS Biol5(8):e203.doi:10.1371/journal.pbio.0050203IntroductionMicroRNAs(miRNAs)have a profound impact on the development of multicellular organisms.Animals lacking the Dicer enzyme,which is responsible for the processing of the precursor miRNA into the mature form,cannot live[1–3]. MiRNA mutants have been described only for Caenorhabditis elegans and Drosophila,reviewed in[4].From these studies,it is clear that invertebrate miRNAs are involved in a variety of cellular processes,such as developmental timing[5,6], apoptosis[7,8],and muscle growth[9].Analysis of conditional Dicer null alleles in mouse has indicated a general role for miRNAs in morphogenesis of the limb,skin,lung epithelium, and hair follicles[10–13].Overexpression studies in mouse have implicated speciﬁc vertebrate miRNAs in cardiogenesis and limb development[14,15].In zebraﬁsh,embryos lacking both maternal and zygotic contribution of Dicer have severe brain defects[2].Strikingly,the brain phenotype of maternal-zygotic Dicer zebraﬁsh can be restored by injection of miR-430,the most abundant miRNA in early zebraﬁsh develop-ment.Despite all these studies describing functions for miRNAs in development,no vertebrate miRNA mutant has been described to date.Genetically,it is challenging to obtain mutant miRNA alleles in zebraﬁsh,because their small size makes them less prone to mutations by mutagens,and for many miRNAs,there are multiple alleles in the genome or they reside in families of related sequence.Temporal inhibition of miRNAs by antisense molecules provides another strategy to study miRNA function.29-O-methyl oligonucleotides have been successfully used in vitro and in vivo to knock down miRNAs[16–18].Morpholinos are widely applied to knock down genes in zebraﬁsh development [19]and have recently been used to target mature miR-214in zebraﬁsh[20].However,off-target phenotypes are often associated with the use of antisense inhibitors.Here,we show that morpholinos targeting the miRNA precursor can knock down miRNAs in the zebraﬁsh embryo. Several independent morpholinos can knock down the same miRNA,and these serve as positive controls toﬁlter out off-target effects.Morpholinos can block miRNA maturation at the step of Drosha or Dicer cleavage,and they can inhibit the activity of the mature miRNA.We show that inhibition of miR-375,which is expressed in the pancreatic islet and pituitary gland of the embryo[21],results in dispersed islet cells in later stages of embryonic development,whereas no effects were observed in the pituitary gland.The morpholino-mediated miRNA knockdown strategy presented here,is an extremely fast and well-controlled method to study miRNA function in development.Academic Editor:James C.Carrington,Oregon State University,United States of AmericaReceived October13,2006;Accepted May22,2007;Published July24,2007Copyright:Ó2007Kloosterman et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Abbreviations:dpf,days postfertilization;GFP,green fluorescent protein;hpf, hours postfertilization;LNA,locked nucleic acid;miRNA,microRNA;MO, morpholino oligonucleotide;RT-PCR,reverse transcriptase PCR*To whom correspondence should be addressed.E-mail:rene@niob.knaw.nlP L o S BIOLOGYResultsMorpholinos Targeting the Mature miRNA Deplete the Embryo of Specific miRNAsSince it is difﬁcult to obtain a genetic mutant for a miRNA in zebraﬁsh,we looked for alternative strategies to deplete the embryo of speciﬁc miRNAs.Antisense molecules such as 29-O-methyl and locked nucleic acid(LNA)oligonucleotides have been used to inhibit miRNAs in cell lines[16,18,22], Drosophila embryos[23],and adult mice[17].We tried to use these molecules to inhibit the function of endogenous miRNAs in the zebraﬁsh embryo.Although they can be used to suppress the effects of miRNA overexpression[24], injection of higher concentrations required to obtain good knockdown of endogenous miRNAs resulted in toxic effects, when injecting1nl solution at a concentration of approx-imately10l M and50l M for LNA and29-O-methyl oligonucleotides,respectively(unpublished data).Therefore, we switched to morpholinos because these are widely used to inhibit mRNA translation and splicing in zebraﬁsh embryos [19],and have also been shown to target miRNAs in the embryo[2,20,24].We injected1nl of600l M morpholino solution with a morpholino complementary to the mature miR-206in one-or two-cell–stage embryos.Subsequently, embryos were harvested at24,48,72,and96hours postfertilization(hpf),and subjected to in situ hybridization and Northern blotting(Figure1A and1B).This analysis showed that the mature miRNA signal is suppressed up to4d after injection of the morpholino.The knockdown effect was speciﬁc for this miRNA;parallel in situ analysis of the same embryos with a probe for miR-124did not show any effects on expression of this miRNA(Figure1B).Thus,miRNA detection can be speciﬁcally and efﬁciently suppressed during embryonic and early larval stages of zebraﬁsh development using morpholinos antisense to the mature miRNA.The zebraﬁsh embryo can be used to monitor the effect of miRNAs on greenﬂuorescent protein(GFP)reporters fused to miRNA target sites[24].To determine the effect of a morpholino in this assay system,we constructed a GFP reporter for miR-30c and tested it in the presence and absence of a mature miR-30c duplex.Injected miR-30c silences this GFP reporter,which is in line with previous reports using similar strategies in the embryo(Figure1C) [2,20,24].Co-injection of the miR-30c duplex and a morpho-lino targeting mature miR-30c rescues the reporter signal, whereas injection of a control morpholino did not reverse the silencing by miR-30c.These data indicate that a morpholino can block the activity of a mature miRNA duplex in a functional assay.There are three possible explanations for the observed reduction in the detection signal for a miRNA that is targeted by a morpholino.First,the hybridization of a morpholino could disturb isolation of the miRNA.Second,the morpho-lino could destabilize the miRNA.Third,the morpholino could inhibit the maturation of the miRNA.To examine the effect of a morpholino on the isolation of a mature miRNA,we incubated a mature miR-206duplex and a control duplex(miR-205)with a morpholino against miR-206 in vitro.After isolation,samples were analyzed by Northern blotting for the presence of miR-206and miR-205.We could still detect miR-206,indicating that there is no effect of the morpholino on the RNA isolation procedure(Figure1D). However,when morpholino and miRNA duplex were incubated together in vitro and loaded on a denaturing gel without isolation,we observed a decrease in the signal for miR-206,indicating that the morpholino can bind to the miRNA in vitro and still does so in the denaturing gel. Next,we wanted to know whether a morpholino could affect the stability of a mature miRNA in vivo.Therefore,we injected a mature miR-206and a control duplex(miR-205) together with a morpholino against miR-206in the embryo. After incubation for8h,RNA was isolated and subjected to Northern blot analysis to probe for injected miR-206and injected miR-205.In contrast to the data obtained for endogenous miR-206,there was no decrease observed in the amount of injected miR-206in the morpholino-injected embryos(Figure1D)(endogenous miR-206is not yet ex-pressed at this stage).Since these data show that there is no effect of a morpholino on miRNA isolation or stability,we conclude that morpholinos deplete the embryo of miRNAs by inhibit-ing miRNA maturation.If this is the case,then we expect morpholinos targeting other regions of the miRNA precursor to act as well as the morpholinos designed against the mature miRNA,and this is indeed what weﬁnd(see next section). Morpholinos Targeting the miRNA Precursor Interfere with Primary miRNA ProcessingInjection of antisense oligos in embryos might result in off-target effects.Thus,phenotypic data retrieved from antisense knockdown experiments should be treated with caution.In Drosophila,29-O-methyl oligo–mediated knockdown of embry-onically expressed miRNAs caused defects that clearly differed from the phenotype of the corresponding knockout ﬂy[9,23].In sea urchin experiments,off-target effects of morpholino knockdowns are well documented,though low incubation temperatures favor off-target interactions[25]. Toﬁlter out off-target effects,we sought a control strategy that would allow us to compare effects of morpholinos withAuthor SummaryThe striking tissue-specific expression patterns of microRNAs (miRNAs)suggest that they play a role in tissue development. These small RNA molecules(;22bases in length)are processed from long primary transcripts(pri-miRNA)and regulate gene expression at the posttranscriptional level.There are hundreds of different miRNAs,many of which are strongly conserved.Vertebrate embryonic development is most easily studied in zebrafish,but genetically disrupting miRNA genes to see which miRNA does what is technically challenging.In this study,we interfere with miRNA function during the first few days of zebrafish embryonic develop-ment by introducing specific antisense morpholino oligonucleotides (morpholinos have been used previously to interfere with the synthesis of the much larger mRNAs).We show that morpholinos targeting the miRNA precursor can block processing of the pri-miRNA or directly inhibit the activity of the mature miRNA.We also used morpholinos to study the developmental effects of miRNA knockdown.Although we did not observe gross phenotypic defects for many miRNAs,we found that zebrafish miR-375is essential for formation of the insulin-secreting pancreatic islet.Loss of miR-375 results in dispersed islet cells by36hours postfertilization, representing one of the first vertebrate miRNA loss-of-function phenotypes.independent sequences targeted to the same miRNA.Because our data on morpholinos targeting the mature miRNA suggested that miRNA biogenesis might be affected,we designed morpholinos targeting the Drosha and Dicer cleavage sites of the precursor miRNA (Figure 2A).We decided to test this strategy on miR-205,since it is expressed relatively early,and there are only two,but identical,copies in the ﬁsh genome.Four different morpholinos were designed to inhibit miR-205biogenesis:two targeting the Drosha cleavage site complementary to either the 59or 39arm of the stem,and two morpholinos similarly targeting the Dicer cleavage site (Figure S1).These morpholinos were injected under similar conditions as described for miR-206and compared to the morpholino targeting mature miR-205.Interestingly,all ﬁve morpholinos induced complete or near-complete loss of miR-205(Figure 2B).Many miRNAs are highly expressed during later stages of embryonic development [21].Therefore,we tested how long the effect of the morpholinos would last.Although for this series of morpholinos the knockdown is best at 24hpf,the effect is still signiﬁcant up to 72hpf (Figure 2C).Next,we tested a similar series of morpholinos against the miR-30c precursor and analyzed miR-30c expression by Northern blotting (Figure S2).However,we only observed knockdown for the morpholino targeting mature miR-30c,but not for the other four morpholinos targeting the miR-30c precursor.This could be because miR-30c resides in a family of closely related species,with more sequence variability in the regions outside of the mature miRNA.The precursors ofthe family members might not all be targeted by these morpholinos (Figure S2).Thus,not all miRNAs are equally prone to knockdown by morpholinos that target the miRNA precursor.To investigate the effect of morpholinos on exogenously introduced pri-miR-205,we injected mRNA derived from a GFP construct with pri-miR-205in the 39UTR.Again,we could not detect mature miR-205derived from this construct after targeting by morpholinos (Figure 2D).Interestingly,the miR-205precursor also could not be detected in the embryos co-injected with morpholinos,whereas pre-miR-205could be detected in the absence of morpholinos (Figure 2D).Because pri-miR-205was cloned in the 39UTR of GFP,we monitored GFP ﬂuorescence after injection of this construct.In the presence of a morpholino,GFP ﬂuorescence increased (Figure 2E),suggesting accumulation of the primary miRNA.Therefore,we performed reverse transcriptase PCR (RT-PCR)on 8-h-old embryos injected with GFP-pri-miR-205and a control mRNA (luciferase)(Figure 2F).In the presence of a morpholino,the GFP-pri-miR-205mRNA level is higher compared to control embryos that were not injected with morpholinos.This experiment conﬁrms the GFP data and shows that morpholinos targeting the miRNA precursor inhibit Drosha cleavage.Next,we tested whether processing of the pre-miRNA might also be inhibited by morpholinos.Therefore,we injected a miR-205precursor in the one-cell–stage embryo.Northern analysis showed that the precursor was processed into mature miRNA in the embryo (Figure 2G).However,co-Figure 1.Morpholinos Targeting the Mature miRNA Deplete the Zebrafish Embryo of Specific miRNAs(A)Northern blot for miR-206in wild-type and MO miR-206–injected embryos at 24,48,and 72hpf.5S RNA serves as a loading control.(B)In situ analysis of miR-206and miR-124expression in different stage embryos after injection of MO miR-206.(C)Effect of a morpholino targeting miR-30c on a silencing assay with miR-30c and a responsive GFP sensor construct.(D)In vivo and in vitro effects of a morpholino on the stability and RNA extraction of a synthetic miR-206duplex.miR-205serves as a loading control.doi:10.1371/journal.pbio.0050203.g001Figure2.Morpholinos Targeting the Precursor miRNA Interfere with miRNA Maturation(A)Design of morpholinos targeting the precursor miRNA.(B)Northern blot analysis of miR-205in30-h-old embryos injected with different morpholinos against pri-miR-205.5S RNA serves as a loading control.(C)Time series of miR-205expression after injection of mature,no lap loop,and drosha star morpholinos against pri-miR-205.(D)Northern blot analysis of miR-205derived from embryos injected with a GFP-pri-miR-205transcript and four different morpholinos targeting pri-miR-205.Co-injected miR-206serves as an injection and loading control.Embryos were collected8h after injection.(E)GFP expression in24-h embryos injected with morpholinos and a GFP-pri-miR-205construct as used in(C).Pri-miR-205is positioned just upstream of the polyA signal in the39UTR of the GFP mRNA.Red fluorescent protein(RFP)serves as an injection control.(F)RT-PCR analysis of injected GFP-pri-miR-205mRNA with(þ)and without(À)co-injected morpholinos.Luciferase serves a an injection control. Embryos were collected8h after injection.(G)Northern analysis of the effect of morpholinos on an injected miR-205precursor.Embryos were collected8h after injection.WT,wild type.doi:10.1371/journal.pbio.0050203.g002injection of the overlap loop and non-overlapping loop morpholinos blocked processing completely.There was only a little effect of morpholinos targeting the Drosha cleavage site,probably because they only partially overlap the precursor.A similar analysis was performed for miR-375,which is expressed in the pancreatic islet and pituitary gland[21],and has two copies in the zebraﬁsh genome,which differ in the regions outside the mature miRNA.Overlap loop and loop morpholinos were designed for both miR-375–1and miR-375–2,and a morpholino against the miRNA star sequence could be used to target both copies of miR-375simultaneously(Figure3A).The efﬁcacy of all morpholinos was assessed by determining their effect on injected pri-miR-375–1or pri-miR-375–2transcripts(Figure 3B).As expected,each morpholino targeted the transcript to which it was directed.However,the star miR-375morpholino did not knock down miR-375completely.In addition, morpholino oligonucleotide(MO)miR-375did not interfere with processing of miR-375from pri-miR-375–1,possibly because this primary transcript forms a more stable hairpin. In all cases,the lack of a signal for mature miR-375coincided with the absence of pre-miR-375,which could be detected in the absence of a complementary morpholino.Next,all morpholinos were injected separately and in combination,and embryos were subjected to Northern blotting to determine endogenous miR-375expression at24 and48hpf(Figure3C).In contrast to the results obtained by in situ hybridization(see last section),the morpholino to mature miR-375only slightly decreased the expression of miR-375.However,MO miR-375could inhibit the activity of a mature miR-375duplex in a GFP-miR-375-target reporter assay(Figure3E).The morpholinos targeting only one copy of miR-375reduced miR-375expression,with the strongest effect for the morpholinos targeting pri-miR-375–1.How-ever,simultaneous injection of morpholinos targeting pri-miR-375–1and pri-miR-375–2completely knocked down mature miR-375,indicating that both transcripts are ex-pressed.To further determine the contribution of each transcript to mature miR-375accumulation,we performed in situ hybridization for pri-miR-375–1and pri-miR-375–2(Figure 3D).Both transcripts could not be detected in wild-type embryos.However,pri-miR-375–1was detected in the pancreatic islet and the pituitary gland in embryos injected with the miR-375–1loop morpholino and the morpholino to miR-375star.Similarly,pri-miR-375–2was only detected in embryos injected with the miR-375–2loop morpholino,the morpholino to miR-375star and mature miR-375.Thus,both transcripts are expressed in the pituitary gland and the pancreatic islet,similar to miR-1in the developing mouse heart[15].Together,this indicates that these morpholinos inhibit primary miRNA processing and result in primary miRNA accumulation,as we described for miR-205.In conclusion,our data demonstrate that morpholinos targeting the miRNA precursor can interfere with primary miRNA processing at either the Drosha or Dicer cleavage step and that morpholinos targeting the mature miRNA can inhibit their activity in a functional assay.Taken together, our data show that different morpholinos targeting the same miRNA may serve as positive controls for miRNA knockdown phenotypes in the embryo.Knockdown of Many miRNAs Does Not Result in Any Observed Developmental DefectsTo identify functions for individual miRNAs in zebraﬁsh embryonic development,we knocked down a series of11 conserved vertebrate miRNAs(Table S1)and analyzed their expression after morpholino knockdown.Injected embryos were monitored phenotypically by microscopic observation until four days postfertilization(dpf).Knockdown of most miRNAs resulted in loss of in situ staining for the respective miRNA.However,we could not observe gross morphological malformations after knockdown of these miRNAs(Figure4A). Therefore,we analyzed embryos injected with morpholinos against miR-182,miR-183,or miR-140in more detail,because we could easily stain the tissues that express these miRNAs (Figure4B).Embryos injected with morpholinos against miR-182or miR-183,which are expressed in the lateral line neuromasts and hair cells of the inner ear,were treated with DASPEI,which stains hair cells.Embryos injected with a morpholino against miR-140,which is expressed in cartilage, were subjected to Alcian Blue staining,a cartilage marker. However,staining of these speciﬁc cell types that express the miRNA did not uncover any defects upon knockdown(Figure 4B).In conclusion,knockdown of many miRNAs does not appear to signiﬁcantly affect zebraﬁsh embryonic develop-ment,at least not to the extent that can be visualized by the methods used in these examples.Knockdown of miR-375Affects Pancreatic Islet MorphologyMiR-375is known to be expressed in the pancreatic islet and the pituitary gland,and wasﬁrst isolated from pancreatic beta cells[21,26].This miRNA is conserved in vertebrates and may regulate insulin secretion by inhibiting myotrophin[26]. We injected a morpholino against mature miR-375into the one-cell–stage embryo.This morpholino effectively knocked down miR-375in theﬁrst4d of development(Figure5A),and it could also block the activity of an injected miR-375duplex, as monitored by its effect on a GFP reporter silenced by miR-375(Figure3E).During theﬁrst5dpf,there was no clear developmental defect except for a general delay in development.At around7 dpf,approximately80%of the injected embryos died.Next, we analyzed the development of both the pituitary gland and the pancreatic islet,by in situ hybridization with pit1and insulin markers.This analysis revealed no change in the formation of the pituitary gland(Figure5B).However, analysis of insulin expression showed a striking malformation of the islet cells in3-d-old morphant embryos(Figure5B). Wild-type embryos have a single islet at the right side of the midline,whereas the miR-375knockdown embryos have dispersed insulin-positive cells.The effect is sequence speciﬁc,because a morpholino complementary to the mature miR-375morpholino inhibited the pancreatic islet pheno-type(Figure5E).The pancreatic islet consists of four cell types,a,b,d,and PP,expressing glucagon,insulin,somatostatin,and pancre-atic polypeptide,respectively.Insulin is theﬁrst hormone expressed,and somatostatin co-localizes partially with in-sulin,whereas glucagon-expressing cells are distinct[27].A more detailed analysis using somatostatin and glucagon asmarker genes revealed a similar pattern of scattered islet cells in the miR-375morphant (Figure 5C).In zebraﬁsh,insulin is ﬁrst expressed at the 12-somite stage in a few scattered cells located at the midline,dorsal to the yolk [28].Insulin-positive cells migrate posteriorly and converge medially to form an islet by 24hpf.To look at the development of the pancreatic islet in time,we collected MO miR-375and noninjected control embryos at different stages,Figure 3.Specific Morpholinos Deplete the Embryo of miR-375(A)Sequence alignment of the two miR-375genes from zebrafish and design of morpholinos targeting the dre-miR-375–1and dre-miR-375–2precursors.(B)Northern blot analysis of the effect of morpholinos on the expression of miR-375derived from injected pri-miRNA mRNAs for miR-375–1and miR-375–2.MO-375–1overlap loop and loop morpholinos target exclusively the pri-miR-375–1construct,and MO-375–2overlap loop and loop morpholinos target exclusively the pri-miR-375–2construct.Co-injected miR-206serves as a loading and injection control.Embryos were collected 8h after injection.(C)Northern blot analysis of the effect of morpholinos on endogenous miR-375expression at 24hpf and 48hpf.MiR-206serves as loading control.(D)In situ hybridization for pri-miR-375–1and pri-miR-375–2on wild-type (WT)and morpholino-injected embryos.Arrowheads indicate the pituitary gland and the pancreatic islet.(E)Analysis of GFP expression in 24-h embryos injected with a miR-375GFP sensor construct,a synthetic miR-375duplex and MO miR-375.Red fluorescent protein (RFP)serves as an injection control.NIC,noninjected control.doi:10.1371/journal.pbio.0050203.g003Figure 4.Knockdown of Many miRNAs Does Not Affect Zebrafish Embryonic Development(A)Phenotypes and in situ analysis of 3-and 4-d-old embryos after injection of morpholinos against 11different mature miRNAs.(B)Daspei staining of 72-h-old embryos injected with MO miR-182and MO miR-183,and wild-type control (upper panel).Alcian Blue staining of 72-h-old embryos injected with MO miR-140and noninjected control (lower panel).doi:10.1371/journal.pbio.0050203.g004and investigated the expression of insulin (Figure 5D).At the 16-somite stage,insulin-positive cells are scattered at the midline in both noninjected and MO miR-375–injected embryos,and a presumptive islet is formed by 24hpf.Subsequently,when the insulin-positive islet is moving to the right side of the embryo in later stages,the islet breaks apart and insulin-positive cells become scattered in morphantembryos (Figure 5D).Also,in later stages,the phenotype persists,although miR-375is re-expressed at approximately 5dpf in morpholino-injected embryos (Figure 5A).Next,we analyzed the effect of all miR-375control morpholinos described in the previous section,by staining for insulin (Figure 6A).Both the dispersion phenotype and the knockdown were striking for embryos injected withMOFigure 5.Knockdown of miR-375Results in Aberrant Migration of Pancreatic Islet Cells(A)In situ analysis of miR-375knockdown in MO miR-375–injected embryos and noninjected controls at 24,48,72,and 120hpf.Arrowheads indicate the pituitary gland and the pancreatic islet.(B)In situ analysis of the pancreatic islet (insulin staining)and the pituitary gland (pit1staining)in miR-375morphants and noninjected controls.Arrowheads indicate the pituitary gland and the pancreatic islet.(C)In situ analysis of pancreatic islet development in wild-type and morphant embryos using insulin,somatostatin,and glucagon as markers.(D)Time series of insulin expression in wild-type and morphant embryos injected with MO miR-375.(E)Insulin expression in 72-hpf embryos injected with MO miR-375and a complementary morpholino.doi:10.1371/journal.pbio.0050203.g005Figure6.Specific Effects of miR-375Knockdown on the Development of the Endocrine Pancreas(A)In situ analysis of miR-375and insulin expression in72-hpf embryos injected with morpholinos against the miR-375precursor and negative control morpholinos for let-7and miR-124.(B)Expression of islet1,foxa2,and ptf1a in wild-type and miR-375knockdown embryos.Arrows indicate the pancreatic islet.WT,wild typedoi:10.1371/journal.pbio.0050203.g006。

改进的CRISPR基因组编辑使用小型高活性和特异性的工程RNA引导核酸酶说明书

Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases Moritz J. Schmidt1, Ashish Gupta1, Christien Bednarski1, Stefanie Gehrig-Giannini1, Florian Richter1, Christian Pitzler1, Michael Gamalinda1, Christina Galonska1, Ryo Takeuchi2, Kui Wang2, Caroline Reiss2, Kerstin Dehne1, Michael J Lukason3, Akiko Noma2, Cindy Park-Windhol2, Mariacarmela Allocca2, Albena Kantardzhieva2, Shailendra Sane2, Karolina Kosakowska2, Brian Cafferty2, Jan Tebbe1, Sarah J Spencer3, Scott Munzer2, Christopher J. Cheng2, Abraham Scaria2, Andrew M. Scharenberg2, André Cohnen1* and Wayne M. Coco1* Supplementary Figure 1. Selection of four small, novel Staphylococcus Cas9s.(a) Relatedness and genomic context of four uncharacterized Staphylococcus Cas9 genes: S. hyicus (Shy), S. lugdunensis (Slu), S. microti (Smi) and S. pasteuri (Spa). Each Cas9 locus includes several direct repeats (DRs) and a tracr sequence upstream of the nuclease. Loci not drawn to scale. (b) Sequences and structure of RNA components. The associated elements shared 98.8 % (tracrs) and 97.2 % (DRs) sequence identity with the corresponding SauCas9 sequences1. Top left: Complementarity between Slu genome locus DRs and tracr sequence. Bottom left: Alignment of the four DR sequences. Blue box: non-identities. Right: sgRNA design for SluCas9 with GAAA tetraloop (Loop 1) fusing DR and tracr2. Secondary structure predictions indicated similar folding for the four tracrRNAs. (c) Alignment of known and putative PAM interacting motifs in the selected Staphylococcus Cas9s suggests PAM-interacting residues. Amino acid numbering based on SauCas9. Strictly conserved amino acids are inblue, chemically similar amino acids are green. Red boxes correspond to residues responsible for PAM recognition in SauCas93. (d) PAM sequences as web logos. PAMs of the Cas9s were identified by in vitro-cleavage assays on a heptanucleotide (N7) DNA library 3’ of VEGFA_T24 and confirmed by bacterial survival assays carrying the same libraries. In contrast to SauCas9, 3 of the 4 chosen Cas9s recognized the short, non-degenerate 5’-NNGG-3’ PAM. PAM numbering begins with the first position after the last 3’ guide nucleotide. Source data are pr ovided in the source d ata file. (e) Alignment to SauCas9 nuclease active site residues. Corresponding amino acids for each orthologue are highlighted with red boxes.Supplementary Figure 2. Genome editing with four Cas9s in HEK293T cells assayed by amplicon sequencing. Shy, Smi and Slu Cas9 activity on endogenous loci in HEK293T cells using SluCas9 tracrRNA. Spa, Smi and Slu were tested for 2 targets with 5’-NNGG-3’ PAM (guide_87 and guide_102 targeting the HBB_R01_T2 and VEGFA_T22 loci, respectively) and Shy with 5’-NNARMM-3’ PAM (guide_1-4 targeting the HBB_R01_T1, VEGFA_T1 and FANCF_T1 and FANCF_T2 loci). Cas9s were delivered as RNPs via nucleofection and editing was analyzed via amplicon sequencing (AmpSeq). For negative controls, the respective nucleases were nucleofected in absence of sgRNA. Editing values were normalized against background, n = 2 independent biological replicates, source data are provided in the source data file.Supplementary Figure 3. Screening assays for functional sRGN variants. (a) Screening approach used to identify improved sRGNs by protein engineering. The succession of screening assays is shown on the left with the corresponding number of sRGN variants that progressed on the right. 25 sRGN variants were identified as top hits in the final BFP disruption screen in HEK293T cells5. (b) Schematic for “live/dead” (L/D) bacterial survival assay. Cells were generated harbouring an arabinose-inducible toxic ccdB gene reporter plasmid, a second plasmid harbouring a transcription cassette for the corresponding sgRNA, and a third plasmid encoding an IPTG-inducible, Trc promot er-controlled nuclease gene. Active Cas9/sgRNA complexes successfully cleave the toxic reporter, which is inactivated by cleavage at the VEGFA-T2 target site (guide_113), and cells survive under selection conditions. Open circles are cartoons of plasmids, green circle depicts ccdB gene product, purple circle represents nuclease. (c) L/D assay validation using SpyCas9 and SpyCas9 mutants. WT = SpyCas9; D10A and H840A = nickases; and dSpyCas9 = catalytically inactive SpyCas9. Upon botharabinose and IPTG induction, SpyCas9-or D10A-expressing cells survive while those that express H840A or dSpyCas9 do not, WTSpyCas9 n = 2 independent biological replicates, all other data n =3 independent biological replicates, data are presented as mean ± SD. Source data are provided in the source data file. (d) Fluorescence polarization assay (FP Assay) schematic. Biotinylated and ATTO647N-labelled oligonucleotide duplexes were immobilized on streptavidin coated plates. RNP complexes were formed, and cleavage of the dsDNA was monitored by following decreasing anisotropy and increasing fluorescence intensity, arb.unit = arbitrary units.Supplementary Figure 4. Rational exchange of PAM-interacting domains. (a) Shuffling fragment architecture of screening hits sRGN1, sRGN2, sRGN3 and sRGN4. Dark blue = SluCas9 segments, light blue = ShyCas9 segments, green = SmiCas9 segments, pink = SpaCas9 segments. (b) Rationally swapping PI-domains (PID) and WEDGE/PI-domains from the NNGG-recognizing SluCas9 to ShyCas9 alters PAM motif recognition and creates functionally active proteins. Chimera 1 and 2 substitute SluCas9 WEDGE/PI domain amino acids 739-1053 and 910-1053, respectively, into the ShyCas9 gene. Bacterial live/dead growth analysis on the VEGFA_T2 target sequence (guide_113) with a NNGG-PAM, indicates that both chimeric constructs gained the ability to effectively employ an NNGG PAM motif, while the unaltered ShyCas9, as expected, cannot. Data presented is the mean of n =2 independent biological replicates, source data are provided in the source data file. PLL = phosphate lock loop, CTD = C-terminal domain, WED = WEDGE domain, TOPO = topoisomerase domain, Ara = arabinose.Supplementary Figure 5. Catalytic turnover and indel patterns. (a) Plasmid containing the on-target sequence, 5'-TCGTAAAGTGGTGCGTTCTC-3', was mixed with the indicated nuclease at a DNA:RNP molar ratio of 2:1. At the indicated times, reactions were quenched and analyzed, data presented are n =1 biological replicate. Stoichiometric product formation ratios greater than 1 indicate that single nuclease molecules cleave d multiple substrate molecules. (b) Insertion and deletion (indel) pattern for SpyCas9, SluCas9 and sRGN3.1 on eight target sites within the VEGFA locus (for guide sequences, see Supplementary Table 1). Amplicon sequencing was performed and indel identity was calculated by CRISPResso. Data are presented as mean of n =2-3 independent biological replicates, except Spy (guide_37) n = 1 independent biological replicate, source data are provided in the source data file. Unaltered reads (indel = 0 bp, black squares) were excluded from the analysis.Supplementary Figure 6. Genome editing in a murine hepatoma cell line. Genome editing performance of SpyCas9, SluCas9, sRGN3.1, sRGN3.3, sRGN1, sRGN2, and sRGN4 variants in murine cells was assessed by transfection of Hepa1-6 cells with the respective albumin locus-targeting mRNAs (see Supplementary Table 2) and sgRNAs (Alb-T1 target, guide 112, see Supplementary Table 1 for sequence), n = 3 independent biological replicates for sRGNs and SluCas9, data are presented as mean ± SD, and n = 2 for SpyCas9). Source data are provided in the source data file.Supplementary Figure 7. Extended activity and specificity assessments. (a) Activity comparison plots of sRGN3.1 vs. SpyCas9 and SluCas9 on 96 targets. 48 therapeutically relevant targets and 48 rationally designed targets (engineered to explore GC-content between 20% and 80 %, see Supplementary Table 1 for sequences) were probed with SpyCas9, SluCas9 and sRGN3.1 in the FP cleavage assay. Each dot depicts the mean initial slope for each reaction, arb.unit = arbitrary units. Line visualizes x = y; data are presented in mean and are n = 2 independent biological replicates. (b) Left: Nuclease specificity assessment by cell-free FP assay at all possible single nucleotide target::gRNA mismatches. Heatmap ranges from white (no off-target cleavage) to dark blue (extensive off-target cleavage), purple box = WT nucleotide. PAM (not depicted) is to the right of the displayed sequences. Middle: alternative depiction of these data with relative initial rate values (slope) for each mismatch position, colors indicate the respective mutation at each position. Overall specificity for SpyCas9 and SluCas9 was about equal, while overall specificity of sRGN3.1 was 15% higher than SpyCas9. Right: on-target cleavage in these experiments relative to SpyCas9, data are presented as mean with n =2 independent biological replicates. (c) On-target editing observed with the nuclease concentrations selected by titration as input for the GUIDE-Seq experiment, with or without double-stranded oligodeoxynucleotide (dsODN), required for capturing of off-targets in GUIDE-Seq experiments. 60 pmol SpyCas9 and 30 pmol each of SluCas9 and sRGN3.1 were used for targeting HBB-R01, except for sRGN3.1 with 22nt guide, 8pmol were used. For targeting VEGFA_T2, 60 pmol SpyCas9, 8 and 18 pmol sRGN3.1 (for 20nt and 22nt guide, respectively) and 6 and 10 pmol SluCas9 (for 20nt and 22nt guide, respectively), n = 1.(d) Quantification of off-targets retrieved for each nuclease by GUIDE-Seq by number of mismatches (left) or overall number of off-targets (right). Results for both targets (HBB_R01 and VEGFA_T2) were combined for these plots. Source data for all subfigures are provided in the source data file.Supplementary Figure 8. LNP-mediated in vivo editing of sRGNs. (a)UPLC analysis of sRGN3.1 and SpyCas9 LNPs. Left: Representative chromatogram. sRGN3.1 mRNA LNPs had a size of 74 nm, polydispersity index (PDI) of 0.11, and RNA entrapment of 96%; SpyCas9 mRNA LNPs had a size of 74 nm, PDI of 0.13, and RNA entrapment of 91%. Lipid standards (right) were analyzed to identify peaks. sRGN3.1 LNPs showed a slight reduction in amino lipid(AL) and an increase in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1(Δ9-Cis)PE (DOPE) phospho-lipid (Phos) content compared to SpyCas9 LNPs, suggesting altered lipid-RNA associations compared to SpyCas9 mRNA, arb.unit = arbitrary units, CH = cholesterol, PEG =poly-ethylene-glycol, black line = background. (b) CryoTEM morphology analysis of sRGN3.3 and SpyCas9 LNPs. sRGN LNPs displayed improved circularity and multilamellar structure. One representative image of preparations shown in 8 c. (c) SpyCas9-LNP and sRGN3.3-LNP functional stability at 4°C. Liver editing in mice was assessed on indicated days upon intravenous administration. Left: dose of 2 mg/kg, n = 4 independent biological replicates, mean ± S.D. Right: Normalization of the data to day 1 showed 3.6-fold activity reduction for SpyCas9-LNPs and 1.3-fold activity reduction for sRGN3.3-LNPs. (d) Functional in vivo evaluation via TIDE analysis of uridine depletion for sRGN3.1 and sRGN3.3 mRNA constructs with different uridine-substituted base modifications. sRGN3.3 mRNA with (N1)-methylpseudouridine (m1Ψ) or sRGN3.1 with pseudouridine (Ψ) showed no significant difference with uridine depletion; whereas sRGN3.1 mRNA with m1Ψ, 5-methoxyuridine (5moU), and no modification showed significantly increased editing with uridine depletion, (dose of 1 mg/kg, n = 4 independent biological replicates, mean ± SD). For all other in vivo studies m1Ψ modification and the non-uridine depleted constructs were used. Significance was determined using the Mann Whitney test, (*) = p < 0.05, ns = not significant. (e) In vivo evaluation of sgRNA modification approaches in mice via TIDE analysis. Tested were internal chemical modifications (Internal mod 1 and 2) and increasing protospacer length from 20 to 23 nt (Supplementary Table 1). Liver editing showed a dose response at 0.5, 0.75, and 1.5 mg/kg of total LNP-encapsulated RNA. Both modification strategies showed improved potency compared to standard modified sgRNAs. Protospacer length of 23 nt (standard modifications) showed highest potency. N = 4 independent biological replicates, mean ± SD. Source data are provided in the source data file.Supplementary Figure 9. Editing at the intron 40 of the USH2A gene locus. (a) The 7595-2144A > G Usher disease mutation in intron 40 of USH2A (IVS40) results in an additional exon being incorporated in the USH2A mRNA. The T429 IVS50 guide (guide_110) targets the mutant IVS40 allele, while WT-IVS40 targets the WT (Supplementary Table 1, guide_111). GUIDE-seq revealed no sRGN off-target sites above background for T428-IVS40-guide and confirmed via AmpSeq (source data file, Supplementary Fig. 9). A plasmid carrying the sRGN3.1 gene and T428-IVS40-guide was nucleofect ed into the homozygous IVS40 293FT (293FT-IVS40) cell line together with dsODN. Break sites in which dsODN was inserted were identified by NGS. Only genomic sequences quite distant (> 7 mismatches and multiple alignment gaps) from the on-target site for T428-IVS40-guide were captured by dsODN at relevant read counts, suggesting that these sites were not cleaved as off-targets by sRGN3.1 complexed with T428-IVS40-guide. Background was 0.26 % for SpyCas9 and 0.07 % for sRGN3.1. Three replicates for sRGN3.1are shown, identical scales for each replicate. (b) Comparison of editing with mutant IVS40 allel e targeting guide and its surrogate guide. 293FT-IVS40 cell line and its WT parent cell line were transfected with a plasmid carrying either SpyCas9 or sRGN3.1 and sgRNA that matched either WT (guide_111) or the IVS40 SNP allele(guide_110). WT-IVS40-guide differs from T428-IVS40-guide by a single nucleotide and completely matches the wild type USH2A intronic sequence of NHP and human. Insertions and deletions (indels) were quantified using cells harvested 7 days after transfection via TIDE analysis, n = 2 biologically independent experiments, 2 technical replicas each, each datapoint is shown. Source data are provided in the source data file.Supplementary Figure 10. Gating strategy for FACS analysis of BFP-disruption. HEK293T cells, harboring a BFP cassette in the AAVS1 locus were transfected with nuclease expression plasmid (with T2A-GFP) and guide expression plasmid (guide 5-9 and 11-16 for sRGNs and guide_19-23and 25-30 for SpyCas9). Cells were FACS-analyzed 7 days post transfection. Shown is the gating strategy for the control (nuclease only) and a nuclease plus guide treated sample, as used for evaluation of % BFP disruption as presented in Figure 2. A signal from minimum of 10,000 cells was assayed using the V450 filter set in the BD FACS canto II and software diva 8.0.1 and FlowJo 10.7.2.Supplementary References1. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature520,186–191 (2015).2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptivebacterial immunity. Science337, 816–821 (2012).3. Nishimasu, H. et al. Crystal Structure of Staphylococcus aureus Cas9. Cell162, 1113–1126 (2015).4. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases inhuman cells. Nature Biotechnology31, 822–826 (2013).5. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAMspecificities. Nature523, 481–485 (2015).。

MicroRNA-33b downregulates the differentiation

MicroRNA-33b downregulates the differentiation and development of porcine preadipocytesMasaaki Taniguchi •Ikuyo Nakajima •Koichi Chikuni •Misaki Kojima •Takashi Awata •Satoshi MikawaReceived:11February 2013/Accepted:20December 2013/Published online:8January 2014ÓThe Author(s)2014.This article is published with open access at Abstract Sterol regulatory element binding transcription factor (SREBF)is a key transcription regulator for lipid homeostasis.MicroRNA-33b (miR-33b)is embedded in intron 16of porcine SREBF1and is conserved among most mammals.Here,we investigated the effect of miR-33b on adipocyte differentiation and development in porcine sub-cutaneous pre-adipocytes (PSPA).PSPA were transiently transfected with miR-33b,and adipose differentiation was then induced.Delayed adipose differentiation and decreased lipid accumulation were observed in miR-33b-transfected putational predictions suggested that miR-33b may target early B cell factor 1(EBF1),an adipocyte activator of lipogenesis regulators such as CCAAT-enhancer binding protein alpha (C/EBP a )and peroxisome proliferator-activated receptor gamma (PPAR c ).Both gene and protein expression of EBF1were downregulated in miR-33b-transfected PSPA,followed by considerable decreases in the expression of C/EBP a and PPAR c and their downstream lipogenic genes.However,miR-33b transfection did not markedly affect mRNA and protein expression of SREBF1.We also investigated dif-ferences in the expression of miR-33b and lipogenic genesin subcutaneous fat tissues between 5-month-old crossbred gilts derived from Landrace (lean-type)and Meishan (fatty-type)ndrace-derived crossbred gilts expressed more miR-33b and less lipogenic genes than did gilts derived from Meishan.Our results suggest that miR-33b affected the differentiation and development of PSPA by attenuating the lipogenic gene expression cascade through EBF1to C/EBP a and PPAR c .The differential expression of miR-33b observed in crossbred gilts may in part account for differences in lipogenic gene expression and the fat:lean ratio between pig breeds.Keywords Adipocyte ÁGene expression ÁLipogenesis ÁMicroRNA ÁPPAR ÁSREBFIntroductionPork,together with chicken and beef,is an important protein source for humans.In the livestock industry,including pork production,carcass fat quantity and quality are major determinants of the productivity and palatability of meat.Previous work has shown that the molecular pathway of fat metabolism is regulated in a species-speciﬁc and fat-depot-speciﬁc manner [1,2].Therefore,it is essential to understand the molecular mechanisms that underlie adipogenesis and lipogenesis in porcine fat tissues.Previous studies have investigated the transcriptional regulation of genes associated with adipogenesis [3,4].Among the transcription regulators,sterol regulatory ele-ment-binding transcription factor (SREBF)is known to regulate the transcriptional activation of genes involved in the uptake and synthesis of cholesterol,fatty acids,tri-glycerides,and phospholipids [5–7].In addition to tran-scription factors,microRNAs (miRNA),which are *22-ntElectronic supplementary material The online version of this article (doi:10.1007/s11033-013-2954-z )contains supplementary material,which is available to authorized users.M.Taniguchi ÁM.Kojima ÁT.Awata ÁS.Mikawa (&)Animal Genome Research Unit,Agrogenomics Research Center,National Institute of Agrobiological Sciences,2-1-2Kannondai,Tsukuba,Ibaraki 305-8602,Japan e-mail:mikawa@affrc.go.jpI.Nakajima ÁK.ChikuniAnimal Products Research Division,National Institute of Livestock and Grassland Science,2Ikenodai,Tsukuba,Ibaraki 305-0901,JapanMol Biol Rep (2014)41:1081–1090DOI 10.1007/s11033-013-2954-znon-coding RNAs generated from the sequential process-ing of long RNA transcripts[8],are considered to play key roles in the regulation of gene expression at the post-transcriptional level in the diverse regulatory pathways of many cellular processes.MirBase,a public database of miRNA,contains331miRNAs that have been identiﬁed in the porcine genome(Release19available since August 2012,)[9].Recent studies[10,11] reported that miRNA(miR-33a)embedded in SREBF2 gene inﬂuences cholesterol metabolism in murine hepato-cytes and human macrophages by repressing adenosine-triphosphate-binding cassette transporter A1(ABCA1).In addition,Da´valos et al.[12]reported that miR-33a/b is associated with the repression of fatty acid oxidation and insulin signaling in hepatocytes.Further,identiﬁcation and functional studies of miRNAs have been done in livestock species including pig and cattle[13,14].These studies suggest that fat cell development is achieved via a com-plicated mechanism that is potentially regulated by miR-NAs in a species-speciﬁc manner.Therefore,to clarify the porcine-speciﬁc pathways in adipocyte differentiation and development,we asked whether miR-33b inﬂuences the regulation of adipogenesis and lipogenesis in porcine preadipocytes.First,we deter-mined the full-length nucleotide sequence of the porcine SREBF1gene.Second,we transfected miR-33b into por-cine subcutaneous pre-adipocytes(PSPA)to examine whether the transcriptional regulation of genes relevant to adipocyte differentiation and development was affected by miR-33b.We also investigated the mRNA and protein expression levels of an miR-33b target gene that was identiﬁed in an miRNA target prediction search and,as such,assumed to be involved in adipocyte differentiation and st,we discuss the possible effect of miR-33b on differences in backfat(BF)thickness and blood triglyceride levels between representative lean and fat crossbred pigs.Materials and methodsAnimal samplesEuropean crossbred(E)dams,including Landrace,Large White,and Duroc,were mated with Landrace(L)or Meishan(M)boars.Meishan pigs are substantially fatter, particularly in subcutaneous adipose tissue,than are typical European pig breeds[15,16].Seven each of EL and EM gilts were fed under the same nutritional con-ditions until slaughter at5months old at the National Institute of Livestock and Grassland Science(NILGS). Body weights(mean±SD)of the EL and EM at slaughter were82.0±3.8and82.8±6.2,respectively.There was no signiﬁcant body weight difference between the breed types(P=0.782by Student t test).Tissue samples for total RNA extraction were collected from the liver,longissimus dorsi(LD)muscle,and subcuta-neous adipose tissues at the mid-dorsal area of these animals.BF thickness measurements at the shoulder, back,and lumber positions were averaged and are pre-sented as such.All animal care and use in this study was in accordance with the animal experimentation guideline of the NILGS and was approved by the NILGS Animal Care Committee.Cell culturePSPA were cultured in DMEM growth medium containing a low glucose concentration(1.0g/L)and10%fetal bovine serum(FBS)(Invitrogen,Carlsbad,CA,USA).The subconﬂuent cells were passed every3days.To produce mature adipocytes,we plated the PSPA at2.19104cells/cm2 and grew them for3days to reach conﬂuence.After the cells reached conﬂuence(denoted as0day),adipose con-version was carried out in high-glucose(4.5g/L)DMEM containing10%FBS,5l g/mL insulin(Sigma-Aldrich, Basel,Switzerland),0.25l M dexamethasone(Sigma-Aldrich),33l M biotin(Wako Pure Chemicals,Osaka, Japan),17l M pantothenate(Sigma-Aldrich),and5mM octanoate(Sigma-Aldrich).The medium was changed every other day.To investigate the effect of miR-33b on these cells,we subjected groups of cells to the following three experimental conditions:(1)growth medium alone;(2)differentiation with a negative control miRNA (miR-NC)transfection;and(3)differentiation with miR-33b transfection.All cells were cultured at37°C in a humidiﬁed incubator in5%CO2.TransfectionTo investigate the effect of miR-33b,we transfected a commercially available mature miRNA molecule,Pre-miR TM miRNA Precursor(hsa-miR-33b)(Ambion,Austin, TX,USA),into conﬂuent cells.The transfectant hsa-miR-33b is not a hairpin construct but a mature miRNA molecule,so that although it was designed for use with human cells,it can be applicable to pigs,cattle,or dogs,because the mature miRNA sequence of miR-33b is100%identical among these species.For the miR-NC in experimental group2,we used Pre-miR TM Negative Control#1,which is a Pre-miR TM molecule designed to produce no identiﬁable effects on known miRNA function(Ambion).These transfectants were transfected by using the DharmaFECT1 transfection reagent according to the manufacturer’s instructions(ThermoFisher Scientiﬁc,Waltham,MA, USA).Four replicate samples of cells were harvested at2,4,8,12,and16days after transfection and induction of adipocyte differentiation.Cloning of the porcine SREBF1geneA porcine bacterial artiﬁcial chromosome(BAC)library constructed from a boar of Large White/Landrace/Duroc composite was screened by means of a PCR-based method with primers derived from the porcine SREBF1mRNA sequence(GenBank ID:NM_214157.1).BAC clones were sequenced to determine the full-length porcine SREBF1 gene in accordance with the previously established method [17].Gene expression assayTotal RNA including small RNA was extracted from por-cine tissue samples and PSPA by using ISOGEN IITable1Nucleotide sequences of primers and probes used for real-time PCR Gene Nucleotide sequence of primers and probes(50–30)Accession numberSREBF1Forward CGGACGGCTCACAATGCAB686492 Reverse GCAAGACGGCGGATTTATTCProbe TCAACGACAAGATCATCGAGPPAR c1Forward CTCGGACACCGGAGCTGGAJ006756 Reverse CAACCATGGTCACCTCGCTAAProbe CGCCAGGCCACCACCGCAGATTPPAR c2Forward GGTGAAACTCTGGGAGATTCTCTTAAF059245 Reverse CAACCATGGTCACCTCTTGTGAProbe CGATGCCTTCGACACGCTGTCTGCAAC/EBP aForward AGGAGGACGAGTCGAAGCAXM_003127015 Reverse GGCGGAGGGTGTGAATGCProbe CTTTCCCTACCAGCCACCGCCGCEBF1Forward ATGTTTGTCCATAATAACTCCAAGCXM_003359834 Reverse CTTTGATACAGGGAGTAGCATGTTProbe ACCCCTCGGAAGGTACGCCCTCTTATCFASNForward GCTGGCCTACACGCAGAGNM_001099930 Reverse GGCCCTGGAGCGGTATCAProbe CGCCTCCAGCACCTTGCCTTGCaP2Forward CAGGAATTTGATGAAGTCACTGCNM_001002817 Reverse GTGGTTGTCTTTCCATCCCACProbe TGACAGGAAAGTCAAGAGCACCATAACCTTADIPOQForward CACCACTGGCAAATTCCACTGNM_214370 Reverse CCTTCACATCCTTCAAGTAGACCProbe CCTGGGCTGTACTACTTCTCCTTCCACGCD36Forward CCTACTGGCTGAGTTATTGTGACNM_001044622 Reverse CACAGCATAGATTGACCTGCAAProbe TGGTACAGATGCAGCCTCATTTCCACCTSCD1Forward ACGGATATCGCCCTTATGACAAGNM_213781 Reverse CGCTGGCAGAATAGTCATAGGGProbe TGGAAGCCCTCACCCACAGCTCCC(NIPPON GENE,Toyama,Japan).Total RNAs were reverse-transcribed to synthesize single-strand DNA by using ReverTra Ace(TOYOBO,Osaka,Japan).Real-time PCR was performed with the TaqMan system(Applied Biosystems,Foster City,CA,USA)to examine the relative gene expression of SREBF1,C/EBPa,EBF1,fatty acid synthase(FASN),peroxisome proliferator-activated protein gamma1and2(PPAR c1and PPAR c2),stearoyl-CoA desaturase1(SCD1),adipocyte-fatty acid binding protein (aP2),adiponectin(ADIPOQ),and fatty acid translocase (CD36)by using primers and gene-speciﬁc probes (Table1).The TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assays(Applied Biosystems) designed for hsa-miR-33b were used for relative quantiﬁ-cation of miR-33b.The TaqMan Endogenous Control Eukaryotic18S rRNA gene(Applied Biosystems)was used for the relative quantiﬁcation of all of the genes examined. The comparative threshold cycle method(DD Ct)was employed to calculate the relative quantiﬁcation of gene expression based on the formula,2(-DD Ct)whereDD Ct¼ðCt targert geneÀCt18S rRNAÞtestÀðCt targert geneÀCt18S rRNAÞcalibrator[18,19].The DD Ct values obtained from0day,non-trea-ted control PSPA of experimental group1and from LD muscle were used as calibrators for the relative quantiﬁ-cation of gene expression in the PSPA and5-month-old gilts,respectively.Western blot analysisNuclear and cytoplasmic proteins were prepared by using the CelLytic NuCLEAR Extraction Kit(Sigma-Aldrich), according to the manufacturer’s instructions.The protein content was determined by using a bicinchoninic acid protein assay(Pierce,Rockford,IL,USA).Fifteen micro-grams of nuclear or cytoplasmic protein were separated by electrophoresis through12.5%SDS-polyacrylamide gels (ATTO,Tokyo,Japan).After they were electro-transferred onto nitrocellulose membranes by using the iBlot gel transfer system(Invitrogen),the proteins underwent Pon-ceau S staining(Sigma-Aldrich)to verify equal loading of the lanes.The membranes were then blocked overnight at 4°C in phosphate buffered saline(PBS)containing5% skim milk and0.1%Tween20,followed by a1-h incu-bation at room temperature with primary polyclonal anti-bodies speciﬁc to EBF1(Abcam,Cambridge,UK),the transcriptionally active form of SREBF1in the nucleus (Abnova,Aachen,Germany),and b-actin(ACTB)(Ana-Spec,San Jose,CA,USA).Horseradish peroxidase-con-jugated anti-rabbit antibodies(GE Healthcare, Buckinghamshire,UK)served as secondary antibodies. Antigen–antibody complexes were visualized by use of the ECL detection system(GE Healthcare),and the chemilu-minescence signal was scanned with a LAS-3000imaging system and quantiﬁed with Multi Gauge ver2.0,with which the imaging system was equipped(Fujiﬁlm,Tokyo,Japan). Lipid accumulationAt each time point of the culture period,PSPA were washed with PBS,ﬁxed with10%formaldehyde,and stained withﬁltered oil-red O solution(0.3%oil-red O in 60%isopropanol).The triglycerides in the PSPA and blood from the crossbred gilts were extracted with chlo-roform–methanol(2:1,v/v)and quantiﬁed enzymatically by using the Triglyceride E Test(Wako).Data analysisMeasurements of gene expression,protein expression and triglyceride(TG)content were repeated in three indepen-dent experiments.Differences in gene expression,protein expression,and TG content among the cell culture treat-ments were analyzed with Tukey’s multiple comparison tests(P\0.05).Body weight,gene expression levels,BF thickness,and blood triglyceride concentration were com-pared between crossbred gilts by using the Student t test (P\0.05).Results and discussionCharacterization of the full-length sequenceof the porcine SREBF1geneFirst,we determined the20,099-nt sequence of the full-length porcine SREBF1gene including the50-untranslated region(UTR),the19exons that encode the3,456-nt open reading frame,the18introns,and the30-UTR(GenBank ID:AB686492).Unlike the human SREBF1gene(Gen-Bank ID:NG_029029.1),the porcine SREBF1gene sequence that we determined,together with NCBI refer-ence sequence(RefSeq)information,revealed that there is only one SREBF1isoform,and no transcriptional variants have been identiﬁed in the pig genome(GenBank ID: NM_214157.1)or in cattle[20].Porcine SREBF1shares 86and83%homology to the human SREBF1c mRNA and amino acid sequences,respectively.In the full-length porcine SREBF1gene sequence which we determined in this study,intron16included a sequence that is highly conserved among mammals and contains a pre-miRNA sequence(GenBank ID:AB686493)(Supplementary Fig.1),while the corresponding region in recently pub-lished porcine genome sequence(NW_003540979)is notidentiﬁed.The pre-miRNA sequence expressed mature porcine miR-33b (ssc-miR-33b),which shared high homology with several other mammalian species except for mouse (Supplementary Fig.1).Effect of miR-33b on lipid accumulation in PSPA To investigate the effect of miR-33b on lipid homeo-stasis,we transfected miR-33b into PSPA,which repre-sent an ideal experimental model to study porcine adipocyte physiology [21].Non-transfection/non-differ-entiation-induced control cells continued to proliferate and showed no signs of morphological change or lipid accumulation throughout the time course,whereas dif-ferentiation induction clearly induced lipogenesis after 4days (Fig.1).Transfection of miR-33b at differentia-tion induction decreased lipid accumulation in PSPA after 8days compared with transfection of miR-NC (negative control microRNA),suggesting that miR-33b affected the differentiation and development of the PSPA (Fig.1).Consistent with the observation of lipid accu-mulation by oil-red O staining,the TG content of the miR-33b-transfected cells was signiﬁcantly (P \0.05)decreased by 70.3%(4days),71.4%(8days),67.6%(12days)and 77.4%(16days)compared with that of the miR-NC-transfected cells (Fig.2).These results clearly demonstrate that miR-33b attenuates the differ-entiation and development of PSPA.MiR-33b target gene predictionTo investigate the biological function of miR-33b in PSPA,we explored putative miR-33b target genes by using algo-rithms including PITA [22],TargetScan v6.0[23],and [24].These computational predictions sug-gested that miR-33b may target porcine early B-cell factor 1(EBF1)mRNA,because multiple possible miR-33b recog-nition sites were found in the 30-UTR of porcine EBF10 d (A)control2 d 4 d 8 d 12 d 16 d(B)miR-NC(C)miR-33bFig.1Morphological characterization of PSPA.PSPA were treated with:a growth medium;b differentiation medium with negative-control miRNA (miR-NC)transfection;c Differentiation mediumwith miR-33b transfection.Lipid accumulation was visualized with oil-red O staining.Scale bar 50l mTime, day2481216T r i g l y c e r i d e , m g /d L2004008001,0001,2001,400a ab a bb a bc a b ca b c Fig.2Differences in triglyceride content in PSPA.PSPA were treated as follows:growth medium (white ),differentiation medium with negative-control miRNA transfection (shaded ),and differenti-ation medium with miR-33b transfection (black ).Triglyceride content was measured at least three parisons of relative gene expression were performed by using Tukey’s multiple comparison tests.Different letters show a signiﬁcant difference (P \0.05)mRNA,one of which was 100%identical to that of numer-ous other mammalian species (Supplementary Fig.2a,b).In contrast,none of the master regulators of adipocyte dif-ferentiation and development were predicted to be miR-33b targets with these methods.These ﬁndings suggest that ssc-miR-33b has a conserved effect on the post-transcrip-tional regulation of EBF1expression in porcine adipocytes.The BioSystems Database describing ‘‘Transcriptional Regulation of White Adipocyte Differentiation’’in the NCBI [24]suggested that EBF1binds and activates the PPAR c promoter (/biosystem s?term=205243).In addition,Jimenez et al.[26]reported that EBF1promotes adipogenesis by inducing the expres-sion of the C/EBP a and PPAR c 1promoters,and repressing GATA-2,which is considered to negatively affect adipo-genesis in mice [4].Like SREBF1,C/EBP a and PPAR c are master regulators that are recognized as molecular markers of adipocyte differentiation and development [27–29].Fig.3Changes in adipogenic and lipogenic genes in PSPA.PSPA were treated as follows:growth medium (white ),differentiation medium with negative-control miRNA transfection (shaded ),and differentiation medium with miR-33b transfection (black ).Relativegene expression was measured by using the 2-DD Ct method at least three parisons of relative gene expression were performed by using Tukey’s multiple comparison tests.Different letters show a signiﬁcant difference (P \0.05)Therefore,we assumed that the observed decrease in lipogenesis in PSPA was caused by ssc-miR33b via the attenuation of adipogenic and lipogenic pathways that are regulated by C/EBP a and PPAR c ,which are ordinarily activated by EBF1.We examined the promoter regions of the porcine PPAR c 1,c 2,and C/EBP a genes by using TFSERACH ver1.3based on TRANSFAC [28]and found that the promoters of C/EBP a and PPAR c genes contained EBF1and C/EBP a binding sites,respectively (Supple-mentary Figs.3,4,5).These results suggest that porcine EBF1may be associated with the transcriptional regulation of C/EBP a and,indirectly,of PPAR c genes.Effect of miR-33b on SREBF1and EBF1expression Transfection of precursor miRNAs into PSPA successfully induced the transitional expression of miR-33b.The expression level of miR-33b in miRNA precursor-transfectedPSPA was signiﬁcantly higher (P \0.05)than those in the control and in miR-NC-transfected PSPA,although the endogenous miR-33b level was relatively low and did not ﬂuctuate over time (Fig.3).Because of the genomic localization of ssc-miR-33b,we examined whether miR-33b inﬂuences its host SREBF1gene.SREBF1gene expression did not appear to be ele-vated during the PSPA differentiation induction time course.The mRNA level of SREBF1upon miR-33b transfection tended to be lower throughout the time course,but no signiﬁcant differences were detected (Fig.3).In addition,the relative protein expression level of SREBF1after miR-33b transfection was not different from that after transfection of miR-NC (Fig.4).These results suggest miR-33b had relatively little effect on SREBF1expression.In contrast,the mRNA levels of EBF1in cells with miR-33b transfection were signiﬁcantly (P \0.05)decreased by 66.9%(4days),70.2%(8days),and 80.8%(12days)compared with those after miR-NC transfection (Fig.3).In addition to this mRNA abundance,the protein expression level of EBF1in miR-33b-transfected PSPA was signiﬁ-cantly (P \0.05)decreased by 80.8%(2days),61.2%(8days),and 84.8%(16days)compared with that after miR-NC transfection (Fig.4).These results suggest that miR-33b affects EBF1expression at both the transcrip-tional and post-transcriptional levels.Effect of miR-33b on C/EBP a ,PPAR c and their downstream adipo/lipogenic genesGiven our ﬁnding of decreased EBF1expression upon miR-33b transfection,we investigated the gene expression of the adipogenic and lipogenic master regulators C/EBP a and PPAR c and their downstream genes,including aP2,CD36,ADIPOQ ,SCD1,and FASN (Fig.3).The relative mRNA level of PPAR c 2gradually increased in the differentiation-induced PSPA.However,the PPAR c 2mRNA level after miR-33b transfection sig-niﬁcantly decreased compared with that after miR-NC transfection from the 2-day time point onward (P \0.05).In agreement with the PPAR c 2mRNA expression,the relative gene expression of PPAR c 1,another isoform of PPAR c broadly expressed in various tissue types,was also decreased in miR-33b-transfected PSPA compared with that in miR-NC-transfected PSPA (P \0.05).Similarly,the relative gene expression of C/EBP a in miR-33b-trans-fected PSPA was signiﬁcantly decreased compared with that in miR-NC-transfected PSPA from the 2-day time point onward (P \0.05).These results demonstrate that the changes in the expression patterns of PPAR c and C/EBP a were similar,although the levels of gene expres-sion were different.The results of the gene expression assays for PPAR c 2and C/EBP a suggest that thedecreaseFig.4Changes in SREBF1and EBF1protein expression levels in PSPA.PSPA were treated as described above.Relative gene expression was determined by Western blotting at least three times.The image shown is a representative parisons of relative protein expression were performed by using Tukey’s multiple comparison tests.1H:Positive control (nucleoprotein extracted from HeLa cells).2miR-NC:negative control miRNA.Different alphabets show a signiﬁcant difference (P \0.05)in expression of these genes was affected by EBF1deg-radation,as indicated in the BioSystems Database of the NCBI and by Jimenez et al.[26].Genes known to be regulated by PPAR c ,such as aP2,CD36,and ADIPOQ ,were highly expressed in adipose differentiation-induced PSPA,whereas their expression was signiﬁcantly decreased in miR-33b-transfected PSPA (Fig.3).In addition,the same ﬂuctuating expression pat-tern was observed for the PPAR c 2gene,suggesting that the decreased expression of the aP2,CD36,and ADIPOQ genes was the result of decreased PPAR c 2in miR-33b-transfected PSPA.In human adipocytes,transcription of aP2,CD36,and ADIPOQ is reported to be regulated by PPAR c 2,and these genes are associated with fat formation and metabolism [31–33].The mRNA level of SCD1was generally increased in line with the degree of lipogenesis in adipose differentia-tion-induced PSPA,but the mRNA level after miR-33b transfection was at least 30%less than that after miR-NC transfection from the 4-day time point onward (P \0.05)(Fig.3).The observed decrease in SCD1mRNA expres-sion levels was similar to that of the PPAR c 2mRNA level,because SCD1gene transcription can be regulated bySREBF1or PPAR c 2[5,34].The mRNA level of FASN after miR-33b transfection was 41%less than that after miR-NC transfection at 4days (P \0.05).In contrast to the SCD1mRNA level,changes in the FASN mRNA level were small,suggesting that FASN transcription may be modestly regulated by PPAR c 2,as demonstrated in pre-vious studies [35,36].Taken together,these results suggest that expression of adipogenic and lipogenic genes is well regulated by the master regulators PPAR c 2and C/EBP a in PSPA and that the observed decrease of lipid accumulation in PSPA can be explained by the decrease in expression of these key genes.Furthermore,the delay in adipogenesis and decrease in lipogenesis observed in PSPA after miR-33b transfection was characterized by the degradation of PPAR c 2and C/EBP a but not that of SREBF1,possibly due to decreased EBF1transcriptional regulation.Possible effect of miR-33b on fat formation differences between pig breedsTo investigate the effect of miR-33b on fat formation in fattening pigs and on the generation of different fat traitsinFig.5Comparison of miR-33b,lipogenic genes,and fat formation indices betweenMeishan-and Landrace-derived crossbred gilts.Expression of miR-33b and lipogenic genes in muscle (LD muscle),liver and Sc fat (subcutaneous fat)tissues.Open squares and shaded circles indicate individual crossbred gilts derived from Landrace (EL)and Meishan (EM),respectively.Mean values are denoted with horizontal barspigs that were crossbred with typical lean(Landrace)-and fatty(Meishan)-type breeds,we analyzed differences in BF thickness and blood TG levels and also in miR-33b, SREBF1,EBF1,PPAR c,and C/EBP a gene expression in the subcutaneous tissues of EL and EM crossbred gilts.We found that EM gilts showed signiﬁcantly higher blood TG levels(P\0.01)and BF thickness(P\0.001) than did EL gilts,in agreement with a previousﬁnding [37].In addition to this difference in physiological char-acteristics between these pig breeds,gene expression assays indicated that miR-33b expression in EL tended to be higher than that in EM(P=0.08)(Fig.5).In contrast, EL gilts showed signiﬁcantly lower expression of lipogenic genes,including SREBF1(P\0.01),EBF1(P\0.01), PPAR c2(P\0.01),and C/EBP a(P\0.05)than did EM gilts.These results suggest that the difference in miR-33b expression between these pig breeds is associated with differences in the transcriptional regulation of lipogenic genes,leading to observed differences in fat-related traits such as BF thickness and blood TG levels.It is important to note that only seven gilts were used to obtain these data; therefore,given the small sample size,it is difﬁcult to draw conclusions regarding correlations between miR-33b and lipogenic gene regulation and fat-related traits.In addition, genetic polymorphisms in SREBF1and miR-33b may have certain effect on their expressions,although so far there is no evidence for a difference in the SREBF1gene sequences between Landrace and Meishan pigs.As described in a review by Dodson et al.[2],meat animals have recently begun to be utilized as good experimental models for lipid metabolic research.In addition,the lack of miR-33b in Srebf1gene in traditional model animals such as mouse and rat(Supplementary Fig.1)[11,12]may mean that the pig is a better model for biomedical research on lipid metabolism.Future studies of miR-33b will include evaluating cor-relations between miR-33b,lipogenic genes,and pork carcass characteristics related to fat tissues by using a larger number of pigs.Acknowledgments This work was supported by KAKENHI (22780256).Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use,dis-tribution,and reproduction in any medium,provided the original author(s)and the source are credited.References1.Hishikawa D,Hong YH,Roh SG,Miyahara H,Nishimura Y,Tomimatsu 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21世纪是生命科学的世纪20世纪后叶分子生物学的突破性...

第一章绪论一简答题1. 21世纪是生命科学的世纪。

20世纪后叶分子生物学的突破性成就，使生命科学在自然科学中的位置起了革命性的变化。

试阐述分子生物学研究领域的三大基本原则，三大支撑学科和研究的三大主要领域？答案：（1）研究领域的三大基本原则：构成生物大分子的单体是相同的；生物遗传信息表达的中心法则相同；生物大分子单体的排列（核苷酸，氨基酸）导致了生物的特异性。

（2）三大支撑学科：细胞学，遗传学和生物化学。

（3）研究的三大主要领域：主要研究生物大分子结构与功能的相互关系，其中包括DNA和蛋白质之间的相互作用；激素和受体之间的相互作用；酶和底物之间的相互作用。

2. 分子生物学的概念是什么？答案：有人把它定义得很广：从分子的形式来研究生物现象的学科。

但是这个定义使分子生物学难以和生物化学区分开来。

另一个定义要严格一些，因此更加有用：从分子水平来研究基因结构和功能。

从分子角度来解释基因的结构和活性是本书的主要内容。

3 二十一世纪生物学的新热点及领域是什么？答案：结构生物学是当前分子生物学中的一个重要前沿学科，它是在分子层次上从结构角度特别是从三维结构的角度来研究和阐明当前生物学中各个前沿领域的重要学科问题，是一个包括生物学、物理学、化学和计算数学等多学科交叉的，以结构（特别是三维结构）测定为手段，以结构与功能关系研究为内容，以阐明生物学功能机制为目的的前沿学科。

这门学科的核心内容是蛋白质及其复合物、组装体和由此形成的细胞各类组分的三维结构、运动和相互作用，以及它们与正常生物学功能和异常病理现象的关系。

分子发育生物学也是当前分子生物学中的一个重要前沿学科。

人类基因组计划，被称为“21世纪生命科学的敲门砖”。

“人类基因组计划”以及“后基因组计划”的全面展开将进入从分子水平阐明生命活动本质的辉煌时代。

目前正迅速发展的生物信息学，被称为“21世纪生命科学迅速发展的推动力”。

尤应指出，建立在生物信息基础上的生物工程制药产业，在21世纪将逐步成为最为重要的新兴产业；从单基因病和多基因病研究现状可以看出，这两种疾病的诊断和治疗在21世纪将取得不同程度的重大进展；遗传信息的进化将成为分子生物学的中心内容”的观点认为，随着人类基因组和许多模式生物基因组序列的测定，通过比较研究，人类将在基因组上读到生物进化的历史，使人类对生物进化的认识从表面深入到本质；研究发育生物学的时机已经成熟。

Metatranscriptom...

Metatranscriptomic Sequencing of a Cyanobacterial Soil-Surface Consortium with and without a Diverse Underlying Soil MicrobiomeTerrence H.Bell,a,b Ryan V.Trexler,a Xin Peng,b,c Marcel Huntemann,d Alicia Clum,d Brian Foster,d Bryce Foster,dSimon Roux,d Krishnaveni Palaniappan,d Neha Varghese,d Supratim Mukherjee,d T.B.K.Reddy,d Chris Daum,dAlex Copeland,d Natalia N.Ivanova,d Nikos C.Kyrpides,d Christa Pennacchio,d Emiley A.Eloe-Fadrosh,d Mary Ann Bruns b,ca Department of Plant Pathology and Environmental Microbiology,The Pennsylvania State University,University Park,Pennsylvania,USAb Intercollege Graduate Degree Program in Ecology,Huck Institutes of the Life Sciences,The Pennsylvania State University,University Park,Pennsylvania,USAc Department of Ecosystem Science and Management,The Pennsylvania State University,University Park,Pennsylvania,USAd Department of Energy Joint Genome Institute,Walnut Creek,California,USAABSTRACT Soil surface consortia are easily observed and sampled,allowing exami-nation of their interactions with soil microbiomes.Here,we present metatranscrip-tomic sequences from Dark Green1(DG1),a cyanobacterium-based soil surface con-sortium,in the presence and absence of an underlying soil microbiome and/or urea.M icrobial inoculants can establish unpredictably in soils,due to factors includingcompetition with established microorganisms(1);however,inoculants thatform visible surfaceﬁlms provide unique opportunities to track survival.In2013,cyanobacterium-based soil surface consortia from Pennsylvania were enriched to de-velop surfaceﬁlm-forming inoculants(2).One consortium,Dark Green1(DG1),wasenriched in culture over2years without added nitrogen or carbon,and abundantmembers include Cylindrospermum spp.and six nonphotosynthetic taxa(3).We introduced DG1to soils containing low-or high-diversity microbiomes,with orwithout urea added.Soil was collected from Penn State’s Agronomy Research Farm(4),sieved to2mm,and twice autoclaved(45min,24-h interval).To one portion,nonau-toclaved soil was reintroduced at5%(vol/vol)to establish a high-diversity microbiome.Inoculated and uninoculated soil was dispensed into12petri dishes each(10by15mm;25g dry soil/dish).An even fructose/maltose/glucose/galactose/ribose mixture was added to microcosms at2g carbon/kg dry soil.Six microcosms from each soil type received urea at150(start of incubation)and50mg nitrogen/kg dry soil(pre-DG1 addition),generating four treatments.The microcosms were dark incubated for43 weeks at21°C.DG1was grown in modiﬁed BG-11medium under continuousﬂuorescent lighting (average1,865lux)and moderate agitation at21°C(4).The cultures were pelleted at 5,500rpm in50-ml Falcon tubes,the medium was removed,and sterile deionized(DI) water was added(3:1[vol/vol])to resuspend the mixture.We pipetted3-ml suspension across the surface of each soil sample and incubated the microcosms under constant ﬂuorescent light for5weeks at21°C.RNA was extracted from the excised bioﬁlms using the RNeasy PowerSoil total RNA kit(Qiagen),assessed on an Agilent BioAnalyzer at the Penn State Genomics Core(RNA integrity no.[RIN],Ͼ7),and shipped to the Joint Genome Institute(JGI).Metatranscrip-tome library preparation was performed on a Sciclone NGS robot(PerkinElmer)using Illumina’s Ribo-Zero rRNA removal kits(equimolar bacteria/yeast/plant root)and the TruSeq stranded total RNA high-throughput(HT)kit,with100ng/sample RNA and10 PCR cycles for library ampliﬁcation.Libraries were quantiﬁed with KAPA library quan-Citation Bell TH,Trexler RV,Peng X,Huntemann M,Clum A,Foster B,Foster B,Roux S,Palaniappan K,Varghese N,Mukherjee S,Reddy TBK,Daum C,Copeland A,Ivanova NN, Kyrpides NC,Pennacchio C,Eloe-Fadrosh EA, Bruns MA.2020.Metatranscriptomic sequencing of a cyanobacterial soil-surface consortium with and without a diverse underlying soil microbiome.Microbiol Resour Announc9:e01361-19.https:///10.1128/ MRA.01361-19.Editor Vincent Bruno,University of Maryland School of MedicineThis is a work of the ernment and isnot subject to copyright protection in theUnited States.Foreign copyrights may apply. Address correspondence to Terrence H.Bell,*************.Received30October2019Accepted20November2019Published2January2020OMICS DATA SETStiﬁcation kits on a Roche LightCycler 480.Sequencing was performed on an Illumina NovaSeq using XP v1reagent kits following a 2ϫ150-nucleotide (nt)indexed run recipe.Default parameters were used for all software unless otherwise noted.BBDUK (v38.26)removed (i)contaminants,(ii)adapter sequences and right read segments where quality was equal to 0,(iii)reads with N bases,a mean quality score of Ͻ10,or minimum length of Յ51bp or 33%of full length,and (iv)rRNA (5).The ﬁltered reads were assembled using MEGAHIT v1.1.2(–k list,23,43,63,83,103,123)(6).The ﬁltered reads were mapped to contigs using BBMap (v38.25,ambiguous ϭrandom)to estimate coverage (5).Genes were identiﬁed and annotated in IMG/M v4(7,8).Taxonomic assignments for transcripts were determined by selecting the options “compare ge-nomes”and “phylogenetic distribution”at a percent identity of ୑60%and normalized by estimated gene copies.Table 1presents the annotation statistics for the metatran-scriptomes.Initial analysis suggests fewer cyanobacterium transcripts when high-diversity mi-crobiomes are present,particularly with urea.Of interest will be the frequency of transcripts indicating interspeciﬁc interactions.Data availability.Metatranscriptome sequences are available through the JGI Genomes OnLine Database (GOLD)under project identiﬁer Gs0132857.ACKNOWLEDGMENTSIn-lab work was supported by the USDA National Institute of Food and Agricul-ture,Hatch projects 1016233and 1003346,and the Penn State University College of Agricultural Sciences Research Applications for Innovation program.Funding for metatranscriptomic sequencing was provided through the Joint Genome Institute’s Community Science Program (CSP 503310).The work conducted by the U.S.De-partment of Energy (DOE)Joint Genome Institute,a DOE Ofﬁce of Science userplus urea2231,138,23048.219,28179,20998,70355.35595,64896.964.7421.133000314953225,572,28221.25,78529,05539,26057.06437,55995.6763.5919.4733000314994195,630,98816.24,63522,23929,75856.12228,28795.0663.5119.3233000315025199,215,54818.34,97925,84233,51754.72531,75294.7364.2618.5433000315036208,163,37617.78,06429,99437,21359.16735,17894.5364.721.333300031504High diversity 1186,588,01023.313,14943,86751,45458.6949,06595.3660.3519.6133000314842197,781,21422.88,20135,74645,00655.91743,03295.6162.9919.1833000314873247,118,63213.34,93521,23625,86653.51323,99192.7562.4618.633000314914232,297,57223.47,46135,59345,66455.47643,66695.6264.1919.3233000314905177,035,36458.625,71797,967124,26760.664121,38597.6867.623.2933000314936185,064,55637.715,32061,97578,18358.32475,87197.0466.421.893300031476Low diversity plus urea1290,517,45416.53,48119,78729,35053.73728,86298.3470.1624.133000314882187,827,806 5.21,4256,6569,25748.7039,08398.1267.9124.2533000314833227,368,660 5.71,3686,8879,57049.2149,39998.2170.2423.1233000314754207,822,51410.91,76610,91217,26751.58717,00798.4970.4224.0233000314945225,974,35625.23,57824,95741,64558.97641,12798.7670.5725.8133000314926199,479,2467.81,1847,12011,58148.02511,37098.1868.7922.293300031479Low diversity 1213,606,58212.61,04810,67018,25150.18818,02898.7869.5623.5733000314982222,910,45813.783111,11719,56050.39319,24498.3868.8422.8933000314893243,278,23219.82,15519,35330,79454.18730,42498.868.1423.8733000314774205,533,538109578,59414,31247.5714,08098.3868.0421.6833000314825196,564,51211.11,22110,28416,35748.78216,09598.468.8922.533000314976228,387,13412.11,84112,24219,16151.53118,89198.5969.2524.083300031480aCDS,coding DNA sequence.Bell et al.facility,is supported by the Ofﬁce of Science of the U.S.Department of Energy under contract no.DE-AC02-05CH11231.We thank Timothy Peoples for assistance with DNA extraction,and we thank Roxanne Lease for help in establishing our incubation conditions for the microcosms.REFERENCES1.Kaminsky LM,Trexler RV,Malik RJ,Hockett KL,Bell TH.2019.The inherentconﬂicts in developing soil microbial inoculants.Trends Biotechnol37: 140–151.https:///10.1016/j.tibtech.2018.11.011.2.Peng X.2016.Potential use of N2-ﬁxing cyanobacteria for establishing re-newable biological soil crusts and modulating soil nitrogen in agroecosys-tems.PhD dissertation.The Pennsylvania State University,University Park,PA.3.Peng X,Bruns MA.2019.Development of a nitrogen-ﬁxing cyanobacterialconsortium for surface stabilization of agricultural soils.J Appl Phycol 31:1047–1056.https:///10.1007/s10811-018-1597-9.4.Peng X,Bruns MA.2019.Cyanobacterial soil surface consortia mediate Ncycle processes in agroecosystems.Front Environ Sci6:156.https://doi .org/10.3389/fenvs.2018.00156.5.Bushnell B.2014.BBMap.https:///projects/bbmap/.6.Li D,Liu C-M,Luo R,Sadakane K,Lam T-W.2015.MEGAHIT:an ultra-fastsingle-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.Bioinformatics31:1674–1676.https:///10 .1093/bioinformatics/btv033.7.Chen I-MA,Chu K,Palaniappan K,Pillay M,Ratner A,Huang J,HuntemannM,Varghese N,White JR,Seshadri R,Smirnova T,Kirton E,Jungbluth SP, Woyke T,Eloe-Fadrosh EA,Ivanova NN,Kyrpides NC.2019.IMG/M v.5.0: an integrated data management and comparative analysis system for microbial genomes and microbiomes.Nucleic Acids Res47:D666–D677.https:///10.1093/nar/gky901.8.Huntemann M,Ivanova NN,Mavromatis K,Tripp HJ,Paez-Espino D,Palaniappan K,Szeto E,Pillay M,Chen I-MA,Pati A,Nielsen T,Markowitz VM,Kyrpides NC.2015.The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline(MGAP v.4).Stand Genomic Sci 10:86.https:///10.1186/s40793-015-0077-y.Microbiology Resource Announcement。

On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform

On-chip quantitative detection of pathogen genes by autonomousmicroﬂuidic PCR platformHiroaki Tachibana a,b,Masato Saito a,n,Shogo Shibuya b,Koji Tsuji b,Nobuyuki Miyagawa b,Keiichiro Yamanaka a,Eiichi Tamiya aa Department of Applied Physics,Graduate School of Engineering,Osaka University,2-1Yamadaoka,Suita,Osaka575-0871,Japanb Eco Solutions Company,Panasonic Corporation,1048Kadoma,Kadoma,Osaka571-8686,Japana r t i c l e i n f oArticle history:Received14March2015Received in revised form24June2015Accepted4July2015Available online9July2015Keywords:Continuous-ﬂow polymerase chain reactionQuantitative real-time PCRMicroﬂuidic chipCapillary forceOn-site pathogen detectiona b s t r a c tPolymerase chain reaction(PCR)-based genetic testing has become a routine part of clinical diagnosesand food testing.In theseﬁelds,rapid,easy-to-use,and cost-efﬁcient PCR chips are expected to be ap-peared for providing such testing on-site.In this study,a new autonomous disposable plastic microﬂuidicPCR chip was created,and was utilized for quantitative detection of pathogenic microorganisms.Tocontrol the capillaryﬂow of the following solution in the PCR microchannel,a driving microchannel wasnewly designed behind the PCR microchannel.This allowed the effective PCR by simply dropping the PCRsolution onto the inlet without any external pumps.In order to achieve disposability,injection-moldedcyclo-oleﬁn polymer(COP)of a cost-competitive plastic was used for the PCR chip.We discovered thatcoating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface fordriving the capillaryﬂow through the1250-mm long microchannel.As a result,quantitative real-timePCR with the lowest initial concentration of human,Escherichia coli(E.coli),and pathogenic E.coli O157genomic DNA of4,0.0019,0.031pg/μl,respectively,was successfully achieved in less than18min.Ourresults indicate that the platform presented in this study provided a rapid,easy-to-use,and low-costreal-time PCR system that could be potentially used for on-site gene testing.&2015Elsevier B.V.All rights reserved.1.IntroductionPolymerase chain reaction(PCR)plays a central role in geneticanalysis since its invention in the mid-1980s by Kary Mullis(Mullis and Falona,1987).PCR allows the detection of low levels ofpathogens by amplifying their deoxyribo nucleic acid(DNA)withhigh accuracy and precision.Therefore,PCR is used for diagnosinginfectious diseases,for testing bacterial food poisoning,and inbiodefense(Christopoulos,1999).TaqMan-based quantitative real-time PCR allows simultaneous detection of target sequences byusing primers and dual-labeledﬂuorogenic probes.Real-time PCRhas been used to detect many bacterial pathogens,such as Es-cherichia coli(E.coli)(Frydendahl et al.,2001;Ståhl et al.,2011;Silkie et al.,2008),pathogenic E.coli O157(Call et al.,2001;Leeet al.,2006),Salmonella(Lee et al.,2006),Staphylococcus aureus(Lee et al.,2006),Campylobacter jejuni(Best et al.,2003),andCampylobacter coli(Best et al.,2003).Although PCR is a usefuldiagnostic tool because of its high sensitivity and accuracy,it istime-consuming when performed using laboratory-scale thermalcyclers.Kopp et al.(1998)and coworkers demonstrated theﬁrst con-tinuous-ﬂow PCR(CF-PCR)by using a microﬂuidic chip for high-speed ampliﬁcation of target sequences.Three temperature zones,corresponding to denaturation,annealing,and extension tem-peratures,respectively,were established in a glass-based micro-ﬂuidic chip by using temperature-ﬁxed heaters.Only the PCR so-lution underwent thermal cycling in singleﬂuidic microchannelamong3temperature zones.This allowed the PCR solution toswitch rapidly among different temperatures compared with thatin conventional thermal cyclers.After this epochal research,somerelated studies were performed to improve the design and efﬁcacyof microﬂuidic chips such as tuning the cross-sectional size of themicrochannels(Li et al.,2006;Cao et al.,2011),a short cycle length(Crews et al.,2008a,2008b).Reverse transcription PCR(RT-PCR)targeting an inﬂuenza virus(Yamanaka et al.,2011),and integra-tion of PCR chips withﬂuorescence detection systems(Obeid andChristopoulos,2003;Nakayama et al.,2006,2010)were alsoContents lists available at ScienceDirectjournal homepage:/locate/biosBiosensors and Bioelectronics/10.1016/j.bios.2015.07.0090956-5663/&2015Elsevier B.V.All rightsreserved.n Corresponding author.Fax:þ81668797840.E-mail addresses:tachibana@ap.eng.osaka-u.ac.jp(H.Tachibana),saitomasato@ap.eng.osaka-u.ac.jp(M.Saito),shibuya.shogo@(S.Shibuya),tsuji.koji-001@(K.Tsuji),miyagawa.nobuyuki@(N.Miyagawa),k-yamanaka@ap.eng.osaka-u.ac.jp(K.Yamanaka),tamiya@ap.eng.osaka-u.ac.jp(E.Tamiya).Biosensors and Bioelectronics74(2015)725–730performed.However,these modi ﬁcations still required the use of complex external pumps such as syringe pumps to control the ﬂow of PCR solution in the PCR microchannel.To eliminate the use of external pumps,we developed a ca-pillary-driven,self-propelled CF-PCR (SP-CF-PCR)micro ﬂuidic chip (Tachibana et al.,2015).The PCR microchannel was prepared using a Si substrate and was covered by a glass substrate.The surface of the microchannel was oxidized to form a hydrophilic ﬁlm.PCR solution dropped onto the inlet of the micro ﬂuidic chip was transported through the microchannel by capillary ﬂow to achieve the desired ampli ﬁcation.The theoretical approach of capillary ﬂow involving more than one temperature was developed based on previous studies (Juncker et al.,2002;Delamarche et al.,1998,2005;Zhu and Petkovic-Duran,2010).We used this SP-CF-PCR micro ﬂuidic chip to amplify the genomic DNA of some pathogens.However,the ampli ﬁcation was inef ﬁcient in the head area of the ﬂow because of the evaporation of PCR solution or adsorption of reagents.Moreover,the use of Si for preparing micro ﬂuidic chips is associated with 2major disadvantages.One of the disadvantages is longer intermediate temperature zone between the high and low temperature zones due to the high thermal conductivity of Si.This intermediate temperature zone increases the microchannel length and decrease PCR ef ﬁciency at an unnecessary temperature.An-other disadvantage is its high cost.The material used for making micro ﬂuidic chips should be cost-competitive to allow mass pro-duction for on-site use such as screening of pathogens.From this point of view,plastics are very promising for devel-oping micro ﬂuidic chips because they have low thermal con-ductivity and are inexpensive.However,one of the dif ﬁculties of using plastics for developing SP-CF-PCR chips is the achievement of hydrophilic surface on the microchannel walls because most plastics are hydrophobic.Some dry processes such as plasma (Gervais et al.,2011)or ultraviolet (UV)ozone (Novo et al.,2011,2013)treatment have been used to activate and deliver hydro-philic groups on the surface of the microchannel walls in poly-dimethylsiloxane (PDMS)micro ﬂuidic chips.However,because the activated surfaces are unstable (Eddington et al.,2006),hydro-philicity disappears during the subsequent fabrication process,and because of time-associated degradation or heating.Therefore,some active hydrophilic layers should be developed on the mi-crochannel walls.In the present study,a plastic-based micro ﬂuidic real-time quantitative PCR chip was developed as shown in Fig.1(a).A driving microchannel,which was placed behind the PCR micro-channel,was designed to exclude the head area of the ﬂow from the PCR microchannel and to maintain capillary force for con-trolling the ﬂow.The microchannel of the SP-CF-PCR chip was formed in a cyclo-ole ﬁn polymer (COP)plate by injection molding.Water-soluble polymers and surfactants were examined for pro-ducing the hydrophilic layer on the microchannel walls.Moreover,the PCR micro ﬂuidic chip was integrated with a ﬂuorescence de-tection system containing a laser and photomultiplier tube (PMT)to analyze the ampli ﬁed product within the chip.For verifying our concept,human β-actin,E.coli DNA,and E.coli O157DNA were ampli ﬁed using the new SP-CF-PCR chip and the ﬂuorescence detectionsystem.Fig.1.Design of the self-propelled continuous-ﬂow PCR (SP-CF-PCR)chip.(a)Concept diagram.The PCR chip is placed on 2heater blocks having temperatures 95°C and 60°C,respectively.The PCR solution dropped onto the inlet hole is loaded into the microchannels autonomously by capillary force.The driving microchannel placed behind the PCR microchannel maintains the capillary force to control the following ﬂow of the solution.(b)Simulated temperature distribution in the COP (ﬁlled circle with solid line)and glass/Si (Tachibana et al.,2015)(ﬁlled triangle with dashed line)micro ﬂuidic chips in planes where the microchannels are formed.The microchannels were formed between a lower and an upper plate.The lower plates were attached to the heater blocks.The origin of displacement was set at the center between the high and low temperature zones on the left (negative direction)and right (positive direction),respectively.The block heaters were ﬁxed with 1-mm gap,and the temperatures at both the ends of the chip were ﬁxed at 99°C and 60°C,respectively.H.Tachibana et al./Biosensors and Bioelectronics 74(2015)725–7307262.Materials and methods2.1.Thermal simulation analysisThe cross-sectional temperature distributions in planes inwhich the microchannels were formed were simulated usingﬁniteelement method(FEM)with ANSYS©.The microchannels wereformed between upper and lower plates of20mm-width.Thelower plate was attached on2aluminum heater blocks whosewidth and height were both15mm.The blocks wereﬁxed with a1-mm gap.In the thermal simulations,the blocks were consideredas heating element and were heated in order that the tempera-tures at both the ends of the chip becameﬁxed at99°C and60°C,respectively.Si,glass and COP plates whose thermal conductivitiesare 1.5Â102, 1.0,0.60W/(m K),respectively,were used in thesimulations.2.2.Chip fabricationThe PCR chip included two57Â25mm2COP plates(ZeonCorp.,Tokyo,Japan).One of the plates was1.5-mm thick and in-cluded150-μm deep and150-μm wide rectangular cross-sectional microchannels.The other one was a0.7-mm thickﬂat cover plate.The PCR chip was fabricated as follows.First,the COP plate withthe microchannels was fabricated by injection molding.The pat-tern of the microchannels was fabricated in Si substrate by usingmicromachining technologies as a mold.The microchannels wereformed by photolithography and deep etching process(Tachibanaet al.,2015).Note that the microchannels were convex shape.Theinjection molding of the COP plate using this Si mold was out-sourced(Richel Corp.,Toyama,Japan).The inlet and drain holeswere punched at both the ends of the microchannels.The platewas then attached to the cover plate by heating and applyingpressure by exposing the bonding surfaces of the plates to O2plasma for surface cleaning and activation.After fabricating thechip structure,a hydrophilic layer was produced on the micro-channel walls as follows.The microchannels wereﬁlled with asolution containing the hydrophilic medium by using a syringe.Next,the solvent inside the microchannel was evaporated.Threesolutions,namely,1%(w/w)solution of water-soluble polymercarboxymethyl cellulose(CMC)(Wako Pure Chemical Industries,Ltd.Osaka,Japan)in distilled water,1%(w/w)solution of anionsurfactant sodium cocoyl sarcosinate(SCS)(Soypon SCE,KawakenFine Chemicals Co.Ltd.,Tokyo,Japan)in ethanol,and1%(w/w)solution of non-ionic surfactant polyoxyethylene(20)sorbitanmonolaurate(Tween20)(Wako Pure Chemical Industries,Ltd.Osaka,Japan)in ethanol were used for coating the microchannelwalls in separate chips.2.3.PCR reagentsGenomic DNA of humans,E.coli,and pathogenic E.coli O157were ampliﬁed by performing quantitative PCRs with the SP-CF-PCR chip.As a common reagents of all the targets,10Âfast buffer I(SpeedSTAR s HS DNA Polymerase kit;Takara Bio Inc.,Tokyo,Ja-pan),0.2mM dNTP mixture(Takara Bio Inc.),0.15U/μl SpeedSTAR HS DNA Polymerase(Takara Bio Inc.),1μg/μl bovine serum albu-min(BSA)were used.The PCR mixture for amplifying human genomic DNA included300nM each of forward and reverse primers,and TaqManﬂuor-escence probe containingﬂuorescein amidite(FAM)(TaqManβ-Actin Detection Reagents kit;Life Technologies Inc.,Carlsbad,USA). Human genomic DNA concentrations of4,40,400,and3200pg/μl (Life Technologies Inc.)were used as the templates.The mixture for amplifying E.coli genomic DNA included10Âfast buffer I,0.2mM dNTP mixture,0.15U/μl SpeedSTAR HS DNA Polymerase,and1μg/μl BSA.The mixture also included300nM each of forward(5′-CGGAAGCAACGCGTAAACTC-3′),and reverse (5′-TGAGCGTCGCAGAACATTACA-3′)primers,and300nM TaqMan probe(5′-FAM-CGCGTCCGATCACCTGCGTC-BHQ1-3′)targeting uidA(Silkie et al.,2008).Genomic DNA extracted from E.coli DH5α(Takara Bio Inc.)by using PureLink Genomic DNA Mini Kit(Life Technologies Inc.)was used as the template.The mixture for amplifying E.coli O157genomic DNA included 10Âfast buffer I,0.2mM dNTP mixture,0.15U/μl SpeedSTAR HS DNA Polymerase,and1μg/μl BSA.In addition,the mixture in-cluded300nM each of forward(5′-CAATTTTTCAGGGAATAA-CATTG-3′),reverse(5′-AAAGTTCAGATCTTGATGACATTG-3′)pri-mers,and300nM TaqMan probe(5′-FAM-TCAA-GAGTTGCCCATCCTGCAGCAA-BHQ1-3′)targeting eaeA(Call et al., 2001).Genomic DNA of E.coli O157extracted from GTC01061was obtained from Gifu University,School of Medicine,Pathogenic Microorganism Genetic Resource Stock Center(GMGC)and used as the template.2.4.On chip quantitative PCRThe PCR chip was placed on two15Â15Â80-mm3aluminum heater blocks whose temperatures were controlled individually. The aluminum heater blocks were set in parallel with1-mm gap and included cartridge heaters and thermocouples(Kyushu-Nis-sho Co.Ltd,Fukuoka,Japan).One of the heaters was controlled at denaturation temperature while the other was controlled at an-nealing and extension temperatures by using proportional-in-tegral-derivative controller(Shimaden Co.Ltd.,Tokyo,Japan). 50μl of PCR solution was dropped on the inlet hole.Displacement of capillaryﬂow was determined from the time of PCR solution loading by visually monitoring the position of theﬂuid front until all the microchannels wereﬁlled.Once the microchannels were ﬁlled,optical component was scanned over the PCR microchannel, andﬂuorescence of the solution was detected.The detail of the ﬂuorescence detection system and the analysis of the ampliﬁca-tion curves withﬂuorescence are described in Supporting in-formation on page2–3and Fig.S1.3.Results and discussion3.1.Design of the PCR microﬂuidic chipOne drawback of the SP-CF-PCR is the decrease in PCR efﬁ-ciency at theﬂuid front(Tachibana et al.,2015).Theﬂuid front of the PCR solution is continuously in contact with the fresh surface of the microchannel.Therefore,adsorption of PCR reagents such as polymerase or ampliﬁed DNA occurs ineluctably(Nakayama et al., 2006).Moreover,evaporation of the PCR solution occurs at the ﬂuid front because it is constantly exposed at high temperature.To overcome these issues,we newly designed a driving mi-crochannel that was placed behind the PCR microchannel(Fig.1). The velocity of capillaryﬂow in CF-PCR is described as(Tachibana et al.,2015)v xR P xx x8d1 h2c∫η()=()(‵)‵() According to Eq.(1),the velocity of the capillaryﬂow decreases as the solutionﬂows through the microchannel but never becomes 0.Therefore,the driving microchannel controls the capillary forces required to maintain theﬂow of the PCR solution even after the PCR microchannel isﬁlled.Because of the driving microchannel, the head area of theﬂow,with the high initialﬂow velocity and low PCR efﬁciency,can be removed to the driving microchannel.H.Tachibana et al./Biosensors and Bioelectronics74(2015)725–730727Thus,the driving microchannel allows in achieving a stableﬂow velocity and high PCR efﬁciency.The SP-CF-PCR chip having meander shaped PCR microchannel was set on2aluminum block heaters in order to form2tem-perature zones in the same plane.This results in the ineluctable formation of an intermediate temperature zone between the 2temperatures zones.A longer intermediate zone not only in-creases the total microchannel length but also decreases PCR ef-ﬁciency at an unnecessary temperature.The intermediate tem-perature zone depends largely on the thermal conductivity of the PCR chips.In this study,intermediate temperature zones were investigated by comparing between a glass/Si chip having high thermal conductivity(Tachibana et al.,2015)and a COP chip having low thermal conductivity.For evaluating the intermediate temperature zones,cross-sectional temperature distributions in planes in which the microchannels were formed were simulated using FEM analysis.Fig.1(b)shows the results of the comparison between the glass/Si PCR chip and the COP PCR chip.The origin of displacement was set at the center between the high and low temperature zones,which were placed on the left(negative di-rection)and right(positive direction),respectively.The micro-channels of the glass/Si PCR chip were formed between a0.5-mm thick Si lower plate and a0.5-mm thick glass upper plate.The microchannels of the COP PCR chip were formed between a0.7-mm thick lower COP plate and a1.5-mm thick upper plate.High and low temperature zones were deﬁned as95–99°C for dena-turation and60–65°C for annealing and extension,respectively. Hence,the temperature of the intermediate zone was between 65°C and95°C.The intermediate temperature zone in the glass/Si PCR chips was7.8mm long,which was twice its length in the COP PCR chips(3.6mm).Although the formation of the microchannels and hydrophilic surfaces on the microchannel walls by thermal oxidation is easier in the glass/Si PCR chips,heat insulation in these chips is more difﬁcult than that in COP chips because the thermal conductivity of Si is two orders of magnitude higher than that of COP.Materials used to make the PCR chip should have low thermal conductivity to allow the temperature gradient to con-centrate in the gap between2heaters.According to this simula-tion,the microchannel length in each turn,which corresponded to half the length of each PCR cycle,was set as7mm to achieve sufﬁcient length.The designed microchannels were820mm(53 cycles)of PCR microchannel and440mm of driving microchannel.3.2.Capillaryﬂow in the microchannelsHydrophilic layers can be developed on hydrophobic surfaces by using water-soluble polymers(Kargl et al.,2012)and surfac-tants(Aegerter and Menning,2004)because they contain both hydrophobic and hydrophilic groups.In this study,we examined suitable surface treatments to develop a hydrophilic layer on the surface of COP plates,such as treatment with CMC,a water-soluble polymer;SCS,an anion surfactant;and Tween20,a non-ionic surfactant.Contact angle of the hydrophilic layer formed on the COPﬂat plate was15°with CMC and below10°with SCS and Tween20.The hydrophilicity of all the microchannels was main-tained for more than10days.The positions of theﬂuid fronts of the capillaryﬂow in PCR chips whose microchannel walls were covered with CMC,SCS,and Tween20are shown in Fig.2.In the PCR chip whose microchannel walls were covered with CMC,the PCR solution was loaded only several millimeters,indicating that the CMC coating on the mi-crochannel walls was non-homogeneous.This could be because CMC was dissolved in distilled water;therefore,CMC solution was localized in the microchannel during evaporation because the surface of the COP plate repelled water.In contrast,PCR solutions were successfully loaded into PCR chips whose microchannel walls were covered with either SCS or Tween20because homogeneous coatings could be obtained.Surfactants can be applied easily on the microchannel walls because they contain both hydrophilic and hydrophobic groups(Aegerter and Menning,2004).Moreover,the surfactants did not agglomerate during evaporation because they were liquid at room temperature.Tween20was found to be sui-table for producing the hydrophilic layer in this study because the PCR solution reached the drain in less than20min.On the other hand,in the SCS-coated chip,the PCR solution could not reach to the drain.These results indicated that surface hydrophilicity of Tween20was higher than that of SCS.This could be because Tween20has more hydrophilic groups than SCS and because it is generated by polar ether linkage compared with SCS,which only contains a carboxyl group at the end of a long-chain hydrocarbon.In the Tween20-coated chip,the PCR solution initially followed the PCR microchannel andﬁlled it within7min(Fig.2).The PCR solution thenﬁlled the driving microchannel.The capillary force present at theﬂuid front in the driving microchannel helped the ﬂow of the PCR solution through the PCR microchannel.The total time forﬁlling the microchannels was18min in this study.InthisFig.2.Characteristics of capillaryﬂow.(a)Position of theﬂuid front of the PCR solution with respect to time in the PCR chip whose microchannel walls are treated with polyoxyethylene(20)sorbitan monolaurate(Tween20),a non-ionic surfactant(ﬁlled circle with solid line);sodium cocoyl sarcosinate(SCS),an anion surfactant(ﬁlled triangle with dashed line);and carboxymethyl cellulose(CMC),a water-soluble polymer(ﬁlled rectangles with dotted line).The dashed line at820mm indicates the boundary between the PCR and the driving microchannels.(b)Photographs of capillaryﬂow in the PCR chip whose microchannel walls are treated with Tween20by using the new coccine dye.The arrows indicate the position of theﬂuid front.H.Tachibana et al./Biosensors and Bioelectronics74(2015)725–730728experiment,the volume per cycle in the PCR microchannel was 0.35μl.On the other hand,the volume of the meander-shaped driving microchannel was9.8μl,which corresponded to the vo-lume of28PCR cycles.Therefore,PCR solution of28PCR cycle in theﬂuid front was eventually moved to the driving microchannel. As a result,effective PCR was initiated when theﬂuid front was at the positions of the28th cycle.The averageﬂow velocity during effective PCR was0.87mm/s,which corresponded to16s per cy-cle.The time for each cycle was sufﬁcient to achieve an efﬁcient PCR.The possibility of controlling the capillaryﬂow by using the driving microchannel was successfully observed in the present study.Other optimal designs such as capillary pump structure (Zimmermann et al.,2007)will enable us to obtain a faster and steadyﬂow velocity.3.3.Quantitative PCR detectionQuantitative real-time PCR was completed once the driving microchannel wasﬁlled.Therefore,the PCR time was18min as shown in Fig.2.The ampliﬁcation curves of the PCR targeting the humanβ-actin are shown in Fig.3(a).Clear ampliﬁcations were obtained for the initial human genomic DNA concentrations of4, 40,400,and3200pg/μl.The ampliﬁcation curves were shifted to the low cycle as the initial concentration of the human genomic DNA increased.Speciﬁc ampliﬁcation of a295-bp fragment was achieved as a representative result of gel electrophoresis(Fig.S2). The standard curve(threshold cycles(C t values)versus initial concentrations of human genomic DNA)of the human genomic DNA is shown in Fig.3(b).The deﬁnition of C t value is described in the Supporting information on page1–2.As expected,the C t va-lues had a linear characteristic with respect to the logarithmic values of the initial DNA concentrations.The obtained C t value including the reproducibility for initial DNA concentration of4pg/μl was42.273.2,which indicates that the corresponding coefﬁ-cient of variance(CV)was7.5%.The lowest initial concentration of human genomic DNA was4pg/μl,which corresponded to ap-proximately0.6genome/μl(Doležel et al.,2003).These results indicated that our PCR chip and detection system were applicable for quantitative DNA analysis with sufﬁcient high sensitivity and reproducibility.Next,we performed the quantitative PCR targeting uidA of E. coli with initial genomic DNA concentrations of0.0019,0.019,0.19, and1.9pg/μl.The ampliﬁcation curves and a result gel electro-phoresis are shown in Fig.S3.Standard curve of E.coli genomic DNA were obtained from the C t values obtained for each initial DNA concentration(Fig.3(c)).The obtained C t value including the reproducibility for initial DNA concentration of0.0019pg/μl was 45.672.9,which indicates that the corresponding coefﬁcient of variance(CV)was6.3%.The lowest initial concentration of E.coli genomic DNA of0.0019pg/μl corresponded to approximately 0.4genome/μl.Finally,we performed quantitative PCR by using the genomic DNA of a pathogenic microorganism.E.coli O157is one of the most notorious foodborne pathogens that colonizes the intestinal tract of cattle and is present in beef and other related products.The infectivity of E.coli O157is extremely high;therefore,infection can be caused by only a few hundred cells(Karmali,2004).Therefore, rapid,highly accurate,and highly sensitive on-site detection is required to prevent an outbreak of food poisoning.QuantitativeFig.3.On-chip quantitative real-time PCR results.(a)Ampliﬁcation curves withﬂuorescence targeting humanβ-actin.(b)Standard curve of PCR targeting humanβ-actin.(c)Standard curve of PCR targeting ui dA of E.coli.(d)Standard curve of quantitative real-time PCR targeting eaeA of E.coli O157.H.Tachibana et al./Biosensors and Bioelectronics74(2015)725–730729PCR targeting eaeA of E.coli O157was performed with initial genomic DNA concentrations of0.031,0.31,3.1,and31pg/μl.The ampliﬁcation curves and a representative result of gel electro-phoresis are shown in Fig.S4.Standard curve of E.coli O157 genomic DNA were obtained from the C t values calculated from the corresponding ampliﬁcation curves.The obtained C t value in-cluding the reproducibility for initial DNA concentration of 0.031pg/μl was41.772.8,which indicates that the corresponding coefﬁcient of variance(CV)was6.7%.This standard curve indicated that this technique could be used to quantitatively analyze E.coli O157genomic DNA for practical purpose such as food testing. Moreover,the lowest initial concentration of E.coli O157genomic DNA was0.031pg/μl in this experiment,which corresponded to approximately6.9genome/μl,indicating that this technique could detect DNA from less than500cells in a practical sample.4.ConclusionsIn this study,we have successfully developed an autonomous microﬂuidic PCR platform for on-chip quantitative detection of pathogen genes.The newly designed driving microchannel en-abled us to maintain theﬂow of the PCR solution.The excellent thermal controllability was obtained in the COP PCR chip.More-over,we have achieved optimal surface hydrophilicity of the mi-crochannel walls by detailed investigation of non-ionic surfac-tants.As the results,the PCR solution was successfully transported through the whole microchannels autonomously and quantitative real-time PCR was successfully performed in less than18min.Our results indicated that the SP-CF-PCR chips could be used for rapid, low-cost and easy-to-use gene testing in the medical and en-vironmentalﬁelds.Appendix A.Supplementary materialSupplementary data associated with this article can be found in the online version at /10.1016/j.bios.2015.07.009.ReferencesAegerter,M.A.,Menning,M.,2004.Sol–gel technologies for glass producers and users.Kluwer Academic Publishers,Norwell.Best,E.L.,Powell,E.J.,Swift,C.,Grant,K.A.,Frost,J.A.,2003.FEMS Microbiol.Lett.229,237–241.Call,D.R.,Brockman,F.J.,Chandler,D.P.,2001.Int.J.Food Microbiol.67,71–80. Cao,Q.,Kim,M.,Klapperich,C.M.,2011.Biotechnol.J.6,177–184. Christopoulos,T.K.,1999.Anal.Chem.71,425R–438R.Crews,N.,Wittwer,C.,Gale,B.,2008a.Biomed.Microdev.10,187–195.Crews,N.,Wittwer,C.,Palais,R.,Gale,B.,b Chip8,919–924. Delamarche,E.,Bernard,A.,Schmid,H.,Bietsch,A.,Michel,B.,Biebuyck,H.,1998.J.Am.Chem.Soc.120,500–508.Delamarche,E.,Juncker,D.,Schmid,H.,2005.Adv.Mater.17,2911–2933.Doležel,J.,Bartos,J.,Voglmayr,H.,Greilhuber,J.,2003.Cytometry A51,127–128. Eddington,D.T.,Puccinelli,J.P.,Beebe,D.J.,2006.Sens.Actuators B114,170–172. Frydendahl,K.,Imberechts,H.,Lehmann,S.,2001.Mol.Cell.Probes15,151–160. Gervais,L.,Hitzbleck,M.,Delamarche,E.,2011.Biosens.Bioelectron.27,64–70. Juncker,D.,Schmid,H.,Drechsler,U.,Wolf,H.,Wolf,M.,Michel,B.,Rooij,N.,De-lamarche,E.,2002.Anal.Chem.74,6139–6144.Kargl,R.,Mohan,T.,Brac i c,M.,Kulterer,M.,Dolis k a,A.,Stana-Kleinschek,K.,Ri-bitsch,V.,ngmuir28,11440–11447.Karmali,M.A.,2004.Mol.Biotechnol.26,117–122.Kopp,M.U.,Mello,A.J.,Manz,A.,1998.Science280,1046–1048.Lee,D.-Y.,Shannon,K.,Beaudette,L.A.,2006.J.Microbiol.Method65,453–467. Li,S.,Fozdar,D.Y.,Ali,M.F.,Li,H.,Shao,D.,Vykoukal,D.M.,Vykoukal,J.,Floriano,P.N.,Olsen,M.,McDevitt,J.T.,Gascoyne,P.R.C.,Chen,S.,2006.J.Microelec-tromech.Syst.15,223–236.Mullis,K.B.,Falona,F.A.,1987.Methods Enzymol.155,335–350.Nakayama,T.,Kurosawa,K.,Furui,S.,Kerman,K.,Kobayashi,M.,Rao,S.R.,Yone-zawa,Y.,Nakano,K.,Hino,A.,Takamura,Y.,Tamiya,E.,2006.Anal.Bioanal.Chem.386,1327–1333.Nakayama,T.,Hiep,H.M.,Furui,S.,Yonezawa,Y.,Saito,M.,Takamura,Y.,Tamiya,E., 2010.Anal.Bioanal.Chem.396,457–464.Novo,P.,Prazeres,D.M.F.,Chu,V.,Conde,J.P.,b Chip11,4063–4071. Novo,P.,Volpetti,F.,Chu,V.,Conde,J.P.,b Chip13,641–645.Obeid,P.J.,Christopoulos,T.K.,2003.Anal.Chim.Acta494,1–9.Silkie,S.S.,Tolcher,M.P.,Nelson,K.L.,2008.J.Microbiol.Method72,275–281.Ståhl,M.,Kokotovic,B.,Hjulsager,C.K.,Breum,S.Ø.,Angen,Ø.,2011.Vet.Microbiol.151,307–314.Tachibana,H.,Saito,M.,Tsuji,K.,Yamanaka,K.,Hoa,L.Q.,Tamiya,E.,2015.Sens.Actuators B206,303–310.Yamanaka,K.,Saito,M.,Kondoh,K.,Hossain,M.M.,Koketsu,R.,Sasaki,T.,Nagatani, N.,Ikuta,K.,Tamiya,E.,2011.Analyst136,2064–2068.Zhu,Y.,Petkovic-Duran,K.,2010.Microﬂuid Nanoﬂuid8,275–282. Zimmermann,M.,Schmid,H.,Hunziker,P.,Delamarche,E.,b Chip7, 119–125.H.Tachibana et al./Biosensors and Bioelectronics74(2015)725–730 730。

牡荆素调控miR-219a-5p表达对LPS诱导的支气管上皮细胞凋亡和炎症反应的影响

牡荆素调控miR-219a-5p表达对LPS诱导的支气管上皮细胞凋亡和炎症反应的影响王伟平王君邓飞①赵彩杰韩景新②（唐山职业技术学院护理系，唐山063300）中图分类号R562.2文献标志码A文章编号1000-484X（2022）03-0324-04慢性气道炎症性疾病是多种细胞因子引起的慢性呼吸系统疾病，支气管上皮细胞在维持气道微环境稳态中起重要作用，支气管上皮细胞损伤与感染性疾病的发生密切相关［1-2］。

牡荆素是一种天然黄酮类化合物，可通过抗神经凋亡、调节炎症因子对神经系统起保护作用［3］。

牡荆素还可通过减少心肌细胞凋亡，降低炎症因子水平保护心肌炎症细胞免受CVB3病毒诱导的损伤［4］。

但牡荆素对支气管上皮细胞凋亡和炎症反应的影响及机制尚不清楚。

脂多糖（lipopolysaccharide，LPS）可诱导机体炎症反应，因此，本研究采用LPS刺激支气管上皮细胞，观察牡荆素对LPS诱导的支气管上皮细胞凋亡和炎症反应的影响及机制，为慢性气道炎症性疾病治疗提供新思路和新靶点。

1材料与方法1.1材料人支气管上皮细胞系16HBE购自上海酶研生物科技有限公司；RPMI1640培养基购自美国Gibco公司；LPS、牡荆素（纯度≥95%）购自美国Sigma公司；TUNEL检测试剂盒购自北京中山生物技术有限公司；凋亡检测试剂盒、RIPA蛋白裂解液购自上海碧云天生物技术有限公司；IL-6、IL-13、肿瘤坏死因子-α（TNF-α）ELISA试剂盒购自南京建成生物工程研究所；Trizol试剂、反转录试剂盒、SYBR Premix ExTaq TM试剂盒购自日本TaKaRa公司。

1.2方法1.2.1肺损伤小鼠模型构建15只C57BL/6健康雄性小鼠随机分为对照组、模型组、牡荆素低、中、高剂量组，每组3只。

所有小鼠腹腔注射10%水合氯醛进行麻醉，仰卧位固定于滴注板，吸除小鼠口咽部分泌物后将带针芯的静脉留置针插入气管内，立即拔出针芯，模型组小鼠向气管内快速滴注LPS （1000μg/ml，4mg/kg），对照组注入等量生理盐水，并立即注入空气0.5ml，牡荆素低、中、高剂量组先用LPS滴注，损伤模型成功建立后分别滴注1.5、3、6mg/kg牡荆素。

聚二甲基硅氧烷芯片自由酶反应器检测葡萄糖

聚二甲基硅氧烷芯片自由酶反应器检测葡萄糖作者：仲海燕周洁余晓冬陈洪渊【摘要】应用电泳中介微分析（EMMA）技术，构建聚二甲基硅氧烷（PDMS）芯片自由酶反应器，在线检测葡萄糖（Glu），在十字形的芯片通道上，采用自制的碳纤维微电极检测葡萄糖氧化酶（GOD）催化氧化Glu生成的H2O2，并对检测电位、GOD浓度、GOD进样时间、分离电压等参数进行了优化，测定了该自由酶反应器的线性范围和检出限，考察了其重现性及稳定性。

结果表明，此自由酶反应器制作方便，操作简单，重现性好，Glu浓度在0.1～20 mmol/L之间有较好的线性关系（r=0.997），检出限为19.8 μmol/L（S/N=3）。

【关键词】自由酶反应器；葡萄糖；电泳中介微分析；聚二甲基硅氧烷芯片The Key Lab of Analytical Chemistry for Life Science, Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093)Abstract Microfluidic enzyme based reactor could be used to detect the biomolecules with high sensitivity and selectivity. Based on the electrophoretic mediated microanalysis (EMMA) method, a new kind of free enzyme based poly(dimethylsiloxane) (PDMS) microchip was developed to detect glucose (Glu). On line catalysis reaction of Glu by glucose oxidase (GOD) was carriedout on the chip. The product H2O2 was detected using single carbon fibre cylindrical electrode. Factors influencing the separation and detection, such as detection potential, GOD concentration, GOD injection time and separation voltage, were investigated and optimized. Results showed that the peak current had a good linear relationship with Glu concentration in the range of 0.1－20 mmol/L (R=0.997). The detection limit of Glu was 19.8 μmol/L (S/N=3). In addition, the PDMS enzyme based reactor had long term stability and excellent reproducibility (RSD=2.02%, n=10). It was easy to fabricate and operate, which showed great potential application in bioanalysis.Keywords Free enzyme based microchip; Glucose; Electrophoretic mediated microanalysis; Poly(dimethylsiloxane)1 引言近年来，基于酶催化反应的微流控芯片备受关注。

11111人类 microRNA转录物的上游元件DOC

学校代码：1 0 0 6 2学号：2006062108本科毕业论文（设计）UNDERGRADUATE DISSERTATION论文题目：人类microRNA转录物的上游元件预测与分析TITLE The Prediction and Analysis of theUpstream Elements of MicroRNAin Human Genome院系生物医学工程学院专业生物医学工程年级 2006级学生姓名李娟指导教师王兆月副教授2010年6月目录中文摘要 (1)英文摘要 (2)1. 前言 (3)2. 研究对象和方法 (5)2.1 提取数据 (5)2.1.1 pre-miRNA获取、聚类和提取上游侧翼序列 (5)2.1.2 获取随机序列 (5)2.2 TSS预测 (6)2.3 TFBS预测 (7)2.4 CpG岛预测 (9)2.5 数据处理与统计 (9)3. 实验结果 (10)3.1 TSS结果 (10)3.2 TFBS结果 (12)3.3 CpG岛结果 (14)4. 讨论 (14)5. 结论 (17)致谢 (18)参考文献 (19)附件 (21)目的：microRNA是具有转录后调控作用的内源小分子RNA，约20~24nt（少数是20nt），其初级转录本的注释对于我们了解miRNA的生物功能及其调节靶位非常重要。

本论文预测microRNA的转录起始位点（TSS）、转录因子结合位点（TFBS）、CpG岛，对以上三方面进行统计分析，得到microRNA上游结构。

方法：从MirBase数据库中获取基因间隔区的miRNA共290个，按照其在基因组中相互之间的距离进行聚类，共分为0kb、1kb、3kb、5kb、10kb五类；使用biosmart和galaxy分别获取基因间的miRNA 上游序列和随机的基因间序列；使用the Eponine TSS prediction track of Ensembl、UCSC 的TFBS conserved、EMBOSS CpGplot分别对TSS、TFBS、CpG岛进行预测；使用SAS 9.2对预测结果进行频数统计和核密度估计。

pmirGLO vector

Promega Corporation2800 Woods Hollow Road Madison, WI 53711-5399USA Telephone 608-274-4330Toll Free 800-356-9526Fax 608-277-2516InternetPRODUCT USE LIMITATIONS, WARRANTY,DISCLAIMER Promega manufactures products for a number of intended uses. Please refer to the product label for the intended use statements for specific products.Promega products contain chemicals which may be harmful if misused. Due care should be exercised with all Promega products to prevent direct human contact.Each Promega product is shipped with documentation stating specifications and other technical information.Promega products are warranted to meet or exceed the stated specifications. Promega's sole obligation and the customer's sole remedy is limited to replace-ment of products free of charge in the event products fail to perform as warranted. Promega makes no other warranty of any kind whatsoever, and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WAR-RANTIES OF ANY KIND OR NATURE WHATSOEVER,DIRECTLY OR INDIRECTLY, EXPRESS OR IMPLIED,INCLUDING, WITHOUT LIMITATION, AS TO THESUITABILITY, PRODUCTIVITY, DURABILITY, FITNESS FOR A PARTICULAR PURPOSE OR USE, MER-CHANTABILITY, CONDITION, OR ANY OTHER MAT-TER WITH RESPECT TO PROMEGA PRODUCTS. In no event shall Promega be liable for claims for any other damages, whether direct, incidental, foresee-able, consequential, or special (including but not lim-ited to loss of use, revenue or profit), whether based upon warranty, contract, tort (including negligence) or strict liability arising in connection with the sale or the failure of Promega products to perform in accordance with the stated specifications.Part# 9PIE133Revised 10/09Part# 9PIE133Printed in USA Revised 10/09pmirGLO Dual-Luciferase miRNA Target Expression Vector:Cat.#Size E133020µgC a t .# E 1330 c o n t a i n s :P a r t N o .N a m e E133A pmirGLO Vector 20µg C838A Oligo Annealing Buffer1mlDescription: The pmirGLO Dual-Luciferase miRNA Target Expression Vector (a–e)is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites 3´ of the firefly luciferase gene (luc2). These target sites can be introduced by cloning putative miRNA binding sites alone, or the 3´ untranslated region (UTR) of a gene of interest, to study the influence of these sites on transcript stability and activity. Firefly luciferase is the primary reporter gene;reduced firefly luciferase expression indicates the binding of endogenous or introduced miRNAs to the cloned miRNA target sequence. This vector is based on Promega dual-luciferase technology, with firefly luciferase (luc2) used as the primary reporter to monitor mRNA regulation and Renilla luciferase (hRluc-neo ) acting as a control reporter for normalization and selection. This vector contains the following features:•Human phosphoglycerate kinase (PGK) promoter provides low translational expression, which is advantageous when reduction of signal is the desired response. The PGK promoter is a nonviral universal promoter, which functions across cell lines (yeast, rat, mouse and human).•Firefly luciferase reporter gene (luc2) inversely reports miRNA activity in mammalian cells.•Multiple cloning site (MCS) is located 3´ of the firefly luciferase reporter gene (luc2).•Humanized Renilla luciferase-neomycin resistance cassette (hRluc -neo) is used as a control reporter for normalization of gene expression and stable cell line selection.•Amp r gene allows bacterial selection for vector amplification.•SV40 late poly(A) signal sequence is positioned downstream of luc2to provide efficient transcription termination and mRNA polyadenylation.•Synthetic poly(A) signal/transcription stop site.Concentration: 1µg/µl in 10mM Tris-HCl, 1mM EDTA; final pH 7.4.GenBank ®Accession Number:FJ376737.Storage Conditions:See the storage temperature and expiration date on the Product Information Label.Functional AssaysIdentity Assay: The vector has been sequenced completely and has 100% identity with the published sequence availableat: ww w w .p r o m e g a .c o m /v e c t o r s /Restriction Digestion:The functional purity of this vector DNA is verified by complete digestion with restriction enzymes at the optimal temperature for 1 hour. Samples are examined by agarose gel electrophoresis, comparing cut and uncut vector DNA with marker DNA.Contaminant AssaysContaminating Nucleic Acids: RNA, single-stranded DNA and chromosomal DNA are not evident in specified quantities of this vector as determined by agarose gel electrophoresis.Nuclease Assay: Following incubation of 1µg of this vector in Restriction Enzyme Buffer at 37°C for 16–24 hours, no evidence of nuclease activity is detected by agarose gel electrophoresis.Physical Purity:A 260/A 280≥1.80, A 260/A 250≥1.05.© 2008, 2009 Promega Corporation. All Rights Reserved.Dual-Glo is a registered trademark of Promega Corporation. GeneClip and PureYield are trademarks of Promega Corporation.GenBank is a registered trademark of US Department of Health and Human Services.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.All specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.AF9PI E133 1009E133(a)READ THIS FIRST BEFORE OPENING PRODUCTThe sale of this product and its use by the purchaser are subject to the terms of a limited use label license, the full text of which is shippedwith this product and also available at: ww w w .p r o m e g a .c o m \L U L L . That text must be read by the purchaser prior to opening this product to determine whether the purchaser agrees that all use of the product shall be in accordance with the license terms. If the purchaser is not willing to accept the terms of the limited use label license, Promega is willing to accept the return of the unopened product and provide the purchaser with a full refund. However, if the product is opened for any reason, then the purchaser agrees to be bound by the terms of the limited use label license.(b)U.S. Pat. No. 5,670,356.(c)Australian Pat. No. 2001 285278 and other patents pending.(d)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673. A license (from Promega for research reagent products and from The Regents of the University of California for all other fields) is needed for any commercial sale of nucleic acid contained within or derived from this product.(e)Licensed from University of Georgia Research Foundation, Inc., under U.S. Pat. Nos. 5,292,658, 5,418,155, Canadian Pat. No.2,105,984 and related patents.S i g n e d b y :J. Stevens, Quality AssurancePromega Corporation2800 Woods Hollow Road·Madison, WI 53711-5399 U.S.A. Toll Free in the USA 800-356-9526 Telephone 608-274-4330 Features List and Map for the pmirGLO VectorSV40 late poly(A) signal106–327SV40 early enhancer/promotor426–844hRluc -neo fusion protein coding region 889–2664Synthetic polyadenylation signal 2728–2776β-lactamase (Amp r ) coding region3037–3897Col E1-derived plasmid origin of replication 4052–4088Human phosphoglycerate kinase promoter 5094–5609luc2reporter gene5645–7297Multiple cloning site (MCS, Figure 1)7306–7350I.Sample ProtocolA.Vector Cloning1.Design oligonucleotides: Order oligonucleotide pairs that contain the desired miRNAtarget region and, when annealed and ligated into the pmirGLO Vector, result in the miRNA target region in the correct 5´ to 3´ orientation. Insure that the overhangs created by oligonucleotide annealing are complementary to those generated by restric-tion enzyme digestion of the pmirGLO Vector in Step 2. Add an internal restriction site to your oligonucleotides for clone confirmation (e.g., NotI in Figure 3 creates a ~125bp insert when digested with NotI because of a NotI site at position 93 in the vector).2.Digest vector: Linearize the pmirGLO Vector with the appropriate restriction enzymes togenerate overhangs that are complementary to the annealed oligonucleotide overhangs.3.Anneal oligonucleotides: Dilute both oligonucleotides (supplied by user) to 1µg/µl.Combine 2µl of each oligonucleotide with 46µl of Oligo Annealing Buffer. Heat at 90°C for 3 minutes, then transfer to a 37°C water bath for 15 minutes. Use the annealed oligonucleotides immediately, or store at –20°C.B.Ligation and Transformation1.Dilute annealed oligonucleotides 1:10 in nuclease-free water to a final concentrationof 4ng/µl per oligonucleotide. Ligate 4ng of annealed oligonucleotides and 50ng of linearized vector using a standard ligation protocol. Transform ligated pmirGLO Vector using high-efficiency JM109 competent cells (e.g., Cat.# L2001).2.Select clones on ampicillin-containing plates, then select clones containing theoligonucleotides by digesting miniprep-purified DNA (e.g., purified using thePureYield™ Plasmid Miniprep System, Cat.# A1221) using the unique restriction site in the oligonucleotide pair. The purified plasmid DNA can be transfected directly or expanded to generate more DNA.Additional information about annealing, ligation, transformation and oligonucleotide design can be found in the GeneClip ™ U1 Hairpin Cloning Systems Technical Manual ,C.An Example of Detecting mi-R21 Activity Using the pmirGLO Vector:miR-21 ConstructAn overview describing the use of the pmirGLO Vector to interrogate endogenous mi-R21microRNA is shown in Figure 2.The presence of broadly endogenous microRNA mi-R21 was monitored in HeLa cells.Constructs contained either an exact match to the 21bp mi-R21 target sequence or a mismatched version of that target site (1) as well as PmeI, XbaI and NotI restriction sites (Figure 3; mismatched sequence is in italics). Twenty-four hours after transfection with the mi-R21 pmirGLO Vector constructs, cells were analyzed for luciferase activity using the Dual-Glo ®Luciferase Assay System (Cat.# E2920) and a MicroLumatPlus LB96V lumino-meter (Berthold). Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO Vector no-insert control. For each transfection, luciferase activity was averaged from six replicates.II.Reference1.Zeng, Y. and Cullen, B.R. (2003) Sequence requirements for micro RNA processing-neo SV40 late PGK ...GCAAG ATCGC CGTGT AATTC TAGTT GTTTA AACGA GCTCG CTAGCCTCGA GTCTA GAGTC GACCT GCAGG...PmeI DraI 5´EcoICRI Sac INheIXhoI SalI AccIXbaI3´F i g u r e 1. p m i rG L O V e c t o r m u l t i p l e c l o n i ng s i te .gene firefly luciferase translation In absence of mi-R21activity.proteinmRNA destablized;F i g u r e 2. M e c h a n i s m o f a c t i o n o f t h e p m i r G L O V e c t o r .5´ CTAGA TAGCTTATC TT CTGATGTTGA ACTA GCGGCCGC TA GTTT 3´XbaImi-R21 mismatch sense, PmeI and XbaImi-R21 antisense, PmeI and XbaImi-R21 sense, PmeI and XbaImi-R21 mismatch antisense, PmeI and XbaI5´ AAAC TA GCGGCCGC TAGT TCAACATCAG TCT GATAAGCTA T 3´5´ CTAGA TAGCTTATC AGA CTGATGTTGA ACTA GCGGCCGC TA GTTT 3´5´ AAAC TA GCGGCCGC TAGT TCAACATCAG AA GATAAGCTA T 3´PmeI NotI internal sitemi-R21 target sequence XbaIPmeINotI internal site mi-R21 target sequence F i g u r e 3. S a m p l e o l i g o n u c l e o t i d e s f o rm i -R 21.mismatchP e r c e n t f i r e f l y :R e n i l l a l u c i f e r a s e a c t i v i t y c o m p a r e d t o n o -i n s e r t c o n t r o lF i g u r e 4. N o r m a l i z e d l u c i f e r a s e a c t i v i t y u s i n g t h e p m i rG L O V e c t o r w i t h a n m i -R 21 t a r g e t s e q u e n c e .。

AP-1 Transcription Factor Complex Is a Target of Signals

DOI: 10.1126/science.281.5384.1860, 1860 (1998);281 Science , et al.Howard Y. Chang Adapter Protein Daxx Activation of Apoptosis Signal-Regulating Kinase 1 (ASK1) by theThis copy is for your personal, non-commercial use only.clicking here.colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to othershere.following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles): January 15, 2012 (this infomation is current as of The following resources related to this article are available online at/content/281/5384/1860.full.html version of this article at:including high-resolution figures, can be found in the online Updated information and services, /content/281/5384/1860.full.html#ref-list-1, 4 of which can be accessed free:cites 20 articles This article 386 article(s) on the ISI Web of Science cited by This article has been /content/281/5384/1860.full.html#related-urls 100 articles hosted by HighWire Press; see:cited by This article has been/cgi/collection/cell_biol Cell Biologysubject collections:This article appears in the following registered trademark of AAAS.is a Science 1998 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mActivation of Apoptosis Signal–Regulating Kinase 1(ASK1)by the Adapter Protein DaxxHoward Y.Chang,*Hideki Nishitoh,*Xiaolu Yang,†Hidenori Ichijo,‡David Baltimore ‡The Fas death receptor can activate the Jun NH 2-terminal kinase (JNK)pathway through the receptor-associated protein Daxx.Daxx was found to activate the JNK kinase kinase ASK1,and overexpression of a kinase-deﬁcient ASK1mutant inhibited Fas-and Daxx-induced apoptosis and JNK activation.Fas activation induced Daxx to interact with ASK1,which consequently relieved an inhibitory intramolecular interaction between the amino-and carboxyl-termini of ASK1,activating its kinase activity.The Daxx-ASK1connection completes a signaling pathway from a cell surface death receptor to kinase cascades that modulate nuclear transcription factors.Fas is a cell surface receptor that induces apoptosis upon oligomerization (1).Fas be-longs to a family of related death receptors,including the receptors for tumor necrosis factor–␣(TNF-␣)and the cytotoxic ligand TRAIL (1,2).Fas-induced apoptosis has a critical role in maintaining peripheral im-mune tolerance (1).Fas can activate two in-dependent signaling pathways.One well-characterized pathway involves the adapter protein FADD,which recruits procaspase-8and activates a protease cascade leading to apoptosis (1,3).The second pathway is me-diated by Daxx,which can enhance Fas-in-duced apoptosis by activating the JNK kinase cascade,culminating in the phosphorylation and activation of transcription factors such as c-Jun (4,5).Because Daxx might activate JNK through a mitogen-activated protein (MAP)kinase kinase kinase (MAP3K)(6–8),we focused on ASK1.It is a MAP3K that can activate apoptosis,is activated by TNF-␣,and the dominant negative form of which can block TNF-␣Ϫinduced death (8).Us-ing an immunoprecipitation (IP)-kinase as-say after expression in human embryonic kidney 293cells (8,9),we found that ASK1activity was potentiated by coex-pression with Daxx (Fig.1A).Another MAP3K that can activate the same kinase cascade,TAK1(6),was not activated by Daxx.The Daxx domain that encodes its JNK activation and apoptotic activities (amino acids 501to 625)and fragments incorporating it (4),but not other parts of Daxx,also increased ASK1activity (Fig.1B).These data implicate ASK1as a down-stream target of Daxx.Consistent with this notion,endogenous ASK1activity was ac-tivated rapidly by Fas cross-linking in a dose-dependent manner in Jurkat cells;lowbut detectable ASK1activation was evident 5min after Fas cross-linking (Fig.1C).To determine the functional role of ASK1in Daxx and Fas signaling,we tested the effect of altering ASK1activity on the apo-ptotic activities of Daxx and Fas (Fig.2).An activated deletion mutant of Daxx,DaxxC501,can induce cell death in a Fas-independent manner in 293cells but not in HeLa cells (4).However,coexpression of ASK1and DaxxC501in HeLa cells synergistically induced apoptosis (Fig.2A).A conser-vative point mutation in the ATP binding loop of ASK1(K709R)completely abro-gated cell killing (Fig.2A).ASK1(K709M),which has less residual kinase activity than ASK1(K709R),inhibited apoptosis by Fas and DaxxC501in a dose-dependent manner (Fig.2,B and C).ASK1(K709M)also in-hibited the ability of DaxxC501and Fas to activate JNK,whereas the caspase inhibitor crmA did not (Fig.2D).Collectively,these results imply a critical role for the ASK1kinase in JNK activation and apoptosis in-duced by Fas binding of Daxx.Because MAP3Ks such as Raf directly interact with upstream signaling proteins (5),we assayed physical interaction between Daxx and ASK1by coimmunoprecipitation from transfected 293T cells.Full-length hu-man Daxx specifically coimmunoprecipitated with ASK1(Fig.3A),indicating that these two proteins physically interact in mammali-H.Y.Chang and X.Yang,Department of Biology,Massachusetts Institute of Technology,Cambridge,MA 02138,USA.H.Nishitoh and H.Ichijo,Depart-ment of Biomaterials Science,Faculty of Dentistry,Tokyo Medical and Dental University,1-5-45Yushi-ma,Bunkyo-ku,Tokyo 113-8549,and Department of Biochemistry,Cancer Institute,Tokyo,Japanese Foun-dation for Cancer Research,1-37-1Kami-Ikebukuro,Toshima-ku,Tokyo 170,Japan.D.Baltimore,Depart-ment of Biology,Massachusetts Institute of Technol-ogy,Cambridge,MA 02138,and California Institute of Technology,Pasadena,CA 91125,USA.*These authors contributed equally to this work.†Present address:Department of Molecular and Cel-lular Engineering and Institute for Human Gene Ther-apy,University of Pennsylvania,Philadelphia,PA 19104,USA.‡To whom correspondence should beaddressed.tion of ASK1.(A )Daxx activates ASK1.pcDNA3-Myc-ASK1(0.5␮g)or pCS3-Myc-TAK1(0.5␮g)was cotransfected with pEBB-Daxx (1.5␮g)into 293cells (23).ASK1and TAK1were im-munoprecipitated by anti-Myc.The immune complex was incubated with GST-MKK6and GST-SAPK/p38␥,and the kinase activity was measured with the substrate ATF2(1–109)peptide.(Top)Phosphorylation of ATF2after in vitro kinase (IVK)assay.(Bottom)Immunoblotting (WB)of immunopre-cipitated Myc-ASK1and Myc-TAK1.Fold activation of ASK1and TAK1kinase activities is indicated below.Kinase activities relative to the amount of ASK1or TAK1proteins were calculated,and the activities are shown as fold activation relative to the activities of ASK1or TAK1from Daxx-negative cells.(B )ASK1activation by Daxx deletion mutants.pcDNA3-FLAG-ASK1(0.5␮g)and each Daxx mutant (1.5␮g)were cotransfected into 293cells (left)or HeLa cells (right),and ASK1was immuno-precipitated with anti-FLAG.The immune complex was incubated with GST-MKK6,and then the kinase activity was measured with the substrate GST-SAPK/p38␥(KN).The sequences incorporated in each Daxx construct are as follows:Daxx [amino acids (aa)1to 739],Daxx ⌬C (aa 1to 625),Daxx1–501(aa 1to 501),DaxxC501(aa 501to 739),Daxx501–625(aa 501to 625),DaxxC (aa 626to 739).(Top)Phosphorylation of GST-SAPK3/p38␥(KN).(Bottom)Expression of FLAG-ASK1.Fold activation of ASK1kinase activities is indicated below.(C )Fas-induced activation of ASK1.Jurkat cells (5ϫ106)were treated with CH-11anti-human Fas (MBL,Nagoya,Japan)(100ng/ml)for the indicated times (left)or with the indicated concentrations for 30min (right).The endogenous ASK1was immunoprecipitated with anti-ASK1(DAV)(24),and the ASK1kinase activity was measured as described in (B).18SEPTEMBER 1998VOL 281SCIENCE 1860 o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o man cells.FLAG-tagged FADD was not copre-cipitated by ASK1under the same condition.To evaluate the observed Daxx-ASK1inter-action under more physiological conditions,we examined the association of endogenous Daxx and ASK1by coimmunoprecipitationin L/Fas cells,a mouse fibroblast cell line expressing murine Fas (4).Daxx became as-sociated with ASK1after Fas ligation byanFig.2.Role of ASK1in Daxx-and Fas-induced apoptosis and signaling.(A )Synthetic lethality of ASK1with DaxxC501.HeLa cells were transfected with 0.5␮g of pcDNA3-ASK1or pcDNA3-ASK1(K709R)(23)and 1.0␮g of pEBB-DaxxC501along with 0.5␮g of pCMV-lacZ reporter by calcium phosphate precipitation.Total amount of transfected DNA was made con-stant by adding vector DNA.Twenty-four hours after transfection,the cells were stained with X-Gal and scored for apoptotic morphology (4).Speciﬁc apoptosis was calculated as the percentage of apoptotic blue cells in each experimental condition minus the percentage of apoptotic blue cells (ϳ5%)in parallel vector-transfected cells.The data shown are the mean ϮSD of two to four independent experiments.(B )Inhibition of Fas-induced apopto-sis by ASK1(K709M).HeLa cells were transfected with 0.5␮g of pEBB-Fas and pCMV-lacZ and the indicated amount (in micrograms)of ASK1(K709M).Jo2antibody (12.5ng/ml)was added 16hours later.X-Gal staining was done at 24hours after transfection.Speciﬁc apoptosis was calculated as in (A).(C )Inhibition of DaxxC501-induced apoptosis by ASK1(K709M).pEBB-DaxxC501(2.0␮g)and the indicated amount (in micro-grams)of pcDNA3-ASK1(K709M)were cotransfected with 0.5␮g of pCMV-lacZ in 293cells.Twenty hours after transfection the cells were stained with X-Gal and speciﬁc apoptosis scored as in (A).(D )Inhibition of DaxxC501-and Fas-induced JNK activation by ASK1(K709M).Expression constructs for each indicated protein (1.0␮g)were cotransfected with 1.0␮g of pCMV-FLAG-JNK1in 293cells.Cells in lanes 7to 10were treated with Fas mAb (Jo2,0.5␮g/ml)for 30min before assay.JNK1was immunoprecipitated with anti-FLAG,and in vitro kinase assay with 1␮g of GST-cJun(1–79)was performed as described (4).(Top)Phospho-rylation of GST-cJun(1–79).(Bottom)Immunoblotting of immunoprecipitated FLAG-JNK1.Fig.3.Daxx interacts with ASK1.(A )As-sociation of Daxx and ASK1in 293T cells.Four micrograms of pRK5-FLAG-hDaxx,pcDNA3,or pcDNA3-Myc-ASK1(23)were cotransfected with 2.0␮g of pRK5-crmA in 293T cells by calcium phosphate precipitation.(CrmA prevents the induction of apoptosis and allows the accumulation of tranfected proteins.)After 24hours,cells were extracted in IP-lysis buffer (25),immunoprecipitated with anti-Myc coupled to agarose beads (Santa Cruz)for 3hours at 4°C,and washed three times with 500␮l of IP-lysis buffer.The IP samples as well as portions of the extracts (10%of IP input)were resolved by SDS-PAGE and immunoblotted with M2anti-FLAG (Kodak)as described (4).(B )Fas-induced interaction of Daxx and ASK1.(Left)Identiﬁcation of endogenous Daxx protein in L/Fas cells.Lysate from 3ϫ107L/Fas cells was immunoprecipitated with poly-clonal anti-Daxx (DSS)(24)in the absence or presence of blocking peptide (5␮g/ml)and immunoblotted with DSS.(Right)L/Fas cells (3ϫ107)were treated with mAb Jo2(immunoglobulin G,100ng/ml)(26)for the indicated times (lanes 4to 7)or left untreated (lane 3).Cell lysates were immunoprecipitated with anti-ASK1(lanes 3to 7)and immunoblotted with DSS (top)(25).Equivalent IP of ASK1was conﬁrmed by immunoblotting of the same membrane with anti-ASK1(bottom).(C )Recruitment of endogenous ASK1to Fas.L/Fas cells (1.5ϫ107)were incubated in the presence or absence of Jo2(2␮g/ml)for 30min at 37°C.Cells were washed once with ice-cold PBS and lysed in IP-lysis buffer.The postnuclear supernatant was immunoprecipitated with 40␮l of protein A/G-agarose (Santa Cruz)for 3hours at 4°C.In samples that were not ﬁrst incubated with Jo2,isotype-matched control antibody (2␮g/ml,lane 1)or Jo2(lane 2)were added after cell lysis.Immunopre-cipitates were washed ﬁve times with lysis buffer,resolved by 7.5%SDS-PAGE,and immunoblotted for ASK1with the DAV antiserum.Positions of molecular size standards (in kilodaltons)are shown on the left.(D )Requirement of Daxx for Fas-ASK1interaction.Two micrograms of pcDNA3-ASK1(K709R),1.0␮g of pCI-AU1-hFas,and 4.0␮g of pRK5-hDaxxC (23)in the indicated combinations were transfected into 293T cells along with 2.0␮g of pRK5-crmA and vector DNA as needed to equalize total DNA.Transfected cells were extracted,immunoprecipitated with anti-AU1(Babco)and protein A/G-agarose (Santa Cruz),and immunoblotted for HA-ASK1as in (A).(E )Schematic diagram of ASK1mutants.Amino acid number of domain boundaries is indicated.pcDNA3-⌬N,⌬C,and kinase each contain a COOH-terminal hemagglutinin (HA)epitope tag.pcDNA3-ASKN contains an NH 2-terminal Myc epitope tag (23).(F )Daxx interacts with the NH 2-terminus of ASK1.Four micrograms of each ASK1mutant was cotransfected with 4.0␮g of pRK5-FLAG-hDaxx and 2.0␮g of pRK5-crmA in 293T cells.Samples:ASK1(lanes 1and 5);⌬N (lanes 2and 6);⌬C (lanes 3and 7);kinase (lanes 4and 8).Twenty-four hours after transfection cells were extracted in IP-lysis buffer and immunoprecipitated with M2anti-FLAG coupled to agarose beads (Kodak).IP samples and extract aliquots were immunoblotted by anti-HA as in (A).Positions of molecular size standards (in kilodaltons)are shown on theright. SCIENCE VOL 28118SEPTEMBER 19981861o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o magonistic monoclonal antibody (mAb);this interaction peaked after 15min and decreased thereafter (Fig.3B).The Daxx-ASK1inter-action raised the possibility that ASK1may interact indirectly with Fas through Daxx.In L/Fas cells,the endogenous ASK1was spe-cifically coimmunoprecipitated with Fas after mAb cross-linking (Fig.3C,lane 3),indicat-ing that ASK1does interact with Fas and therefore may be a component of the Fas receptor signaling complex.In contrast,ad-dition of mAb to Fas after cell lysis,which immunoprecipitates monomeric Fas (10),did not coprecipitate ASK1(Fig.3C,lane 2).The Fas-ASK1interaction is apparently mediated by Daxx because coexpression of DaxxC,the COOH-terminal 112amino acid Fas-binding domain of Daxx,blocked the Fas-ASK1in-teraction,presumably by competing out en-dogenous Daxx (Fig.3D,lane 3).The ability of DaxxC to block ASK1recruitment to Fas can explain the documented dominant nega-tive effects of DaxxC on both Fas-induced apoptosis and JNK activation (4).In the yeast two-hybrid system,ASK1interacted with Daxx but not with Fas (11),suggesting that Daxx interacts directly with ASK1and bridg-es ASK1and Fas.Deletion mutagenesis showed that the NH 2-terminal 648amino ac-ids of ASK1,termed ASKN,could interactwith Daxx (Fig.3E and F,lane 7),whereas other parts of ASK1could not interact.Deletion of the NH 2-terminal 648amino acids of ASK1,forming ASK1⌬N,caused the constitutive activation of kinase activity (12)as it does in other MAP3Ks (6).Purified recombinant glutathione S-transferase (GST)–ASKN inhibited the in vitro kinase activity of ASK1but not ASK1⌬N immunoprecipitated from cells (Fig.4A),suggesting that one or more interacting cellular factors regulate ASKN autoinhibition.ASK1⌬N exhibited constitutive cell death activity in HeLa cells in the absence of added Daxx (Fig.4B).Apoptosis induced by ASK1⌬N was quanti-tatively similar to that induced by ASK1plus DaxxC501and was not enhanced by coex-pression with DaxxC501(Fig.4B).These results indicate that an activated allele of ASK1functions as a genetic bypass of Daxx and suggests that with regard to ASK1acti-vation,the function of Daxx is to relieve the inhibition caused by the NH 2-terminal regu-latory domain.We tested this model directly by in vivo interaction assays.ASKN interact-ed with Daxx (Fig.4C,lane 2).It also spe-cifically coimmunoprecipitated ASK1⌬N (Fig.4C,lane 4),implying an intramolecular interaction in full-length ASK1.Importantly,when an excess of Daxx was coexpressedwith ASKN and ASK1⌬N,ASKN associated with Daxx but not ASK1⌬N (Fig.4C,lane 6).This supports a model whereby Daxx activates ASK1activity by displacing an in-hibitory intramolecular interaction between the NH 2-and COOH-termini of the kinase and “opening up”the kinase into an active conformation.In support of this model,ASKN can inhibit the constitutive apoptotic activity of ASK1⌬N in trans,and this inhibi-tion is fully reversed by the coexpression of Daxx (Fig.4D).The present results suggest a Fas-Daxx-ASK1axis in activating JNK and p38MAP kinase cascades.The mechanism by which ASK1is activated by Daxx is similar to that described for the activation of Byr2,a MAP3K in the Schizosaccharomyces pombe mating pheromone pathway,by its activators Ste4and Shk1(13).Fas activation has been reported to activate JNK by caspase-depen-dent (14)and -independent pathways (4,15).During apoptosis,caspases can cleave and activate PAK2and MEKK (16,17),two kinases that can activate the JNK pathway;JNK activation in this context is believed to effect morphologic changes associated with apoptosis (16).The Daxx-ASK1connection provides a mechanism for caspase-indepen-dent activation of JNK by Fas and perhaps other stimuli.In mice deficient for JNK3,hippocampal neurons are protected from ap-optosis after excitotoxic injury,illustrating that in certain circumstances JNK is essential for the apoptotic program (18).In this study,we have used several tumor-derived cell lines where JNK activation by the Fas-Daxx-ASK1axis led to apoptosis.Because FADD -deficient embryonic fibroblasts and T cells are blocked for Fas-induced apoptosis (19),at least in these cells Daxx does not provide an independent death pathway.The physiologic role of the Daxx-ASK1axis and its cell spec-ificity in vivo remain to be addressed.References and Notes1.S.Nagata,Cell 88,355(1997);A.K.Abbas,ibid.84,655(1996).2.P.Golstein,Curr.Biol.7,R750(1997).3.M.P.Boldin,T.M.Goncharov,Y.V.Goltsev,D.Wallach,Cell 85,803(1996);M.Muzio,et al.,ibid.,p.817.4.X.Yang,R.Khosravi-Far,H.Y.Chang,D.Baltimore,ibid.89,1067(1997).5.J.M.Kyriakis and J.Avruch,BioEssay 18,567(1996);C.J.Marshall,Cell 80,279(1995).6.K.Yamaguchi et al.,Science 270,2008(1995).7.L.A.Tibbles et al.,EMBO J.15,7026(1996).8.H.Ichijo et al.,Science 275,90(1997).9.293and HeLa cells were maintained in Dulbecco’s minimum essential medium (DMEM)supplemented with 10%fetal bovine serum (FBS),glucose (4.5␮g/ml),and penicillin (100U/ml)and transfected with Tfx-50(Promega).Jurkat cells were cultured in RPMI 1640medium containing 10%FBS and antibi-otics in an atmosphere of 5%CO 2at 37°C.SAPK3/p38␥and ATF2peptide (1–109)were provided by M.Goedert and Z.Yao,respectively.Cells were extracted and immunoprecipitated with Myc mAb (Ab-1,Calbiochem),FLAG mAb (M2,Kodak),or antiserum to ASK1(DAV)(19)with protein G(forFig.4.Mechanism of Daxx activation of ASK1.(A )ASKN inhibition of ASK1activity in vitro.pcDNA3-ASK1-HA orpcDNA3-ASK1⌬N-HA were transfected into 293cells and immunoprecipitated with anti-HA (12CA5)and protein A–Sepharose.Equalized input kinase activities were incubated with the indicated amount of GST or GST-ASKN for 60min at 4°C,and subjected to the immune complex kinase assay as described in Fig.1B.G-ASKN,GST-ASKN.(B )Constitutive apoptotic activity of ASK1⌬N.HeLa cells were transfected with 0.5␮g of each indicated ASK1mutant,1.0␮g of pEBB or pEBB-DaxxC501,and 0.5␮g of pCMV-lacZ reporter.Twenty-four after transfection,cells were stained with X-Gal and scored for speciﬁc apoptosis as in Fig.2A.(C )Daxx releases the COOH-terminus of ASK1from the NH 2-terminus of ASK1.293T cells were transfected with pcDNA3-Myc-ASKN,pcDNA3-ASK1⌬N,and pRK5-FLAG-hDaxx as indicated along with 2.0␮g of pRK5-crmA and vector DNA as needed.Two micrograms of each indicated DNA was transfected in lanes 1to 4;in lanes 5and 6,0.5␮g of ASKN,2.0␮g of ASK1⌬N,and 4.0␮g of Daxx were transfected.Transfected cells were extracted,immunoprecipitated with anti-Myc coupled to agarose beads,and immunoblotted with anti-HA and anti-FLAG as in Fig.3A.(D )One microgram each of pcDNA3-ASK1⌬N,pcDNA3-ASKN,and pRK5-FLAG-hDaxx was cotransfected as indicated with 0.5␮g of pCMV-lacZ and vector DNA as needed in HeLa cells.Twenty-four hours after transfection cells were stained with X-Gal and scored for speciﬁc apoptosis as in Fig.2A.18SEPTEMBER 1998VOL 281SCIENCE 1862 o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mAb-1or M2)–or protein A (for DAV)–Sepharose.Immune complex assays were performed essential-ly as described (12).Phosphorylation of ATF2pep-tide or GST-SAPK3/p38␥was analyzed by a Fuji BAS2000image analyzer.ASK1or TAK1protein was detected by immunoblotting and enhanced chemiluminescence (ECL),which in exposures less than 10min did not detect 32P radioactivity from kinase autophosphorylation.Protein levels from immunoblot were quantiﬁed by densitometry (Quantity One program,pdi).10.F.C.Kischkel et al .,EMBO J.14,5579(1995).11.EGY48yeast strain was tranformed with EG202-ASK1(K709R),pJG4-5vector,pJG4-5-mFas(192–295),or pJG4-5-mDaxx,and JK101reporter plasmids,and quantitative liquid ␤-galactosidase (␤-Gal)assay was performed (4).Relative ␤-Gal units ϮSD for ASK1(KR)alone,2.4Ϯ0.2;ASK1(KR)plus Daxx,119Ϯ8.8;ASK1(KR)plus Fas,1.8Ϯ0.4.12.M.Saitoh et al .,EMBO J.17,2596(1998).13.H.Tu,M.Barr,D.L.Dong,M.Wigler,Mol.Cell.Biol.17,5876(1997).14.M.A.Cahill et al.,Oncogene 13,2087(1996);F.Toyoshima,T.Moriguchi,E.Nishida.J.Cell Biol.139,1005(1997);S.Huang et al.,Immunity 6,739(1997).15.E.Goillot et al .,Proc.Natl.Acad.Sci.U.S.A.94,3302(1997);H.Wajant et al .,Curr.Biol.8,113(1998).16.T.Rudel and G.M.Bokoch,Science 276,1571(1997);N.Lee et al .,Proc.Natl.Acad.Sci.U.S.A.94,13642(1997).17.M.H.Cardone,G.S.Salvesen,C.Widmann,G.John-son,S.M.Frisch,Cell 90,315(1997).18.D.D.Yang et al .,Nature 389,865(1997).19.W.-C.Yeh,et al .Science 279,1954(1998);J.Zhang,D.Cado,A.Chen,N.H.Kabra,A.Winoto,Nature 392,296(1998).20.H.Hsu,J.Xiong,D.V.Goeddel,Cell 81,495(1995).21.K.Tobiume et al .,mun.239,905(1997).22.Abbreviations for the amino acid residues are as follows:A,Ala;C,Cys;D,Asp;E,Glu;F,Phe;G,Gly;H,His;I,Ile;K,Lys;L,Leu;M,Met;N,Asn;P,Pro;Q,Gln;R,Arg;S,Ser;T,Thr;V,Val;W,Trp;and Y,Tyr.23.pEBB-Daxx (4),Daxx mutants (4),pEBB-Fas (4),pcDNA3-ASK1(8),pcDNA3-ASK1(K709R)(8),Myc-TAK1(6),pCMV-FLAG-JNK1(4),pRK5-crmA (20),EG202-ASK1(K709R)(12),and pJG4-5-mDaxx (4)were as described.ASK1⌬N,⌬C,kinase,FLAG-ASK1,Myc-ASK1,ASK1(K709M)-HA,and Myc-ASKN were constructed in pcDNA3(Invitrogen)by polymerase chain reaction (PCR).FLAG-tagged human Daxx and hDaxxC were derived from EST clone AA085057and constructed in pRK5(20)by PCR.pCI-AU1-hFas was constructed by J.Wang and M.J.Lenardo.The plas-mids of GST–human MKK6,GST SAPK3/p38␥(KN),and GST-ASKN for bacterial fusion protein were con-structed in pGEX-4T-1(Pharmacia Biotech)by PCR.24.Antiserum to ASK1(DAV)was raised to the peptide sequence DAVATSGVSTLSSTVSHDSQ,amino acids 1217to 1236in human ASK1,as described (21).Rabbit polyclonal antibody to mouse Daxx (DSS)was raised against the peptide sequence DSSTRVDSP-SHELVTSSLC (amino acids 680to 698)(22).25.293T cells (2ϫ106)[grown in DMEM supplemented with 10%FBS,penicillin-streptomycin (100U/ml),and glutamine (1mM)]were plated onto a 60-mm dish the day before transfection.Twenty-four hours after transfection,cells were washed once in ice-cold phosphate-buffered saline (PBS)and lysed in 300␮l of IP-lysis buffer [50mM Hepes (pH 7.4),1%NP-40,150mM NaCl,10%glycerol,1mM EDTA,2mM dithiothreitol]supplemented with 1mM phenyl-methylsulfonyl ﬂuoride and 1%aprotinin.Extract (50␮l)was diluted in IP-lysis buffer (500␮l)and immu-noprecipitated with antibody reagents as described in the ﬁgure legends.In Fig.3B,L/Fas cells were lysed in 1ml of lysis buffer.Cell lysates were immunoprecipi-tated with antiserum to ASK1through use of protein A–Sepharose.The beads were washed twice with the washing buffer,separated by SDS—polyacrylamide gel electrophoresis (PAGE),and immunoblotted with anti-Daxx (DSS).26.J.Ogasawara et al .,Nature 364,806(1993).27.We are grateful to P.Svec for technical support.We thank S.Nagata,K.Matsumoto,J.Wang,M.J.Le-nardo,D.V.Goeddel,M.Goeddert,and Z.Yao for reagents,and A.Hoffmann for valuable advice and critical review of the manuscript.H.I.thanks K.Miya-zono for valuable discussion.H.Y.C.is supported by the Medical Scientist Training Program at Harvard Medical School.H.I.is supported by Grants-in-Aid forscientiﬁc research from the Ministry of Education,Science,and Culture of Japan.X.Y.is a fellow of the Leukemia Society of America.Supported by NIH grant CA51462.16March 1998;accepted 13August 1998Promotion of Dendritic Growth by CPG15,an Activity-InducedSignaling MoleculeElly Nedivi,*†Gang-Yi Wu,Hollis T.ClineActivity-independent and activity-dependent mechanisms work in concert to regulate neuronal growth,ensuring the formation of accurate synaptic con-nections.CPG15,a protein regulated by synaptic activity,functions as a cell-surface growth-promoting molecule in vivo.In Xenopus laevis ,CPG15enhanced dendritic arbor growth in projection neurons,with no effect on interneurons.CPG15controlled growth of neighboring neurons through an intercellular sig-naling mechanism that requires its glycosylphosphatidylinositol link.CPG15may represent a new class of activity-regulated,membrane-bound,growth-promoting proteins that permit exquisite spatial and temporal control of neu-ronal structure.The cpg15gene was identified in a forward genetic approach designed to isolate activity-regulated genes that mediate synaptic plasticity (1).In the adult rat,cpg15is induced in the brain by kainate (KA)and in visual cortex by light (2).During development,cpg15expres-sion is correlated with times of afferent in-growth,dendritic elaboration,and synaptogen-esis (2).Sequence analysis predicts a small,secreted protein (2)that is membrane-bound by a glycosylphosphatidylinositol (GPI)linkage (3).Antiserum generated against bacterially ex-pressed rat CPG15recognizes a protein from rat brain dentate gyrus extracts (Fig.1A)(4)of the size predicted by sequence analysis.A pro-tein of similar size is induced in Xenopus laevis after KA injections into the brain ventricle (Fig.1A)(5).In situ hybridizations using a partial clone of Xenopus cpg15indicate that the CPG15mRNA is expressed in retinal ganglion cells and in differentiated neurons throughout the central nervous system (CNS)of stage-47tadpoles (6).Xenopus CPG15protein is present in neurons and axons throughout the CNS (7,8).In the optic tectum,differentiated neurons label in a honeycomb pattern similar to N-CAM (neural cell adhesion molecule)and other cell-surface antigens,while cells in the proliferativezone have undetectable levels of CPG15(Fig.1C).To investigate the cellular function of CPG15,we used a recombinant vaccinia virus (VV)to express CPG15in optic tectal cells of albino Xenopus tadpoles (9,10).Tadpoles were infected by ventricular injection with VV car-rying rat cpg15and ␤-galactosidase (␤-gal)cDNAs in a dual promoter vector,or with a control virus containing only the ␤-gal cDNA (11).Two days after viral infection and approx-imately 24hours after the beginning of expres-sion of foreign protein (9),single tectal cells were labeled with DiI (10,12).Confocal imag-es through the entire structure of each neuron were collected at 24-hour intervals over a peri-od of 3days,and three-dimensional (3D)im-ages were reconstructed from this (13).The most prominent effect of CPG15on the morphology of tectal projection neurons was that the dendritic arbors of neurons from CPG15VV-infected animals increased their total dendritic branch length (TDBL)and be-came more complex than arbors of neurons from ␤-gal–infected or uninfected animals (Fig.2)(14).This effect was quantified as an increase in averaged TDBL (Fig.3A)and by Sholl analysis (Fig.3B).We measured the distribution of dendritic arbor sizes,expressed as TDBL,within the population of neurons from CPG15VV-infect-ed animals and from control animals (Fig.3C).All three populations of neurons showed a gradual shift toward larger TDBLs as their dendritic arbors grow.The shift toward larger TDBLs was greatest in neurons from CPG15VV-infected animals.This analysis also demonstrates the presence of a subpopulationCold Spring Harbor Laboratory,1Bungtown Road,Cold Spring Harbor,NY 11724,USA.*To whom correspondence should be addressed.E-mail:nedivi@†Present address:Department of Brain and Cognitive Sciences and Center for Learning and Memory,Mas-sachusetts Institute of Technology,Cambridge,MA 02139,USA. SCIENCE VOL 28118SEPTEMBER 19981863o n J a n u a r y 15, 2012w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

智能手机成像的晶片上基于逆转录环介导等温扩增(RT-LAMP讲解

智能手机成像的晶片上基于逆转录环介导等温扩增ＧｒｅｇｏｒｙＬ．Ｄａｍｈｏｒｓｔ(RT-LAMP１，２，ＣａｒｌｏｓＤｕａｒｔｅ-Ｇｕｅｖａｒａ技术的全血中２，３，ＷｅｉｌｉＣｈｅｎ２，３，ＴａｎｍａｙＧｈｏｎｇｅHIV-1１，２，ＢｒｉａｎＴ．Ｃｕｎｎｉｎｇｈａｍ检测１，２，３，ＲａｓｈｉｄＢａｓｈｉｒ１，２，３*摘要：期临床护理来说是一个必不可少的工具。

然而，考虑到病毒载病毒载量测量对于人类免疫缺陷病毒(HIV阳性患者长人数已达到3690万[1]。

从HIV 大流行的出现到现在的近量测量所需的仪器体积、成本和操作的复杂性，在医疗基础设四十年间，抗逆转录病毒疗法已经将HIV 感染从一个“死刑判决”转变成一种可控的慢性疾病：若HIV 感染能够普及标准的病毒载量测量仪器是较为困难的。

为提高该检测方施较差的偏远地区(尤其是在被HIV 感染人群比例较高的地区被有效控制，则其对患者预期寿命的影响很小[2]。

从人法的普及性，人们已经开始开发可以进行即时检测的病毒载量口层面来看，无论是新感染病例数还是母婴传播病例数，或是与HIV 有关的死亡率都在下降[2]。

然而，在HIV 常易于操作等多种实际要求。

本文通过运用微流体和微型硅晶片检测平台，然而尚没有解决办法能够同时满足低成本、便携、规处理中缺少可普及的、适当的诊断技术来指导治疗仍平台，对经过最低程度处理的含有然是数以百万计的HIV 阳性患者，尤其是在发展较为落录环介导等温扩增HIV 的全血样本进行了逆转后、医疗资源稀缺地区的患者，在接受标准化治疗中面测。

集成实验检测结果表明，一滴约(RT-LAMP，并利用智能手机进行了荧光检临的最大障碍。

的CD4+ T淋巴细胞计数和血浆中病毒载量检测是HIV 每微升全血样品中只有3个病毒依然可以通过670RT-LAMP 个病毒。

该技术在数字化技术被检测到，这相当于60 nL的反应液滴中仅有治疗中两个核心诊断方法。

这两个指标对每一个感染患方法上具有重要意义，扩展该技术能够实现对RT-LAMP者治疗的启动和治疗方案的确立起到非常重要的指导作临床护理中采集指血进行病毒载量检测。

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2000;60:5012-5016. Published online September 1, 2000.Cancer ResGeorge I. Salti, Taline Manougian, Miguel Farolan, et al.in Intermediate-thickness Cutaneous Malignant MelanomaMicropthalmia Transcription Factor: A New Prognostic MarkerUpdated Version/content/60/18/5012Access the most recent version of this article at:Cited Articles/content/60/18/5012.full.html#ref-list-1This article cites 14 articles, 8 of which you can access for free at:Citing Articles/content/60/18/5012.full.html#related-urls This article has been cited by 18 HighWire-hosted articles. Access the articles at:E-mail alerts related to this article or journal.Sign up to receive free email-alerts Subscriptions Reprints and .pubs@ Department at To order reprints of this article or to subscribe to the journal, contact the AACR Publications Permissions.permissions@ Department at To request permission to re-use all or part of this article, contact the AACR Publications[CANCER RESEARCH60,5012–5016,September15,2000]Advances in BriefMicropthalmia Transcription Factor:A New Prognostic Marker inIntermediate-thickness Cutaneous Malignant Melanoma1George I.Salti,2Taline Manougian,Miguel Farolan,Anne Shilkaitis,Dibyen Majumdar,and Tapas K.Das Gupta Departments of Surgical Oncology[G.I.S.,T.M.,A.S.,T.K.D.G.]and Pathology[M.F.],College of Medicine,and Department of Mathematics,Statistics,and Computer Sciences[D.M.],University of Illinois at Chicago,Chicago,Illinois60612AbstractMicropthalmia transcription factor(Mitf)is involved in melanocyte development and differentiation.The current study was undertaken to determine whether there is a relationship between Mitf expression and survival in patients with intermediate-thickness(1.0–4.0mm)melanoma. Original paraffin blocks or slides of the primary tumor were accessible in 63such patients.Mitf expression was evaluated by immunocytochemistry and analyzed visually.Slides were graded as follows according to the percentage of cells whose nuclei stained positive for Mitf:(a)0,0%;(b)؉1,1–25%;(c)؉2,26–50%;(d)؉3,51–75%;and(e)؉4,>75%.Median follow-up was50months.Mean thickness was2.2؎0.7mm.Mean overall survival was171.90؎13.12months.Mean disease-free survival was 168.53؎13.96months.Fifty-two melanomas(82.5%)stained positive for Mitf.By univariate analysis,mean overall survival and disease-free sur-vival in patients whose melanomas did not express Mitf were 80.89؎17.98months(median,51months)and71.36؎19.87months (median,40months),respectively.This compares with187.90؎13.41 months(median,not reached)and186.78؎13.84months(median,not reached),respectively,for patients whose melanomas expressed Mitf (P؍0.0086and P؍0.0054).These findings persisted in multivariate analysis.In addition,patients with>50%Mitf expression had signifi-cantly fewer nodal metastases after node dissection than patients with <50%Mitf expression(P؍0.04).Our data suggest that Mitf may be a new molecular prognostic marker in patients with intermediate-thickness melanoma.IntroductionThe incidence of cutaneous malignant melanoma continues to rise. It is estimated that in the year2000,47,700new cases will be diagnosed,with7,700deaths(1).The prognosis of primary cutaneous melanoma is usually estimated by several histopathological criteria, such as tumor thickness,ulceration,levels of invasion,and mitoses/ mm2(2).However,limitations in the accuracy of these prognostic markers have led investigators to search for additional clues in the microenvironment in primary cutaneous melanomas.Notable cellular and molecular markers include expression of various types of inte-grins(3,4).Although these markers have helped define several subsets of patients with different natural histories,the search contin-ues for additional prognostic markers.A number of genetic and cell biological models indicate that pigment cell development and function depend on both cell-extrin-sic and-intrinsic factors.Although how these factors are function-ally integrated is poorly understood,it is generally agreed that the basic helix-loop-helix-zipper Mitf3relates to the downstream targets responsible for cell proliferation,fate determinism,and melanin biosynthesis(5,6).Mutations in Mitf are found from fishes to man.In fact,Mitf is critical not only for neural crest-derived melanocytes of the integument and inner ear(7)but also for the cells of the retinal pigment layer,which develop from the neural epithelium of the optic cup.Mouse mutations at the micropthalmia locus(mi)are characterized by small nonpigmented eyes,lack of integumentary melanocytes,and deficiencies in the inner ears.Also,these mutations are associated with a deficiency in mast cells and osteoclasts,resulting in a condition similar to osteopetrosis in humans.In humans,Mitf mutation is characterized by hearing deficiencies,white forelock,and ocular anomalies(Waardenburg syndrome IIA;Ref.8).It is believed that Mitf can induce pigment cell-specific transactivation of tyrosinase and tyrosinase-related protein1(9–11).In addition,␣-MSH has been shown to up-regulate the pigment gene expression via a cyclic AMP-mediated signal transduction pathway that stimulates the expression of Mitf(12).Moreover,it was reported that the c-Kit signaling pathway in melanocytes targets transcription factor for micropthalmia simultaneously for short-lived activation and net degradation(13). Recently,King et al.(14)reported that Mitf is a sensitive and specific melanocyte marker for melanoma diagnosis.Because Mitf appears to be involved in melanocyte development and differentiation,we hypothesized that its expression correlates with survival in patients with intermediate-thickness(1–4mm)melanoma. Our findings suggest that Mitf expression in the primary tumor correlates with patient survival.Materials and MethodsPatients and Tumor Specimens.We analyzed the primary lesions of63 patients with intermediate-thickness(1.0–4.0mm)malignant melanomas treated in the Department of Surgical Oncology at the University of Illinois at Chicago.The slides and blocks were obtained from our melanoma tissue and sera bank.All patients have been managed by the Department of Surgical Oncology at the University of Illinois at Chicago.The clinical and follow-up data of these patients are maintained in the melanoma research registry.Immunohistochemistry.Four-␮m-thick sections were used for immuno-cytochemical studies.Standard avidin-biotin-peroxidase immunohistochemis-try was performed with the mouse monoclonal IgG1micropthalmia antibody Ab-2(Neomarkers,Union City,CA).Antigen retrieval was accomplished with 1m M EDTA buffer.We used1%dried skim milk to block nonspecific binding before incubation with the primary antibody for2h(1:25dilution).Amin-oethylcarbazine was used as a chromagen.Evaluation of Staining.The stained slides were graded as follows accord-ing to the percentage of cells whose nuclei stained positive for Mitf:(a)0,0%;(b)ϩ1,1–25%;(c)ϩ2,26–50%;(d)ϩ3,51–75%;and(e)ϩ4,Ͼ75%. Cytoplasmic staining was excluded from the analysis.Representative immuno-histochemical staining of two different primary melanomas is shown in Fig.1.Received6/1/00;accepted8/3/00.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solely to indicate this fact.1Supported by NIH Training Grant T32-CA09432and a grant from the Cancer Federation,Inc.2To whom requests for reprints should be addressed,at Department of SurgicalOncology(M/C820),College of Medicine,University of Illinois at Chicago,840South Wood Street,Chicago,IL60612.Phone:(312)996-3288;Fax:(312)996-9365;E-mail: geosalti@.3The abbreviations used are:Mitf,micropthalmia transcription factor;OS,overall survival;DFS,disease-free survival.Statistical Analysis.Survival curves were generated by the Kaplan-Meier product limit estimate.Statistical comparisons were performed using the log-rank test.Outcomes evaluated were DFS and OS.We evaluated the effect of Mitf staining (graded from 0to ϩ4)on both DFS and OS.In addition,we arbitrarily evaluated the effect of three different cutoff levels of Mitf staining on survival:(a )0(negative)versus ϩ1to ϩ4(positive);(b )0to ϩ1(Յ25%)versus ϩ2to ϩ4(Ͼ25%);and (c )0to ϩ2(Յ50%)versus ϩ3to ϩ4(Ͼ50%).Pairwise association between Mitf staining and known prognostic variables such as thickness,histology,location of primary tumor,number of mitoses/mm 2,ulceration,and nodal status was also evaluated.Differences in distribu-tion of variables were assessed by the Mann-Whitney and Kruskal-Wallis tests.Multivariate analysis was performed by using the Cox proportional hazards model.P Ͻ0.05was considered statistically significant.Death from melanoma was considered the only event for OS.DFS events included regional recur-rences and distant metastases.ResultsThere were 36males and 27females.The mean age at diagnosis was 53.25Ϯ15.49years (age range,17–81years).The primary cutaneous melanomas were located on the trunk in 23patients (36.5%),the lower extremities in 23patients (36.5%),the upper extremities in 10patients (15.9%),and the head and neck in 7patients (11.1%).Thirty-one (49.2%)melanomas were superficial spreading melanomas,18(28.5%)were nodular melanomas,9(14.3%)were acral lentiginous melanomas,and 5(7.9%)were lentigo maligna melanomas.Mean thickness was 2.2Ϯ0.7mm (range,1–4mm).Twenty-one melanomas were ulcerated (33.3%),and 42(66.7%)were nonulcerated.The mean number of mitoses/mm 2per high-power field was 4.3Ϯ2.62(range,1–12).The majority of melanomas had Clark’s level IV invasion (52of 63melanomas,82.5%).Of 63patients,11(17.5%)did not stain forMitf,17(27%)had ϩ1staining,13(20.6%)had ϩ2staining,13(20.6%)had ϩ3staining,and 9(14.3%)had ϩ-plete node dissection was performed in 56of 63(88.8%)patients.Twenty-six (46.4%)of these patients were node positive,and 37(58%)were node negative.The median follow-up was 50months.The mean DFS (Fig.2A )was 168.53Ϯ13.96months (5-year DFS,74.1%).Mean OS (Fig.2B )was 171.9Ϯ13.12months (5-year OS,74.5%).Nodal status significantly affected survival in our cohort of patients.Mean DFS and OS in those patients who had nodal disease were 105.37months (5-year DFS,58.4%)and 109.90months (5-year OS,58.6%).In contrast,patients without nodal disease had a DFS of 192.55months (5-year DFS,82%;P ϭ0.04)and an OS of 193.54months (5-year OS,82.2%;P ϭ0.05).In this small series of 63patients,only thickness significantly influenced survival when evaluated by univariate analysis.The sur-vival advantage was most marked when patients had primary mela-noma thicker than 2.6mm.In those instances,the DFS and OS were 66.48months (5-year DFS,60%)and 67.1months (5-year OS,58%),respectively,whereas the DFS and OS were 180.52months (5-year DFS,76.5%;P ϭ0.067)and 183.24months (5-year OS,78%;P ϭ0.039%),respectively,in patients with primary lesions Ͻ2.6mm thick.Curiously,when stratified according to the presence or absence of ulceration in the primary tumor,ulceration was significant only in patients whose melanomas were Ͼ2mm thick.In patients with ulceration,the DFS and OS were 119.41months (5-year DFS,58.9%)and 123.20months (5-year OS,60%),respectively,com-pared with 160months (5-year DFS,80.7%;P ϭ0.086)andFig.1.Representative immunostaining of dif-ferent levels of Mitf expression in two primary cutaneous malignantmelanomas.Fig.2.Kaplan-Meier survival curves of the entire popu-lation.A ,DFS.B ,OS.165.14months (5-year OS,82.7%;P ϭ0.05)in patients with nonulcerated primaries.Expression of Mitf as determined immunohistochemically in the primary lesion appears to significantly influence both DFS and OS(Table 1;Fig.3).Moreover,by applying semiquantitative methods,it appears that the amount of Mitf expression could be directly correlated to both DFS and OS.Mean DFS and OS in the 11patients whose melanomas did not stain positive for Mitf were 71.36months (median,40months;5-year DFS,37.6%)and 80.89months (median,51months;5-year OS,42.1%),respectively.These figures are significantly lower than those for patients who showed evidence of Mitf expression,for whom the mean DFS and OS were 186.78months (5-year DFS,83.1%;P ϭ0.0086)and 187.9months (5-year OS,84%;P ϭ0.0054),respectively.Mean DFS and OS with Ͼ25%of the melanoma cells staining positive for Mitf expression were 137.84months (5-year DFS,92.2%;P ϭ0.023)and 147.32months (5-year OS,92%;P ϭ0.039)compared with cells in which Ͻ25%of the cells stained positive for Mitf expression,in which the DFS and OS were 127.14months (5-year DFS,55.8%)and 136.68months (5-year OS,58%),re-spectively.This observation was most marked in DFS where Ͼ50%of the cells expressed Mitf.DFS was 212.57months(5-yearFig.3.Kaplan-Meier survival curves that compare patients with different levels of Mitf expression.DFS and OS were significantly better for those patients whose melanomas stained positive for Mitf [A (DFS),P ϭ0.0086;B (OS),P ϭ0.0054)]and for those patients whose melanomas expressed Ͼ25%Mitf [C (DFS),P ϭ0.023;D (OS),P ϭ0.039].DFS was significantly better for those patients whose melano-mas expressed Ͼ50%Mitf (E ,P ϭ0.035).OS tended to be better for those patients whose melanomas ex-pressed Ͼ50%Mitf (F ,P ϭ0.054).Table 1Influence of Mitf expression on DFS and OSMitf expression N DFS aOS a01171.36(P ϭ0.048)80.89(P ϭ0.077)ϩ117162.32172.73ϩ213119.62120.05ϩ313bbϩ49105.8106.8Absent 1171.36(P ϭ0.0086)80.89(P ϭ0.0054)Present 52186.78187.9Յ25%28127.14(P ϭ0.023)136.68(P ϭ0.039)Ͼ25%35137.84147.32Յ50%41127.27(P ϭ0.035)133.37(P ϭ0.054)Ͼ50%22212.57212.64a Mean values in months.bAll observations were censored;mean values were not calculated.DFS,92.3%)in these patients as compared with patients in whom Ͻ50%of the cells expressed Mitf,where the mean DFS was 127.27months(5-year DFS,64.8%).The P between these two survival figures was0.035.In this series,location of the primary tumor,age,sex,histological subtype,and level of invasion did not play any role in either DFS or OS.When multivariate analysis was performed on known primary prognostic variables,patients whose melanomas wereϾ2.6mm thick had worse DFS(Pϭ0.005)and OS(Pϭ0.032).When the nodal status was included as a covariate,both thickness and nodal status significantly influenced survival:(a)DFS,Pϭ0.078and Pϭ0.05,respectively;and(b)OS,Pϭ0.005and Pϭ0.048, respectively.When Mitf expression with a cutoff value of25%was included with the other primary tumoral prognostic variables, it significantly affected DFS(Pϭ0.014)and OS(Pϭ0.018). When all covariates were analyzed together(Table2),Mitf expression emerged as the most significant variable influencing DFS(Pϭ0.008).By stepwise Cox regression,the final model consisted of Mitf expression(Pϭ0.015)and thickness (Pϭ0.035)influencing DFS,and regional nodal status (Pϭ0.048)influencing OS.A significant relationship was noted between quantitative Mitf expression and the presence or absence of metastases in the regional nodes.WhenՆ50%of melanoma cells expressed Mitf,the incidence of nodal metastases was significantly lower.Of the26patients with nodal metastases,6(23%)had primary lesions withϾ50%Mitf expression,whereas20(77%)of the primary melanomas hadϽ50% of the cells expressing Mitf(Pϭ0.04).DiscussionWe have evaluated the biological significance of Mitf expression in primary cutaneous melanoma.It appears that Mitf expression is a molecular marker that can be used clinically along with such estab-lished markers as thickness of the primary lesion,ulceration,and incidence of regional node metastases.Our data suggest that Mitf expression is a marker for improved survival.This assumption is substantiated by the fact that6of11patients whose primary tumors did not stain positive for Mitf died of melanoma,and the mean DFS in these patients was40months.Furthermore,using a semiquantita-tive immunocytochemical analysis,it appears that the higher the number of melanoma cells showing Mitf expression in a given pri-mary tumor,the better the prognosis.For example,when25%of the melanoma cells expressed Mitf,both DFS and OS increased signifi-cantly compared with the primary lesions in which no Mitf was expressed.If the cutoff point for positivity of Mitf expression is assumed to be50%of the cells expressing Mitf,then the DFS of this group of patients increased significantly,and OS tended to increase significantly.Clinically,nodal status is probably the most important prognostic marker in cutaneous melanoma,and in the primary lesion,thickness and ulceration are considered the most significant markers for sur-vival.In this study,we confirmed that all of these three aspects are clinically reliable markers.However,in this small series,thickness and ulceration became statistically significant when the lesions were Ն2mm.Mitf expression,in contrast,remains significant in all in-stances when compared by univariate analysis.In multivariate anal-ysis,using the stepwise Cox regression model,Mitf expression and nodal status emerged as the most important variables influencing DFS and OS.This prognostic correlation between nodal status and Mitf ex-pression suggests that Mitf expression or lack thereof in primary lesions is an indicator of regional node metastases.This certainly was the case in our study.Patients with Mitf expression ofϾ50% had a significantly lower incidence of nodal disease than those with a Mitf expression ofՅ50%.The biological role of Mitf in melanocyte differentiation continues to show new facets.Our findings suggest that increased Mitf expression in patients with intermediate-thickness melanomas may lead to more differentiated tumors.This,in turn,leads to decreased relapse and nodal metas-tases and improved survival.In the study reported by King et al.(14),all melanomas evaluated stained positive for Mitf,but not for other melanocytic markers evaluated.In our study,82.5%of melanomas stained positive for Mitf.It should be noted that in addition to tissue blocks,we examined archival specimens only for which slides from outside institutions were sent to us for evaluation.The difference in tissue handling may explain,in part,the difference in staining.In addition,we have used a commercially available antibody for immunostaining.However,it is germane to emphasize that Mitf expression is a sign of differentiation; 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