Computer-Aided Cytogenetic Method of Breast Cancer Diagnosis. Part II- Test Criteria R.I.An
深度学习在视网膜疾病中的应用
化,不断完善后的 DL模型可广泛应用于临床,促进 眼科疾病的 早 诊 断、早 发 现、早 治 疗,也 可 以 进 入 一 些医疗资源 匮 乏 的 偏 远 地 区,弥 补 专 科 医 师 数 量 和 医师经验的不足。 2.2 黄斑病变 2.2.1 AMD AMD包括干性年龄相关性黄斑变性 (dAMD)和湿 性 年 龄 相 关 性 黄 斑 变 性 (wAMD),d AMD以后极部视网膜外层、视网膜色素上皮(RPE) 层、玻璃膜及 脉 络 膜 毛 细 血 管 缓 慢 进 行 性 变 性 萎 缩 为特征,进展缓慢,目前无有效干预方法。而 wAMD 是由于玻 璃 膜 的 变 性 损 害 诱 发 脉 络 膜 新 生 血 管 形 成,新生血管长入 RPE或神经视网膜下,引发渗出 性或出血性视网膜脱离[12],其特点是视网膜毛细血 管内液体渗漏,并在视网膜内积聚,目前有效且最常 用的治疗 方 法 为 玻 璃 体 内 注 射 抗 血 管 内 皮 生 长 因 子。早期发现、定期监测、定期治疗是控制疾病进展 的关键因素,针对疾病的早期诊断,亟需一种迅速有 效的识 别 方 法 来 进 行 筛 查 和 确 诊。2017年 Fang 等[13]提出了 CNN结合图形搜索方法应用于 dAMD 视网膜各层的分割上,结果显示 CNN结合图形搜索 的方 法 能 够 有 效 分 割 视 网 膜 各 层 结 构。2020年 Vaghefi等[14]利用 OCT、OCTA和彩色眼底照片的图 像分析,使用多种模式输入的 DL方法来诊断中度 dAMD,发现 OCT单独输入训练的 CNN的诊断准确 率为 94%,OCTA单独输入训练的 CNN的诊断准确 率为 91%。当多种模式结合使用时,诊断准确率提 高到 96%。多模式输入为网络提供了更多特征,能 强化网络 学 习,可 以 获 得 更 高 的 诊 断 准 确 率。 同 年 Alsaih等[15]利用图像分割的四种 DL模型 (FCNN、 UNet、SegNet和 Deeplabv3+)对视网膜内液(IRF)、 视网膜下液(SRF)和视网膜色素上皮脱离(PED)三 种 wAMD视网膜液体进行分割,结果显示,DL模型 能以高 Dice系数分割 SRF、IRF和 PED三类视网膜 积液,进行参 数 微 调 后 模 型 的 表 现 较 从 头 开 始 训 练 的网络更精准、更快速。 2.2.2 中心性浆液性脉络膜视网膜病变 中心性 浆液性脉络膜视网膜病变 (CSC)是以 RPE功能障 碍、脉络膜高通透性和增厚引起的浆液性 SRF积聚, 从而导致的以视网膜神经上皮脱离为特征的脉络膜 视网膜病变[16],虽然急性 CSC通常具有自限性,很少 遗留视觉后遗症,但复发性的 CSC和慢性 CSC患者有 可能会发展成 RPE萎缩,导致永久性的视力损伤[17]。
巴戟天-续断-益母草组药干预肾虚血瘀型多囊卵巢综合征网络药理学作用机制
巴戟天-续断-益母草组药干预肾虚血瘀型多囊卵巢综合征网络药理学作用机制∗袁仁智1,2,3,肖卫琼1,3,唐鹏1,2,康开彪2,潘文21甘肃省中医院,甘肃兰州730050;2甘肃省中医药研究院;3邵阳学院[摘要]目的:运用网络药理学方法和技术研究“巴戟天-续断-益母草”组药治疗多囊卵巢综合征(polycystic ovarian syndrome,PCOS )的主要活性成分、靶点、通路等药理学作用机制。
方法:通过数据挖掘获得PCOS 肾虚血瘀型核心组药巴戟天-续断-益母草;检索中药系统药理学数据库分析平台(traditional Chinese medicine systems pharmacology database and analysis platform,TCMSP ),筛选出巴戟天-续断-益母草的活性成分和潜在靶点;查询NCBI数据库获得PCOS疾病靶点,找到药物和疾病的共靶点;通过DAVID 数据库对共靶点进行基因功能GO 富集分析和KEGG 富集分析;采用Cytoscape 软件构建成分-靶标、靶标-通路网络。
结果:从“巴戟天-续断-益母草”组药中筛选得到26个候选活性分子,作用于719个靶点,找到PCOS 疾病靶点228个,两者共有靶点22个,主要涉及炎性反应、细胞因子、雌激素、胰岛素抵抗等生物学过程中的多条信号通路。
结论:本研究初步揭示“巴戟天-续断-益母草”组药的主要活性成分、靶点及协同作用机制,为进一步深入探讨其药理学作用提供了参考。
[关键词]多囊卵巢综合征;肾虚血瘀;网络药理学;巴戟天;续断;益母草[中图分类号]R271[文献标识码]A[文章编号]2096-9600(2021)05-0014-08Network Pharmacological Mechanism of Bajitian -Xuduan -Yimucao Drug Combinationin the Internvetion for PCOS of Kidney-deficiency Blood-stasis PatternYUAN Renzhi 1,2,3,XIAO Weiqiong 1,3,TANG Peng 1,2,KANG Kaibiao 2,PAN Wen 21Gansu Provincial Hospital of Traditional Chinese Medicine,Lanzhou 730050,China;2Gansu Provincial Academy of Chinese Medicine;3Shaoyang UniversityAbstractObjective:To study main active ingredients,target spot,pathway and others of "Bajitian -Xuduan-Yimucao "drug combinations in the treatment for polycystic ovarian syndrome (PCOS)using network pharmcological method and technology.Methods:Core drug combinations "Bajitian -Xuduan -Yimucao "for PCOS of kidney-deficiency blood-stasis pattern were obtained by data mining;active ingredients and potiential target spot of "Bajitian -Xuduan -Yimucao "were screened by searching traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP);PCOS disease target spots,common target spots of drugs and diseases were obtained by searching NCBI database;Gene function Go concentration analysis and KEGG concentration analysis of the common target spots were performed using DAVID database;ingredient-target spot,target spot-pathway network were constructed by adopting Cytoscape software.Results:All 26candidate bioactive molecules were selected from "Bajitian -Xuduan -Yimucao "drug combinations,and they acted on 719target spots,228PCOS disease target spots were found,22common target spots,and many signal channels about inflammtory reaction,cytokines,estrogen,insuslin resistance and others in the biological process were involved mainly.Conclusion:The study preliminarily reveals main active ingredients,target spots and synergistic mechanism of the drug combinations,and it could provide the reference for further exploration into its pharmacology.KeywordsPCOS;kidney-deficiency blood-stasis pattern;network pharmacology;Bajitian ;Xuduan ;YimucaoDOI:10.12174/j.issn.2096-9600.2021.05.05网络出版时间:2019-11-1808:32:11网络出版地址:/kcms/detail/62.1204.R.20191118.0832.002.html多囊卵巢综合征(polycystic ovarian syn-drome,PCOS)是影响女性最常见的内分泌紊乱疾病,在育龄女性中发病率约5%~20%,且呈不断上升趋势[1]。
KEGG数据库在生物合成研究中的应用
#技术与方法#生物技术通报BI OTEC HNOLOG Y BULLETI N2011年第1期KEGG 数据库在生物合成研究中的应用韩增叶 田平芳(北京化工大学生命科学与技术学院,北京100029)摘 要: KEGG (K yoto Encyc l oped i a o f G enes and G eno m es)提供了一个操作平台,即以基因组信息(GEN ES)和化学物质信息(L I GAND )为构建模块,通过代谢网络(PATHW AY )将基因组和生物系统联系起来,然后根据功能等级进行归纳分类(BR ITE)。
KEGG 还为各种组学研究提供相关软件,用于代谢途径重建、遗传分析和化合物比对。
作为一个综合数据库,KEGG 不仅指导生物燃料、药物和新材料等生物基化学品的合成,而且致力于研究日趋严重的环境问题。
系统介绍了K EGG 数据库的结构、功能及其相关工具的最新进展,并展望在生物合成中的应用前景。
关键词: KEGG 基因 化合物 代谢途径 生物合成Applicati ons of KEGG Dat abase in Researc h of Biosynt hesisH an Zengye T i an P ingfang(Colle ge of L i fe Science and T ec hnology,B eiji ng Uni versit y of Che m ical T ec hnology,Beijing 100029)Abstrac:t KEGG set a stage for bio l og ists t o accom plish reprogra mm ing cell behav i o r or even crea ti ng a novel o rganis m.K EGGbasica ll y consi sts o f f our daughter da tabases :GENES ,L I GAND,PATHWAY,and BR ITE .GENES is a co llection of gene ca talogs for all comp l e te g eno m es and so m e partia l genomes .L I GAND cove rs both endogenous and ex og enous che m i ca l substances .PATHWAY pro -v i des m o l ecular i nteracti on net w orks such as path w ay s and m o l ecular comp l exes .BR I TE is an onto l ogy database representi ng functiona l h i erarch ies of va ri ous b i o l og ical objects .In additi on ,KEGG prov ides ana l y si s t oo ls for om ics research ,i nclud i ng m etabo lic path w ay re -constructi on ,gene ti c ana lysis ,and comparison o f chem ical co m pounds .A s a comprehensi ve da tabase ,K EGG not only gu i des t he bi o syn -t hesis o f bio -based chem icals ,such as bio -f ue l s ,drugs or nove l bio m ate rials ,but a l so ill um ina tes our perception o f pressi ng env iron m en -ta l issues .In t h is paper ,t he KEGG co m position ,functi on ,and re l evant soft w are w ere rev ie w ed and its potential u tilizations in biosynthe -si s w ere a l so tentati v ely env isi oned .K ey words : KEGG G eneCo m pound M etabo lic pa t hway B i osynthes i s 收稿日期:2010-05-25基金项目:国家自然科学基金项目(20876009)作者简介:韩增叶,男,硕士研究生,研究方向:分子生物学;E-m ai:l hanzengye @163.co m 通讯作者:田平芳,男,博士,副教授,研究方向:合成生物学;E-m ai:l ti anp @f m ai.l buct .edu .cn如何使细胞和有机体在计算机上完整地表达和演绎,是后基因组时代的重大挑战。
2003-Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins
DOI: 10.1126/science.1088755, 1884 (2003);301 Science , et al.Jwa-Min Nam ProteinsNanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection ofThis 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): February 16, 2012 (this infomation is current as of The following resources related to this article are available online at/content/301/5641/1884.full.html version of this article at:including high-resolution figures, can be found in the online Updated information and services, /content/suppl/2003/09/25/301.5641.1884.DC1.htmlcan be found at:Supporting Online Material /content/301/5641/1884.full.html#related found at:can be related to this article A list of selected additional articles on the Science Web sites 718 article(s) on the ISI Web of Science cited by This article has been /content/301/5641/1884.full.html#related-urls 29 articles hosted by HighWire Press; see:cited by This article has been/cgi/collection/chemistry Chemistrysubject collections:This article appears in the following registered trademark of AAAS.is a Science 2003 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 F e b r u a r y 16, 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 mnumber corresponds to a bulk resistivity of 2.4ϫ10Ϫ6ohm ⅐m for the silver nanowire.This nanowire is easily reproducible and has mark-edly higher conductivity than previously report-ed double-helix DNA–templated silver nanow-ires (20).The 4ϫ4DNA tile can be easily pro-grammed by varying the sticky ends to form more sophisticated arrays for applications in construction of logical molecular devices;for instance,quantum-dot cellular automata arrays may be constructed by specifically incorporat-ing metal nanoparticles into the nanogrids.The cavities can also be used as pixels in a uniform pixel array,which could be applied to AFM visual readout of self-assembly DNA computa-tions such as a binary counting lattice (30).References and Notes1.N.C.Seeman,Nature 421,427(2003).Bean,in Computational Biology and Genome Informatics ,J.T.L.Wang,C.H.Wu,P.P.Wang,Eds.(World Scientific,River Edge,NJ,2003),p.35.3.E.Winfree,F.Liu,L.A.Wenzler,N.C.Seeman,Nature 394,539(1998).Bean et al.,J.Am.Chem.Soc.122,1848(2000).5.C.Mao,W.Sun,N.C.Seeman,J.Am.Chem.Soc.121,5437(1999).6.R.Sha,F.Liu,N.C.Seeman,Chem.Biol.7,743(2000).7.H.Yan,Bean,L.Feng,J.H.Reif,Proc.Natl.Acad.Sci.U.S.A.100,8103(2003).8.C.Mao,W.Sun,Z.Shen,N.C.Seeman,Nature 397,144(1999).9.B.Yurke et al.,Nature 406,605(2000).10.H.Yan,X.Zhang,Z.Shen,N.C.Seeman,Nature 415,62(2002).11.J.J.Li,W.Tan,Nano Lett.2,315(2002).12.L.Feng,S.H.Park,J.H.Reif,H.Yan,Angew.Chem.Int.Ed.,in press.13.L.M.Adleman,Science 266,1021(1994).14.Q.Liu et al.,Nature 403,175(2000).15.C.Mao,Bean,J.H.Reif,N.C.Seeman,Nature407,493(2000).16.Y.Benenson et al.,Nature 414,430(2001).17.B.S.Ravinderjit et al.,Science 296,499(2002).18.C.A.Mirkin,Inorg.Chem 39,2258(2000).19.A.P.Alivisatos et al.,Nature 382,609(1996).20.E.Braun,Y.Eichen,U.Sivan,G.Ben-Yoseph,Nature391,775(1998).21.K.Keren et al.,Science 297,72(2002).22.F.Patolsky,Y.Weizmann,O.Lioubashevski,I.Willner,Angew.Chem.Int.Ed.41,2323(2002).23.C.F.Monson,A.T.Woolley,Nano Lett.3,359(2003).24.W.E.Ford,O.Harnack,A.Yasuda,J.M.Wessels,Adv.Mater.13,1793(2001).25.J.Richter et al.,Adv.Mater.12,507(2000).26.C.M.Niemeyer,W.Burger,J.Peplies,Angew.Chem.Int.Ed.37,2265(1998).27.S.Xiao et al.,J.Nanopart.Res.4,313(2002).28.N.C.Seeman,Trends Biotechnol.17,437(1999).29.Materials and methods are available as supportingmaterial on Science Online.30.E.Winfree,J.Biomol.Struct.Dyn.11,263(2000).31.We thank E.Winfree,P.Rothemund,and N.Papadakisforhelpful advice with AFM underaqueous buffer ;D.L iu for development of the metallization procedure and L.Feng for technical assistance in the thermal profile experiment;and J.Liu for providing access to his AFM instrument.This work was supported by grants from the National Science Foundation (H.Y.,J.H.R.,and T.H.L.)and Defense Advanced Research Projects Agency (J.H.R.)and by an industrial partners arrangement with Taiko Denki Co.,Ltd.(J.H.R.and T.H.L.).Supporting Online Material/cgi/content/full/301/5641/1882/DC1Materials and Methods Figs.S1to S6References18July 2003;accepted 21August 2003Nanoparticle-Based Bio–Bar Codes for the Ultrasensitive Detection of ProteinsJwa-Min Nam,*C.Shad Thaxton,*Chad A.Mirkin †An ultrasensitive method for detecting protein analytes has been developed.The system relies on magnetic microparticle probes with antibodies that spe-cifically bind a target of interest [prostate-specific antigen (PSA)in this case]and nanoparticle probes that are encoded with DNA that is unique to the protein target of interest and antibodies that can sandwich the target captured by the microparticle probes.Magnetic separation of the complexed probes and target followed by dehybridization of the oligonucleotides on the nanoparticle probe surface allows the determination of the presence of the target protein by identifying the oligonucleotide sequence released from the nanoparticle probe.Because the nanoparticle probe carries with it a large number of oligonucle-otides per protein binding event,there is substantial amplification and PSA can be detected at 30attomolar concentration.Alternatively,a polymerase chain reaction on the oligonucleotide bar codes can boost the sensitivity to parable clinically accepted conventional assays for detecting the same target have sensitivity limits of ϳ3picomdar,six orders of magnitude less sensitive than what is observed with this method.The polymerase chain reaction (PCR)and other forms of target amplification have en-abled rapid advances in the development of powerful tools for detecting and quantifying DNA targets of interest for research,forensic,and clinical applications (1–3).The develop-ment of comparable target amplification methods for proteins could substantially im-prove medical diagnostics and the developing field of proteomics (4–7).Although one can-not yet chemically duplicate protein targets,it is possible to tag such targets with oligonu-cleotide markers that can be subsequently amplified with PCR and then use DNA de-tection to identify the target of interest (8–13).This approach,often referred to as im-muno-PCR,allows the detection of proteins with DNA markers in a variety of different formats.Thus far,all immuno-PCR ap-proaches involve initial immobilization of a target analyte to a surface and subsequent detection with an antibody (Ab)with a DNA marker.The DNA marker is typically strong-ly bound to the Ab (either through covalent interactions or streptavidin-biotin binding).Although these approaches are considerable advances in protein detection,they have sev-eral drawbacks:(i)a low ratio of DNA iden-tification sequence to detection Ab,which limits sensitivity,(ii)slow target-binding ki-netics because of the heterogeneous nature of the target-capture procedure,which increasesassay time and decreases assay sensitivity,(iii)complex conjugation chemistries that are required to link the Ab and DNA markers,and (iv)PCR requirements (14).Herein,we report a nanoparticle-based bio–bar-code approach to detect a protein target,free prostate-specific antigen (PSA),at low at-tomolar concentrations (Fig.1).PSA was cho-sen as the initial target for these studies because of its importance in the detection of prostate and breast cancer,the most common cancers and the second leading cause of cancer death among American men and women,respectively (15–18).Identification of disease relapse after the surgical treatment of prostate cancer using PSA as a marker present at low levels (10s of copies)could be extremely beneficial and en-able the delivery of curative adjuvant therapies (17,19).Furthermore,PSA is found in the sera of breast cancer patients,and it is beginning to be explored as a breast cancer screening target (16).Because the concentration of free PSA is much lower in women’s serum as compared to that of men,an ultrasensitive test is needed for breast cancer screening and diagnosis.The bio–bar-code assay reported herein uses two types of probes,magnetic micropar-ticles (MMPs,1-m diameter polyamine par-ticles with magnetic iron oxide cores)func-tionalized with PSA monoclonal antibodies (mAbs)(Fig.1A)(20)and gold nanoparticles (NP)heavily functionalized with hybridized oligonucleotides (the bio–bar codes;5ЈAC-ACAACTGTGTTCACTAGCGTTGAACGT-GGATGAAGTTG 3Ј)(7,21,22)and poly-clonal detection Abs to recognize PSA (Fig.1A)(20).In a typical PSA detection experi-ment (Fig.1B),the gold NPs and the MMPs sandwich the PSA target,generating a com-Department of Chemistry and Institute for Nanotech-nology,Northwestern University,2145Sheridan Road,Evanston,IL 60201,USA*These authors contributed equally to the work.†To whom correspondence should be addressed.E-mail:camirkin@R E P O R T S26SEPTEMBER 2003VOL 301SCIENCE 1884 o n F e b r u a r y 16, 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 mplex with a large ratio of bar-code DNA to protein target (23).Application of a magnetic field draws the MMPs to the wall of the reaction vessel in a matter of seconds,allow-ing the separation of all of the MMPs but only the reacted NPs from the reaction mix-ture.Washing the aggregate structures in NANOpure water (18megohm;Barnstead International,Dubuque,IA)dehybridizes bar-code DNA from NP-immobilized com-plements.With the use of the magnetic sep-arator,we readily removed the aggregate from the assay solution to leave only the bar-code DNA,which can be quickly identi-fied by standard DNA detection methodolo-gies [e.g.,gel electrophoresis,fluorophore-labeling,and scanometric (24)approaches]that may or may not rely on PCR (Fig.1B).Although gel electrophoresis was routine-ly used to analyze the results of the assay (20),in general the scanometric method pro-vided higher sensitivity and was easier to implement than the gel-based method.There-fore,the results of the scanometric assay are reported herein.In the case of PCR-less de-tection,30-nm gold particles were used dur-ing the detection step instead of 13-nm gold particles to increase the amount of detectable bar-code DNA (Fig.1B,step 2).For bar-code DNA identification,chip-immobilized DNA20-mers[5ЈSH-(CH 2)6-A 10-CAACTTCATC-CACGTTCAAC 3Ј],which are complemen-tary with half of the target bar-code sequence,were used to capture the isolated bar-code DNA sequences,and oligonucleotide-modi-fied 13-nm gold NPs ϭ[5ЈGCTAGTGAA-CACAGTTGTGT-A 10-(CH 2)3-SH 3Ј-Au]were used to label the other half of the se-quence in a sandwich assay format.Chips with hybridized NP probes are then subjected to silver amplification (25),which results in gray spots that can be read with a Verigene ID (identification)system (Nanosphere,In-corporated,Northbrook,IL)that measures light scattered from the developed spots.Tar-get PSA concentrations from 300fM to 3aMFig.1.The bio–bar-code assay method.(A )Probe design and preparation.(B )PSA detection and bar-code DNA amplification and identification.In a typical PSA-detectionexperiment,an aqueous dispersion of MMP probes functionalized with mAbs to PSA (50l of 3mg/ml magnetic probe solution)was mixed with an aqueous solution of free PSA (10l of PSA)and stirred at 37°C for30min (Step 1).A 1.5-ml tube containing the assay solution was placed in a BioMag microcentrifuge tube separator (Polysciences,Incorporated,Warrington,PA)at room temperature.After 15s,the MMP-PSA hybrids were concentrated on the wall of the tube.The supernatant (solution of unbound PSA molecules)was removed,and the MMPs were resuspended in 50l of 0.1M phosphate-buffered saline (PBS)(repeated twice).The NP probes (for 13-nm NP probes,50l at 1nM;for30-nm NP pr obes,50l at 200pM),functionalized with polyclonal Abs to PSA and hybridized bar-code DNA strands,were then added to the assay solution.The NPs reacted with the PSA immobilized on the MMPs and provided DNA strands for signal amplification and protein identification (Step 2).This solution was vigorously stirred at 37°C for 30min.The MMPs were then washed with 0.1M PBS with the magnetic separator to isolate the mag-netic particles.This step was repeated four times,each time for 1min,to remove everything but the MMPs (along with the PSA-bound NP probes).After the final wash step,the MMP probes were resuspended in NANOpure water (50l)for 2min to dehybridize bar-code DNA strands from the nanoparticle probe surface.Dehybridized bar-code DNA was then easily separated and collected from the probes with the use of the magnetic separ ator(Step 3).Forbar -code DNA amplification (Step 4),isolated bar-code DNA was added to a PCR reaction mixture (20-l final volume)containing the appropriate primers,and the solution was then thermally cycled (20).The bar-code DNA amplicon was stained with ethidium bromide and mixed with gel-loading dye (20).Gel electrophoresis or scanometric DNA detection (24)was then performed to determine whether amplifica-tion had taken place.Primer amplification was ruled out with appro-priate control experiments (20).Notice that the numberof bound NP probes for each PSA is unknown and will depend upon target proteinconcentration.Fig.2.Scanometric de-tection of PSA-specific bar-code DNA.PSA con-centration (sample vol-ume of 10l)was var-ied from 300fM to 3aM and a negative control sample where no PSA was added (control)is shown.Forall seven samples,2l of antidi-nitrophenyl (10pM)and 2l of -galactosidase (10pM)were added as background proteins.Also shown is PCR-less detection of PSA (30aM and control)with 30nm NP probes (inset).Chips were imaged with the Verigene ID system (20).R E P O R T S SCIENCE VOL 30126SEPTEMBER 20031885o n F e b r u a r y 16, 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 mwere detected with the use of the PCR-cou-pled approach (Fig.2).The use of this ap-proach in a more complicated medium such as goat serum provided a detection sensitivity of 30aM,with clear differentiation from background signal (fig.S4).The selectivity for the bar-code DNA sequence was excel-lent,as evidenced by the lack of signal from the control spots with noncomplementary capture DNA [5ЈSH-(CH 2)6-A 10-GGCAGC-TCGTGGTGA 3Ј]and the observation that there is little discernible signal when PSA is absent (Fig.2).Importantly,one can eliminate the PCR step and still obtain a high sensitivity assay by using larger nanoparticles (30nm),which can support larger absolute amounts of bar-code DNA.With such an assay,it was pos-sible to detect PSA at 30aM concentration in a 10-l sample (Fig.2,inset).This substan-tially simplifies the overall complexity of the assay and still yields a sensitivity that is five orders of magnitude greater than the cited commercial assay sensitivity (19)and two orders of magnitude greater than that cited for immuno-PCR on the same target under near-identical conditions (13).The bio –bar-code method offers several ad-vantages over current protein detection meth-ods.First,the target-binding portion of the as-say is homogeneous (in the sense that the cap-ture antibodies on the MMPs are dispersed in solution as opposed to the flat surface of a microarray or titer plate).Therefore,we can add a large quantity of MMPs to the reaction vessel to facilitate the binding kinetics between the detection antibody and target analyte.Homoge-neous mixing makes this assay faster than het-erogeneous immuno-PCR systems and also can increase sensitivity because the capturing step is more efficient (the equilibrium can be pushed toward the captured protein state by increasing the concentration of MMP probe,which cannot be done in the heterogeneous assay).Second,the use of the NP bio –bar codes provides a high ratio of PCR-amplifiable DNA to labeling Ab that can substantially increase assay sensitivity.Third,this assay obviates the need for compli-cated conjugation chemistry for attaching DNA to the labeling Abs.Bar-code DNA is bound to the NP probe through hybridization at the start of the labeling reaction and liberated for subse-quent identification with a simple wash step.Because the labeling Ab and DNA are present on the same particle,there is no need for the addition of further antibodies or DNA-protein conjugates before the identification of bar-code DNA.In addition,the bar-code DNA is re-moved from the detection assay,and direct detection or PCR is carried out on samples of bar-code DNA that are free from PSA,most of the biological sample,microparticles,and nano-particles.This step substantially reduces back-ground signal.Finally,this protein detection scheme has the potential for massive multiplex-ing and the simultaneous detection of many analytes in one solution,especially in the PCR-less form.Although the PSA system is used for the proof of concept,the approach should be general for almost any target with known Abs,and,by using the NP-based bio –bar-code approach (7),one could prepare a distinct identifiable bar code for nearly every target of interest.References and Notes1.R.K.Saiki et al .,Science 230,1350(1985).2.R.A.Gibbs,Curr.Opin.Biotechnol.2,69(1991).3.S.A.Bustin,J.Mol.Endocrinol.29,23(2002).4.G.MacBeath,S.L.Schreiber,Science 289,1760(2000).5.H.Zhu et al .,Science 293,2101(2001);published online 26July 2001;10.1126/science.1062191.6.B.B.Haab,M.J.Dunham,P.O.Brown,Genome Biol .2(2):research0004.1(2001).7.J.-M.Nam,S.-J.Park,C.A.Mirkin,J.Am.Chem.Soc.124,3820(2002).8.T.Sano,C.L.Smith,C.R.Cantor,Science 258,120(1992).9.A.McKie,D.Samuel,B.Cohen,N.A.Saunders,J.Im-munol.Methods 270,135(2002).10.H.Zhou,R.J.Fisher,T.S.Papas,Nucl.Acids Res.21,6038(1993).11.E.R.Hendrickson,T.M.Hatfield-Truby,R.D.Joerger,W.R.Majarian,R.C.Ebersole,Nucl.Acids Res.23,522(1995).12.C.M.Niemeyer et al .,Nucl.Acids Res.27,4553(1999).13.B.Schweitzer et al .,Proc.Natl.Acad.Sci.U.S.A.97,10113(2000).14.C.M.Niemeyer,Trends Biotechnol.20,395(2002).15.J.L.Stanford et al .,Prostate Cancer Trends 1973–1995[National Institutes of Health (NIH)Pub.No.99-4543,Surveillance,Epidemiology,and End Results (SEER)Program,National Cancer Institute,Bethesda,MD,1999].16.M.H.Black et al .,Clin.Cancer Res.6,467(2000).17.R.A.Ferguson,H.Yu,M.Kalyvas,S.Zammit,E.P.Diamandis,Clin.Chem.42,675(1996).18.L.A.G.Ries et al .,SEER Cancer Statistics Review 1973–1999(SEER Program,National Cancer Institute,Bethesda,MD,2002).19.H.Yu,E.P.Diamandis,A.F.Prestigiacomo,T.A.Stamey,Clin.Chem.41,430(1995).20.Detailed materials and methods can be found on Science Online.21.C.A.Mirkin,R.L.Letsinger,R.C.Mucic,J.J.Storhoff,Nature 382,607(1996).22.Y.C.Cao,R.Jin,C.A.Mirkin,Science 297,1536(2002).23.For13-nm NPs,each NP can suppor t up to 100strands of DNA with the proteins present (26);this value represents the upper limit,and the exact num-berforthese par ticles has not yet been deter mined.24.T.A.Taton,C.A.Mirkin,R.L.Letsinger,Science 289,1757(2000).25.Silver enhancement kit was purchased from Ted Pella (Redding,CA),and silverenhancement time was 6min.26.L.M.Demers et al .,Anal.Chem.72,5535(2000).27.C.A.M.acknowledges the AirFor ce Office of Scientific Research,the Defense Advanced Research Projects Agency,and the NSF for support of this research.C.S.T.acknowledges the Howard Hughes Medical In-stitute fora Tr aining fellowship.A.Schaefferis ac-knowledged forhelpful discussions,and V.evenson assisted in PCR optimization.Supporting Online Material/cgi/content/full/301/5641/1884/DC1Materials and Methods Figs.S1to S5References and Notes3July 2003;accepted 28August 2003Particle Formation by Ion Nucleation in the Upper Troposphere andLower StratosphereS.-H.Lee,1*J.M.Reeves,1J.C.Wilson,1D.E.Hunton,2A.A.Viggiano,ler,2J.O.Ballenthin,it 3Unexpectedly high concentrations of ultrafine particles were observed over a wide range of latitudes in the upper troposphere and lower stratosphere.Particle number concentrations and size distributions simulated by a numerical model of ion-induced nucleation,constrained by measured thermodynamic data and observed atmospheric key species,were consistent with the obser-vations.These findings indicate that,at typical upper troposphere and lower stratosphere conditions,particles are formed by this nucleation process and grow to measurable sizes with sufficient sun exposure and low preexisting aerosol surface area.Ion-induced nucleation is thus a globally important source of aerosol particles,potentially affecting cloud formation and radiative transfer.Atmospheric aerosols affect climate direct-ly by altering the radiative balance of the Earth (1)and indirectly by acting as cloud condensation nuclei (CCN)(2),which inturn change the number and size of cloud droplets and the cloud albedo.Homoge-neous nucleation (HN)(formation of solid or liquid particles directly from the gas phase)is an important source of new par-ticles in the atmosphere (3,4),but the process is poorly understood and alone is unable to explain the observed particle for-mation.Homogeneous nucleation includes binary homogeneous nucleation (BHN)of sulfuric acid –water (H 2SO 4-H 2O)(3,4)and ternary homogeneous nucleation1Department of Engineering,University of Denver,Denver,CO 80208,USA.2Air Force Research Labora-tory,Space Vehicle Directorate,Hanscom Air Force Base,MA 01731,USA.3National Aeronautics and Space Administration,Goddard Space Flight Center,Greenbelt,MD 20771,USA.*To whom correspondence should be addressed.E-mail:shanlee@R E P O R T S26SEPTEMBER 2003VOL 301SCIENCE 1886 o n F e b r u a r y 16, 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。
J. Comput. Chem.
2D Depiction of Nonbonding Interactions forProtein ComplexesPENG ZHOU,1FEIFEI TIAN,2ZHICAI SHANG11Institute of Molecular Design&Molecular Thermodynamics,Department of Chemistry,Zhejiang University,Hangzhou310027,China2College of Bioengineering,Chongqing University,Chongqing400044,ChinaReceived7May2008;Revised25June2008;Accepted22July2008DOI10.1002/jcc.21109Published online22October2008in Wiley InterScience().Abstract:A program called the2D-GraLab is described for automatically generating schematic representation of nonbonding interactions across the protein binding interfaces.The inputfile of this program takes the standard PDB format,and the outputs are two-dimensional PostScript diagrams giving intuitive and informative description of the protein–protein interactions and their energetics properties,including hydrogen bond,salt bridge,van der Waals interaction,hydrophobic contact,p–p stacking,disulfide bond,desolvation effect,and loss of conformational en-tropy.To ensure these interaction information are determined accurately and reliably,methods and standalone pro-grams employed in the2D-GraLab are all widely used in the chemistry and biology community.The generated dia-grams allow intuitive visualization of the interaction mode and binding specificity between two subunits in protein complexes,and by providing information on nonbonding energetics and geometric characteristics,the program offers the possibility of comparing different protein binding profiles in a detailed,objective,and quantitative manner.We expect that this2D molecular graphics tool could be useful for the experimentalists and theoreticians interested in protein structure and protein engineering.q2008Wiley Periodicals,Inc.J Comput Chem30:940–951,2009Key words:protein–protein interaction;nonbonding energetics;molecular graphics;PostScript;2D-GraLabIntroductionProtein–protein recognition and association play crucial roles in signal transduction and many other key biological processes. Although numerous studies have addressed protein–protein inter-actions(PPIs),the principles governing PPIs are not fully under-stood.1,2The ready availability of structural data for protein complexes,both from experimental determination,such as by X-ray crystallography,and by theoretical modeling,such as protein docking,has made it necessary tofind ways to easily interpret the results.For that,molecular graphics tools are usually employed to serve this purpose.3Although a large number of software packages are available for visualizing the three-dimen-sional(3D)structures(e.g.PyMOL,4GRASP,5VMD,6etc.)and interaction modes(e.g.MolSurfer,7ProSAT,8PIPSA,9etc.)of biomolecules,the options for producing the schematic two-dimensional(2D)representation of nonbonding interactions for PPIs are very scarce.Nevertheless,a few2D graphics programs were developed to depict protein-small ligand interactions(e.g., LIGPLOT,10PoseView,11MOE,12etc.).These tools,however, are incapable of handling the macromolecular complexes.Some other available tools presenting macromolecular interactions in 2D level mainly include DIMPLOT,10NUCPLOT,13and MON-STER,14etc.Amongst,only the DIMPLOT can be used for aesthetically visualizing the nonbinding interactions of PPIs. However,such a program merely provides a simple description of hydrogen bonds,hydrophobic interactions,and steric clashes across the binding interfaces.In this article,we describe a new molecular graphics tool, called the two-dimensional graphics lab for biosystem interac-tions(2D-GraLab),which adopts the page description language (PDL)to intuitively,exactly,and detailedly reproduce the non-bonding interactions and energetics properties of PPIs in Post-Script page.Here,the following three points are the emphasis of the2D-GraLab:(i)Reliability.To ensure the reliability,the pro-grams and methods employed in2D-GraLab are all widely used in chemistry and biology community;(ii)Comprehensiveness. 2D-GraLab is capable of handling almost all the nonbonding interactions(and even covalent interactions)across binding Additional Supporting Information may be found in the online version of this article.Correspondence to:Z.Shang;e-mail:shangzc@interface of protein complexes,such as hydrogen bond,salt bridge,van der Waals(vdW)interaction,hydrophobic contact, p–p stacking,disulfide bond,desolvation effect,and loss of con-formational entropy.The outputted diagrams are diversiform, including individual schematic diagram and summarized sche-matic diagram;(iii)Artistry.We elaborately scheme the layout, color match,and page style for different diagrams,with the goal of producing aesthetically pleasing2D images of PPIs.In addi-tion,2D-GraLab provides a graphical user interface(GUI), which allows users to interact with this program and displays the spatial structure and interfacial feature of protein complexes (see .Fig.S1).Identifying Protein Binding InterfacesAn essential step in understanding the molecular basis of PPIs is the accurate identification of interprotein contacts,and based upon that,subsequent works are performed for analysis and lay-out of nonbonding mon methods identifyingprotein–protein binding interfaces include a Voronoi polyhedra-based approach,changes in solvent accessible surface area(D SASA),and various radial cutoffs(e.g.,closest atom,C b,andcentroid,etc.).152D-GraLab allows for the identification of pro-tein–protein binding interfaces at residue and atom levels.Identifying Binding Interfaces at Residue LevelAll the identifying interface methods at residue level belong toradial cutoff approach.In the radial cutoff approach,referencepoint is defined in advance for each residue,and the residues areconsidered in contact if their reference points fell within thedefined cutoff ually,the C a,C b,or centroid are usedas reference point.16–18In2D-GraLab,cutoff distance is moreflexible:cutoff distance5r A1r B1d,where r A and r B are residue radii and d is set by users(as the default d54A˚,which was suggested by Cootes et al.19).Identifying Binding Interfaces at Atom LevelAt atom level,binding interfaces are identified using closestatom-based radial cutoff approach20and D SASA-basedapproach.21For the closest atom-based radial cutoff approach,ifthe distance between any two atoms of two residues from differ-ent chains is less than a cutoff value,the residues are consideredin contact;In the D SASA-based approach,the SASA is calcu-lated twice to identify residues involved in a binding interface,once for the monomers and once for the complex,if there is achange in the SASA(D SASA)of a residue when going from themonomers to the dimer form,then it is considered involved inthe binding interface.In2D-GraLab,three manners are provided for visualizing thebinding interfaces,including spatial structure exhibition,residuedistance plot,and residue-pair contact map(see .Figs.S2–S4).Analysis and2D Layout of NonbondingInteractionsThe inputfile of2D-GraLab is standard PDB format,and the outputs are two-dimensional PostScriptfile giving intuitive and informative representation of the PPIs and their strengths, including hydrogen bond,salt bridge,vdW interaction,desolva-tion effect,ion-pair,side-chain conformational entropy(SCE), etc.The outputs are in two forms as individual schematic dia-gram and summarized schematic diagram.The individual sche-matic diagram is a detailed depiction of each nonbonding profile,whereas the summarized schematic diagram covers all nonbonding interactions and disulfide bonds across the binding interface.To produce the aesthetically high quality layouts,which pos-sess reliable and accurate parameters,several widely used pro-grams listed in Table1are employed in2D-GraLab to perform the core calculations and analysis of different nonbonding inter-actions.2D-GraLab carries out prechecking procedure for pro-tein structures and warns the structural errors,but not providing revision and refinement functions.Therefore,prior to2D-GraLab analysis,protein structures are strongly suggested to be prepro-cessed by programs such as PROCHECK(structure valida-tion),27Scwrl3(side-chain repair),28and X-PLOR(structure refinement).29Individual Schematic DiagramHydrogen BondThe program we use for analyzing hydrogen bonds across bind-ing interfaces is HBplus,23which calculates all possible posi-tions for hydrogen atoms attached to donor atoms which satisfy specified geometrical criteria with acceptor atoms in the vicinity. In2D-GraLab,users can freely select desired hydrogen bonds involving N,O,and/or S atoms.Besides,the water-mediated hydrogen bond is also given consideration.Bond strength of conventional hydrogen bonds(except those of water-mediated Table1.Standalone Programs Employed in2D-GraLab.Program FunctionReduce v3.0322Adding hydrogen atoms for proteinsHBplus v3.1523Identifying hydrogen bonds and calculatingtheir geometric parametersProbe v2.1224Identifying steric contacts and clashes at atomlevelMSMS v2.6125Calculating SASA values of protein atoms andresiduesDelphi v4.026Calculating Coulombic energy and reactionfield energy,determining electrostatic energyof ion-pairsDIMPLOT v4.110Providing application programming interface,users can directly set and executeDIMPLOT in the2D-GraLab GUI9412D Depiction of Nonbonding Interactions for Protein ComplexesFigure1.(a)Schematic representation of a conventional hydrogen bond and a water-mediated hydro-gen bond across the binding interface of IGFBP/IGF complex(PDB entry:2dsr).This diagram was produced using2D-Gralab.The conventional hydrogen bond is formed between the atom N(at the backbone of residue Leu69in chain B)and the atom OE1(at the side-chain of residue Glu3in chain I);The water-mediated hydrogen bond is formed between the atom ND1(at the side-chain of residue His5in chain B)and the atom O(at the backbone of residue Asp20in chain I),and because hydrogen positions of water are almost never known in the PDBfile,the water molecule,when serving as hydrogen bond donor,is not yet determined for its H...A length and D—H...A angle,denoted as mark ‘‘????.’’In this diagram,chains,residues,and atoms are labeled according to the PDB format.(b)Spa-tial conformation of the conventional hydrogen bond.(c)Spatial conformation of the water-mediated hydrogen bond.hydrogen bonds)is calculated using Lennard-Jones 8-6potential with angle weighting.30D U HB¼E m 3d m 8À4d m6"#cos 4h ðh >90 Þ(1)where d is the separation between the heavy acceptor atom andthe donor hydrogen atom in angstroms;E m ,the optimum hydro-gen-bond energy for the particular hydrogen-bonding atoms con-sidered;d m ,the optimum hydrogen-bond length for the particu-lar hydrogen-bonding atoms considered.E m and d m vary accord-ing to the chemical type of the hydrogen-bonding atoms.The hydrogen bond potential is set to zero when angle h 908.31Hydrogen bond parameters are taken from CHARMM force field (for N and O atoms)and Autodock (for S atom).32,33Figure 1a is the schematic representation of a conventional hydrogen bond and a water-mediated hydrogen bond across the binding interface of insulin-like growth factor-binding protein (IGFBP)/insulin-like growth factor (IGF)complex.In this dia-gram,abundant information about the hydrogen bond geometry and energetics properties is presented in a readily acceptant manner.Figures 1b and 1c are spatial conformations of the cor-responding conventional hydrogen bond and water-mediated hydrogen bond.Van der Waals InteractionThe small-probe approach developed in Richardson’s laboratory enables us to detect the all atom contact profile in protein pack-ing.2D-GraLab uses program Probe 24to realize this method to identity steric contacts and clashes on the binding interfaces.Word et al.pointed out that explicit hydrogen atoms can effec-tively improve Probe’s performance.24However,considering calculations with explicit hydrogen atoms are time-consuming,and implicit hydrogen mode is also possibly used in some cases;therefore,in 2D-GraLab,both explicit and implicit hydrogen modes are provided for users.In addition,2D-GraLab uses the Reduce 22to add hydrogen atoms for proteins,and this programis also developed in Richardson’s laboratory and can be wellcompatible with Probe.According to previous definition,vdW interaction between two adjacent atoms is classified into wide contact,close contact,small overlap,and bad overlap.24Typically,vdW potential function has two terms,a repulsive term and an attractive term.In 2D-GraLab,vdW interaction is expressed as Lennard-Jones 12-6potential.34D U SI ¼E m d m d 12À2d md6"#(2)where E m is the Lennard-Jones well depth;d m is the distance at the Lennard-Jones minimum,and d is the distance between two atoms.The Lennard-Jones parameters between pairs of different atom types are obtained from the Lorentz–Berthelodt combina-tion rules.35Atomic Lennard-Jones parameters are taken from Probe and AMBER force field.24,36Figure 2a was produced using 2D-GraLab and gives a sche-matic representation of steric contacts and clashes (overlaps)between the heavy chain residue Tyr131and two light chain res-idues Ser121and Gln124of cross-reaction complex FAB (the antibody fragment of hen egg lysozyme).By this diagram,we can obtain the detail about the local vdW interactions around the residue Tyr131.In contrast,such information is inaccessible in the 3D structural figure (Fig.2b).Desolvation EffectIn 2D-GraLab,program MSMS 25is used to calculate the SASA values of interfacial residues at atom level,and four atomic radii sets are provided for calculating the SASA,including Bondi64,Chothia75,Li98,and CHARMM83.32,37–39Bondi64is based on contact distances in crystals of small molecules;Chothia75is based on contact distances in crystals of amino acids;Li98is derived from 1169high-resolution protein crystal structures;CHARMM83is the atomic radii set of CHARMM force field.Desolvation free energy of interfacial residues is calculated using empirical additive model proposed by Eisenberg andFigure 2.(a)Schematic representation of steric contacts and overlaps between the residue Tyr131in heavy chain (chain H)and the surrounding residues Ser121and Gln124in light chain (chain L)of cross-reaction complex FAB (PDB entry:1fbi).This diagram was produced using 2D-Gralab in explicit hydrogen mode.In this diagram,interface is denoted by the broken line;Wide contact,close contact,small overlap,and bad overlap are marked by blue circle,green triangle,yellow square,and pink rhombus,respectively;Moreover,vdW potential of each atom-pair is given in the histogram,with the value measured by energy scale,and the red and blue indicate favorable (D U \0)and unfav-orable (D U [0)contributions to the binding,respectively;Interaction potential 20.324kcal/mol in the center circle denotes the total vdW contribution by residue Tyr131;Chains,residues,and heavy atoms are labeled according to the PDB format,and hydrogen atoms are labeled in Reduce format.(b)Spatial conformation of chain H residue Tyr131and its local environment.Green or yellow stands forgood contacts (green for close contact and yellow for slight overlaps \0.2A˚),blue for wide contacts [0.25A˚,hot pink spikes for bad overlaps !0.4A ˚.It is revealed that Tyr131is in an intensive clash with chain L Gln124,while in slight contact with chain L Ser121,which is well consistent with the 2D schematic diagram.9432D Depiction of Nonbonding Interactions for Protein Complexes944Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure2.(Legend on page943.)Maclachlam,40and the conformation of interfacial residues is assumed to be invariant during the binding process.D G dslv¼Xic i D A i(3)where the sum is over all the atoms;c i and D A i are the atomic solvation parameter(ASP)and the changes in solvent accessible surface area(D SASA)of atom i,respectively.Juffer et al.41 found that although desolvation free energies calculated from different ASP sets are linear correlation to each other,the abso-lute values are greatly different.In view of that,2D-GraLab pro-vides four ASP sets published in different periods:Eisenberg86, Kim90,Schiffer93,and Zhou02.40,42–44As shown in Figure3,the D SASA and desolvation free energy of interfacial residues in chain A of HLA-A*0201pro-tein complex during the binding process are reproduced in a rotiform diagram form using2D-GraLab.In this diagram,the desolvation free energy contributed by chain A is28.056kcal/ mol,and moreover,the D SASA value of each interfacial residue is also presented clearly.Ion-PairThere are six types of residue-pairs in the ion-pairs:Lys-Asp, Lys-Glu,Arg-Asp,Arg-Glu,His-Asp,and ually,ion-pairs include three kinds:salt bridge,NÀÀO bridge,and longer-range ion-pair,and found that most of the salt bridges are stabi-lizing toward proteins;the majority of NÀÀO bridges are stabi-lizing;the majority of the longer-range ion-pairs are destabiliz-ing toward the proteins.45The salt bridge can be further distin-guished as hydrogen-bonded salt bridge(HB-salt bridge)and nonhydrogen-bonded salt bridge(NHB-salt bridge or salt bridge).46In2D-GraLab,the longer-range ion-pair is neglected, and for short-range ion-pair,four kinds are defined:HB-salt bridge,NHB-salt bridge or salt bridge,hydrogen-bonded NÀÀO bridge(HB-NÀÀO bridge),and nonhydrogen-bonded N-O bridge (NHB-NÀÀO bridge or NÀÀO bridge).Although both the N-terminal and C-terminal residues of a given protein are also charged,the large degree offlexibility usually experienced by the ends of a chain and the poor structural resolution resulting from it.47Therefore,we preclude these terminal residues in the 2D-GraLab.A modified Hendsch–Tidor’s method is used for calculating association energy of ion-pairs across binding interfaces.48D G assoc¼D G dslvþD G brd(4)where D G dslv represents the sum of the unfavorable desolvation penalties incurred by the individual ion-pairing residues due to the change in their environment from a high dielectric solvent (water)in the unassociated state;D G brd represents the favorable bridge energy due to the electrostatic interaction of the side-chain charged groups.We usedfinite difference solutions to the linearized Poisson–Boltzmann equations in Delphi26to calculate the D G dslv and D G brd.Centroid of the ion-pair system is used as grid center,with temperature of298.15K(in this way,1kT50.593kcal/mol),and the Debye-Huckel boundary conditions are applied.49Considering atomic parameter sets have a great influ-ence on the continuum electrostatic calculations of ion-pair asso-ciation energy,502D-GraLab provides three classical atomic parameter sets for users,including PARSE,AMBER,and CHARMM.51–53Figure4is the schematic representation of four ion-pairs formed across the binding interface of penicillin acylase enzyme complex.This diagram clearly illustrates the information about the geometries and energetics properties of ion-pairs,such as bond length,centroid distance,association energy,and angle. The ion-pair angle is defined as the angle between two unit vec-tors,and each unit vector joins a C a atom and a side-chain charged group centroid in an ion-pairing residue.54In this dia-gram,the four ion-pairs,two HB-salt bridges,and two HB-NÀÀO bridges formed across the binding interface are given out. Association energies of the HB-salt bridges are both\21.5 kcal/mol,whereas that of the HB-NÀÀO bridges are all[20.5 kcal/mol.Therefore,it is believed that HB-salt bridge is more stable than HB-NÀÀO bridge,which is well consistent with the conclusion of Kumar and Nussinov.45,46Side-Chain Conformational EntropyIn general,SCE can be divided into the vibrational and the con-formational.55Comparison of several sets of results using differ-ent techniques shows that during protein folding process,the mean conformational free energy change(T D S)is1kcal/mol per side-chain or0.5kcal/mol per bond.Changes in vibrational entropy appear to be negligible compared with the entropy change resulted from the loss of accessible rotamers.56SCE(S) can be calculated quite simply using Boltzmann’s formulation.57S¼ÀRXip i ln p i(5)where R is the universal gas constant;The sum is taken over all conformational states of the system and p i is the probability of being in state i.Typical methods used for SCE calculations, include self-consistent meanfield theory,58molecular dynam-ics,59Monte Carlo simulation,60etc.,that are all time-consum-ing,thus not suitable for2D-GraLab.For that,the case is sim-plified,when we calculate the SCE of an interfacial residue,its local surrounding isfixed(adopting crystal conformation).In this way,SCE of each interfacial residue is calculated in turn.For the20coded amino acids,Gly,Ala,Pro,and Cys in disulfide bonds are excluded.57For other cases,each residue’s side-chain conformation is modeled as a rotamer withfinite number of discrete states.61The penultimate rotamer library used was developed by Lovell et al.,62as recommended by Dun-brack for the study of SCE.63For an interfacial residue,the potential E i of each rotamer i is calculated in both binding state and unbinding state,and subsequently,rotamer’s probability dis-tribution(p)of this residue is resulted by Boltzmann’s distribu-tion law,then the SCE in different states are solved out using eq.(5).The situation of rotamer i is defined as serious clash or nonclash:serious clash is the clash score of rotamer i more than a given threshold value,and then E i511;whereas for the9452D Depiction of Nonbonding Interactions for Protein Complexes946Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure3.Schematic representation of desolvation effect for interfacial residues in chain A of HLA-A*0201complex(PDB entry:1duz).This diagram was produced using2D-GraLab.In this diagram,the pie chart is equally divided,with each section indicates an interfacial residue in chain A;In a sec-tor,red1blue is the SASA of corresponding residue in unbinding state,the blue is in binding state,and the red is thus of D SASA;The green polygonal line is made by linking desolvation free energy ofeach interfacial residue,and at the purple circle,desolvation free energy is0(D U50),beyond thiscircle indicates unfavorable contributions to binding(D U[0),otherwise is favorable(D U\0);Inthe periphery,residue symbols are colored in red,blue,and black in terms of favorable,unfavorable,and neutral contributions to the binding,respectively;The SASA and desolvation free energy for eachinterfacial residue can be measured qualitatively by the horizontally black and green scales.[Colorfigure can be viewed in the online issue,which is available at .]Figure4.Four ion-pairs formed across the binding interface of penicillin acylase enzyme complex (PDB entry:1gkf).In thisfigure,left is2D schematic diagram produced using2D-GraLab,and posi-tively and negatively charged residues are colored in blue and red,respectively;Bridge-bonds formed between the charged atoms of ion-pairs are colored in green,blue,and yellow dashed lines for the hydrogen-bonded bridge,nonhydrogen-bonded bridge,and long-range interactions,respectively;The three parameters in bracket are ion-pair type,angle,and association energy.The right in thisfigure is the spatial conformations of corresponding ion-pairs.[Colorfigure can be viewed in the online issue, which is available at .]Figure5.(a)Loss of side-chain conformational entropy of chain B interfacial residues in HIV-1 reverse transcriptase complex(PDB entry:1rt1).This diagram was produced using2D-GraLab.In this diagram,the pie chart is equally divided,with each section indicates an interfacial residue in chain B; In a sector,side-chain conformational entropies in unbinding and binding state are colored in yellow and blue,respectively;The green polygonal line is made by linking conformational free energy of each interfacial residue;The conformational entropy and conformational free energy for each interfa-cial residue can be measured qualitatively by the horizontally black and green scales,respectively;In the periphery,residue symbols are colored in yellow,blue,and black in terms of favorable,unfavora-ble,and neutral contributions to binding,respectively.(b)The rotamers of chain B interfacial residues Lys20,Lys22,Tyr56,Asn136,Ile393,and Trp401in HIV-1reverse transcriptase complex.These rotamers were generated using2D-GraLab.[Colorfigure can be viewed in the online issue,which is available at .]9472D Depiction of Nonbonding Interactions for Protein Complexes948Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure5.(Legend on page947.)Figure6.The summarized schematic diagram of nonbonding interactions and disulfide bond across the interface of AIV hemagglutinin H5complex(PDB entry:1jsm).Length of chain A and chain B are321and160,represented as two bold horizontal lines.Interface parts in the bold lines are colored in orange,and residue-pairs in interactions are linearly linked;Conventional hydrogen bond,water-mediated hydrogen bond,ionpair,hydrophobic force,steric clash,p–p stacking,and disulfide bond are colored in aqua,bottle green,red,blue,purple,yellow,and brown,respectively;In the‘‘dumbbell shape’’symbols,residue-pair types and distances are also presented.[Colorfigure can be viewed in the online issue,which is available at .]9492D Depiction of Nonbonding Interactions for Protein Complexescase of nonclash,four potential functions are used in2D-Gra-Lab:(i)E i5E0,a constant61;(ii)statistical potential,the poten-tial energy E i of rotamer i is calculated from database-derived probability61;(iii)coarse-grained model,E i of rotamer i is esti-mated by atomic contact energies(ACE)64;and(iv)Lennard-Jones potential.58Loss of binding entropy of chain B interfacial residues in HIV-1reverse transcriptase complex is schematically repre-sented in Figure5a.Similar to desolvation effect diagram,loss of binding entropy is also presented in a rotiform diagram form. This diagram reveals that during the process of forming HIV-1 reverse transcriptase complex,the total loss of conformational free energy of chain B is9.14kcal/mol,indicating a strongly unfavorable contribution to binding(D G[0),and the average loss of conformational free energy for each residue is about0.3 kcal/mol,much less than those in protein folding(about1kcal/ mol56).Figure5b shows the rotamers of six interfacial residues in chain B.Summarized Schematic DiagramFigure6illustrates nonbonding interactions and disulfide bond formed across the binding interface of avian influenza virus (AIV)hemagglutinin H5.This protein is a dimer linked by a disulfide bond.In this diagram,conventional hydrogen bond, water-mediated hydrogen bond,ion-pair,hydrophobic force, steric clash,p–p stacking,and disulfide bond are represented in different colors.Hydrogen bonds,colored in aqua,are calculated by program HBplus.23Data in this diagram are the separation between the acceptor atom and the heavy donor atom.Water-mediated hydrogen bonds are colored in bottle green, also calculated by HBplus.23Ion-pairs,colored in red,include salt bridge and NÀÀO bridge,determined by the Kumar’s rule.45,46Data in this dia-gram are centroid distance of ion-pair.Hydrophobic forces are colored in blue.According to the D SASA rule,if the two apolar and/or aromatic interfacial resi-dues(Leu,Ala,Val,Ile,Met,Cys,Pro,Tyr,Phe,and Trp)are within the distance d\r A1r B12.8(r A and r B are side-chain radii,2.8is the diameter of water molecule),they are considered in hydrophobic contact.Data in this diagram are centroid–cent-roid separation between the two residues.Steric clashes are colored in purple.Here,only bad overlaps calculated by Probe24are presented.In2D-GraLab,explicit and implicit hydrogen modes are provided,hydrogen atoms in explicit hydrogern mode are added using Reduce.22Data in this diagram are the centroid–centroid separation when the two atoms are badly overlapped.p–p stacking are colored in yellow.Presently,studies on pro-tein stacking interactions are in lack.In2D-GraLab,p–p stack-ing is identified using the McGaughey’s rule,65i.e.,if the cent-roid–centroid separation between two aromatic rings is within 7.5A˚,they are regarded as p–p stacking(aromatic residues are Phe,Tyr,Trp,and His).This rule has been successfully adopted to study the p–p stacking across protein interfaces by Cho et al.66Besides,2D-GraLab also sets the constraints of stacking angle(dihedral angel between the planes of two aromatic rings).Data in this diagram are centroid–centroid separations between two aromatic rings in stacking state.Disulfide bonds are colored in brown,taken from the PDB records.Data in this diagram are the separations of two sulfide atoms.ConclusionsMost,if not all,biological processes are regulated through asso-ciation and dissociation of protein molecules and essentially controlled by nonbonding energetics.67Graphically-intuitive vis-ualization of these nonbonding interactions is an important approach for understanding the mechanism of a complex formed between two proteins.Although a large number of software packages are available for visualizing the3D structures,the options for producing schematic2D summaries of nonbonding interactions for a protein complex are comparatively few.In practice,the2D and3D visualization methods are complemen-tary.In this article,we have described a new2D molecular graphics tool for analyzing and visualizing PPIs from spatial structures,and the intended goal is to schematically present the nonbonding interactions stabilizing the macromolecular complex in a graphically-intuitive manner.We anticipate that renewed in-terest in automated generation of2D diagrams will significantly reduce the burden of protein structure analysis and make insights into the mechanism of PPIs.2D-GraLab is written in C11and OpenGL,and the output-ted2D schematic diagrams of nonbinding interactions are described in PostScript.Presently,2D-GraLab v1.0is available to academic users free of charge by contacting us. References1.Chothia,C.;Janin,J.Nature1974,256,705.2.Jones,S.;Thornton,J.M.Proc Natl Acad Sci USA1996,93,13.3.Luscombe,N.M.;Laskowski,R.A.;Westhead,D.R.;Milburn,D.;Jones,S.;Karmirantzoua,M.;Thornton,J.M.Acta Crystallogr D 1998,54,1132.4.DeLano,W.L.The PyMOL Molecular Graphics System;DeLanoScientific:San Carlos,CA,2002.5.Petrey,D.;Honig,B.Methods Enzymol2003,374,492.6.Humphrey,W.;Dalke,A.;Schulten,K.J Mol Graphics1996,14,33.7.Gabdoulline,R.R.;Wade,R.C.;Walther,D.Nucleic Acids Res2003,31,3349.8.Gabdoulline,R.R.;Hoffmann,R.;Leitner,F.;Wade,R.C.Bioin-formatics2003,19,1723.9.Wade,R. C.;Gabdoulline,R.R.;De Rienzo, F.Int J QuantumChem2001,83,122.10.Wallace, A. C.;Laskowski,R. A.;Thornton,J.M.Protein Eng1995,8,127.11.Stierand,K.;Maaß,P.C.;Rarey,M.Bioinformatics2006,22,1710.12.Clark,A.M.;Labute,P.J Chem Inf Model2007,47,1933.13.Luscombe,N.M.;Laskowski,R. A.;Thorntonm J.M.NucleicAcids Res1997,25,4940.14.Salerno,W.J.;Seaver,S.M.;Armstrong,B.R.;Radhakrishnan,I.Nucleic Acids Res2004,32,W566.15.Fischer,T.B.;Holmes,J.B.;Miller,I.R.;Parsons,J.R.;Tung,L.;Hu,J.C.;Tsai,J.J Struct Biol2006,153,103.950Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational Chemistry。
细菌学:第十章 细菌基因组学课件
意外的发现
• 另外,此前科学界一致认为鸡没有嗅觉 ,但是分析结果表明鸡具有大量的嗅觉 基因,味觉基因却很缺乏。
• 分析还发现,鸡缺乏人类所具有的产生 乳汁、唾液和牙齿的基因。
鸡基因组研究的意义
• 鸡是研究低等脊椎动物和人类等哺乳动物 的一种比较理想的中介。
• 将人类基因组与鸡等其他生物的基因组进 行比较,有助于更深入理解人类基因的结 构和功能,进而开发治疗疾病的新手段, 对于培育优质鸡种、改善食品安全、控制 禽流感病毒的蔓延也有重要意义。
1. 原核生物基因组的大小--基因组较大的原
• 1997 年9 月,大肠杆菌的完整基因图谱已绘制成 功, 基因组全序列完成, 全长为5Mb ,共有4 288 个基因,同时也搞清了所有基因产物的氨基酸序 列.
• 人们常说,每个分子生物学家都对两种生物感 兴趣,一种是所研究的物种,另一种就是E. coli。研究人员可以利用实验室中的E. coli菌株 克隆DNA、表达蛋白质、分离目的基因等,如 果没有E. coli,实验室将无法工作。
测序微生物的类别
• 几乎所有类别的病毒 • 模式微生物 • 极端环境微生物 • 病原原核生物 • 环境降解微生物 • 其他
Viruses
微生物基因组的特点
类别
特征
染色体结构 基因组大小 编码序列
多为一条环状闭合双链DNA 从0.16-13Mb 占基因组总长度的90%,平均为1Kb左 右
GC含量
鸡的进化研究
• 鸡是种常见的家禽,长期受到进化生物学家的 青睐。它的基因序列也有助于科学家了解农业 和进化学上重要特性的遗传学基础。
转基因小鸡
• 对鸡和人类的基因组进行比较后发现约 七千万个碱基对是共有的。
• 这暗示着在大约三亿一千万年前二个物 种从共同祖先分化出来的时候,遗传物 质具有守恒性。
研究基因功能的遗传方法
研究基因功能的遗传方法Studying the genetic function of genes is crucial in understanding how our bodies work and how diseases develop. 研究基因功能对于了解人体的运作方式以及疾病如何发展具有至关重要的意义。
Genetic methods provide a powerful tool through which we can investigate the roles of specific genes in various biological processes. 遗传方法为我们提供了一个强大的工具,可以研究特定基因在各种生物过程中的作用。
From classical genetic studies involving crosses of different organisms to modern molecular techniques like CRISPR/Cas9, there are a multitude of approaches available to researchers. 从涉及不同组织交叉的经典遗传研究到像CRISPR/Cas9这样的现代分子技术,研究人员有许多方法可供选择。
One of the fundamental genetic methods is gene knockout, where a specific gene is intentionally deactivated to study its function. 基因敲除是基本的遗传方法之一,它是一种有意使某个特定基因失活以研究其功能的方法。
In this technique, researchers can create knockout animals or cell lines to observe the effects of lacking that particular gene. 在这种技术中,研究人员可以制造敲除动物或细胞系,以观察缺乏该特定基因的影响。
Tc-99m-MIBI serve as a predictor of chemotherapy response in malignant lymphomas compared with P-gP
British Journal of Haematology ,2001,113,369±374Technetium-99m-sestamethoxyisobutylisonitrile scan as a predictor of chemotherapy response in malignant lymphomas compared with P-glycoprotein expression,multidrug resistance-related protein expression and other prognosis factorsChia-Hung Kao,1Shih-Chuan Tsai,2Jhi-Joung Wang,3Yung-Jen Ho,4Shung-Tai Ho 5and Sheng-Ping Changlai 61Department of Nuclear Medicine,Taichung Veterans General Hospital,Taichung,2Department of Nuclear Medicine,Show-Chwan Memorial Hospital,Chunghua,3Department of Medical Research,Chi-Mei Medical Centre,Tainan,4Department of Radiology,Jen-Ai Hospital,Taichung,5School of Medicine,National Defence Medical Centre,Taipei,and 6Department of Nuclear Medicine,Chung-Shan Medical and Dental Hospital,Taichung,TaiwanReceived 13November 2000;accepted for publication 14January 2001Summary .The purpose of the present study was to predict the response of malignant lymphomas (MLs)to chemotherapy usingtechnetium-99m methoxyisobutylisonitrile (Tc-MIBI)scan and to compare it with the predictive ability of P-glycoprotein (P-gp)expression,multidrug resistance-related protein (MRP)expression and other prognosis factors.Twenty-five ML patients were enrolled in this study prior to initiation of chemotherapy .Images were obtained 10min after intravenous injection of Tc-MIBI,interpreted visually and the tumour-to-background (T/B)ratios calculated.Immunohistochemical analyses were performed on sections of the biopsy specimens to determine P-gp and MRP expression.Chemotherapy response was evaluated in the first 1±2years after completion of chemotherapy .The mean T/B ratio of the 15patients with agood response (3´3^0´6)was significantly higher than that of the 10patients with a poor response (1´2^0´1).All 15patients with a good chemotherapy response had positive Tc-MIBI scan results and negative P-gp and MRPexpression.All 10patients with a poor response had negative Tc-MIBI scan results and either positive P-gp or MRP expression.Other prognosis factors showed no significant difference in the incidence of good and poor responses.Tc-MIBI scan results represent P-gp or MRP expression more accurately than other prognosis factors and predict the chemotherapy response in ML patients.Keywords:malignant lymphoma,technetium-99m methoxy-isobutylisonitrile,chemotherapy response,P-glycoprotein expression,multidrugresistance-related protein expression.Chemotherapy is the primary therapeutic modality for many malignant lymphomas (MLs)including all non-Hodgkin's lymphoma (NHL)cases and many cases of Hodgkin's disease (HD)(Barr et al ,1997;Neal &Hoskin,1997;Wilson &Chabner,1998).As resistance to chemotherapeutic agents is a major cause of treatment failure,the goal of chemotherapy for ML is to avoid possible resistance and achieve the highest response.The mechanism of tumour uptake of technetium-99m methoxyisobutylisonitrile (Tc-MIBI)may involve bindingto the cytosol of the tumour cell (Hassan et al ,1989).The cationic charge and lipophilicity of Tc-MIBI,mitochondrial and plasma membrane potentials of tumour cells,and cellular mitochondrial content can all play a significant role in tumour uptake of this agent (Chiu et al ,1990),or the uptake may be caused by indirect phenomena such as increased tumour blood flow and capillary permeability .Tc-MIBI scans have been used to successfully predict the chemotherapy response of MLs (Kapucu et al ,1997;Shih et al ,1998).However,the previous studies have not compared the relationship between Tc-MIBI scan resultsq 2001Blackwell Science Ltd369Correspondence:Chia-HungKao,MD,Department of Nuclear Medicine,TaichungVeterans General Hospital,160Taichung Harbour Road,Section 3,Taichung407,Taiwan.E-mail:kaoch@ .twand P-glycoprotein(P-gp)or multidrug resistance-related protein(MRP)expression in predictingthe chemotherapy response of MLs.Therefore,the aim of this study was to compare Tc-MIBI scan results,immunohistochemical ana-lyses of P-gp and MRP expression,and other prognosis factors as predictors of chemotherapy response in ML patients.PATIENTS AND METHODSPatients.Twenty-five patients(13men,12women;age range25±65years;mean age:46´2^12´3years)with ML(11with HD and14with NHL)were included in the study and underwent Tc-MIBI scans prior to chemotherapy (Table I).The classification of ML followed the Lukes and Butler and updated Kiel systems(Jaffe,1998).After Tc-MIBI scans,the11HD patients received chemotherapy regimens with nitrogen mustard(mechlorethamine),vincristine, procarbazine and prednisone(MOPP),alternatingwith doxorubicin,bleomycin,vinblastine and dacarbazine (ABVD);the14NHL patients received chemotherapy regimens with cyclophosphamide,doxorubicin,vincristine and prednisone(CHOP)protocols(Barr et al,1997;Neal& Hoskin,1997;Wilson&Chabner,1998).Technetium-99m methoxyisobutylisonitrile scan.The ima-ging procedure began30min after oral intake of500mg of perchlorate to prevent any abnormal uptake of free Tc-99m pertechnetate.A commercial MIBI preparation(max. 5´56GBq in approximately1±3ml)was obtained from The Du Pont Merck Pharmaceutical Company(Cardiolite, Billerica,MA,USA).The labellingand quality control procedures were carried out accordingto the manufac-turer's bellingefficiencies were all.95%. Each patient was place in a supine position on the imaging table with the chest strapped to prevent motion. Because of physiological Tc-MIBI accumulation in abdom-inal and pelvic organs,visualization of MLs located in abdominal and pelvic regions is unreliable.In this study, images of supradiaphragmatic MLs were obtained10min after intravenous injection of740MBq Tc-MIBI in the anterior and posterior projection.The equipment consisted of a large field-of-view gamma camera fitted with a low-energy,high-resolution collimator.A single20%energy window was set at140keV and500K counts were obtained for each static image.Tumour-to-background(T/ B)ratios were calculated as the mean counts over the region of interest(ROI)of the tumour outlined in the largest lesion4the mean counts over the ROI ofTable I.Detailed data of patients in this study.Case Tc-MIBI scan results Immunohistochemical stainingAge BChemotherapyresponsenumber Sex T/B ratio Visual interpretation P-gp expression MRP expression(years)Type Stage symptoms results1Female1´0Negative Positive Negative33HD I Yes Poor2Female1´0Negative Negative Positive53NHL III No Poor3Female1´1Negative Positive Negative40HD II Yes Poor4Male1´1Negative Positive Negative51NHL III No Poor5Male1´1Negative Negative Positive62HD IV No Poor6Male1´2Negative Positive Negative35NHL IV No Poor7Male1´2Negative Negative Positive55HD IV Yes Poor8Female1´3Negative Positive Negative27NHL III Yes Poor9Male1´3Negative Negative Positive65NHL III Yes Poor10Male1´4Negative Positive Negative43NHL II Yes Poor11Female2´4Positive Negative Negative43HD II Yes Good12Male2´7Positive Negative Negative56HD IV No Good13Male2´8Positive Negative Negative37NHL III No Good14Male2´9Positive Negative Negative55HD I Yes Good15Male2´9Positive Negative Negative62HD IV No Good16Male3´0Positive Negative Negative61NHL III No Good17Female3´2Positive Negative Negative31NHL III No Good18Male3´2Positive Negative Negative47HD IV No Good19Female3´3Positive Negative Negative60HD III Yes Good20Male3´3Positive Negative Negative25NHL II Yes Good21Female3´6Positive Negative Negative30HD II Yes Good22Female3´6Positive Negative Negative58NHL IV No Good23Male4´0Positive Negative Negative35HD IV No Good24Female4´1Positive Negative Negative50NHL III No Good25Male4´5Positive Negative Negative42NHL II Yes GoodHD,Hodgkin's disease;NHL,non-Hodgkin's lymphoma;P-gp,P-glycoprotein;MRP,multidrug resistance-related protein;Tc-MIBI, technetium-99m methoxyisobutylisonitrile;T/B,tumour-to-background.370 C.-H.Kao et alq2001Blackwell Science Ltd,British Journal of Haematology113:369±374background,defined as the contralateral normal side for the neck and axilia lesions or normal soft tissue of the thorax for mediastinal lesions.Tc-MIBI uptake in the lesions $axillary soft tissue background,based on the visual interpretation of at least two experienced nuclear medicine physicians,was considered a positive Tc-MIBI scan result (Figs 1and 2).Immunohistochemical staining.Formalin-fixed paraffin sections (5m m)were deparaffinized in an oven at 508C for 40min,then hydrated with varyingconcentrations of ethanol±water dilutions.For MRP immunohistochemical staining,antigen retrieval was performed by treatment in citrate buffer in a 700W microwave oven for 5min.Endogenous peroxidase was blocked by 3%hydrogen peroxide for 15min,followed by 5min in phosphate-buffered saline (PBS).The sections were incubated over-night in a moist chamber at 48C with primary antibody MRP QCRL-1(10m g/ml,Signet Laboratories,Dedham,MA,USA)at 1:100concentration.For P-gp immunohis-tochemical staining,endogenous peroxidase was blocked by 3%hydrogen peroxide for 15min.Antigen retrieval was performed by treatment with enzyme digestion in 0´1%trypsin in PBS for 5min at room temperature and inhibited with 10%skimmed milk in PBS for 5min.The sections were incubated for 2h in a moist chamber at 378C with primary antibody JSB-1(50m g/ml,Boehringer Mannheim Biochemica,Germany)at 1:50concentration.After three 5min washes in PBS,detection of the primary antibody was performed with a link antibody accordingto the manufacturer's instructions (DAKO LSAB_2System,Peroxidase,Dako Corporation,Carpinteria,CA,USA)(Niehans et al ,1992;Marie,1995;Yamaguchi et al ,1995;Kostakoglu et al ,1998;Webb et al ,1998).P-gp and MRP expressions were interpreted by an experienced pathologist blind to clinical outcome as follows:negati-ve less than 10%,positive 10%or more stained tumour cells (Figs 3and 4).Chemotherapy response evaluation.In this study ,the chemotherapy response of each patient was evaluated for the first 1±2years after completion of treatment using clinical and radiological methods such as plain chest X-ray ,chest computerized tomography (CT)or magnetic resonance imaging (MRI),as well as head and neck CT or MRI,accordingto the followingscale:(1)Complete response no evidence of disease,(2)Partial respon-se at least 50%decrease in the sum of the products of the maximum perpendicular diameters of all measurable lesions,no evidence of progression in any lesion and no new lesions,(3)No response less than 25%increase in the sum of the products of the maximum perpendicular diameters of all measurable lesions,no evidence of progression in any lesion and no new lesions,and (4)Progressive disease at least 25%increase in the sum of the products of the maximum perpendicular diameters of all measurable lesions and/or the appearance of new lesions.We defined complete and partial responses as good response,while no response and progressive disease were defined as poor response.Statistical analyses.The T/B ratio was expressed as meanT a b l e I I .D i s t r i b u t i o n s o f T c -M I B I s c a n r e s u l t s ,P -g p e x p r e s s i o n ,M R P e x p r e s s i o n ,a g e ,t u m o u r t y p e ,t u m o u r s t a g e a n d B s y m p t o m s r e l a t e d t o c h e m o t h e r a p y r e s p o n s e r e s u l t s .I m m u n o h i s t o c h e m i c a l s t a i n i n gC h e m o t h e r a p y r e s p o n s e T c -M I B I s c a n r e s u l t s P -g p e x p r e s s i o nM R P e x p r e s s i o nA g e T y p e S t a g eB s y m p t o m sr e s u l t s P o s N e g P -v a l u eP o s N e g P -v a l u e P o s N e g P -v a l u e#40y e a r s .40y e a r s P -v a l u e H D N H L P -v a l u e I ±I I I I I ±I V P -v a l u e Y e s N o P -v a l u e G o o d1500150155107851069P o o r 010,0´0164,0´0146,0´01460´73460´74370´86640´33q 2001Blackwell Science Ltd,British Journal of Haematology 113:369±374Tc-MIBI Predicts Chemotherapy Response in HD and NHL371^standard deviation (SD).A Mann±Whitney U -test was used to evaluate the difference in T/B ratios between patients with a good versus a poor response.The difference in incidence of good and poor response was evaluated for eight possible prognosis factors:positive versus negative Tc-MIBI scan results,positive versus negative P-gp expression,positive versus negative MRP expression,HD versus NHL,stage I±II versus stage III±IV ,age .40years versus #40years,and with B symptoms versus without B symptoms (night sweats,fever .388C for three consecutive days and unexplained weight loss of .10%body weight)(Barr et al ,1997;Neal &Hoskin,1997).A Chi-square test was used to determine if the frequency of good and poor response was the same for each pair.If the P -value was ,0´05,the difference was considered significant.RESULTSDetailed patient data are shown in Table I.The mean T/B ratio of the 15patients with a good response (3´3^0´6)was significantly (P ,0´01)higher than that of the 10patients with a poor response (1´2^0´1).All 15(100%)patients with a good response had positive Tc-MIBI scan results and negative P-gp and MRP expression.All 10(100%)patients with a poor response had negativeTc-MIBI scan results,six (60%)of whom had positive P-gp expression while the other four (40%)had positive MRP expression.Tc-MIBI scan results,P-gp expression and MRP expression all showed significant differences in the rate of good and poor responses.However,no significant difference in the incidences of good and poor responses was found for lymphoma type,stage,age or B symptoms (Table II).DISCUSSIONOur review of previous literature found only one paper that reported that 17ML children with positive Tc-MIBI scan results and a higher mean T/B ratio had a better response to chemotherapy than seven ML children with negative Tc-MIBI scan results and a lower mean T/B ratio (Kapucu et al ,1997).Our results support their findings.However,their study did not examine the relationship between other prognosis factors,P-gp or MRP expression,and chemo-therapy response.The mechanism of chemotherapy resistance in ML is thought to involve expression of P-gp and MRP (Niehans et al ,1992;Yamaguchi et al ,1995;Zhan et al ,1997;Webb et al ,1998).The retention of Tc-MIBI in tumour cells depends on P-gp and MRP expression and they function as ATP-dependent efflux pumps for manychemotherapyFig 1.Case no.25had a goodchemotherapy response result.(A)Tc-MIBI scan reveals significant tracer uptake in the right neck and the result is positive (T/B ratio 4´5)(arrow).(B)Neck computerized tomography shows a mass in the same area(arrow).Fig 2.Case no.4had a poorchemotherapy response result.(A)Tc-MIBI scan reveals no definitely abnormal tracer MIBI uptake in the chest and neck and the result is negative (T/B ratio 1´1).(B)Gallium-67citrate scan shows multiple abnormal tracer uptake in the chest and neck (arrows).372 C.-H.Kao et alq 2001Blackwell Science Ltd,British Journal of Haematology 113:369±374agents (Hendrikse et al ,1998,1999;Vergote et al ,1998;Sun et al ,2000).Therefore,in this study we used the Tc-MIBI scan to predict the response of MLs to chemother-apy .We found that positive Tc-MIBI scan results accurately predicted all good chemotherapy results,which were also related to negative P-gp and MRP expression.Moreover,negative Tc-MIBI scan results accurately predicted poor chemotherapy results in all patients with positive P-gp or MRP expression (Table I).In our previous studies,only early chest images performed 10min after intravenous injection of Tc-MIBI proved to be accurate enough to predict chemotherapy response in lung and breast cancer (Kao et al ,1998,2000;Vergote et al ,1998).Therefore,in this study ,we did not consider it necessary to perform delayed chest imaging to calculate the tumour washout rate or retention index of Tc-MIBI to predict the chemotherapy response.mRNA expression is not fully corrected with P-gp or MRP expression in the tumour cell membrane,Tc-MIBI tumour uptake is directly based onthe P-gp or MRP expression in the tumour cell membrane,and it was impossible to extract mRNA from the formalin-fixed paraffin sections of biopsy specimens (Wang et al ,1997;Dexter et al ,1998;Yokogami et al ,1998).Therefore,we directly detected P-gp or MRP expression using immunostainingto correct with Tc-MIBI tumour uptake (T/B ratio)in our study .Based on our findings,we conclude that Tc-MIBI scan results can represent P-gp and MRP expression for predict-ingthe chemotherapy response in ML patients.However,further studies includinglarg er case numbers and patients who have relapsed followingchemotherapy are necessary to confirm our findings.ACKNOWLEDGMENTSThis work was supported in part by grants from Taichung Veterans General Hospital (TCVGH-896708D)andNationalFig 3.Immunohistochemistry performed on sections of malignant lymphomaspecimens from two different groups reveals (A)negative and (B)positive P-gp expression (Â500).Fig 4.Immunohistochemistry performed on sections of malignant lymphomaspecimens from two different groups reveals (A)negative and (B)positive MRP expression (Â1000).q 2001Blackwell Science Ltd,British Journal of Haematology 113:369±374Tc-MIBI Predicts Chemotherapy Response in HD and NHL 373Science Council(NSC89±2314-B-075A-015,89±2320-B-075A-001,88±2314-B-075A-006),Taiwan.REFERENCESBarr,L.,Cowan,R.&Nicolson,M.(1997)Haematological Malignancies.In:Oncology(ed.by L.Barr,R.Cowan&M. Nicolson),p.150.Churchill Livingstone,New York.Chiu,M.L.,Kronauge,J.F.&Piwnica-Worms,D.(1990)Effect of mitochondrial and plasma membrane potentials on accumula-tion of hexakis(2-methoxyisobutylisonitrile)technetium(I)in cultured mouse fibroblast.Journal of Nuclear Medicine,31,1646±1653.Dexter,D.W.,Reddy,R.K.,Geles,K.G.,Bansal,S.,Myint,M.A., Rogakto,A.,Leighton,J.C.&Goldstein,L.J.(1998)Quantitative reverse transcriptase-polymerase chain reaction measured expression of MDR1and MRP in primary breast carcinoma. Clinical Cancer Research,4,1533±1542.Hassan,I.M.,Sahweil,A.,Constantinides,C.,Mahmoud,A.,Nair, M.,Omar,Y.T.&Abdel-Dayem,H.M.(1989)Uptake and kinetics of Tc-99m hexakis2-methoxy isobutyl isonitrile in benign and malignant lesions in the lungs.Clinical Nuclear Medicine,14, 333±340.Hendrikse,N.H.,Franssen,E.J.,van-der-Graaf,W.T.,Meijer,C., Piers, D.A.,Vaalburg,W.&de-Vries, E.G.(1998)99mTc-sestamibi is a substrate for P-glycoprotein and the multidrug resistance-associated protein.British Journal of Cancer,77,353±358.Hendrikse,N.H.,Franssen,E.J.,van-der-Graaf,W.T.,Vaalburg,W.& de-Vries,E.G.(1999)Visualization of multidrugresistance in vivo.European Journal of Nuclear Medicine,26,283±293. Jaffe,E.S.(1998)Histopathology of the non-Hodgkin's lymphomas and Hodgkin's disease.In:The Lymphomas(ed.by G.P.Canellos, T.A.Lister&J.L.Sklar),p.77.Saunders,Philadelphia.Kao, C.H.,ChangLai,S.P.,Chieng,P.U.&Yen,T.C.(1998) Technetium-99m methoxyisobutylisonitrile chest imaging of small cell lungcarcinoma.Relationship to patient prog nosis and chemotherapy response±a preliminary report.Cancer,83, 64±68.Kao,C.H.,Hsieh,J.F.,Tsai,S.C.,Ho,Y.J.&Lee,J.K.(2000)Quickly predictingchemotherapy response to paclitaxel-based therapy in non-small cell lungcancer by early technetium-99m methox-yisobutylisonitrile chest single-photon-emission computed tomo-graphy.Clinical Cancer Research,6,820±824.Kapucu,L.O.,Akyuz, C.,Vural,G.,Oguz, A.,Atasever,T., Buyukpamukcu,M.&Unlu,M.(1997)Evaluation of therapy response in children with untreated malignant lymphomas using technetium-99m-sestamibi.Journal of Nuclear Medicine,38,243±247.Kostakoglu,L.,Guc,D.,Canpinar,H.,Kars,A.,Alper,E.,Kiratli,P., Hayran,M.,Gunduz,U.&Kansu,E.(1998)P-glycoproteinexpression by technetium-99m-MIBI scintigraphy in hematologic malignancy.Journal of Nuclear Medicine,39,1191±1197. Marie,J.P.(1995)P-glycoprotien in adult hematologic malignan-cies.Hematology Oncology Clinics of North America,9,239±249. Neal,A.J.&Hoskin,P.J.(1997)Lymphoma.In:Clinical Oncology: Basic Principle and Practice(ed.by A.J.Neal&P.J.Hoskin),p.162. Arnold,London.Niehans,G.A.,Jaszca,W.,Brunetto,V.,Perri,R.T.,Gajl-Peczalska, K.,Wick,M.R.,Tsuruo,T.&Bloomfield,C.D.(1992)Immuno-histochemical identification of P-glycoprotein in previously untreated,diffuse large cell in immunoblastic lymphomas.Cancer Research,52,3768±3775.Shih,W.J.,Rastogi,A.,Stipp,V.,Magoun,S.&Coupal,J.(1998) Functional retention of Tc-99m MIBI in mediastinal lymphomas as a predictor of chemotherapeutic response demonstrated by consecutive thoracic SPECT imaging.Clinical Nuclear Medicine, 23,505±508.Sun,S.S.,Hsieh,J.F.,Tsai,S.C.,Ho,Y.J.,Lee,J.K.&Kao,C.H.(2000) Expression of mediated p-glycoprotein multidrug resistance related to Tc-99m MIBI scintimamography results.Cancer Letters, 153,95±100.Vergote,J.,Moretti,J.L.,de-Vries,E.G.&Garnier-Suillerot,A.(1998) Comparison of the kinetics of active efflux of99mTc-MIBI in cells with P-glycoprotein-mediated and multidrug-resistance protein-associated multidrug-resistance phenotypes.European Journal of Biochemistry,252,140±146.Wang,C.S.,LaRue,H.,Fortin,A.,Gariepy,G.&Tetu,B.(1997) mdr1mRNA expression by RT-PCR in patients with primary breast cancer submitted to neoadjuvant therapy.Breast Cancer Research and Treatment,45,63±74.Webb,M.,Brun,M.,McNiven,M.,Le-Couteur,D.&Craft,P.(1998) MDR1and MRP expression in chronic B-cell lymphoproliferative disorders.British Journal of Haematology,102,710±717. Wilson,W.H.&Chabner,B.A.(1998)Principles of chemotherapy for lymphomas.In:The Lymphomas(ed.by G.P.Canellos,T.A. Lister&J.L.Sklar),p.235.Saunders,Philadelphia. Yamaguchi,M.,Kita,K.,Miwa,H.,Nishii,K.,Oka,K.,Ohno,T., Shirakawa,S.&Fukumoto,M.(1995)Frequent expression of P-glycoprotein/MDR1by nasal T-cell lymphoma cells.Cancer,76, 2351±2356.Yokogami,K.,Kawano,H.,Moriyama,T.,Uehara,H.,Sameshima,T., Oku,T.,Goya,T.,Wakisaka,S.,Nagamachi,S.,Jinnouchi,S.& Tamura,S.(1998)Application of SPET usingtechnetium-99m sestamibi in brain tumours and comparison with expression of the MDR-1gene:is it possible to predict the response to chemotherapy in patients with gliomas by means of99mTc-sestamibi SPET? European Journal of Nuclear Medicine,25,401±409.Zhan,Z.,Sandor,V.A.,Gamelin,E.,Regis,J.,Dickstein,B.,Wilson, W.,Fojo,A.T.&Bates,S.E.(1997)Expression of the multidrug resistance-associated protein gene in refractory lymphoma: quantitation by a validated polymerase chain reaction assay. 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【课件】生物化学合工大第三章酶精编版
• 1913Michaelis和Menten提出米氏学 说—酶促动力学原理。
• 1926Sumner首次从刀豆中提出脲酶结 晶,并证明具有蛋白质性质。
• 1930年Northrop等得到了胃蛋白酶、 胰蛋白酶和胰凝乳蛋白酶的结晶,并进 一步证明了酶是蛋白质。
J.B.Sumner
辅助因子
辅基 与酶蛋白结合紧密的小分子有机物。 金属离子 金属离子作为辅助因子。
酶蛋白和辅助因子单独存在均无催化活性,只有二者 结合为全酶才有催化活性。
• 参与的酶促反应主要为氧化-还原反 应或基团转移反应。
• 大多数辅酶的前体主要是水溶性 B 族维生素。许多维生素的生理功能 与辅酶的作用密切相关。
J.H.Northrop
•
1969 化学合成核糖核酸酶。 1967-1970 从E.coli 中发现第I、第II类限制性
核酸内切酶。
•
20世纪80年代发现某些RNA有催化活性, 还有一些抗体也有催化活性,甚至有些DNA 也有催化活性,使酶是蛋白质的传统概念受 到很大冲击。
·某些RNA有催化活性( ribozyme,核酶)
1982年美国T. Cech等人发现•四膜虫的rRNA前 体能在完全没有蛋白质的情况下进行自我加工,发 现RNA有催化活性 。
Thomas Cech University of Colorado at Boulder, USA
•
·1983年美国S.Altman等研究RNaseP(由20%蛋 白质和80%的RNA组成),发现 R前体加工。
……
二、酶的概念
酶是一类由活性细胞产生的具有催化作 用和高度专一性的特殊蛋白质或核酸。 简单说,酶是一类由活性细胞产生的生 物催化剂。
铁死亡调节剂与顺铂毒性研究的最新进展
铁死亡调节剂与顺铂毒性研究的最新进展摘要:顺铂(CDDP)是第一种用于抗肿瘤药的重金属化合物,但它所导致的顺铂毒性(cisplatin-induced toxicity)使患者的生活质量大打折扣,这一点严重局限了CDDP的临床应用。
故预防和治疗肿瘤化疗后顺铂毒性成为了一个重要课题。
铁死亡(ferroptosis)是科学家们近几年发现的一种新型的非凋亡调控的细胞死亡方式,其特点是铁依赖的脂质过氧化物的积累。
越来越多的文献报道铁死亡参与了多种恶性肿瘤顺铂化疗过程,应用铁死亡调节剂(ferroptosis regulator)可以在一定程度上影响顺铂毒性。
因此,本文将对铁死亡调节剂的相关分子机制及其在顺铂诱导的耳毒性、肾毒性和细胞毒性研究中的应用和影响进行综述,旨在为临床实践中预防和治疗顺铂毒性寻找理论依据和靶标。
关键词:顺铂毒性;铁死亡调节剂;分子机制众所周知,顺铂(CDDP)是一种广泛应用于临床的抗肿瘤药物,在卵巢癌,乳腺癌,骨肉瘤,肺癌,头颈癌等恶性肿瘤都表现出显著疗效[1]。
伴随着化疗剂量的升高,顺铂的治疗效果越来越好,但其毒副作用也越来越强,这严重影响癌症患者的愈后,大大限制了它的临床应用。
顺铂毒性(cisplatin-induced toxicity)主要表现为肾毒性(nephrotoxicity)、耳毒性(ototoxicity)和细胞毒性(cytotoxicity)[2,3,4]。
导致顺铂毒性很难治愈或预防的主要原因就是它的具体分子机制尚不清楚。
因此,进一步探究顺铂毒性发生机制,发现新的治疗靶点,寻找有效的防治方法和药物已经迫在眉睫。
2012年Dixon等人提出了一种在形态学,生物化学和遗传学上与细胞凋亡和坏死不同的新型细胞死亡方式—铁死亡(ferroptosis),它是铁依赖性的,由活性氧(ROS)和脂质过氧化物(LPO)的积累介导[5]。
铁死亡可以由许多小分子化合物诱导和抑制,涉及参与许多疾病,在恶性肿瘤顺铂化疗过程中也发挥重大作用。
Method and apparatus for computer modeling of the
专利名称:Method and apparatus for computermodeling of the interaction between andamong cortical and subcortical areas in thehuman brain for the purpose of predictingthe effect of drugs in psychiatric andcognitive diseases发明人:Hugo Geerts,Athan Spiros申请号:US13412626申请日:20120306公开号:US08332158B2公开日:20121211专利内容由知识产权出版社提供专利附图:摘要:A computer model of a diseased human brain includes inputs representing a drug and outputs representing the clinical effect of that drug on psychiatric and cognitive diseases. Diseases that can be modeled include psychiatric disorders, such as schizophrenia, bipolar disorder, major depression, ADHD, autism, obsessive-compulsive disorder, substance abuse and cognitive deficits therein and neurological disorders such as Alzheimer's disease, Mild Cognitive impairment, Parkinson's disease, stroke, vascular dementia, Huntington's disease, epilepsy and Down syndrome. The computer model preferably uses the biological state of interactions between and among cortico and subcortical areas of the human brain, to define the biological processes related to the biological state of the generic synapse model, the striatum, Locus Coeruleus, Dorsal raphe, hippocampus, amygdala and cortex, as well as certain mathematical relationships related to interactions among biological variables associated with the biological processes and to correlations between the biological variables and clinical effects on a clinical scale.申请人:Hugo Geerts,Athan Spiros地址:Berwyn PA US,Portland OR US 国籍:US,US代理机构:Berliner & Associates更多信息请下载全文后查看。
Chapter_02_lecture
2-3
Outline of Griffith’s Transformation Experiments
2-4
DNA: The Transforming Material
In 1944 Avery, Macleod and McCarty used a transformation test similar to Griffith’s procedure taking care to define the chemical nature of the transforming substance
•
•
End of 19th century – DNA and RNA separated from proteins Levene, Jacobs, et al. characterized basic composition of DNA and RNA
2-2
Molecular Foundation: Early experiments that explored the question: What is the genetic material?
2-6
Confirmation for DNA as the genetic material
• In the 1940s geneticists doubted the use of DNA as the genetic material as it appeared to be monotonous repeats of 4 bases • By 1953 Watson & Crick published the doublehelical model of DNA structure and Chargaff demonstrated that the 4 bases were not present in equal proportions • In 1952 Hershey and Chase demonstrated that bacteriophage infection comes from DNA, adding more evidence to support that DNA is the genetic material
科学故事蛋白质化学
第一章氨基酸科学故事:第二十二种标准氨基酸生物学家因发现新的氨基酸带来的喜悦不亚于物理学家发现了新的粒子或化学家发现了新的元素。
1986年以前,人们一直认为,出现在蛋白质分子中的由遗传密码编码的标准氨基酸残基只有20种。
到了1986年,科学家们终于在含硒蛋白中发现第二十一种标准氨基酸——含硒半胱氨酸。
时隔16年之后,来自美国俄亥俄州立大学的两个研究小组在产甲烷细菌里发现了第二十二种标准氨基酸——吡咯赖氨酸。
俄亥俄州立大学由Joseph A. Krzycki领导的研究小组一直在研究一种属于古细菌的产甲烷微生物——巴氏甲烷八叠球菌(Methanosarcina barkeri)。
此微生物能够将单甲胺(monomethylamine)、二甲胺(dimethylamine)和三甲胺(trimethylamine)转变成甲烷。
1995年,Krzycki的研究小组分离得到一些与甲烷生成有关的特殊蛋白质。
两年以后,他们分离得到编码其中一个蛋白质的基因,并测定出了它的核苷酸序列。
1998年,他们发表了这个基因的全序列,结果显示其阅读框架内含有一个反常的琥珀型终止密码子(amber codon)。
密码子是决定氨基酸的三字母核苷酸序列(参看“第三十八章蛋白质的生物合成与细胞内降解”),琥珀型终止密码子的核苷酸序列是TGA,它通常不决定任何氨基酸,它的出现一般是多肽链合成结束的标志。
然而,让Krzycki吃惊的是,该终止密码子竟然编码一种氨基酸,而且这种奇怪的现象还出现在其他几种与甲烷产生有关的基因上。
与此同时,由Michael Chan领导的研究小组开始研究由琥珀密码子编码的氨基酸的结构。
他们意识到这个古怪的密码子也许编码一种新的氨基酸,但也有其他的可能性。
Krzycki及其同事决定测定原来蛋白质的氨基酸序列。
当得到蛋白质的氨基酸序列以后,他们发现由琥珀密码子决定的氨基酸似乎仅仅是一个赖氨酸。
但是,Krzycki仍然要求Chan和他的一个博士研究生Bing Hao对含有这个氨基酸的蛋白质晶体结构进行研究以确定那个氨基酸的性质。
乳胶免疫层析法定量检测猪尿液中的盐酸克伦特罗
乳胶免疫层析法定量检测猪尿液中的盐酸克伦特罗管 笛1,2,魏 颖1,2,王 旗1,2,李俊博1,2,武会娟1,2,*(1.北京市理化分析测试中心,北京 100089;2.北京市基因测序与功能分析工程技术研究中心,北京 100089)摘 要:目的:建立以彩色乳胶微球为标记物的免疫层析技术检测猪尿液中的盐酸克伦特罗(clenbuterol ,CLB )。
方法:彩色乳胶微球标记CLB 抗体并真空冷冻干燥,盐酸克伦特罗-牛血清白蛋白(CLB-bovine serum albumin ,CLB-BSA )人工抗原与羊抗鼠抗体分别喷到硝酸纤维素膜上作检测线和质控线,猪尿液样本与冻干微球混匀后,插入试纸条,通过读条仪进行测定。
结果:通过理化参数的优化,吐温-20为最优表面活性剂,选用300 nm 乳胶微球,层析时间为9 min 。
方法的检出限为0.013 ng /mL ,回收率范围在97.8%~106.0%之间,相对标准偏差不高于9.6%。
结论:本研究成功建立了一种灵敏、准确的检测猪尿液中CLB 的彩色乳胶免疫层析方法。
关键词:免疫层析试纸;彩色乳胶;盐酸克伦特罗;猪尿液;定量检测Development of Dyed Latex Bead-Based Immunochromatographic Assay for Quantitative Detection ofClenbuterol in Swine UrineGUAN Di 1,2, WEI Ying 1,2, WANG Qi 1,2, LI Junbo 1,2, WU Huijuan 1,2,*(1. Beijing Center for Physical and Chemical Analysis, Beijing 100089, China;2. Beijing Engineering Technique Research Center for Gene Sequencing & Function Analysis, Beijing100089, China)Abstract: Objective: Aiming at the development of an immunochromatographic method based on dyed latex beads for the detection of clenbuterol (CLB) in swine urine. Methods: Dyed latex beads were conjugated with anti-CLB antibody and vacuum freeze-dried. CLB-bovine serum albumin (CLB-BSA) conjugate and goat anti-mouse IgG antibodies were coated onto nitrocellulose membranes to establish test and control lines, respectively. The test was carried out by placing swine urine into the sample well and mixed with dyed latex beads. After incubation, the optical density was measured by a microplate reader. Results: After optimizing the physical and chemical parameters, the best surfacant was Tween-20, 300 nm dyed latex beads were chosen, and the incubation time was 9 minutes. The limit of detection (LOD) was 0.013 ng /mL. The recoveries from spiked swine urine varied from 97.8% to 106.0%, with relative standard deviation of less than 9.6%. Conclusion: A sensitive and accurate method for the detection of CLB was successfully developed in this paper.Key words: immunochromatographic strip; dyed latex beads; clenbuterol; swine urine; quantitative detection DOI:10.7506/spkx1002-6630-201712042中图分类号:TS207.3 文献标志码:A 文章编号:1002-6630(2017)12-0273-05引文格式:管笛, 魏颖, 王旗, 等. 乳胶免疫层析法定量检测猪尿液中的盐酸克伦特罗[J]. 食品科学, 2017, 38(12): 273-277. DOI:10.7506/spkx1002-6630-201712042. GUAN Di, WEI Ying, WANG Qi, et al. Development of dyed latex bead-based immunochromatographic assay for quantitative detection of clenbuterol in swine urine[J]. Food Science, 2017, 38(12): 273-277. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-201712042. 收稿日期:2016-07-22基金项目:北京市优秀人才项目(2014400685627G277);北京市科学技术研究院青年骨干计划项目(201415)作者简介:管笛(1983—),男,副研究员,博士,研究方向为分析化学。
PDCD4_自噬信号通路及凋亡在大鼠癫痫持续状态中的变化
PDCD4/自噬信号通路及凋亡在大鼠癫痫持续状态中的变化1内科学专业研究生郑雪姣指导老师狄政莉教授摘要背景:癫痫是一种神经系统常见的慢性脑部疾病,是仅次于脑卒中的第二大疾病。
癫痫持续状态(SE)是癫痫最严重的发作形式之一,年发病率为(10-41)/10万,病死率在7.6%至39%之间。
癫痫的持续发作不仅可以引起神经元损伤,造成永久性脑损害,而且可以引起内环境紊乱、呼吸和循环衰竭造成死亡。
研究表明,细胞自噬和细胞凋亡参与了中枢神经系统疾病神经元损伤过程,包括脑外伤、脑卒中和神经系统变性疾病,但是其在癫痫中的作用尚不明确。
自噬是机体对各种生理刺激作出的应答反应,起到维持内环境稳定和自我保护的作用,但是过度的自噬对机体是有害的,可以引起细胞死亡。
据报道细胞凋亡与神经元死亡密切相关。
自噬与凋亡通路共享一些重要组件,二者即独立又相互影响,共同调控细胞的生存。
程序性细胞死亡因子4(PDCD4)是细胞程序性死亡中重要的原因,是胚胎发育、正常组织更新等许多生物过程中必不可少的事件。
有研究显示PDCD4在脑血管病、中枢神经系统变性等疾病中能作为自噬和凋亡通路的上游分子来调控细胞自噬和凋亡,减轻细胞损伤产生的影响。
这提示我们在SE中自噬和凋亡通路是否也能通过PDCD4来调节,从而减轻癫痫后脑神经元的损伤,但具体的作用机制还需要进一步的研究和论证。
本研究旨在观察PDCD4/自噬信号通路及凋亡在大鼠SE中的表达变化,为进一步探究PDCD4在SE中的作用提供研究基础。
方法:本实验采用腹腔注射氯化锂-匹鲁卡品诱导癫痫持续状态模型,将大鼠随机分为正常对照组、癫痫组(SE组),正常对照组4只,SE组Racine评分≥IV级的大鼠进一步随机分为SE后6h(n=4)、12h(n=4)、24h(n=4)、48h(n=4)、72h(n=4)五个亚组。
应用qRT-PCR检测对照组和SE组不同时间点大鼠海马组织中PDCD4、ATG5、LC3、Beclin-1在mRNA水平的表达。
电力系统稳定器PSS参数优化整定的研究
上海交通大学硕士学位论文
3
《电力系统稳定器(PSS)参数优化整定的研究》
This thesis mainly focuses on optimization design of PSS parameters. Some contributions are summarised as follows. 1、The thesis proposes an approach called Adaptive genetic algorithm (AGA) for optimization of PSS parameters. The method solves problems through improved crossover and mutation operators which changed from time to time according to the fitness. However they are constants in simple genetic algorithm, which is not helpful to the population of the algorithm, so simple genetic algorithm (SGA) has a poor convergence and bad stability. Using new crossing operators and mutation operators, AGA can make every generation has a good fitness value. The example shows us that low frequency oscillation was suppressed after PSS parameters were optimized by improved GA. 2、Immune Algorithm (IA) is developed on the base of Immune theorem, and it is used to optimize PSS parameters in this thesis.Ordinary evolution algorithms develop under a certain probability and these algorithms seek optimal values at a random probability and without guiding. So on the one hand they offer evolution chances to the offspring of population and on the other hand they make lots of drawbacks inevitable, such as precocious phenomenon etc. Immune algorithm can overcome these drawbacks and strengthen the searching ability. In the thesis, immune secondary response supress degeneration phenomenon during crossing and
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n ≥ 150 .
But
mathematical simulations show that the interval
ˆ contains not less than 95% of the values J from G when n ≥ 11 .
The 3s-rule is closely connected with the 3s1-rule, which allows us to calculate a confidence interval for unknown mathematical expectation m ( x ) on the basis of the sample with significance level not exceeding 0.05. At first, consider the problem of the constructing of the confidence interval on the basis of 3σ-rule, in the case when the value of the random variable x and its variance σ2 ( x ) are known. By virtue of the inequality (2) we have:
Department of Mathematical Sciences and Center for Applied Mathematics and Statistics, New Jersey Institute of Technology, Newark, NJ, USA
Abstract. In this part we describe the statistical test criteria which are used in Part I in the construction of computer-aided cytogenetic method of breast cancer diagnosis. Keywords: breast cancer, fibroadenomatosis, buccal epithelium, discriminant analysis.
P x − m ( x ) ≥ 3σ ( x ) ≤ 0.05
(
)
(1)
1 n ∑ xk , n k =1 1 n 2 σ2 ( x ) ≈ s 2 = ( xk − x ) . ∑ n − 1 k =1 m ( x) ≈ x =
These estimations have good properties. They are unbiased, i.e. their mathematical expectations coincide with the exact value of the estimated parameters m ( x ) and D ( x ) :
m ( x ) = m ( x) ,
m ( s2 ( x )) = D ( x ) .
In constructing the confidence interval J containing the bulk of the general population G on the basis of the sample x1 , x2 ,..., xn it is quite naturally to replace the mathematical expectation m ( x ) and the variance σ2 ( x ) by their estimations x and s 2 respectively. So, we can formulate the so-called 3s-rule:the is a源自interval random
confidence interval for unknown mathematical expectation m ( x ) with significance level 0.05 (by virtue of 3σ-rule). In prevalent number of cases we can put x = x , so that
8 4 1 p ( x − m( x ) ≥ k σ ( x ) ) ≤ ⋅ 2 , k ≥ (2) 9 k 3
2
The 3s-rule
In order to construct the confidence interval
ˆ = ( x − 3s, x + 3s ) , J
where
Therefore, the
Computer-Aided Cytogenetic Method of Breast Cancer Diagnosis. Part II - Test Criteria
R.I.Andrushkiw D.A.Klyushin, K.N.Golubeva, M.Pokoyovy, A.V.Romanov Kyiv National Taras Shevchenko University, Kyiv, Ukraine
when
does not exceed 0.05, i.e.
σ ( x) σ( x) p m ( x) ∈ x − 3 ,x +3 ≥ 0.95 n n
. It is easy to see that the following estimation of the variance of the sample mean is unbiased, and has the same properties as the estimation s 2 ( x ) :
where m(x) is the expectation and σ(x) is the standard deviation of x. The value of the constant 0.05 is stipulated by the fact that in many applied sciences (for example, biology and medicine) the 5% significance level is the most widely used. The justification of the 3σrule was given in paper [1]. There also exist several different proofs of this empirical rule [2–4]. Theorem 1. For all k > 0, the following inequality holds for an arbitrary random variable x having a unimodal distribution and finite variance σ2 ( x ) > 0
s12 ( x ) =
n 1 2 1 2 s ( x) = ( xk − x ) . ∑ n n ( n − 1) k =1
Replacing σ2 ( x ) by its estimation s12 ( x ) , we obtain the 3s1-rule that states that the confidence interval
2 sn respectively.
1
The 3σ–rule
The empirical 3σ-rule, which is well known in mathematical statistics, states that for the overwhelming majority of commonly encountered random variables x the following inequality holds:
p x − m ( x ) ≤ 3σ ( x ) = = p ( −3σ ( x ) ≤ m ( x ) − x ≤ 3σ ( x ) ) =
(
)
= p ( x − 3σ ( x ) ≤ m ( x ) ≤ x + 3σ ( x ) ) ≥ 0.95
Hence, it follows that
J = ( x − 3σ ( x ) , x + 3σ ( x ) )
n n
significance
level
of
the
x=
1 1 2 xk , s 2 = ( xk − x ) .When ∑ ∑ n k =1 n − 1 k =1
confidence
interval
σ σ ,x +3 x −3 n n
n is large, this interval contains not less than 95% of the values from G .Now, let us consider the following question: under what n the 3srule holds. According to practical recommendations, the estimation x almost coincides with m ( x ) when n ≥ 30 , and s2 ( x ) ≈ D ( x )
x1 , x2 ,..., xn
3s ( x ) 3s ( x ) ,x + J1 = x − n n
contains unknown mathematical expectation m ( x ) with the probability not exceeding 0.95, when n is large. Since the estimation s 2 ( x ) has practically the same value as σ2 ( x ) if n ≥ 150 , we can assume that the estimation s12 ( x ) coincides with the variance σ2 ( x ) and that the 3s1-rule holds when n ≥ 150 . Nevertheless, this rule may be applied even for n ≥ 11 . In mathematical statistics samples are classified by their size: 1) small samples, when n ≤ 30 ; 2) middle samples, when 30 < n < 150 , and 3) large samples, when n ≥ 150 . To summarize, we can state that the 3s and 3s1-rules hold for middle and large samples, and even for small samples, if their size exceeds n = 11 .