Wall characterization via TSVD in through-the-wall imaging

生物工程学报Chin J Biotech2009, May 25; 25(5): 745-753 Chinese Journal of Biotechnology ISSN 1000-3061cjb@© 2009 Institute of Microbiology, CAS & CSM, All rights reserved小鼠睾丸支持细胞体外培养特性师冰洋1, 张淑香1, 郭美锦1, 王永红1, 张嗣良1, 史小林21 华东理工大学生物反应器工程国家重点实验室, 上海 2002372 首都医科大学生殖医学中心, 北京 100069摘要:支持细胞是睾丸的重要组成部分, 其主要功能是为生精细胞提供适宜的生长环境。

从幼鼠睾丸中分离得到支持细胞, 并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。

通过对原代小鼠睾丸支持细胞的贴壁、生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的研究, 发现支持细胞的贴壁时间主要集中在接种后2~4 h; 当培养液中氨根离子浓度高于 2.3 mmol/L, 渗透压高于326 mosm/kg, pH≤6.8时支持细胞生长进入衰亡期; 在氨基酸代谢方面, 发现培养过程中丙氨酸和谷氨酸浓度迅速增加,缬氨酸、亮氨酸和异亮氨酸浓度略有降低, 丝氨酸、精氨酸和甘氨酸浓度基本保持不变。

因此培养液中铵根离子浓度的过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。

研究结果为支持细胞大规模培养及工艺优化奠定了基础。

关键词:支持细胞, 分离与鉴定, 体外培养, 代谢特性Metabolic characterization of rat sertoli cell in vitro cultureBingyang Shi1, Shuxiang Zhang1, Meijin Guo1, Yonghong Wang1, Siliang Zhang1, and Xiaolin Shi21 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China2 Reproductive Medical Center of Capital Medical University, Beijing 100069, ChinaAbstract: Sertoli cell (SC) is intrinsic to the testis and provides an appropriate growth environment for the germ cells. It was separated from rat’s testis and identified by hematoxylin and eosin staining(HE) and immunocytochemical reaction, then cultivated invitro. Culture conditions such as pH, osmotic pressure and metabolic parameters that include consumption rates of glucose, glutamine,amino acids and formation rates of lactic acid, ammonium ion were investigated. It was showed that adhesion process of SCs was accomplished within 2−4 hours after inoculation. It was also observed that the SCs entered into the decline phase when the concentration of ammonium ion and lactic acid were above 2.3 mmol/L and 14 mmol/L, respectively, which caused osmotic pressureabove 326 mosm/kg and pH below 6.8 in the medium. As the changes of amino acids during culture were concerned, Glu and Ala accumulated rapidly, while Val, Leu, Ile reduced slightly and at the same time Ser, Arg, and Gly were stable. The restrict factors forSCs grown in static culture might be high osmotic pressure and low pH, which were generated when glutamine and glucose were metabolized into lactic acid. The findings could be fundamental in the process optimization of large scale Sertoli cells in vitro culture.Received:December 9, 2008; Accepted:February 19, 2009Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2007AA02Z216), ShanghaiBiotechnology Industrialized Platform (No. 07DZ22914), National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204). Corresponding author: Shuxiang Zhang. E-mail: zhangsx1976@Meijing Guo. E-mail: guo_mj@国家高技术研究发展计划(863计划)(No. 2007AA02Z216), 上海市生物技术产业化平台(No. 07DZ22914), 国家重点实验室专项经费(No. 2060204)资助。

化工进展CHEMICAL INDUSTRY AND ENGINEERING PROGRESS2019年第38卷第1期动态现场原位（operando ）表征技术在多相催化反应中的应用与进展孙杨1，丁豆豆1，林昌1，刘向林1，张超1，田鹏飞1，曹晨熙1，杨子旭1，徐晶1，韩一帆1，2（1华东理工大学化学工程联合国家重点实验室，上海200237；2郑州大学化工与能源学院，河南郑州450001）摘要：动态现场原位（operando ）表征是在接近过程工业反应条件下，揭示催化反应机理及工业催化剂结构演变的新兴动态结构解析技术。

本文综述了operando 表征技术在多相催化反应中的应用及发展趋势，从operando 红外、operando 拉曼、operando X 射线衍射、operando 穆斯堡尔谱、operando X 射线吸收谱及operando X 射线光电子能谱6个方面概述了operando 技术的最新进展。

此外，还介绍了正在兴起的operando 联用技术，该技术综合多种operando 技术为一体，能够在反应过程中对催化剂的结构全貌进行深度表征，实现工业催化剂的理性设计，将成为未来多相催化研究的重要手段。

然而，目前operando 技术的时间分辨率和空间分辨率仍需进一步提升，其巨大潜力依然有待开发。

关键词：动态现场原位技术；催化；原位表征；催化剂构-效关系；反应；表面中图分类号：TQ02；TQ03文献标志码：A文章编号：1000-6613（2019）01-0260-18Advances in operando techniques for the heterogeneouscatalytic reactionsSUN Yang 1，DING doudou 1，LIN Chang 1，LIU Xianglin 1，ZHANG Chao 1，TIAN Pengfei 1，CAO Chenxi 1，YANG Zixu 1，XU Jing 1，HAN Yifan 1，2(1State Key Laboratory of Chemical Engineering,East China University of Science and Technology,Shanghai 200237,China;2School of Chemical Engineering and Energy,Zhengzhou University,Zhengzhou 450001,Henan,China)Abstract:Operando characterization is a cutting-edge technique for revealing the catalytic reaction mechanism and the dynamic structure evolution of industrial catalysts under the conditions close to industrial reaction.The development of operando technique and its application on heterogeneous catalysis have been reviewed,and the latest progress on operando infrared spectroscopy (IR),operando Raman,operando X-ray diffraction (XRD),operando Mössbauer,operando X-ray adsorption spectra (XAS),and operando X-ray photoelectron spectroscopy (XPS)have been summarized.In addition,the coupling of various operando characterizations has also been introduced.Through this technique,the overall structure of the catalyst during reaction process can be characterized more deeply,and thus can realize the rational design of industrial catalysts.Therefore,we believe this coupling technique will become an important and prospective way to study heterogeneous catalysis.Nevertheless,the time and space resolution of current特约评述DOI ：10.16085/j.issn.1000-6613.2018-1196收稿日期：2018-06-07；修改稿日期：2018-09-26。

V o l.21高等学校化学学报 N o.1 2000年1月 CH E M I CAL JOU RNAL O F CH I N ESE UN I V ERS IT IES 1～4　导向剂法合成Β沸石的晶化机制研究周　群裘式纶　庞文琴(苏州大学化学化工学院,苏州215006)(吉林大学化学系,长春130023)摘要　采用导向剂法合成了Β沸石.在导向剂中存在Β沸石微晶核,晶化过程中Β沸石只能在一个较窄的硅铝比范围内形成;模板剂T EAOH仅存在于Β沸石微晶核中.在Β沸石合成过程中,水合钠离子不仅起着平衡沸石骨架负电荷的作用,并且在Β沸石的晶体生长过程中起到一定程度的结构导向作用.关键词　导向剂;Β沸石;I R光谱;T GA27热重分析中图分类号　O612 文献标识码　A 文章编号　025120790(2000)0120001204Β沸石于1967年由M obil公司[1]首次合成.由于其独特的拓扑结构[2],优良的热稳定性及特有的疏水性而具有独特的催化性能[3～6].传统合成方法中需要大量价格昂贵的有机模板剂四乙基氢氧化铵(T EAOH),极大限制了Β沸石在工业中的应用.近年来,人们对Β沸石的合成进行了大量研究,试图寻找降低其合成成本的新途径[7,8].我们首先开发了导向剂合成法[9,10],极大地降低了合成中T EAOH 的用量.本文考察了Β沸石导向剂的I R光谱及用导向剂法合成Β沸石过程中固、液相的组成和固相物质的热重行为,发现导向剂内含有经液相成核机理而形成的Β沸石微晶核.Β沸石的晶体生长经液相转化机理,并被局限在一个较窄的硅铝比范围内,体系中水合钠离子不仅在Β沸石形成过程中起平衡骨架负电荷的作用,并且在沸石生长中起着结构导向作用.1　实验部分1.1　样品的合成导向剂及Β沸石的合成方法见前文[9].Β沸石导向剂的制备:将铝酸钠、氢氧化钠、四乙基氢氧化铵(T EAOH)和白碳黑按n(A l2O3)∶n(Si O2)∶n[(T EA)2O]∶n(N a2O)∶n(H2O)=1∶80∶11∶215∶850混合均匀,于413K下陈化4h,即得Β沸石导向剂.Β沸石的合成:分别将铝酸钠、氢氧化钠、白碳黑、水和自制的Β沸石导向剂按n(A l2O3)∶n(Si O2)∶n(N a2O)∶n(H2O)=1∶30∶9.5∶825混合均匀,导向剂的加入量占合成混合物总体积的3%～8%,于413K下晶化一定时间得Β沸石.实验中所用铝酸钠和氢氧化钠均为分析纯,T EAOH和白碳黑(Si O2)为工业品.1.2　样品的表征样品物相和结晶度用X射线粉末衍射仪(D m ax2A 型)测定;在氧气气氛下(30c m3 m in)用T GA27热重仪进行热重分析;采用溴化钾压片法,在N icolet5DX FT I R光谱仪上测定样品的红外光谱;用等离子发射光谱(P las m a2s pec型)和化学分析法测定样品的组成.2　结果与讨论2.1　Β沸石导向剂的I R光谱分析制备Β沸石导向剂时,反应混合物首先形成一次凝胶,在陈化期间,一次凝胶完全溶解为略有粘稠的无色液体.导向剂陈化过程中液相物质I R谱图的跟踪测试见图1.收稿日期:1999203208.基金项目:国家教育部吉林大学无机合成与制备化学开放研究实验室资助.联系人简介:周　群(1965年出生),女,博士,讲师,从事无机合成研究.　F i g .1　Ti m e dependence of I R spectra of structure di recti n g agen t a .A ged after 1h;b .A ged after 4h;c .Zeolite .比较图1谱线a 和c 可知,导向剂陈化初期(谱线a ),在520和601c m -1处未出现Β沸石骨架的特征振动峰,表明此时体系中的Si 和A l 物种基本上以无定形存在.当导向剂陈化4h 后,液相的I R 光谱发生了明显变化(图1谱线b ),在520和601c m -1处出现了Β沸石的特征振动峰,说明此时液相中已有Β沸石微晶核形成.Pereze 等[11]在考察模板剂法合成Β沸石过程中也发现,诱导期固相物质是以无定形硅铝酸盐形式存在的,因此认为固相中不存在Β沸石微晶核.我们的结果则直接证实Β沸石微晶核由液相生成,并存在于液相中.亦即在一次凝胶溶解后,反应混合物首先经由液相成核机理形成Β沸石微晶核,而Β沸石进一步的晶体生长只是围绕这些微晶核进行的.2.2　合成Β沸石过程中固、液相物质的组成分析以导向剂替代T EAOH 模板剂合成Β沸石过程中,配制初始反应混合物时形成一次凝胶,此凝胶在晶化过程中很快转化为固相和液相两部分.图2示出了导向剂法合成Β沸石过程中固相和液相中Si O 2含量随晶化时间的变化情况.由图2可见,在诱导期,固相和液相中Si O 2含量基本不变,但进入晶体生长期后,固相中Si O 2含量从67%升高到74%;而液相中Si O 2含量仅略有降低,且固相和液相中Si O 2含量的变化与晶化曲线一致,达到最大结晶度时不再变化.固相和液相中A l 2O 3含量的检测结果表明,液相中A l 2O 3含量非常少,几乎很难测出,只是在进入晶体生长期后,才能观察到液相中A l 2O 3含量略有增加的趋势.F i g .2　The changes of Si O 2con ten t i n sol i d and l i qui dpha ses w ith cryst a ll i za ti on ti m eD ash line :crystallizati on curve ;a .SiO 2content in s olidphase ;b .Si O 2content in liquid phase .F i g .3　The changes of A l 2O 3con ten t and Si A l ra ti o i n sol i d pha se w ith cryst a ll i za ti on ti m e D ash line :crystallizati on curve ;a .A l 2O 3content ;b .n (Si ) n (A l )rati o .固相中A l 2O 3含量的变化则较为明显(图3曲线a ),A l 2O 3含量在诱导期也无明显变化,当晶化过程进入晶体生长期时,才从12%下降至10%,并趋于恒定,这与Si O 2的变化相对应,从而导致了固相中n (Si ) n (A l )比在晶体生长期间的增加(图3曲线b ).以上结果表明,导向剂法合成Β沸石时,无论在诱导期或晶体生长期,绝大部分铝存在于固相中,在进入晶体生长期后,固相中的铝一旦溶解,立刻被结合进入Β沸石晶体中,同时,这一过程还伴随着液相中的硅补充进入Β沸石晶体,从而导致了固相的n (Si ) n (A l )比逐渐增高,表明Β沸石晶体生长经由液相转化机理.由图3可见,固相和液相物质在整个晶化过程中变化不大,即在导向剂法合成条件下,Β沸石晶体生长所需的n (Si ) n (A l )比被限制在一个较小的范围内.正如前文[9]所报道的,无论2 高等学校化学学报V ol.21投料时反应混合物的n (Si ) n (A l )比如何变化,所生成的Β沸石n (Si ) n (A l )比却变化不大.2.3　Β沸石合成过程中固相物质的TEAOH 变化图4曲线a 为导向剂法合成Β沸石的热重谱图.为便于比较,同时测定了以T EAOH 为模板剂合成Β沸石的热重谱图(图4曲线b ).模板剂法合成的Β沸石表现出两个明显的有机模板剂失重温度区域:573～773K 和773～943K .这与文献[11,12]的报道结果一致,分别对应于孔道中填充的T EAOH 分子和阳离子位置上平衡沸石骨架负电荷的T EA+离子脱附.导向剂法合成的Β沸石(图4曲线a )仅在623～773K 内有一归属于有机模板剂T EAOH 分子的脱附峰,而几乎观察不到归属于T EA +阳离子的脱附峰,与用传统模板剂法合成Β沸石的热重谱图存在很大区别.由等离子发射光谱和化学分析测定的导向剂法合成Β沸石的化学组成进一步阐明了这种差异的本质,测得样品的组成为:N a 0.6A l 1.0Si 4.5O 11.0,即样品中存在大量N a +离子,表明导向剂法合成的Β沸石骨架负电荷主要由N a +离子来平衡,而不是传统模板剂法合成Β沸石中的T EA +阳离子.F i g .4　TGA curves of zeol ite Βs am plesa .Zeolite Βsynthesized w ith nuclei gel ;b.Zeolite Βsynthesized w ith te mp late.F i g .5　Ti m e dependence of TEAOH and H 2O i n sol i d pha sea .Percentage of TEAOH ;b .Percentage of H 2O .为进一步确定T EAOH 在晶化过程中的存在形式,通过热重分析考察了处于晶化过程不同时期固相物质中T EAOH 的含量变化(图5).对处于诱导期的无定型硅铝酸盐,未观察到任何对应于T EAOH 或T EA +的脱附峰,表明在诱导期,有机模板剂仅存在于液相中.只有进入Β沸石晶体生长期后才在固相中观测到T EAOH 的脱附.在生长期的前期,Β沸石结晶度还非常小时,固相中T EAOH 的含量就迅速达到了最大值,此后,随Β沸石结晶度的增加,T EAOH 的含量有所减少.固相中T EAOH 含量的变化,可能与导向剂法合成Β沸石的晶化机制密切相关.如前文[9]所述,在导向剂中已存在Β沸石微晶核,T EAOH 主要存在于这些微晶核中,这些微晶核在随后的Β沸石晶体生长过程中起着至关重要的作用.晶体生长期间,当Β沸石微晶核周围的n (Si ) n (A l )比达到一个合适值时,这些微晶核迅速生长,并很快沉积到固相中,从而使得固相中T EAOH 含量在很短时间内达到了最大值.而在随后的晶体生长过程中,继续溶解的无定形硅铝酸盐仅仅促进了那些Β沸石晶体的进一步生长,此时将不再有含T EAOH 的Β沸石微晶核沉积出来,因而,固相中T EAOH 的含量相应减少.2.4　Β沸石的晶化机制Pereze 等[11]在研究模板剂法合成Β沸石的成核机理时提出,T EA +首先在液相中与硅酸根或多硅酸根结合形成化合物,再与溶解的铝结合,在T EAOH 的协同作用下,经由一系列中间过渡态而形成Β沸石微晶核.其中T EAOH 及其阳离子诱导了Β沸石的成核和晶体生长并起着平衡Β沸石骨架负电荷的作用.我们的实验结果表明(图1和图4),导向剂法合成中Β沸石微晶核主要来源于导向剂,制得的Β沸石中几乎不含有T EA +阳离子,样品中存在大量的N a +离子(元素分析),表明在此条件下微晶核的形成可能与T EAOH 以及水合钠离子有关.有研究[13,14]表明,T EAOH 的分子大小与Β沸石的直孔道尺寸十分相近.因此,我们认为T EAOH 3N o .1周　群等:导向剂法合成Β沸石的晶化机制研究 直接诱导了Β沸石十二元环直孔道的形成,所形成微晶核的骨架负电荷则由水合钠离子来平衡.而在随后的合成过程中,仅仅是这些微晶核在合适的条件下逐渐生长形成了Β沸石,此时正四面体的水合钠离子以及水分子可能以某种聚集方式填充在逐渐生长的Β沸石孔道中,并在一定程度上起着促进Β沸石晶体生长的结构导向作用.水合钠离子的结构导向作用已在ZS M 25沸石的合成中得到证实[15].但是,在我们的合成体系中,显然水合钠离子和水分子的这种特殊聚集体并不十分稳定,一旦合成条件或者沸石形成过程中的化学环境有所改变,沸石骨架就可能向更为致密的形态转变.事实上,我们在以前的报道[10]中曾指出,当合成温度高于453K 或延长合成时间时,所得的沸石则为致密的ZS M 25和丝光沸石.另一方面,如图2和图3所示,Β沸石的生长被局限在一个相对较窄的n (Si ) n (A l )比范围内,这可能也与水合钠离子特殊的结构导向作用有关.参　考　文　献　1　W adlinger R .F .,Kerr G .T .,RosinskiB .J ..U S 3308069[P ],1967　2　WAN G Ying 2X ia (王颍霞),M EN G X ian 2P ing (孟宪平),L IN eng (李　能)et al ..Che m .J .Chinese U niversities (高等学校化学学报)[J ],1998,19(8):1185—1190　3　N e w sa m S .F .,Fell m ann J .D .,Kilner P .H ..U S 4774364[P ],1988　4　T sai T .C .,W ang I ..A pp l .Catal.[J ],1991,77:209—222　5　Loenders R .,Jacobs P .A .,M artens J .A ..J .Catal .[J ],1998,176(2):545—551　6　H arding R .H .,Gatte R .R .,Pereira C .J ..J .Catal .[J ],1993,140:41—52　7　Cannan T .R .,H inchey R .J ..U S 5139759[P ],1992　8　Borade R. B.,C learfield A..Catal .L ett .[J ],1994,26:285—289　9　ZHOU Q un (周　群),L IBao 2Zong (李宝宗),Q I U Shi 2L un (裘式纶)et al ..Che m.J.Chinese U niversities (高等学校化学学报),1999,20(5):693—695　10　ZHOU Q un (周　群).Ph .D .Thesis [D ],J ilin U niversity ,1994　11　Pereze 2pariente J .,M artens J .A .,Jacobs P .A ..A pp l .Catal .[J ],1987,31:35—64　12　L a m i E .B .,Renzo F .D .,Fajula F ..J .Phys .Che m .[J ],1992,96:3807—3811　13　T reacy M .M .J .,N e w sa m J .M ..N ature [J ],1988,332:249—251　14　Conw ay B .E .,V erall R .E .,D esnoyers J .E ..T rans .Faraday Soc .[J ],1966,62:2738—2745　15　S ON G T ian 2You (宋天佑).Ph .D .Thesis [D ],J ilin U niversity ,1989Studi es on the Cryst a ll i za ti on M echan is m of ΒZeol iteSyn thesi zed w ith Nuclea ti on GelZHOU Q un 3(D ep art m ent of Che m istry and Che m ical E ng ineering ,S uzhou U niversity ,S uzhou 215006,Ch ina )Q I U Sh i 2L un ,PAN G W en 2Q in(D ep art m ent of Che m istry ,J ilin U niversity ,Chang chun 130023,Ch ina )Abstract Zeo lite Βw as synthesized w ith a nucleati on gel.T he I R s pectro scop ic characterizati on s confir m the existence of the m icrocrystal nuclei in the nucleati on gel.Che m ical analysis of s olid and liquid phases de monstrates that zeo lite Βcan grow w ith in a relatively narrow n (Si ) n (A l )rati o range .T GA analysis re 2veals that T EAOH only ex ists in the m icrocrystal nuclei for m ed in the nucleati on gel.It w as als o found that the negative charges of the zeo lite fra m e wo rk are actually balanced by the hydrated N a +i on s ,w h ichm ay direct the grow th of zeolite Βduring the crystallizati on p rocess.Keywords N ucleati on gel ;Zeolite Β;I R s pectroscopy ;T GA 27analysis(Ed .:M ,G )4 高等学校化学学报V ol .21。

2Industrial Weighing and MeasuringDairy & CheeseNewsIncrease productivitywith efficient filling processesThe new IND560 weighing terminal enables you to boost speed and precision during the filling process. Choose from a wide range of scales and weigh modules to connect to the terminal.The versatile IND560 excels in control-ling filling and dosing applications, delivering best-in-class performance for fast and precise results in manual, semi-automatic or fully automatic operations. For more advanced filling, the Fill-560 application software adds additional sequences and component inputs. Without complex and costly programming you can quickly con-figure standard filling sequences, or create custom filling and blending applications for up to four compo-nents, that prompt operators for action and reduce errors.Ergonomic design Reducing operator errors is achieved through the large graphic display which provides visual signals.SmartTrac ™, the METTLER TOLEDO graphical display mode for manual operations, which clearly indicate sta-tus of the current weight in relation to the target value, helps operators to reach the fill target faster and more accurately.Connectivity and reliabilityMultiple connectivity options are offered to integrate applications into your con-trol system, e.g. Allen-Bradley ® RIO, Profibus ®DP or DeviceNet ™. Even in difficult vibrating environments, the TraxDSP ™ filtering system ensures fast and precise weighing results. High reli-ability and increased uptime are met through predictive maintenance with TraxEMT ™ Embedded MaintenanceTechnician.METTLER TOLEDO Dairy & Cheese News 22Speed up manual operations with flexible checkweighingB e n c h & F l o o r S c a l e sHygienic design, fast display readouts and the cutting-edge color backlight of the new BBA4x9 check scales and IND4x9 terminals set the standard for more efficient manual weigh-ing processes.Flexibility through customizationFor optimal static checkweighing the software modules ‘check’ and‘check+’ are the right solutions. They allow customization of the BBA4x9 and the IND4x9 for individual activi-ties and needs, e.g. manual portion-ing or over/under control. Flexibility is increased with the optional battery which permits mobility. Hygienic design Easy-to-clean equipment is vital in food production environments. Both the BBA4x9 scale and the IND4x9 ter-minal are designed after the EHEDGand NSF guidelines for use in hygi-enically sensitive areas.Even the back side of the scale stand has a smooth and closed surfacewhich protects from dirt and allowstrouble-free cleaning.Fast and preciseThe colorWeight ® display with a colored backlight gives fast, clear indication when the weight is with-in, below or above the tolerance.The color of the backlight can be chosen (any mixture of red, greenand blue) as well as the condition itrefers to (e.g. below tolerance). The ergonomic design enables operators to work more efficiently due to less exhaustion.Short stability time, typically between 0.5s and 0.8s, ensures high through-put and increased productivity.PublisherMettler-Toledo GmbH IndustrialSonnenbergstrasse 72CH-8603 Schwerzenbach SwitzerlandProductionMarCom IndustrialCH-8603 Schwerzenbach Switzerland MTSI 44099754Subject to technical changes © 06/2006 Mettler-Toledo GmbH Printed in SwitzerlandYellow – weight above toleranceGreen – weight within toleranceRed – weight below toleranceYour benefits• Fast and precise results and operations • Higher profitability• Ergonomic design, simple to operate • Mobility up to 13h due to optional batteryFast facts BBA4x9 and IND4x9• 6kgx1g, 15kgx2g, 30kgx5g (2x3000d), for higher capacity scales: IND4x9 terminal • Weights and measures approved versions 2x3000e • Functions: simple weighing, static checkweighing, dispensing • Color backlight, bar graph • Tolerances in weight or %• 99 Memory locations • Optional WLAN, battery• Meets the IP69k protection standardsagainst high-pressure and steam cleaning • Complete stainless steel construction Immediate checkweighing resultswith color Weight®EHEDGThe colored backlight of the LC display provides easy-to- recognize indication whether the weight is within the tolerancelimits or not.WLANMETTLER TOLEDO Dairy & Cheese News 23HACCP programs, GMP (Good Manufacturing Practice), pathogen monitoring and good cleaning practices are essential for effective food safety plans. Our scales are constructed for compliance with the latest hygienic design guidelines.Hygienic design to improve food safetyMETTLER TOLEDO supports you in complying with the latest food safety standards like BRC, IFS or ISO 22000 by offering solutions which are:• Compliant with EHEDG (European Hygienic Engineering & Design Group) and NSF (National Sanitation Foundation) guidelines • Full V2A stainless steel construc-tions, optional V4A load plates • Smooth surface (ra < 0.8μm)• Easy-to-clean construction, no exposed holes • Radius of inside corners > 3mm• Ingress protection rating up to IP69k• Hermetically sealed loadcellsYour benefits• Reduce biological and chemical contamination risks • Fast and thorough cleaning procedures • Fulfillment of hygiene regulations • Long equipment life thanks to rugged designGuaranteed serviceKeep your business runningAvoid unnecessary downtime with our wide range of service packages.With a range of innovative service solutions offering regulatory compli-ance, equipment calibration, train-ing, routine service and breakdown assistance, you can profit from sus-tained uptime, together with ongoing performance verification and long life of equipment. There is a range of contract options designed to comple-ment your existing quality systems. Each one offers its own unique set of benefits depending on the equipment and its application.4/serviceFast facts PUA579 low profile scale • 300kgx0.05kg – 1500kgx0.2kg • Open design• Lifting device for easy cleaning • EHEDG conform(300 and 600kg models -CS/-FL)• Free size scale dimensions • Approach rampsExample:PUA579 first EHEDG conform floor scaleEHEDGW e i g h P r i c e L a b e l i n gChallenges faced in the packaging area are:• Responding quickly to retailer demands while improving margins • Improving pack presentation • Minimizing downtime and product giveawaysWith a complete offering of cutting-edge weighing technology, high-per-formance printing, and smart soft-ware solutions, we can help you tackle your labeling challenges whether they are very simple or highly demanding. Intuitive human-machine interfaceTouch-screen operator displays withgraphical icons guide the operator intuitively and reduce nearly every operation to just one or two key-strokes. This interface allows reduced operator training as well as increased operating efficiency.Advanced ergonomics and sani-tary designOur weigh-price labelers are made out of stainless steel for extensive pro-tection against food contamination. Careful attention to hygienic design requirements, with no dead spots and few horizontal parts, ensure that the labelers are easy to clean.Modular designOur product offering includes both manual and automatic weigh-price labelers constructed of flexible “build-ing blocks.” Different combinations and configurations can meet specific budget and operational requirements. METTLER TOLEDO will help you toselect the right:• Scale performance • Display technology • Memory capacity • IT connections • Degree of automation• Integration kitsA large range of options and peripher-als give flexibility for meeting unique requirements e.g. wireless network, hard disks, external keyboards, bar code scanners, RFID transponder, dynamic checkweighing, or metal detection.Weigh-price-labeling Ergonomic, modular, fastEtica 2300 standard manual labelerFor individual weight labeling of various products, high- speed weighing, smart printing and fast product changes are essential. METTLER TOLEDO offers static and automated solutions for both manual and high-speed prepack applica-tions. Choose from our Etica and PAS product range.METTLER TOLEDO Dairy & Cheese News 24Etica 2400i combination with automatic stretch wrappersEtica 2430G multi-conveyer weigh-price labeler rangeEfficient label applicatorsThe unique Etica label applicator (Etica G series) does not require an air compressor, allowing savings on initial equipment expense and ongo-ing maintenance costs. Labels are gently applied in any pre-memorized orientation.PAS systems provide motorized height adjustment and places the label in any corner of the package. Users will have a new degree of freedom in planning their case display layouts to maximize both product presentation and consumer impact.Smart label design tools Retailers want labels to carry clear, correct information, in accordance with their traceability and style requirements. Our solutions are equipped with labeldesign software tools which facili-tate the design of labels customizedfor retailers demands. A touch-screenallows the user to create specific labels– even with scanned elements such aslogos and graphics, pre-programmedlabel templates, or RFID.Versatile integration capabilitiesThe engineers at METTLER TOLEDOworked closely with Europe’s leadingautomatic stretch wrapper suppliersto design performance-enhancingand cost-effective weigh-wrap-labelsystem solutions. Achieving a smallsystem footprint means the systemsrequire only slightly more floor spacethan the wrapper alone.The PAS and Etica weigh-price label-ers can be integrated via TCP/IP ina METTLER TOLEDO scale network,in host computer systems and goodsmanagement systems.Etica weigh-price-labeling systems• Static and automatic weigh-price-labeling up to 55 pieces/min.• Operator displays:– 5.7” color back-lighted LCD (Etica 2300 series)– 10.4” high resolution touch screen (Etica 4400 series)• 3 inch graphic thermal printer (125 to 250mm/sec) withfully programmable label format (max. size 80x200mm)• Data memory:– 64 to 256 Mb RAM– 128 Mb to 10 Gb mass storage– Unlimited number of logo graphics and label descriptions• Interfaces:– 1 serial RS232 interface– Optional second RS232 + RS485 + Centronics port– Ethernet network communication interface(10baseT), TCP/IP, 2 USB ports (1)– Optional: hand-held bar code scanner for automatictraceability data processingGarvens PAS 3008/3012 price labelersEtica 4400METTLER TOLEDO Dairy & Cheese News 2FlexMount ® weigh moduleFast, reproducible and reliable batch-ing and filling are key success factors for your production process. Various factors can affect precision: foam can compromise optical/radar sensors, and solids do not distribute evenly in a tank or silo. Our weighing techno-logy is not affected by these condi-tions and provides direct, accurate and repeatable measurement of mass without media contact. In addition our range of terminals and transmit-ters/sensors enable easy connectivity to your control systems.Key customer benefits• Increased precision and consistencyof your material transfer processes• Faster batching process throughsupreme TraxDSP ™ noise and vibration filtering • Minimal maintenance cost Fast facts terminals/transmitters: PTPN and IND130• Exclusive TraxDSP ™ vibration rejection and superior noise filter-ing system • Easy data integration through a variety of interfaces, including Serial, Allen-Bradley ® RIO, Modbus Plus ®, Profibus ® and DeviceNET • IP65 stainless harsh versionsProcess terminal PTPN• Local display for weight indication and calibration checks • Panel-mount or stainless steel desk enclosureIND130 smart weight transmitter• Direct connectivity where no local display is required • Quick setup and run via PC tool • CalFREE ™ offers fast and easy cali-bration without test weights • DIN rail mounting versionPLCIND1306Tank and silo weighing solutions master your batching processesT a n k & S i l o W e i g h i n g S o l u t i o n sBoost your productivity and process uptime with reliable weighing equipment – improved batching speed and precision, maximum uptime at low maintenance cost.TraxDSP ™ ensures accurate results evenin difficult environments with vibrationPTPN process terminalMETTLER TOLEDO Dairy & Cheese News 27Quality data under control?We have the right solutionConsistently improving the quality of your products requires the ability of efficiently controlling product and package quality parameters in a fast-changing and highly competi-tive environment.Competition in the food industry –with high volumes but tight margins – causes demands for efficient quality assurance systems. Statistical Quality Control (SQC) systems for permanent online information and documenta-tion about your key quality para-meters convert into real cost savings.Our solutions for Statistical Quality Control (SQC) combine ease of opera-tion, quality data management and analysis functionality.• We offer mobile compact solutions with embedded SQC intelligence up to networked systems with an SQL database.• The systems are upgradeable and can be expanded and adapted to meet changing customer needs.• Simple and intuitive prompts guide the user through the sample proc-ess, reducing training costs as well as sampling errors.• Realtime analysis and alarms help to take immediate corrective measures and to save money by reducing overfilling.Throughout the manufacturing pro-cess, METTLER TOLEDO SQC solu-tions analyze your important product and package quality parameters andpresent them the way you want, help-ing to comply to legislation, to control and document your product qualityand your profitability.Metal detectionCheckweigher Sample check ® onlinequality data analysis/dairy-cheeseFor more informationMettler-Toledo GmbH CH-8606 Greifensee SwitzerlandTel. +41 44 944 22 11Fax +41 44 944 30 60Your METTLER TOLEDO contact:1. SevenGo ™ portable pH-meter2. In-line turbidity, pH and conductivity sensors3. DL22 Food and beverage analyzer4. Halogen moisture analyzersA wide range of solutions to improve processes1. Statistical Quality Control/Statistical Process Control2. Process weighing3. Predictive maintenance4. Methods of moisture content determinationShare our knowledgeLearn from our specialists – our knowledge and experience are at your disposal in print or online.Learn more about all of our solutions for the dairy and cheese industry at our website. You can find information on a wide range of topics to improve your processes, including case studies,application stories, return-on invest-ment calculators, plus all the product information you need to make aninformed decision.1 2 341423。

Characterization of particulate-bound polycyclic aromatic hydrocarbons and trace metals composition of urban air in Delhi,IndiaD.P.Singh a ,Ranu Gadi a ,*,Tuhin K.Mandal ba Indira Gandhi Institute of Technology,GGS Indraprastha University,Delhi 110006,India bRASD,National Physical Laboratory,New Delhi,Indiaa r t i c l e i n f oArticle history:Received 17June 2010Received in revised form 22February 2011Accepted 22February 2011Keywords:Airborne particulate matterPolycyclic aromatic hydrocarbons Trace metalsVehicular emissions Enrichment factor Diagnostic ratioa b s t r a c tThe concentrations of sixteen polycyclic aromatic hydrocarbons (PAHs)and trace metals adsorbed to respirable particulate matter (PM 10m m)and the fine fraction of particulate matter (PM 2.5m m)were determined at a site in Delhi (India)during the winter and summer periods in 2007e 2008.The annual mean concentrations for PM 10and PM 2.5were 138.5Æ40.4m g m À3and 50.6Æ20.4m g m À3,respectively,with higher concentrations during winter than summer period.Concentrations of PM 10and PM 2.5have been found that were higher than the prescribed limits of the WHO and the NAAQS given by CPCB,India.The trace metals detected in the PM 10and PM 2.5were Al,Ca,Cd,Cr,Cu,Fe,Mn,Ni,Pb,V and Zn and their concentrations were similar to those observed in heavily polluted urban areas from local traf fic and other anthropogenic emissions.Total PAH concentrations for PM 10and PM 2.5were much higher in winter (81.5and 96ng m À3,respectively)compared to summer (33.1and 45.8ng m À3,respectively)with high molecular weight homologues (4e 6ring PAHs),which account for 80e 95.8%of total PAHs.In general,the PM 2.5PAH concentrations were higher than PM 10particles.The results of diagnostic ratio and enrichment factor analyses showed that vehicular and anthropogenic emissions related to combustion,industrial processes as well as natural sources associated with the transport of dust from the roadside area were the main pollutant sources for PAHs and trace metals.Ó2011Elsevier Ltd.All rights reserved.1.IntroductionAirborne particulate matter (PM)within most urban areas of developing countries derives from a wide variety of sources such as road traf fic emissions,secondary particles and coarse particles (deriving mainly from marine aerosol),resuspension of soils and road dusts (APEG,1999).Recent studies in urban atmospheres showed that the concentrations of PM 10and PM 2.5aerosols exhibit good agreement with traf fic-related pollutants (both diesel and gasoline vehicles)and their health effects through respiratory and cardiac diseases (Lee et al.,2006).Ambient particulate matter consists of various organic compounds,which includes volatile or semi-volatile organic compounds,such as polycyclic aromatic hydrocarbons (PAHs)compounds,which are formed due to incomplete combustion of organic materials and these,are carcinogenic and/or mutagenic in nature (Larsen and Larsen,1998).Some studies in India have examined PAHs associated with Total Suspended Particulate (TSP)and PM 10aerosols in urban and remote areas (Sharma et al.,2007;Khillare et al.,2008)but study on determination of PAHs associated with PM 2.5has not been done so far in India.Heavy metals constituting the PM,arising from different envi-ronmental sources are an important group to be considered due to their toxic characters.Many studies have been done in India addressing the problem of pollution by heavy metals either based on short-term studies of coarse suspended particulates or TSP (Tripathi et al.,2004;Srivastava et al.,2009;Sridhar et al.,2010)and on metal composition of PM 10(Karar and Gupta,2006;Nair et al.,2006;Kulshrestha et al.,2009)but there are very few studies on metal composition of PM 2.5(Kulshrestha et al.,2009).Large amount of data on concentration levels of SPM,NO x ,SO 2,CO and PAHs associated with TSP are available for prominent urban areas in India;studies on the simultaneous measurements of trace metals and PAHs from PM 10and PM 2.5in ambient air of major cities are rather limited.In the present study,the seasonal variations of particle associated PAHs and trace metals in Delhi is evaluated in order to assess the behaviour of these compounds and their risk for human health.The seasonal distribution of the PM fraction in selected major cities of various countries has also been compared with ones in Delhi.Diagnostic ratios and Enrichment factors are also determined to evaluate the different pollution sources contributing to the trace metals and PAHs.*Corresponding author.Tel.:þ911123900363;fax:þ911123869872.E-mail address:ranugadi@ (R.Gadi).Contents lists available at ScienceDirectAtmospheric Environmentjournal homep age:www.elsevi/locate/atmosenv1352-2310/$e see front matter Ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.atmosenv.2011.02.058Atmospheric Environment 45(2011)7653e 76632.Experiments2.1.Study area and site descriptionDelhi,the capital of India,has a geographic area of1483km2 and is situated at latitude of28 2401700N e28 530N and the longitude of76 2003700E e77 2003700E with an altitude of216m above mean sea level.Delhi is surrounded by four different climatic zones i.e.,the Himalayas in the north,central hot plains in the south,the Thar Desert in west and the Gangetic plain in the east.Delhi lies in the semi-arid zone of India.The summers and winters are extreme with maximum and minimum temperatures as45.6 C and27.6 C in summers(March e June)and22.2 C and 2 C in winters(November e February),respectively.The air over Delhi is dry during the greater part of the year with April and May being the driest months.The monitoring site(G.G.S.I.P University campus)located in the eastern part of Delhi is just50m away from the busy road with unique transportation which includes buses,gasoline passenger vehicles,taxis and trucks fuelled by diesel.Around100m from the sampling site,many local roads interweave together and have a large amount of motor vehicles. The traffic density at the sampling site was approximately 500e600vehicles per hour during the sampling period.There are no other large combustion sources nearby besides vehicular emissions.The map of Delhi showing the sampling site is given in supplementary information;Fig.S1.2.2.Ambient samplingThe samples for particulate matter were collected by Respirable Dust Sampler APM460BL,(flow rate:1.2Lpm)and Fine Particu-late Sampler APM550(flow rate:16.6Lpm)(both supplied by Envirotech Instruments Pvt.Ltd.,Okhla,Delhi)for PM10and PM2.5 respectively.Whatman GF/A glass-fibrefilters were used for collection of PM10and PM2.5(size800Â1000and47mm,respec-tively).The instruments were kept at15m above the ground level. Collection for PM was performed in two periods:winter(22nd November2007e26th February2008)and summer(2nd April e25th June2008),with16and14samples for winter and summer,respectively.Glass-fibrefilters(GFFs)used for the particulate matter sampling were pre-cleaned by baking at550 C for10h before use to minimize PAH and trace metals blank in the GFFs.Before and after sampling thefilters were kept in a Secador desiccator(Tarson)under controlled temperature(5e30 C;with automatic controller)and35e40%relative humidity for at least 24h to prevent hydration of thefilter surface.Thefilters were weighed twice before and after sampling using four digit micro-balance with0.010m g sensitivity(Sartorius,Model:GC1603S-OCE) to obtain PM10and PM2.5concentration respectively.After weigh-ing,thefilter papers were packed in aluminium foil to protect them from sunlight and they were stored under refrigeration(À16 C)to prevent volatilization of PAHs of lower molecular weight,until extraction and analysis could be completed.It is pertinent to mention here that only the particulate PAHs associated with PM10 and PM2.5may be trapped on the glass-fibrefilter.Since,in the present study no collection media(sorbent)was used down-stream of thefilters to collect the gaseous component of PAHs;the measured values of PAHs would have in all probability a low bias (USEPA,Method TO-13A).2.3.Extraction of PAHs and analysis by GC e MSThe PAHs were extracted from the PM collected on thefilter paper using appropriate solvents.For extracting the PAHs,a quarter of thefilter paper was taken(both in PM10and PM2.5) and further cut into strips and was extracted with30mL of HPLC-grade dichloromethane(DCM)using ultrasonic agitation for 30min,the extraction procedure was repeated two times more to attain maximum extraction.The extract was evaporated to small volume(w5mL)using a rotary evaporator at a temperature between30e40 C and then underflow of ultra pure nitrogen gas, it was further reduced andfinally adjusted to exactly1mL by DCM afterfiltering through membranefilter(PVDF0.5m m micro syringe).The extract was then injected into GC e MS for analysis.0.5ng of semi-volatile internal standard supplied by Supelco was added to all extracts prior to chromatographic analysis.The particulate PAHs were quantified by Hewlett e Packard5890Gas Chromatograph-5971A Mass selective Detector(GC e MS)system equipped with a fused silica capillary DB-5column (30Â0.25mm i.d.,0.25m mfilm thickness).The Carrier gas was helium(0.5ml minÀ1).The ionization was carried out in the electron impact mode at70eV and the mass range scanned was from50to550amu under full scan acquisition mode.The temperature program was as follows:initial temperature of50 C was held for2min,increased at rate of4 C minÀ1e280 C,then held for20min.1microlitre volume of each sample was injected in the splitless mode and the purge time was1min.Identification and quantification of16PAHs compounds were based on matching their retention time with a mixture of PAH standard (Supelco-EPA610PAH mix,Supelco,USA).The16PAHs compounds were Naphthalene(Naph),Acenaphthylene(Acy), Acenaphthene(Ace),Fluorene(Flu),Phenanthrene(Phen), Anthracene(Anth),Fluoranthene(Flt),Pyrene(Pyr),Benzo[a] anthracene(BaA),Chrysene(Chr),Benzo[b]fluoroanthene(BbF), Benzo[k]fluoroanthene(BkF),Benzo[a]pyrene(BaP),Dibenzo[a,h] anthracene(DahA),Benzo[ghi]perylene(BghiP),and Indeno[123-cd]pyrene(IcdP).Blank samples(including two GFFs)were prepared,treated and analyzed in the same manner as the real samples to eval-uate analytical bias and precision.The method detection limit (MDL)was used to determine the lowest concentration level that can be detected which is statistically different from a blank. The MDL was determined by selecting the concentration slightly higher than the lowest concentration of the standard line.This blank concentration was repeated several times to estimate the standard deviation.Then,the MDL was based on three times the standard deviation of the blank concentration. Method detection limits were between0.039ng and0.531ng for BghiP and Chr,respectively.Recoveries of PAHs were calculated by spiking a standard PAH solution containing the16 PAHs(16PAH-mix,Supelco,U.S.A)in a quarter piece of a blank filter and following the same experimental procedure used for the sample treatment.For the16spiked individual PAHs,the recoveries from Naph to BghiP were from60to115%for the GFF. Reproducibility was calculated on replicate analyses giving an error of aboutÆ12%.2.4.Analysis of heavy metalsAnother quarter of thefilter was taken for the estimation of heavy metals by Wavelength Dispersive X-Ray Fluorescence Spectrometer(WD-XRF),S4Pioneer(Bruker AXS)equipped with Rh X-Ray tube,a2.7kW generator,an eight-position automatic crystal changer,a gasflow-proportional counter and a scintilla-tion detector.The spectral data were processed with Spectra Plus software.The following metals were quantified:Al,Ca,Cr,Cu,Fe, Mn,V,Ni,Pb,Cd and Zn.Minimum detection limits(MDL)for most of the elements varied from4to35ng mÀ3.Some of the other elements(Co,Zr,As,Mo and Sn),although principallyD.P.Singh et al./Atmospheric Environment45(2011)7653e7663 7654detectable,were found to be below the MDL.At least10%of the samples were evaluated by spiking with a known amount of metal to calculate the recovery efficiencies.The analytical procedure for the recovery test was the same as that forfield samples.The range of recovery efficiency was89.5e99%.3.Results and discussionThis environmental study had two main objectives.First,to know the PM10and PM2.5levels in Delhi atmosphere,and secondly,to know about the nature and quantity of the PAHs and trace metals contained in the PM10and PM2.5.In addition,the origin and their sources were also analyzed.Weekly concentra-tions of PM,particulates-bound PAHs and trace metals are given in supplementary information,Table S2.3.1.PM10and PM2.5concentrationsTable1presents the statistical summary of particulate concentration along with temperature,relative humidity and wind speed during the total sampling days covering two seasons (2007e2008)in order to observe the levels of airborne particles and seasonal changes during winter and summer period.Results (Table1)show that PM10and PM2.5concentration in winter were almost two times of the summer concentrations.PM10mass determined in our study varied within the range80e214.5m g mÀ3 in winter(2008)and55e135.4m g mÀ3in summer period (2007e08),whereas the PM2.5concentration ranged between 30.2e73.5m g mÀ3and16.3e63.2m g mÀ3in winter and summer respectively.The concentration of PM10was higher than PM2.5in both the seasons(winter e summer).On applying t-test to the above PM mass concentration,the average PM10and PM2.5 concentration was similar statistically(p¼0.70at95%signifi-cance).The results compiled in Table1show that the yearly mean average of PM10and PM2.5concentrations in Delhi,are 138.5Æ40.4and50.6Æ20.4m g mÀ3respectively which exceeded 2.3times(60m g mÀ3annual average)and1.3times(40m g mÀ3 annual average)for PM10and PM2.5respectively of NAAQS as specified by Central Pollution Control Board,India.When these values are compared with World Health Organisation(WHO)air-quality guidelines(WHO,2005)(20m g mÀ3and10m g mÀ3,annual average for PM10and PM2.5respectively)it was found that average annual mean concentrations were6.9times higher for PM10and 5.1times higher for PM2.5.Winters are characterized with mostly higher PM10and PM2.5. This is probably due to low wind speed and high humidity during the season so the removal of aerosol particles by wet scavenging is reduced.Moreover during summers the prevailing winds that are due to thermal circulations are stronger and the mixing height is deeper.Wind turbulence with large mixing height results in proper dilution and dispersion of pollutants during summer(Mantis et al., 2005).Measurements for the15years in Delhi showed30e50% reduction in SO2,NO x,CO and Pb,but airborne PM was reduced by only15%compared to high levels in the1990s (146e578m g mÀ3;Sharma et al.,2003).Daily PM10concentra-tions exceeded the NAAQS(100m g mÀ3).Some earlier studies at Delhi(Mohan et al.,2007;Khillare et al.,2008)for PM10and PM2.5are comparable with our study and indicated that traffic is a major contributor to airborne particulate matter(PM).The PM10concentrations measured by CPCB for the year2008at ITO (traffic intersection site)ranged from127to228m g mÀ3,which are in the same range as ours(55e213.4m g mÀ3).It has been estimated that traffic may be responsible for more than80%of total suspended PM in urban areas(Danielis and Chiabai,1998). In particular,diesel emissions may account for up to30%of pollution byfine particles.In this study,concentrations of PM10 were higher than the values found by different researchers in European countries(Pakkanen et al.,2001;Querol et al.,2001; Gotschi et al.,2002;Farmer et al.,2003;Harrison et al.,2003; Rajsic et al.,2004;Valavandis et al.,2006)but PM2.5 (61.8Æ20.7m g mÀ3)concentration was somewhat similar to studies done at Milan(53.7m g mÀ3)and Sofia(89.8m g mÀ3) during winter period by Lonati et al.(2005)and Farmer et al. (2003)respectively.Our results for PM10and PM2.5are also higher than the other studies done in different parts of India.The higher concentrations of PM in Delhi are probably related to heavy vehicular trafficflow,especially from diesel vehicles, emissions from nearby industries and heating of biomass during winter months,despite the alternative daily restrictions of private cars and better public transport.The compilations of these recent studies are given in supplementary information; Table S2.3.2.PAHs concentrationsMean concentrations of16PAHs in PM(PM10and PM2.5) during winter and summer period(2007e2008)in ambient air of Delhi are presented in Table2.Concentrations of PAHs bound to PM showed a seasonal trend,with higher concentrations during the winter period than summer period.Particle-bound PAH concentrations varied inversely with temperature e i.e., higher concentrations in winter and lower in the summer months(Table1).Although wind speed varied little between the seasons,other meteorological parameters such as low temper-ature and moderate relative humidity pointed towards the poor dilution of pollutants during winter season(Table1).The increase in particulate PAH during winter and the dependence of PAH concentration on atmospheric temperature have been reported in a number of previously published studies(Eiguren-Fernandez et al.,2004;Tsapakis and Stephanou,2005;Li et al., 2006).Low atmospheric temperature can affect the distribu-tion of PAHs between the particle and gas phases and result in relatively larger portion of PAH portioning to the particle phase. The elevated concentrations of PAHs in winter can be attributed to increased emissions from higher consumption of fossil fuels, biomass fuels more traffic volumes and less dispersion due to meteorological conditions prevailing in winter,whereas inTable1Summary of meteorological parameters and particle mass concentrations during study period(2007e2008).Season Years Particulate matter Temperature( C)Relative humidity(%)Wind speed(m sÀ1)MeanÆSD(m g mÀ3)Median RangeWinter(Nov.e Jan.)2007e2008PM10 1.6e25.224e93.20e4.1182Æ32.5173.280e214.5 PM2.561.8Æ11.759.430.2e73.5 Summer(April e June)2008PM1029.8e45.614e54 2.1e16.195.1Æ22.285.455e135.4 PM2.539.4Æ13.936.416.3e63.2D.P.Singh et al./Atmospheric Environment45(2011)7653e76637655summer high wind speeds and high temperature along with enhanced photodecomposition of PAHs,lead to increased dispersion and decrease in the concentration of PAHs.The particle-bound PAH levels and seasonal trend in the current study have been compared with other studies and it was found that our concentrations are higher than those reported for urban environments in Los Angeles,CA,USA (Eiguren-Fernandez et al.,2004);Brazil (Bourette et al.,2005);Croatia (Sisovic et al.,2005),Greece (Tsapakis and Stephanou,2005);China (Li et al.,2006),and Japan (Tham et al.,2008).In earlier studies also the concentrations of particle associated PAHs in urban areas have been found to peak in winter (Harrison et al.,1996;Park et al.,2002;Guo et al.,2003).While explaining the winter/summer (w/s)ratios,Tsai et al.(1995)found that the wind speed and wind direction are the most signi ficant parameters affecting PAH concentrations.As seen from Table 2,in our study w/s ratio is 2.47and 2.1for PM 10and PM 2.5respectively.Other studies carried out in Europe and in the USA found that PAHs in winter were generally higher by a factor of 1.5e 10to that in summer (Harrison et al.,1996;Caricchia et al.,1999;Ohura et al.,2004).In a recent study,Guo et al.(2003)found rather high w/s ratios of 8.6and 7.5at two sites in the city of Hong Kong.The 4e 6ring PAHs (IcdP,BghiP,DahA,BkF,BbF,BaP,Chr,Anth and Pyr)have dominance during winter in the PM 10and PM 2.5which are mostly related with diesel exhaust combustion including wood combustion in residential heating.Previous studies showed that IcdP,BghiP,DahA and Pyr dominate in diesel exhaust emissions (Cecinato et al.,1999).Although after implementation of Compressed Natural Gas (CNG),Delhi government has placed restrictions on the number of diesel taxis,there are a large number of diesel vehicles still in use in Delhi which are contributing substantially to the air pollution in the city.It is well documented in previous study that most of the PAHs are released due to a variety of combustion processes and were predominantly (70e 80%)found in the fine particles (Cecinato et al.,1999).Seven carcinogenic PAH (Chr,BaA,BbF,BkF,BaP,DahA and IcdP)concentrations in PM 10and PM 2.5were also calculated and given in Table 2.The concentrations of seven PAHs in PM 10and PM 2.5during winter period were 1.2e 3times higher than the summer period.The highest concentration ofseven carcinogenic PAHs in PM 10(winter samples)was in decreasing order IcdP >DahA >BkF >BbF >BaP >Chr >BaA (1.4e 17.3ng m À3),whereas,in the summer samples the order was BkF >DahA >IcdP >BbF >BaP >Chr >BaA (1.3e 8.5ng m À3).In case of PM 2.5,the concentration of PAHs were in the order of IcdP >kF >BaP >BbF >Chr >BaA >DahA (2.2e 25.8ng m À3),whereas,in the summer samples the order was BkF >IcdP >BaP >BbF >Chr >DahA >BaA (1.4e 8.9ng m À3).It was also found that seven carcinogenic PAHs have maximum loading out of total PAHs with almost two-third of the total PAHs concentration,probably due to vehicular parisons of PM 10and PM 2.5associated 4e 6ring PAH values in this study with those reported values for other major cities of the world (Supplementary information;Tables S3and S4).It was observed that values reported in the present study,are much higher than those reported in other parts of the world,PAH values in PM 10samples (BbF,BaP,IcdP and BghiP)are comparable to earlier data over Delhi (Sharma et al.,2003)and Mumbai (Kulkarni and Venkataraman,2000).PAHs in the present study could be classi fied according to their number of aromatic rings as follows:2e 3-rings include Naph,Ace,Acy,Flu,Phen,Anth;4-rings include Flt,Pyr,BaA,Chr;5-rings include BbF,BkF,BaP,DahA;6-rings include BghiP and IcdP.The percentage of different rings to total PAHs in PM 10and PM 2.5samples during winter and summer was shown in Fig.1.Low molecular weight PAHs homologues (including 2e 3ring PAHs)contribute a lower fraction in total PAHs,whereas high molecular weighed PAHs homologues (including 4e 6ring PAHs)have higher fraction,ranging from 80.1%to 95.2%.The larger fraction of high molecular weight PAHs in both PM 10andPM 1051015202530354045502-3 ring 4-ring 5-ring 6-ring P e r c e n t i n t o t a l P A H (%)SummerWinterPM 2.5510152025303540452-3 ring 4-ring 5-ring 6-ringP e r c e n t i n t o t a l P A H (%)Summer WinterFig.1.Distribution of grouped PAH by ring size (a)in PM 10(b)in PM 2.5.Table 2Mean concentration (ng m À3)and standard deviation (ÆSD)of PAHs in PM 10and PM 2.5.PAH compoundsPM 10PM 2.5WinterSummerWinter Summer Naphthalene 1.4Æ0.60.2Æ0.1 2.1Æ0.6 1.8Æ1.4Acenaphthylene 0.6Æ0.20.3Æ0.1 2.2Æ1.1 1.3Æ1.0Acenaphthene 1.1Æ0.40.6Æ0.30.6Æ0.40.4Æ0.2Fluorene0.4Æ0.20.2Æ0.1 1.2Æ0.60.8Æ0.5Phenanthrene 0.8Æ0.40.5Æ0.3 1.2Æ0.6 1.0Æ0.5Anthracene 2.4Æ1.40.8Æ0.4 3.2Æ2.1 1.6Æ0.8Fluoranthene 1.7Æ1.20.6Æ0.2 3.8Æ2.2 2.7Æ1.8Pyrene 4.1Æ2.1 2.5Æ1.4 2.1Æ1.1 1.5Æ1.0Chrysene4.4Æ1.5 2.2Æ0.8 3.7Æ1.4 2.8Æ1.3Benzo[a]anthracene 3.2Æ1.8 2.1Æ0.8 3.6Æ2.2 1.8Æ0.6Benzo[b]fluoranthene 8.8Æ3.5 4.4Æ2.211.8Æ5.5 5.4Æ2.6Benzo[k]fluoranthene 9.6Æ3.6 3.9Æ2.813.6Æ4.66.9Æ1.8Benzo[a]pyrene6.9Æ2.1 3.1Æ1.99.9Æ3.2 5.1Æ2.9Dibenzo[ah]anthracene 9.8Æ4.6 3.7Æ1.6 2.8Æ0.6 2.1Æ0.8Benzo[ghi]perylene 12.5Æ5.3 4.4Æ2.515.5Æ6.3 6.4Æ2.5Indeno[123-cd]pyrene 13.8Æ3.53.6Æ2.418.7Æ7.14.2Æ2.4S 16PAH 81.5Æ32.433.1Æ18.996Æ39.645.8Æ22.1S 7PAH a56.5Æ20.323Æ12.564.1Æ24.628.3Æ12.4aSeven carcinogenic PAHs includes:Chr,BaA,BbF,BkF,BaP,DahA and BghiP.D.P.Singh et al./Atmospheric Environment 45(2011)7653e 76637656PM2.5samples are mostly due to the combustion of petroleum fuels.Similar observations are also reported by Liu and Cheng (2007).3.3.Trace metals concentrationThe trace elements were analyzed to establish the connection of airborne PM with different sources and are presented in Table3.Average mass of analyzed metals constitutes10.1%and 11.2%of PM10and PM2.5samples respectively.For residential sites there are standard set values prescribed by NAAQS for Pb, Ni and As as1000ng mÀ3,20ng mÀ3and6ng mÀ3,respectively (CPCB,2009).The annual average concentration of Pb in present study did not exceed the national standard of1.0m g mÀ3.Our results indicated that the metallic elements Al,Ca,Fe,Cu,Zn and Cr were the largest components in the airborne PM10and PM2.5 fractions.Fe probably originating from natural sources(soil dust),was almost equally distributed in the two fractions(PM10 and PM2.5)of PM but the level of Fe is also much higher than expected.It is due to the fact that the ambient air of Delhi contains high levels of Fe,with crustal origin and present in abundant amount in loosely bound dust of Delhi.When Fe is associated with Zn,pollution may be related to anthropogenic emission sources,fossil fuel combustion,incineration,or natural origin(Manalis et al.,2005).Trace metals(Al,Ca,Fe,Zn,Pb and Cu)comprised90%of all metals in PM10and PM2.5,suggesting three main sources:traffic,static combustion and crustal matter. Results were consistent with values reported by Manalis et al. (2005).The mean values of trace metals in the PM10and PM2.5 samples in winter was in the order of Ca>Al>Zn>Fe>Cr>V>Mn>Pb>Cu>Ni>Cd and in summer as: Ca>Al>Fe>Zn>Cr>Ni>Cu>Pb>Mn>V>Cd. Concentrations of metals in our study are found to be higher for both PM10and PM2.5than other studies but lesser than the study done at Agra(industrial site)(Supplementary information; Table S5).The trace metals V,Ni and Cd found in PM10and PM2.5 might be originated from diesel and gasoline exhaust,these elements were within the permissible levels of WHO(WHO, 2005).Trace metals(e.g.Cd,Cu,Pb,Zn,Ni and V)originating from anthropogenic sources,have exhibited predominant occurrence in the PM2.5fraction and low enrichment factors(EF) values in the PM10particles.On the other hand,elements such as Cr,originating also from natural sources and soil dust,were equally distributed in both fractions.The common sources attributed to this metal at this site are resuspension of road dust due to vehicular activity,which is rich in Ni and Zn.It may be due to activities like wear and tear of tyres,oil burning,abrasion of mechanical parts of road vehicles,oil lubricants.In India Pb has been phased out of gasoline from year2000;however it still persists in road dust from earlier vehicular exhaust emissions as it has longer residence time in environment.The mean concentrations of Zn,V,Mn,Cu,Cr,Ni in PM10and PM2.5 (Table3)show that predominance of these above metals at monitoring sight might be due to fuel combustion(petrol and diesel),industrial sources,wearing of break pads of vehicles and oil burning.Metal concentration in summer is less as compared to winter because of better dispersing conditions due to increase in the wind speed in these months.4.Source identification4.1.Diagnostic ratioThe diagnostic ratio method for PAH source identification involves comparing ratios of pairs of frequently found PAHsTable3Concentration of heavy metals in particulate matter(PM10and PM2.5).PM Season Al(ng mÀ3)Ca(ng mÀ3)Cd(ng mÀ3)Cu(ng mÀ3)Fe(ng mÀ3)Ni(ng mÀ3)Zn(ng mÀ3)V(ng mÀ3)Pb(ng mÀ3)Cr(ng mÀ3)Mn(ng mÀ3)PM10Summer52519452 1.822.3202131.2431.5 2.2210.5144.719.1 Winter369041529.236.2288415.3612.360.4420.7128.544.7PM2.5Summer2454917.2 2.465.2612.729.5477.2 1.8500.274.133.7 Winter2880424.112.6125.3244615.3825.1 5.6630.880.525.6Table4Diagnostic ratio of PAHs in ambient atmosphere of Delhi.Diagnostic ratios Our study Value Sources ReferencesPM10PM2.5IcdP/(IcdPþBghiP)0.18Gasoline Kavouras et al.(1999)0.510.510.37e0.70DieselBaP/(BaPþChr)0.600.690.73Gasolineemissions Guo et al. (2003)BaP/BghiP0.590.680.5e0.6Trafficemissions Park et al. (2002)IcdP/BghiP0.27e0.4Gasolineengine Caricchia et al. (1999)1.02 1.051Diesel engine1.06e1.12Coal/CokeFlt/(FltþPyr)0.270.650.4e0.7Automobileemissions Ravindra et al. (2008)BaA/(BaAþChr)0.5Industrial Kavouras et al.(1999)0.450.450.38e0.65Diesel motors0.43Gasoline motorsPhen/(PhenþAnth)0.300.31<0.7Biomass burning Kavouras et al.(1999)>0.7Fossil fuels0.11101001000EnrichmentFactorFig.2.Enrichment factor values using the metal concentrations of Delhi sampling from November2007to June2008.D.P.Singh et al./Atmospheric Environment45(2011)7653e76637657。

[文章编号]　100723949(2004)1220220151204・实验研究・球囊拉伤致家兔动脉粥样硬化斑块破裂及血栓形成陈文强,张运,季晓平,张梅,朱永锋,殷乐,姚桂华(山东大学齐鲁医院心内科,山东省济南市250012)[关键词]　内科学;　药物触发斑块破裂及血栓形成;　斑块破裂及血栓形成的兔模型;　动脉粥样硬化;　血栓形成;　球囊;　组胺[摘　要]　为建立动脉粥样硬化的兔模型并用药物触发造成斑块破裂及血栓形成,将60只雄性纯种新西兰兔随机平均分成三组:球囊损伤+高脂(1%胆固醇)组、高脂喂养组及普通饲料喂养组,喂养3个月后分别给予鲁塞尔蝰蛇毒和组胺药物触发以造成斑块破裂及血栓形成。

结果发现,球囊损伤+高脂组与高脂喂养组均形成动脉粥样硬化斑块,其中球囊损伤+高脂组所形成的粥样斑块为具有较大脂质核的软斑块;药物触发后球囊损伤+高脂组存活的18只中有11只共15处发生斑块破裂及血栓形成;高脂喂养组中的19只经药物触发后仅5只共7处发生斑块破裂及血栓形成;普通饲料喂养组中未见斑块破裂及血栓形成。

结果提示,在构建的动脉粥样硬化斑块的动物模型基础上,应用药物触发后能够造成斑块破裂及血栓形成。

[中图分类号]　R5[文献标识码]　AR esearch on T riggering Plaque Disruption and Arterial Thrombosis in VivoCHE N Wen 2Qiang ,ZH ANG Y un ,J I X iao 2Ping ,ZH ANG Mei ,ZH U Y ong 2Feng ,YI N Y ue ,and Y AO G ui 2Hua(Department o f Cardiology ,Qilu Hospital o f Shandong University ,Jinan 250012,China )[KE Y WOR DS ] Atherosclerosis ;　Thrombosis ;　Balloon ;　Histamine ;　Pharmacological T riggering ;　Disruption [ABSTRACT ] Aim T o build an animal m odel of plaque disruption and arterial thrombosis by pharmacological triggering atherosclerotic rabbits. Methods S ixty New Z ealand White rabbits were equally divided into 3groups at random :balloon 2in 2jury +high lipid diet group ,high lipid diet group and regular diet group.　Rabbits in balloon 2injury +high lipid diet group un 2derwent balloon 2induced arterial wall injury and then were fed on a diet of 1%cholesterol ;rabbits in high lipid diet group were only given 1%cholesterol ;rabbits in regular diet group were fed on a regular diet.　They were all fed for 3m onths.　Then the three groups underwent pharmacological triggering with Russell ’s viper venom (RVV )and histamine. R esults Atherosclero 2tic plaques were found in balloon 2injury +high lipid diet group and high lipid diet group ;the lipid cores in the first group were larger than those in the second group.　In balloon 2injury +high lipid diet group ,it was found that plaques disruption and throm 2bosis occurred in 11out of the 18rabbits and in total 15lesions occurred plaque disruption and thrombi after triggering.　In high lipid diet group ,only 5rabbits dem onstrated plaques disruption and thrombosis and there were in all 7thrombi.　H owever ,rab 2bits in the regular diet group did not have plaques disruption and thrombosis. Conclusions With atherosclerotic plaque ani 2mal m odels ,we could pharmacologically trigger plaque disruption and arterial thrombosis.[收稿日期]　2003205230 [修回日期]　2003212222[基金项目]　卫生部临床学科重点项目基金(20012943)资助[作者简介]　陈文强,心内科博士研究生,主治医师,主要从事冠心病方面的研究工作。

Adsorption behavior of reactive dye in aqueous solutionon chemical cross-linked chitosan beadsM.S.Chiou *,H.Y.LiDepartment of Chemical Engineering,National Lien Ho Institute of Technology,Miao Li 36003,Taiwan,ROCReceived 25October 2001;received in revised form 30September 2002;accepted 30September 2002AbstractA batch system was applied to study the adsorption of reactive dye (reactive red 189)from aqueous solutions by cross-linked chitosan beads.The ionic cross-linking reagent sodium tripolyphosphate was used to obtain more rigid chitosan beads.To stabilize chitosan in acid solutions,chemical cross-linking reagent epichlorohydrin (ECH),glu-taraldehyde and ethylene glycol diglycidyl ether was used and ECH shows a higher adsorption capacity.The Langmuir and Freundlich adsorption models were applied to describe the equilibrium isotherms at different particle sizes and isotherm constants were determined.The Langmuir model agrees very well with experimental data and its calculated maximum monolayer adsorption capacity has very large value of 1802–1840(g/kg)at pH 3.0,30°C.The kinetics of the adsorption with respect to the initial dye concentration,temperature,pH,ionic strength,and wet/dry beads were in-vestigated.The pseudo-first-order,second-order kinetic models and intraparticle diffusion model were used to describe the kinetic data and the rate constants were evaluated.The dynamical data fit well with the second-order kinetic model,except for the dry beads fitting better with the first-order model.The adsorption capacity increases largely with de-creasing solution pH or with increasing initial dye concentration.Thermodynamic parameters such as change in free energy (D G 0),enthalpy (D H 0),entropy (D S 0)and activation energy were also determined.The adsorption mechanism is shown to be the electrostatic interactions between the dye and chitosan beads.The desorption data shows that the removal percent of dye RR 189from the cross-linked chitosan beads is 63%in NaOH solutions at pH 10.0,30°C.The desorbed chitosan beads can be reused to adsorb the dye and to reach the same capacity as that before desorption.Ó2002Elsevier Science Ltd.All rights reserved.Keywords:Adsorption capacity;Adsorption rate;Reactive dye;Chitosan;Cross-linking1.IntroductionThe effluents of wastewater in some industries such as dyestuff,textiles,leather,paper,plastics,etc.contain various kinds of synthetic dyestuffs.A very small amount of dye in water is highly visible and can be toxic to creatures in water.Hence,the removal of color from process or waste effluents becomes environmentally im-portant.Among several chemical and physical methods,adsorption process is one of the effective methods to re-move dyes from wastewater.Many studies have been undertaken to find suitable adsorbents to lower dye concentrations from aqueous solutions.They included activated carbon (McKay,1983;Allen,1996),peat (Ramakrishna and Viraraghavan,1997;Ho and McKay,1998),chitin (McKay et al.,1982,1983;Juang et al.,1997),silica (McKay,1984),and others (Hu,1996;Namasivayam et al.,1998;Aksu,2001).For both re-generative and non-regenerative systems,high adsorp-tion capacity is essential for adsorbent selection.However,the amount (g)of dyes adsorbed on theaboveChemosphere 50(2003)1095–1105/locate/chemosphere*Corresponding author.Tel.:+886-37-352840x62;fax:+886-37-332397.E-mail address:chiou@.tw (M.S.Chiou).0045-6535/03/$-see front matter Ó2002Elsevier Science Ltd.All rights reserved.PII:S 0045-6535(02)00636-7adsorbents(kg)are not high enough,some have capac-ities of200–600g/kg and some even lower than50g/kg. To improve adsorption performance researches on new absorbents are still under development.Chitosan is the deacetylated form of chitin,which is a linear polymer of acetylamino-D-glucose and contains high contents of amino and hydroxyl functional groups. Chitosan has been reported for the high potentials of the adsorption of dyes(Yoshida et al.,1993;Wu et al.,2000; Chiou and Li,2002),metal ions(Guibal et al.,1998), and proteins(Zeng and Ruckenstein,1998).Other useful features of chitosan include its abundance,non-toxicity, hydro-philicity,biocompatibility,biodegradability,and anti-bacterial property(Kumar,2000).The adsorption of reactive dyes in neutral solutions using chitosan shows large adsorption capacities of1000–1100g/kg (Wu et al.,2000).In acid aqueous solutions,the amino groups of chitosan are much easier to be cationized and they adsorb the dye anions strongly by electrostatic at-traction(Kumar,2000).However,chitosan formed gels below pH5.5and could not be evaluated.Some cross-linking reagents are used(Zeng and Ruckenstein,1996; Ngah et al.,2002)to stabilize chitosan in acid solutions. Yoshida et al.(1993)used Denacol EX841as a cross-linking reagent and exhibited a high adsorption capacity (1200–1700g/kg)of Acid Orange II(acid dye)on the cross-linked chitosanfibers in acid solutions of pH3.0 and4.0.Chiou and Li(2002)cross-linked chitosan by epichlorohydrin(ECH)and obtained a high adsorption capacity(1600–1900g/kg)of reactive dye(RR189)on the cross-linked chitosan beads in acid aqueous solu-tions at pH3.0.It appears technically feasible to remove acid and reactive dyes from acid aqueous solutions by cross-linked chitosan.In this work,we want to shorten the preparation time and to improve the mechanical strength of the chitosan beads comparing with our previous work(Chiou and Li, 2002).The solution of sodium tripolyphosphate(TPP) was used to produce more rigid beads via ionic cross-linking reaction(Mi et al.,1999).Also three reagents ECH,glutaraldehyde(GA),and ethylene glycol diglyc-idyl ether(EGDE)were used in chemical cross-linking and compared with their adsorption behaviors.Chitosan with different molecular weight and different degree of deacetylation was used to evaluate the adsorption ca-pacity.We investigated the equilibrium and kinetics of adsorption of RR189on the chitosan in solutions of pH 1–9.The Langmuir and Freundlich equations were used tofit the equilibrium isotherm.The dynamical behaviors of the adsorption were investigated on the effect of initial dye concentration,temperature,solution pH value,ionic strength(NaCl),and wet/dry chitosan beads.The ad-sorption rates were determined quantitatively and com-pared by the pseudo-first-order,second-order models and the intraparticle diffusion equation.The dynamical adsorption behavior were compared between the chito-san and chitin to demonstrate the adsorption mecha-nism.The desorption data indicated the possibility of reuse of the cross-linked chitosan beads.2.Materials and methods2.1.ChemicalsChitosan(a-type;degree of deacetylation:84.5–85.5%,95%;was supplied by Fluka and OHKA Enter-prises Co.Reagents ECH(P98%),GA(50%),EGDE (50%)and TPP(P98%)were purchased from Fluka. The commercial dye RR189(C.I.18210)was supplied by local manufacturer,Dyexel Chemical Corp.and used as received(purity>90%).Fig.1displays the structure of dye RR189.2.2.Preparation of chitosan beadsThe preparation of chitosan beads involves three steps:1.chitosan dissolution,2.beads formation(Mi et al.,1999),and3.chemical cross-linking(Zeng and Ruckenstein,1996,1998;Guibal et al.,1999):1.10.0g of chitosan were dissolved in300cm3,5wt.%of acetic acid solution.The aqueous solution was di-luted to1.0dm3by strong stirring over night,and then let it stay still for24h.2.The above solution was poured into a buretteequipped with a micro-pipette tip.10cm3of the chitosan solution were dropped from the burette into a100-cm3aqueous solution of TPP(1wt.%).The dia-meters of the droplets were controlled via various tips of the micro-pipette.The chitosan beads werefiltered out,washed with deionized water,and stored in dis-tilled water for using.3.The wet non-cross-linked chitosan beads(containing0.1g dry basis of chitosan)and50cm3,1N NaOHsolution were poured together in a125-cm3flask.Cross-linking reagent ECH was added into the above solution,and shaken for6h at50°C with a water bath(Deng Yng Corp.,Taiwan).(Cross-linking re-agent GA and EGDE were used to cross-link in dis-tilled water,instead of NaOH solution.)Different1096M.S.Chiou,H.Y.Li/Chemosphere50(2003)1095–1105molar ratios of cross-linking reagents/chitosan were carried out in this work.The cross-linked chitosan beads werefiltered out,washed with deionized water, and stored in distilled water for using.(Dry cross-linked chitosan beads were used in some experiments.They were obtained from the wet cross-linked beads after staying in a vacuumed chamber at room tem-perature for24h.)2.3.Batch equilibrium studiesDye RR189was dissolved in deionized water to the required concentrations.The pH of dye solutions was adjusted by buffer solutions of acetic acid/acetate.In experiments of equilibrium adsorption isotherm,0.1g chitosan beads and50cm3dye solution were put in a 125-cm3flask,which contained acetic acid buffer solu-tion with desired pH value,and were shaken for5days using a bath to control the temperature at30Æ1°C. (The solution pH was adjusted to1.0or2.0by con-centrated HCl,and to9.0by NaOH.)In order to mea-sure the dye concentration,the solutions were adjusted to pH6.0and analyzed at wavelength534nm by an UV/ Visible spectrometer(JASCO V-530).2.4.Batch kinetic studiesIn experiments of batch kinetic adsorption,0.1g dry basis of wet chitosan beads and50cm3dye solution were poured in a125-cm3flask and stirred using a shaker with a water bath to control temperature.Every other period of time,0.1cm3of dye solution was taken out to dilute to10cm3,was adjusted to pH6.0,and determined its concentration.The experimental parameters included initial dye concentration,temperature,pH,the cross-linking ratio,NaCl concentration,and the wet/dry chi-tosan beads.3.Results and discussion3.1.Cross-linking reagent and chitosan3.1.1.Effect of ionic cross-linkingIn the bead formation step,the ionic cross-linking reagent TPP was used to strengthen the structure of chitosan pared with our previous method using NaOH,the chitosan beads ionic cross-linked by TPP in this work have several advantages.These beads are more rigid because of the ionic attractions betweenP3O5À10(TPP)and the–NHþ3group of chitosan in acidsolutions(Mi et al.,1999).This improves the mechanical properties of the chitosan beads.The time required to form beads decreases largely from24h(NaOH)to4h (TPP).After chemical cross-linking and drying,the chitosan beads made from TPP shrink to small and hard spheres,which are suitable for packing and transport. After soaking in water,these dry cross-linked chitosan beads swell to smaller size as the original ones.However, the chitosan beads made from NaOH break intoflakes after chemical cross-linking and drying.According to Mi et al.(1999),chitosan-TPP com-plexes were formed only by the ionic interaction between the positively charged amino group and negatively charged counter ions.In acid solutions,this interaction between chitosan and TPP is higher cross-linking,due to the high contents of the positively charged amino group. However,in strong basic solution,the–NHþ3groups are deprotonated and the ionic interactions between chito-san and TPP are disappeared.In our experiments,the ionic cross-linked TPP was washed out from the chito-san bead by1N NaOH solution in the step3to prepare chemical cross-linked chitosan beads.Therefore,TPP has no effect on dye adsorption.3.1.2.Effect of chemical cross-linking reagentThree chemical cross-linking reagents ECH,GA and EGDE were evaluated in this work.Fig.2shows the effect of the cross-linking reagent with various molar ratios to chitosan on the adsorption capacity of RR189 onto cross-linked chitosan beads at particle size2.3–2.5 mm,pH3.0,30°C,and initial dye concentration4290g/ m3.The result indicates that the cross-linking ratio af-fects slightly the equilibrium adsorption capacity for the three cross-linking reagents under the range we studied. The maximum q e appears at molar ratio0.87,0.17and 0.87for ECH,GA and EGDE,respectively.Fig.2also shows that the maximum adsorption capacity for using ECH is69%and83%higher than that of GA and EGDE,respectively.This result may be explained by that the different functional groups of chitosan were in-volved in the chemical cross-linking reaction asdifferentFig.2.The effects of chemical cross-linking reagent and cross-linking ratio on the adsorption capacity of RR189onto cross-linked chitosan beads.M.S.Chiou,H.Y.Li/Chemosphere50(2003)1095–11051097reagents were used.ECH cross-links chitosan molecules by connecting mostly with the–OH group of chitosan (Zeng and Ruckenstein,1996;Ngah et al.,2002), whereas GA and EGDE connecting with more–NH2 group(Zeng and Ruckenstein,1998;Guibal et al.,1999). Therefore GA and EGDE reduce the major adsorp-tion site(–NHþ3)on chitosan attracting the anion of dyeRR189via electrostatic interaction.The reagent ECH (with cross-linking ratio0.52)was used as the chemi-cal cross-linking reagent for all other studies in this work.3.1.3.Effect of chitosanChitosan with different molecular weight and degree of deacetylation was used to evaluate adsorption ability (Figure not shown).The equilibrium adsorption capac-ities of dye RR189on cross-linked chitosan beads made of molecular weight150k,220k,400k,and600k(degreeof deacetylation84.5–85.5%)are almost the same ($1750g/kg)at particle size2.3–2.5mm,pH3.0,30°C, and initial dye concentration4571g/m3.Since the total number of amino groups,the adsorption sites,remain unchanged for the same weight of chitosan beads no matter what its molecular weight is.This explains our experimental results that the molecular weight of chitosan has no effect on adsorption capacity of reactive dye.The dye-binding capacity of cross-linked chitosan with degree of deacetylation95%(M W200k)is close to that of deacetylation$85%.Since both molecular weight and degree of deacetylation of chitosan in the range we studied have almost no effect on its equilibrium adsorption capacity,the chitosan of M W220k and85.5% deacetylation was used in the following experiments for convenience.3.1.4.Effect of particle sizeFig.3shows the equilibrium adsorption of RR189 at pH3.0,30°C on the cross-linked chitosan beads for three different particle sizes and one size of non-cross-linked chitosan beads(pH6.0).It is seen that the satu-ration adsorption of cross-linked chitosan beads at pH 3.0is almost90%greater than that of non-cross-linked chitosan beads at pH6.This may suggest that at low pH chitosanÕs free amino groups are protonated,causing them to attract anionic dye.The shape of the isotherms looks rectangular because at low equilibrium dye con-centrations C e,the equilibrium adsorption densities q e of cross-linked chitosan beads are almost the same as those at high C e.It indicates that the cross-linked chitosan beads have high adsorption density even at low C e.The rectangular shape of the isotherms was also observed in Yoshida et al.(1993).Fig.3also indicates that the ad-sorption capacity at smaller C e decreases slightly with increasing the diameter of the chitosan bead,while they are close to each others at higher C e(1500–2000g/m3). The experiment also show that the time to achieve equilibrium decreases with reducing the bead diameter. Thus,the chitosan beads of particle size2.3–2.5mm were used in the following experiments.The isotherms in Fig.3werefitted by linear Langmuir and Freundlich equations.Table1shows the computa-tion results.The Langmuir isothermfits quite well with the experimental data for the three different particle sizes at pH3.0(correlation coefficient,R2>0:999),whereas the low correlation coefficients(R2<0:742)show the poor agreements of Freundlich isotherm with the ex-perimental data.Table1also indicates that the computed maximum monolayer capacity Q of RR189on the cross-linked chitosan beads has large value,ranging from1802 to1840g/kg.3.2.Adsorption dynamicsIn order to investigate the mechanism of adsorption, the pseudo-first-order adsorption,the pseudo-second-order adsorption and the intraparticle diffusion model were used to test dynamical experimental data.Thefirst-order rate expression of Lagergren is given aslogðq eÀqÞ¼log q eÀk12:303tð1Þwhere q e and q are the amounts of dye adsorbed on adsorbent(g/kg)at equilibrium and at time t,respec-tively and k1is the rate constant offirst-order adsorption (minÀ1).The slopes and intercepts of plots of logðq eÀqÞvs.t were used to determine thefirst-order rate constant k1.In many cases thefirst-order equation of Lagergren does notfit well to the whole range of contact time and is generally applicable over the initial stage of the ad-sorption processes(McKay and Ho,1999a).The second-order kinetic model(McKay and Ho, 1999b)is expressedasFig.3.Equilibrium adsorption isotherm of RR189on chitosan beads at different particle sizes.1098M.S.Chiou,H.Y.Li/Chemosphere50(2003)1095–1105t q ¼1k 2q 2e þt q e ð2Þand h ¼k 2q 2eð3Þwhere k 2(kg/g min)is the rate constant of second-order adsorption and h is the initial adsorption rate (g/kg min).The slopes and intercepts of plots of t =q vs.t were used to calculate the second-order rate constant k 2and q e .It is more likely to predict the behavior over the whole range of adsorption and is in agreement with an ad-sorption mechanism being the rate-controlling step (McKay and Ho,1999a,b).The intraparticle diffusion equation can be described as q ¼k i t 0:5ð4Þwhere k i is intraparticle diffusion rate constant (g/kg min À0:5).The k i is the slope of straight-line portions of plot of q vs.t 0:5.3.2.1.Effect of initial dye concentrationFig.4shows that the effect of initial RR 189con-centration on the adsorption kinetics of the cross-linked chitosan at pH 3.0,30°C.An increase in initial dye concentration leads to an increase in the adsorption capacity of dye on chitosan.The adsorption capacity for 10h with initial dye concentration at 5096and 2900g/m 3is 93%and 53%greater than that at 1910g/m 3,respectively.This indicates that the initial dye concen-tration plays an important role in the adsorption ca-pacity of RR 189on the cross-linked chitosan beads.Table 2lists the results of rate constant studies for different initial dye concentrations by the pseudo-first-order,second-order models and the intraparticle diffu-sion model.The correlation coefficient R 2for the pseu-do-second-order adsorption model has extremely high value (>0.998),and its calculated equilibrium adsorp-tion capacities q e ;cal fit well with the experimental data.These suggest that the pseudo-second-order adsorption mechanism is predominant and that the overall rate of the dye adsorption process appears to be controlled by the chemical process (McKay and Ho,1999a,b).The similar phenomena have also been observed in biosorp-tion of dye RB2,RY2,and Remazol Black B on biomass (Aksu and Tezer,2000;Aksu,2001).The results in Table 2also show k 2,h and q e ,as a function of initial dye concentration.For the pseudo-second-order model,the rate constant decreases with an increasing of initial dye concentration,while the initial adsorption rate increases with an increasing of initial dye concentration.An in-crease in initial dye concentration results in significant increasing the q e ;cal .The q e ;cal for initial dye concentra-tion at 5096and 2900g/m 3is 93%and 54%greater than that of at 1910g/m 3,respectively.Table 1Langmuir and Freundlich isotherm constants at different particles sizes (30°C)Particle sizes (mm)Langmuir a Freundlich b Q (g/kg)b (m 3/g)R 2Q f n R 2pH 3.0(cross-linked)Small:2.3–2.518340.146 1.000114715.00.553Medium:2.5–2.718400.0660.9998919.80.742Large:3.5–3.818020.0340.99986510.10.722pH 6.0(non-cross-linked)Small:2.3–2.59500.0610.9952314.40.716aLinear Langmuir equation:C e =q e ¼ð1=Qb ÞþðC e =Q Þ,C e :equilibrium concentration (g/m 3)of dye left in aqueous solution;q e :amount of adsorption at equilibrium (g/kg);Q and b are the Langmuir constants related to the capacity and energy of adsorption respectively.bLinear Freundlich equation:log q e ¼log Q f þð1=n Þlog C e ,Q f and n are the Freundlich constants related to the adsorption ca-pacity and intensity of the sorbentrespectively.Fig.4.Adsorption kinetics of RR 189on cross-linked chitosan beads at different initial dye concentrations.M.S.Chiou,H.Y.Li /Chemosphere 50(2003)1095–110510993.2.2.Effect of temperatureFig.5shows the effect of temperature on adsorption of RR 189onto the cross-linked chitosan at pH 3.0and initial dye concentration 4330g/m 3.An increase in the temperature leads to an increase in initial adsorption rate,but the adsorption capacities at 6h are close to each other.Below and above equilibrium time,the adsorption density q in Fig.6shows different trend at different temperatures.Below equilibrium time,an increase in the temperature leads to an increase in dye adsorption rate d q =d t and q ,which indicates a kinetically controlling process.After equilibrium,the uptake decreases with increasing the temperature indicates that the adsorptionof RR 189onto chitosan is controlled by an exothermic process (Kumar,2000).Similar temperature effect on the adsorption trend has also been shown in the case of sorption of Acid Blue 25onto peat (Ho and McKay,1998).However,it can be seen from Fig.5that these effects are insignificant.Normal wastewater temperature variations do not significantly affect the overall decol-orization performance (Kumar,2000).However,a sig-nificant effect of temperature on the equilibrium isotherm was observed in the low equilibrium dye concentration range (<1mol/m 3)for adsorption of Acid Orange II (acid dye)on cross-linked chitosan (Yoshida et al.,1993).Table 2Comparison of the first-order,second-order adsorption,and intraparticle diffusion rate constants,calculated q e and experimental q t values for different initial dye concentrations,temperatures,pH,and wet/dry beadsParametersq t (g/kg)First-order kinetic model Second-order kinetic model Intraparticle diffusion model k 1(min À1)q e ;cal (g/kg)R 2k 2(kg/g min)h(g/kg min)q e ;cal (g/kg)R 2k i(kg/g min)R 2Initial dye concentration (g/m 3)t ¼10h (pH3.0)19109411:86Â10À25730.8894:27Â10À542.19950.99884.80.979290014461:16Â10À29860.9982:03Â10À547.91536 1.000121.40.980509616161:39Â10À210700.9381:91Â10À570.919250.999137.20.978Temperature (°C)t ¼6h (pH 3.0)3016801:61Â10À218870.9717:79Â10À631.820200.997120.80.9914016792:21Â10À218130.9871:59Â10À555.918730.997145.90.9965016523:40Â10À212840.8715:03Â10À5148.217150.997182.90.981pH t ¼48h 1.020896:92Â10À311790.9241:56Â10À570.02119 1.000138.50.9663.017061:31Â10À215040.9552:38Â10À569.61711 1.000113.10.9936.012632:56Â10À33130.6892:45Â10À540.11278 1.00091.40.978wet/dry t ¼48h (pH 1.0)Wet beads 20896:92Â10À311790.9241:56Â10À570.02119 1.000138.50.966Dry beads 21602:89Â10À322640.9999:21Â10À76.225850.95279.40.961Fig.5.Adsorption kinetics of RR 189on cross-linked chitosan beads at different temperatures.Fig.6.Adsorption kinetics of RR 189on cross-linked chitosan beads at different pH.1100M.S.Chiou,H.Y.Li /Chemosphere 50(2003)1095–1105Table2lists the results of rate constant studies for different temperatures calculated by the three models. The correlation coefficient R2for the pseudo-second-order adsorption model has the highest value(¼0:997) suggesting the dye adsorption process is predominant by the pseudo-second-order adsorption mechanism.The experimental adsorption capacity q t at6h reaches83%, 90%and96%of the predicted equilibrium adsorption capacities q e;cal at30,40and50°C,respectively.This suggests that the equilibrium time is shorter with in-creasing temperature.It may be explained by that higher temperature increases the reaction rate and decreases the particle density,which forms larger pore size and is easier for dye molecule to diffuse.For the pseudo-sec-ond-order model,both the rate constant and the initial adsorption rate increase significantly with an increasing of temperature from30to50°C.In Table2,the rate constant at50and40°C is,respectively,6.46and2.04 times of that at30°C whereas the initial adsorption rate at50and40°C is,respectively,4.66and1.76times of that at30°C.An increase in temperature from30to50°C leads to an decrease in the calculated q e;cal.The thermodynamic parameters such as change in free energy(D G0),enthalpy(D H0)and entropy(D S0) were determined using the following equations(Catena and Bright,1989;Namasivayam and Yamuna,1995):K C¼C AeC eð5ÞD G0¼ÀRT ln K Cð6Þlog K C¼D S02:303RÀD H02:303RTð7Þwhere K C is the equilibrium constant,C Ae is the amount of dye(g)adsorbed on the adsorbent per dm3of the solution at equilibrium,C e is the equilibrium concen-tration(g/dm3)of the dye in the solution,T is the so-lution temperature(K)and R is the gas constant.D H0 and D S0were calculated from the slope and intercept of vanÕt Hoffplots of log K C vs.1=T.The results are listed in Table3.(The q e;cal of the pseudo-second-order model in Table2was used to obtain C Ae.)The negative values of D G0indicate that the adsorption of dye RR189on the cross-linked chitosan is spontaneous.The negative value of D H0shows that the adsorption is an exothermic process.The negative value of D S0indicates that the randomness decreases at the solid–solution interface during the adsorption of dye on the cross-linked chito-san.The rate constant k for pseudo-second-order reaction shows an Arrhenius dependence on reciprocal temper-ature.The relationship can be expressed byk¼k0expÀE adRTð8Þwhere k is the rate constant of adsorption,k0is the temperature independent factor(kg/g min),and E ad is the activation energy(J/mol)for the adsorption.The values of k0and E ad are calculated from the slope and intercept of a plot of lnðkÞvs.1=T.The results are listed in Table3.The relationship between the activation energies of adsorption,E ad,and desorption,E de,is described byD H0¼E adÀE deð9ÞAccording to the values in Table3,the activation energy of desorption,E de,is estimated to be128.656kJ molÀ1, whose large value might indicate the difficulty for de-sorption of the adsorbed dye RR189from the surface of the cross-linked chitosan into solutions.An effective desorption is required to involve the change of the conditions,such as the solution pH value.The results of desorption will be discussed in Section3.2.7.3.2.3.Effect of pHFig.6shows the effect of pH on adsorption of RR 189onto chitosan at30°C,and initial dye concentration 4571g/m3.It shows that the adsorption capacity in-creases significantly with decreasing the pH.Under the same operating conditions as Figs.6and7shows48-h adsorption capacities of the cross-linked chitosan beads (pH1.0–9.0)and the non-cross-linked chitosan beads (pH6.0–9.0).The pH of solution strongly affects the adsorption capacity of the cross-linked chitosan whereas the capacity nearly remains constant for the non-cross-linked chitosan.The48-h amounts of adsorption of dye RR189on the cross-linked chitosan at pH1.0(HCl),3.0 and 6.0(acetic acid/acetic)are118%,78%and32% higher than that of the non-cross-linked chitosan at pHTable3The thermodynamic and rate constant parameters(pH3.0,particle size2.3–2.5mm,and initial dye concentration4330g/m3) Temperature(°C)Thermodynamics Rate constantK C D G0(kJ/mol)D H0(kJ/mol)D S0(J/mol K)R2k0(kg/g min)E ad(kJ/mol)R23014.0)6.640 6.4)4.8)52.9)153.10.9918:11Â10775.70.977 50 3.8)3.6M.S.Chiou,H.Y.Li/Chemosphere50(2003)1095–11051101。

中国预防兽医学报Chinese Journal of Preventive Veterinary Medicine第42卷第12期2020年12月V ol.42No.12Dec.2020doi ：10.3969/j.issn.1008-0589.202003049猪德尔塔冠状病毒BJ 株的分离鉴定与致病性分析刘秋歌1，王洪峰2，范前进1，冯力1*，陈建飞1*（(1.中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪消化道传染病创新团队，黑龙江哈尔滨150069；2.哈尔滨维科生物技术有限公司，黑龙江哈尔滨150069）摘要：猪德尔塔冠状病毒（PDCoV ）是一种新出现的猪冠状病毒。

本研究将北京某猪场PDCoV 阳性仔猪腹泻样品无菌处理后接种猪睾丸（ST ）细胞并连续传代培养，通过电镜、间接免疫荧光和基因序列测定对病毒培养物进行鉴定。

结果表明分离到一株PDCoV ，并将其命名为BJ 株。

基于全基因组序列的系统发育树分析结果表明BJ 株与我国PDCoV 参考株的亲源关系较近。

利用105TCID 50/mL 的病毒液2mL 口服接种3日龄仔猪并观察临床症状，结果显示BJ 株能够引起3日龄仔猪发病，出现典型的腹泻症状。

此外，病理组织学结果表明感染仔猪的小肠绒毛损伤，免疫组化结果表明在感染仔猪的小肠组织中检测到PDCoV 抗原。

本研究证实了PDCoV BJ 株具有致病力，丰富了与PDCoV 相关的流行病学、致病性和分子进化的信息。

关键词：猪德尔塔冠状病毒；分离鉴定；致病性中图分类号：S852.65文献标识码：A文章编号：1008-0589（2020）12-1287-05Isolation,identification and pathogenesis of porcinedeltacoronavirus BJ strainLIU Qiu-ge 1,WANG Hong-feng 2,FAN Qian-jin 1,FENG Li 1*,CHEN Jian-fei 1*(1.Division of Swine Digestive System Infectious Diseases,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;2.Harbin Veken Biotechnology Co.,Ltd.,Harbin 150069,China)Abstract :Porcine deltacoronvirus (PDCoV)is a novel emerging porcine coronavirus.In this study,a PDCoV strain,designated BJ strain,was isolated from the positive PDCoV samples from a pig farm in Beijing by inoculating with swine testis (ST)cells.The characterization of the isolated virus was identified by immunofluorescence assay,electron microscopy observation and whole genomic sequencing.The phylogenetic tree analysis based on the whole-genome sequences indicated that PDCoV BJ strain had a close phylogenetic relationship with Chinese PDCoV reference strains.Furthermore,3-day-old piglets were inoculated orally with 2×105TCID 50virus suspension to investigate the pathogenicity of PDCoV BJ strain.The PDCoV-inoculated piglets developed diarrhea and intestinal villi damaged by HE staining.And the immunohistochemistry staining results showed that the PDCoV-specific antigen was detected in the small intestine of PDCoV-inoculated piglets.Our results confirmed the pathogenicity of PDCoV BJ strain.This study has enriched the information on the epidemiology,pathogenicity and molecular evolution of PDCoV .Key words :Porcine deltacoronavirus;isolation and identification;pathogenesis收稿日期：2020-03-29基金项目：国家重点研发计划课题（2016YFD0500705）；黑龙江省科学基金项目（TD2020C002）作者简介：刘秋歌（1994-），女，河南鹿邑人，硕士研究生，主要从事猪冠状病毒研究.*通信作者：E-mail ：**************；********************Corresponding author中国预防兽医学报2020年猪德尔塔冠状病毒（Porcine deltacoronavirus，PDCoV）是一种新发的猪冠状病毒，能够引起猪肠道疾病。

Rac Activation and InactivationControl Plasticityof Tumor Cell MovementVictoria Sanz-Moreno,1Gilles Gadea,1Jessica Ahn,1Hugh Paterson,1Pierfrancesco Marra,1Sophie Pinner,2Erik Sahai,2and Christopher J.Marshall1,*1Institute of Cancer Research,Cancer Research UK Centre for Cell and Molecular Biology,237Fulham Road,London SW36JB,UK 2Tumour Cell Biology Laboratory,London Research Institute Cancer Research UK,44Lincoln’s Inn Fields,London WC2A3PX,UK *Correspondence:chris.marshall@DOI10.1016/j.cell.2008.09.043SUMMARYTumor cells exhibit two different modes of individual cell movement.Mesenchymal-type movement is characterized by an elongated cellular morphology and requires extracellular proteolysis.In amoeboid movement,cells have a rounded morphology,are less dependent on proteases,and require high Rho-kinase signaling to drive elevated levels of acto-myosin contractility.These two modes of cell move-ment are interconvertible.We show that mesenchy-mal-type movement in melanoma cells is driven by activation of the GTPase Rac through a complex containing NEDD9,a recently identified melanoma metastasis gene,and DOCK3,a Rac guanine nucleo-tide exchange factor.Rac signals through WAVE2to direct mesenchymal movement and suppress amoe-boid movement through decreasing actomyosin contractility.Conversely,in amoeboid movement, Rho-kinase signaling activates a Rac GAP,ARH-GAP22,that suppresses mesenchymal movement by inactivating Rac.We demonstrate tight interplay between Rho and Rac in determining different modes of tumor cell movement,revealing how tumor cells switch between different modes of movement. INTRODUCTIONAbnormal cell migration and invasion is a characteristic of malig-nant cancer cells and is one component of metastasis,the major clinical problem in cancer.To metastasize,tumor cells must move through tissues and cross tissue boundaries,which re-quires cell motility,remodeling of cell-cell contacts,and interac-tions with the extracellular matrix(Fidler,1999).Tumor cells can move as individual cells or as collective groups(Wolf et al.,2007). Recent work has demonstrated that individual tumor cells have two different modes of movement:a mesenchymal mode char-acterized by an elongated morphology that requires extracellular proteolysis localized at cellular protrusions and an amoeboid mode in which movement is independent of proteases and cells have a rounded morphology with no obvious polarity(Wolf et al., 2003).In amoeboid movement,the cell surface undergoes very active blebbing driven by actomyosin contractility(Sahai and Marshall,2003;Wyckoff et al.,2006).It is also clear that these two modes of tumor cell movement are interconvertible,de-pending on environmental conditions(De Wever et al.,2004; Wolf et al.,2003).Consequently,the ability of tumor cells to switch between different modes of motility may limit the effec-tiveness of single therapeutic agents aimed at reducing invasion. Members of the Rho family of small GTPases are key regula-tors of cell movement through their actions on actin assembly, actomyosin contractility,and microtubules(Ridley,2001).Ex-pression of Rho family proteins is deregulated in some tumors and correlates with progression of disease(Sahai and Marshall, 2002).The three prototypical members of the family,Rho,Rac, and Cdc42,have all been linked to cell movement.Rac1drives motility by promoting lamellipodia formation(Ridley et al., 1992),whereas RhoA signals to the kinases ROCKI and II,pro-moting the formation of actin stressfibers and generation of the actomyosin contractile force required for cell movement (Amano et al.,1997;Kimura et al.,1996).Previous work shows that amoeboid and mesenchymal modes of movement are dis-tinguished by their different usage of signaling pathways.The amoeboid mode involves signaling through ROCK,whereas the mesenchymal mode does not have an obligate requirement for ROCK but requires extracellular proteolysis(Sahai and Mar-shall,2003)for Rac-dependent actin protrusions to be pushed through channels in the extracellular matrix(Wolf et al.,2003). Amoeboid movement is characterized by high levels of actomy-osin contractility driven by ROCK(Wilkinson et al.,2005),consis-tent with a mechanism whereby cells squeeze through voids and deform the matrix(Wolf et al.,2003;Wyckoff et al.,2006).To investigate mechanisms of different modes of cell move-ment,we have studied how the regulation of Rho family GTPases determines the mode of tumor cell movement.Guanine nucleo-tide exchange factors(GEFs)and GTPase accelerating proteins (GAPs)are key regulators of small GTPases,with GEFs promot-ing activation to the GTP bound state and GAPs promoting inac-tivation by stimulating GTP hydrolysis(Bos et al.,2007).We car-ried out a systematic siRNA screen of GEFs and GAPs to identify regulators of Rho family GTPases affecting modes of tumor cell510Cell135,510–523,October31,2008ª2008Elsevier Inc.movement.We show that a Rac-specific GEF,DOCK3,in asso-ciation with the adaptor molecule NEDD9,regulates Rac and WAVE2to drive mesenchymal movement and suppress amoe-boid movement.Conversely,in amoeboid movement,Rho sig-naling via ROCK to a Rac GAP,ARHGAP22,inactivates Rac and suppresses mesenchymal movement.RESULTSDOCK3Is a Rac GEF Regulating the Transition between Amoeboid and Mesenchymal MovementTo investigate Rho family GTPase signaling pathways involved in tumor cell morphology and movement,we initially used A375M2melanoma cells that show amoeboid movement in vivo (Pinner and Sahai,2008)and in 3D tissue culture (Sahai and Marshall,2003;Wilkinson et al.,2005).Cells were plated on top of a thick deformable layer of collagen I,and cell morphology andmove-Figure 1.DOCK3Is Required for the Amoe-boid-Mesenchymal Transition(A)Upper panel:images of Mock or DOCK3siRNA-transfected A375M2cells plated on a thick layer of collagen for 24hr.The scale bar repre-sents 100m m.Lower panel:DOCK3immunoblot.(B)Upper panel:images of Mock or DOCK3siRNA-transfected cells plated on collagen and treated with Y27632for 16hr.Lower panel:per-centage of elongated cells after Y27632,H1152,or blebbistatin treatment (200cells/experiment,n =3,error bars indicate +SE).(C)Upper panel:percentage of elongated cells af-ter transfection with four different DOCK3oligos (200cells/experiment,n =3,error bars indicate +SE).Lower panel:DOCK3immunoblot.(B and C)Student’s t test was used to generate p values;**p <0.01,*p <0.05.(D)Migration speeds of cells on collagen in 1%serum treated with H1152where indicated.Student’s t test was used to generate p values;**p <0.01.(E)Rac activation (GTP-bound Rac1/total Rac)in A375M2cells transfected with siRNAs on colla-gen.Upper panels:left,representative pulldown;right,quantification (n =3,error bars indicate +SE).Lower panels:A375M2stably transfected with DELDOCK3;left,representative pulldown;right,quantification.(n =3,error bars indicate +SE;Student’s t test was used to generate p values,**p <0.01,*p <0.05.)ment were monitored by time-lapse mi-croscopy.In this system,cell adhesion is at least partially dependent on integrin b 1(Figure S1A available online,)and cells show similar adhesion to when plated on a thin,rigid layer of collagen (Figure S1B).Approximately 90%of A375M2cells dis-played a rounded morphology on thick collagen (Figures 1A and 1B,Movie S1),and higher magnifications show that any one time about 20%of the rounded cellsmove randomly in an amoeboid fashion with dynamic blebbing plasma membranes (Movie S2,Figure 2F)with velocities of ap-proximately 0.1m m per min.These velocities of amoeboid move-ment on top of a deformable collagen layer are similar to those observed in a 3D collagen environment (Figure S1C).About 10%of A375M2cells show an elongated morphology (Figures 1A and 1B),and of these about 40%are moving in a mesenchy-mal fashion with long protrusions at speeds similar to amoeboid cells (Figures 1D and 2F).Treatment with cytochalasin D to block actin polymerization showed that both amoeboid movement and elongated,mesenchymal-type movement require actin assem-bly (data not shown).To examine the interconvertibility of cell morphologies and movement,we made time-lapse movies from reconstructions of confocal sections of cells contained within a 3D collagen ma-trix (Movie S3).These showed that individual A375M2cells inter-convert between a rounded and elongated morphology overCell 135,510–523,October 31,2008ª2008Elsevier Inc.511a 24hr time period.Examination of other melanoma cell lines on thick collagen showed that several were like A375M2,consisting of a majority of rounded cells that move in an amoeboid fashion;another group had similar proportions of rounded and elongated cells,whereas a third group consisted of a majority of elongated cells with mesenchymal-type movement (Figure S1D).To identify GEFs that regulate cell morphology and movement,we transfected A375M2cells with siRNA oligonucleotide du-plexes (Dharmacon Smart Pools)targeting 83GEFs for Rho fam-ily GTPases (Table S1)and monitored them on thick collagen layers.Silencing of DOCK3/MOCA resulted in almost complete elimination of cells with an elongated morphology and blocked the interconversion between amoeboid and mesenchymal movement (Figures 1A and 1B)without affecting adhesive prop-erties (Figure S1B,lower panel).We have previously shownthatFigure 2.Rac Regulates the Amoeboid-Mesenchymal Transition(A)Left panels:images of A375M2cells trans-fected with siRNAs or treated with 50m M NSC23766,photographed at 24hr on collagen.Right panel:percentage of elongated cells (200cells/experiment,n =3,error bars indicate +SE);inset,Rac1immunoblot.(B)Rac activation on collagen after treatment for the indicated times with Y27632,H1152,or bleb-bistatin (blebbis).Left:representative pulldown.Right:quantification (n =5,error bars indicate +SE;Student’s t test was used to generate p values,**p <0.01,*p <0.05).(C)Percentage of elongated cells after Mock or Rac1siRNA transfection at 24hr after treatment with vehicle,Y27632,H1152,or blebbistatin (200cells/experiment,n =3,error bars indicate +SE).(A and C)Student’s t test was used to generate p values;**p <0.01.(D)Images of A375P cells on collagen stably trans-fected with Rac1T17N,Rac1shRNA,or control vectors.(E)Rac1and RhoA activation in A375M2and A375P cells.(F)Histogram showing the percentage of moving cells classified by cell shape on collagen after transfection with siRNAs and treated with H1152where indicated (n =3,error bars indicate +SE;Student’s t test was used to generate p values,**p <0.01,*p <0.05).(G)A375M2cells on collagen stably expressing Rac1Q61L.Upper panel:images.Lower panel:percentage of elongated cells (200cells/experi-ment,n =3,error bars indicate +SE;Student’s t test was used to generate p values,**p <0.01).(H)Migration speeds of cells on collagen.Stu-dent’s t test was used to generate p values;**p <0.01.treatment of A375M2cells with low concentrations of the myosin II ATPase inhibitor blebbistatin resulting in partial inhibition of actomyosin contractility leads to an elongated morphology and mesenchymal movement (Wilkinsonet al.,2005);therefore,we investigated whether this conversion requires DOCK3.Furthermore,because Rho-ROCK signaling to phosphorylation of myosin light chain 2(MLC2)has been shown to be a key determinant of actomyosin contractility,we investi-gated whether inhibition of ROCK led to an elongated morphol-ogy and whether this was dependent on DOCK3.Figure 1B shows that treatment with blebbistatin or two different ROCK in-hibitors (Y27632and H1152)resulted in conversion to elongated cells moving in a mesenchymal manner (Movies S6and S7)and that this was abrogated by siRNAs targeting DOCK3(Figure 1B;Movie S8).These results were confirmed with Dharmacon ‘‘On-Target Plus’’oligonucleotides,which use a modified chemistry to reduce off-target effects and are directed at different sequences of the gene (Figure 1C).Interestingly,although blebbistatin or ROCK inhibition increased the proportion of elongated cells,it512Cell 135,510–523,October 31,2008ª2008Elsevier Inc.did not affect the speed of migration(Figure1D).Knockdown of other GEFs from the DBL or the DOCK families did not affect cell elongation in response to ROCK inhibition(Figure S2).DOCK3has been previously characterized as a GEF for Rac (Namekata et al.,2004);therefore,we investigated whether the conversion from amoeboid to mesenchymal movement involved the activation of Rac.Silencing of DOCK3expression with two different oligonucleotides showed that Rac activity in A375M2 is dependent on DOCK3(Figure1E).Moreover,expression of an internal deletion mutant,DELDOCK3(aa1062–1393)(Name-kata et al.,2004),which lacks the DHR2exchange factor domain but retains protein-protein interaction domains and could act as a dominant negative,also decreased Rac activation(Figure1E, lower panel)and blocked the conversion of rounded cells to elon-gated morphology(Figure S3).To determine whether Rac activa-tion is required for the amoeboid-mesenchymal transition,we silenced Rac1by siRNA transfection.Figures2A and2C and Movies S4and S9show that silencing of Rac1abolished elonga-tion of A375M2cells.To confirm these results,we used treatment with compound NSC23766,which blocks Rac1activation by in-terfering with complex formation between GEFs and Rac(Gao et al.,2004)or expression of the interfering Rac mutant Rac1T17N.NSC23766treatment reduced the proportion of elon-gated cells to1.5%,compared with8%in control A375M2cells (Figure2A).Because NSC23766has not been shown to affect Rac activation mediated by DOCK family members,we showed that NSC23766blocks Rac activation after overexpression of DOCK3(Figure S4).Only rounded cells were found to express Rac1T17N after transfection(data not shown).To determine whether cell elongation after inhibition of actomyosin contractility or ROCK led to Rac activation,we measured Rac-GTP levels af-ter treatment with blebbistatin or ROCK inhibitors.Figure2B shows that these treatments increase Rac activation.This activa-tion of Rac is dependent on DOCK3(data not shown).However, treatment with ROCK inhibitors or blebbistatin did not increase the proportion of elongated cells if Rac had been depleted by siRNA(Figure2C)or by Rac1T17N transfection(data not shown). A375M2was derived from a low metastatic melanoma cell line,A375P(Clark et al.,2000),that has a more elongated mor-phology(40%elongated)with fewer blebs than A375M2cells (Figure2D).Interestingly,we could show that A375P cells have lower levels of Rho-GTP and higher levels of Rac-GTP than do A375M2cells(Figure2E).In A375P cell lines stably expressing either Rac1T17N or shRNA-Rac1,the proportion of elongated cells was reduced(Figure2D).These results show that Rac sig-naling is required for the elongated/mesenchymal phenotype. Because amoeboid movement of A375M2cells is dependent on ROCK(Sahai and Marshall,2003)but inhibition of ROCK leads to Rac-dependent mesenchymal-type movement,we in-vestigated whether inhibition of ROCK in combination with blockage of Rac activation would prevent cells from moving. Figure2F shows that the combination of ROCK-inhibitor treat-ment with silencing of either Rac or DOCK3markedly reduces cell movement.To determine whether activation of Rac signaling is sufficient to convert rounded cells to elongated cells,we ex-pressed a constitutively activated Rac1Q61L in A375M2cells; this resulted in the proportion of elongated cells increasing to 40%,compared to4%in controls(Figure2G).Blocking Rac Signaling Suppresses Mesenchymal Movement and Enhances Amoeboid MovementThese experiments show that activation of Rac by DOCK3in A375M2cells results in an elongated morphology and mesen-chymal movement but do not address the issue of whether Rac is required for amoeboid movement.Movie S9shows that cells in which Rac has been silenced move solely in an amoeboid fashion.Time-lapse movies from reconstructions of confocal sections of A375M2cells suspended within a collagen matrix show that blockage of Rac signaling either by siRNA transfection or treatment with NSCC2376prevents conversion of rounded, amoeboid cells to elongated mesenchymal-type movement (Movies S4and S5;compare with control cells in Movie S3).Sur-prisingly,analysis of the speed of cells plated on top of a collagen matrix showed that rounded cells in which Rac signaling was ab-lated moved up to1.7-fold faster(Figure2H).These results dem-onstrate that not only is Rac activation by DOCK3required for an elongated morphology and mesenchymal movement,but it also suppresses amoeboid movement.NEDD9Complexes with DOCK3to Regulate Rac Activity DOCK3is a member of the DOCK180family of guanine nucleo-tide exchange factors,and individual members of the family ac-tivate either Rac or Cdc42(Cote and Vuori,2002).The prototyp-ical member of this family,DOCK180,is activated via a complex consisting of the adaptors Crk and p130Cas(Kiyokawa et al., 1998).We speculated that DOCK3may complex with a member of the p130Cas family.Expression studies by Q-PCR showed that p130Cas and NEDD9/HEF1but not Efs/Sin were expressed in A375M2human melanoma cells.Interestingly,NEDD9is over-expressed in35%–50%of metastatic human melanomas and is overexpressed and involved in invasion in a mouse model of metastatic melanoma(Kim et al.,2006).To investigate the roles of NEDD9and p130Cas,we examined whether silencing of ex-pression of p130Cas or NEDD9caused a similar phenotype to si-lencing of DOCK3expression.NEDD9but not p130Cas ablation reduced the number of elongated cells after treatment with ROCK inhibitors(Figure3A;knockdown in Figure S5A).To show that NEDD9is required for Rac activation,we transfected A375M2with siRNA oligonucleotides.Figure3B shows that silencing NEDD9expression reduces Rac activity.To confirm that Rac activation by DOCK3requires NEDD9,we overexpressed DOCK3in HEK293T cells.Figure3C shows that overexpression of DOCK3led to Rac activation that was blocked by silencing of endogenous NEDD9(knockdown in Figure S5C). Similarly,Rac activation by overexpressed NEDD9was depen-dent on endogenous DOCK3(Figure3C,knockdown in Figure S5D).These results suggest that DOCK3functions together with NEDD9to activate Rac.Therefore,we used coimmunopre-cipitation to look for evidence that NEDD9and DOCK3are in a signaling complex.We were able to detect an endogenous complex of DOCK3with NEDD9in A375M2cells(Figure3D). These results were confirmed by overexpression analysis;en-dogenous DOCK3was found in immunoprecipitates isolated with Flag-tag antibodies from cells expressing Flag-epitope-tagged NEDD9(Figure3D,lower-left panel),whereas endoge-nous NEDD9could be detected in complexes with HADOCK3af-ter expression of an HA-epitope-tagged DOCK3(Figure3D).Cell135,510–523,October31,2008ª2008Elsevier Inc.513Rac Signaling Suppresses Actomyosin Contractility through WAVE2Because amoeboid movement is associated with elevated levels of MLC2phosphorylation and actomyosin contractility,we investigated whether Rac acts by controlling actomyosin contractility.Reduction of Rac1expression with Rac1siRNA in A375M2cells (Figure 4A)or Rac1shRNA in A375P cells (Figure 4B)increased phosphorylation of MLC2on threonine18and serine19.Similarly,silencing of DOCK3or NEDD9led to in-creased MLC2phosphorylation (Figures 4C and 4D).Con-versely,expression of constitutively activated Rac1Q61L in A375M2cells that leads to an elongated morphology resulted in reduced levels of MLC2phosphorylation (data notshown).Figure 3.NEDD9Complexes with DOCK3to Regulate Rac Activity(A)Upper panel:images of siRNA-transfected A375M2cells on collagen treated with Y27632for 16hr.Lower panel:percentage of elongated cells (200cells/experiment,n =3,error bars indi-cate +SE;Student’s t test was used to generate p values,**p <0.01).The scale bar represents 100m m.(B)Rac1activation on collagen of siRNA-trans-fected A37M2cells.Left panel:representative pulldown and NEDD9immunoblot.Right panel:quantification of pulldowns (n =3,error bars indi-cate +SE).(C)Rac1activation in 293T cells transfected with HADOCK3or FlagNEDD9and siRNAs plated on plastic.Upper panels:representative pulldowns and immunoblots.Lower panels:quantification of pulldowns (n =3,error bars indicate +SE).(B and C)Student’s t test was used to generate p values;**p <0.01,*p <0.05.(D)Upper-left panel:anti-NEDD9immunoprecipi-tates immunoblotted for DOCK3.Lower-left panel:anti-Flag immunoprecipitates from A375M2cells transfected with Flag-NEDD9immunoblotted for DOCK3.Upper-right panel:anti-HA immunopre-cipitates from A375M2cells transfected with HA-DOCK3immunoblotted for NEDD9.Lower-right panel:anti-NEDD9immunoprecipitates from A375M2cells transfected with HA-DOCK3immu-noblotted for HA-DOCK3.IP,immunoprecipitated.These results show that Rac negatively regulates MLC2phosphorylation and thereby actomyosin contractility.To find the Rac effector responsible for regulating MLC2phosphorylation,we used siRNAs targeting the Rac effectors PAK1,2,3,and 4as well as WAVE1,2,and 3;only WAVE2depletion led to ele-vated levels of MLC2phosphorylation (Figure 4E).Consistent with its role in Rac-dependent Arp2/3promotion of ac-tin assembly and the formation of cell protrusions characteristic of the mesen-chymal type of cell movement (Leng et al.,2005),WAVE2silencing reducedthe proportion of elongated A375M2cells on thick collagen (data not shown)and abolished Rac-dependent elongation after ROCK inhibition (Figure 4F).We were not able to detect any de-fect in elongation after ROCK inhibition or change in the levels of MLC2phosphorylation when depleting WAVE1or WAVE3(Figure S6).To confirm the results obtained for WAVE2,we used individual Dharmacon On-Target Plus oligos targeting different regions of WAVE2.As shown in Figure 4G,there is a strong correlation be-tween the degree of knockdown for WAVE2protein and the re-duction in the percentage of elongated cells after ROCK-inhibitor treatment.These results therefore show that elongation of A375M2cells requires Rac signaling to WAVE2and that514Cell 135,510–523,October 31,2008ª2008Elsevier Inc.WAVE2negatively regulates MLC2phosphorylation,thereby suppressing rounded morphology and amoeboid movement.ROCK Signaling Downregulates Rac Activity via ARHGAP22We have shown that Rac opposes amoeboid movement while in-hibition of ROCK in amoeboid cells leads to Rac activation,sug-gesting that ROCK signaling in rounded,amoeboid cells sup-presses Rac activation.A potential mechanism is through ROCK regulation of a Rac GAP (Ohta et al.,2006).We investi-gated whether a Rac GAP regulates the mesenchymal transition by screening an siRNA library consisting of 72Rho family GAPs (Table S2).Silencing of ARHGAP22led to elongation of A375M2rounded cells (Figures 5A and 5B,kd in Figure S5B)without alter-ing the adhesion properties of the cells (Figure S1B,lowerpanel).Figure 4.Rac Suppression of Amoeboid Movement Is Mediated by WAVE2(A)Phospho-MLC (p-T18/S19-MLC2)in A375M2cells on collagen after transfection with Rac1siRNA (n =3,error bars indicate +SE).Inset:repre-sentative immunoblot.(B)Phospho-MLC in A375P cells on collagen sta-bly transfected with Rac1shRNA (n =3,error bars indicate +SE).Inset:representative immunoblot.(C)Phospho-MLC in A375M2cells on collagen after transfection with siRNAs.Left panel:repre-sentative immunoblot.Right panel:quantification (n =3,error bars indicate +SE).(D)Phospho-MLC in A375M2cells on collagen af-ter transfection with NEDD9siRNAs.Upper panel:representative immunoblot.Lower panel:quantifi-cation (n =3,error bars indicate +SE).(E)Phospho-MLC in A375M2cells on collagen af-ter transfection with WAVE2siRNA.Upper panel:representative immunoblot.Lower panel:quantifi-cation (n =3,error bars indicate +SE).(F)Mock or WAVE2siRNA-transfected A375M2cells on collagen treated with Y27632,H1152,or blebbistatin for 24hr.Left panel:images of cells.Right panel:histogram showing the percentage of elongated cells (200cells/experiment,n =3,error bars indicate +SE).(G)Left panel:percentage of elongated A375M2cells on collagen after transfection with four differ-ent On Target siRNAs against WAVE2and treated with Y27632(200cells/experiment,n =3,error bars indicate +SE).Right panel:WAVE2immuno-blot.Student’s t test was used to generate p values;**p <0.01,*p <0.05.We did not observe this increase in the number of elongated cells when knocking down the expression of GAPs belonging to the same subfamily such as ARH-GAP24or ARHGAP25or other GAPs (Figure S7).To show that ARHGAP22regulates Rac-GTP levels,we assayed Rac activity in A375M2cells in which expression ofARHGAP22was abrogated with two different sets of oligonucle-otides.RacGTP levels were increased 2-fold when ARHGAP22expression was silenced (Figure 5C).Silencing of ARHGAP22led to decreased MLC2phosphorylation (Figure 5D),consistent with our observations that the Rac-WAVE2pathway downregu-lates MLC phosphorylation.Silencing of a subset of other Rac GAPs did not affect Rac activity (Figure S7C).To demonstrate that ROCK regulates ARHGAP22,we used A375P cells expressing a ROCK-estrogen receptor fusion pro-tein to provide a system whereby ROCK activity could be con-trolled (Croft and Olson,2006).Activation of ROCK-ER-WT (wild-type)by 16hr treatment with tamoxifen led to cells acquir-ing a rounded morphology with associated blebbing similar to A375M2cells (Figure S8)and a substantial decrease in the level of Rac-GTP (Figure 5E,kd in Figure S5E).This decrease wasCell 135,510–523,October 31,2008ª2008Elsevier Inc.515not observed if cells were depleted of ARHGAP22by siRNA (Figure 5E).In contrast,tamoxifen treatment of cells expressing a kinase-inactive version of ROCK fused to ER (kinase-dead K121G)did not lead to significant changes in Rac activation or cell morphology (Figure S8).These experiments show that ROCK suppresses Rac activation through ARHGAP22.A375M2cells have high levels of actomyosin contractility driven by ROCK,and because treatment with low doses of the myosin II inhibitor blebbistatin leads to the activation of Rac (Figure 2B),we investigated whether ROCK signals to ARHGAP22via actomyosin contractility.Blebbistatin treatment blocked the fall in Rac-GTP levels stimulated by increased ROCK activity,suggesting that ROCK does not regulate ARHGAP22directly but does so via modulating actomyosin contractility (Figure 5F).Figure 5.ROCK Signaling Downregulates Rac via ARHGAP22(A)Images of Mock or ARHGAP22siRNA-trans-fected A375M2cells plated on collagen for 24hr.(B)Percentage of elongated cells (200cells/exper-iment,n =3,error bars indicate +SE;Student’s t test was used to generate p values,**p <0.01,*p <0.05).(C)Rac activation in ARHGAP22siRNA-trans-fected cells on collagen.Upper panels:represen-tative pulldown.Lower panel:quantification (n =3,error bars indicate +SE;Student’s t test was used to generate p values,*p <0.05).(D)Phospho-MLC in ARHGAP22siRNA-trans-fected A375M2cells on collagen.Upper panel:representative immunoblot.Lower panel:quantifi-cation (n =3,error bars indicate +SE;Student’s t test was used to generate p values,**p <0.01).(E)Rac activation in A375P-ROCKER cell lines (wild-type [WT]or kinase dead [KD])transfected with siRNAs plated on plastic and treated with 4-OH-tamoxifen or vehicle for 16hr.Left panels:representative pulldowns.Right panels:quantifi-cation (n =5,error bars indicate +SE).(F)Rac activation in A375P-ROCKERWT cells treated with 4-OH-tamoxifen and blebbistatin where indicated for 16hr.Upper panel:represen-tative pulldown.Lower panel:quantification (n =3,error bars indicate +SE.(E and F)Student’s t test was used to generate p values,**p <0.01,*p <0.05.(G)Percentage of elongated cells on collagen at 24hr from A375M2cells transfected with siRNAs against ARHGAP22,DOCK3,or both,(200cells/experiment,n =3,error bars indicate +SE;Stu-dent’s t test was used to generate p values,**p <0.01,*p <0.05).Inset:immunoblot for DOCK3.Taken together,our data show that the Rac GEF DOCK3activates Rac to pro-mote an elongated,mesenchymal-type phenotype,whereas ROCK signaling to the Rac GAP ARHGAP22opposes this phenotype.Consistent with this model,we find that silencing of ARHGAP22,which results in increased number ofelongated cells,could be overcome by simultaneous silencing of DOCK3(Figure 5G,kd in Figure S5F).These results confirm that DOCK3and ARHGAP22act in opposite ways to regulate the mesenchymal-amoeboid transition.Roles of NEDD9,DOCK3,and ARHGAP22in 3D Cell MovementTo examine the roles of the NEDD9-DOCK3-Rac and ROCK-ARHGAP22signaling pathways in 3D cell movement,we tested invasion into a 3D collagen I matrix.We used two cell lines,A375M2and WM1361,in the presence of serum or IGF1,a known motility factor for melanoma cells produced by dermal fibroblasts (Neudauer and McCarthy,2003).Knockdown of NEDD9,DOCK3,or Rac1alone or inhibition of ROCK had little effect on the numbers of invading cells (Figure 6A,kd in516Cell 135,510–523,October 31,2008ª2008Elsevier Inc.。