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ferrocenecompounds.xxxix

metal-organic compoundsm328#2003International Union of CrystallographyDOI:10.1107/S0108270103011703Acta Cryst.(2003).C 59,m328±m330Ferrocene compounds.XXXIX.11-FerrocenylisochromaneMario Cetina,a Senka akovicÂ,b Vladimir Rapic Âb *and Amalija GolobicÏc aFaculty of Textile Technology,University of Zagreb,Pierottijeva 6,HR-10000Zagreb,Croatia,b Laboratory of Organic Chemistry,University of Zagreb,Faculty of Food Technology and Biotechnology,Pierottijeva 6,HR-10000Zagreb,Croatia,and cLaboratory of Inorganic Chemistry,Faculty of Chemistry and Chemical Technology,University of Ljubljana,PO Box 537,SI-1001Ljubljana,Slovenia Correspondence e-mail:*************Received 14April 2003Accepted 27May 2003Online 22July 2003In the title compound,[Fe(C 5H 5)(C 14H 13O)],the plane of the heterocyclic ring is almost perpendicular to the plane of the substituted cyclopentadienyl ring,and the heterocyclic ring adopts a half-chair conformation.The conformation of the nearly parallel cyclopentadienyl (Cp)rings [the dihedral angle between their planes is 2.7(1) ]is almost halfway between eclipsed and staggered,and the rings are mutually twisted by about 19.4(2) (mean value).The mean lengths of the CÐC bonds in the substituted and unsubstituted cyclopentadienylring are 1.420(2)and 1.406(3)AÊ,respectively,and the FeÐC distances range from 2.029(2)to 2.051(2)AÊ.The phenyl and unsubstituted cyclopentadienyl rings are involved in CÐH ÁÁÁ%interactions,with intermolecular H ÁÁÁcentroiddistances of 2.85and 3.14AÊfor CÐH ÁÁÁ%(Ph),and 2.88A Êfor CÐH ÁÁÁ%(Cp).In two of these interactions,the CÐH bond points towards one of the ring bonds rather than towards the ring centroid.In the crystal structure,the CÐH ÁÁÁ%interactions connect the molecules into a three-dimensional framework.CommentOptically active ferrocene derivatives are widely employed as chiral ligands in asymmetric reactions,and there is continuing interest in the development of ef®cient procedures for the preparation of these derivatives in enantiopure forms (Gonsalves &Chen,1995;Bolm et al.,1998;Pioda &Togni,1998;Perea et al.,1999).Ferrocene derivatives exhibiting centro-and planar chirality are very convenient substrates forbiotransformations (KoÈllner et al.,1998;Richards &Locke,1998;Schwink &Knochel,1998;Patti &Nicolosi,1999;akovicÂet al.,2003).In the course of our research on enzyme-catalyzed resolution of centrochiral ferrocene compounds,racemic 2-( -hydroxyferrocenyl)benzenethanol and 1-ferro-cenylisochromane,(I),were prepared by reduction of methyl2-(ferrocenoyl)benzeneacetate ( akovicÂ,2000).The molecular structure of (I)is the ®rst reported structure to contain an isochromanyl group attached to the ferrocenyl moiety (Fig.1).Moreover,the Cambridge Structural Database (Allen,2002)lists only three structures containing an iso-chromanyl group at all (Yamato et al.,1984;Unterhalt et al.,1994;Eikawa et al.,1999).The heterocyclic six-membered ringActa Crystallographica Section CCrystal Structure CommunicationsISSN0108-2701Figure 2Part of the crystal structure of (I),showing the formation of (010)sheets built from C14ÐH14A ÁÁÁCg 1i and C18ÐH18ÁÁÁCg 2ii interactions (Cg 1and Cg 2are the centroids of rings C12/C13/C16±C19and C6±C10,respectively).CÐH ÁÁÁ%interactions are indicated by dashed lines.[Symmetry codes:(i)x ,12Ày ,z À12;(ii)1+x ,y ,1+z.]Figure 1A view of (I),with the atom-numbering scheme.Displacement ellipsoids for non-H atoms are drawn at the 20%probability level.1Part XXXVIII:Cetina et al.(2003).adopts a distorted half-chair conformation,in which atoms O1and C15are 0.426(1)and À0.348(2)AÊfrom the plane of the other ring atoms (C11±C14);the C11ÐC12ÐC13ÐC14torsion angle is 2.4(2) .The bond lengths in the heterocyclic and fused phenyl rings (Table 1)mostly agree with the equivalent bond lengths in the structures of 1,1H -oxybis(iso-chromane)(Eikawa et al.,1999)and (S )-1-(phenyl)ethyl-ammonium (S )-isochromane-1-carboxylate (Unterhalt et al.,1994).The exception is the C12ÐC13bond,which is shorter($0.04AÊ)in the latter structure.Heterocyclic ring atoms O1,C11and C15and cyclopentadienyl (Cp)ring atom C1lie in the same plane,the C15ÐO1ÐC11ÐC1torsion angle being 178.95(14) .The dihedral angle between the mean plane of these four atoms and the C1±C5Cp ring is 47.8(1) .Furthermore,the plane of the heterocyclic ring is almost perpendicular to the plane of the C1±C5ring and is parallel to the plane of the fused phenyl ring.The corresponding dihedral angles are 87.3(1)and 4.0(1) .The exocyclic C2ÐC1ÐC11bond angle is larger than the C5ÐC1ÐC11angle (Table 1).The Cp rings are planar and almost parallel to each other [the dihedral angle between their planes is 2.7(1) ],and the FeÐC distances are in the range2.034(2)±2.051(2)AÊfor the substituted (C1±C5)and 2.029(2)±2.049(2)AÊfor the unsubstituted (C6±C10)ring,the average values being 2.042(2)and 2.038(2)AÊ,respectively.The CÐC bonds are slightly longer in the substituted ring than in the unsubstituted ring [1.410(3)±1.429(2)versus 1.397(3)±1.414(3)AÊ],and the bond angles in both rings range from 107.52(15)to 108.33(18) .The geometry of the ferrocenyl moiety agrees well with the structures of ferrocene (Seiler &Dunitz,1979)and of theferrocene derivatives we have reported previously (Cetina et al.,2002,2003).The main conformational difference was observed in the orientation of the Cp rings.In (I),the rings are twisted from an eclipsed conformation by 19.4(2) (mean value).The values of the corresponding CÐCg 3ÐCg 2ÐC pseudo-torsion angles (Cg 3and Cg 2are the centroids of the C1±C5and C6±C10rings,respectively),de®ned by joining two eclipsing Cp C atoms through the ring centroids,range from 19.0(2)to 19.7(2) .The conformation is almost exactly halfway between eclipsed and staggered,as demonstrated by the C1ÐCg 3ÐCg 2ÐC9torsion angle of 163.4(1) .This angle would be 180 for a staggered conformation and 144 for a fully eclipsed conformation.The centroids of the Cp rings are almost equidistant from the Fe atom;the FeÐCg 3and FeÐCg 2distances are 1.647(1)and 1.650(1)AÊ,respectively,while the Cg 3ÐFeÐCg 2angle is 178.2(1) .There are a number of CÐH ÁÁÁ%interactions (Table 2and Fig.2).Atom H14A of the heterocyclic ring is positioned almost perpendicularly above the phenyl-ring centroid (Cg 1)of the adjacent molecule.The six relevant H ÁÁÁC distances fallin the narrow range 3.06±3.28AÊ,and the H ÁÁÁCg i distance is signi®cantly shorter than any of the H ÁÁÁC distances[symmetry code:(i)x ,12Ày ,z À12;Table 2].The CÐH ÁÁÁ%interaction between phenyl atom H18and the unsubstituted Cp ring exhibits a completely different geometry.The H18ÁÁÁC7ii distance is shorter than the H ÁÁÁCg ii distance [symmetry code:(ii)1+x ,y ,1+z ].The second shortest H ÁÁÁC contact is that to atom C6,and the CÐH bond points towards the C6ÐC7bond of the Cp ring rather than towards the ring centroid (Cg 2).Similarly,the longest interaction,C5ÐH5ÁÁÁCg 1iii [symmetry code:(iii)1Àx ,Ày ,2Àz ],points towards the C12ÐC13bond.Both the H5ÁÁÁC12iii and the H5ÁÁÁC13iii contacts are shorter than the H ÁÁÁCg iii distance.The molecules linked by these CÐH ÁÁÁ%interactions build a three-dimensional framework (Fig.3).ExperimentalNaBH 4(253mg,6.7mmol)was added gradually to a solution of methyl 2-(ferrocenoyl)benzeneacetate (326mg,0.9mmol)in a mixture of EtOH and Et 2O (1:1v /v ;5ml).The mixture was re¯uxed for 2h and worked up in the usual manner.Separation by preparative thin-layer chromatography on silica gel (Merck,Kieselgel 60HF 254)yielded 2-( -hydroxyferrocenyl)benzeneethanol (237mg;yield 78%)and orange crystals of 1-ferrocenylisochromane (57mg;yield 20%;m.p.365±366K).Single crystals of the title compound were obtained by slow evaporation from a cyclohexane solution at room tempera-ture.IR (CH 2Cl 2,cm À1):)3081(w )and 3020(w )(CÐH,ferrocene),2942(m )(CÐH,aliphatic),1278(m )(CÐOÐC);1H NMR (DMSO,p.p.m.): 7.18(d ,1H,H16),7.12(d ,1H,H17),7.14(d ,1H,H18),7.16(d ,1H,H19),4.23(s ,5H,unsubstituted ferrocene ring),4.13±4.20(m ,4H,substituted ferrocene ring),3.97(m ,1H,H15A ),3.77(m ,1H,H15B ),2.78(m ,2H,H14),5.58(s ,1H,H11);13C NMR (DMSO,p.p.m.): 137.29(C12),132.91(C13),128.5(C17),126.25(C18),125.98(C16),125.36(C19),90.21(C1),73.63(C11),68.62(unsub-stituted ferrocene ring),68.56±66.31(substituted ferrocene ring),61.55(C15),27.99(C14).Acta Cryst.(2003).C 59,m328±m330Mario Cetina et al.[Fe(C 5H 5)(C 14H 13O)]m329metal-organic compoundsFigure 3Part of the crystal structure of (I),showing the cyclic motif generated by the C5ÐH5ÁÁÁCg 1iii interaction (Cg 1is the centroid of ring C12/C13/C16±C19),which links the (010)sheets into a three-dimensional framework.CÐH ÁÁÁ%interactions are indicated by dashed lines,and the unit-cell box has been omitted for clarity.[Symmetry code:(iii)1Àx ,Ày ,2Àz .]metal-organic compoundsm330Mario Cetina et al.[Fe(C 5H 5)(C 14H 13O)]Acta Cryst.(2003).C 59,m328±m330Crystal data[Fe(C 5H 5)(C 14H 13O)]M r =318.18Monoclinic,P 21a ca =11.5053(2)A Êb =18.5095(3)A Êc =7.1941(1)AÊ =106.933(1)V =1465.62(4)AÊ3Z =4D x =1.442Mg m À3Mo K radiationCell parameters from 3411re¯ections =2.6±27.5 "=1.02mm À1T =293(2)K Prism,orange0.80Â0.40Â0.15mm Data collectionNonius KappaCCD area-detector diffractometer 9and 3scansAbsorption correction:multi-scan (DENZO±SMN ;Otwinowski &Minor,1997)T min =0.630,T max =0.85716885measured re¯ections3334independent re¯ections 2662re¯ections with I >2'(I )R int =0.064 max =27.4 h =À14314k =À23323l =À939Re®nementRe®nement on F 2R [F 2>2'(F 2)]=0.030wR (F 2)=0.076S =1.033334re¯ections 190parametersH-atom parameters constrainedw =1/['2(F 2o )+(0.0341P )2+0.3545P ]where P =(F 2o +2F 2c )/3(Á/')max =0.001Á&max =0.25e A ÊÀ3Á&min =À0.23e AÊÀ3All H atoms were included in calculated positions as riding atoms,with SHELXL 97(Sheldrick,1997)defaults viz.CÐH =0.93AÊfor aromatic H atoms,0.98AÊfor methine H atoms,and 0.97A Êfor methylene H atoms.For all H atoms,the isotropic displacement parameters were set at 1.2times the equivalent anisotropic displacement parameters of the attached non-H atoms.Data collection:COLLECT (Nonius,2000);cell re®nement:DENZO±SMN (Otwinowski &Minor,1997);data reduction:DENZO±SMN ;program(s)used to solve structure:SHELXS 97(Sheldrick,1997);program(s)used to re®ne structure:SHELXL 97(Sheldrick,1997);molecular graphics:PLATON (Spek,2003);soft-ware used to prepare material for publication:SHELXL 97.The re¯ection data were collected at the Faculty of Chem-istry and Chemical Technology,University of Ljubljana,Slovenia.We acknowledge with thanks the ®nancial contri-bution of the Ministry of Education,Science and Sport of the Republic of Slovenia (grant Nos.X-2000and PS-511-103),which made the purchase of the apparatus possible.Supplementary data for this paper are available from the IUCr electronic archives (Reference:GD1251).Services for accessing these data are described at the back of the journal.ReferencesAllen,F.H.(2002).Acta Cryst.B 58,380±388.Bolm,C.,MunÄiz-Ferna Ândez,K.,Seger,A.,Raabe,G.&Gunther,K.(1998).Chem.63,7860±7867.Cetina,M.,Hergold-BrundicÂ,A.,Nagl,A.,Jukic Â,M.&Rapic Â,V .(2003).Struct.Chem.14,289±293.Cetina,M.,MrvosÏ-Sermek,D.,Jukic Â,M.&Rapic Â,V .(2002).Acta Cryst.E 58,m676±m678.akovicÂ,S.(2000).PhD thesis,University of Zagreb,Croatia. akovicÂ,S.,Lapic Â,J.&Rapic Â,V .(2003).Biocatal.Biotransform.In the press.Eikawa,M.,Sakaguchi,S.&Ishii,Y.(1999).Chem.64,4676±4679.Gonsalves,K.E.&Chen,X.(1995).Ferrocenes ,edited by A.Togni &T.Hayashi,ch.10,pp.497±527.Weinheim:VCH.KoÈllner,C.,Pugin,B.&Togni,A.(1998).J.Am.Chem.Soc.120,10274±10275.Nonius (2000).COLLECT.Nonius BV ,Delft,The Netherlands.Otwinowski,Z.&Minor,W.(1997).Methods in Enzymology ,Vol.276,Macromolecular Crystallography ,Part A,edited by C.W.Carter Jr &R.M.Sweet,pp.307±326.New York:Academic Press.Patti,A.&Nicolosi,G.(1999).Tetrahedron :Asymmetry ,10,2651±2654.Perea,J.J.A.,Lotz,M.&Knochel,P .(1999).Tetrahedron :Asymmetry ,10,375±384.Pioda,G.&Togni,A.(1998).Tetrahedron :Asymmetry ,9,3903±3910.Richards,C.J.&Locke,A.(1998).Tetrahedron :Asymmetry ,9,2377±2407.Schwink,L.&Knochel,P .(1998).Chem.Eur.J.4,950±968.Seiler,P .&Dunitz,J.D.(1979).Acta Cryst.B 35,2020±2032.Sheldrick,G.M.(1997).SHELXS 97and SHELXL 97.University ofGoÈttingen,Germany.Spek,A.L.(2003).J.Appl.Cryst.36,7±13.Unterhalt,B.,Krebs,B.,Lage,M.&Nocon,B.(1994).Arch.Pharm.327,799±804.Yamato,M.,Hashigaki,K.,Kokubu,N.&Nakato,Y.(1984).J.Chem.Soc.Perkin Trans.1,pp.1301±1304.Table 2Hydrogen-bonding geometry (AÊ, ).Cg 1and Cg 2are the centroids of rings C12/C13/C16±C19and C6±C10,respectively.D ÐH ÁÁÁA D ÐH H ÁÁÁA D ÁÁÁA D ÐH ÁÁÁA C14ÐH14A ÁÁÁCg 1i 0.97 2.85 3.627(2)138C18ÐH18ÁÁÁCg 2ii 0.93 2.88 3.660(2)142C18ÐH18ÁÁÁC7ii 0.93 2.86 3.760(3)163C18ÐH18ÁÁÁC6ii 0.93 3.03 3.874(3)151C5ÐH5ÁÁÁCg 1iii 0.93 3.14 3.984(2)152C5ÐH5ÁÁÁC12iii 0.93 2.98 3.840(2)154C5ÐH5ÁÁÁC13iii0.933.023.949(2)177Symmetry codes:(i)x Y 12Ày Y z À12;(ii)1 x Y y Y 1 z ;(iii)1Àx Y Ày Y 2Àz .Table 1Selected geometric parameters (AÊ, ).O1ÐC111.4236(19)O1ÐC15 1.432(2)C1ÐC11 1.503(2)C11ÐC12 1.523(2)C12ÐC16 1.388(2)C12ÐC13 1.397(2)C13ÐC19 1.392(3)C13ÐC14 1.504(3)C14ÐC15 1.501(3)C16ÐC17 1.384(3)C17ÐC18 1.380(3)C18ÐC19 1.379(3)C11ÐO1ÐC15110.39(13)C2ÐC1ÐC11127.61(14)C5ÐC1ÐC11124.78(14)O1ÐC11ÐC1109.37(13)O1ÐC11ÐC12111.64(13)C1ÐC11ÐC12112.14(13)C13ÐC12ÐC11119.77(14)C12ÐC13ÐC14120.38(16)C15ÐC14ÐC13111.24(15)O1ÐC15ÐC14110.04(15)。

The crystal structure of the β″ phase in Al–Mg–Si alloys

THE CRYSTAL STRUCTURE OF THE b0PHASE INAl±Mg±Si ALLOYSS.J.ANDERSEN1,2,H.W.ZANDBERGEN2,J.JANSEN2,3,C.TRáHOLT2,U.TUNDAL4and O.REISO41SINTEF Materials Technology,Applied Physics,7034Trondheim,Norway,2National Centre for HREM,Laboratory of Materials Science,Delft University of Technology,Rotterdamseweg137,2628 AL Delft,The Netherlands,3Laboratory for Crystallography,University of Amsterdam,Nieuwe Achtergracht166,1018WV Amsterdam,The Netherlands and4HYDRO Aluminium,Metallurgical Rand D Centre,Sunndalsùra,Norway(Received17November1997)AbstractÐThe crystal structure of b0,one of the strengthening phases in the commercially important Al±Mg±Si alloys,is determined by use of high resolution electron microscopy(HREM)and electron di raction(ED).A trial structure was established from exit wave phase reconstructed HREM images.A least-square re®nement of the model coordinates was done using data from digitally recorded ED patterns.A recently developed computer program(MSLS)was applied,taking into account dynamic scattering.The atomic unit cell contains two units of Mg5Si6.It is C-centred monoclinic,space group C2/m, a=1.51620.002nm,b=0.405nm,c=0.67420.002nm,b=105.320.58.The atomic packing may be regarded as a hard ball packing using clusters,the clusters being(1)centred tetragons of Mg atoms and(2) so-called twin icosacaps where Mg atoms are centred above and below pentagonal rings of four Si atoms an one Mg atom.A growth related stacking fault in the structure is explained by a de®ciency of Mg atoms.A model for the b0/Al interface is given.#1998Acta Metallurgica Inc.1.INTRODUCTION1.1.GeneralThe discovery of the precipitation hardening mech-anism in the beginning of this century in an Al±Cu alloy has had great implications for all technologies requiring light alloys with some strength,and es-pecially for the aerospace and construction technol-ogies.The increase in hardness that the commercial Al alloys achieve upon hardening is usually a factor of2or more.In the Al±Mg±Si(6xxx)alloys such a tremendous increase in strength is caused by pre-cipitates formed from solution,of merely1wt%of Mg and Si that is added to the aluminium.The maximum hardness is achieved when the alloy con-tains a combination of very®ne fully coherent so-called Guinier Preston(GP-I)zones with diameters about2.5nm,and the semicoherent,larger needles, b0(GP-II zones)with a typical size4Â4Â50nm3. The density of these phases is very high.For the b0 needles,a number density in the matrix of about 104/m m3is normal.This is equal to a volume of nearly1%in the material.The6xxx series alloys are not among the strongest aluminium alloys,but they represent a high share of the aluminium pro-ducts in the world(H20%).In1989,about90%of the tonnage extruded in western Europe,was Al±Mg±Si alloys[1].1.2.The precipitation/transformation sequenceThe phases occurring in the Al±Mg±Si alloys have been studied for more than50years due to the commercial importance of these materials.In1948 Geisler and Hill[2]and Gunier and Lambot[3] reported that X-ray Laue pattern zones indicated the formation of small(H2Â2Â10nm3)needles or Guinier Preston(GP)zones,when the temperature was raised to2008C.Further heating caused the zones to thicken into rods,called b',and®nally a large plate-shaped equilibrium phase,b,was seen to form.The latter was known to be of the f.c.c.CaF2 type with a composition Mg2Si.The alloys that were studied were close to the Al±Mg2Si section of the Al±Mg±Si phase diagram;therefore it was assumed that the composition of all the Mg±Si con-taining phases was ter experiments have shown that the precipitation and transformation is quite complicated and that except for the equili-brium phase,b,the phases involved do not have the stoichiometric ratio Mg2Si.In Table1the transformation sequence at low ageing temperatures for alloys near the quasi-binary section Al±Mg2Si of the phase diagram is summar-ised.The range of existence and sizes of the b'rods and b plates depend not only on the heat-treatment, but on several other factors as well,such as cooling rate from homogenisation or extrusion and the number of Al±Fe(+Mn)±Si containing phases (dispersoids)in the material.This will not be dis-cussed in this paper.In the following a discussion of the precipitation/ transformation sequence shown in Table1is given.Acta mater.Vol.46,No.9,pp.3283±3298,1998#1998Acta Metallurgica Inc.Published by Elsevier Science Ltd.All rights reservedPrinted in Great Britain1359-6454/98$19.00+0.00 PII:S1359-6454(97)00493-X32831.2.1.Atomic clusters.After rapid cooling from homogenisation or extrusion the material is super-saturated with Mg and Si.Due to the higher solubi-lity of Mg in Al,when stored at room temperature or heated,Si ®rst goes out of solution and forms small clusters,but there are also some indications of clustering of Mg [5].The nucleation of Si-clusters will occur at quenched-in vacancies at temperatures as low as À508,below which the vacancy movement becomes very low [6].Storing or heating above À508will cause Mg to di use to the clusters,and Mg±Si phases will pre-cipitate.The di usion of Mg to the Si clusters has been veri®ed through APFIM [5,7]where the ratio of Mg/Si in the average cluster was found to increase with time when heated at 708.Since the number of Si clusters formed will be important for the precipitation of the strengthening GP zones,the storing time at a low temperature before arti®cial ageing is important concerning the material proper-ties.1.2.2.GP zones and the b 0phase .The ®rst phase to precipitate on the small clusters is the GP zones.Based on a TEM study of Al±Mg 2Si [8]Thomas proposed a model for these particles;Mg and Si replace Al in such a ratio that the occupied volume is about the same.He proposed a simple substi-tution along 110-directions with strings of atoms in the sequence Mg±Si±Mg±Mg±Si±Mg.Here two di-ameters of Mg (2Â0.32nm)and one of Si (0.235nm)amounts to 0.874nm,as compared with three diameters of Al (0.859nm).In more recent research the evolution of GP zones in several Al±Mg 2Si alloys was studied by calorimetry [6],in 6061by calorimetry and TEM [5],and by atom-probe ®eld-ion microscopy (APFIM)and TEM/HREM [5,7].These works support the view that there are at least two phases in the size range of the GP-zones,called GP-I and GP-II.For the GP-I type the size is in the range 1±3nm.The crystal structure is unknown.The zones are fully coherent and probably have a spherical shape.Dutta and Allen [9]observed by TEM small spot-like features of ``unresolved''shape of about 2.5nm that should be the GP-I zones.Particles investigated by APFIM [5]with comparable dimensions to these zones seem to have Mg/Si ratios usually less than 1.This composition is therefore di erent from that of the model proposed by Thomas [8].The GP-II zone is the same phase as the currently investigated b 0phase.This phase has the shape of ®ne needles,typically about 4Â4Â50nm 3when the material is in the aged-hardened condition [7,10].In this condition the number density of the nee-dles is high;typically 104/m m 3[10].The b 0phase is fully coherent only along the b -axis.Edwards et al.[7]managed to determine the unit cell of the b 0phase by electron di raction.It was found to be a monoclinic C-centred structure with a =0.153420.012nm,b =0.405nm,c =0.68320.015nm,b =10621.58.The b -axis is along the needle-axis.It is the full coherency of GP-I zones,the semi-coherency of the GP-II zones together with their high number densities that introduce in the alu-minium matrix strain and resistance against move-ment of dislocations,that gives the material its mechanical strength.1.2.3.The b 'phase .The next phase in the trans-formation sequence after the GP-I zones and the b 0phase is the b 'phase.This has a lower Mg/Si ratio than the equilibrium b phase.Lynch et al.found by X-ray microanalysis evidence for a ratio of Mg/Si in the b 'rods in an overaged material to be about 1.73[11],while Matsuma et al.[12]later determined the ratio to be about 1.68.For materials with excess silicon relative to Al±Mg 2Si there may be very small precipitates also of the b 'and a so-called B 'phase that is richer in silicon,or even Si particles [4].Because of this such particles with sizes comparable to b 0[7,4]may be mistaken for the b 0phase.The b 'and the B 'phase are reported as having the hexa-gonal unit cells a =0.705nm,c =0.405nm and a =0.104nm,c =0.405nm,respectively.In Refs [7,4]the relative number of b 0as compared with the smallest b '(and B ')particles was not deter-mined.It was recently suggested that b 'is a h.c.p.structure with a =0.405nm,c =0.67nm [12,13].1.2.4.The b phase.The b phase is the equilibrium phase in this system.It is the only phase up to now with a known structure.It is a CaF 2type f.c.c.structure with a =0.639nm having formula Mg 2Si.The structure may be described as strings of three atoms,Mg±Si±Mg,on the corners and faces of a cube,directed along the diagonals.Table 1.The evolution of Mg±Si phases near the quasi-binary section Al±Mg 2Si (top to bottom)Transformation/precipitation sequence Crystal type Size (nm)Composition Clusters of Si and fewer of Mg unknown unknown Si (Mg)Clusters containing Si and Mg unknown unknown Mg/Si <1Coherent spherical GP-I zonesunknown H 1±3Mg/Si H 1Semi-coherent GP-II zones (b 0needles)monoclinic H 4Â4Â50Mg/Si r 1b 'rods (and B 'rods)hexagonal H 20Â20Â500Mg/Si H 1.7b -Mg 2Si platescubicmicronsMg/Si =2The B 'phase is observed with alloys having excess Si relative to Al±Mg 2Si.It contains more Si than b '[4].ANDERSEN et al.:Al±Mg±Si ALLOY32841.3.SummationSumming up the information above,it appears that the phases that evolve from the very®ne Si-clusters into coarser particles take up progressively more magnesium during the coarsening and trans-formation processes,until an equilibrium compo-sition Mg2Si for the b phase®nally is reached.In this paper we report the structure determi-nation of the b0phase,which must be one of the important hardening phases in the commercial6xxx alloys.The technique used in the structure determi-nation is the through focus exit wave reconstruction technique in high resolution electron microscopy,in combination with quantitative electron di raction.2.EXPERIMENTAL2.1.Material and sample preparationThe as-received material was in the shape of extruded sections.It was supplied by HYDRO Aluminium AS(Sunndalsùra).The composition of the material was Al±0.2Fe±0.5Mg±0.53Si±0.01Mn (wt%).The material is from the same batch and extruded sections as investigated in Refs[10,14], there labelled as A and C,respectively.Specimen preparation and location in the extruded section of the samples for TEM are described in Refs[10,14]. Prior to the arti®cial ageing(5h at1858)the ma-terial had undergone a rather standard processing for an extrusion product.After the jet-polishing, specimens were stored in methanol.Most of the TEM experiments were performed within a day after specimen preparation.2.2.TEM equipment and experimental dataAll TEM work was performed using a PHILIPS CM30-ST/FEG electron microscope operated at 300kV.The microscope is equipped with a Photometrix1024Â1024slow scan CCD camera (12bits dynamical range),enabling a linear record-ing of HREM and ED puter control of the CCD camera and the microscope is handled with a Tietz software package.In this way series of 15±20HREM images with focus increments of typi-cally 5.2nm were recorded for each exit wave reconstruction.For the high resolution work suitable aluminium grains were selected and tilted into a h100i zone axis.HREM images were recorded at room tem-perature on as thin areas as possible,typically4±10nm.Needles were selected that could be viewed along their[010]zone axis.In this situation,the needles usually extend through the whole thickness of the specimen,such that no image blurring occurs due to overlap with the matrix.For a single image, the exposure time was usually about1s.For the di raction experiments a small spot-size (5±10nm)was used with exposure times of1±5s. Two zone-axes of the needles were chosen;[010]and[001].For the latter,the aluminium grain was tilted to a h310i zone axis,where statistically one out of six needles is in the correct orientation. Many of the needles contain stacking-faults or sec-ond phases.For a reliable structure determination it is important that the area where a di raction pat-tern is taken is free of defects.Given the resolution of the microscope it should be relatively easy to select single crystalline b0particles.However,to prevent the rapid contamination of the illuminated area that is typical for this kind of specimen at room temperature,the specimen was cooled to about100K.The sample cooling holder has a much poorer mechanical stability resulting in such a loss of resolution that selection of single crystal b0particles was di cult.Because of this ED pat-terns were taken from each particle encountered. Therefore quite many di raction patterns had to be discarded because of streaking and twinning prob-ably caused by the stacking-faults or sometimes extra spots caused by a intergrown phase that was determined to be b'.Five[010]di raction patterns were selected.For the[001]zone axis there is a greater chance of``cross-talk''due to more overlap of the matrix with the crystal,and suitable di rac-tion patterns for the re®nement were more di cult to®nd.Here®ve of the16recorded patterns were from the correct projection or particle.Only two of these patterns could later be re®ned.In addition to the problem with overlap spots from the b'phase, the reason was also the strong interference with the aluminium matrix in this projection that made sub-traction of the background di cult.The thickness of the investigated areas were somewhat larger for the di raction experiments than for the HREM ex-periments.The subsequent re®nements showed that the thickness usually exceeded10nm.In Fig.6, parts of two of the digitally recorded di raction images are shown.This®gure also shows some streaking caused by oversaturation of the CCD camera,which was not equipped with over¯ow pro-tection.The streaks and the aluminium di raction re¯ections were excluded from the images prior to data reduction.The exit wave reconstruction of the HREM focus series were done with a software package based on algorithms developed by Van Dyck and Coene[15±17].Given the coherency of the presently available ®eld emission guns the structural information in ordinary HREM images goes well beyond the point-to-point resolution in the electron microscope. The reconstruction method takes advantage of the knowledge about the transfer function,e.g.how the microscope optics distorts the electron wave after leaving the crystal(the exit wave)on its way to the image plane.This distortion is also a function of defocus.A series of HREM images are recorded at intervals of known defocus.The amplitude and phase information that is mixed up in the HREM images is retrieved through digital processing,andANDERSEN et al.:Al±Mg±Si ALLOY3285corrections for focus and spherical aberration are done.Furthermore,since typically15±20images are used in the reconstruction a considerable reduction in noise is attained.The exit wave is thus independent of various aberrations of the electron microscope, but it is still dependent on the specimen thickness. Only for very small specimen thicknesses is the exit wave very similar to the projected potential,viz.the projected atomic structure.For thicker sections,e.g. more than about10nm for the presently presented exit wave image,the local contrast in the exit wave can be quite di erent from the local scattering poten-tial.Thus,for such thicknesses a higher brightness at a certain point in the phase image of the exit wave as compared to other points,does not have to imply the presence of a locally more strongly scattering atom at this point.The good news is that the positions of the bright dots should correlate well with the location of the atoms.In the presently used electron microscope the res-olution is enhanced from0.20nm to about0.14nm. The HREM images presented in this work are recombined exit wave phase images.See Coene et al.[17],Zandbergen et al.[18]and Op de Beeck et al.[19]for examples and discussion of the method. The re®nement of the structure was done using the computer programme package MSLS[20].The CCD images with the di raction patterns were cor-rected for the¯at®eld(variation in the pixel sensi-tivity)and over¯ow during read-out of the CCD camera.Spurious X-ray signals and the Al di rac-tion spots were omitted.Automatic indexing and data reduction on the patterns were done.The obtained two-dimensional indices of the images were next transformed into the correct hkl indices so that the di raction data sets could be combined. MSLS was used for re®nement of the trial structure coordinates as obtained from the reconstructed exit wave.This program re®nes coordinates based on the least-squares procedure using the multi-slice al-gorithm to account for the dynamic di raction.The parameters re®ned were the thickness,the scaling factor,the centre of the Laue circle for each of the data sets,and the atomic coordinates and tempera-ture factors.The R-value used as measure of the correctness of the structure is de®ned as R=a(I calcÀI obs)2/a(I obs)2.Only the signi®cant re¯ections(I obs>2s(I obs))were used.R-values between2and6%are being quoted for the most reliably determined structures.3.RESULTS/DISCUSSION3.1.Conventional HREM/TEMConventional TEM shows the interior of the Al grains to mainly contain particles having a®ne nee-dle shape.The needles lay along h100i Al directions. Figure1gives an example.It is a bright®eld image in an Al h100i zone axis where the needles clearly point in two normal directions.The dark spots are needles pointing in the viewing direction.The exper-imental di raction patterns as well as HREM images show that the needle shaped particles mostly are of one kind,the monoclinic phase that is usually referred to as the b0phase.Figure2shows a HREM image with one such needle.Such images show the precipitates to be coherent along the nee-dle direction(their b-axis)with a h100i Al direction. This con®rms that their cell parameter is the same as aluminium,b=0.405nm.Many of the b0precipitates were found to con-tain stacking faults.In some precipitates an inter-growth of b0with another phase was observed.It is most probably the b'phase which has the hexago-nal axis along the needle direction.Sometimes this phase was found to exist alone.The cell parameter a=0.705nm has been con®rmed from exit wave simulated images.These images will be published later.In the same material coarser rods of the b' phase have earlier been investigated;It was reported that they nucleate on®ne Al±Fe±Si particles[14].It may be expected that much of the b'particles nucle-ate on b0since with longer arti®cial ageing times the micro-structure will contain an increasing amount of rods of b'.By selected area electron dif-fraction the coarse b'phase in this material was determined to have a hexagonal structure with a H0.71nm,c H0.41nm.The a-axis therefore®ts well with the phase intergrown with b0.The struc-ture of the small and large b'is therefore probably the same.We did not observe any B'phase in the material.3.2.Elemental analysis of the b0phaseWe performed several X-ray analyses of the small precipitates with the spot along the needle axis. Due to the very thin specimen areas(10±40nm)the spectra obtained should in principle not be signi®-cantly in¯uenced by absorption in the specimen, which is the most important reason for deviations from the actual concentration.In spite of the small size of the spot(1±2nm),there was always an Al peak present in the spectrum,of varying height. This is partly caused by stray electrons travelling down the column of the electron microscope which are not focused with the rest of the electrons in the beam probe and therefore many hit aluminium. Secondly,because during analysis the beam is par-allel to the needle axis,i.e.to the[010]zone axis of b0,this implies an e ective beam broadening by the elastic scattering of some electrons into aluminium. For some of the recordings there is also an e ect of specimen drift during recording.Another e ect is the contamination layer and the(aluminium)oxide layer on the surface of the particle which primarily contains Al.The EDS experiments could therefore not rule out that some Al is contained in the precipitate.As a standard for determining the K-ratios a mineral forsterite was used whose mainANDERSEN et al.:Al±Mg±Si ALLOY 3286components are MgO and SiO 2with a composition so that the Mg/Si atomic ratio is 2.Not taking into account the possible systematic deviations,the EDS experiments indicated that the atomic ratio for Mg/Si was close to or even below 1.The accuracy of these measurements were on the order of 10%.However,they ruled out the earlier accepted ratio of 2for the b 0phase.EDS measurements were also performed on larger particles of the b 'and b -Mg 2Si phases which had been extracted from the alu-minium matrix.These phases gave compositions near the expected,as listed in Table 1.The accuracy here was much better for thin sections since the alu-minium matrix could be avoided entirely.3.3.Exit wave reconstruction3.3.1.The unit cell.Coherency of the b 0phase with the matrix .In Fig.3a reconstructed exit wave (phase)of a b 0particle in the [010]orientation embedded in aluminium is shown.The b 0[010]direction is parallel to a h 100i type aluminium zone axis and is along the needle.Atomic columns in the viewing direction in the image appear as bright dots.The columns in the Al matrix are clearly resolved;in this projection the separation between nearest neighbor columns are 0.2025nm,or half the Al unit cell length.Due to the face centering of alu-minium the nearest neighbor atom columns are also shifted 0.2025nm in the viewing direction relative to each other.In the ®gure circles are drawn that indicate the two di erent height positions of the atoms in the viewing direction.The lattice image of the Al matrix changes over the image due to local variations in tilt.The b 0unit cell is outlined in the particle.Due to the C-centering,the a -axis is twice the apparent periodicity.By calibrating the magni®cation of the image using the aluminium lattice,the unit cell was established to be a =1.51620.002nm,c =0.67420.002nm and b H 105±1068.HREM of other nee-dles lying in the normal direction (Fig.2)have shown that there is a full coherence between the crystal along the b -axis with the same periodicity as the aluminium matrix;therefore b =0.405nm.In the re®nement of di raction images for this zone axis,the monoclinic angle is calculated.It was found to have a mean value b =105.320.58when averaged over 7di raction patterns.The b 0unit cell is closely related to the alu-minium lattice.From di raction patterns (Fig.5)asFig.1.A typical low magni®cation micrograph of b 0needles in a h 001i Al zone axis.Needles are directed along the three h 100i Al directions and appear therefore point-like (dark spots)in the viewing direction.The needles have a mean diameter of about 4nm,and an average length about 50nm.Alarger b 'rod (white appearance)is directed in the viewing direction in the centre of the image.ANDERSEN et al.:Al±Mg±Si ALLOY 3287well as from the exit wave (Fig.3)the following relationship between the phases can be found; 001 Al k 010 b 0,"310 Al k 001 b 0,230 Al k 100 b 0This relationship is the same as found earlier byEdwards et al.[7].A corresponding super cell in aluminium can be de®ned by real vectors ~ab 0 2~a Al 3~b Al ,~b b 0~c Al ,~c b 0 À32~a Al 12~b Alwith respective lengths 1.46,0.405and 0.64nm witha monoclinic angle of 105.38.Half of this super cellis outlined in Fig.3on the left side of the b 0par-ticle.The super cell is also C-centred monoclinicsince two neighbor corners of the half cell along ~ab 0fall on Al atoms in di erent layers.The unit cell for b 0is slightly larger than this Al super cell;3.8%along ~ab 0and 5.3%along ~c b 0.The half super cell (asymmetric unit)contains 11Al atoms.The coherency between b 0and aluminium aids in quantifying the shift of the stacking fault (sf)in the particle that is indicated in Fig.3;By using the Al matrix as reference it can be veri®ed that Al atoms at the left interface,at the upper part (e.g.near the white corners of the unit cell of b 0)are at a di er-ent height relative to similar atoms of b 0on the lower part (here with a black ®ll){.This is illus-trated by the two outlined (half)super cells in the Al matrix that are related to the unit cell of b 0in the upper and lower part of the particle.These super cells are shifted a vector a Al [101]/2relative to each other,which indicates that the shift across the stacking fault in the particle is nearly the same.This shift vector is a Burgers vector of the most common dislocation in aluminium.A model of the fault is given in Section5.Fig.2.Ordinary HREM image of b 0-needle in an h 001i zone axis in Al.The c -axis of the needle is in the plane,and the coherency with h 100i Al in the needle direction is evident.As expected,there is no exact zone axis of b 0along the viewing direction h 001i Al zone axis.The left part of the picture was fourier ®ltered;A high pass ®lter was applied to the upper part and a low pass ®lter to the lower partto extract the periodic information from Al (upper)and the b 0-phase (lower)only.{Alternatively,assume the corners of the outlined unit cells of b 0on each side of the stacking fault to be at thesame heights along ~cb 0.The atoms to the left of Ðand in the matrix outside Ðthese corners must then necessarily have similar heights,since the atomic con®guration and distances to the left of these corners are similar,whether above or below the stacking fault.This assumption must be wrong;When keeping track of the atomic columns in the matrix it leads to the conclusion of an Al atom being at two heights at the same time.Therefore,the corners ofthe unit cells along ~cb 0have di erent heights across the stacking fault.ANDERSEN et al.:Al±Mg±Si ALLOY3288In Fig.4the coherency between the two phases can be studied in more detail.This image is a Fast Fourier Transformation (FFT)of part of Fig.3.Only the lower part of the b 0precipitate is included to reduce streaking caused by the stacking fault.After applying a Fourier ®lter (selecting the con-tents inside the circles superposed on the FFT of Fig.4)the Al re¯ections plus the 610,610,403and 403re¯ections of b 0contribute to the image in Fig.5.The white arrows indicate interface dislo-cations between the particle and matrix.For example,the b 0(601)lattice planes with a spacing d 601=0.211nm are parallel with the Al (200)planes with a spacing of 0.203nm.Therefore,one interface dislocation is expected for each 25Al d 200spacings (normal to the [100]axis in the ®gure).Similarly,for the 403planes,for each 20Al d 020spacing one expects an interface dislocation.The spacings between dislocations observed in Fig.5are di erent from the theoretical ones.The reason for this devi-ation is probably variation in local strain in the particle caused by the stacking fault.Although the exact dislocation is not clear in the image,a matrix dislocation found (marked ``d '')also complicates the situation concerning the mis®t dislocations.This dislocation is found to have a Burgers vector b =0.5a Al [101],as was found when a Burgers vec-tor loop was performed around the particle.This is indicated by the open arrow (d).In Fig.6,two ex-perimental di raction images from the [010]and [001]zone axes are shown.The b 0610and 403re¯ections that coincide with the 200and 020Al matrix re¯ections can also be seen in Fig.6(a).In Fig.6(b)the perfect coherency relation of the (010)lattice planes of the b 0phases with (200)lattice planes can be seen from the overlap of the respect-ive di raction spots.3.3.2.Extraction of the atomic coordinates for b 0from the exit wave images .Figure 7(a)is an increased magni®cation of part of Fig.3.Here the atomic columns are represented as white dots.From this image the atomic positions were esti-mated using the following assumptions:(1)The number of atoms in the unit cell is 22,just as the number of atoms in the similar super cell in aluminium.The number ®ts the apparentnumberFig.3.Phase of an reconstructed exit wave of a typical b 0needle in Al is shown.The needle is viewed head-on along its [010]axis,and along an Al h 001i zone axis.Atomic columns appear white.The b 0unit cell and half the corresponding super cell in Al are outlined.Similarly ®lled circles in the matrix or in the precipitate are atoms (Al or Mg)at the same height.A stacking fault (sf)is indicated.The shiftacross the stacking fault can be determined to be a Al [101]/2.ANDERSEN et al.:Al±Mg±Si ALLOY 3289。

2013-cancer cell-Chromatin-Bound IκBα RegulatesPolycomb Target Genes in Differentiation and Cancer

Cancer CellArticleChromatin-Bound I k B a Regulates a Subset of Polycomb Target Genes in Differentiation and CancerMarı´a Carmen Mulero,1Dolors Ferres-Marco,2Abul Islam,3,4Pol Margalef,1Matteo Pecoraro,5Agustı´Toll,6Nils Drechsel,8Cristina Charneco,8Shelly Davis,9Nicola´s Bellora,3Fernando Gallardo,6Erika Lo ´pez-Arribillaga,1Elena Asensio-Juan,1Vero´nica Rodilla,1Jessica Gonza ´lez,1Mar Iglesias,7Vincent Shih,10M.Mar Alba `,3,11Luciano Di Croce,5,11Alexander Hoffmann,10Shigeki Miyamoto,9Jordi Villa`-Freixa,8,12Nuria Lo ´pez-Bigas,3,11William M.Keyes,5Marı´a Domı´nguez,2Anna Bigas,1,13and Lluı´s Espinosa 1,13,*1Program in Cancer Research,Institut Hospital del Mar d’Investigacions Me`diques (IMIM),Barcelona 08003,Spain 2DevelopmentalNeurobiology,Instituto de Neurociencias de Alicante,CSIC-UMH,Alicante 03550,Spain3Research Program on Biomedical Informatics,Universitat Pompeu Fabra,IMIM-Hospital del Mar,Barcelona 08003,Spain 4Department of Genetic Engineering and Biotechnology,University of Dhaka,Dhaka 1000,Bangladesh 5Gene Regulation,Stem Cells and Cancer,Centre de Regulacio ´Geno `mica (CRG),Barcelona 08003,Spain 6Dermatology Department 7Pathology DepartmentHospital del Mar,Barcelona 08003,Spain8Computational Biochemistry and Biophysics Laboratory,IMIM-Hospital del Mar and Universitat Pompeu Fabra,Barcelona 08003,Spain 9McArdle Laboratory for Cancer Research,University of Wisconsin Carbone Cancer Center,University of Wisconsin-Madison,6159Wisconsin Institute for Medical Research,1111Highland Avenue,Madison,WI 53705,USA 10Signaling Systems Laboratory,UCSD,La Jolla,CA 92093-0375,USA 11Institucio ´Catalana de Recerca i Estudis Avanc ¸ats (ICREA),Barcelona 08003,Spain 12Escola Polite `cnica Superior (EPS),Universitat de Vic,Barcelona 08500,Spain 13These authors contributed equally to this work *Correspondence:lespinosa@imim.es/10.1016/r.2013.06.003SUMMARYHere,we demonstratethat sumoylated and phosphorylated of keratinocytes and interacts with histones H2A and H4at the regulatory region of HOX and IRX genes.Chromatin-bound I k B a modulates Polycomb recruitment and imparts their competence to be activated by TNF a .Mutations in the Drosophila I k B a gene cactus enhance the homeotic phenotype of Polycomb mutants,which is not counteracted by mutations in dorsal/NF-k B .Oncogenic trans-formation of keratinocytes results in cytoplasmic I k B a translocation associated with a massive activation of Hox .Accumulation of cytoplasmic I k B a was found in squamous cell carcinoma (SCC)associated with IKK activation and HOX upregulation.INTRODUCTIONNF-k B plays a crucial role in biological processes,such as native and adaptive immune responses,organ development,cell proliferation,apoptosis,or cancer (Naugler and Karin,2008;Vallabhapurapu and Karin,2009).NF-k B activation de-pends on the IKK-mediated degradation of the NF-k B inhibitors,I k B proteins,that takes place in the cytoplasm and results in the translocation of the NF-k B transcription factor to the nucleus,where it activates gene expression.Recent studies demonstrate the existence of alternative nuclear functions for regulatory ele-ments of the pathway (reviewed in Espinosa et al.,2011),but their biological implications remain poorly understood.Recently,it has been demonstrated that nuclear I k B b binds the promoterCancer Cell 24,151–166,August 12,2013ª2013Elsevier Inc.151角质形成细胞Cancer CellI k B a Is a Modulator of Polycomb Function(legend on next page) 152Cancer Cell24,151–166,August12,2013ª2013Elsevier Inc.of NF-k B target genes following lipopolysaccharide (LPS)stimu-lation to prevent I k B a -mediated inactivation,thereby sustaining cytokine expression in immune cells (Rao et al.,2010).Numerous studies have reported nuclear translocation of I k B a (Aguilera et al.,2004;Arenzana-Seisdedos et al.,1997;Huang and Miyamoto,2001;Wuerzberger-Davis et al.,2011)and various partners for nuclear I k B a ,including histone deacetylases (HDACs)and nuclear corepressors,have been identiﬁed (Agui-lera et al.,2004;Espinosa et al.,2003;Viatour et al.,2003).In ﬁbroblasts,nuclear I k B a associates with the promoter of Notch target genes correlating with their transcriptional repression,which is reverted by TNF a (Aguilera et al.,2004).Nevertheless,the mechanisms that regulate association of I k B to the chromatin and its repressive function remain unknown.I k B a -deﬁcient mice die around day 5because of skin inﬂam-mation associated with high levels of IL1b and IFN-g in the dermis,CD8+T cells,and Gr-1+neutrophils inﬁltrating the epidermis,as well as altered keratinocyte differentiation (Beg et al.,1995;Klement et al.,1996;Rebholz et al.,2007),similar to keratinocyte-speciﬁc I k B a -deﬁcient mice (I k B a k5D /k5D )(Re-bholz et al.,2007).In all cases,disruption of TNF a signaling rescued the skin phenotype (Shih et al.,2009),suggesting that lethality was associated with an excessive inﬂammatory response,likely due to increased NF-k B activity.However,mice expressing different I k B a mutants that are equally able to repress NF-k B in the skin showed divergent phenotypes.Speciﬁcally,mice expressing the nondegradable I k B a mutant,I k B a S32-36A ,developed skin tumors resembling SCC (van Hoger-linden et al.,1999),whereas mice carrying a predominantly nuclear form of I k B a show no overt skin defects (Wuerzberger-Davis et al.,2011).Skin differentiation depends on the correct establishment and maintenance of speciﬁc gene expression patterns,including genes of the HOX family,which in the basal progenitor cells are repressed by EZH2,the catalytic subunit of the Polycomb repressive complex 2(PRC2)(Ezhkova et al.,2009,2011).PRC2is composed by EZH2,the WD-repeat protein EED,RbAp48,and the zinc-ﬁnger protein SUZ12(Zhang and Reinberg,2001).Methylation of lysine 27on histone H3(H3K27me3)by EZH2imposes gene silencing in part by trig-gering recruitment of PRC1(Cao et al.,2002;Min et al.,2003)and histone deacetylases (HDACs).Here,we investigate analternative function for I k B a in the regulation of skin homeosta-sis,development,and cancer.RESULTSPhosphorylated and Sumoylated I k B a Localizes in the Nucleus of KeratinocytesTo investigate the physiological role for nuclear I k B a ,we per-formed an initial screen to determine its subcellular distribution in human tissues.We found that I k B a localizes in the cytoplasm of most tissues and cell types as expected (Figure S1A available online);yet,a distinctive nuclear staining of I k B a was found in human (Figure 1A)and mouse skin sections (Figures 1A,S1A,and S1C),more prominently in the keratin14+basal layer kerati-nocytes.I k B a distribution became more diffused in the supra-basal layer of the skin and gradually disappeared in the more differentiated cells.Speciﬁcity of nuclear I k B a staining was conﬁrmed using skin sections from newborn I k B a -knockout (KO)mice (Figure S1B)and different anti-I k B a antibodies and blocking peptides (Figure S1C).By immunoﬂuorescence (IF)and immunoblot (IB),we detected I k B a protein in both the cyto-plasmic and the nuclear/chromatin fractions of human (Figures 1B and 1C)and mouse (Figure S1D)keratinocytes.Interestingly,nuclear I k B a displayed a shift in its electrophoretic mobility (z 60kDa)detected by different anti-I k B a antibodies,including the anti-phospho-S32-36-I k B a antibody.We next precipitated I k B a from nuclear and cytoplasmic keratinocyte extracts and determined whether this low I k B a mobility was a result of ubiq-uitin or SUMO modiﬁcations.We found that nuclear I k B a was speciﬁcally recognized by anti-SUMO2/3,but not anti-SUMO1or anti-ubiquitin antibodies (Figure 1D;data not shown).Here-after,we will refer to this nuclear I k B a species as phospho-SUMO-I k B a (PS-I k B a ).By cotransfection of different SUMO plasmids in HEK293T cells,we demonstrated that SUMO2was integrated to HA-I k B a at K21,22(Figure S1E),independently of S32,36phosphorylation (Figure 1E).By subcellular fractionation,we found that most HA-PS-I k B a was distributed in the nucleus of HEK293T cells (data not shown),and both K21,22R and S32,36A I k B a mutants showed reduced association with the chromatin (Figure 1F).These results suggest that phosphorylation and sumoylation are both required for I k B a nuclear functions in vivo.Of note,PS-I k B a levels were always low in HEK293T cells whenFigure 1.Phosphorylated and Sumoylated I k B a Is Found in the Nucleus of Normal Basal Keratinocytes(A)Immunodetection of I k B a (green)in normal human skin and detail of basal layer.B,basal;S,spinous,G,granular;and C,corniﬁed layers of epidermis.Dashed line indicates the dermis interphase.DAPI was used for nuclear staining.(B)IF of I k B a in primary human keratinocytes.(C)Subcellular fractionation of human keratinocytes followed by IB with the indicated antibodies.(D)I k B a was immunoprecipitated from primary murine keratinocyte extracts followed by IB with the indicated antibodies.(E)IB analysis of His-tag precipitates from HEK293T cells transfected with the indicated plasmids.SUMO2is incorporated in I k B a when K21,22are present.(F)HEK293T cells were transfected with the indicated I k B a plasmids and processed following the ChIP protocol to obtain the whole chromatin fraction that was analyzed by IB.(G)IF of I k B a and P-IKK in skin sections.Cells with P-IKK staining do not contain nuclear I k B a .(H)IB analysis of keratinocytes transduced with myc-IKK a EE or control.(I)IF analysis of the indicated differentiation markers in skin sections of WT and I k B a KO newborn mice.(J)IB analysis of indicated proteins in control or Ca 2+-treated murine keratinocytes.Total and nuclear/chromatin fractions are shown.(K)Determination of Filaggrin ,K10,and p63mRNA levels in control and I k B a KD keratinocytes following Ca 2+treatment.Expression levels are relative to Gapdh and compared to control cells.Error bars indicate SD.I k B a protein levels were analyzed by IB.Data correspond to one representative of three experiments.N,nuclear;C,cytoplasmic.See also Figure S1.Cancer CellI k B a Is a Modulator of Polycomb FunctionCancer Cell 24,151–166,August 12,2013ª2013Elsevier Inc.153Cancer CellI k B a Is a Modulator of Polycomb FunctionFigure2.I k B a Binds Histones H2A and H4(A)PD experiment using GST-I k B a and native(lane2)or denatured-renatured(lanes3–4)human keratinocyte nuclear extracts.One representative gel stained with Coomassie blue is shown(n=3).(B)Puriﬁcation and analysis of B and C bands identiﬁed as histones H2A and H4by mass spectrometry.Table indicates the number of peptides identiﬁed and their score factor.The highest score is highlighted.(C)Coprecipitation from DSP-crosslinked nuclear extracts from human keratinocytes.(D and E)PD using different GST-H2A proteins and total lysates from HEK293T cells expressing the indicated proteins.(legend continued on next page) 154Cancer Cell24,151–166,August12,2013ª2013Elsevier Inc.compared with keratinocytes,even in overexpression conditions and cell lysates directly obtained under denaturing conditions (see inputs in Figures 1E and S1E).It is well known that IKK activity regulates the cytoplasmic levels of I k B a .By double staining of skin sections,we found that the few cells that were positive for active IKK contained I k B a ,but this I k B a was excluded from the nucleus (Figure 1G).To directly investigate whether IKK regulates subcellular distri-bution of I k B a ,we transduced primary murine keratinocytes with lentiviral IKK a EE .We found that active IKK a induced a decrease in the nuclear levels of PS-I k B a as determined by IB (Figure 1H)and IF (Figure S1G).Additional experiments comparing the effects of both IKK isoforms demonstrated that IKK a EE was more efﬁcient than IKK b EE in decreasing nuclear PS-I k B a levels (60%±5%compared with 16%±9%reduction;p <0.001)(Figure S1F).To directly address whether I k B a was required for normal skin differentiation,we performed IF analysis using different markers comparing I k B a wild-type (WT)and KO newborn skins.Consis-tent with previous reports,I k B a -deﬁcient mice do not show any obvious skin defect at birth with a normal K5-positive basal layer,although we observed a slight reduction in the thickness of the K10-positive suprabasal epidermal layer.Most importantly,I k B a mutant skins showed a severe reduction of the more differ-entiated layer of cells identiﬁed by the accumulation of ﬁlaggrin granules (Figure 1I).This is a cause for impaired barrier function (Palmer et al.,2006).Next,we aimed to distinguish between cell-autonomous and non-cell-autonomous effects of I k B a deﬁ-ciency by using an in vitro system for keratinocyte differentiation induced by high Ca 2+exposure (Hennings et al.,1980).In this model,we found that keratinocyte differentiation was associated with a decrease in both I k B a and PS-I k B a levels and activation of nuclear IKK (Figures 1J and S1H).Notably,knockdown (KD)of I k B a disturbs in vitro keratinocyte differentiation as indicated by the impaired K10and ﬁlaggrin induction in response to Ca 2+,which was accompanied by sustained expression of the progenitor marker p63(Figure 1K).Together,these results strongly suggest that I k B a plays a cell-autonomous function in skin differentiation.I k B a Directly Binds to the N-Terminal Tail of Histones H2A and H4To further investigate the mechanisms underlying nuclear I k B a functions,we searched for nuclear proteins that directly asso-ciate with I k B a .Using GST-I k B a and human keratinocyte nuclear extracts in pull-down (PD)experiments,we isolated pro-teins of estimated molecular weights of 15and 14kDa that were identiﬁed by mass spectrometry as histones H2A and H4(Fig-ures 2A and 2B).Interaction between histones H2A and H4and I k B a was further conﬁrmed by coprecipitation of endoge-nous proteins from keratinocyte nuclear extracts.Of note,the NF-k B subunit p65was absent from nuclear I k B a precipitates but coprecipitated in the cytoplasmic fraction (Figure 2C).By PD assays,we determined the speciﬁcity of I k B a binding compared to other I k B homologs (Figure 2D)and mapped the I k B a -binding domain of histone H2A to be between amino acids 2and 35(Figure 2E).Preincubation of I k B a with p65prevented its association with histones (Figure S2A),suggestive of mutually exclusive complexes.Comparative sequence analysis of the I k B a -binding region of histone H2A (AA1–36)and the homologous region of H4revealed the presence of a motif (3KXXXK/R)that was absent from other histone and nonhistone proteins (Figure 2F).To further study I k B a binding speciﬁcity,we screened a histone peptide array using nuclear HA-I k B a expressed in HEK293T cells as bait.We found that I k B a bound to peptides containing AA11–30of his-tone H4,but not the corresponding region of histone H3.Most importantly,binding of I k B a to H4was prevented by the combi-nation K12/K16Ac and K20Ac or Me2(Figure 2G).Because the equivalent peptides from histone H2A were not included in the array,we performed parallel precipitation experiments using biotin-tagged peptides (AA5–23)of histone H2A and H4(Figures 2H and 2I).We found that I k B a association was prevented by K12Ac,K16Ac,and K20me2of histone H4or the equivalent modiﬁcations of the H2A peptide (Figure 2H)and also when all K/R residues in the 3KXXXK motif were changed into A (Figure 2I).Of note that in these experiments histone-bound HA-I k B a was mostly identiﬁed as a nonsumoylated band,which opens the possibility that posttranslation modiﬁcations are not essential to mediate this interaction in vitro.However,parallel binding experiments using keratinocyte extracts,PS-I k B a ,showed a preferential binding to the histone peptides compared with the cytoplasmic 37kDa I k B a form (Figure S2B).Together,these re-sults strongly suggest that only PS-I k B a can bind the chromatin,but in HEK293T cells this molecule is then desumoylated in vivo or as a consequence of the experimental processing.To gain further insights into the molecular basis of I k B a binding to histones,we completed the structure of I k B a obtained from the Protein Data Bank (ID code 1IKN),which lacked part of the ankyrin repeat (AR)1,using RAPPER (Depristo et al.,2005)and performed docking studies with AutoDock Vina (Trott and Olson,2010)of the histone H4peptide,GKGGAKRHRKV,that contains most of the KXXXK domain.Docking calculations showed two deep pockets for K interaction in I k B a located between ARs 1-2and 2-3and an additional shallower patch between AR3and 4.Overall,the peptide bound in a clearly nega-tive region on the I k B a surface (Figure 2J),with higher afﬁnity than the modiﬁed peptide that was acetylated in the ﬁrst and(F)ClustalW alignment of the conserved KXXXK/R motifs in the N terminus of histones H2A and H4.Conserved K and R residues are in green.Red triangle indicates the last AA included in GST-H2A 2-35.(G)Histone peptide array hybridized with nuclear HEK293T extracts expressing HA-I k B a .One informative area of the blot image and the relative binding of selected peptides are shown.(H)Coprecipitation of cytoplasmic and nuclear HA-I k B a expressed in HEK293T cells with the indicated histone H2A and H4peptides.(I)HA-I k B a was precipitated using the indicated histone H2A peptide,the K/A mutant,or scrambled peptide.In (G),(H),and (I),cell lysates were denatured-renatured previous to the precipitation to disrupt preformed complexes.(J)Model for binding of the histone H4peptide (unmodiﬁed or modiﬁed)to consecutive ankyrin repeats of I k B a (3KXXXK).See also Figure S2.Cancer CellI k B a Is a Modulator of Polycomb FunctionCancer Cell 24,151–166,August 12,2013ª2013Elsevier Inc.155Cancer CellI k B a Is a Modulator of Polycomb FunctionFigure3.Analysis and Identiﬁcation of I k B a Target Genes(A)Developmentally related genes selected from I k B a targets identiﬁed in ChIP-seq analysis.Fold change over the random background is indicated.(B)Functional enrichment of target genes with p value cutoff%10À5based on gene ontology(GO)as extracted from Ensembl database using GiTools.Enriched categories are represented in heatmap with the indicated color-coded p value scale.(legend continued on next page) 156Cancer Cell24,151–166,August12,2013ª2013Elsevier Inc.second K residues (K12and K16).We experimentally validated that ARs of I k B a participate in histone binding because the I k B aD 55-106mutant,lacking part of AR1,failed to bind GST-H2A (Figure S2C).Similarly,this association was prevented by 1%deoxycholate (Figure S2D),as described for interactions involving the ARs of I k B a (Baeuerle and Baltimore,1988;Savi-nova et al.,2009).I k B a Is Speciﬁcally Recruited to the Regulatory Regions of Developmental GenesTo identify putative PS-I k B a target genes,we performed chromatin immunoprecipitation sequencing (ChIP-seq)using chromatin extracts from primary human keratinocytes and anti-I k B a antibody.We identiﬁed 2,778enriched peaks,correspond-ing to 2,433Ensembl genes that were signiﬁcantly enriched with p values %10À5.Gene ontology analysis showed that a signiﬁ-cant proportion of genes participate in biological processes associated with embryonic development and cell differentiation.I k B a targets included genes of the HOX and IRX families,ASCL4,CDX2,NEUROD4,OLIG3,and NEURL ,among others (Figures 3A and 3B).Annotation of the peak genomic positions to the closest gene demonstrated that many peaks were positioned immediately after the transcription start site (TSS),with a sharp decrease near the transcription termination site (TTS)(Figure 3C),whereas others were located far from promoter regions.Some of the latter overlapped with regions enriched in H3K4me1,a his-tone mark associated with enhancer regions (data not shown).Randomly selected I k B a targets were conﬁrmed by conventional chromatin immunoprecipitation (ChIP)using primers ﬂanking the regions identiﬁed in the ChIP-seq experiment (Figure 3D).Consistent with its overall effects on I k B a levels,sustained Ca 2+treatment caused the loss of I k B a from all tested gene pro-moters (Figure 3E).Similarly,short treatment with TNF a released chromatin-bound I k B a in keratinocytes,as we previously found in ﬁbroblasts (Aguilera et al.,2004).However,we did not detect a general effect of TNF a on PS-I k B a levels,but we consistently found a partial redistribution of PS-I k B a to the soluble nuclear fractions (Figure S3A).Next,we investigated whether TNF a and Ca 2+modulated HOX and IRX transcription in keratinocytes.All tested I k B a targets (n =12)were robustly induced by TNF a treatment (up to 12-fold)following different kinetics (Figures 3F)and to a lesser extent (up to 3-fold)by Ca 2+treatment (Fig-ure S3B)or I k B a KD (Figure S3C).Interestingly,1hr of TNF a treatment prevented Ca 2+-induced differentiation of murine keratinocytes (Figure S3D),supporting the notion that PS-I k B a integrates inﬂammatory signals with skin homeostasis (see Dis-cussion ).We also tested whether p65participated in HOX or IRX gene activation by TNF a .By ChIP analysis,we did not ﬁnd anyrecruitment of p65to I k B a target genes after TNF a treatment,in contrast to a canonical NF-k B target gene promoter (Fig-ure S3E).However,we detected low amounts of p65at HOX genes under basal conditions that might contribute to gene repression (Dong et al.,2008),although the function of chromatin-bound p65at regions distant from the TSS of both NF-k B targets and nontargets is unresolved.Binding of p65to HOX and IRX was reduced after TNF a treatment,suggesting that p65was redistributed from noncanonical to canonical NF-k B targets once activated.Silencing of HOX genes in keratinocytes involves PRC2and its core component the H3K27methyltransferase EZH2(Ezhkova et al.,2009;Mejetta et al.,2011).To explore a putative associa-tion between I k B a and PRC2function,we crossed our list of 2,433I k B a targets with available ChIP-seq data from keratino-cytes.Approximately 50%of I k B a targets corresponded to genes enriched for the H3K27me3mark (Figures 3G and S3F),although I k B a targeted only 13%of the H3K27trimethylated genes.Most importantly,genomic sequences occupied by I k B a essentially overlapped with those regions containing high H3K27me3levels (Figure 3G).We also found a statistically signif-icant overlap (p <10À16)between I k B a target genes and PRC tar-gets in ES cells (Birney et al.,2007;Ku et al.,2008)(Figure S3G).I k B a Interacts with and Regulates Association of PRC2to Target Genes in Response to TNF aIn the mass spectrometry analysis of proteins that associate with GST-I k B a ,we identiﬁed a few peptides corresponding to chro-matin modiﬁers,such as EZH2and SUZ12,and SIN3A (Figure S4A).Speciﬁcity of I k B a interactions with PRC2elements,but also I k B a association to the PRC1protein BMI1,was conﬁrmed by PD assays (Figure S4B).SUZ12was able to interact with nuclear I k B a ,whereas p65speciﬁcally associated with cyto-plasmic I k B a in the IP experiments (Figure 4A).Importantly,exogenous wild-type I k B a ,but not an I k B a mutant that failed to bind histones,facilitated the association of SUZ12to GST-H4(Figure 4B).Moreover,ChIP experiments demonstrated that TNF a treatment induced the dissociation of SUZ12from I k B a target regions,but not non-I k B a targets (Figure 4C).Sequential ChIP experiments demonstrated that I k B a and SUZ12simultaneously bound to I k B a target genes (Figure 4D).To test the functional relevance of I k B a in PRC-mediated repression,we used WT murine embryonic ﬁbroblast (MEFs),which expressed detectable levels of PS-I k B a (Figure 4E)and I k B a KO MEFs.By ChIP-on-chip experiments using three different I k B a antibodies,we conﬁrmed that several Hox genes were also targets of I k B a in MEFs (Table S1).By ChIP,we found that SUZ12and EZH2bound I k B a targets efﬁciently in WT MEFs(C)Graphs show the relative distance to the nearest ChIPed region,3kb upstream and downstream of the RefSeq gene’s TSS and TTS.(D)Validation of the identiﬁed DNA regions (À222to À200for HOXA10,À9,380to À9,360for HOXB2,+6,166to +6,186for HOXB5,+4,451to +4,471for HOXB3,and À18,820to À18,800for IRX3)by conventional ChIP.Ampliﬁcation of 2kb distant regions was used as negative controls.(E)ChIP analysis of I k B a after 20min of TNF a or 48hr Ca 2+treatments.In (D)and (E),graphs represent mean enrichment relative to nonspeciﬁc immunoglobulin G (IgG)(n =2).(F)Expression levels of I k B a target genes following TNF a treatment analyzed by qRT-PCR.Gene represented is in black,whereas genes following the same kinetics are indicated in red.(G)ChIP-seq proﬁles of endogenous I k B a occupancy in three enriched loci (HOXA ,HOXB ,and IRX5)and one negative locus (JARID1B/KDM5B )compared to H3K27me3(from the UW ENCODE Project)in keratinocytes.(D–F)Bars represent mean,and error bars indicate SD.See also Figure S3.Cancer CellI k B a Is a Modulator of Polycomb FunctionCancer Cell 24,151–166,August 12,2013ª2013Elsevier Inc.157Figure 4.I k B a Interacts with and Regulates Association of PRC2in Response to TNF a(A)IB analysis of I k B a precipitates from nuclear and cytoplasmic primary murine keratinocyte extracts.Five percent of the input and 25%of the IP was loaded in all cases except for detection of I k B a input that represents 0.5%.(B)PD using GST-H4and cell lysates from HEK293T expressing different combinations of HA-I k B a and SUZ12.(C)Relative recruitment assessed by ChIP of SUZ12to different genes 40min after TNF a in primary murine keratinocytes.(legend continued on next page)Cancer CellI k B a Is a Modulator of Polycomb Function158Cancer Cell 24,151–166,August 12,2013ª2013Elsevier Inc.but only weakly in I k B a KO MEFs (Figure 4F,time 0).In WT cells,TNF a treatment induced a signiﬁcant but temporary release of SUZ12and EZH2from these loci,which peaked after 30–60min of treatment (Figure 4F).The binding of PRC2proteins at Hox genes inversely correlated with the expression of these Hox genes (Figure 4G).In contrast,I k B a KO cells failed to activate Hox transcription in response to TNF a (Figure 4G),which is consistent with a defective release of PRC2proteins (Figure 4F).Unexpectedly,we did not detect changes in H3K27me3levels in these Hox genes upon TNF a treatment at any of the time points studied (20min,2hr,and 7hr)(data not shown),likely reﬂecting the high stability of this histone modiﬁca-tion (De Santa et al.,2007).Supporting the possibility that activa-tion of I k B a targets is independent of the enzymatic activity of EZH2,a 24hr treatment with the EZH2inhibitor DZNep does not affect Hoxb8or Irx3messenger RNA (mRNA)levels in kera-tinocytes (data not shown).Together,these results suggest that transcriptional repression-activation of these genes does not strictly depend on EZH2enzymatic activity but rather PRC2release might modulate the dissociation of PRC1or HDACs (van der Vlag and Otte,1999)that associate with more dynamic chromatin modiﬁcations.In agreement with this possibility,his-tone H3is rapidly acetylated following TNF a treatment at different Hox gene promoters (Figure S4C).To further study the involvement of NF-k B in the regulation of I k B a targets by TNF a ,we attempted to use different mutant MEFs,including the p65,Ikk a ,Ikk b ,and the triple p65;p50;c-Rel KO.We found that TNF a induced Hox and Irx expression in both p65and Ikk b KO cells (Figure S4D)suggesting that it was NF-k B independent.However,speciﬁc mutants contained vari-able levels of I k B a and PS-I k B a (Figures S4E and S4F),which make a more accurate quantitative analysis unproductive.Inter-estingly,phosphorylation of nuclear I k B a was not reduced in the Ikk a or Ikk b KO cells (Figures S4F),indicating that other kinases are involved in generating PS-I k B a .Only triple KO cells,which essentially lacked I k B a (Figure S4G)and the Ikk a -deﬁcient MEFs,showed a strong defect on Hox and Irx transcription (Figures S4D and S4H).To better understand the contribution of NF-k B to Hox regulation,we next performed luciferase re-porter assays measuring the ability of different I k B a mutant pro-teins to repress a Hoxb8-promoter construct compared with a reporter containing three consensus sites for NF-k B (3x k B).We found that WT I k B a and the nuclear I k B a NES mutant (Huang et al.,2000)signiﬁcantly repressed both promoters.However,mutations that affect chromatin-association (Figure 1F)pre-vented Hoxb8repression but still inhibited the expression of the 3x k B reporter (Figure 4H).Consistently,Hoxb8mRNA levels were signiﬁcantly reduced in the skin of mice expressing I k B a NES(Wuerzberger-Davis et al.,2011)(Figure 4I).Moreover,these animals showed an expansion of the K14-positive basal layer of keratinocytes containing nuclear I k B a (Figure 4J),associated with increased proliferation measured by ki67staining and impaired differentiation as indicated by the reduced thickness of the suprabasal K10-expressing layer (Figure 4K).Genetic Interaction between I k B a /cactus and polycomb in DrosophilaPolycomb group (PcG)I k B and NF-k B proteins are conserved from ﬂies to humans.In addition,Drosophila contains one Hox cluster,compared with four clusters in vertebrates,which facili-tates studying genetic interactions.We ﬁrst conﬁrmed that the single Drosophila I k B homolog,cactus (cact)(Geisler et al.,1992),maintained the capacity to associate with histones (Figure 5A).By IF,we detected colocalization of cactus and Polycomb (Pc),a PRC1protein that is essential for the repressive PRC2function,in speciﬁc bands of polytene chromosomes (Figure 5B).Most of the cactus staining overlapped with Pc,but only a few Pc-positive bands contained cactus.Based on our mammalian data,our ﬁrst attempt was to generate single PRC2mutants and combine them with cactus -deﬁcient mutants.All mutants were tested in heterozygosis because homozygous mutations in cactus or PcG genes are lethal.We found that heterozygous mutations in PRC2genes (e.g.,the null mutations of E (z ))as well as the composed E (z )and cact exhibit no overt homeotic phenotypes.In contrast,hetero-zygous Pc mutants exhibit a variety of characteristic homeotic transformations,including the partial transformation of the sec-ond and third (mesothoracic and metathoracic)legs toward ﬁrst (also known as prothoracic)legs that in males are characterized by the presence of sex combs.Modiﬁcations of this phenotype (also called ‘‘extra sex comb’’)have been extensively used as a functional assay to validate new PcG proteins in vivo.Thus,we tested the effect of reducing cact on Pc -induced homeotic transformation using 12recessive mutations of cact and two independently generated Pc alleles.All cact mutant alleles,but more prominently cact 1,enhanced the ‘‘extra sex comb’’pheno-type of Pc mutations (Pc 3and Pc XT109)(Figure 5C;Table S2).Because cells lacking cact exhibit massive nuclear localization of the transcription factor dorsal (dl,Drosophila NF-k B/Rel/p65ortholog)during postembryonic stages (Lemaitre et al.,1995),we tested whether enhancement of homeotic defect of Pc by cact mutations is due to increased dorsal activity.Because sta-bility of cact is under NF-k B/Dorsal control (Kubota and Gay,1995),we anticipated that for phenotypes due to increased dorsal,dl mutations would counteract cact mutations,whereas for phenotypes independent of dorsal,reducing dl should yield(D)Sequential ChIP using the indicated combinations of antibodies.An analysis of two different Hox regulatory regions is shown.(E)IB analysis of WT ﬁbroblasts showing the presence of cytoplasmic and nuclear I k B a .(F)Relative chromatin binding of PRC2and I k B a in WT and I k B a KO MEFs treated with TNF a .ChIP values were normalized by IgG precipitation.(G)Relative levels of the indicated genes in WT and I k B a KO MEFs.(H)Luciferase assays to determine the effect of different I k B a constructs on the activity of HoxB8compared to the 3x k B reporter.Lower panels show expression levels of different constructs.(I)Expression levels of HoxB8in the skin of WT and I k B a NES/NES mice by qRT-PCR (n =2).(J and K)Analysis of skin sections from 7-to 8-week-old WT and I k B a NES/NES mice by IF.K14labels the basal layer keratinocytes (J).Immunostaining of ki67,the suprabasal marker K10,and ﬁlaggrin (K).Throughout the ﬁgure,bars represent mean,and error bars indicate SD.See also Figure S4and Table S1.Cancer CellI k B a Is a Modulator of Polycomb FunctionCancer Cell 24,151–166,August 12,2013ª2013Elsevier Inc.159。

AVVIO RAPIDO Router mobili 5G 安装指南说明书

Per caricare la batteria, collegare il cavo USB al router mobile, quindi collegarlo a una presa a muro utilizzando l'adattatore di alimentazione CA o una porta USB del computer.Assicurarsi che l'orientamento della scheda nano SIM coincida con l'orientamento indicato sull'etichetta del dispositivo e inserirla delicatamente, quindi posizionare la batteria e il coperchio posteriore.NOTA: utilizzare solo le dita per inserire o rimuovere la scheda nano SIM. L'utilizzo di altri oggetti potrebbe danneggiare il dispositivo.1. COM'È FATTO IL DISPOSITIVO2. INSTALLAZIONE DELLA SIM E DELLA BATTERIAIl router mobile viene fornito con i seguenti componenti:• Router mobile Nighthawk® M6 o M6 Pro 5G*• Coperchio della batteria • Batteria• Cavo USB Tipo C• Alimentatore (varia in base all’area geografica)• Adattatori con presa Tipo C (per la maggior parte dei Paesi europei)•Adattatori con presa Tipo G (per il Regno Unito)*Illustrazioni del modello Nighthawk M6 per scopi illustrativi.antenna esterna (TS-9)antenna esterna (TS-9)USB Tipo CEthernetCONFORMITÀ NORMATIVA E NOTE LEGALIPer informazioni sulla conformità alle normative, compresala Dichiarazione di conformità UE, visitare il sito Web https:///it/about/regulatory/.Prima di collegare l'alimentazione, consultare il documento relativo alla conformità normativa.Può essere applicato solo ai dispositivi da 6 GHz: utilizzare il dispositivo solo in un ambiente al chiuso. L'utilizzo di dispositivi a 6 GHz è vietato su piattaforme petrolifere, automobili, treni, barche e aerei, tuttavia il suo utilizzo è consentito su aerei di grandi dimensioni quando volano sopra i 3000 metri di altezza. L'utilizzo di trasmettitori nella banda 5.925‑7.125 GHz è vietato per il controllo o le comunicazioni con sistemi aerei senza equipaggio.SUPPORTO E COMMUNITYDalla pagina del portale di amministrazione Web, fare clic sull'icona con i tre puntini nell'angolo in alto a destra per accedere ai file della guida e del supporto.Per ulteriori informazioni, visitare il sito netgear.it/support per accedere al manuale dell'utente completo e per scaricare gli aggiornamenti del firmware.È possibile trovare utili consigli anche nella Community NETGEAR, alla pagina /it.GESTIONE DELLE IMPOSTAZIONI TRAMITE L'APP NETGEAR MOBILEUtilizzare l'app NETGEAR Mobile per modificare il nome della rete Wi-Fi e la password. È possibile utilizzarla anche per riprodurre e condividere contenutimultimediali e accedere alle funzioni avanzate del router mobile.1. Accertarsi che il dispositivo mobile sia connesso a Internet.2. Eseguire la scansione del codice QR per scaricare l'appNETGEAR Mobile.Connessione con il nome e la password della rete Wi-Fi 1. Aprire il programma di gestione della rete Wi‑Fi deldispositivo.2. Individuare il nome della rete Wi‑Fi del router mobile(NTGR_XXXX) e stabilire una connessione.3. Only Connessione tramite EthernetPer prolungare la durata della batteria, l'opzione Ethernet è disattivata per impostazione predefinita. Per attivarla, toccare Power Manager (Risparmio energia) e passare a Performance Mode (Modalità performance).4. CONNESSIONE A INTERNETÈ possibile connettersi a Internet utilizzando il codice QR del router mobile da uno smartphone oppure selezionando manualmente il nome della rete Wi‑Fi del router e immettendo la password.Connessione tramite codice QR da uno smartphone 1. Toccare l'icona del codice QR sulla schermata inizialedello schermo LCD del router mobile.NOTA: quando è inattivo, lo schermo touch si oscura per risparmiare energia. Premere brevemente e rilasciare il pulsante di alimentazione per riattivare lo schermo.3. CONFIGURAZIONE DEL ROUTER MOBILETenere premuto il pulsante di accensione per due secondi, quindi seguire le istruzioni visualizzate sullo schermo per impostare un nome per la rete Wi‑Fi e una password univoci.La personalizzazione delle impostazioni Wi‑Fi consente di proteggere la rete Wi‑Fi del router mobile.Impostazioni APNIl router mobile legge i dati dalla scheda SIM e determina automaticamente le impostazioni APN (Access Point Name) corrette con i piani dati della maggior parte degli operatori. Tuttavia, se si utilizza un router mobile sbloccato con un operatore o un piano meno comune, potrebbe essere necessario immettere manualmente le impostazioni APN.Se viene visualizzata la schermata APN Setup Required (Configurazione APN richiesta), i dati APN dell’operatore non sono presenti nel nostro database ed è necessario inserirli manualmente. Immettere i valori fornitidall’operatore nei campi corrispondenti, quindi toccare Save (Salva) per completare la configurazione.NOTA: l’operatore determina le proprie informazioni APN e deve fornire le informazioni per il proprio piano dati. Si consiglia di contattare il proprio operatore per le impostazioni APN corrette e di utilizzare solo l’APN suggerito per il piano specifico.Schermata inizialeAl termine della configurazione, il router visualizza la schermata iniziale:Wi‑FiPotenza Carica Rete Codice QR connessione rapida Wi‑FiNome e Wi‑FiIcona del codice QR。

T.W. ANDERSON (1971). The Statistical Analysis of Time Series. Series in Probability and Ma

425 BibliographyH.A KAIKE(1974).Markovian representation of stochastic processes and its application to the analysis of autoregressive moving average processes.Annals Institute Statistical Mathematics,vol.26,pp.363-387. B.D.O.A NDERSON and J.B.M OORE(1979).Optimal rmation and System Sciences Series, Prentice Hall,Englewood Cliffs,NJ.T.W.A NDERSON(1971).The Statistical Analysis of Time Series.Series in Probability and Mathematical Statistics,Wiley,New York.R.A NDRE-O BRECHT(1988).A new statistical approach for the automatic segmentation of continuous speech signals.IEEE Trans.Acoustics,Speech,Signal Processing,vol.ASSP-36,no1,pp.29-40.R.A NDRE-O BRECHT(1990).Reconnaissance automatique de parole`a partir de segments acoustiques et de mod`e les de Markov cach´e s.Proc.Journ´e es Etude de la Parole,Montr´e al,May1990(in French).R.A NDRE-O BRECHT and H.Y.S U(1988).Three acoustic labellings for phoneme based continuous speech recognition.Proc.Speech’88,Edinburgh,UK,pp.943-950.U.A PPEL and A.VON B RANDT(1983).Adaptive sequential segmentation of piecewise stationary time rmation Sciences,vol.29,no1,pp.27-56.L.A.A ROIAN and H.L EVENE(1950).The effectiveness of quality control procedures.Jal American Statis-tical Association,vol.45,pp.520-529.K.J.A STR¨OM and B.W ITTENMARK(1984).Computer Controlled Systems:Theory and rma-tion and System Sciences Series,Prentice Hall,Englewood Cliffs,NJ.M.B AGSHAW and R.A.J OHNSON(1975a).The effect of serial correlation on the performance of CUSUM tests-Part II.Technometrics,vol.17,no1,pp.73-80.M.B AGSHAW and R.A.J OHNSON(1975b).The inﬂuence of reference values and estimated variance on the ARL of CUSUM tests.Jal Royal Statistical Society,vol.37(B),no3,pp.413-420.M.B AGSHAW and R.A.J OHNSON(1977).Sequential procedures for detecting parameter changes in a time-series model.Jal American Statistical Association,vol.72,no359,pp.593-597.R.K.B ANSAL and P.P APANTONI-K AZAKOS(1986).An algorithm for detecting a change in a stochastic process.IEEE rmation Theory,vol.IT-32,no2,pp.227-235.G.A.B ARNARD(1959).Control charts and stochastic processes.Jal Royal Statistical Society,vol.B.21, pp.239-271.A.E.B ASHARINOV andB.S.F LEISHMAN(1962).Methods of the statistical sequential analysis and their radiotechnical applications.Sovetskoe Radio,Moscow(in Russian).M.B ASSEVILLE(1978).D´e viations par rapport au maximum:formules d’arrˆe t et martingales associ´e es. Compte-rendus du S´e minaire de Probabilit´e s,Universit´e de Rennes I.M.B ASSEVILLE(1981).Edge detection using sequential methods for change in level-Part II:Sequential detection of change in mean.IEEE Trans.Acoustics,Speech,Signal Processing,vol.ASSP-29,no1,pp.32-50.426B IBLIOGRAPHY M.B ASSEVILLE(1982).A survey of statistical failure detection techniques.In Contribution`a la D´e tectionS´e quentielle de Ruptures de Mod`e les Statistiques,Th`e se d’Etat,Universit´e de Rennes I,France(in English). M.B ASSEVILLE(1986).The two-models approach for the on-line detection of changes in AR processes. In Detection of Abrupt Changes in Signals and Dynamical Systems(M.Basseville,A.Benveniste,eds.). Lecture Notes in Control and Information Sciences,LNCIS77,Springer,New York,pp.169-215.M.B ASSEVILLE(1988).Detecting changes in signals and systems-A survey.Automatica,vol.24,pp.309-326.M.B ASSEVILLE(1989).Distance measures for signal processing and pattern recognition.Signal Process-ing,vol.18,pp.349-369.M.B ASSEVILLE and A.B ENVENISTE(1983a).Design and comparative study of some sequential jump detection algorithms for digital signals.IEEE Trans.Acoustics,Speech,Signal Processing,vol.ASSP-31, no3,pp.521-535.M.B ASSEVILLE and A.B ENVENISTE(1983b).Sequential detection of abrupt changes in spectral charac-teristics of digital signals.IEEE rmation Theory,vol.IT-29,no5,pp.709-724.M.B ASSEVILLE and A.B ENVENISTE,eds.(1986).Detection of Abrupt Changes in Signals and Dynamical Systems.Lecture Notes in Control and Information Sciences,LNCIS77,Springer,New York.M.B ASSEVILLE and I.N IKIFOROV(1991).A uniﬁed framework for statistical change detection.Proc.30th IEEE Conference on Decision and Control,Brighton,UK.M.B ASSEVILLE,B.E SPIAU and J.G ASNIER(1981).Edge detection using sequential methods for change in level-Part I:A sequential edge detection algorithm.IEEE Trans.Acoustics,Speech,Signal Processing, vol.ASSP-29,no1,pp.24-31.M.B ASSEVILLE, A.B ENVENISTE and G.M OUSTAKIDES(1986).Detection and diagnosis of abrupt changes in modal characteristics of nonstationary digital signals.IEEE rmation Theory,vol.IT-32,no3,pp.412-417.M.B ASSEVILLE,A.B ENVENISTE,G.M OUSTAKIDES and A.R OUG´E E(1987a).Detection and diagnosis of changes in the eigenstructure of nonstationary multivariable systems.Automatica,vol.23,no3,pp.479-489. M.B ASSEVILLE,A.B ENVENISTE,G.M OUSTAKIDES and A.R OUG´E E(1987b).Optimal sensor location for detecting changes in dynamical behavior.IEEE Trans.Automatic Control,vol.AC-32,no12,pp.1067-1075.M.B ASSEVILLE,A.B ENVENISTE,B.G ACH-D EVAUCHELLE,M.G OURSAT,D.B ONNECASE,P.D OREY, M.P REVOSTO and M.O LAGNON(1993).Damage monitoring in vibration mechanics:issues in diagnos-tics and predictive maintenance.Mechanical Systems and Signal Processing,vol.7,no5,pp.401-423.R.V.B EARD(1971).Failure Accommodation in Linear Systems through Self-reorganization.Ph.D.Thesis, Dept.Aeronautics and Astronautics,MIT,Cambridge,MA.A.B ENVENISTE and J.J.F UCHS(1985).Single sample modal identiﬁcation of a nonstationary stochastic process.IEEE Trans.Automatic Control,vol.AC-30,no1,pp.66-74.A.B ENVENISTE,M.B ASSEVILLE and G.M OUSTAKIDES(1987).The asymptotic local approach to change detection and model validation.IEEE Trans.Automatic Control,vol.AC-32,no7,pp.583-592.A.B ENVENISTE,M.M ETIVIER and P.P RIOURET(1990).Adaptive Algorithms and Stochastic Approxima-tions.Series on Applications of Mathematics,(A.V.Balakrishnan,I.Karatzas,M.Yor,eds.).Springer,New York.A.B ENVENISTE,M.B ASSEVILLE,L.E L G HAOUI,R.N IKOUKHAH and A.S.W ILLSKY(1992).An optimum robust approach to statistical failure detection and identiﬁcation.IFAC World Conference,Sydney, July1993.B IBLIOGRAPHY427 R.H.B ERK(1973).Some asymptotic aspects of sequential analysis.Annals Statistics,vol.1,no6,pp.1126-1138.R.H.B ERK(1975).Locally most powerful sequential test.Annals Statistics,vol.3,no2,pp.373-381.P.B ILLINGSLEY(1968).Convergence of Probability Measures.Wiley,New York.A.F.B ISSELL(1969).Cusum techniques for quality control.Applied Statistics,vol.18,pp.1-30.M.E.B IVAIKOV(1991).Control of the sample size for recursive estimation of parameters subject to abrupt changes.Automation and Remote Control,no9,pp.96-103.R.E.B LAHUT(1987).Principles and Practice of Information Theory.Addison-Wesley,Reading,MA.I.F.B LAKE and W.C.L INDSEY(1973).Level-crossing problems for random processes.IEEE r-mation Theory,vol.IT-19,no3,pp.295-315.G.B ODENSTEIN and H.M.P RAETORIUS(1977).Feature extraction from the encephalogram by adaptive segmentation.Proc.IEEE,vol.65,pp.642-652.T.B OHLIN(1977).Analysis of EEG signals with changing spectra using a short word Kalman estimator. Mathematical Biosciences,vol.35,pp.221-259.W.B¨OHM and P.H ACKL(1990).Improved bounds for the average run length of control charts based on ﬁnite weighted sums.Annals Statistics,vol.18,no4,pp.1895-1899.T.B OJDECKI and J.H OSZA(1984).On a generalized disorder problem.Stochastic Processes and their Applications,vol.18,pp.349-359.L.I.B ORODKIN and V.V.M OTTL’(1976).Algorithm forﬁnding the jump times of random process equation parameters.Automation and Remote Control,vol.37,no6,Part1,pp.23-32.A.A.B OROVKOV(1984).Theory of Mathematical Statistics-Estimation and Hypotheses Testing,Naouka, Moscow(in Russian).Translated in French under the title Statistique Math´e matique-Estimation et Tests d’Hypoth`e ses,Mir,Paris,1987.G.E.P.B OX and G.M.J ENKINS(1970).Time Series Analysis,Forecasting and Control.Series in Time Series Analysis,Holden-Day,San Francisco.A.VON B RANDT(1983).Detecting and estimating parameters jumps using ladder algorithms and likelihood ratio test.Proc.ICASSP,Boston,MA,pp.1017-1020.A.VON B RANDT(1984).Modellierung von Signalen mit Sprunghaft Ver¨a nderlichem Leistungsspektrum durch Adaptive Segmentierung.Doctor-Engineer Dissertation,M¨u nchen,RFA(in German).S.B RAUN,ed.(1986).Mechanical Signature Analysis-Theory and Applications.Academic Press,London. L.B REIMAN(1968).Probability.Series in Statistics,Addison-Wesley,Reading,MA.G.S.B RITOV and L.A.M IRONOVSKI(1972).Diagnostics of linear systems of automatic regulation.Tekh. Kibernetics,vol.1,pp.76-83.B.E.B RODSKIY and B.S.D ARKHOVSKIY(1992).Nonparametric Methods in Change-point Problems. Kluwer Academic,Boston.L.D.B ROEMELING(1982).Jal Econometrics,vol.19,Special issue on structural change in Econometrics. L.D.B ROEMELING and H.T SURUMI(1987).Econometrics and Structural Change.Dekker,New York. D.B ROOK and D.A.E VANS(1972).An approach to the probability distribution of Cusum run length. Biometrika,vol.59,pp.539-550.J.B RUNET,D.J AUME,M.L ABARR`E RE,A.R AULT and M.V ERG´E(1990).D´e tection et Diagnostic de Pannes.Trait´e des Nouvelles Technologies,S´e rie Diagnostic et Maintenance,Herm`e s,Paris(in French).428B IBLIOGRAPHY S.P.B RUZZONE and M.K AVEH(1984).Information tradeoffs in using the sample autocorrelation function in ARMA parameter estimation.IEEE Trans.Acoustics,Speech,Signal Processing,vol.ASSP-32,no4, pp.701-715.A.K.C AGLAYAN(1980).Necessary and sufﬁcient conditions for detectability of jumps in linear systems. IEEE Trans.Automatic Control,vol.AC-25,no4,pp.833-834.A.K.C AGLAYAN and R.E.L ANCRAFT(1983).Reinitialization issues in fault tolerant systems.Proc.Amer-ican Control Conf.,pp.952-955.A.K.C AGLAYAN,S.M.A LLEN and K.W EHMULLER(1988).Evaluation of a second generation reconﬁgu-ration strategy for aircraftﬂight control systems subjected to actuator failure/surface damage.Proc.National Aerospace and Electronic Conference,Dayton,OH.P.E.C AINES(1988).Linear Stochastic Systems.Series in Probability and Mathematical Statistics,Wiley, New York.M.J.C HEN and J.P.N ORTON(1987).Estimation techniques for tracking rapid parameter changes.Intern. Jal Control,vol.45,no4,pp.1387-1398.W.K.C HIU(1974).The economic design of cusum charts for controlling normal mean.Applied Statistics, vol.23,no3,pp.420-433.E.Y.C HOW(1980).A Failure Detection System Design Methodology.Ph.D.Thesis,M.I.T.,L.I.D.S.,Cam-bridge,MA.E.Y.C HOW and A.S.W ILLSKY(1984).Analytical redundancy and the design of robust failure detection systems.IEEE Trans.Automatic Control,vol.AC-29,no3,pp.689-691.Y.S.C HOW,H.R OBBINS and D.S IEGMUND(1971).Great Expectations:The Theory of Optimal Stop-ping.Houghton-Mifﬂin,Boston.R.N.C LARK,D.C.F OSTH and V.M.W ALTON(1975).Detection of instrument malfunctions in control systems.IEEE Trans.Aerospace Electronic Systems,vol.AES-11,pp.465-473.A.C OHEN(1987).Biomedical Signal Processing-vol.1:Time and Frequency Domain Analysis;vol.2: Compression and Automatic Recognition.CRC Press,Boca Raton,FL.J.C ORGE and F.P UECH(1986).Analyse du rythme cardiaque foetal par des m´e thodes de d´e tection de ruptures.Proc.7th INRIA Int.Conf.Analysis and optimization of Systems.Antibes,FR(in French).D.R.C OX and D.V.H INKLEY(1986).Theoretical Statistics.Chapman and Hall,New York.D.R.C OX and H.D.M ILLER(1965).The Theory of Stochastic Processes.Wiley,New York.S.V.C ROWDER(1987).A simple method for studying run-length distributions of exponentially weighted moving average charts.Technometrics,vol.29,no4,pp.401-407.H.C S¨ORG¨O and L.H ORV´ATH(1988).Nonparametric methods for change point problems.In Handbook of Statistics(P.R.Krishnaiah,C.R.Rao,eds.),vol.7,Elsevier,New York,pp.403-425.R.B.D AVIES(1973).Asymptotic inference in stationary gaussian time series.Advances Applied Probability, vol.5,no3,pp.469-497.J.C.D ECKERT,M.N.D ESAI,J.J.D EYST and A.S.W ILLSKY(1977).F-8DFBW sensor failure identiﬁcation using analytical redundancy.IEEE Trans.Automatic Control,vol.AC-22,no5,pp.795-803.M.H.D E G ROOT(1970).Optimal Statistical Decisions.Series in Probability and Statistics,McGraw-Hill, New York.J.D ESHAYES and D.P ICARD(1979).Tests de ruptures dans un mod`e pte-Rendus de l’Acad´e mie des Sciences,vol.288,Ser.A,pp.563-566(in French).B IBLIOGRAPHY429 J.D ESHAYES and D.P ICARD(1983).Ruptures de Mod`e les en Statistique.Th`e ses d’Etat,Universit´e deParis-Sud,Orsay,France(in French).J.D ESHAYES and D.P ICARD(1986).Off-line statistical analysis of change-point models using non para-metric and likelihood methods.In Detection of Abrupt Changes in Signals and Dynamical Systems(M. Basseville,A.Benveniste,eds.).Lecture Notes in Control and Information Sciences,LNCIS77,Springer, New York,pp.103-168.B.D EVAUCHELLE-G ACH(1991).Diagnostic M´e canique des Fatigues sur les Structures Soumises`a des Vibrations en Ambiance de Travail.Th`e se de l’Universit´e Paris IX Dauphine(in French).B.D EVAUCHELLE-G ACH,M.B ASSEVILLE and A.B ENVENISTE(1991).Diagnosing mechanical changes in vibrating systems.Proc.SAFEPROCESS’91,Baden-Baden,FRG,pp.85-89.R.D I F RANCESCO(1990).Real-time speech segmentation using pitch and convexity jump models:applica-tion to variable rate speech coding.IEEE Trans.Acoustics,Speech,Signal Processing,vol.ASSP-38,no5, pp.741-748.X.D ING and P.M.F RANK(1990).Fault detection via factorization approach.Systems and Control Letters, vol.14,pp.431-436.J.L.D OOB(1953).Stochastic Processes.Wiley,New York.V.D RAGALIN(1988).Asymptotic solutions in detecting a change in distribution under an unknown param-eter.Statistical Problems of Control,Issue83,Vilnius,pp.45-52.B.D UBUISSON(1990).Diagnostic et Reconnaissance des Formes.Trait´e des Nouvelles Technologies,S´e rie Diagnostic et Maintenance,Herm`e s,Paris(in French).A.J.D UNCAN(1986).Quality Control and Industrial Statistics,5th edition.Richard D.Irwin,Inc.,Home-wood,IL.J.D URBIN(1971).Boundary-crossing probabilities for the Brownian motion and Poisson processes and techniques for computing the power of the Kolmogorov-Smirnov test.Jal Applied Probability,vol.8,pp.431-453.J.D URBIN(1985).Theﬁrst passage density of the crossing of a continuous Gaussian process to a general boundary.Jal Applied Probability,vol.22,no1,pp.99-122.A.E MAMI-N AEINI,M.M.A KHTER and S.M.R OCK(1988).Effect of model uncertainty on failure detec-tion:the threshold selector.IEEE Trans.Automatic Control,vol.AC-33,no12,pp.1106-1115.J.D.E SARY,F.P ROSCHAN and D.W.W ALKUP(1967).Association of random variables with applications. Annals Mathematical Statistics,vol.38,pp.1466-1474.W.D.E WAN and K.W.K EMP(1960).Sampling inspection of continuous processes with no autocorrelation between successive results.Biometrika,vol.47,pp.263-280.G.F AVIER and A.S MOLDERS(1984).Adaptive smoother-predictors for tracking maneuvering targets.Proc. 23rd Conf.Decision and Control,Las Vegas,NV,pp.831-836.W.F ELLER(1966).An Introduction to Probability Theory and Its Applications,vol.2.Series in Probability and Mathematical Statistics,Wiley,New York.R.A.F ISHER(1925).Theory of statistical estimation.Proc.Cambridge Philosophical Society,vol.22, pp.700-725.M.F ISHMAN(1988).Optimization of the algorithm for the detection of a disorder,based on the statistic of exponential smoothing.In Statistical Problems of Control,Issue83,Vilnius,pp.146-151.R.F LETCHER(1980).Practical Methods of Optimization,2volumes.Wiley,New York.P.M.F RANK(1990).Fault diagnosis in dynamic systems using analytical and knowledge based redundancy -A survey and new results.Automatica,vol.26,pp.459-474.430B IBLIOGRAPHY P.M.F RANK(1991).Enhancement of robustness in observer-based fault detection.Proc.SAFEPRO-CESS’91,Baden-Baden,FRG,pp.275-287.P.M.F RANK and J.W¨UNNENBERG(1989).Robust fault diagnosis using unknown input observer schemes. In Fault Diagnosis in Dynamic Systems-Theory and Application(R.Patton,P.Frank,R.Clark,eds.). International Series in Systems and Control Engineering,Prentice Hall International,London,UK,pp.47-98.K.F UKUNAGA(1990).Introduction to Statistical Pattern Recognition,2d ed.Academic Press,New York. S.I.G ASS(1958).Linear Programming:Methods and Applications.McGraw Hill,New York.W.G E and C.Z.F ANG(1989).Extended robust observation approach for failure isolation.Int.Jal Control, vol.49,no5,pp.1537-1553.W.G ERSCH(1986).Two applications of parametric time series modeling methods.In Mechanical Signature Analysis-Theory and Applications(S.Braun,ed.),chap.10.Academic Press,London.J.J.G ERTLER(1988).Survey of model-based failure detection and isolation in complex plants.IEEE Control Systems Magazine,vol.8,no6,pp.3-11.J.J.G ERTLER(1991).Analytical redundancy methods in fault detection and isolation.Proc.SAFEPRO-CESS’91,Baden-Baden,FRG,pp.9-22.B.K.G HOSH(1970).Sequential Tests of Statistical Hypotheses.Addison-Wesley,Cambridge,MA.I.N.G IBRA(1975).Recent developments in control charts techniques.Jal Quality Technology,vol.7, pp.183-192.J.P.G ILMORE and R.A.M C K ERN(1972).A redundant strapdown inertial reference unit(SIRU).Jal Space-craft,vol.9,pp.39-47.M.A.G IRSHICK and H.R UBIN(1952).A Bayes approach to a quality control model.Annals Mathematical Statistics,vol.23,pp.114-125.A.L.G OEL and S.M.W U(1971).Determination of the ARL and a contour nomogram for CUSUM charts to control normal mean.Technometrics,vol.13,no2,pp.221-230.P.L.G OLDSMITH and H.W HITFIELD(1961).Average run lengths in cumulative chart quality control schemes.Technometrics,vol.3,pp.11-20.G.C.G OODWIN and K.S.S IN(1984).Adaptive Filtering,Prediction and rmation and System Sciences Series,Prentice Hall,Englewood Cliffs,NJ.R.M.G RAY and L.D.D AVISSON(1986).Random Processes:a Mathematical Approach for Engineers. Information and System Sciences Series,Prentice Hall,Englewood Cliffs,NJ.C.G UEGUEN and L.L.S CHARF(1980).Exact maximum likelihood identiﬁcation for ARMA models:a signal processing perspective.Proc.1st EUSIPCO,Lausanne.D.E.G USTAFSON, A.S.W ILLSKY,J.Y.W ANG,M.C.L ANCASTER and J.H.T RIEBWASSER(1978). ECG/VCG rhythm diagnosis using statistical signal analysis.Part I:Identiﬁcation of persistent rhythms. Part II:Identiﬁcation of transient rhythms.IEEE Trans.Biomedical Engineering,vol.BME-25,pp.344-353 and353-361.F.G USTAFSSON(1991).Optimal segmentation of linear regression parameters.Proc.IFAC/IFORS Symp. Identiﬁcation and System Parameter Estimation,Budapest,pp.225-229.T.H¨AGGLUND(1983).New Estimation Techniques for Adaptive Control.Ph.D.Thesis,Lund Institute of Technology,Lund,Sweden.T.H¨AGGLUND(1984).Adaptive control of systems subject to large parameter changes.Proc.IFAC9th World Congress,Budapest.B IBLIOGRAPHY431 P.H ALL and C.C.H EYDE(1980).Martingale Limit Theory and its Application.Probability and Mathemat-ical Statistics,a Series of Monographs and Textbooks,Academic Press,New York.W.J.H ALL,R.A.W IJSMAN and J.K.G HOSH(1965).The relationship between sufﬁciency and invariance with applications in sequential analysis.Ann.Math.Statist.,vol.36,pp.576-614.E.J.H ANNAN and M.D EISTLER(1988).The Statistical Theory of Linear Systems.Series in Probability and Mathematical Statistics,Wiley,New York.J.D.H EALY(1987).A note on multivariate CuSum procedures.Technometrics,vol.29,pp.402-412.D.M.H IMMELBLAU(1970).Process Analysis by Statistical Methods.Wiley,New York.D.M.H IMMELBLAU(1978).Fault Detection and Diagnosis in Chemical and Petrochemical Processes. Chemical Engineering Monographs,vol.8,Elsevier,Amsterdam.W.G.S.H INES(1976a).A simple monitor of a system with sudden parameter changes.IEEE r-mation Theory,vol.IT-22,no2,pp.210-216.W.G.S.H INES(1976b).Improving a simple monitor of a system with sudden parameter changes.IEEE rmation Theory,vol.IT-22,no4,pp.496-499.D.V.H INKLEY(1969).Inference about the intersection in two-phase regression.Biometrika,vol.56,no3, pp.495-504.D.V.H INKLEY(1970).Inference about the change point in a sequence of random variables.Biometrika, vol.57,no1,pp.1-17.D.V.H INKLEY(1971).Inference about the change point from cumulative sum-tests.Biometrika,vol.58, no3,pp.509-523.D.V.H INKLEY(1971).Inference in two-phase regression.Jal American Statistical Association,vol.66, no336,pp.736-743.J.R.H UDDLE(1983).Inertial navigation system error-model considerations in Kalmanﬁltering applica-tions.In Control and Dynamic Systems(C.T.Leondes,ed.),Academic Press,New York,pp.293-339.J.S.H UNTER(1986).The exponentially weighted moving average.Jal Quality Technology,vol.18,pp.203-210.I.A.I BRAGIMOV and R.Z.K HASMINSKII(1981).Statistical Estimation-Asymptotic Theory.Applications of Mathematics Series,vol.16.Springer,New York.R.I SERMANN(1984).Process fault detection based on modeling and estimation methods-A survey.Auto-matica,vol.20,pp.387-404.N.I SHII,A.I WATA and N.S UZUMURA(1979).Segmentation of nonstationary time series.Int.Jal Systems Sciences,vol.10,pp.883-894.J.E.J ACKSON and R.A.B RADLEY(1961).Sequential and tests.Annals Mathematical Statistics, vol.32,pp.1063-1077.B.J AMES,K.L.J AMES and D.S IEGMUND(1988).Conditional boundary crossing probabilities with appli-cations to change-point problems.Annals Probability,vol.16,pp.825-839.M.K.J EERAGE(1990).Reliability analysis of fault-tolerant IMU architectures with redundant inertial sen-sors.IEEE Trans.Aerospace and Electronic Systems,vol.AES-5,no.7,pp.23-27.N.L.J OHNSON(1961).A simple theoretical approach to cumulative sum control charts.Jal American Sta-tistical Association,vol.56,pp.835-840.N.L.J OHNSON and F.C.L EONE(1962).Cumulative sum control charts:mathematical principles applied to their construction and use.Parts I,II,III.Industrial Quality Control,vol.18,pp.15-21;vol.19,pp.29-36; vol.20,pp.22-28.432B IBLIOGRAPHY R.A.J OHNSON and M.B AGSHAW(1974).The effect of serial correlation on the performance of CUSUM tests-Part I.Technometrics,vol.16,no.1,pp.103-112.H.L.J ONES(1973).Failure Detection in Linear Systems.Ph.D.Thesis,Dept.Aeronautics and Astronautics, MIT,Cambridge,MA.R.H.J ONES,D.H.C ROWELL and L.E.K APUNIAI(1970).Change detection model for serially correlated multivariate data.Biometrics,vol.26,no2,pp.269-280.M.J URGUTIS(1984).Comparison of the statistical properties of the estimates of the change times in an autoregressive process.In Statistical Problems of Control,Issue65,Vilnius,pp.234-243(in Russian).T.K AILATH(1980).Linear rmation and System Sciences Series,Prentice Hall,Englewood Cliffs,NJ.L.V.K ANTOROVICH and V.I.K RILOV(1958).Approximate Methods of Higher Analysis.Interscience,New York.S.K ARLIN and H.M.T AYLOR(1975).A First Course in Stochastic Processes,2d ed.Academic Press,New York.S.K ARLIN and H.M.T AYLOR(1981).A Second Course in Stochastic Processes.Academic Press,New York.D.K AZAKOS and P.P APANTONI-K AZAKOS(1980).Spectral distance measures between gaussian pro-cesses.IEEE Trans.Automatic Control,vol.AC-25,no5,pp.950-959.K.W.K EMP(1958).Formula for calculating the operating characteristic and average sample number of some sequential tests.Jal Royal Statistical Society,vol.B-20,no2,pp.379-386.K.W.K EMP(1961).The average run length of the cumulative sum chart when a V-mask is used.Jal Royal Statistical Society,vol.B-23,pp.149-153.K.W.K EMP(1967a).Formal expressions which can be used for the determination of operating character-istics and average sample number of a simple sequential test.Jal Royal Statistical Society,vol.B-29,no2, pp.248-262.K.W.K EMP(1967b).A simple procedure for determining upper and lower limits for the average sample run length of a cumulative sum scheme.Jal Royal Statistical Society,vol.B-29,no2,pp.263-265.D.P.K ENNEDY(1976).Some martingales related to cumulative sum tests and single server queues.Stochas-tic Processes and Appl.,vol.4,pp.261-269.T.H.K ERR(1980).Statistical analysis of two-ellipsoid overlap test for real time failure detection.IEEE Trans.Automatic Control,vol.AC-25,no4,pp.762-772.T.H.K ERR(1982).False alarm and correct detection probabilities over a time interval for restricted classes of failure detection algorithms.IEEE rmation Theory,vol.IT-24,pp.619-631.T.H.K ERR(1987).Decentralizedﬁltering and redundancy management for multisensor navigation.IEEE Trans.Aerospace and Electronic systems,vol.AES-23,pp.83-119.Minor corrections on p.412and p.599 (May and July issues,respectively).R.A.K HAN(1978).Wald’s approximations to the average run length in cusum procedures.Jal Statistical Planning and Inference,vol.2,no1,pp.63-77.R.A.K HAN(1979).Someﬁrst passage problems related to cusum procedures.Stochastic Processes and Applications,vol.9,no2,pp.207-215.R.A.K HAN(1981).A note on Page’s two-sided cumulative sum procedures.Biometrika,vol.68,no3, pp.717-719.B IBLIOGRAPHY433 V.K IREICHIKOV,V.M ANGUSHEV and I.N IKIFOROV(1990).Investigation and application of CUSUM algorithms to monitoring of sensors.In Statistical Problems of Control,Issue89,Vilnius,pp.124-130(in Russian).G.K ITAGAWA and W.G ERSCH(1985).A smoothness prior time-varying AR coefﬁcient modeling of non-stationary covariance time series.IEEE Trans.Automatic Control,vol.AC-30,no1,pp.48-56.N.K LIGIENE(1980).Probabilities of deviations of the change point estimate in statistical models.In Sta-tistical Problems of Control,Issue83,Vilnius,pp.80-86(in Russian).N.K LIGIENE and L.T ELKSNYS(1983).Methods of detecting instants of change of random process prop-erties.Automation and Remote Control,vol.44,no10,Part II,pp.1241-1283.J.K ORN,S.W.G ULLY and A.S.W ILLSKY(1982).Application of the generalized likelihood ratio algorithm to maneuver detection and estimation.Proc.American Control Conf.,Arlington,V A,pp.792-798.P.R.K RISHNAIAH and B.Q.M IAO(1988).Review about estimation of change points.In Handbook of Statistics(P.R.Krishnaiah,C.R.Rao,eds.),vol.7,Elsevier,New York,pp.375-402.P.K UDVA,N.V ISWANADHAM and A.R AMAKRISHNAN(1980).Observers for linear systems with unknown inputs.IEEE Trans.Automatic Control,vol.AC-25,no1,pp.113-115.S.K ULLBACK(1959).Information Theory and Statistics.Wiley,New York(also Dover,New York,1968). K.K UMAMARU,S.S AGARA and T.S¨ODERSTR¨OM(1989).Some statistical methods for fault diagnosis for dynamical systems.In Fault Diagnosis in Dynamic Systems-Theory and Application(R.Patton,P.Frank,R. Clark,eds.).International Series in Systems and Control Engineering,Prentice Hall International,London, UK,pp.439-476.A.K USHNIR,I.N IKIFOROV and I.S AVIN(1983).Statistical adaptive algorithms for automatic detection of seismic signals-Part I:One-dimensional case.In Earthquake Prediction and the Study of the Earth Structure,Naouka,Moscow(Computational Seismology,vol.15),pp.154-159(in Russian).L.L ADELLI(1990).Diffusion approximation for a pseudo-likelihood test process with application to de-tection of change in stochastic system.Stochastics and Stochastics Reports,vol.32,pp.1-25.T.L.L A¨I(1974).Control charts based on weighted sums.Annals Statistics,vol.2,no1,pp.134-147.T.L.L A¨I(1981).Asymptotic optimality of invariant sequential probability ratio tests.Annals Statistics, vol.9,no2,pp.318-333.D.G.L AINIOTIS(1971).Joint detection,estimation,and system identiﬁrmation and Control, vol.19,pp.75-92.M.R.L EADBETTER,G.L INDGREN and H.R OOTZEN(1983).Extremes and Related Properties of Random Sequences and Processes.Series in Statistics,Springer,New York.L.L E C AM(1960).Locally asymptotically normal families of distributions.Univ.California Publications in Statistics,vol.3,pp.37-98.L.L E C AM(1986).Asymptotic Methods in Statistical Decision Theory.Series in Statistics,Springer,New York.E.L.L EHMANN(1986).Testing Statistical Hypotheses,2d ed.Wiley,New York.J.P.L EHOCZKY(1977).Formulas for stopped diffusion processes with stopping times based on the maxi-mum.Annals Probability,vol.5,no4,pp.601-607.H.R.L ERCHE(1980).Boundary Crossing of Brownian Motion.Lecture Notes in Statistics,vol.40,Springer, New York.L.L JUNG(1987).System Identiﬁcation-Theory for the rmation and System Sciences Series, Prentice Hall,Englewood Cliffs,NJ.。

OJIC20120300003_67452463

1. INTRODUCTION
The reticular design and synthesis of metal-organic frameworks (MOFs) are currently the hot topic for discovery of the materials with potential applications in magnetism [1-6], catalysis [7-10], luminescence and chemical sensing [11-15], as well as gas adsorption and/or separation [16-18]. However, any of a successful construction of the target MOFs shall practically depend on a deliberate design or a judicious selection of the organic ligand incurporating with a proper geometrical metal. For example, the MOFs with zeolitic or zeolite-like structures are constructed by using imidazole and its derivates incorporating with tetrahedral coordinated metals [19-20]. To create novel MOFs having the 4-connected zeolitic structures, the MOFs based on imidazole derivate of 4-(1Himidaozol-1-yl)benzoic acid (HIBA) and divalent sixcoordinated metals have been reported in our previous work [21,22], where the ligand presents two ((2.21)syn-syn

趋化因子受体CCR研究概述

趋化因子受体CCR研究概述趋化因子要发挥生物学作用，必须与相应的受体结合才行。

趋化因子受体(Chemokine Receptor)属于G蛋白偶联受体，具有7个富含疏水氨基酸的α螺旋穿膜区结构，主要表达于骨髓来源的各白细胞亚群，同时也表达于上皮细胞、血管内皮细胞、神经细胞等类型的细胞。

趋化因子受体根据其结合的配体不同也分为4个亚家族：CCR、CXCR、XCR和CX3CR。

其中CCR亚族已克隆11种（CCR1-CCR11），CXCR亚族6种（CXCR1-CXCR6），另俩个亚族分别各有1种：XCR1和CX3CR1。

本文主要论述CCR的研究进展。

CCR1对多种人类CC趋化因子有反应，包括钙动员、腺苷酸环化酶抑制、细胞外酸化和趋化性增加。

CCR1已经从多个种属获得克隆，如恒河猴、兔、小鼠和大鼠，并且这些序列之间存在高度的序列同源性，人和恒河猴的相似性为87％。

人CCR1序列的大部分显著特征是保守的，其存在的变化主要限于N末端和细胞外环，可能参与配体结合的区域。

人和小鼠CCR1蛋白以高亲和力结合人和小鼠CCL3和CCL5。

通过靶向基因破坏研究和通过有效的CCR1拮抗剂的研究，提供了对CCR1的生理和病理生理作用的了解。

CCR2的cDNA可以编码两个蛋白质CCR2A和CCR2B。

CCR2B是主要表达形式，并且在慢性炎症中起作用，特别是动脉粥样硬化和多发性硬化疾病。

CCR2的mRNA可以在单核细胞、血源性树突状细胞、天然杀伤细胞和T淋巴细胞中检测到，但不能在嗜中性粒细胞或嗜酸性粒细胞中检测到。

抗体研究显示CCR2B在单核细胞、活化记忆T细胞、B细胞和嗜碱性粒细胞中表达。

CCR2通过与配体结合，产生许多生物学信号，包括腺苷酸环化酶的抑制、细胞内钙动员和细胞趋化性的增加。

CCR2已从许多物种克隆，包括小鼠、大鼠和恒河猴。

序列高度同源并且显示与人CCR2的78-95％氨基酸一致。

小鼠CCR2特异性结合了具有高亲和力的与MCP-1和MCP-3。

心脏MR_组织追踪技术定量评估心肌淀粉样变性患者心肌应变

·方法技术学·Cardiac MR tissue tracking technique for quantitativelyevaluating myocardial strain of cardiacamyloidosis patientsHE　Jiangkai1， CUI　Chen1， MA　Wei2， WANG　Zhi2， LIU　Jia1， LI　Wei1，ZHAO　Kai1， NAI　Rile1， XU　Shasha1， QIU　Jianxing1*（1.Department of Radiology，2.Department of Cardiovascular， PekingUniversity First Hospital， Beijing 100034， China）［Abstract］Objective　To observe the feasibility of cardiac MR tissue tracking （CMR-TT）technique for quantitatively evaluating myocardial strain of patients with myocardial amyloidosis （CA）.Methods　Cardiac MRI were collected from20 patients of immunoglobulin amyloid light-chain CA （AL-CA，group A），20 cases of transthyretin CA （ATTR-CA，group B） and 20 healthy subjects （group C）， and myocardial strain parameters were obtained using CMR-TT technique.Left ventricular cardiac function parameters were compared among 3 groups， so were strain parameters of each myocardial segment of left ventricle and global myocardium，including 3D longitudinal strain （LS），3D radial strain （RS）and 3D circumferential strain （CS）.Results　Compared with those in group C，significant differences of left ventricular cardiac function parameters were found in both group A and B （all P＜0.01），while no statistical difference was found between group A and B （all P＞0.05）. Except for apical segment RS （P=0.81）， strain parameters in group A and B were both lower than those in group C （all P＜0.01）， while no significant difference was detected between group A and B （all P＞0.05）.Conclusion　CMR-TT technique could be used to quantitatively evaluate left ventricular myocardial strain of CApatients.［Keywords］myocardium； amyloidosis； magnetic resonance imaging； tissue tracking techniqueDOI：10.13929/j.issn.1672-8475.2024.01.009心脏MR组织追踪技术定量评估心肌淀粉样变性患者心肌应变何江凯1，崔晨1，马为2，王智2，刘佳1，李玮1，赵凯1，奈日乐1，徐莎莎1，邱建星1*（1.北京大学第一医院医学影像科，2.心血管内科，北京 100034）［摘要］目的　观察利用心脏MR组织追踪（CMR-TT）技术定量评估心肌淀粉样变性（CA）患者心肌应变的可行性。

Development and Applications of CRISPR-Cas9 for Genome Engineering

Leading EdgeReviewDevelopment and Applications ofCRISPR-Cas9for Genome EngineeringPatrick D.Hsu,1,2,3Eric nder,1and Feng Zhang1,2,*1Broad Institute of MIT and Harvard,7Cambridge Center,Cambridge,MA02141,USA2McGovern Institute for Brain Research,Department of Brain and Cognitive Sciences,Department of Biological Engineering, Massachusetts Institute of Technology,Cambridge,MA02139,USA3Department of Molecular and Cellular Biology,Harvard University,Cambridge,MA02138,USA*Correspondence:zhang@/10.1016/j.cell.2014.05.010Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9are enabling the systematic interrogation of mammalian genome function.Analogous to the search function in modern word processors,Cas9can be guided to speciﬁc locations within complex genomes by a short RNA search ing this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice.Cas9-mediated genetic perturbation is simple and scalable,empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review,we describe the development and applications of Cas9for a variety of research or translational applications while highlighting challenges as well as future directions.Derived from a remarkable microbial defense system,Cas9is driving innovative applications from basic biology to biotechnology and medicine.IntroductionThe development of recombinant DNA technology in the1970s marked the beginning of a new era for biology.For theﬁrst time,molecular biologists gained the ability to manipulate DNA molecules,making it possible to study genes and harness them to develop novel medicine and biotechnology.Recent advances in genome engineering technologies are sparking a new revolution in biological research.Rather than studying DNA taken out of the context of the genome,researchers can now directly edit or modulate the function of DNA sequences in their endogenous context in virtually any organism of choice, enabling them to elucidate the functional organization of the genome at the systems level,as well as identify causal genetic variations.Broadly speaking,genome engineering refers to the process of making targeted modiﬁcations to the genome,its contexts (e.g.,epigenetic marks),or its outputs(e.g.,transcripts).The ability to do so easily and efﬁciently in eukaryotic and especially mammalian cells holds immense promise to transform basic sci-ence,biotechnology,and medicine(Figure1).For life sciences research,technologies that can delete,insert, and modify the DNA sequences of cells or organisms enable dis-secting the function of speciﬁc genes and regulatory elements. Multiplexed editing could further allow the interrogation of gene or protein networks at a larger scale.Similarly,manipu-lating transcriptional regulation or chromatin states at particular loci can reveal how genetic material is organized and utilized within a cell,illuminating relationships between the architecture of the genome and its functions.In biotechnology,precise manipulation of genetic building blocks and regulatory machin-ery also facilitates the reverse engineering or reconstruction of useful biological systems,for example,by enhancing biofuel production pathways in industrially relevant organisms or by creating infection-resistant crops.Additionally,genome engi-neering is stimulating a new generation of drug development processes and medical therapeutics.Perturbation of multiple genes simultaneously could model the additive effects that un-derlie complex polygenic disorders,leading to new drug targets, while genome editing could directly correct harmful mutations in the context of human gene therapy(Tebas et al.,2014). Eukaryotic genomes contain billions of DNA bases and are difﬁcult to manipulate.One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination(HR),which integrates exogenous repair templates that contain sequence homology to the donor site(Figure2A)(Capecchi,1989).HR-mediated targeting has facilitated the generation of knockin and knockout animal models via manipulation of germline competent stem cells, dramatically advancing many areas of biological research.How-ever,although HR-mediated gene targeting produces highly pre-cise alterations,the desired recombination events occur extremely infrequently(1in106–109cells)(Capecchi,1989),pre-senting enormous challenges for large-scale applications of gene-targeting experiments.To overcome these challenges,a series of programmable nuclease-based genome editing technologies havebeen1262Cell157,June5,2014ª2014Elsevier Inc.developed in recent years,enabling targeted and efﬁcient modi-ﬁcation of a variety of eukaryotic and particularly mammalian species.Of the current generation of genome editing technolo-gies,the most rapidly developing is the class of RNA-guided endonucleases known as Cas9from the microbial adaptive im-mune system CRISPR (clustered regularly interspaced short palindromic repeats),which can be easily targeted to virtually any genomic location of choice by a short RNA guide.Here,we review the development and applications of the CRISPR-associated endonuclease Cas9as a platform technology for achieving targeted perturbation of endogenous genomic ele-ments and also discuss challenges and future avenues for inno-vation.Programmable Nucleases as Tools for Efﬁcient and Precise Genome EditingA series of studies by Haber and Jasin (Rudin et al.,1989;Plessis et al.,1992;Rouet et al.,1994;Choulika et al.,1995;Bibikova et al.,2001;Bibikova et al.,2003)led to the realization that tar-geted DNA double-strand breaks (DSBs)could greatly stimulate genome editing through HR-mediated recombination events.Subsequently,Carroll and Chandrasegaran demonstrated the potential of designer nucleases based on zinc ﬁnger proteins for efﬁcient,locus-speciﬁc HR (Bibikova et al.,2001,2003).Moreover,it was shown in the absence of an exogenous homol-ogy repair template that localized DSBs can induce insertions or deletion mutations (indels)via the error-prone nonhomologous end-joining (NHEJ)repair pathway (Figure 2A)(Bibikova et al.,2002).These early genome editing studies established DSB-induced HR and NHEJ as powerful pathways for the versatileand precise modiﬁcation of eukaryotic genomes.To achieve effective genome editing via introduction of site-speciﬁc DNA DSBs,four major classes of customizable DNA-binding proteins have been engineered so far:meganucleases derived from microbial mobile genetic elements (Smith et al.,2006),zinc ﬁnger (ZF)nucleases based on eukaryotic transcrip-tion factors (Urnov et al.,2005;Miller et al.,2007),transcription activator-like effectors (TALEs)from Xanthomonas bacteria (Christian et al.,2010;Miller et al.,2011;Boch et al.,2009;Mos-cou and Bogdanove,2009),and most recently the RNA-guided DNA endonuclease Cas9from the type II bacterial adaptive im-mune system CRISPR (Cong et al.,2013;Mali et al.,2013a ).Meganuclease,ZF,and TALE proteins all recognize speciﬁc DNA sequences through protein-DNA interactions.Although meganucleases integrate its nuclease and DNA-binding domains,ZF and TALE proteins consist of individual modules targeting 3or 1nucleotides (nt)of DNA,respectively (Figure 2B).ZFs and TALEs can be assembled in desired combi-nations and attached to the nuclease domain of FokI to direct nucleolytic activity toward speciﬁc genomic loci.Each of these platforms,however,has unique limitations.Meganucleases have not been widely adopted as a genome engineering platform due to lack of clear correspondence between meganuclease protein residues and their target DNA sequence speciﬁcity.ZF domains,on the other hand,exhibit context-dependent binding preference due to crosstalk between adjacent modules when assembled into a larger array (Maeder et al.,2008).Although multiple strategies have been developed to account for these limitations (Gonzaelz et al.,2010;Sander et al.,2011),assembly of functional ZFPs with the desired DNA binding speciﬁcity remains a major challenge that requires an extensive screening process.Similarly,although TALE DNA-binding monomers are for the most part modular,they can still suffer from context-dependent speciﬁcity (Juillerat et al.,2014),and their repetitive sequences render construction of novel TALE arrays labor intensive and costly.Given the challenges associated with engineering of modular DNA-binding proteins,new modes of recognition would signiﬁ-cantly simplify the development of custom nucleases.The CRISPR nuclease Cas9is targeted by a short guide RNA that recognizes the target DNA via Watson-Crick base pairing (Figure 2C).The guide sequence within these CRISPR RNAs typically corresponds to phage sequences,constituting the nat-ural mechanism for CRISPR antiviral defense,but can be easily replaced by a sequence of interest to retarget the Cas9nuclease.Multiplexed targeting by Cas9can now be achieved at unprecedented scale by introducing a battery of short guideFigure 1.Applications of Genome EngineeringGenetic and epigenetic control of cells with genome engineering technologies is enabling a broad range of applications from basic biology to biotechnology and medicine.(Clockwise from top)Causal genetic mutations or epigenetic variants associated with altered biological function or disease phenotypes can now be rapidly and efﬁciently recapitulated in animal or cellular models (Animal models,Genetic variation).Manipulating biological circuits could also facilitate the generation of useful synthetic materials,such as algae-derived,silica-based diatoms for oral drug delivery (Materials).Additionally,precise genetic engineering of important agricultural crops could confer resistance to envi-ronmental deprivation or pathogenic infection,improving food security while avoiding the introduction of foreign DNA (Food).Sustainable and cost-effec-tive biofuels are attractive sources for renewable energy,which could be achieved by creating efﬁcient metabolic pathways for ethanol production in algae or corn (Fuel).Direct in vivo correction of genetic or epigenetic defects in somatic tissue would be permanent genetic solutions that address the root cause of genetically encoded disorders (Gene surgery).Finally,engineering cells to optimize high yield generation of drug precursors in bacterial factories could signiﬁcantly reduce the cost and accessibility of useful therapeutics (Drug development).Cell 157,June 5,2014ª2014Elsevier Inc.1263RNAs rather than a library of large,bulky proteins.The ease of Cas9targeting,its high efﬁciency as a site-speciﬁc nuclease,and the possibility for highly multiplexed modiﬁcations have opened up a broad range of biological applications across basic research to biotechnology and medicine.The utility of customizable DNA-binding domains extends far beyond genome editing with site-speciﬁc endonucleases.Fusing them to modular,sequence-agnostic functional effector domains allows ﬂexible recruitment of desired perturbations,such as transcriptional activation,to a locus of interest (Xu and Bestor,1997;Beerli et al.,2000a;Konermann et al.,2013;Maeder et al.,2013a;Mendenhall et al.,2013).In fact,any modular enzymatic component can,in principle,be substituted,allowing facile additions to the genome engineering toolbox.Integration of genome-and epigenome-modifying enzymes with inducible protein regulation further allows precise temporal control of dynamic processes (Beerli et al.,2000b;Konermann et al.,2013).CRISPR-Cas9:From Yogurt to Genome EditingThe recent development of the Cas9endonuclease for genome editing draws upon more than a decade of basic research into understanding the biological function of the mysterious repetitive elements now known as CRISPR (Figure 3),which are found throughout the bacterial and archaeal diversity.CRISPR loci typically consist of a clustered set of CRISPR-associated (Cas)genes and the signature CRISPR array—a series of repeat sequences (direct repeats)interspaced by variable sequences (spacers)corresponding to sequences within foreign genetic elements (protospacers)(Figure 4).Whereas Cas genes are translated into proteins,most CRISPR arrays are ﬁrst tran-scribed as a single RNA before subsequent processing into shorter CRISPR RNAs (crRNAs),which direct the nucleolytic activity of certain Cas enzymes to degrade target nucleic acids.The CRISPR story began in 1987.While studying the iap enzyme involved in isozyme conversion of alkaline phosphatase in E.coli ,Nakata and colleagues reported a curious set of 29nt repeats downstream of the iap gene (Ishino et al.,1987).Unlike most repetitive elements,which typically take the form of tandem repeats like TALE repeat monomers,these 29nt repeats were interspaced by ﬁve intervening 32nt nonrepetitive sequences.Over the next 10years,as more microbial genomes were sequenced,additional repeat elements were reported from genomes of different bacterial and archaeal strains.Mojica and colleagues eventually classiﬁed interspaced repeat sequences as a unique family of clustered repeat elements present in >40%of sequenced bacteria and 90%of archaea (Mojica et al.,2000).These early ﬁndings began to stimulate interest in such micro-bial repeat elements.By 2002,Jansen and Mojica coined the acronym CRISPR to unify the description of microbial genomic loci consisting of an interspaced repeat array (Jansen et al.,2002;Barrangou and van der Oost,2013).At the same time,several clusters of signature CRISPR-associated (cas )genes were identiﬁed to be well conserved and typically adjacent to the repeat elements (Jansen et al.,2002),serving as a basis for the eventual classiﬁcation of three different types of CRISPR systems (types I–III)(Haft et al.,2005;Makarova et al.,2011b ).Types I and III CRISPR loci contain multiple Cas proteins,now known to form complexes with crRNA (CASCADE complex for type I;Cmr or Csm RAMP complexes for type III)to facilitate the recognition and destruction of target nucleic acids (BrounsFigure 2.Genome Editing Technologies Exploit Endogenous DNA Repair Machinery(A)DNA double-strand breaks (DSBs)are typically repaired by nonhomologous end-joining (NHEJ)or homology-directed repair (HDR).In the error-prone NHEJ pathway,Ku heterodimers bind to DSB ends and serve as a molecular scaffold for associated repair proteins.Indels are introduced when the complementary strands undergo end resection and misaligned repair due to micro-homology,eventually leading to frameshift muta-tions and gene knockout.Alternatively,Rad51proteins may bind DSB ends during the initial phase of HDR,recruiting accessory factors that direct genomic recombination with homology arms on an exogenous repair template.Bypassing the matching sister chromatid facilitates the introduction of precise gene modiﬁcations.(B)Zinc ﬁnger (ZF)proteins and transcription activator-like effectors (TALEs)are naturally occurring DNA-binding domains that can be modularly assembled to target speciﬁc se-quences.ZF and TALE domains each recognize 3and 1bp of DNA,respectively.Such DNA-binding proteins can be fused to the FokI endonuclease to generate programmable site-speciﬁc nucleases.(C)The Cas9nuclease from the microbial CRISPR adaptive immune system is localized to speciﬁc DNA sequences via the guide sequence on its guide RNA (red),directly base-pairing with the DNA target.Binding of a protospacer-adjacent motif (PAM,blue)downstream of the target locus helps to direct Cas9-mediated DSBs.1264Cell 157,June 5,2014ª2014Elsevier Inc.et al.,2008;Hale et al.,2009)(Figure 4).In contrast,the type II system has a signiﬁcantly reduced number of Cas proteins.However,despite increasingly detailed mapping and annotation of CRISPR loci across many microbial species,their biological signiﬁcance remained elusive.A key turning point came in 2005,when systematic analysis of the spacer sequences separating the individual direct repeats suggested their extrachromosomal and phage-associated ori-gins (Mojica et al.,2005;Pourcel et al.,2005;Bolotin et al.,2005).This insight was tremendously exciting,especially given previous studies showing that CRISPR loci are transcribed (Tang et al.,2002)and that viruses are unable to infect archaeal cells carrying spacers corresponding to their own genomes (Mojica et al.,2005).Together,these ﬁndings led to the specula-tion that CRISPR arrays serve as an immune memory and defense mechanism,and individual spacers facilitate defense against bacteriophage infection by exploiting Watson-Crick base-pairing between nucleic acids (Mojica et al.,2005;Pourcel et al.,2005).Despite these compelling realizations that CRISPR loci might be involved in microbial immunity,the speciﬁc mech-anism of how the spacers act to mediate viral defense remained a challenging puzzle.Several hypotheses were raised,including thoughts that CRISPR spacers act as small RNA guides to degrade viral transcripts in a RNAi-like mechanism (Makarova et al.,2006)or that CRISPR spacers direct Cas enzymes to cleave viral DNA at spacer-matching regions (Bolotin et al.,2005).Working with the dairy production bacterial strain Strepto-coccus thermophilus at the food ingredient company Danisco,Horvath and colleagues uncovered the ﬁrst experimental evidence for the natural role of a type II CRISPR system as an adaptive immunity system,demonstrating a nucleic-acid-based immune system in which CRISPR spacers dictate target speci-ﬁcity while Cas enzymes control spacer acquisition and phage defense (Barrangou et al.,2007).A rapid series of studies illumi-nating the mechanisms of CRISPR defense followed shortly and helped to establish the mechanism as well as function of all three types of CRISPR loci in adaptive immunity.By studying the type I CRISPR locus of Escherichia coli ,van der Oost and colleagues showed that CRISPR arrays are transcribed and converted into small crRNAs containing individual spacers to guide Cas nuclease activity (Brouns et al.,2008).In the same year,CRISPR-mediated defense by a type III-A CRISPR system from Staphylococcus epidermidis was demonstrated to block plasmid conjugation,establishing the target of Cas enzyme activity as DNA rather than RNA (Marrafﬁni andSontheimer,Figure 3.Key Studies Characterizing and Engineering CRISPR SystemsCas9has also been referred to as Cas5,Csx12,and Csn1in literature prior to 2012.For clarity,we exclusively adopt the Cas9nomenclature throughout this Review.CRISPR,clustered regularly interspaced short palindromic repeats;Cas,CRISPR-associated;crRNA,CRISPR RNA;DSB,double-strand break;tracrRNA,trans -activating CRISPR RNA.Cell 157,June 5,2014ª2014Elsevier Inc.12652008),although later investigation of a different type III-B system from Pyrococcus furiosus also revealed crRNA-directed RNA cleavage activity(Hale et al.,2009,2012).As the pace of CRISPR research accelerated,researchers quickly unraveled many details of each type of CRISPR system (Figure4).Building on an earlier speculation that protospacer-adjacent motifs(PAMs)may direct the type II Cas9nuclease to cleave DNA(Bolotin et al.,2005),Moineau and colleagues high-lighted the importance of PAM sequences by demonstrating that PAM mutations in phage genomes circumvented CRISPR inter-ference(Deveau et al.,2008).Additionally,for types I and II,the lack of PAM within the direct repeat sequence within the CRISPR array prevents self-targeting by the CRISPR system.In type III systems,however,mismatches between the50end of the crRNA and the DNA target are required for plasmid interference(Marraf-ﬁni and Sontheimer,2010).By2010,just3years after theﬁrst experimental evidence for CRISPR in bacterial immunity,the basic function and mecha-nisms of CRISPR systems were becoming clear.A variety of groups had begun to harness the natural CRISPR system for various biotechnological applications,including the generation of phage-resistant dairy cultures(Quiberoni et al.,2010)and phylogenetic classiﬁcation of bacterial strains(Horvath et al., 2008,2009).However,genome editing applications had not yet been explored.Around this time,two studies characterizing the functional mechanisms of the native type II CRISPR system elucidated the basic components that proved vital for engineering a simple RNA-programmable DNA endonuclease for genome editing. First,Moineau and colleagues used genetic studies in Strepto-coccus thermophilus to reveal that Cas9(formerly called Cas5,Csn1,or Csx12)is the only enzyme within the cas gene cluster that mediates target DNA cleavage(Garneau et al.,2010).Next,Charpentier and colleagues revealed a key component in the biogenesis and processing of crRNA in type II CRISPR systems—a noncoding trans-activating crRNA(tracrRNA)that hybridizes with crRNA to facilitate RNA-guided targeting of Cas9(Deltcheva et al.,2011).This dual RNA hybrid,together with Cas9and endogenous RNase III,is required for processing the CRISPR array transcript into mature crRNAs(Deltcheva et al.,2011).These two studies suggested that there are at least three components(Cas9, the mature crRNA,and tracrRNA)that are essential for recon-stituting the type II CRISPR nuclease system.Given the increasing importance of programmable site-speciﬁc nucleases based on ZFs and TALEs for enhancing eukaryotic genome editing,it was tantalizing to think that perhaps Cas9could be developed into an RNA-guided genome editing system. From this point,the race to harness Cas9for genome editing wason.Figure4.Natural Mechanisms of Microbial CRISPR Systems in Adaptive Immunity Following invasion of the cell by foreign genetic elements from bacteriophages or plasmids(step 1:phage infection),certain CRISPR-associated (Cas)enzymes acquire spacers from the exoge-nous protospacer sequences and install them into the CRISPR locus within the prokaryotic genome (step2:spacer acquisition).These spacers are segregated between direct repeats that allow the CRISPR system to mediate self and nonself recognition.The CRISPR array is a noncoding RNA transcript that is enzymatically maturated through distinct pathways that are unique to each type of CRISPR system(step3:crRNA biogenesis and processing).In types I and III CRISPR,the pre-crRNA transcript is cleaved within the repeats by CRISPR-asso-ciated ribonucleases,releasing multiple small crRNAs.Type III crRNA intermediates are further processed at the30end by yet-to-be-identiﬁed RNases to produce the fully mature transcript.In type II CRISPR,an associated trans-activating CRISPR RNA(tracrRNA)hybridizes with the direct repeats,forming an RNA duplex that is cleaved and processed by endogenous RNase III and other unknown nucleases.Maturated crRNAs from type I and III CRISPR systems are then loaded onto effector protein complexes for target recognition and degradation.In type II systems, crRNA-tracrRNA hybrids complex with Cas9to mediate interference.Both type I and III CRISPR systems use multi-protein interference modules to facilitate target recognition.In type I CRISPR,the Cascade com-plex is loaded with a crRNA molecule,constituting a catalytically inert surveillance complex that rec-ognizes target DNA.The Cas3nuclease is then recruited to the Cascade-bound R loop,mediatingtarget degradation.In type III CRISPR,crRNAs associate either with Csm or Cmr complexes that bind and cleave DNA and RNA substrates,respectively.In contrast,the type II system requires only the Cas9nuclease to degrade DNA matching its dual guide RNA consisting of a crRNA-tracrRNA hybrid.1266Cell157,June5,2014ª2014Elsevier Inc.In2011,Siksnys and colleaguesﬁrst demonstrated that the type II CRISPR system is transferrable,in that transplantation of the type II CRISPR locus from Streptococcus thermophilus into Escherichia coli is able to reconstitute CRISPR interference in a different bacterial strain(Sapranauskas et al.,2011).By 2012,biochemical characterizations by the groups of Charpent-ier,Doudna,and Siksnys showed that puriﬁed Cas9from Strep-tococcus thermophilus or Streptococcus pyogenes can be guided by crRNAs to cleave target DNA in vitro(Jinek et al., 2012;Gasiunas et al.,2012),in agreement with previous bacte-rial studies(Garneau et al.,2010;Deltcheva et al.,2011;Sapra-nauskas et al.,2011).Furthermore,a single guide RNA(sgRNA) can be constructed by fusing a crRNA containing the targeting guide sequence to a tracrRNA that facilitates DNA cleavage by Cas9in vitro(Jinek et al.,2012).In2013,a pair of studies simultaneously showed how to suc-cessfully engineer type II CRISPR systems from Streptococcus thermophilus(Cong et al.,2013)and Streptococcus pyogenes (Cong et al.,2013;Mali et al.,2013a)to accomplish genome editing in mammalian cells.Heterologous expression of mature crRNA-tracrRNA hybrids(Cong et al.,2013)as well as sgRNAs (Cong et al.,2013;Mali et al.,2013a)directs Cas9cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated genome editing.Multiple guide RNAs can also be used to target several genes at once.Since these initial studies,Cas9has been used by thousands of laboratories for genome editing applications in a variety of experimental model systems(Sander and Joung,2014).The rapid adoption of the Cas9technology was also greatly accelerated through a com-bination of open-source distributors such as Addgene,as well as a number of online user forums such as http://www. and . Structural Organization and Domain Architecture ofCas9The family of Cas9proteins is characterized by two signature nuclease domains,RuvC and HNH,each named based on homology to known nuclease domain structures(Figure2C). Though HNH is a single nuclease domain,the full RuvC domain is divided into three subdomains across the linear protein sequence,with RuvC I near the N-terminal region of Cas9and RuvC II/IIIﬂanking the HNH domain near the middle of the pro-tein.Recently,a pair of structural studies shed light on the struc-tural mechanism of RNA-guided DNA cleavage by Cas9. First,single-particle EM reconstructions of the Streptococcus pyogenes Cas9(SpCas9)revealed a large structural rearrange-ment between apo-Cas9unbound to nucleic acid and Cas9in complex with crRNA and tracrRNA,forming a central channel to accommodate the RNA-DNA heteroduplex(Jinek et al., 2014).Second,a high-resolution structure of SpCas9in complex with sgRNA and the complementary strand of target DNA further revealed the domain organization to comprise of an a-helical recognition(REC)lobe and a nuclease(NUC)lobe consisting of the HNH domain,assembled RuvC subdomains,and a PAM-interacting(PI)C-terminal region(Nishimasu et al.,2014) (Figure5A and Movie S1).Together,these two studies support the model that SpCas9 unbound to target DNA or guide RNA exhibits an autoinhibited conformation in which the HNH domain active site is blocked by the RuvC domain and is positioned away from the REC lobe (Jinek et al.,2014).Binding of the RNA-DNA heteroduplex would additionally be sterically inhibited by the orientation of the C-ter-minal domain.As a result,apo-Cas9likely cannot bind nor cleave target DNA.Like many ribonucleoprotein complexes,the guide RNA serves as a scaffold around which Cas9can fold and orga-nize its various domains(Nishimasu et al.,2014).The crystal structure of SpCas9in complex with an sgRNA and target DNA also revealed how the REC lobe facilitates target binding.An arginine-rich bridge helix(BH)within the REC lobe is responsible for contacting the308–12nt of the RNA-DNA het-eroduplex(Nishimasu et al.,2014),which correspond with the seed sequence identiﬁed through guide sequence mutation ex-periments(Jinek et al.,2012;Cong et al.,2013;Fu et al.,2013; Hsu et al.,2013;Pattanayak et al.,2013;Mali et al.,2013b). The SpCas9structure also provides a useful scaffold for engi-neering or refactoring of Cas9and sgRNA.Because the REC2 domain of SpCas9is poorly conserved in shorter orthologs, domain recombination or truncation is a promising approach for minimizing Cas9size.SpCas9mutants lacking REC2retain roughly50%of wild-type cleavage activity,which could be partly attributed to their weaker expression levels(Nishimasu et al., 2014).Introducing combinations of orthologous domain re-combination,truncation,and peptide linkers could facilitate the generation of a suite of Cas9mutant variants optimized for different parameters such as DNA binding,DNA cleavage,or overall protein size.Metagenomic,Structural,and Functional Diversity of Cas9Cas9is exclusively associated with the type II CRISPR locus and serves as the signature type II gene.Based on the diversity of associated Cas genes,type II CRISPR loci are further subdivided into three subtypes(IIA–IIC)(Figure5B)(Makarova et al.,2011a; Chylinski et al.,2013).Type II CRISPR loci mostly consist of the cas9,cas1,and cas2genes,as well as a CRISPR array and tracrRNA.Type IIC CRISPR systems contain only this minimal set of cas genes,whereas types IIA and IIB have an additional signature csn2or cas4gene,respectively(Chylinski et al.,2013). Subtype classiﬁcation of type II CRISPR loci is based on the architecture and organization of each CRISPR locus.For example,type IIA and IIB loci usually consist of four cas genes, whereas type IIC loci only contain three cas genes.However, this classiﬁcation does not reﬂect the structural diversity of Cas9proteins,which exhibit sequence homology and length variability irrespective of the subtype classiﬁcation of their parental CRISPR locus.Of>1,000Cas9nucleases identiﬁed from sequence databases(UniProt)based on homology,protein length is rather heterogeneous,roughly ranging from900to1600 amino acids(Figure5C).The length distribution of most Cas9 proteins can be divided into two populations centered around 1,100and1,350amino acids in length.It is worth noting that a third population of large Cas9proteins belonging to subtype IIA,formerly called Csx12,typically contain around1500amino acids.Despite the apparent diversity of protein length,all Cas9pro-teins share similar domain architecture(Makarova et al.,2011a;Cell157,June5,2014ª2014Elsevier Inc.1267。

核磁共振光谱NMR光谱

可见，弛豫决定处于高能级核寿命。而弛豫时间长，核磁共振信号窄；反之，谱线宽。
弛豫可分为纵向弛豫和横向弛豫。
32
纵向弛豫：
处于高能级的核将其能量及时转移给周围分子骨架(晶格)
中的其它核，从而使自己返回到低能态的现象。又称自旋
-晶格弛豫。
其半衰期用T1表示
横向弛豫：当两个相邻的核处于不同能级，但进动频率相同时，高能级核与低能级核通过自旋状态的交换而实现能量转移所发生的弛豫现象。又称自旋-自旋弛豫。
N NH i N NL
j
E
h
e kT e kT
通过计算，在常温下， 1H 处于 B0 为 2.3488T的磁场中，处于低能级的1H 核数目仅比高能级的核数目多出百万分之十六！
会造成什么后果？
27
随实验进行，低能级核越来越少，最后高、低能级上的核数目相等--------饱和-----从低到高与从高到低能级的跃迁的数目相同---体系净吸收为0-----共振信号消失！
问世，NMR开始广泛应用
4
第二阶段 70年代：Fourier Transform的应用
13C-NMR技术（碳骨架）（GC，TLC，HPLC技术的发展）第三阶段 80年代：Two-dimensional (2D) NMR诞生（COSY，碳骨架连接顺序，非键原子间距离，生物大分子结构，……）
5
这个过程称之弛豫过程(Relaxation),即高能态的核以非辐射的形式放出能量回到低能态重建Boltzmann分布。
30
两种弛豫过程：
N
h
Relaxation
N+
31
谱线宽度
据Heisenberg测不准原理，激发能量E与体系处于激发态的平均时间(寿命)成反比，与谱线变宽成正比，即：

crochralski法 -回复

crochralski法-回复关于crochralski法的相关问题。

1. 什么是crochralski法？Crochralski法（Czochralski method）是一种用于晶体生长的工艺方法，通过在高温下将一个称作“种子”（seed）的晶体与熔融的物质接触，使晶体逐渐增长并形成完整的晶体。

2. crochralski法的历史和背景是什么？Czochralski法最早由波兰科学家Jan Czochralski在1916年发现并提出，该方法最初用于金属材料的晶体生长。

后来，随着材料科学的发展，crochralski法被广泛用于半导体、光电子学和光纤等领域的晶体生长。

3. crochralski法的工艺过程是怎样的？crochralski法的工艺过程分为准备、熔炼、成型、冷却和修整等步骤。

（1）准备：选取合适的种子晶体，并将其固定在晶体生长装置的底部。

（2）熔炼：将适量的原料加入熔炉中进行熔化，并控制温度和熔体成分。

（3）成型：将种子晶体与熔体接触，通过拉出晶体生长装置使种子晶体缓慢向上拉伸，同时转动，并通过准确控制温度差和拉速度来控制晶体的尺寸和纯度。

（4）冷却：当晶体达到所需尺寸后，停止拉晶过程，让晶体自然冷却。

（5）修整：将冷却后的晶体切割、研磨和抛光，形成所需的形状和表面质量。

4. crochralski法的原理是什么？crochralski法的原理是基于熔体与晶体之间的凝固反应。

在拉晶过程中，种子晶体和熔体接触，形成一个凝固界面。

通过控制凝固界面与种子晶体之间的温度差和拉伸速度，可以控制晶体生长的尺寸和质量。

当熔体冷却过程中，熔体中的原子逐渐排列成有序结构，形成晶体的晶格结构。

5. crochralski法有哪些优点和局限性？优点：（1）可以生长大尺寸和高质量的晶体。

（2）生长过程可控性强，晶体的尺寸和纯度可以通过调节温度差和拉速度来控制。

（3）可以控制晶体的掺杂和杂质含量。

局限性：（1）晶体生长速度相对较慢，通常需要数小时甚至数天。

Investigation of the Effect of Paclitaxel on MCF-7

Corresponding Author: Mustafa Nisari, Dr., Asst. Prof.,research fields:biochemistry, cancer.
nontoxic and act at various stages of angiogenesis [6]. Various chemotherapeutic drugs have been
investigated for anticancer activity. Paclitaxel, also known as taxol, is a chemotherapeutic drug with a high medicinal property and is one of the most frequently used chemotherapeutics in the clinic. It is a broad spectrum anticancer drug that is effective in various solid tumors such as ovarian and breast cancer, lung cancer, melanoma, head and neck cancer, and bladder cancer. Paclitaxel has a strong anti-proliferative effect against tumor cells and by stabilizing microtubules, blocking mitosis and inducing apoptosis. Angiogenesis involves a complex sequence of events. With angiogenic stimulation, vascular endothelial cells stimulate cell proliferation to break up the extracellular and tissue matrix, increase endothelial cell motility and provide the required number of cells for growing vessels [7]. The aim of this study is to investigate the effects of different doses of paclitaxel against in vitro cell proliferation of the human breast cancer cell line MCF-7.

阴离子对鼠尾草提取物中Ni盐前体绿色合成的NiO纳米粒子磁性和结构性能的影响

阴离子对鼠尾草提取物中Ni盐前体绿色合成的NiO纳米粒子磁性和结构性能的影响Kubra Zenkin;Aslihan Dalmaz;Mesut Ozdincer;Sefa Durmus【期刊名称】《无机化学学报》【年(卷),期】2024(40)2【摘要】采用绿色合成方法,将鼠尾草提取物用作还原和封端剂合成氧化镍纳米颗粒(NiO NPs)。

此外,使用各种前驱体合成了NiO NPs,并使用扫描电子显微镜对其形貌进行了分析。

利用傅里叶变换红外光谱和粉末X射线衍射(PXRD)对NiO NPs 的结构进行了表征,使用振动样品磁强计测量了它们的磁性。

PXRD研究表明,所有合成的NiO NPs都表现出具有高结晶度的面心立方相,并且形成了具有高纯度相的NiO。

磁化研究结果表明,3种镍盐(乙酸盐、氯化物和硫酸盐)前驱体合成的NiO NPs分别表现出超顺磁、软铁磁和顺磁行为。

【总页数】12页(P451-462)【作者】Kubra Zenkin;Aslihan Dalmaz;Mesut Ozdincer;Sefa Durmus【作者单位】Department of Chemistry Education Institute University 81620;Department of Natural and Herbal Products/Cosmetic Products Education Institute University 81620;Department of Composite-Materials Education Institute University 81620;Department of Chemistry of Art and Science University 81620【正文语种】中文【中图分类】TB34;O614.813【相关文献】1.核壳结构Ni@SiO_2纳米粒子的可控合成及磁性能研究2.快速微波燃烧法制备的Cu—Ni合金纳米粒子结构和磁性能3.溶胶凝胶燃烧合成纳米NiO对太阳盐微结构和热性能的影响4.化学法合成Nd-Fe-B磁性纳米粒子的评述—微结构与磁性能因版权原因，仅展示原文概要，查看原文内容请购买。

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Alkali –aggregate reaction in activated fly ash systemsI.García-Lodeiro ⁎,A.Palomo ⁎,A.Fernández-JiménezEduardo Torroja Institute (CSIC),Serrano Galvache 4-28033Madrid,SpainReceived 22November 2005;accepted 2November 2006AbstractCertain aspects of the durability of a new cementitious material,alkali activated fly ash,are addressed in this article;specifically,a series of findings relating to the alkali-silica reaction are reported.The approach adopted in the study was to compare the new cementitious systems to analogous Portland cement mortars using aggregates of differing reactivity and a procedure based on the test described in ASTM standard C 1260.The results of SEM/EDX and XRD analysis of the materials showed that activated fly ash mortars performed better than the Portland cement equivalents.©2006Elsevier Ltd.All rights reserved.Keywords:Alkali-activated fly ash;Alkali –aggregate reaction;NBRI test1.IntroductionToday Portland cement concrete is the construction material par excellence .Its cost-effectiveness,mechanical strength and durability have made it indispensable to the construction industry.The exceptionally high temperatures (1400–1500°C)required,however,make Portland cement production a very costly,energy-intensive process.In addition,large amounts of CO 2,one of the main greenhouse gases,are released into the atmosphere during clinker manufacture.Finally,the durability of traditional Portland cement concrete is limited by a number of factors,most prominently the developments associated with the alkali –aggregate reaction.In light of these problems,the scientific community has undertaken to seek new processes,technologies and materials to provide the construc-tion industry with alternative binders.The alkali –aggregate reaction is a chemical process involving alkaline oxides generally deriving from the alkalis in the cement and certain forms of reactive silica present in the aggregate.Silex is one of the many types of aggregate that may give rise to the ASR.The mineralogical composition of this compound may vary from opaline to cryptocrystalline silica andfrom chalcedony to microcrystalline quartz.In alkaline environments,these forms of silica are susceptible to attack,ultimately leading to the dissolution of the aggregate.Such mineralogical forms are,in fact,regarded to be potentially reactive [1–3].And there is evidence to suggest that a certain concentration or proportion of reactive aggregate,if combined with a given amount of inert aggregate,prompts maximum expansion giving rise to what is known as the concept of the “pessimum ”proportion effect [1,4,5].One measure that has been successfully used to control ASR-induced expansion is the partial replacement of Portland cement with fly ash [6–9].The role of the ash in curbing expansion is attributed to a number of factors,including the reduced alkalinity in the solution saturating the concrete pores,the lower available calcium content in the system and so on.The presence of calcium has long been controversial and even today the role played by that element in alkali –aggregate reaction mechanisms is poorly understood [10–13].The present article reports on research focusing on a new binder known as alkali-activated fly ash (AAFA).In mortar and concrete made with such binders,Portland cement can be 100%replaced with fly ash and the mixing water with an alkaline solution [14,15].Alkali activation of fly ash is a chemical process in which the vitreous component of this industrial by-product is converted into a compact material with cementitious properties.The primary reaction product is athree-dimensionalCement and Concrete Research 37(2007)175–183⁎Corresponding authors.Tel.:+34913020440;fax:+34913026047.E-mail addresses:iglodeiro@ietcc.csic.es (I.García-Lodeiro),palomo@ietcc.csic.es (A.Palomo).0008-8846/$-see front matter ©2006Elsevier Ltd.All rights reserved.doi:10.1016/j.cemconres.2006.11.002alkaline aluminosilicate gel whose silicon tetrahedra are co-ordinated with aluminium tetrahedra.Crystalline zeolites such as herschelite,hydroxysodalite and so on are obtained as secondary products[16–18].Alkaline cements and concretes,in general,are characterized by high alkali content but also by low calcium content.As a result of all the foregoing,these materials might be expected toexhibit a different behaviour than Portland cement with respect to the alkali–aggregate reaction.The purpose of the present study,then,was to evaluate the performance of these new alkaline binders(made with a class F, low calcium content fly ash)in the context of the ASR and compare their behaviour to the findings for traditional Portland cement concrete.The experimental approach involved using aggregates with different degrees of reactivity,among them a “pessimum”proportion aggregate mix.2.Experimental2.1.MaterialsThe materials used in this study were as follows:➢Class F(according to ASTM standard C618-03)fly ash from a Spanish steam power plant.➢Type I low alkali content Portland cement from a Spanish cement factory.➢Arcosic sandstone with carbonatic cement(Siliceous aggregate)commonly used to manufacture concrete.➢Reactive opal aggregate supplied by CELITE,S.A.The aggregate was crushed,ground and sieved to the particle size distribution shown in Table1.Table2gives the chemical characteristics of the raw materials used.Ash composition was determined as recommended in Spanish standards UNE80-125-88and UNE80-225-93.Cement and aggregate chemical composition was established with a PHILIPS PW1404X-ray fluorescence spectrometer.The prime materials were also characterized for mineralogy and microstructure with a PHILIPS PW1710-LINK X-ray diffractometer.2.2.Method2.2.1.Mortars preparationThe method used to evaluate the possible appearance of the ASR in the specimens was based on ASTM standard C1260-94. This method estimates the deterioration that the ASR may potentially cause in cement mortars in16days.The procedure described in the standard was modified slightly in the present study in terms of the aggregate grain size distribution used,(see Table1),and the temperature,which was85°C instead of the recommended80°C.The mortar specimens were made with the aggregates described above.Three series of specimens were prepared.The first series contained100%siliceous aggregate,the second 100%opal aggregate and the third a siliceous+opal aggregate mix in a proportion of90:10by weight to induce the “pessimum”proportion effect.Prismatic(2.5×2.5×28.5cm)specimens were made from the materials to be tested,Portland cement mortars(as a control) and(Portland cement-free)alkali-activated fly ash mortars. 2.2.2.Mortars curingOPC Mortars:The(deionized)water/cement ratio used in the Portland cement mortars was0.47(see Table3).After an initial curing in a chamber at21°C and99%relative humidity for24h,the prisms were de-moulded,immersed in water and stored in sealed containers in an oven at85°C for a further 24h.Table1The granulometry distribution of the aggregate usedSieve sizePassing Retained on Mass,%4mm2mm102mm1mm251mm500μm25500μm250μm25250μm125μm15Table2Chemical composition of the raw materialsMgO Al2O3SiO2P2O5SO3CaO Fe2O3Na2O e⁎Insol.L.O.I.Total Fly ash 1.7926.4254.420.01 3.217.01 2..570.78 2.1999.44 Pórtland cement 1.01 3.3121.50.08 2.9366.2 2.670.46 1.4499.80 Siliceus aggregate0.93 6.9448.50.060.0621.1 2.58 1.5717.499.62 Opal aggregate0.980.4984.00.030.02 6.020.440.068.44100⁎Na2Oe:weight percentage Na2O+0.658weight percentage K2O.Table3Mortar featuresMix(1)SAcontent(g)(2)RAcontent(g)OPCcontent(g)Fly ashcontent(g)Activator(3)L/SratioOPC1135006000–0.47OPC2013506000–0.47⁎OPC312151356000–0.47FA1135000600Na(OH)8M0.47FA2013500600Na(OH)8M0.47⁎FA312151350600Na(OH)8M0.47(1)Siliceous aggregate;(2)opal aggregate(3)water/cement ratio or solution/ashratio in fly ash systems.⁎In OPC3and FA3mortars,the siliceous and opalaggregates were mixed in a proportion of90:10by weight to induce the“pessimum”proportion effect[5,19].176I.García-Lodeiro et al./Cement and Concrete Research37(2007)175–183AAF A Mortars:An8M solution of NaOH was used as the activator to make the fly ash mortar,which was mixed in the same way as the Portland cement.The“solution/ash”ratio was 0.47.After mixing,the specimens(in their respective moulds) were placed in an oven where the mortars were cured for20h at 85°C in high humidity conditions.Finally,they were removed from their moulds.2.2.3.Accelerated mortar bar methodAfter their respective curing processes,both the Portland cement and activated fly ash mortar specimens were immersed in a1M NaOH solution and stored in sealed containers in an oven at85°C.The specimens were routinely measured for length throughout the duration of the test to determine ASR-induced expansion.Finally,at the ages of16and90days,the specimens were studied and characterized by SEM/EDX(with a JEOL JSM 5400microscope fitted with a LINKS-ISIS energy dispersive microanalysis system)and XRD with the PHILIPS diffractom-eter mentioned above.3.Results3.1.XRD characterization of initial materialsXRD analysis(Fig.1(a))showed that the siliceous aggregate (SA)consisted primarily of quartz and calcite,together with small quantities of plagioclases and micas.A small amount of dolomite was also observed.The diffractogram for the opal aggregate(RA)showed opal(amorphous silica)to be the prevailing phase.Small amounts of quartz and calcite were likewise detected in this aggregate,see Fig.1(a).Unsurprisingly,the XRD analysis of the Portland cement revealed that the majority phase was alite(tricalcium silicate). The other phases normally present in Portland cement were also observed:belite,gypsum and the interstitial phase(aluminates and ferrites)(Fig.1(b),OPC).XRD analysis of the fly ash,in turn,showed that it consisted essentially in a vitreous phase,although minority quartz and mullite crystalline phases were also identified,along with smallquantities of hematite(Fig.1(b)FA).3.2.Determination of mortar prism expansionExpansion patterns were determined for each and every oneof the mortars listed in Table3.The expansion curves for thecement and activated fly ash mortars with siliceous aggregate(OPC1and FA1)are given in Fig.2(a),(b)depicts the results forcement mortars made with opal aggregate(due to the immediatedeterioration of the specimens,no data could be obtained formortar FA2).Lastly,the expansion curves for cement andactivated ash mortars made with a mixed siliceous-opalaggregate(90:10by weight,or“pessimum”proportion)areshown in Fig.2(c).All the prisms were tested as stipulated in thestandard(ASTM C1260-94).A comparison of Fig.2(a),(b),(c)shows that for Portlandcement mortar,the greatest expansion was recorded with the “pessimum”proportion aggregate mix,(system OPC3).After only4days of immersion in the1M NaOH solution at85°C,thematerial had expanded more than the limit allowed in the standard(0.1%).Moreover,even when100%theoretically non-reactivesiliceous aggregate was used(system OPC1);the expansion limitwas also exceeded in a short period of time(9days).After90days(see Fig.2(d)),typical ASR map cracking could be seen across theentire surface of the specimen.This should be interpreted to meanthat,contrary to the initial hypothesis,the siliceous aggregate is “moderately”reactive(this was verified by a petrographic analysis of the aggregate conducted in a previous study,when the presence of small amounts of stressed quartz was detected [19]).Finally,when100%opal aggregate was used(system OPC2),the expansion limit was reached on the16th day.Initially, expansion in this system appeared later than in system OPC1. However,at the age of56days,the specimens were so severely deteriorated that expansion could not be measured(Fig.2(f)).When fly ash mortars were prepared with100%siliceousaggregate(FA1),by contrast,the expansion limit was notexceeded even after90days,see Fig.2(a).Fig.2(e)shows thatin fact the fly ash system specimens were in perfect conditionat Fig.1.(a)XRD patterns of siliceous aggregate(SA)and opal aggregate(RA)(b)XRD patterns of Portland cement(OPC)and fly ash(FA);m mica a plagioclases;c calcite;d dolomite;Q quartz;o opal;Y gypsum;A alite;B belite;C3A tricalcium aluminate;M mullite;G hematite;C4AF tetracalcium aluminoferrite;CaO lime.177I.García-Lodeiro et al./Cement and Concrete Research37(2007)175–183this age,with no surface cracks.When fly ash mortars were prepared with“pessimum”proportion aggregate(FA3),under-went slight expansion,but much more moderately than the respective Portland cement mortars,see Fig.2(c).The NaOH-activated fly ash specimens made with100% opaline aggregate(FA2)were so severely deteriorated after24h of immersion in1M NaOH that matrix expansion could not be measured.Nonetheless,generally speaking the activated ash systems performed better than the Portland cement systems.3.3.XRD and SEM/EDX studies of the mortarsThe different mortars were studied with XRD and SEM/ EDX for mineralogical composition and microstructure after 16and90days of storage in the1M NaOH solution at 85°C.EDX studies were also run at the above ages to determine the elementary composition of the different phases, (see Table4).3.3.1.Mortar prepared with100%siliceous aggregateFig.3(a)shows the diffractograms for OPC1mortar at16and 90days.According to these results,the aggregate underwent significant change,with a substantial decline in the intensity of the plagioclase signals.The intensity of the portlandite peaks also decreased considerably,which may be interpreted to be a sign of lime uptake by the gel formed in the alkali–silica reaction.The XRD results for the activated ash systems,in turn,see Fig.3(b)revealed that they were characterized,among others, by the absence of portlandite and the presence of a series of zeolitic crystalline compounds(herschelite,hydroxysodalite Fig.2.Expansion curves with the time(a)OPC1and FA1mortars(100%of SA)(b)OPC2mortars(100%of RA);FA2mortars(No data)(c)OPC3y FA3mortars (“pessimum”proportion aggregate).Image of the mortars(d)OPC1(100%of SA)on the90th day(e)FA1(100%of SA)on the90th day(f)OPC2(100%of RA)on the56th day.The features of all mortars are shown in Table3.178I.García-Lodeiro et al./Cement and Concrete Research37(2007)175–183and zeolite P).Moreover,the intensity of the peaks for these crystalline phases grew with the time of immersion in NaOH at 85°C while analcime,a new zeolite phase not detected at the age of 16days,appeared in the 90-day analyses.At the same time,the 16-day SEM/EDX study of the OPC1mortars revealed the presence of a gel with a high sodium content in the aggregate –matrix interface,(see Fig.4(a)and Table 4),point 1),along with portlandite plates (Fig.4(a)and Table 4,point 2).After 90days,this same mortar exhibited numerous inward-growing microcracks.A “typical ”ASR product was detected in the aggregate –matrix interface with a “rod-type ”morphology [20]and Ca/Si and Na/Si ratios of 0.2and 0.77,respectively,(see Fig.4(b)and Table 4),point 3.By contrast,after 16days of immersion in the 1M NaOH solution at 85°C (FA1mortars),the activated fly ash mortars exhibited only the reaction product typically generated in alkaline activation,see Fig.5(a),with Si/Al and Na/Al ratios of0.97and 0.43,respectively (Table 4,point 4).No calcium was detected in these analyses.The presence of zeolitic crystalline compounds (Fig.5(b),p.5)was confirmed after 90days.These crystals,which had high Si/Al and Na/Al ratios (point 5,Table 4),were normally found in gaps in the matrix.Alkali –aggregate reaction products with a pseudo-rosette type morphol-ogy and Ca/Si and Na/Si ratios of 0.15and 0.67,respectively (point 6,Table 4),were also detected,albeit very sporadically.3.3.2.Mortar prepared with 100%opal aggregateFig.6gives the X-ray diffraction pattern for OPC2mortar at the age of 16days and the pattern of the original opal aggregate.The disappearance of the characteristic peaks for opal (2θ:22°and 35.8°)in this cement mortar at the age of 16days provides evidence that reactive silica was attacked and subsequently dissolved.Furthermore,the amount of portlandite in this mortar at the age of 16days was semi-quantitatively observed to beTable 4Elemental composition determinate by EDXDaysP ⁎Na 2O ⁎⁎(%)SiO 2⁎⁎(%)CaO ⁎⁎(%)Al 2O 3⁎⁎(%)Ca/Si Na/Si Si/Al Na/Al OPC116124.8128.1736.05 1.3 1.37 1.70––2 2.5110.5978.16–7.90.46––90324.6761.6610.90–0.180.77––FA116410.2245.06–39.08––0.970.4390515.8859.62–17.67–– 2.86 1.47622.0863.4010.20–0.170.67––OPC216719.8965.8412.52–0.20.58––OPC316832.1039.6822.86–0.61 1.56––90921.1064.8911.84–0.190.63––FA3901010.9562.17–17.96–– 2.9311119.8265.7111.93–0.190.58––P ⁎:Analysis number.(1:gel with a high sodium content;2:portlandite plates;3:ASR product with a “rod-type ”morphology;4:product of alkaline activation (quasi-amorphous gel)5:zeolitic crystalline compounds;6:ASR product with a “rosette/type ”morphology;7:ASR product with a “rosette/type ”morphology;8:ASR product with a “rod-type ”morphology;9:ASR product with a “rosette/type ”morphology;10:zeolitic crystalline compounds 11:ASR product with a “pseudo rosette/type ”morphology.⁎⁎Content in%.Fig.3.(a)XRD patterns at 16and 90days of OPC1mortars (b)XRD patterns at 16and 90days of FA1mortars (q quartz;c calcite;m moscovite;b biotite;A alite;p portlandite;a plagioclases;cl chlorite;hs hydroxysodalite;anl analcime;zp zeolite P;hr herschelite).179I.García-Lodeiro et al./Cement and Concrete Research 37(2007)175–183much smaller than the content in the OPC1mortar at the same age (see Fig.3(a)).The 16-day SEM/EDX analysis clearly revealed the existence of ASR product,here with a rosette-type morphology,(see Fig.7)previously described in the literature [21].The Ca/Si and Na/Si ratios for these crystals were 0.2and 0.58,respectively.3.3.3.Mortars prepared with the “pessimum ”proportion aggregate mixFig.8(a)shows the XRD traces for the OPC3cement (“pessimum ”proportion)mortars.As in the case of OPC1mortar,the portlandite peaks declined steadily throughout the test,very likely indicating calcium uptake by the gel generated in the alkali –silica reaction.Immersion in 1M NaOH at 85°C prompted a decline in plagioclase signal intensity,indicating that the aggregate was under attack.Furthermore,these diffracto-grams contained no peaks corresponding to opal (reactive silica),which is therefore believed to be attacked very rapidly.Fig.8(b)shows the diffractograms for mortar FA3at 16and 90days,in which zeolitic compounds were identified,as in the case of the FA1mortar.The zeolites present at the age of 16days were the same as in mortar FA1,but semi-quantitative findings indicated much larger amounts of these crystals in FA3.At this age zeolite P was found to be the predominant crystalline species (with a higher Si/Al ratio than the zeolites observed earlier).This may be explained by the 10%of highly reactivesilica in this mortar,which greatly increased the amount of silica available in the initial system.As in the preceding case,analcime was the predominant species at the age of 90days.The OPC3aggregates were observed to be slightly affected in the 16-day SEM/EDX study.Considerable microcracking was detected throughout the matrix,along with an ASR product with rod-type morphology;see Fig.9(a),and high silica content (Table 4,point 8).At the age of 90days,when the expansion limit had been vastly exceeded,see Fig.2(c),the specimens were severely cracked and rge amounts of ASR products with different morphologies (rod and rosette)were detected.The only species found in 16-day FA3mortars,in turn,was a typical zeolite precursor generated in alkaline activation,while at 90days substantial amounts of zeolitic crystalline products were observed,primarily in the gaps in the matrix.Very occasional ASR products with pseudo-rosette morphology (Fig.9(c))were also seen at this age,which might explain the slight expansion detected in these mortars.4.DiscussionThe ASR-induced deterioration of concrete is a serious problem with no simple solution.While there is no lack of traditional methods that attempt to prevent the alkali –silica reaction,such as the choice of appropriatelynon-reactiveFig.4.Photomicrograph of OPC1mortar (a)At 16days.(P1)Gel with a high sodium content (P2)Portlandite plates (b)at 90days.(P3)Cracks in the matrix and ASR product with a “rod-type ”morphology.Fig.5.Photomicrograph of FA1mortar (a)at 16days.(P4)product of alkaline activation;(b)At 90days,zeolitic crystalline compounds in the matrix (P5);ASR product with a “rosette-type ”morphology (P6).180I.García-Lodeiro et al./Cement and Concrete Research 37(2007)175–183aggregate,the use of cementitious materials with a low alkaline content,the addition of pozzolanic materials and so on,the fact is that this problem is increasingly present in Portland cement concrete [22].Furthermore,repair of the structures affected is a very costly undertaking.In this regard,the research reported hereunder is amply justified.Perhaps the first feature of this study to merit comment is the “extreme ”aggressiveness of the ASTM C1260test to which the samples were subjected,which caused expansion in the Portland cement systems even when a potentially non-reactive aggregate was used.A previous study found that this aggregate actually contained a certain amount of moderately reactive silica [19].Despite the intensity of the attack,however,the activated fly ash systems exhibited no significant expansion.A considerable number of very varied test methods to determine the potential reactivity of aggregates can be found in the extensive literature on the ASR.Berra et al.[23],for instance,conducted a comparative study of different accelerated test methods:chemical tests based on ASTM standard C 289;tests based on XRD analysis and petrographic examination of the aggregate;mortar specimen trials based on the NBRI test (ASTM C 1260)and so on.This last method,which was employed in the present study,affords several advantages:it detects potential aggregate reactivity in a short period of time (16days)and provides quantitative data on expansion.In short,although fairly aggressive,the test is very useful for the relatively rapid comparison of different systems.In addition,the interpretation of the results described in this study also entailed an in-depth review of the role of calcium in the formation of expansive ASR products.The role of calcium is the object of a good deal of controversy,although the presence of this element would appear to be fundamental.Several authors [7,8]proved that the addition of Ca(OH)2promoted ASR-induced expansion and others [13]showed that there is a direct relationship between portlandite uptake and the formation of ASR gels.In the present case,all the conditions required for the formation of expansive products were found in the Portland cement (control)system media.A sufficient supply of alkalis was provided by the NaOH solution,while the chief source of calcium was the portlandite released during cement hydration.The intensity of the quartz and plagioclase signals in the materials made with Portland cement were found to decline significantly throughout the test,much more acutely in theOPC3mortars.This is a clear indication that the aggregate was attacked by the alkaline solution in which the mortars were immersed.Similarly,the amount of portlandite present in both systems (OPC1and OPC3)at the age of 16days was observed to drop significantly,reaching undetectable levels after 90days.The calcium in the portlandite was incorporated into ASR-generated expansive gels.The SEM/EDX study corroborated the formation of alkali –silica reaction products with different morphologies,very similar to the findings described by other authors [20,21]:rod-shaped (typical of gels with high sodium content,normally associated with the early stages of the alkali –silica reaction)and rosette-type crystals (described by Davis and Oberholster [21]).While these crystals had different Ca/Si and Na/Si ratios,the calcium content was fairly high in all (10–12%,(see Table 4)).The alkali-activated fly ash systems,on the contrary,tended to form a quasi-amorphous gel (Fig.5(a))with high alkali content as the majority reaction product and zeolite crystals such as herschelite,analcime and so on as minority phases,see Fig.5(b).Since zeolites normally “precipitate ”into pre-existing pores in the matrix,their growth generates no stress that might lead to microcracking.As in the case of the cement systems,the waning intensity of the X-ray diffraction peaks for the various crystalline phases present in FA1and FA3mortars provided evidence that the aggregates were under attack.But new zeolite crystalline phases were also found to appear with time.SEM/EDX analysis of the composition of the phases comprising the FA1and FA3mortars tested as described in ASTM standard C 1260showed that,unlike the products generated by Portland cement hydration,they had a very low calcium content.All the foregoing is believed to be largely responsible for attenuating and retarding the expansion in such systems see Fig.2(a),(c).In fly ash-based systems,then,the alkalis were found to be able to interact in two competitive reactions:in the first,they are taken up to activate the vitreous component of the initial ash and convert it into a cementitious material or even form zeolite crystals;but at the same time they may be involved in a second reaction that attacks the aggregate.In the fly ash systems studied here,the primary reaction was clearly alkali activation that led to the formation of an inorganic polymer and a few zeolitic crystalline phases from the vitreous component of theoriginalFig.6.XRD patterns of opal aggregate and OPC2mortar at 16days Q Quartz;o opal;p portlandite;ccalcite.Fig.7.Photomicrograph of OPC2mortar at 16days.ASR product with a “rosette-type ”morphology.181I.García-Lodeiro et al./Cement and Concrete Research 37(2007)175–183ash with respect the alkali –aggregate reaction it also runs along the time.Fig.8is an evidence of how quartz from aggregate is dissolved with time.However due to lack of calcium in these systems,the AAR product is not expansive.Consequently,all the ash systems tested expanded less than the corresponding Portland cement systems,(see Fig.2).Indeed,only system FA3exceeded the 0.1%expansion limit,and only after 90days.Three months in such an aggressive medium may suffice for all the ash to have reacted,leaving the aggregate with no “protection ”against the alkaline environment.But even then,since the amount of calcium in the ash systems is minimal,only tiny volumes of expansive ASR gels are formed.Furthermore,Fig.8(b)shows that in this case,after 90days many zeolitic crystalline compounds are formed,with the zeolite analcime ultimately predominating.Such harsh system conditions (with specimens immersed in NaOH for 90days at 85°C)induce the formation of zeolitic compounds.Given that some of these,such as analcime,are icositetrahedral crystals that can grow to a very large size inside the matrix,the possible generation of internal stresses by the crystals themselves cannot be ruled out.But this only occurs when the conditions are as aggressive as those tested here.Under real life cir-cumstances the inorganic polymer crystallizes into zeolite very slowly and as these minerals are usually found in gaps in the matrix,the existence of stress that might generate cracking is unlikely.Finally,another finding that reveals the differences in reactivity between the two cementitious systems in the face of an alkaline attack is the complete destruction of FA2mortar specimens (ash +100%opal aggregate)when subjected to the ASTM standard C 1260test.The key issue to be considered here is that the ash (essentially vitreous silica)was mixed with a large amount of opal,a highly reactive aggregate.The conditions prevailing in the system were therefore already very extreme when the specimens were immersed in 1M soda at a temperature of 85°C.Inasmuch as these mortars consisted practically entirely of reactive silica,they were attacked in the alkaline medium.5.ConclusionsThe primary conclusion to be drawn from this study is that cementitious systems based on activated fly ash are less susceptible to generate expansion by alkali –silica reactionthanFig.8.(a)XRD patterns of OPC3mortar at 16and 90days.(b)XRD patterns of FA3mortar at 16and 90days.(q quartz;c calcite m ;moscovite;b biotite;A alite;p portlandite;a plagioclases;cl chlorite;hs hydroxysodalite;anl analcime;zp zeolite P;hrherschelite).Fig.9.Photomicrograph of OPC3mortar (a)At 16days.ASR product with a “rod-type ”morphology (b)at 90days.Matrix aggregate interface.ASR product.Photomicrograph of FA3mortar (c)at 90days.ASR product with a “pseudo rosette-type ”morphology.182I.García-Lodeiro et al./Cement and Concrete Research 37(2007)175–183。