Gradings of non-graded Hamiltonian Lie algebras
Sufficient Conditions for a Digraph to be Hamiltonian

1 Introduction
For convenience of the reader we provide all necessary terminology and notation in one section, Section 2. There are many conditions which guarantee that an undirected graph is Hamiltonian. Almost all of them deal with degrees or degree sums of certain subsets of vertices, or with sizes of unions of neighbourhoods of certain subsets. Some conditions involve only certain pairs of non-adjacent vertices.
The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely

Theor Appl Genet (2011) 122:1017–1028DOI 10.1007/s00122-010-1506-3ORIGINAL PAPERThe Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genesBin Yuan · Chun Zhai · Wenjuan Wang ·Xiaoshan Zeng · Xiaoke Xu · Hanqiao Hu ·Fei Lin · Ling Wang · Qinghua PanReceived: 14 August 2010 / Accepted: 22 November 2010 / Published online: 12 December 2010© Springer-Verlag 2010Abstract The blast resistance gene Pik-p, mapping to the Pik locus on the long arm of rice chromosome 11, was iso-lated by map-based in silico cloning. Four NBS-LRR genes are present in the target region of cv. Nipponbare, and a presence/absence analysis in the Pik-p carrier cv. K60 excluded two of these as candidates for Pik-p. The other two candidates (KP3 and KP4) were expressed in cv. K60.A loss-of-function experiment by RNAi showed that both KP3 and KP4 are required for Pik-p function, while a gain-of-function experiment by complementation test revealed that neither KP3 nor KP4 on their own can impart resis-tance, but that resistance was expressed when both were introduced simultaneously. Both Pikp-1 (KP3) and Pikp-2 (KP4) encode coiled-coil NBS-LRR proteins and share, respectively, 95 and 99% peptide identity with the two alle-les, Pikm1-TS and Pikm2-TS. The Pikp-1 and Pikp-2 sequences share only limited homology. Their sequence allowed Pik-p to be distinguished from Pik, Pik-s, Pik-m and Pik-h.Both Pikp-1 and Pikp-2 were constitutively expressed in cv. K60 and only marginally induced by blast infection.IntroductionPlants have developed a multifarious defense system against their various viral, bacterial, fungal, nematode and insect pathogens. The W rst line of their defense is raised by pattern recognition receptors, which respond to pathogen-associated molecular patterns by initiating a basal defense response (Nurnberger et al. 2004; Zipfel and Felix 2005). This response is generally able to prevent infection by non-host pathogens. Some pathogens have evolved e V ector pro-teins and/or toxins, which inhibit this basal defense response, allowing the pathogen to colonize the plant (Jones and Dangl 2006; Zipfel 2008). Their second line of defense is activated by resistance (R)genes, which encode R proteins able to recognize speci W c e V ector molecules produced by the pathogen and then trigger a response within the plant cell (Da Cunha et al. 2006; Jones and Dangl 2006), R proteins typically confer race-speci W c resis-tance, and their expression is frequently associated with a hypersensitive response. Pathogen e V ectors able toCommunicated by B. Keller.B. Yuan andC. Zhai contributed equally to this work.Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1506-3) contains supplementary material, which is available to authorized users.B. Yuan ·C. Zhai · W. Wang · X. Zeng · X. Xu · H. Hu · F. Lin · L. Wang · Q. Pan (&)Laboratory of Plant Resistance and Genetics,College of Resources and Environmental Sciences,South China Agricultural University,Guangzhou 510642, Chinae-mail: panqh@Present Address:B. YuanHubei Academy of Agricultural Science,Wuhan 430064, ChinaPresent Address:X. XuGuangdong Institute of Microbiology,Guangzhou 510070, ChinaPresent Address:H. HuGuangdong Ocean University,Zhanjinag 524088, Guangdong, Chinasuppress this response are the products of avirulence (Avr) genes. Numerous R genes have been isolated from a range of plant species (Fu et al. 2009; Krattinger et al. 2009; Liu et al. 2007a; Martin et al. 1993), and their analysis has shown that they fall into several well-de W ned classes. The nucleotide-binding site leucine-rich repeat proteins (NBS-LRR) is the largest of these (McHale et al. 2006). Their N termini comprises either a Toll-interleukin receptor (TIR)-like domain or a coiled-coil (CC) structure, so allowing for the recognition of the TIR-NBS-LRR and the CC-NBS-LRR subtypes. The Arabidopsis thaliana genome includes 159 NBS-LRR genes (both the TIR and the CC type are represented), while the rice genome includes 535 exclusively CC-NBS-LRR genes (Meyers et al. 2003; Zhou et al. 2004). In both rice and A. thaliana, most of the NBS-LRR genes occur in clusters and some in tandem arrays. Some clusters feature a heterogeneous population of genes, while others contain groups of highly homologous sequences (Leister 2004; Richly et al. 2002). In a few cases, functionality requires the simultaneous presence of two R genes (Ashikawa et al. 2008; Lee et al. 2009; Loutre et al. 2009; Sinapidou et al. 2004).Rice blast (causative pathogen Magnaporthe oryzae) is one of the most devastating diseases of rice (Ou 1985). Over 80 genes encoding resistance to various combina-tions of blast races have been documented (Ballini et al. 2008; Yang et al. 2009) and so far 13 have been isolated and characterized. Except for two (Pi-d2 and pi21), all are of the NBS-LRR type. Pi36 and Pid3 are single copy genes (Liu et al. 2007b; Shang et al. 2009), while Pita, Pib, Pi37 and Pit are members of a gene family (Bryan et al. 2000; Hayashi and Yoshida 2009; Lin et al. 2007; Wang et al. 1999). Pi9, Pi2 and Piz-t map to a single locus, but the Pi9 sequence is only weakly related to that of the allelic pair Pi2/Piz-t(Qu et al. 2006; Zhou et al. 2006). Rice chromosome 11 carries a large number of R genes, with 106 containing the protein kinase domain and 102 the NB-ARC domain (Rice Chromosomes 11 and 12 Sequencing Consortia 2005). Speci W cally, the chromosome carries the blast resistance genes Pik, Pik-p, Pik-s, Pik-g, Pik-h and Pik-m, along with the bacterial blight resistance genes Xa4 and Xa26 (Ashikawa et al. 2008; Kiyosawa 1987; Pan et al. 1998; Sun et al. 2003, 2004; Wang et al. 2009; Xu et al. 2008; Yang et al. 2003). Pik, Pik-p and Pik-m are probably allelic (Hayashi et al. 2006). The resistance spectrum of Pik-m is broader than those of Pik, Pik-p or Pik-s among Japanese, but not necessarily among Chinese pathogen populations (Kiyosawa 1987; Wang et al. 2009). The objective of the present study was to isolate and charac-terize Pik-p, which confers stable and strong resistance against both Japanese and Chinese isolates (Wang et al. 2009).Materials and methodsCandidate gene identi W cationPik-p has been mapped within a 126-kb interval of rice chromosome 11 plus a contig gap (Wang et al. 2009). The gene content in the equivalent interval of cv. Nipponbare was predicted using GENESCAN (/ GENSCAN.html) and FGENESH (http://www.softberry. com). DNA sequence comparisons were performed by pair-wise BLAST (/BLAST/bl2seq/ bl2.html) and protein sequence similarities obtained by BLASTP (Altschul et al. 1997). Transcripts of the genes present in the critical interval were sought in both the GenBank (/blast) and the rice full-length cDNA database (http://cdna01.dna.affrc.go.jp/cDNA). The presence/ absence of candidate genes was established by PCR, wherever there was a large insertion/deletion in the target region between the reference (cv. Nipponbare) and the donor (cv. K60) genomes (Figs.1 and S1).RNAi constructs and transformationTwo Pikp-1 cDNA fragments (1,246–1,730 bp from the start codon, within the NBS domain, and 2,526–3,275 bp corresponding to the LRR domain, see Fig.3a) and three cDNA Pikp-2 fragments (89–833 bp, corresponding to the CC domain, 1,163–1,748 bp, corresponding to NBS domain, and 2,546–3,061 bp, corresponding to LRR domain, see Fig.4a) were ampli W ed using primer sets, KP3i2 F/R, KP3i1 F/R, KP4i1 F/R, KP4i2 F/R and KP4i F/R, respectively (see Table S1). The W rst four of these fragments were ligated into the pDS1301 vector (Chu et al. 2006) and the W fth into the pANDA vector (Miki and Shimamoto 2004). The constructs were introduced into Agrobacterium tumefaciens strain EHA105 by electroporation (GenePulser Xcell, Bio-Rad, Hercules, CA) and then transformed into the Pik-p carrier cv. K60, as described by Lin and Zhang (2005). T0 and T1 plants were inoculated with blast isolate CHL381, and dis-ease reactions were scored as described by Pan et al. (2003). RT–PCR was used to verify that Pik-p expression was absent in susceptible T0 plants. The T1 progeny segregated as one resistant to three susceptibles and were randomly sampled to establish the correlation between disease pheno-type and the presence of the transgene (Fig.2a, b). Statisti-cal testing of the silencing e Y ciency achieved by the various RNAi fragments was carried out using a z test with Excel (Table1; Microsoft Corp., Redmond, WA).Candidate gene cloning and transformationFour overlapping fragments (3.5, 6.5, 5.7 and 6.4 kb; see Fig.S2) were ampli W ed using Phusion, a high-W delityTaq polymerase (NEB, England). After an A-tailing procedure, the 3.5-kb product was ligated into pMD20-T (TaKaRa, Dalian, China), and the other three were digested with Asc I and inserted into the pCAM-BIA1301AscI vector. The sequences of the four resulting recombinant plasmids (KP3-3.5, KP3-6.5, KP34-5.7 and KP4-6.4) were assembled by DNAStar software (http:// ). The Kpn I-Mlu I KP3-3.5, Mlu I-Sal I KP3-6.5 and Sal I-Asc I KP34-5.7 fragments were intro-duced in tandem into pCAMBIA1301AscI to form a full-length Pikp-1 insert of length 12,432 bp (Fig. S2). The full-length Pikp-2 insert (10,459 bp) comprised a combi-nation of KP34-5.7 and KP4-6.4 (Fig. S2). The 16,919 bp KP3+4 construct was built from the Kpn I–Bam HI (KP3), Bam HI–Sal I (KP3), Sal I–Bsp HI (KP4) and Bsp HI–Asc I (KP4) fragments ligated into pCAMBIA1301AscI (Fig.S2). The three constructs were independently trans-formed into the blast-susceptible cv. Q1063 as above. All T0plants were tested for their reaction to blast infection using isolates CHL381 and CHL346. The selected T0 plants were then tested for the presence of both right and left border markers. T1 progeny segregating as three resis-tant to one susceptible were randomly sampled and subjected to co-segregation analysis.Analysis of full-length cDNA and predicted protein sequencesThe cDNA 5Ј end sequences were obtained by RACE-PCR, using a SMART RACE cDNA ampli W cation kit (Clontech, Mountain View, CA), following the manufacturer’s instructions. The KP3 and KP45Ј RACE products were ampli W ed using nested PCR [W rst reaction using primers KP3-5RACE1 and KP4-5RACE1 with the universal primer A mix provided by the kit; the second PCR using KP3-5RACE2 and KP4-5RACE2 primers and the nested univer-sal primer A (NUP) from the same kit (see Table S1; Fig. S2)]. The 3Ј end sequences of KP3 and KP4 were obtained using a GeneRacerTM kit (Invitrogen, Groningen, The Netherlands) as described by Lin et al. (2007). Intermediate RT–PCR fragments were obtained using primer sets KP3ORF F/R and KP4ORF F/R for, respectively, KP3 and KP4, which overlap the 5Ј RACE and 3Ј RACE fragments of each gene. The RACE and intermediate RT–PCR prod-ucts were all cloned into pMD-20 (TaKaRa) for sequenc-ing. Sequence data of Pik-p gene has been deposited in GenBank as accession HM035360.The compute pI/Mw tool (http://www.expasy.ch/ tools/pi_tool.html; Gasteiger et al. 2005) was used topredict the isoelectric point (pI) and molecular weight of each translation product. CC structure was predicted using either COILS (/software/COILS_form.html ; Lupas et al. 1991) or Paircoil2 (/cb/paircoil2; McDonnell et al. 2006)software.Single nucleotide polymorphism (SNP) assayThe coding sequences of the susceptible cv. Q1063 and the Pik -m carrier cv. Tsuyuake, and the Pik -p carrier cv. K60were aligned using Multalin (http://bioinfo.genotoul.fr/multalin/multalin.html ) software. To validate the resulting putative SNP sites, primers were designed to create dCAPS markers able to distinguish Pik -p from Pik , Pik -m , Pik -s and Pik -h .Characterization of the race speci W city of the transgenic linesThe resistance of four T 2 lines carrying the construct KP3+4 was tested for race speci W city. Five reference lines,each carrying one of Pik , Pik -s , Pik -m , Pik -h or Pik -p (Kobayashi et al. 2007), as well as the susceptible recipient cv. Q1063, were used as a control. Eight blast isolates were selected for the test (Table 2). Blast inoculation and disease evaluation were carried out according to Pan et al.(2003).Gene expressionTwo-week-old seedlings of cv. K60 (Pik -p )and cv. Tsu-yuake (Pik -m )were inoculated with isolate CHL381 andPhenot yp eRR S S S S S S S H 2O DNA 2 4 11 15 18K60M 5 17 18 21 23nSR Pikp-1n1 2 3 5 25 5 9 10 11 13H 2ODNAK60M Phenot yp eRSSRSSR SSSSPikp-2M H 2O V213456R S S MS S Sp M H 2O V213456Phenot yp e R R S S S S S 7M H 2O V2134567RRSSSSSRRPhenot yp eSSSSSp 2134567M V Q 1063 1 2 3 4 5 6 7 8 910 MM V Q 1063 1 2 3 4 5 6 1 2 34 5 6 MPhenot yp e R R S R R R S R RSPhenot yp e R S R R R R R S S R R Rheld in the dark at 25°C with 100% relative humidity for 20h in an inoculation incubator. Inoculated leaves were sampled at the time of inoculation and then at 12, 24 and 72h post-inoculation. Total RNA was isolated from leaves using the TRIzol reagent (Invitrogen, Carlsbad, CA),following the manufacturer’s instructions. Quantitative reverse transcription PCR (qRT-PCR) was performed in two steps: W rst, »1 g total RNA was treated with RNase-freeTable 1Characterization of the coupled genes of Pik-p through loss and gain of function analyses **Signi W cant di V erences (at the =0.01 level) in the silencing e Y ciency among constructs containing di V erent fragments of the coupled genes for a 2aAbbreviations for candidates/constructs are KP3, Pikp -1; KP4, Pikp -2; KP3+4, Pikp -1+Pikp -2; RNAi, RNA interference. The detailed informa-tion on the constructs are shown in Figs.3, 4, S2bLoss-of-function-constructs were transformed into the Pik -p carrier cultivar K60, and gain-of-function-constructs were transformed into the highly susceptible cultivar Q1063c The detailed information on the primer sequences and restriction sites for constructing each vector is shown in Table S1d T 0 plants derived from each construct were inoculated with the Pik -p -avirulent isolate, CHL381 and CHL346. R resistant, MR moderately resistant, MS moderately susceptible, S susceptible eThe ratios for loss- and gain-of-function analyses were calculated as (MS +S)/(R +MR +MS +S) and (R +MR)/(R +MR +MS +S),respectivelyCandidate/construct a Recipient cultivar bExpected size (bp)Vector cReaction of T 0 plants d Success ratio (%)eRMRMSSLoss of function KP3 RNAi1K60485pDS130125*********.5**KP3 RNAi2K60750pDS130134123455822.0KP4 RNAi K60745pANDA 1274368147.2**KP4 RNAi1K60585pDS13011707255932.2KP4 RNAi2K60516pDS130131428322915.1Gain of function KP3Q106312,432pCAMBIA1301AscI 0011360KP4Q106310,459pCAMBIA1301AscI 0011080KP3+4Q106316,919pCAMBIA1301AscI7752230120.2Table 2Reactions of the W ve reference lines, each carrying one of the Pik alleles and four transgenic T 2 plants derived from the construct KP3+4,as well as the susceptible recipient cultivar Q1063 to eight isolates of Magnaporthe oryzae S susceptible, R resistant, MS moderately susceptible, MR moderately resistant, ND not determinedHostGeneM. oryzae isolates CHL22CHL346CHL42CHL272CHL446CHL503CHL508CHL995Reference lines IRBLk-Ka Pik S R S S S R ND S IRBLks-S Pik -s S S S S MS S R S IRBLkm-Ts Pik -m R R R S MR R R S IRBLkh-K3Pik -h R R R S R R R S IRBLkp-K60Pik -pRRSSSSSSTransgenic lines KP34-21-2Pikp -1+Pikp -2R R S S S S S S KP34-29-8Pikp -1+Pikp -2R R S S S S S S KP34-33-9Pikp -1+Pikp -2R R S S S S S S KP34-51-7Pikp -1+Pikp -2RRSSSSSSThe recipient cv.Q1063NoneSSSSSSSSDNase I (Promega, Madison, WI) and reverse transcribed by M-MLV (Promega, Madison, WI). Then, a 1- l aliquot of the reaction was used as the template for a qRT-PCR. The primer sets, RRT5 and RRT17 (Ashikawa et al. 2008; see Table S1), were employed to detect Pikp-1 and Pikp-2 expression. Rice Actin1 and the pathogenesis-related probe-nazole-inducible PBZ1 gene were used as internal controls (Ryu et al. 2006; Table S1). The qRT-PCR analysis was performed on a Bio-RAD CFX96 Real-Time PCR Detec-tion System device, using SYBR Premix EX TaqTM (TaKaRa, Dalian, China).ResultsIdenti W cation of candidates for Pik-pThe location of Pik-p has been placed within the genetic interval de W ned by the markers K39 and K28 (Fig.1a). In the cv. Nipponbare genome sequence, this interval is cov-ered by the four BAC clones, OSJNBb0049B20, OSJNB a0047M04, OSJNBa0036K13 and OSJNBb0018L01 (Fig.1b), and contains 25 predicted genes. Of these, four (KP1, KP2, KP3 and KP4) are of the NBS-LRR type, and so were taken as being the most likely candidates for Pik-p(Fig.1c). No ESTs corresponding to KP1 and KP2 could be identi W ed, but the EST CA763104 matched the 3Ј region of KP3, while the full-length cDNA AK073759 matched KP4. Thus, it appeared likely that both KP3 and KP4—but neither KP1 nor KP2—were expressed in cv. Nipponbare. Both KP1 and KP2 are absent in the Pik-m carrier cv. Tsu-yuake (Ashikawa et al. 2008). Ampli W cation of cv. K60 genomic DNA using PCR primer pairs targeted at KP1 and KP2 (sequences based on the cv. Nipponbare sequence, see Fig.1 and Table S1) showed that neither is present in cv. K60 either, leaving KP3 and KP4 as the two strongest can-didates for Pik-p(Figs.1c, d, S1). A number of PCR primer pairs were then designed to amplify both KP3 and KP4 from cv. K60, but the only successful ampli W cation was a 2.6-kb KP4 fragment. Extension of this sequence to »5.2kb was achieved using hi-TAIL PCR (Liu and Chen 2007), and this sequence proved to be 96% homologous to Pikm2-TS, but only 48% to Pikm6-NP (both are alleles of Pikp-2; see Figs.1, S6). Using the sequence of cv. Tsuyuake as the basis for primer design (rather than cv.Nipponbare) led to the successful ampli W cation of both the KP3 and KP4 sequences from cv. K60 (see Table S1 and below).Loss- and gain-of-function analyses for the candidate genes To determine whether KP3 or KP4 (or neither) was Pik-p, RNAi was applied to both candidates. All W ve RNAi constructs were properly expressed and abolished KP3 and KP4 expression in T0 plants (Table1; Figs.2a, S3a, S3b). The presence of the transgene and the response to pathogen infection were fully correlated among the T1 progeny (Fig.2b). The most e V ective RNAi fragment was derived from the 3Ј region of the gene, within its LRR domain (Table1; Figs.3a, 4a). Thus, both KP3 and KP4 appear to be required for Pik-p function.The three constructs KP3, KP4 and KP3+4 (Fig. S2) were then transformed into the highly susceptible japonica cv. Q1063 (Lin et al. 2007) to con W rm Pik-p function by forward complementation. A total of, respectively, 137, 109 and 406 independent T0 plants were generated. When challenged with blast isolates, CHL381 and CHL346 (avir-ulent on cv. K60), all the single gene transformants remained fully susceptible, but 83 of the 406 double gene transformants were resistant. This con W rmed that the pres-ence of both genes is needed to specify Pik-p resistance (Table1; Fig.S4). To test the correlation between pres-ence/absence of both genes and resistance/susceptibility, 53 of the 406 T0 plants were scored for the presence/absence of the right and left border markers (see Table S2; Fig. S2). Of these, 39 resistant and 11 susceptible plants carried both border markers, and two susceptible ones carried one or other of the markers; only one susceptible plant lacked both markers, indicating that the majority of the T0 plants carry-ing the complete construct (KP3+4) did not express any resistance in the recipient cultivar background when driven by their native promoters. On the other hand, the presence of the transgene and the response to pathogen infection were also fully correlated among the T1 progeny (Fig.2c). Molecular characterization of Pik-pFull-length Pikp-1 (KP3) and Pikp-2 (KP4) cDNAs were obtained by a combination of RT- and RACE-PCR (Fig. S2), and compared to their coding sequences. Pikp-1 con-tains an 83-bp 5Ј and a 163-bp 3Ј untranslated region (UTR) and two introns (150 and 2,772 bp) within its open reading frame (ORF) (Fig.3a). It encodes a 1,142-residue polypeptide with an estimated molecular weight of 126.7 kDa and a pI of 6.1. Four characteristic NBS family motifs are present: GLPGGGKTTVAR (beginning at residue 290), KKYLIVIDDIW (beginning at residue 376), DLG-GRIIMTTRLNSI (beginning at residue 402) and EDNPCY DIVNMCYGMPLALIW (beginning at residue 461), which correspond, respectively, to kinase 1a (P-loop), kinase 2, kinase 3a and motif3 (GLPL) (Fig.3b). COILS and Pair-coil2 analyses suggested the presence of a CC domain (P=0.9) between residues 146 and 177. The C-terminal region includes 16 imperfect LRR repeats, composed of »14% leucine, while the remaining 103 residues represent the C-terminal non-LRR (CtNL) region.Pikp-2 contains a 264-bp 5Ј-UTR and a 283-bp 3Ј-UTR, with two introns; the W rst is of length 882 bp and lies within the 5ЈUTR, ending 109 bp upstream of the ATG start codon, while the second is 164-bp long, and interrupts the ORF (Fig.4a). Its predicted gene product is a 1,021-residue polypeptide of molecular weight 114.6 kDa and a pI of 8.6. Its NBS domain contains six conserved motifs: kinase 1a (VLSIVGFGGVGKTTIA: beginning at residue 206), kinase 2 (LEQLLAEKSYILLIDDIW, beginning at residue 323), kinase 3a (GGRIIVTTRFQAV, beginning at residue 358), GLPL (EQVPEEIWKICGGLPLAIV, beginning at residue 415), RNBS-D (CLLYLSIFPKGWK, beginning at residue 488) and MHDV (KTFQVHDMVLEYI, beginning at residue 553) (Fig.4b). Paircoil2, but not COILS,predicted the presence of a CC domain between residues 27 and 57. The C-terminal region of the protein consists of 13 imperfect LRR repeats composed of »17% leucine.The levels of peptide sequence identity between Pikp-1 and Pikm1-TS and Pikm5-NP were 95 and 59%, respec-tively (Fig. S5), while those between Pikp-2 and Pikm2-Ts and Pikm6-NP were 99 and 76%, respectively (Fig. S6). The peptide sequences of Pikp-1 and Pikp-2 could not be aligned, as their level of similarity was lower than 23%. A comparison with the equivalent cDNA sequences ampli W ed from the susceptible cv. Q1063 and the Pik-m carrier cv. Tsuyuake revealed four potential SNPs in Pikp-1 (A222V, V230E, P251D and K261N) and three in Pikp-2 (E230D, S434T and V627M). To determine which of these weregenuinely allele speci W c, a set of reference lines, which are known carriers of Pik, Pik-p, Pik-m, Pik-s and Pik-h, as well as of Pia, Pii, Piz-t and Pita, were genotyped. The out-come was that Pik-p could be distinguished from all the above R genes using a combination of the two SNPs assays, T1-783A/G at Pikp-1 and A2-1879G at Pikp-2 (see Figs.3, 4, 5, S5, S6).Race speci W city of resistance in the transgenic linesThe W ve reference lines for the Pik alleles, Pik, Pik-s, Pik-m, Pik-h and Pik-p, reacted di V erentially to infection by the eight blast isolates (Table2). This showed that these iso-lates were able to successfully distinguish between the vari-ous Pik alleles. Since the disease reaction of the four T2lines carrying both Pikp-1 and Pikp-2 was identical to that of IRBLkp-K60 (Pik-p), the presence of Pikp-1 and Pikp-2 clearly imparts the same phenotype as that of Pik-p.Pikp-1 and Pikp-2 expression in cv. K60The transcription level of both genes was assessed in cv. K60 at four time points after blast inoculation. Both Pikp-1 and Pikp-2 transcripts were detected prior to exposure to the pathogen (Fig.6). The e V ects of mock- and pathogen inoculation were to reduce the transcription level over the W rst 24h, implying a response to stresses resulting from both inoculation and incubation in a humidity chamber (Fig.6). Expression of Pikp-1 and Pikp-2 appeared to increase only marginally during the 72h following eithermock or genuine inoculation. The expression patterns of both Pikm1-TS and Pikm2-TS (the alleles present in cv.Tsuyuake) were similar to those in cv. K60, but in contrast that of the pathogen-inducible gene, PBZ1 (Ryu et al.2006), in cv. Tsuyuake di V ered in several respects (Fig.6).DiscussionTwenty-one R genes have been mapped to date onto rice chromosome 11, including the six rice blast resistance genes Pik , Pik -s , Pik -m , Pik -p , Pik -h and Pik -g , all of which have been shown by classical genetic analysis to be allelic to one another (Ballini et al. 2008; Yang et al. 2009). With regard to the race speci W cities and/or resistance spectra of those alleles, Kiyosawa (1987) found that there are “stair-type” resistances among the alleles in the Japanese blast pathogen population, and ranked the strength of these alle-les in the order Pik -m >Pik >Pik -p >Pik -s .This, in turn,indicated that Pik , Pik -p and Pik -s all form part of the larger or stronger allele, Pik -m (Kiyosawa 1987). Among Chinese isolates, the same ranking was found in Fujian,Yunnan, Jiangsu and Heilongjiang, but not in Guandong,Fig.5Pik -p -speci W c SNP assay. SNP assay for a T1-783A/G and b T2-1879G. Lane 1 cv. K60 (Pik -p ), lane 2 IRBLkp-K60 (Pik -p reference line), lane 3 IRBLkm-Ts (Pik -m reference line), lane 4 IR-BLk-Ka (Pik reference line), lane 5 IRBLks-S (Pik -s reference line),lane 6 IRBLkh-K3 (Pik -h reference line), lane 7 IRBLa-A (Pia refer-ence line), lane 8 IRBLi-F5 (Pii reference line), lane 9 IRBLzt-T (Piz -t reference line) and lane 10 IRBLta-K1 (Pita reference line). M Size marker DL200012345678910MM abExpression analysis of Pik alleles. Transcription of Pik -m in cv. Tsuyuake and Pik -p in cv. K60 as compared by qRT-PCR. Leaf RNA was sampled before inoculation and then at 12, 24 and 72h after inoculation with either blast isolate CHL381 or water. The speci W cRNA content of each sample was estimated from the mean of three rep-licate qRT-PCRs. Outcomes of mock inoculations are shown as black bars and those of pathogen inoculations as hatched ones0 h12 h24 h72 h0 h12 h24 h72 h0 h 12 h 24 h 72 hPikp-1Pikp-2PBZ10 h 12 h 24 h 72 h0 h 12 h 24 h 72 h0 h 12 h 24 h 72 hHunan, Guizhou, Sichuan, Jiangsu, Liaoning or Jiling. In the latter regions, many isolates that were virulent against Pik-m were avirulent against Pik and Pik-p, which has been taken to indicate that the Pik alleles Pik-m, Pik and Pik-p (and perhaps other Pik alleles as well) are, indeed, indepen-dent R genes able to condition di V erential reactions against various isolates (Wang et al. 2009). The isolation of Pik-m and Pik-p has revealed that they are allelic and indepen-dent. They can be distinguished from one another on the basis of both their gene structure and their sequence. Thus, Pikp-1 and Pikm1-TS di V er from one another with respect to the length of their introns and a three base pair indel (Fig.3), while Pikp-2 carries an intron in its 5Ј UTR, which is not present in Pikm2-TS (Fig.4). Furthermore, Pik-p could be distinguished from other alleles, as well as from R genes at other loci with a combination of two SNP assays (Fig.5).Pikp-1 and Pikp-2 are closely linked to one another, but their sequence and structure are quite distinct. The presence of a CC domain in the Pikp-1 product was identi W ed by both COILS and Paircoil2 software, but this was less certain for the Pikp-2 protein. Secondly, the Pikp-2 NBS domain car-ries the motifs RNBS-D and MHDV, which are absent in Pikp-1. Thirdly, the Pikp-1 protein has a CtNL region, which Pikp-2 lacks. Fourthly, although in common with most rice NBS-LRR genes, both Pikp-1 and Pikp-2 contain an intron in their NBS kinase 2 motif (see Figs.3, 4; Bai et al. 2002), and a gene-speci W c intron is present in the Pikp-1 CC domain (Fig.3) and in the Pikp-25ЈUTR (Fig.4). Thus these two genes, although physically closely linked with one another, may well be functionally di V erent and certainly are evolutionarily distant from one another (Ashikawa et al. 2008; Bai et al. 2002; Sinapidou et al. 2004). The Pikp-1 alleles appear to be more polymorphic than the Pikp-2 ones (Figs. S5, S6), suggesting that Pikp-1 may be under greater pathogen selection pressure than Pikp-2. As the CC and NBS regions of Pik-p and Pik-m are so divergent (unlike their LRR region) (Figs. S5, S6), the indication is that race speci W city is probably determined at the CC and/or NBS, rather than at the LRR, a situation which is reproduced at the wheat Lr10 gene (Loutre et al. 2009).The reverse genetics approach RNAi has been widely used to test for loss of function (Chu et al. 2006; Miki et al. 2005; Peart et al. 2005; Peng et al. 2009). To work success-fully, it is necessary for the RNAi sequence to both e V ec-tively and speci W cally inhibit the expression of the target gene. A major e V ect on silencing e Y ciency has been shown by a range of sequences directed at various sites along a single mRNA, but the 3Ј mRNA cleavage acts as the most e V ective siRNAs (Holen et al. 2002). Here, we tested two Pikp-1 and three Pikp-2 fragments for their e Y cacy as RNAi sequences (Figs.3, 4). The most e V ective fragment in each gene was also identi W ed at the 3’ ORF region, cor-responding to the LRR domain (Table1; Figs.3, 4). These results are of general interest in furthering the understand-ing of the function and speci W city of the NBS-LRR R genes.Already, W ve R genes have turned out to be in fact a cou-pled pair. An e V ective allele of the A. thaliana RPP2 gene requires the presence of both RPP2A and RPP2B, separated from one another by »5kb and arrayed in the same orienta-tion (Sinapidou et al. 2004). RPP2A is unusual in that it has only a short LRR domain at its C-terminus, preceded by two incomplete TIR-NBS domains, whereas RPP2B has all the components expected for a full TIR-NBS-LRR class R gene. In both tetraploid and hexaploid wheat, the presence of the two adjacent, but distinct, CC-NBS-LRR genes Lr10 and RGA2 is required to confer leaf rust resistance (Loutre et al. 2009). Whereas Lr10 is a normal CC-NBS-LRR gene show-ing a measurable degree of sequence diversity in its CC domains, RGA2 is similar with RPP2A in its possession of two NBS domains and relative paucity of sequence diversity In rice, Pi5-1 and Pi5-2 are both necessary to confer race-speci W c blast resistance in the line RIL260 (Lee et al. 2009). They are arrayed in opposite orientations, separated from one another by some 15 kb of sequence, and their sequences are highly divergent from one another. The last two examples relate to Pikm1-TS/Pikm2-TS and Pikp-1/Pikp-2, where, in both cases, the pair is arrayed in opposite orientation and sep-arated by only »1kb. This small separation suggests that both genes may be under the control of a common, bidirec-tional promoter, such as the one driving A. thaliana cab1 and cab2 (Mitra et al. 2009). The evidence, such as it is from a small number of examples, is that coupled R genes consist of sequences that are divergent from one another, although both may well belong to the NBS-LRR class. It appears unlikely that these gene pairs can have evolved from one another fol-lowing an initial duplication event (Sinapidou et al. 2004).The allelism of Pik-p with Pik-m raises a number of questions regarding the Pik locus. Are Pik, Pik-h also alle-lic to Pik-p/Pik-m? Do they, like Pik-p/Pik-m, consist of a pair of coupled genes? How do these coupled genes evolve, particularly with respect to generating their allele-speci W c resistance? And how do they interact with one another, and then with their equivalent pathogen Avr genes? Acknowledgments We are grateful to Dr K. Shimamoto for his provision of the panda vector. Financial support was provided by the National 973 project and the National Transgenic Research Projects.ReferencesAltschul SF, Madden TL, Scha V er AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402。
Diminishing returns

In the processed foods industry, manufacturers delight in releasing recipes that require their product. The maker of Ritz crackers, for example, has a “Mock Apple Pie” recipe (see ) that adorned its boxes for years—a recipe that replaces apples with lemon-juice-soaked Ritz crackers. It uses an awful lot of crackers. Very interesting when you consider that apples are easily available in the targeted markets, regardless of season; that the crackers cost as much or more than the required apples; and that few people would prefer the taste of damp, lemon-soaked crackers to real apples.The same behavior occurs in the computer industry.The biggest proponents of computer graphics research these days aren’t the graphics companies, but compa-nies who have invested heavily in the development of general-purpose hardware, software, and operating sys-tems and need a way to sell their “crackers.” The com-puter technology of five years ago performed quite adequately for most household tasks (word processing and tax preparation), so manufacturers need a way to sell in a saturated market.Collaborative virtual reality research is driven simi-larly. The companies making the greatest investment,both through in-house development and funding of external research programs, are those with similar depth of investment in network infrastructure. They need a way to sell their bandwidth.Are next-generation computer graphics and net-worked VEs just modern computing’s version of mock apple pie?Diminishing returnsSpecial-purpose graphics hardware technology has improved steadily, and graphics software has leveraged off of spectacular developments in general-purpose hard-ware. General frameworks and interfaces for graphics production have undergone the scrutiny of the entire community, resulting in a suite of effective standards.Striving towards solving the rendering problem has served computer graphics researchers well for years.Likely another 20 years or more of research remains.The speed at which we’re drawing pictures (or warping depth images, or computing radiosity solutions) will double, and double again, and then again. Drawing today is a million times faster than it was for Sketchpad in 1963, with moderately improved realism.But at some point, when the rendering quality and speed have doubled yet again, no one will notice. The CPU industry is rapidly approaching that point: the pro-cessing speed of desktop computers has doubled eight times in the last 15 years. Most users who purchased first-generation Pentium II’s don’t need an upgrade now,even though available speed has doubled since. The lat-est attraction is the speed of the bus that conveys infor-mation to the processor, rather than the processor itself.Theorem 1 (Corollary to Moore’s Law):There exists a point in time after which Moore’s Law is still applicable but not relevant.The logical extrapolation of the current research focus in computer graphics is smooth, realistic, ubiquitous rendering capability. Clearly, there exists a point of diminishing returns for this line of inquiry. Now is pre-cisely the time to think about the next step.The medium isn’t the messageWe assume, then, that in the future any user’s display platform can render fantastically complex scenes.Having finally shed the concerns related to the computer graphics medium, developers will concentrate on the message. Content will be key—no longer will users accept nonsensical, artistically vacant environments simply because they’re presented in a head-mounted display.Theorem 2 (Technical Darwinism):With cutting-edge technology, someone is bound to get cut.This will also mean that static worlds, no matter how aesthetically pleasing, will come second to environ-ments offering interactive content. The development and provision of dynamic content lie at the heart of the problem we face. For an environment to attract signfi-cant and regular participation, it must react in an intel-ligent and unpredictable fashion. Today, that intelligence can come from only two sources: live human collaboration and computer-generated autonomy.Collaborative VE research combines graphics, net-working, human perception, and distributed computing issues. However, these facets betray a disappointingMichael Capps,Kent Watsen,andMichael Zyda NavalPostgraduate School0272-1716/99/$10.00 © 1999 IEEECyberspace and Mock Apple PieA Vision of the Future of Graphics and Virtual EnvironmentsProjects in VREditors: Michael Macedonia and Lawrence Rosenblum2November/December 1999lack of coordination. Most such pro-jects from graphics researchers con-sider the network a magical transport mechanism, while net-work scientists treat graphics con-tent as no different than any other. You don’t distribute a VE experience by opening sockets and slapping them together, any more than attaching a telephone wire to a microphone makes a conference out of a speech. Myriad issues relate to floor control, session manage-ment, awareness, and persistence, and many more besides.1 Perhaps most telling to the lack of consensus in shared VR research is in the name. We have CVE, net-CVE, shared VR, and shared VE referring to the general topic of multiuser, dis-tributed virtual experiences. We use the acronym CVE here, representing collaborative VEs.Theorem 3 (Law of Names): When a scientific research area has a single, generally acceptedname, the research in that area is either essen-tially unexplored, proprietary, or essentially com-plete.Computer-generated autonomy (CGA) will certainly become inextricably melded with computer graphics. The video-game industry already spends as much as two-thirds of product development effort on intelligence for characters—most of it not reused. Graphics researchers should hardly begin rediscovering the achievements of the artificial intelligence community—the situation calls for cooperation between the producers and consumers of CGA technology. While this article focuses on other aspects of CVEs, the National Research Council’s report on Modeling and Simulation2provides excellent recom-mendations for future avenues of research in CGA, such as behavior adaptability and human representation. Many of the infrastructure requirements for CGA-enhanced systems with a large number of synthetic actors are the same as those needed for large-scale CVEs. The space of spacesHaving demonstrated a logical consequence of the current research phase (rendering) and postulated that the next phase is CVE research, it remains for us only to conjecture on the future of CVEs. All indications are that every CVE, and networked electronic data of all kinds, will converge to a single environment inhabited simul-taneously and persistently by millions. This space of spaces is the real “cyberspace.”The pertinent issue, then, is exactly what fundamen-tal innovations will be required as steps along the way toward making that cyberspace a reality. Cyberspace can never shut down for maintenance, so it must sup-port dynamic extensibility to its control software.Current CVEs comfortably support only a few users, andthe most populated CVE applications provided limitedsupport for a few thousand, so scalability is crucial.Cyberspace will combine many environments and infor-mation sources, authored in a piecemeal fashion, socomposability is equally necessary. Figure 1 illustrates acyberspace consisting of many subspaces, where vastlydifferent participants can interact.Construction on the globally accessible network infra-structure has essentially just begun. Cyberspace awaitsthe ubiquity of realistic rendering and high-speed net-working; CVE systems developed before that time mustfail gracefully as we test the limitations of currentresources.Now we examine each of these requirements in turn,with special attention given to frustrating hindrancesand promising efforts.Dynamic extensibilityThe first worlds to constitute the core of cyberspacewon’t yet be technologically ready for immortality, butthey must be ever-present. The loss of key cyberspaceenvironments could prove just as devastating as the lossof a major World Wide Web gateway site. The failuremight have no far-reaching effects upon other sites, butwould certainly affect user experience. If the beta gen-eration is critical to the long-term success of the tech-nology, then finishing touches must take placetransparently.For successful interaction in cyberspace, its effectsmust persist. Just like a 24-hour eatery, the doors cannever close and the vacuuming must occur with cus-tomers present. And like a shopping mall, renovationsoccur during business hours, behind a privacy screenand a “Pardon Our Dust, but Work We Must” sign.IEEE Computer Graphics and Applications3 CSE-Case CSE-MonaCSE-iPort CSE-bdiMan CSE-iDog1Cyberspacecombines manyvirtual environ-ments,authored sepa-rately but com-posable forinteroperability.Each client-sideentity (CSE) canexport itsgeometry,behaviors, andinteractions.The develop-ment of eachCSE must be assimple as writ-ing a Web page.Supporting such dynamic modification of an execut-ing program, although essential, currently eludes our reach. Most of today’s extensible software simply includes a facility for module integration, allowing sta-tic linking of plug-ins upon initial execution. Figure 2presents a framework for a dynamically extensible client-side entity (CSE) for cyberspace. A key to this development is the abstraction from the hardware and operating system, permitting true cross-platform authoring.This multiple platform support is the single most frus-trating issue in engineering dynamically extensible sys-tems. Support for needed code-linking functionality varies widely across operating systems, as do the meth-ods for performing such feats. Cross-platform efforts such as the Java Virtual Machine, which abstract away the underlying platform, have proven moderately suc-cessful. So far, any such abstraction that aids us with these architectural issues concomitantly frustrates the performance tuning required for scalability and grace-ful degradation.Today’s large-scale Internet games provide excellent experimental data for our extensible CVE architecture.The fantasy world of Ultima Online (), for instance, supports thousands of users whose actions have a persistent effect on the world and the sto-ryline. The client-side software is extensible, with updates fetched over the network and patched in dur-ing initial connection to the world. However, server database maintenance requires daily system-wide downtime, and server software updates cause frustrat-ing (albeit less frequent) open-ended down periods.Ultima Online not only demonstrates the possibilities for remote software updates and persistence, it also exposes the need for true dynamic extensibility.ScalabilityScalability issues arise when we wish to build CVEs that support compelling interaction between large numbers of widely dispersed players. A number of successful enter-tainment-based CVEs support tens of players, and spe-cial-purpose systems (usually military) that can support up to a thousand have had limited success. But a thou-sand users won’t suffice for cyberspace, or even for the needs of current urban or military exercise simulations.Network characteristicsThe most significant hindrances to scalable delivery of content and events in CVEs stem from characteristics of available network technology, namely bandwidth,latency/delay, and reliability. Network infrastructure upgrades are constant but slow. While the typical cor-porate desktop machine has evolved many times in the past 10 years, sadly many of us have had the same 10-Mbit Ethernet network connection to that desktop dur-ing that time. Though we finally see next-generation networks deployed, at no time in the foreseeable future will the network allow pair-wise updates among thou-sands of users. It’s therefore necessary, for both the short and long term, to conserve bandwidth wherever possi-ble in CVE applications.Similarly, while we can reduce network delay, we can’t eliminate it, thanks to the poor signal propagation properties of light. Network delay causes a host of con-cerns for consistent, distributed delivery of contents and actions in a shared virtual world. For instance, an action already experienced by some participants may not yet have been delivered to others; accordingly, the order-ing of events may vary among clients. Causally related to this is the near impossibility of coordinating a discrete action between delayed clients. While some promising efforts attempt to ameliorate the effects of delay on per-ceived fidelity (such as time warping and event predic-tion), scaling a CVE geographically has an unavoidable cost. Note that a major factor in delay is the level of net-work saturation, so bandwidth conservation should con-tribute in a major way to the solution here as well.In the face of unreliable network delivery, CVE sys-tems must deal appropriately with the resulting data inconsistency—similar to the case of excessive delay.Further effort is required in researching dynamically reconfigurable systems to allow continued operation in the face of permanent network or node failure.Promising effortsAwareness management techniques can significantly reduce communications volume in CVEs and are vital for scalability. Given some indication of what informa-tion participants want, we must reduce communications to the minimum required for realism. Choice of tech-nique depends on system architecture, so often the architecture is selected to suit. For instance, message culling at a centralized server can prevent pair-wise communication between mutually uninterested partic-ipants. However, the centralized bottleneck topology isn’t particularly scalable, and it increases the latency of each communication. Performing filtering at the recip-ient eliminates that latency, with concomitant cost in bandwidth and client processing load. Participants can certainly filter messages locally to eliminate that delay,but recipient-side techniques have no effect on band-width consumption.Probably the simplest method for interest management involves regional interest. In such a system, a participant is only aware of (and therefore receiving update messages concerning) other participants within a certain distance or within a predefined grid area of the world. In fact, some systems completely encapsulate these regions as isolat-Projects in VR4November/December 19992Client-sideentity for cyber-space. Dynamic extensiblity requirements include an architecture-independent plug-in system.ed communities and don’t allow direct synchronous inter-action between separated participants. Other awareness managers use visual occlusion (or attenuation in other media), world topology, or the more promising method of task-dependent explicit functions.Computing interest as a continuous value gives the system more flexibility. For example, rather than send-ing full positional updates on unoccluded objects 95 per-cent obscured by fog, systems could instead transmit reduced fidelity (and reduced size) information. In fact, combined with a specification of a client machine’s net-work and processing capacity, this approach can offer different levels of realism to participants as appropriate. Various network transport protocols should be selected on a per-update, per-client basis, letting the system trade between communications overhead and reliability. ComposabilityComposability is the holy grail of the networked VE community. With it we have the ability to dynamically import models and behaviors from one VE into another. We wish to bring an object in over the network and have it instantly work properly in the context of the new vir-tual world. With such capability, we can imagine the entire user population participating in the authoring of cyberspace, similar to the participation we now see in the World Wide Web.Supporting that global participation requires either a single standard for the authoring of all digital infor-mation or established avenues of data interchange. Authors must have the flexibility to create in the mode most compatible with their creativity, and data should be stored in its most appropriate form.The key to supporting composability lies in the prop-er specification of VE components. Given a rich enough understanding of an object, its original environment, and the new environment, it’s perfectly possible to extrapolate the object’s behavior in the new environ-ment. So far it appears possible to provide sufficient description in only the most limited of domains.A popular example involves exchanging a basketball across virtual worlds. In an environment whose behav-ior mimics reality, the ball falls when dropped and bounces when it hits a hard surface. But in a new envi-ronment, the possibilities for interaction become end-less a world so warm that the ball bursts into flames, a world so cold that a dropped ball shatters, or a world so small that the ball brings widespread devastation with every bounce. It may not be possible to specify the semantics of object behavior in a universal manner, but object specification methods look promising for com-posability and interoperability.Representations of object behavior semantics fall into three categories: informal, formal, and analyzable. Informal semantics are text attachments that the partic-ipant is meant to understand, telling the behavior, mean-ing, or purpose of an item in a VE space. In the previous example, “This is a basketball” would serve nicely. Until major problems are solved in natural language under-standing, however, no program or automated agent can use such semantics to assist in navigating or using a VE. Formal semantics are expressed in some notation manipulable by algorithm. Thus, any computer lan-guage can represent formal semantics, and in fact thishappens in practice. The meaning or behavior of anobject is stored as a code block, then the semantics arerealized by execution. However, nearly the same prob-lem of automated understanding exists for this extremeas for semantics in plain text. The portability of code ina programming language is just as questionable asinformal semantics in a particular human language.Analyzable semantics are formal semantics expressedin a notation less powerful than arbitrary code. The goalis to use a notation that some inference engine can oper-ate on, thereby allowing automated agents to “under-stand” the behavior of objects and make decisions basedon the encoded information. If this problem were solved,then other problems in CVE composition could beattacked on a deeper level. We see no broad, effectivesolutions to the analyzable semantics problem, thoughcomponents of a future solution are being researched.Effective definition and use of semantics depends ona common knowledge base and domain of discourse forthe participants in a CVE. The term “ontology” denotesthis shared semantic domain. An ontology specifies con-ceptualizations used to help programs and humansshare knowledge. In practice it consists of definitions of representational vocabulary, including axiomatic the-ories. Such procedures, along with semantics processingtools, must be integrated into upcoming CVE projectsfor composability of VEs to become a reality. Equallyimportant, behavior and interaction protocols must bepresented as abstract data to a VE, rather than buriedthroughout the implementation, if those protocols areto be extended through dynamic composition.End gameWe hope these suggestions give a hearty push towardbuilding the cyberspace of which we can now onlydream. We’re barely on the edge of the developmentswe expect in the next century, and nowhere close towhat science fiction has told us to expect. Now we needto build and discard prototypes, and occasionally fieldour successes.Experience is our apple pie. With the advent of cyber-space we’ll have yet another recipe with a substitutionfor the world around us—essentially, lemon-soaked arti-ficial experience. Still, cyberspace will carry us to realmswe cannot even conceive today, just as television has.We humbly suggest that no one allows their children tolog on for more than two hours a night.IReferences1.S. Singhal and M. Zyda, Networked Virtual Environments—Design and Implementation, Siggraph Series, ACM PressBooks, New York, 1999.2.National Research Council, Modeling and Simulation: Link-ing Entertainment and Defense, M. Zyda and J. Sheehan,eds., National Academy Press, Washington, D.C., 1997.Contact the authors by e-mail: capps@,kent@, and zyda@.IEEE Computer Graphics and Applications5。
Determination of total and unbound warfarin

Journal of Chromatography A,1314 (2013) 54–62Contents lists available at ScienceDirectJournal of ChromatographyAj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o maDetermination of total and unbound warfarin and warfarin alcohols in human plasma by high performance liquid chromatography with fluorescence detectionTommaso Lomonaco a ,Silvia Ghimenti a ,Isabella Piga a ,Massimo Onor b ,Bernardo Melai a ,Roger Fuoco a ,Fabio Di Francesco a ,c ,∗aDepartment of Chemistry and Industrial Chemistry,University of Pisa,Pisa,Italy bInstitute of Chemistry of Organometallic Compounds,CNR,Pisa,Italy cInstitute of Clinical Physiology,CNR,Pisa,Italya r t i c l ei n f oArticle history:Received 23April 2013Received in revised form 23August 2013Accepted 25August 2013Available online 31 August 2013Keywords:WarfarinWarfarin alcoholsHigh performance liquid chromatography Sample preparation techniquesa b s t r a c tTwo analytical procedures are presented for the determination of the total content and unbound fraction of both warfarin and warfarin alcohols in human plasma.Chromatographic separation was carried out in isocratic conditions at 25◦C on a C-18reversed-phase column with a mobile phase consisting of a 70%buffer phosphate 25mM at pH =7,25%methanol and 5%acetonitrile at a flow rate of 1.2mL/min.Fluores-cence detection was performed at 390nm (excitation wavelength 310nm).Neither method showed any detectable interference or matrix effect.Inter-day recovery of the total warfarin and warfarin alcohols at a concentration level of 1000ng/mL was 89±3%and 73±3%,respectively,whereas for their unbound fraction (at a concentration level of 10ng/mL)was 66±8%and 90±7%,respectively.The intra-and inter-day precision (assessed as relative standard deviation)was <10%for both methods.The limits of detection were 0.4and 0.2ng/mL for warfarin and warfarin alcohols,respectively.The methods were successfully applied to a pooled plasma sample obtained from 69patients undergoing warfarin therapy.© 2013 Elsevier B.V. All rights reserved.1.IntroductionWarfarin [3-(␣-acetonylbenzyl)-4-hydroxycoumarin,WAR],the most common anticoagulant drug,is prescribed for the treat-ment of many diseases and the prevention of thromboembolic events [1].WAR is a weakly acidic drug (p K a of 5.19at T =25◦C)with an enolic group (Fig.1a)[2].It has an asymmetric carbon center and is commercially available as a racemic mixture.The two enantiomers (R-WAR and S-WAR)have a different anticoag-ulant potency (S-WAR is 3–4times more potent than R-WAR),metabolism and interaction with other drugs [3,4].WAR is com-pletely absorbed after oral administration (F =1),is highly bound to site I of albumin (≈99%)with a high affinity (K d =3.4±0.7M),has a small volume of distribution (0.11–0.18L/kg),and low clearance (0.0005–0.0079L/h/kg)[5–8].Only the unbound fraction can carry out a therapeutic action and cross the biological membranes (e.g.salivary glands [9]).The drug is metabolized in the liver by cytochrome (CYP)P450to inactive hydroxylated metabolites (OH-WAR)(major pathway)and by ketone reductases to warfarin∗Corresponding author at:Department of Chemistry and Industrial Chemistry,University of Pisa,Pisa,Italy.Tel.:+390502219308;fax:+390502219260.E-mail address:fdifra@dcci.unipi.it (F.Di Francesco).alcohols (WAROHs,Fig.1b)[10].This reduction is more effec-tively catalyzed by nicotinamide adenine dinucleotide phosphate (NADPH)than by nicotinamide adenine dinucleotide (NADH [11]).This latter reduction generates a second chiral center (C-11)and makes two pairs of diastereoisomers possible,namely RS/SR-Warfarin alcohols and RR/SS-Warfarin alcohols [12,13].After the administration of a single dose of WAR,S-7-hydroxywarfarin and RS-Warfarin alcohol are the two major metabolites observed in human plasma [14].Gebauer reported that 7-OH-WAR is an inactive metabolite with a half maximal inhibitor concentration (IC 50)of 250M,whereas WAROHs show a low anticoagulant activity with IC 50of 12.5M (IC 50of WAR is 2.2M)[15].Around 40million patients are currently undergoing WAR ther-apy and this number has been increasing steadily [16].Positive clinical outcomes during WAR therapy depend on maintaining the level of WAR within a narrow therapeutic range.This is challeng-ing due to the large inter-and intra-individual variability in patient responses,as many factors (e.g.diet,liver disease,comorbidities,other drugs,and genetic variability)may interact with the ther-apy [17].Furthermore,after about 60years of clinical use,the anticoagulation mechanism of WAR has never been completely clarified and the possible role of WAROHs has received little atten-tion in the literature.A few studies indicate that the anticoagulant activity of WAROHs may be an additional variable involved in0021-9673/$–see front matter © 2013 Elsevier B.V. All rights reserved./10.1016/j.chroma.2013.08.091T.Lomonaco et al./J.Chromatogr.A1314 (2013) 54–6255Fig.1.Structures of WAR(a)and WAROHs(b)(*asymmetric center).Carbon4 ,10,6,7,8are the hydroxylation sites of WAR by cytochrome P450.the anticoagulant mechanism of WAR[13].However,pharmaco-logical data concerning these metabolites are scarce.Lewis et al. reported that the plasmatic concentrations of WAROHs after a sin-gle dose(100mg)were steady for20days,and the long half-life may strengthen its potency and contribution to the overall antico-agulant activity.Unlike WAR,WAROHs are directly eliminated by renal excretion without any further transformation[13].The international normalized ratio(INR)is the primary assay used in monitoring WAR therapy[18].When a patient is started on WAR therapy,INR monitoring should be performed on a daily basis until the INR is within the therapeutic range and should be performed two or three times a week for one to two weeks[19]. Therapeutic monitoring is essential in some situations to assess anticoagulant affecting factors,especially when the INR is diffi-cult to target.Knowledge of the plasma concentration of WAR and WAROHs is valuable for clinical decisions and enables severe intox-ication due to interference with other drugs and food to be treated effectively[18].Sensitive methods to measure WAR concentrations and its hydroxylated metabolites in biological samples have been pro-posed[19–21],but to our knowledge no study has been published on analytical methods for the determination of the unbound frac-tion of WAROHs in human plasma and other biologicalfluids.Most of the recently reported methods for measuring WAR in biological samples use high performance liquid chromatography(HPLC)with mass spectrometric detection(MS)[22,23].These methods have a high sensitivity,but are too expensive for routine analyses in a clinical laboratory.WAR has the advantage of a nativefluorescence if excited at 310nm,which can be successfully exploited for detection at low concentrations[24].This work illustrates an HPLC method withfluorescence detec-tion(FLD)to determine the total content and unbound fraction of WAR and WAROHs in human plasma.These methods are not expen-sive,show a good recovery and sensitivity,and are thus suitable for use in pharmacokinetic studies and for therapy monitoring.The proposed methods are currently being used in a clinical trial,whose results will be published in a future paper.2.Materials and methods2.1.Chemicals and reagentsRacemic WAR,i.e.3-(␣-acetonylbenzyl)-4-hydroxycoumarin sodium salt(purity≥98.0%),sodium borohydride(purity≥98.0%), sulfuric acid(purity≥99.5%),ethanol(purity≥99.5%)and deuter-ated methanol were purchased from Sigma Aldrich(Milan,Italy). Sodium phosphate monobasic(purity≥99.0%),potassium phos-phate dibasic(purity≥99.0%),dichloromethane(purity≥99.5%), hexane(purity≥95.0%),formic acid(purity≥99.5%),acetoni-trile and methanol were purchased from Sigma Aldrich at HPLC grade.HPLC grade water was produced by a Milli-Q Reagent Water System(Millipore,USA).The thromboplastin reagent (HemosIL RecombiPlasTin2G),thromboplastin diluent(HemosIL RecombiPlasTin2G Diluent),calibration plasma samples,normal control assay,low and high abnormal control assays used both for INR measurements and quality control were supplied by the Instrumentation Laboratory(Milan,Italy).2.2.Synthesis of warfarin alcoholsWAROHs were obtained in our laboratory by reduction of a racemic WAR standard solution with sodium borohydride[25]. WAR(a):1H NMR(CD3OD,ı/ppm):7.98–7.94(1H,m),7.67–7.61 (1H,m),7.41–7.18(7H,m),4.12(1H,dd,J=0.7Hz,CH at C-9),2.22 (2H,ddd,J=30.0,14.0and6.9Hz,CH2at C-10),1.74(3H,s).13C NMR(CD3OD,ı/ppm):207.1(1C,carbonyl at C-11),163.0,159.9, 152.7,143.7,131.7,128.1,127.8,127.1,126.8,125.9,125.7,124.0, 123.9,122.7,99.5,42.8,26.4,24.6.ESI-QTOF-MS:m/z309.1[M+H]+.WAROHs(b):1H NMR(CD3OD,ı/ppm):7.99–7.95(1H,m), 7.60–7.50(1H,m),7.50–7.14(7H,m),4.70–4.63(1H,m,CH at C-11), 3.71–3.68(1H,m,CH at C-9),2.37(2H,m,CH2at C-10),1.27–1.19 (3H,m).13C NMR(CD3OD,ı/ppm):161.2,159.9,152.7,142.9,131.6, 127.9,127.6,127.5,125.8,125.6,125.2,123.7,122.9,122.6,116.0, 65.8(1C,hydroxyl at C-11),40.2,22.8,22.4.ESI-QTOF-MS:m/z 311.1[M+H]+.Carbonyl reduction at C-11generates a second chiral carbon, which makes four stereoisomers(RR,RS,SR,and SS)possible.These can be seen as two pairs of diastereoisomers(RR-SS and RS-SR), with a different chromatographic behavior.Fig.1shows the struc-tures of WAR(1a)and WAROHs(1b).The HPLC separation of the reduced solution revealed that the reaction with sodium borohydride was completed and WAROHs were generated.The product of sodium borohydride reduction was confirmed by comparing the1H NMR and13C NMR spectra of WAR and WAROHs.2.3.Plasma samplesPlasma samples were obtained from the local oral anticoagu-lant center with the permission of the Ethics Committee of the local hospital(AOUP,Pisa,Italy).Human whole blood samples were col-lected into vacuum tubes containing109mM(3.2%)sodium citrate (Vacutest Kima,Italy)and immediately centrifuged at3000rpm for10min to obtain platelet-poor plasma.The plasma sample was divided into two aliquots:thefirst was immediately used for the determination of INR,whereas the second was stored at−80◦C together with the nominally healthy volunteer samples until the WAR and WAROHs were measured.2.4.Preparation of calibration standards and quality control samplesA phosphate buffer solution(PBS)(1M,pH=7.0)was prepared by dissolving18.30g of sodium phosphate monobasic and49.20g56T.Lomonaco et al./J.Chromatogr.A1314 (2013) 54–62of potassium phosphate dibasic in water.This solution was diluted to25mM to obtain the PBS used for the HPLC analyses and for the preparation of standard solutions.Stock solutions of WAR(950g/mL)and WAROHs (1060g/mL)were prepared by dissolving weighed amounts of the pure compounds in water and methanol respectively.The solutions were used to prepare another stock solution containing WAR(96g/mL)and WAROHs(105g/mL)in PBS25mM,which was further diluted with PBS to obtain standard working solutions of WAR and WAROHs at1,2,5,10,15,20,30,100,200,500,1000, 1500,2000and3000ng/mL levels.All the solutions were protected from light and stored in a refrig-erator at4◦C.The stability of the working solutions over2months was investigated.Pooled patient plasma samples(PPPSs)were obtained by pool-ing samples from69patients undergoing WAR therapy,whereas control plasma samples(CPSs)were obtained from10volunteers not taking WAR.Aliquots of these latter samples were spiked with known amounts of WAR and WAROHs to obtain standard plasma samples(SPSs)at different concentration levels.SPSs were stored at−80◦C,and some were used to check the stability over2months and after thaw–freeze cycles.2.5.Sample preparationSeveral preparation procedures were tested to determine the concentrations in plasma of total WAR and WAROHs as well as the unbound fraction.2.5.1.Procedure A:precipitation of plasma proteins(totalWAR/WAROHs)A solution(1mL)containing equal volumes of plasma and ace-tonitrile was vortex-mixed(30s)and centrifuged at5000rpm for 5min at room temperature.The supernatant(10L)was injected into the HPLC system.2.5.2.Procedure B:protein precipitation and ultrafiltration(total WAR/WAROHs)A solution(1mL)containing equal volumes of plasma and ace-tonitrile was vortex-mixed for30sec and centrifuged at5000rpm for5min at room temperature.An aliquot of200L of super-natant was diluted to1mL with PBS25mM at pH=7.This solution was transferred into an Amicon tube(Amicon®Ultra-4,Millipore) with a molecular weight cut-off of3kDa and then centrifuged at 5000rpm for60min at20◦C.25L of the ultrafiltrate sample were injected into the HPLC system without any further treatment.2.5.3.Procedure C:liquid–liquid extraction(total WAR/WAROHs)A modified version of Naidong’s procedure was used[26].In brief,2mL of H2SO40.5M,500L of ethanol,and4mL of a mixture of dichloromethane/hexane(1:5,v/v)were added to a plasma sam-ple(500L).The resulting mixture was vortex-mixed for30s and then centrifuged at5000rpm for5min at room temperature.The upper organic phase was transferred to a screw top V-Vial(Sigma Aldrich,Italy)and evaporated to dryness under a gentle stream of nitrogen at room temperature.The vials were weighed in all steps of this extraction procedure.The residue was dissolved in1mL of PBS25mM at pH=7and then injected(15L)into the HPLC system.2.5.4.Procedure D:ultrafiltration(unbound fractionWAR/WAROHs)An aliquot of the plasma sample(1mL)was transferred into an Amicon tube(Amicon®Ultra-4,Millipore)with a molecular weight cut-off of3kDa and centrifuged at5000rpm for60min at20◦C.25L of the ultrafiltrate sample were injected into the HPLC system without any further treatment.2.6.EquipmentThe HPLC system(Jasco,Japan)was equipped with an autosam-pler(AS2055),a quaternary low-pressure gradient pump(PU 2089),afluorescence detector(FP2020),and an ultraviolet detector (UV2070).The column temperature was controlled by a HT3000 thermostat(ClinLab,USA).The HPLC system was controlled using ChromNAV TM software(Jasco,Japan).Chromatographic separation of WAR and WAROHs was car-ried out with a TC–C18150mm×4.6mm,5m reversed-phase column(Agilent Technologies,USA)connected to a TC–C18 12.5mm×4.6mm,5m guard column(Agilent Technologies, USA).A few selected samples were also analyzed by an HPLC-ESI-Q-TOF to validate the analytical methods.The LC–MS analyses were carried out using a1200Infinity HPLC coupled to a6530Accurate-Mass Quadrupole Time-Of-Flight(Q-TOF)mass spectrometer with a Jet Stream ESI interface(Agilent Technologies,USA).The HPLC system was controlled by MassHunter TM software(Agilent Tech-nologies,USA).Chromatographic separation of WAR and WAROHs was carried out with a Zorbax Extend–C1850mm×2.1mm, 1.8m reversed-phase column(Agilent Technologies,USA)con-nected to an Eclipse Plus12.5mm×4.6mm,5m guard column (Agilent Technologies,USA).NMR spectra were recorded by a Bruker Avence400MHz (Bruker Corporation,USA).Absorption andfluorescence spectra were recorded by a Lambda 25spectrophotometer(PerkinElmer,USA)and a LS45spectrofluo-rimeter(PerkinElmer,USA),respectively.A centrifuge Centrifugette4206(ALC,Italy)and a Centrifuge 5804R equipped with an A-4-44swinging bucket rotor(Eppendorf, Italy)were used for sample centrifugation.The plasma samples were centrifuged using an Amicon®Ultra-4 device(Millipore,Italy),equipped with a cap,afilter device,and a centrifuge tube.The INR measurements were carried out at the clinical chemical laboratory of the local hospital(AOUP,Pisa,Italy)by an automatic ACL TOP700system(Instrumentation Laboratory,USA)equipped with an autosampler.Spectrometric INR measurements were per-formed at a wavelength of671nm to minimize optical interference (e.g.hemoglobin and bilirubin).All data were analyzed using GraphPad Prism v.5(GraphPad Software Inc.,USA).2.7.Chromatographic and detection conditionsChromatographic separation was carried out by the Jasco HPLC in isocratic mode with a mobile phase consisting of25%methanol, 5%acetonitrile,and70%phosphate buffer25mM at pH=7at aflow rate of1.2mL/min.The total run time was20min.The column tem-perature was set at25◦C.Fluorescence detection was performed at excitation and emission wavelengths of310and390nm,respec-tively.A few selected samples were analyzed by HPLC-ESI-Q-TOF. Chromatographic separation was carried out by HPLC in isocratic mode with a mobile phase of35%acetonitrile and65%water con-taining1%formic acid at aflow rate of0.25mL/min at25◦C.The injection volume was4L.The ESI operating conditions were:dry-ing gas(N2,purity>98%):350◦C and10L/min;capillary potential 4.5kV;nebulizer gas35psig;sheath gas(N2,purity>98%):375◦C and11L/min.The fragmentor was kept at100V and the collision energy(CID)for the MS/MS experiments was20V.The collision gas was nitrogen(purity99.999%).High-resolution MS and MS/MST.Lomonaco et al./J.Chromatogr.A1314 (2013) 54–6257Fig.2.Full scan and product ion mass spectra of[M+H]+of WAR(a),RR/SS-Warfarin alcohols(b)and RS/SR-Warfarin alcohols(c).spectra were achieved in positive mode in the range100–350m/z. The protonated molecule[M+H]+with m/z311.1and309.1were monitored for WAROHs and WAR identification,respectively.The total HPLC-ESI-Q-TOF run time was10min.3.Results and discussionValidation of the proposed methods was performed in accor-dance with the IUPAC guidelines[27]and included an evaluation of interferences,matrix effect,calibration curves,limits of detection (LOD)and quantification(LOQ),intra-day and inter-day precision, stability,and recovery.3.1.Sample preparationThree preparation procedures were compared to determine total WAR and WAROHs in plasma samples.The aim was tofind the most simple and reliable sample preparation method to remove any endogenous components(e.g.proteins,salts,lipids)that might interfere with the analyte determination.Three standard plasma samples with a WAR and WAROHs concentration of1000ng/mL were prepared.Each of them was split into three aliquots and processed according to A,B and C procedures.Mean values(n=9) for each aliquots refer to three repeated injections.Procedure A(protein precipitation with1:1addition of ace-tonitrile)showed very good levels of recovery and precision for both WAR(95±2%)and WAROHs(100±2%).However,this treat-ment did not remove all the proteins,thus increasing the pressure in the column and shifting the retention time(data not shown). These drawbacks were avoided in procedure B by ultrafiltrating the samples at3kDa.Lower but still good levels of recovery and a better precision than the previous procedure were observed(WAR: 85±1%and WAROHs:86±1%).A liquid–liquid extraction(proce-dure C)with an acidification step to release WAR and WAROHs from plasma proteins was optimized by comparing single and mul-tiple extractions.The second extraction did not increase recovery58T.Lomonaco et al./J.Chromatogr.A1314 (2013) 54–62Fig. 3.HPLC chromatograms of a standard solution of WAR and WAROHs (100ng/mL).The product ions traces are m/z251.1and175.0for WAROHs(a)and m/z 251.1and163.0for WAR(b).Elution order and retention times:2(RR/SS-Warfarin alcohols,r t:3.8min),3(WAR,r t:6.0min),4(RS/SR-Warfarin alcohols,r t:8.4min).significantly,thus a single extraction was applied for the quantita-tive analysis of WAR and WAROHs in plasma.In fact,procedure C showed the highest efficiency in separating the analytes from the matrix,as well as good levels of recovery and precision(see Section 3.4.3).Procedure C was thus selected for a more in-depth evaluation of the analytical performance.The unbound plasmatic fraction of WAR and WAROHs was determined by sample ultrafiltration with a cut-off membrane. Unlike other studies[20,22,28],the proposed procedure did not require any further treatment.3.2.HPLC–ESI-Q-TOF assay for the determination of warfarin and warfarin alcoholsThe ESI-Q-TOF gave the optimum sensitivity for WAR and WAROHs in positive ionization mode(data not shown).The prod-uct ion scan of the protonated[M+H]+ion for WAR(m/z309.1) displayed clear product ions at m/z251.1and163.0,whereas for WAROHs(m/z311.1),product ions at m/z251.1and175.0were recorded(Fig.2).The HPLC chromatogram of a standard solution of WAR and WAROHs shows three well separated peaks with reten-tion time of3.8,6.0and8.4min(Fig.3).The linear regression of the peak area values versus concentra-tion wasfitted in the concentration range of1–1500ng/mL for both WAR and WAROHs.The seven-point calibration curves(n=3at each concentration)were evaluated by Deming regression analysis, and the best-fit models for WAR and WAROHs were respectively: y=(1750±10)x,(R2=0.9999)and y=(820±10)x,(R2=0.9999).A blank plasma sample spiked at a low level of1ng/mL of WAR and WAROHs was prepared and analyzedfive times with the procedure C.The LOD and LOQ values were calculated,in accordance with IUPAC guidelines[29],as three and ten times the standard deviation(s b)of the“low level spiked blank”,and were 0.6and1.9ng/mL for WAR,and0.8and2.8ng/mL for WAROHs. 3.3.HPLC-fluorescence assay for the determination of warfarinand warfarin alcohols3.3.1.Selection offluorescence wavelengthsWAR shows goodfluorescence when excited at310nm[24]. WAR contain acidic moieties whosefluorescence quantum yields depend on pH,which need to be adjusted to give maximumflu-orescence.Thefluorescence yield of WAR decreases significantly at acidic pH[30],whereas no data were available concerning the dependence of WAROHsfluorescence on pH.Absorbance andfluo-rescence spectra at different pH values(4,5,6and7)were recorded to obtain the maximumfluorescence intensity for WAROHs.Two standard solutions containing10g/mL and0.2g/mL of WAROHs were prepared in PBS25mM at pH=7.Three aliquots of each solu-tion(1mL)were acidified at pH4,5and6by adding10,7and 5L of phosphoric acid1M respectively.The absorbance spectra of the solution at10g/mL(Fig.4a)were recorded in the range 200–400nm with a scan rate of0.2nm/s,whereas thefluorescence spectra of the solution at0.2g/mL(Fig.4b)were recorded by excit-ing at310nm and measuring the emission in the range310–500nm with a scan rate of0.5nm/s.The optimum excitation(310nm)and emission(390nm)wave-lengths were selected for HPLC-FLD measurements.3.3.2.Chromatographic separationFig.5shows the HPLC chromatograms of standard solution of WAR and WAROHs(a),and control plasma samples(CPSs), and pooled patient plasma samples(PPPSs)ultrafiltered(b)and extracted(c).The chromatogram of the standard solutions of WAR and WAROHs shows three well separated peaks with retention times of11.1,13.1and14.6min(Fig.5a).The WAROHs peaks were identified according to Hermans and Thijssen[31].These authors separated the two diastereoisomers of WAROHs by thin-layer chromatography using silica gel and a mobile phase consisting of diethylether and acetic acid(99:1.5,v/v). In these conditions,the RS/SR-Warfarin alcohols traveled faster than the RR/SS-Warfarin alcohols,whereas the opposite is true in reversed phase chromatography[30].Considering the results of these studies and comparing the chro-matograms of PPPSs and standard solutions of WAROHs,the peaks at11.1and14.6min(Fig.5)were assigned to RR/SS-Warfarin alco-hols and the RS/SR-Warfarin alcohols,respectively.3.4.Analyticalfigure of merits3.4.1.Limit of detection(LOD)and quantitation(LOQ)A blank plasma sample spiked at0.5ng/mL of WAR and WAROHs was prepared and analyzedfive times with procedure C.The LOD and LOQ values were calculated in accordance with IUPAC guide-lines[29],as three and ten times the standard deviation(s b)of the “low level spiked blank”,and were0.4and1.3ng/mL for WAR,and 0.2and0.7ng/mL for WAROHs.3.4.2.Calibration curvesA working range of100–3000ng/mL was chosen for the total content determination of both WAR and WAROHs.The WAR plas-matic concentration range reported in the literature is about 0.5–2.5g/mL[21,32],thus this extended range allowed for a safety margin to detect any potential overdose due to drug-drug interactions[16].The seven-point calibration curves(n=3at each concentration)were evaluated by the Deming regression analysis,T.Lomonaco et al./J.Chromatogr.A1314 (2013) 54–6259Fig.4.Absorbance(a)andfluorescence(b)spectra of WAROHs at different pH values.Fluorescence spectra were obtained by exciting at310nm.Fig.5.HPLC chromatograms of a standard solution(10ng/mL)(a),and control plasma samples(CPSs,dashed line)and pooled patient plasma samples(PPPSs,solid line)ultrafiltered(b)and extracted(c).Elution order and retention times:1(7-OH-WAR,r t:6.8min),2(RR/SS-Warfarin alcohols,r t:11.1min),3(WAR,r t:13.1min),4 (RS/SR-Warfarin alcohols,r t:14.6min),and5(10-OH-WAR,r t:15.9min).and the best-fit models for WAR and WAROHs were respectively: y=(7750±10)x,(R2=0.9999)and y=(15,330±10)x,(R2=0.9999).The literature reports that the unbound plasmatic fraction of WAR is about1%of the total[33],thus a working range of 1–30ng/mL was chosen for both WAR and WAROHs.The seven-point calibration curves(n=3at each concentration)were evalu-ated by the Deming regression analysis,and the best-fit models for WAR and warfarin WAROHs were respectively:y=(13,100±10)x, (R2=0.9999)and y=(26,400±10)x,(R2=0.9999).3.4.3.Recovery and precisionProcedure C:liquid–liquid extraction:The extraction recovery of WAR and WAROHs from SPSs was calculated as the percentage of the analyte concentration measured in the extracted sample with respect to the initial spiked concentration.Three samples spiked at three different concentration levels(200,1000and 2000ng/mL)were prepared and each one was evaluated in tripli-cate within the same day and on three consecutive days.The WAR and WAROHs recovery and the corresponding intra-and inter-day relative standard deviation(RSD%)are reported in Table1.The dif-ference between WAR(89%)and WAROHs(73)recovery was likely due to different partition ratios.In fact,in procedure A,which is simply based on protein precipitation with acetonitrile without any partition equilibrium involved,the recovery was similar for both molecules(95for WAR,and100for WAROHs).These results confirmed that procedure C is suitable for a reliable determination of the total concentration of both analytes in human plasma.Procedure D:ultrafiltration:The recovery of WAR and WAROHs with procedure D was calculated in triplicate standard work-ing solutions at three different concentration levels(2,10and 20ng/mL).Standard solutions had to be used in this case because human plasma samples with a known amount of unbound WAR and WAROHs could not be produced and certified standards are not available.In fact,spiking an SPS with a warfarin solution results in a sample in which the unbound fraction is known to be about 1%.Small variations of this percentage,which might be possible due to differences between plasma samples,would result in large errors in the absolute values of the unbound fraction.The WAR and60T.Lomonaco et al./J.Chromatogr.A1314 (2013) 54–62Table1Extraction recovery,intra-and inter-day precision of the determination of WAR and WAROHs in plasma.Concentration(ng/mL)Recovery%Intra-day a RSD Recovery%Inter-day b RSDExpected MeasuredWAR191167862%882%928830894%893%19071670872%882%WAROHs208153705%743%1010740724%733%20801500713%722%a Calculated from three replicates at each concentration value.b Calculated from three replicates at each concentration in three days.Table2Recovery,intra-and inter-day precision of the determination of WAR and WAROHs in standard solutions.Concentration(ng/mL)Recovery%Intra-day a RSD Recovery%Inter-day b RSDExpected MeasuredWAR 1.9 1.3659%6812%9.6 6.3655%668%19.612.9662%664%WAROHs 2.1 1.9872%916%10.49.4859%907%21.419.1911%894%a Calculated from three replicates at each concentration value.b Calculated from three replicates at each concentrations in three days.WAROHs recovery and the corresponding intra-day and inter-day relative standard deviation(RSD%)are reported in Table2.The precision was also estimated in SPSs at the three different concentration levels(200,1000and2000ng/mL).Assuming that the unbound fraction was1%,the estimated WAR and WAROHs concentrations in the ultrafiltrate samples were2,10and20ng/mL. The inter-and intra-day relative standard deviations are reported in Table3.The estimated recoveries of the two methods were used to cal-culate the real concentrations of WAR and WAROHs in plasma samples.3.4.4.InterferenceThe possible effect of endogenous interfering substances on the measured concentrations of WAR and WAROHs(total and unbound fraction)was investigated by comparing results obtained by HPLC-fluorescence and HPLC–ESI-Q-TOF in triplicate on SPSs with a concentration of1000ng/mL and on PPPSs with unknown concentrations.The concentration values obtained by the HPLC–ESI-Q-TOF,assumed as reference values,and the HPLC-fluorescence were in good agreement in all the samples(with differences always lower than10%).Table3Intra-and inter-day precision of the determination of unbound WAR and WAROHs in SPS samples.Estimatedconcentration(ng/mL)Intra-day a RSD Inter-day b RSDWAR 1.94%9%9.33%6%18.74%9%WAROHs 2.14%6%10.13%7%20.42%8%a Calculated from three replicates at each concentration value.b Calculated from three replicates at each concentrations in three days.3.4.5.Matrix effectThe shape and position of the WAR and WAROHs peaks were not affected by the presence of the plasma matrix(Fig.5),which suggests the absence of any detectable matrix effect.This hypothe-sis was confirmed by comparing the slopes of the calibration curves obtained with working solutions,ultrafiltered and extracted PPPSs (Table4).For both methods,these slopes were not significantly different at a confidence level of95%.3.4.6.StabilityThe stability of both standard working solutions and human plasma samples was evaluated following the experimental plan reported in Table5.The concentrations of each analyte at t=0h were used as the reference value.The sample’s stability was eval-uated by analysis of variance(ANOVA)at a confidence level of95%. Standard working solutions were stable throughout the duration of a typical sequence of chromatographic analyses(storage in the autosampler for about24h at room temperature)and for at least2 months at4◦C.In fact,no significant variations in concentrations were observed.The results also showed that the total concentration of WAR and WAROHs in human plasma samples was stable after two cycles of thaw–freeze,whereas the unbound plasmatic fraction showed a decrease of about20%after the second thaw–freeze cycle. No significant degradation occurred in the extracted or ultrafil-tered samples throughout the typical sequence of chromatographic analyses(storage in the autosampler for about24h at room tem-perature)and for at least2months of storage at4◦C.Similar results were obtained for both total and unbound concentrations of plasma samples processed after24h storage at room temperature.The results are consistent with data reported in the literature for WAR [19–34].3.4.7.Robustness of the methodA+10%and−10%variation in the volume of H2SO4(0.5M), ethanol,and the mixture of dichloromethane/hexane(1:5,v/v) were tested in the analysis of a plasma sample spiked with 1000ng/mL of WAR and WAROHs to verify the robustness of the。
Methodological Underestimation of Oceanic Nitrogen

Methodological Underestimation of Oceanic Nitrogen Fixation RatesWiebke Mohr*,Tobias Großkopf,Douglas W.R.Wallace,Julie LaRocheMarine Biogeochemie,Leibniz-Institut fu¨r Meereswissenschaften(IFM-GEOMAR),Kiel,GermanyAbstractThe two commonly applied methods to assess dinitrogen(N2)fixation rates are the15N2-tracer addition and the acetylene reduction assay(ARA).Discrepancies between the two methods as well as inconsistencies between N2fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N2.Here we demonstrate that the15N2-tracer addition method underestimates N2fixation rates significantly when the15N2tracer is introduced as a gas bubble.The injected15N2gas bubble does not attain equilibrium with the surrounding water leading to a15N2concentration lower than assumed by the method used to calculate15N2-fixation rates.The resulting magnitude of underestimation varies with the incubation time,to a lesser extent on the amount of injected gas and is sensitive to the timing of the bubble injection relative to diel N2fixation patterns.Here,we propose and test a modified15N2tracer method based on the addition of15N2-enriched seawater that provides an instantaneous,constant enrichment and allows more accurate calculation of N2fixation rates for both field and laboratory studies.We hypothesise that application of N2fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget.Citation:Mohr W,Großkopf T,Wallace DWR,LaRoche J(2010)Methodological Underestimation of Oceanic Nitrogen Fixation Rates.PLoS ONE5(9):e12583.doi:10.1371/journal.pone.0012583Editor:Zoe Finkel,Mt.Alison University,CanadaReceived July2,2010;Accepted August11,2010;Published September3,2010Copyright:ß2010Mohr et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Funding:This work is a contribution of the Collaborative Research Centre754‘‘Climate-Biogeochemistry Interactions in the Tropical Ocean’’(www.sfb754.de), which is supported by the German Research Association.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.Competing Interests:The authors have declared that no competing interests exist.*E-mail:wmohr@ifm-geomar.deIntroductionBiological dinitrogen(N2)fixation is the major source of fixed nitrogen(N)in the oceanic N budget[1].Current estimates of global oceanic N2fixation are,100–200Tg N a21[2].N2 fixation rates can be assessed through geochemical estimates, modelling of diazotroph abundances and growth rates[3]and direct measurements of N2fixation.Geochemical estimates rely on the measurement of,e.g.,nutrient stoichiometry and estimates or models of ocean circulation[4,5]or the distribution of stable isotope abundances(e.g.,[6]).Direct measurements of N2fixation are obtained either using the15N2-tracer addition method[7,8]or the acetylene reduction assay(ARA)[8,9].However,direct measurements of N2fixation rates account for#50%of the geochemically-derived estimates[10].Furthermore,the sink terms in the oceanic fixed N budget significantly exceed the current estimates of N2fixation and other source terms for fixed N[11,12]. This gap between sources and sinks of fixed N implies an oceanic nitrogen imbalance,which may reflect a non-steady-state of the oceanic fixed-N inventory,or result from over-estimation of loss processes and/or under-estimation of fixed nitrogen inputs [10,13].However,isotopic signatures in sediments suggest that the fixed N budget is in a steady-state[14].The comparison of N2fixation rates measured simultaneously using the15N2-tracer addition and the ARA shows that the15N2-tracer addition generally yields lower rates(for a summary see [15]).In addition,mass balance analyses of15N2-based N2fixation rates measured in experiments with cultured diazotrophs,indicate that the15N2-tracer addition method yields rates that are too low for sustaining the observed growth rates and biomass[16,17].The discrepancies between the two methods and the lack of mass balance in culture experiments have often been attributed to the excretion of recently fixed nitrogen as ammonium(NH4+)or dissolved organic nitrogen(DON).The discrepancies have led to the operational definition of gross and net N2fixation[16,18]as measured by the ARA and the15N2-tracer addition approaches, respectively.However,the measured release of NH4+or DON is rarely sufficient to balance the observed growth in culture,and even invoking recycling of the dissolved fixed N rarely accounts for the observed discrepancies between N2fixation rate and growth rate/biomass[16].The apparent oceanic N imbalance,differences between geochemical estimates and measured rates of N2fixation,and the difficulties in reconciling discrepancies between ARA and 15N2-based estimates of N2fixation in the field and in culture experiments,led us to re-assess the15N2-tracer addition method. This method is based on the direct injection of a15N2gas bubble into a seawater sample[7]sufficient to yield a final enrichment of 2–5atom percent(atom%)and incubation for2–36hours[19]. N2fixation rates are then retrieved from the incorporation of15N2 into the particulate organic nitrogen(PON).The method assumes implicitly that the injected gas fully and rapidly equilibrates with the surrounding water,and this assumption is the basis for calculation of the initial15N enrichment of the dissolved N2pool. Knowledge of this enrichment is pivotal to the calculation of N2 fixation rates with this method as seen in equation1(equationscombined from[8]):N2fixation rate~A finalPN{A t~0PNA N2{A t~0PN|PN½D twhere A=atom%15N in the particulate organic nitrogen(PN)at the end(final)or beginning(t=0)of the incubation or in the dissolved N2pool(N2).In applications of the method,all parameters of the equation are measured except for the atom%15N in the dissolved N2pool(A N2). Equation1shows that calculation of N2fixation rates depends strongly on this value which is calculated from the predicted equilibrium dissolved N2concentration[20,21],its natural15N abundance,and the amount of15N2tracer added with the bubble. The calculation assumes that there is complete isotopic equilibra-tion between the injected bubble of15N2and the surrounding water at the start of the incubation.Here we report results of experiments that were designed to assess the rate of equilibration of an introduced15N2gas bubble with the surrounding water.Based on results of these experiments, we developed a modified approach involving addition of15N2-enriched seawater which assured a well-defined and constant15N enrichment of the dissolved N2gas at the beginning of the incubations.We propose the application of the modified approach for future assessments of N2fixation rates in natural microbial communities and in laboratory cultures.ResultsTime-resolved equilibration of a bubble of15N2in seawaterA first set of experiments(isotopic equilibration experiments) was carried out to assess the time required to attain isotopic equilibrium in the dissolved pool of N2gas after injection of a known amount of15N2gas as a bubble into sterile filtered seawater.A gas bubble of pure15N2was injected directly into incubation bottles which were manually inverted fifty-times (,3min agitation)and left standing for up to24h.Concentration of dissolved15N2was followed over the24h period to assess the degree of equilibration of the15N2gas bubble with the surrounding water as a function of time.Dissolved15N2 concentrations in the seawater increased steadily with the incubation time(Fig.1A).After eight hours,dissolved15N2 concentrations reached about50%of the concentration calculated assuming complete isotopic equilibration of the injected bubblewith the ambient dissolved N2gas in the seawater sample.At the end of the24h incubation,the dissolved15N2concentration had increased to about75%of the calculated concentration.N2fixation rate underestimation due to incomplete15N2 gas bubble equilibrationSimilar results were obtained in the incubation experiments with pure culture of Crocosphaera watsonii(culture experiments), which confirmed the incomplete and time-dependent equilibration of the injected bubble of15N2gas with the surrounding water (Fig.1B).These experiments also demonstrated the associated underestimation of N2fixation rates.Culture experiments were conducted after15N2addition as a gas bubble and also after15N2 addition in the form of15N2-enriched seawater(our modified method,see Methods section).The incubation of C.watsonii after injection of a bubble of15N2gas and without prior incubation of this bubble in algal-free media,gave a N2fixation rate which was only40%of the maximum rate measured in the incubations to which15N2-enriched seawater had been added.In other words,for the12-h incubation period under the described experimental conditions,the N2fixation rate was underestimated by60%when the15N2was introduced as a gas bubble.In contrast,in both the isotopic equilibration and the culture experiments,the concentra-tion of dissolved15N2remained stable at the predicted value throughout the24h in incubations to which15N2-enriched water was added.Factors influencing15N2gas dissolution in N2-saturated seawaterContinuous,vigorous shaking(50rpm)greatly increased the concentration of15N2in the media(Fig.2)reaching,67%of the calculated concentration after30minutes whereas the initial, manual agitation,i.e.inverting bottles50times(,3min),resulted Figure1.Time-dependence of the equilibration of a15N2gas bubble with seawater.Results are presented as a function of the time after bubble injection(white symbols).(A)Measured dissolved 15N2concentrations as percentage of calculated concentration assum-ing rapid and complete isotopic equilibrium.(B)N2fixation rates by C. watsonii as percentage of the maximum rate measured during the experiments.For comparison,the addition of15N2-enriched water to samples yielded a constant15N2enrichment over24h(A,grey symbols) or constant N2fixation rates(B,grey symbols).doi:10.1371/journal.pone.0012583.g001in only ,13%of the calculated rmation on agitation is generally not provided in the published literature,but this is clearly a variable factor in incubations,especially if performed at sea.Continuous,vigorous shaking,as tested here (50rpm;Fig.2),is difficult to achieve in field experiments and may,in addition,be detrimental to some diazotrophs (e.g.Trichodesmium colonies).Increasing the size of the incubation bottles,increasing the amount of gas injected per liter of seawater and the addition of dissolved organic matter (DOM)led in all cases except one to slower equilibration of the 15N 2gas bubble with the surrounding water (Fig.3and 4A),even when bottles were shaken continuously for one hour (Fig.4B).Only with the injection of 8ml of 15N 2gas per liter of water and one hour of continuous,vigorous shaking,was near-complete equilibration achieved (97%of calculated concentration).DiscussionBoth the isotopic equilibration and the culture experiments demonstrated clearly that the equilibration of 15N 2gas injected as a bubble into N 2-saturated seawater is time-dependent and incomplete,even after 24hours.The lack of complete equilibra-tion causes the resulting calculated N 2fixation rates to be variably and significantly underestimated (see Equation 1).The equilibra-tion,i.e.the isotopic exchange between the 15N 2gas in the bubble and the surrounding water is controlled primarily by diffusive processes.The major variables that influence the rate of isotopic exchange include the surface area to volume ratio of the bubble,the characteristics of the organic coating on the bubble surface [22],temperature and the rate of renewal of the water-bubble interface [23].The renewal of the water-bubble interface appears to have the greatest effect on the isotopic exchange,as continuous vigorous shaking of the incubation bottles generated the highest enrichment of 15N 2in the water phase.However,the calculated (equilibrium)enrichment in 15N 2was not attained fully even after one-hour of continuous shaking at 50rpm on a rotary shaker.Incubations carried out on board a research vessel will provide some agitation of the bubble but this will not approach the high and constant agitation tested in our experiments.The implication is that variable sea-state conditions encountered during sea-going incubations,and the details of individual experiments,will lead to variable 15N 2enrichments and hence variable underestimation of N 2fixation rates.Further,N 2fixation studies in the oligotrophic regions of the ocean usually require the use of large incubation volumes (e.g.,2–4L),so that continuous shaking for one hour or more is not practical,and in addition would likely be detrimental to the natural microbial communities.The experiments with variable bottle sizes and DOM additions (Fig.3and 4)demonstrated that there are factors in addition to the bubble incubation time that affect the equilibration.On the other hand,the addition of 15N 2-enriched seawater to the incubations led to a stable enrichment over the 24h incubation time which was instantaneous and independent of the agitation of the bottles.This study was motivated partly by the mismatches between the ARA and 15N 2-based measurements of N 2fixation as well as imbalances between 15N 2-fixation rates and biomass-specific rates (,growth rate)or C:N fixation ratios (Table 1).Such mismatches have been observed in environmental studies and in culture studies,mainly with Trichodesmium .Although it has been shown that Trichodesmium can excrete recently fixed N 2as NH 4+or DON [16,24],the excretion of 15NH 4+or DO 15N rarely accounts for the observed discrepancies [16,17].The operational definition of gross and net N 2fixation as obtained through ARA and 15N 2incubations,respectively,has been mainly based on the mismatch between the rates measured by the two methods.Our results demonstrate that N 2fixation rates,as measured with the 15N 2method [7]are underestimated.Therefore,the magnitude of the exudation of recently fixed nitrogen and the conditions promoting this process should be re-evaluated,taking into account the results presented here.We reviewed published studies that have used the direct injection of a 15N 2gas bubble to assess N 2fixation rates in ordertoFigure 2.Agitation-dependent increase in dissolved 15N 2using bubble incubations.Values are presented as a percentage of the calculated concentration.The manually-shaken (3min)sample was added to the plot for comparison (grey symbol).doi:10.1371/journal.pone.0012583.g002Figure 3.Dissolved 15N 2concentration as a function of bottle size and amount of injected 15N 2gas.Values are presented as a percentage of the calculated concentration.Bottles were incubated for 1hour.Black bars,0.13L bottle and white bars,1.15L bottle.doi:10.1371/journal.pone.0012583.g003evaluate the magnitude of under-estimation.However,first attempts to assess the degree of underestimation of field and culture N 2fixation rates were obscured by a wide range of experimental conditions among the studies.Bottle sizes ranged from 14ml to 10L,the amount of 15N 2injected varied from 0.2to 40.8ml 15N 2per L seawater and incubation times ranged from 0.25to 48hours,with the majority of the field studies using 2–4L bottles and 24h incubations.In addition,information on agitation was,in general,not available.There were no obvious trends of reported N 2fixation rates with either bottle size,incubation time or the amount of injected 15N 2gas probably because of the large variability of geographic locations and environmental conditions prevailing in the individual studies,which would have a dominant effect on the local diazotrophic communities and their N 2fixation rates.An evaluation of the degree of possible underestimation of 15N 2fixation rates in environmental studies is further confounded by diel periodicity of N 2fixation [25–27].The lack of knowledge on the exact timing and magnitude of the individual N 2fixation activity of the different diazotrophs relative to the timing of 15N 2gas injection hinders back-calculation of published N 2fixation data.This can be illustrated,for example,with a hypothetical diazotroph community that is dominated by unicellular cyanobacteria which fix nitrogen during the night period only (Fig.5A).In this microbial community,measure-ments of N 2fixation using the direct injection of a 15N 2gas bubble during a 24hour incubation will lead to a variable underestimation of the true N 2fixation rate,depending on the timing of the incubation start relative to the peak in the nitrogenase activity (Fig.5C,solid lines).The underestimation will be more pronounced if the start of the incubation is coincident with the onset of the active N 2fixation period.In contrast,incubations with enriched 15N 2seawater,will not lead to an underestimate,regardless of the incubation start relative to the diel cycle (Fig.5C,dashed lines).The discrepancies and mismatches/imbalances observed in field and laboratory studies could,in part,be explained by the variable underestimation of the true N 2fixation rate due to the methodological uncertainty reported here.We propose the addition of 15N 2-enriched seawater to incubations to assess N 2fixation rates in laboratory and field studies.We suggest that measurements using this approach are likely to increase measurements and estimates of N 2fixation at species,regional and global level and lead to a reduction in the apparent oceanic nitrogen imbalance.Table 1.Discrepancies observed between15N 2fixation,ARA and carbon fixation or biomass-specific rates a .Organism/areaC 2H 2:15N 2C:N fixation ratio biomass-specific rate [d 21]Reference Trichodesmium /Pacific,Atlantic,north of Australia808b[30]cyanobacterial bloom/Baltic 3–20[18]Trichodesmium IMS 1013–2275–133[17]Trichodesmium IMS 101 1.5–6.90.002–0.011c[16]Trichodesmium /Gulf of Mexico10–107b[31]Trichodesmium /Bermuda Atlantic Time Series station (BATS)13–4370.006–0.03d[32]a C:N fixation ratio is based on 15N 2-fixation measurements.bRatio calculated from DI 13C and 15N 2fixation rates.cCalculated from 15N 2fixation rate divided by PON.dCalculated from doubling time with biomass-specific rate =ln (2)/doubling time.doi:10.1371/journal.pone.0012583.t001Figure 4.Dissolved 15N 2concentration as a function of the amount of injected gas and agitation.Values are presented as a percentage of the calculated concentration (A)after 1hour incubation in manually (3min shaking and 1h subsequent incubation),and (B)in continuously (1h)shaken samples.doi:10.1371/journal.pone.0012583.g004Materials and Methods Culture and growth conditionsThe diazotrophic cyanobacterium Crocosphaera watsonii WH8501was grown in batch cultures in N-free YBCII media [28]at 28u C in a temperature-controlled growth chamber. C.watsonii was subjected to 12:12h dark:light cycles.Direct injection of a15N 2gas bubble in waterWe first examined the rate of equilibration between an injected bubble of 15N 2gas and seawater.Two series of incubations were started by injecting 140m l of 15N 2into 133ml of an artificial seawater media (YBCII)contained in headspace-free,septum-capped glass bottles.In the first series (isotopic equilibration experiments),all bottles were inverted fifty times (,3min)after injection of the 15N 2gas bubble and left at room temperature in the laboratory.One bottle was sampled immediately after the agitation in order to determine how much 15N 2gas had dissolved initially.The other bottles were opened and sampled after standing for periods from 1to 24h.Upon opening of the bottles,samples to measure the dissolved 15N 2were taken and stored in gas-tight glass vials (Exetainer H )until analysis.In the second series (culture experiments),the YBCII media was pre-heated to 28u C in a temperature-controlled chamber before being used to fill septum-capped glass bottles.As with the first series,samples were agitated and left standing for varying periods of time after the injection of a 15N 2gas bubble.Instead of taking subsamples for 15N 2analysis,13ml of media were replaced by C.watsonii WH8501culture upon opening of the bottles.This series of experiments was timed so that the introduction of culture into the media took place at the start of a dark phase of the 12:12h dark:light-adapted C.watsonii culture.The samples with the culture were then incubated for 12h at culture growth conditions (28u C,dark phase,i.e.N 2-fixing)and filtered onto pre-combusted GF/F (Whatman;450u C for 4h)filters at the end of the incubation.Filters were dried immediately after (50u C,6h)and stored at room temperature until analysis.To obtain a measure of underestimation using the direct injection of a 15N 2gas bubble,one bottle containing 13ml of C.watsonii culture was incubated for 12h after the injection of 140m l 15N 2gas at the start of the dark phase and without release of the bubble,essentially resembling a laboratory or field incubation.Direct addition of15N 2tracer-enriched seawaterAn alternative,modified 15N 2tracer addition method was developed,which involved addition of an aliquot of 15N 2-enriched water to incubations.This alternative method was based on earlier approaches used to study oxygen cycling using 18O 2[29]and the release of DON using 15N 2[24].The preparation of the 15N 2-enriched water was started by degassing 0.2m m-filtered artificial seawater (YBCII media).Degassing was carried out by applying vacuum (#200mbar absolute pressure)to continuously stirred (stir bar)media for about 30min.The degassed water was transferred rapidly but gently into septum-capped glass bottles until overflow,and 1ml of 15N 2gas (98at%;Campro Scientific)was injected per 100ml of media.The bottles were shaken vigorously until the bubble disappeared.Aliquots of this 15N 2-enriched water were then added to the incubation bottles,with the enriched water constituting no more than 10%of the total sample volume.This alternative enrichment method was applied to the two series of experiments described above.Assessment of additional factors contributing to variation in 15N 2enrichmentWe assessed possible effects of varying bottle size,amounts of injected gas and different amounts of agitation on their contribution to the equilibration between a bubble of 15N 2gas and the surrounding seawater.For the bottle size comparison,incubations were performed in 0.13L bottles and in 1.15L bottles.The amount of injected gas varied between 1ml 15N 2per 1L seawater up to 8ml 15N 2per 1L seawater.The incubations were agitated either by inverting fifty times manually (,3min)or by continuous agitation on a rotating bench-top shaker (Biometra WT 17)at 50rpm (rotations per minute).We also added marine broth (Difco 2216;0.2m m filter-sterilized;230mg DOM L 21media)to some bottles to examine the effect of dissolved organic matter(DOM).Figure 5.Influence of diel N 2fixation patterns on the magnitude of N 2fixation rates.Schematic diagram illustrating the influence of diel N 2fixation patterns on N 2fixation rates when determined with the direct injection of a 15N 2gas bubble.A hypothetical diel N 2fixation pattern is shown (panel A)with a duration of the N 2-fixing period of 12h.Three possible time periods for 24h incubations are indicated by the solid bars (A–F).The corresponding 15N enrichment in the dissolved N 2pool (panel B)is shown for the three incubation periods using the direct injection of a 15N 2gas bubble (solid lines;A,B and C)and the addition of 15N 2-enriched seawater (dashed line;D,E and F).The resulting cumulative N 2fixation in each of the incubations (panel C)demonstrates that the timing of the incubation relative to diel N 2fixation patterns introduces a variable underestimation in the total N 2fixation rate measured during the incubation after a 15N 2gas bubble is injected (solid lines;A,B and C)as compared to the N 2fixation measured with the addition of 15N 2-enriched seawater (dashed lines;D,E and F).The diagram is based on the observations made in the experiments described in this study.doi:10.1371/journal.pone.0012583.g00515N2analysis in the artificial seawater and15N analysis in the particulate organic nitrogen(PON)Subsamples taken during the equilibration experiments were analysed for15N2concentration with a membrane-inlet mass spectrometer(MIMS;GAM200,IPI)within one week of subsampling.Dried GF/F filters were pelletized in tin cups,and PON as well as isotope ratios were measured by means of flash combustion in an elemental analyser(Carlo Erba EA1108) coupled to a mass spectrometer(Thermo Finnigan Delta S).CalculationsThe expected concentration of15N2following bubble injections was calculated assuming rapid and complete isotopic equilibration between bubble and surrounding seawater and considering atmospheric equilibrium concentrations of dissolved N2[21]. When15N2-enriched aliquots were added,the amount of15N2 originally dissolved in the degassed seawater and the volume of the aliquot added were taken into account.The calculations of N2 fixation rates in the culture incubations were made according to Equation1and are presented as a percentage of the highest rate measured.For the comparison between methods,the measured15N2concentrations are presented as a percentage of the expectedconcentration calculated as followsV15N2 MV|V TOTAL ~mol15N2L{1ÂÃi:e:100%ðÞð2Þfor the direct injection of a15N2gas bubble where V15N2is thevolume of the15N2gas bubble,MV is the molar volume andV TOTAL is the total(water)volume of the incubation.The expectedconcentration was corrected for the amount of15N2gas whichremains in the bubble at isotopic equilibrium with the surroundingwater.For the addition of15N2-enriched water the expectedconcentration isV15N2MV|V DG|V EWV TOTAL~mol15N2L{1ÂÃi:e:100%ðÞð3Þwhere V DG is the volume of degassed water,V EW is the volume of15N2-enriched water added to the incubation and V TOTAL is the total(water)volume of the incubation.AcknowledgmentsWe thank Marcel Kuypers and Hannah Marchant(Max Planck Institutefor Marine Microbiology Bremen)for providing access to and advice onthe membrane-inlet mass spectrometry.The assistance and suggestions ofGert Petrick and Karen Stange(IFM-GEOMAR)concerning the earlyanalyses of15N2is also gratefully acknowledged.Author ContributionsConceived and designed the experiments:WM DWRW JL.Performed theexperiments:WM.Analyzed the data:WM TG DWRW JL.Wrote thepaper:WM.Contributed to figure preparation/schematic diagrams:TG.References1.Gruber N(2008)The marine nitrogen cycle:Overview and challenges.In:Capone DG,Bronk DA,Mulholland MR,Carpenter EJ,eds.Nitrogen in the marine environment.Amsterdam,The Netherlands:Elsevier.pp1–50.2.Karl D,Michaels A,Bergman B,Capone D,Carpenter E,et al.(2002)Dinitrogen fixation in the world’s oceans.Biogeochemistry57/58:47–98.3.Goebel NL,Edwards CA,Church 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cochrane评分表

Risk of biasItem Authors'judgementDescriptionAdequate sequence generation? Yes Prinicipal author stated thatcomputer generatedallocation was usedAllocation concealment? Yes Prinicipal author stated thatallocation was concealedBlinding? Unclear No mention of study personnelor participants being blindto treatment groupIncomplete outcome data addressed? Yes All participants accountedfor, one 'drop out' recordedbut included by us in analysisFree of other bias? Unclear Possible uneven distributionof complete and incompleteparalysis at start of studybetween the two treatmentgroupsCochrane RCT质量评价标准:①随机方法是否正确。
②是否隐蔽分组。
③盲法的使用情况。
④失访或退出描述情况,有无采用意向性(ITT)分析。
以上质量标准中,如所有标准均为“充分”,则发生各种偏倚的可能性很小;如其中一条为不清楚,则有发生相应偏倚的中等度可能性;如其中一条为“不充分”或“未采用”,则有发生相应偏倚的高度可能性。
可参见:RCT的质量评价标准选择总结/bbs/topic/18137535?tpg=1&age=-1Quality assessmentThe quality of the trials was assessed and graded independently by two authors according to the criteria described in The Cochrane Handbook 4.2.6 (Higgins 2006). Gradings were compared and any inconsistencies between the authors in the interpretation of inclusion criteria and their significance to the selected study were discussed and resolved.The selected study was assessed for the following characteristics:1. The adequacy of the randomisation process (possible selection bias). Adequate randomisation includes any one of the following methods: computer generated or table of random numbers, drawing of lots, coin-toss, shuffling cards or throw of a dice. Inadequate methods of randomisation include the following: case record number, date of birth or alternate numbers.2. The adequacy of the allocation concealment (possible selection bias). Adequate methods of allocation concealment include either central randomisation (i.e. separate to other aspects of trial administration) or sequentially numbered sealed opaque envelopes. Inadequate concealment means an open allocation sequence in which either participants or trialists were able to foresee the upcoming assignment.3. The blinding of outcome assessors (i.e. whether the persons assessing the outcome of care were aware of which treatment the participant had received - possible performance bias).4. The extent and handling of losses to follow up (possible attrition bias). Adequate handling of losses to follow up involves a clear description and explanation being given of any significant difference between the losses of the intervention groups. An unacceptable loss in any one intervention group was considered to be loss greater than 20%.Study gradings A, B or C were employed for overall quality as follows.A: Minimisation of bias in all four categories above: i.e. adequate randomisation, few losses to follow up and intention-to-treat analysis, blinding of outcome assessors, high quality outcome assessment;B: Each of the criteria in A partially met;C: One or more of the criteria in A not met.Risk of bias in included studiesWe classified this study as grade C because of the uncertainty about blinding. The possibility of an uneven distribution of complete and incomplete palsies between the two groups is another potential source of bias and we conclude overall that this is a low quality study.Table 8.5.a: The Cochrane Collaboration’s tool for assessi ng risk of biasTable 8.5.c: Criteria for judging risk of bias in the ‘Risk of bias’ assessment toolFigure 8.6.a: Example of a ‘Risk of bias’ table for a single study (fictional)Table 8.7.a: Possible approach for summary assessments of the risk of bias for each important outcome (across domains) within and across studies。
free boundary finance

Solving Free-boundary Problems with Applications in Finance
application on a set of classical problems, of increasing difficulty, in finance. This survey is intended for those are primarily interested in computing numerical solutions to these problems. To this end, we include actual Matlab code for one of the problems studied, namely, American option pric1 and Sunil Kumar2
1 2
McCombs School of Business, University of Texas at Austin, 1 University Sta B6000, Austin, TX 78712, USA, kumar@ Graduate School of Business, Stanford University, 518 Memorial Way, Stanford, CA 94305, USA, skumar@
1.1
Motivation: Controlled Brownian Motion
Glycoprotein of Nonpathogenic Rabies

10.1128/JVI.77.19.10537-10547.2003.2003, 77(19):10537. DOI:J. Virol. and Monique LafonChristophe Préhaud, Stéphanie Lay, Bernhard DietzscholdApoptosisCell Viruses Is a Key Determinant of Human Glycoprotein of Nonpathogenic Rabies/content/77/19/10537Updated information and services can be found at: These include:REFERENCES/content/77/19/10537#ref-list-1at: This article cites 60 articles, 26 of which can be accessed free CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on June 2, 2012 by GUANGXI UNIVERSITY/Downloaded fromJ OURNAL OF V IROLOGY,Oct.2003,p.10537–10547Vol.77,No.19 0022-538X/03/$08.00ϩ0DOI:10.1128/JVI.77.19.10537–10547.2003Copyright©2003,American Society for Microbiology.All Rights Reserved.Glycoprotein of Nonpathogenic Rabies Viruses Is a Key Determinantof Human Cell ApoptosisChristophe Pre´haud,1Ste´phanie Lay,1Bernhard Dietzschold,2and Monique Lafon1* Unite´de Neuroimmunologie Virale,De´partement de Neuroscience,Institut Pasteur,Paris,France,1and Center for Neurology, Department of Microbiology and Immunology,Thomas Jefferson University,Philadelphia,Pennsylvania2Received7February2003/Accepted20June2003We showed that,unlike pathogenic rabies virus(RV)strain CVS,attenuated RV strain ERA triggers thecaspase-dependent apoptosis of human cells.Furthermore,we observed that the induction of apoptosis iscorrelated with a particular virus antigen distribution:the overexpression of the viral G protein on the cellsurface,with continuous localization on the cytoplasmic membrane,and large cytoplasmic inclusions of the Nprotein.To determine whether one of these two major RV proteins(G and N proteins)triggers apoptosis,weconstructed transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression to produce the Gand N proteins of ERA and CVS individually.The induction of ERA G protein(G-ERA)expression but not ofERA N protein expression resulted in apoptosis,and G-ERA was more efficient at triggering apoptosis thanwas CVS G protein.To test whether other viral proteins participated in the induction of apoptosis,human cellswere infected with recombinant RV in which the G protein gene from the attenuated strain had been replacedby its virulent strain counterpart(CVS).Only RV containing the G protein from the nonpathogenic RV strainwas able to trigger the apoptosis of human cells.Thus,the ability of RV strains to induce apoptosis is largelydetermined by the viral G protein.Many viruses have developed strategies to manipulate the survival or the death of the cells that they infect(27).Different viruses use different mechanisms,and these mechanisms have been studied in detail(23).However,a large number of groups are trying to identify proteins that directly trigger apoptosis and to determine the precise roles of these proteins(8,11,14, 34,50).As well as improving knowledge of virus pathogenicity and virulence,the identification of“viral death proteins”may have practical applications for human therapeutics.The induc-tion of apoptosis by peptides or polypeptides might make it possible to eliminate undesirable cells,such as tumor,infected, and autoimmune cells,or to trigger the formation of apoptotic bodies,which are thought to be powerful immune response activators(1,3,32,45,49,51).Rabies virus(RV)is an enveloped,bullet-shaped virus be-longing to the Rhabdoviridae family and the Lyssavirus genus. The viral particle consists of a membrane composed of host lipids and two viral proteins,the G and M proteins,surround-ing a helical nucleocapsid(NC).The NC is composed of a viral negative-strand RNA molecule protected by the N protein,the P protein,and the RNA-dependent RNA polymerase,the L protein.RV proteins are not synthesized in equal amounts in infected cells and are not present at the same ratios in viral particles(13,37).Indeed,the N,G,and M proteins are,in this order,the most prominent species in virions.RV strain CVS is a highly neurotropic virus strain.In con-trast,SAG-2,ERA,and SN-10-SAD are attenuated strains, probably because they have been subjected to several passages in nonneuronal cultures.They grow in vitro in lymphocytes (57).These disabled viruses are candidates for live vaccines. SAG-2,an SN-10-SAD mutant,has been successfully used in oral vaccination programs to eradicate rabies from wildlife reservoirs in western Europe(4,5,19,38).The attenuated live RV vaccine strain,ERA,triggers apoptosis in the human lym-phoblastoid T-cell line Jurkat and activates caspases3,8,and 9.RV-induced apoptosis is blocked by the overexpression of Bcl-2,suggesting that RV induces apoptosis via a mitochon-drial pathway(58).The direct interaction of RV with receptors located on the cytoplasmic membrane is not sufficient to in-duce apoptosis(25,57).Apoptosis requires that the infectious cycle be completed.Apoptosis is correlated with the detection of the G protein,suggesting that viral proteins,the G protein in particular,play a role in this process(15,57).Moreover, Morimoto et al.(43)showed that the accumulation of the G protein,but not the N protein,is correlated with the induction of apoptosis in primary mouse neuron cultures.Nevertheless, differential NC distributions are observed in CVS-and ERA-infected Jurkat T cells(57,58).Thus,the intrinsic properties of the N protein as an apoptosis inducer remain unclear.The goal of this study was to identify the RV protein that causes death in two types of human cells,Jurkat T cells and neuroblastoma SK-N-SH cells.In afirst instance,we decided to focus our investigations on the viral N and G proteins.First, we assessed whether apoptosis,which occurs in Jurkat T cells, could also take place in neuronal cells.Second,we analyzed the expression and the distribution of the G and N proteins in cells infected with apoptotic and nonapoptotic virus strains. Third,we constructed transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression to compare the abilities of the G and N proteins of nonapoptotic RV strain CVS and of proapoptotic RV strain ERA to trigger the apoptosis of Jurkat T cells.Finally,we used recombinant viruses obtained by re-verse genetics to determine whether other proteins from strain*Corresponding author.Mailing address:Institut Pasteur,Unite´de Neuroimmunologie Virale,25Rue du Dr.Roux,75724Paris Cedex15, France.Phone:33-1-45-68-87-52.Fax:33-1-40-61-33-12.E-mail: mlafon@pasteur.fr.10537 on June 2, 2012 by GUANGXI UNIVERSITY / Downloaded fromERA,such as the M,L,and P proteins,were inducers of apoptosis.Our data indicated that the proapoptotic properties of the attenuated RV strain are dependent on the viral G protein in both human lymphoblastoid and human neuronal cell lines and suggest that the accumulation of the viral G protein at the cytoplasmic membrane is an important factor in the triggering of apoptosis.MATERIALS AND METHODSCells and viruses.Jurkat rtTA cells(31)were cultivated in RPMI1640me-dium containing2mM L-glutamine,100U of penicillin/ml,100g of strepto-mycin/ml,10%tetracycline-free fetal bovine serum,and2.5g of puromycin/ml. Cells were cultured at37°C in a5%CO2–95%air atmosphere and split twice a week.The human neuroblastoma cell line SK-N-SH(HTB11;American Type Culture Collection[ATCC])was propagated in Dulbecco modified Eagle me-dium containing2mM L-glutamine,1mM Na pyruvate,10%fetal calf serum,2% Na bicarbonate,100U of penicillin/ml,and100g of streptomycin/ml at37°C and treated with trypsin twice a week.The nonpathogenic RV laboratory strain ERA and the pathogenic RV laboratory strain CVS(ATCC vr332and ATCC vr959,respectively)were propagated as previously described(57).SN-10is a nonpathogenic virus strain derived from SAD B19(53).The recombinant virus R-N2c was used to introduce the entire coding region of the G protein gene from CVS-N2c,a neuroinvasive strain,into the SN-10genome(42,43).SN-10and R-N2c were propagated in mouse neuroblastoma cell line N2a.Abs,MAbs,and reagents.Fluorescein isothiocyanate(FITC)-conjugated af-finity-purified F(abЈ)2fragments of anti-mouse goat immunoglobulin G(IgG) and5-(4,6-dichlorotriazinyl)aminofluorescein-conjugated and Cy3-conjugated streptavidin were purchased from Jackson ImmunoResearch(distributed by Immunotech/Beckman Coulter,Orsay,France).FITC-conjugated NC-specific rabbit antibodies(Abs)were obtained from SanofiDiagnostics(Marnes la Co-quette,France).Phycoerythrin(PE)-conjugated streptavidin was obtained from Dako(Glostrup,Denmark).Biotinylated anti-RV G protein monoclonal anti-body(MAb)8-2(41)was produced in the laboratory.A mixture of three anti-N protein MAbs and anti-G protein MAb15-13were gifts from N.Minamoto (University of Gifu,Gifu,Japan)(35).An Ab specific for a linear RV G protein epitope(6-15-C4)was a gift from A.D.M.E.Osterhaus(Institute of Virology, Erasmus Medical Center,Rotterdam,The Netherlands)(9).The control con-sisted of an irrelevant FITC-conjugated antitrinitrophenol(TNP)IgG1antibody (BD-Pharmingen,Le Pont de Claix,France).A CaspaTag caspase8(leucylglu-tamylthreonylaspartic[LETD])activity kit and Hoechst33342were obtained from Intergen(distributed by Quantum Biogen,Illkirch,France).Cellfix was supplied by Becton-Dickinson Biosciences(Le Pont de Claix,France).Fluoro-mount-G was purchased from Cliniscience.Doxycycline and tetracycline-free fetal bovine serum were obtained from Ozyme(St.Quentin-en-Yvelines, France).Puromycin and hexadimethrine bromide(Polybrene)were purchased from Sigma Aldrich(St.Quentin Fallavier,France).Hygromycin B was obtained from Roche Diagnostics(Meylan,France).Streptomycin,penicillin,and G-418 were purchased from Gibco BRL(Cergy Pontoise,France).Trevigen Apoptotic Cell System annexin V-FITC was obtained from R&D Systems Europe(Abing-don,United Kingdom).Inducible transgenic cell lines.We used the Rev-Tet System,developed by Clontech Laboratories,to construct transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression.All experiments were carried out accord-ing to the manufacturer’s instructions(Rev-Tet System user manual PT3223-1). Briefly,the open reading frames corresponding to the genes for the G protein of ERA(G-ERA)(GenBank accession number AF406693),the G protein of CVS (G-CVS)(AF406694),the N protein of ERA(N-ERA)(AF406695),and the N protein of CVS(N-CVS)(AF406696)were amplified by PCR and inserted into pRevTRE that had been cut with Bam HI and Hpa I and dephosphorylated. Recombinant Moloney murine leukemia viruses(retroviruses)carrying the RV genes were isolated after transfection of RetroPack PT67cells with the resulting plasmids.Hygromycin-resistant clones were subsequently identified.These vi-ruses were used to infect Jurkat rtTA cells(31),and clones resistant to hygro-mycin and puromycin were selected.RV protein expression was monitored by flow cytometry analysis after18h in the presence of1g of doxycycline/ml. Infection of cell cultures.Monolayers of SK-N-SH cells in12-well plates(105 cells)were infected with ERA,CVS,SN-10,and the recombinant virus R-N2c (multiplicity of infection[MOI],3)or mock infected for1h at37°C.The virus inoculum was then replaced by1ml of culture medium,and the cells were incubated for48h at37°C.Jurkat rtTA cells in12-well plates were infected with ERA,CVS,and SN-10 (MOI,3)and with R-N2c(MOI,40)or mock infected.Cells were incubated for 48h at37°C in a5%CO2–95%air atmosphere for further analysis. Immunostaining.For the detection of RV NC and its main constituent,the N protein,which is not expressed on the cell surface,and for the detection of total intracytoplasmic RV G proteins,transgenic or infected Jurkat rtTA cells and SK-N-SH cells werefixed with3%paraformaldehyde(PFA)for20min at4°C. Cells were then incubated for1h at37°C with a mixture of three anti-N protein MAbs,a biotinylated anti-RV G protein MAb,or an FITC-conjugated Ab diluted in permeabilization buffer(1%heat-inactivated fetal calf serum,0.1% [wt/vol]sodium azide,and0.1%[wt/vol]saponin in phosphate-buffered saline). Cells were further incubated with FITC-conjugated affinity-purified F(abЈ)2frag-ments of anti-mouse goat IgG or PE-conjugated streptavidin.For the surface expression of the G protein,PFA treatment was omitted,and cells were incu-bated for1h at37°C with the biotinylated anti-RV G protein MAb diluted in culture medium(Jurkat rtTA or SK-N-SH cells)and then incubated with PE-conjugated or FITC-conjugated streptavidin.Forflow cytometry analysis,cells were washed several times in staining buffer andfixed in Cellfix(1/10in staining buffer[phosphate-buffered saline,1%fetal calf serum,and0.1%sodium azide, pH7.6])(directly for Jurkat rtTA cells and after scraping for SK-N-SH cells). Viral proteins were detected in a cell population(104cells)that was gated to exclude dead cells and cell debris.Viral proteins in cell cultures were measured by determining the frequency of events with FITC or PE signals higher than a given threshold.Assessment of viability by light-scattering analysis.Morphological changes were assessed by side and forward light-scatteringflow cytometry and used to identify apoptotic Jurkat T cells as previously described(28).Detection of nuclear DNA fragmentation by Hoechst staining.The nuclear morphology of normal and apoptotic cells was assayed by staining with Hoechst 33342(1g/ml)and visualized with a Zeiss UV microscope.The percentage of cells with fragmented nuclei in20microscopefields(magnification,ϫ63)was determined with offline software(Metamorph software imaging system;Univer-sal Imaging Corporation,Downingtown,Pa.).Detection of nuclear DNA fragmentation by the TUNEL method.DNA frag-mentation was assessed by using the terminal deoxynucleotidyltransferase-medi-ated dUTP-biotin nick end labeling(TUNEL)technique as previously described (21)with minor modifications(57).Slides were observed with a Zeiss UV microscope.Results are expressed as the number of cells withfluorescent nuclei per500or5,000cells perfield(magnification,ϫ63).Detection of PS exposure.For detection of phosphatidylserine(PS)exposure, SK-N-SH cells adhering to a5-cm2surface were washed with phosphate-buffered saline before being incubated for15min in the dark with25ng of Annexin V-FITC at room temperature.Cells were washed,fixed in3%PFA,and analyzed by confocal microscopy.Detection of caspase activation.Activated caspases were detected by use of FITC-conjugated FAM-LETD-FMK,a caspase inhibitor analog that enters cells and irreversibly binds to activated caspase8and with a reduced efficiency to caspases1,6,9,and10.Live cells(3ϫ105in300l)were incubated with10l of FITC-conjugated FAM-LETD-FMK for1h at37°C in a5%CO2atmosphere in the dark.After two washes,cells were resuspended in500l of staining buffer–50l of Cellfix before being analyzed byflow cytometry.To exclude nonspecific binding of the staining reagents to dead or necrotic cells,flow cy-tometry analyses were gated on a viable population(104cells).Caspase activa-tion following infection or the production of different proteins was determined by measuring the number of events with FITC signals higher than3log units. Confocal microscopy.Confocal microscopy was performed with a Zeiss LSM 510confocal microscope equipped with a helium-neon laser(1mV).Cells were analyzed at488nm with a narrow-bandfilter centered on535nm for FITC signal detection.For each observation,the results for12optical xy sections taken at 0.5-m intervals were recorded.Data were analyzed with Image Browser version 2.8.Images were treated with Adobe Photoshop5.5.RESULTSRV strain ERA triggers apoptosis in infected human SK-N-SH cells.SK-N-SH cells were infected with RV ERA or CVS,and the apoptosis of the infected cells was monitored by costaining with annexin V or Hoechst33342and Abs specificfor virus antigens.Only cell monolayers in which more than10538PRE´HAUD ET AL.J.V IROL.on June 2, 2012 by GUANGXI UNIVERSITY /Downloaded from70%of the cells were infected were included in the subsequent analysis,such that apoptosis was monitored mostly in infected cells.The flux of PS from the inner to the outer lea flet of the plasma membrane is an early marker of apoptosis that can be detected by annexin V staining (18,54).ERA-infected SK-N-SH cells were stained with an RV G protein-speci fic Ab (Fig.1,section I,panel A)and with annexin V (Fig.1,section I,panel B).Costaining (Fig.1,section I,panel C)revealed that infected cells expressing the G protein (red)on the cytoplasmic membrane contained PS on the outer lea flet (green).Annexin V-positive cells were not labeled by an irrelevant Ab (anti-TNP –FITC;data not shown)or by propidium iodide (Fig.1,section I,panel D),a result that can be taken as evidence that PS exposure occurs only on viable cells which have not com-pletely lost their membrane integrity.No annexin V staining was observed in CVS-infected SK-N-SH cells (data not shown),suggesting that CVS-infected cells do not encounter apoptosis.DNA fragmentation is a hallmark of intermediate-stageapo-FIG.1.ERA-infected SK-N-SH cells undergo cell death.(I)Confocal microscopy analysis of PS exposed on the outside of the cytoplasmic membrane of ERA-infected SK-N-SH cells (A)Detection of viral G protein on the cytoplasmic membrane of ERA-infected SK-N-SH cells (red).(B)Detection of PS with FITC-annexin V (green).(C)Annexin V and viral G protein labeling are not strictly colocalized.(D and E)Detection of nuclei after propidium iodide staining on infected cells not treated (D)or treated with PFA (E).These images are representative of 69observations.(II)Nuclear fragmentation in ERA-infected SK-N-SH cells (magni fication,ϫ40).Nonapoptotic strain CVS-infected (panels labeled 1)and proapoptotic strain ERA-infected (panels labeled 2)SK-N-SH cells were stained with Hoechst 33342(blue)and anti-NC Ab (green).Fragmented nuclei,which are characteristic of apoptosis,were observed in ERA-but not in CVS-infected SK-N-SH cells.These images are representative of 70observations.V OL .77,2003RV G PROTEIN MEDIATES APOPTOSIS 10539on June 2, 2012 by GUANGXI UNIVERSITY/Downloaded fromptosis resulting from the caspase-dependent activation of en-donucleases that degrade chromatin into discrete fragments (61).Thus,we assessed DNA fragmentation in ERA-or CVS-infected SK-N-SH cells by costaining with Hoechst 33342and NC-speci fic Abs (Fig.1,section II).The nuclei of ERA-in-fected cells were clearly fragmented (Fig.1,section II,panel 2),whereas those of CVS-infected cells were not (Fig.1,sec-tion II,panel 1).These data indicate that ERA-but not CVS-infected cells undergo apoptosis.We then quanti fied the extent of apoptosis in ERA-infected neuroblastoma populations and compared the results to those for ERA-infected Jurkat rtTA cells.We measured the apopto-sis of ERA-and CVS-infected Jurkat rtTA cells and SK-N-SH cells by means of DNA fragmentation (TUNEL and Hoechst 33342staining)and caspase activation (Fig.2).In addition,for lymphoblastoid cells,apoptosis was quanti fied by light-scatter-ing analysis (28)in order to determine the extent of cells with morphological changes.At similar levels of infection,ERA induced the apoptosis of the human lymphoblastoid cell line Jurkat rtTA (Fig.2A and B)and of the neuroblastoma cell line SK-N-SH (Fig.2C and D),whereas CVS did not (Fig.2).Interestingly,caspases were activated in ERA-infected SK-N-SH cells,in agreement with the presence of the caspase 8protein in these cells (46),a feature that is not shared by all neuroblastoma cells (2,55,56).Altogether,these data con firm that ERA induces apoptosis,whereas CVS does not.The sim-ilarities between the infections caused by the two RV strains in the two cell types indicate that the proapoptotic property of strain ERA does not depend on a speci fic cell environment.The RV G protein and the NC accumulate to different levels in cells infected with proapoptotic and nonapoptotic virus strains.SK-N-SH cells were infected with RV ERA and CVS,and we determined the overall distribution of the NC by im-muno fluorescence.Typical NC staining was observed in both types of infected cells (Fig.1,section II).However,ERA-infected cells exhibited many large inclusions of NC,whereas CVS-infected SK-N-SH cells contained both large and small inclusions together with a more diffuse pattern of staining throughout the cytoplasm.We then investigated the behavior of the RV G protein.The G protein is synthesized in the cytoplasm of infected cells and traf ficked through the endoplasmic reticulum and theGolgiFIG.2.ERA induces the apoptosis of Jurkat rtTA and SK-N-SH cells.(A)Apoptosis in 2-day-old cultures of Jurkat rtTA cells infected withERA or CVS,as measured by determining the percentage of R2cells in the cell population (black bars)or by determining the percentage of cells with fragmented nuclei stained by Hoechst 33342(white bars;500cells counted)or by the TUNEL technique (stippled bars;500cells counted).This experiment is representative of five experiments (means and standard deviations [SD]).(B)Detection of infection (black bars)and caspase activation (white bars)of Jurkat rtTA cells infected with either ERA or CVS.Results are the means and SD of three experiments.(C)Induction of apoptosis in 2-day-old cultures of SK-N-SH cells infected with ERA or CVS,as measured by determining the percentage of cells with fragmented nuclei stained by Hoechst 33342(white bars;500cells counted)or by the TUNEL technique (stippled bars;500cells counted).This experiment is representative of two experiments.(D)Detection of infection (black bars)and caspase activation (white bars)of SK-N-SH cells infected with either ERA or CVS.Results are the means and SD of three experiments.10540PRE´HAUD ET AL.J.V IROL .on June 2, 2012 by GUANGXI UNIVERSITY/Downloaded fromapparatus before being anchored at the cytoplasmic membrane of virus-infected cells.SK-N-SH cells were infected with ERA and CVS,and the level of infection,the amount of G protein inside the cells,and the amount of cytoplasmic membrane-associated G protein were checked by flow cytometry (Fig.3E).Interestingly,we found that the amount of cytoplasmic mem-brane-bound G protein relative to the amount of G protein inside the cells was higher in ERA-than in CVS-infected neuroblastoma cells.The G protein/N protein ratio shows that this difference was not due to a difference in the levels of infection (Fig.3F).To try to explain this difference,we carried out G-protein-speci fic confocal immuno fluorescence studies with living ERA-and CVS-infected SK-N-SH cells and with three different G-protein-speci fic MAbs and an irrelevant MAb.RV-infected neuroblastoma cells were not stained by the irrelevant antibody,showing that the infected cells had not lost their membrane integrity (data not shown).However,in-tense labeling was detected with the three G-protein-speci fic MAbs (Fig.3A to D;data are shown for two of them).The distributions of cytoplasmic membrane-bound G protein were very different in ERA-and CVS-infected cells.In ERA-in-fected cells,the G protein was observed in localized areas of the cytoplasmic membrane,where it formed a fairly large rib-bon-like protein structure.On the contrary,in CVS-infected cells,the G protein was distributed in the cytoplasmic membrane as bright patches.As already illustrated in Fig.1,the ERA-in-fected cells subsequently underwent apoptosis,whereas the CVS infected cells did not.Similar data were obtained with Jurkat rtTA cells (data not shown).It is noteworthy that the morphology of ERA-infected SK-N-SH cells changed following infection.Indeed,they lost their neuronal shape and became rounder (Fig.1,section II,panel 2).This change did not occur in CVS-infected cells.To summarize,SK-N-SH cells infected with proapoptotic strains exhibited patterns of NC and G protein expression different from those of cells infected with nonapoptotic strains (Fig.1and 3).Furthermore,the pro-and antiapoptotic phe-notypes were characterized by different distributions of the RV G protein in the cytoplasmic membrane and morphological modi fications.Doxycycline-inducible expression of RV N and G proteins in Jurkat rtTA cells.To examine the consequences of the over-production of the RV G protein or the RV N protein on apoptosis in a nonviral context,we constructed Jurkat T-cell lines that produced the RV proteins following induction.Jur-kat T cells were selected for production of the reverse tetra-cycline transactivator protein rtTA (puromycin resistant)(31)and then infected with recombinant Moloney murine leukemia viruses (retroviruses)carrying the N or G protein gene of RV ERA and CVS as well as the neomycin resistance gene.TwoFIG.3.Distributions of viral G proteins differ in ERA-and CVS-infected SK-N-SH cells.(A to D)Confocal microscopy analysis of cytoplasmic membrane-anchored G protein in ERA-infected (A and B)and CVS-infected (C and D)SK-N-SH cells.G protein staining is shown for two MAbs:6-15-C4and 8.2.G-ERA formed a large ribbon-like structure.G-CVS was localized on the membrane as patches.(E and F)Quantitative cyto fluorimetry.(E)NC antigens that accumulated in the cytoplasm (black bars)were detected in PFA-treated ERA-or CVS-infected SK-N-SH cells.G protein,which is traf ficked through the endoplasmic reticulum and Golgi apparatus before being exported to the plasma membrane,was measured with an anti-G protein MAb on live cells to detect membrane-bound G protein (mbG;gray bars)or on PFA-treated cells to detect internal G protein (intG;white bars).Error bars indicate standard deviations.(F)mbG/intG (stippled bars),mbG/NC (black bars),or intG/NC (white bars)ratio.Proapoptotic strain ERA had a much higher mbG/intG ratio than nonapoptotic strain CVS.Thus,mbG accumulates on the surface of ERA-infected cells but not on that of CVS-infected cells.V OL .77,2003RV G PROTEIN MEDIATES APOPTOSIS 10541on June 2, 2012 by GUANGXI UNIVERSITY/Downloaded fromtransgenic cell lines that were resistant to hygromycin and puromycin were obtained after infection with the two types of recombinant virus.After doxycycline treatment,RV protein production in PFA-treated cells was monitored by flow cytom-etry (Fig.4).After 18h in the presence of doxycycline,N-ERA (Fig.4A)was present in 57.4%of the live cells,N-CVS was present in 64.3%(Fig.4B),G-ERA was present in 34.4%(Fig.4C),and G-CVS was present in 19.6%(Fig.4D).These data are representative of four independent experiments.Thus,the production of the RV G and N proteins can be induced in this system.These results show that the percentages of cells ex-pressing the G protein from both RV strains were slightly lower than those expressing the N protein and that the pro-duction of G-ERA was more ef ficient than that of G-CVS.The induction of G-ERA production triggers the apoptosis of Jurkat rtTA cells.We then tested whether the production of the RV G and N proteins could induce apoptosis in this sys-tem.Three different methods were used (Fig.5A to C).First,the induction of apoptosis was measured by flow cytometry by comparing side and forward light scattering in G-ERA-trans-genic Jurkat rtTA cells treated with doxycycline (Fig.5A,right panel)or not treated (Fig.5A,left panel).The number of dying Jurkat rtTA cells (R2population)increased when G-ERA was produced.The difference between the levels of ap-optosis in the presence and absence of doxycycline was 31.4%(39.7Ϫ8.3%).Second,we assessed apoptosis by monitoring nuclear fragmentation.Typical nuclear fragmentation (Fig.5B,right panel,arrows)was observed in doxycycline-treated JurkatrtTA cells expressing G-ERA (28.2%)but rarely in doxycy-cline-treated control Jurkat rtTA cells (10%)(Fig.5B,left panel).Finally,we monitored the ability of G-ERA to activate caspases.The detection of cells expressing activated caspases in Jurkat rtTA cells (Fig.5C,left panel)and in Jurkat rtTA cells expressing G-ERA (Fig.5C,right panel)either treated with doxycycline or not treated indicates that G-ERA expres-sion triggers the speci fic activation of caspases in 19%of the population (after subtraction of the value for doxycycline-treated Jurkat rtTA cells).Thus,the production of G-ERA triggers caspase-dependent apoptosis,leading to the fragmen-tation of cell nuclei.As commonly observed,the percentages of apoptosis measured by the different methods were not the same,but they all correlated (5,52).We used the same methods to study apoptosis in three other transgenic Jurkat rtTA cell lines expressing G-CVS,N-ERA,or N-CVS.The production of G-ERA led to the highest level of apoptosis (Fig.5D).The absence of apoptosis in Jurkat rtTA cells expressing G-CVS,N-CVS,or N-ERA could not have resulted from a lower level of expression,since even at a high level of G-CVS expression (Fig.5E)or at a high level of N-ERA or N-CVS expression (Fig.4and 5),apoptosis did not occur.These data strongly suggest that G-ERA is proapop-totic,whereas N-ERA and G-CVS are not.Is the viral G protein the only major apoptosis inducer?A recombinant virus was used to determine whether other RV proteins,namely,the N,P,M,and L proteins,can play major roles in apoptosis induction.Recombinant virus R-N2c was constructed from parental virus SN-10,a nonpathogenic virus that is closely related to ERA.In R-N2c,the G protein gene of strain SN-10is replaced by the G protein gene of strain CVS-N2c (Fig.6A)(42).R-N2c is transcribed and replicates as ef ficiently as SN-10,as shown by its rate of virus production and by the amount of N protein mRNA (42).Infection of Jurkat rtTA and SK-N-SH cells with nonpatho-genic strain SN-10triggered apoptosis,as determined by the percentage of cells in the R2population,the percentage of cells with fragmented nuclei,and the percentage of cells har-boring activated caspases (Fig.6B).In contrast,R-N2c did not induce apoptosis.The absence of apoptosis in R-N2c-infected cultures was associated with an absence of caspase activation (Fig.6B).The absence of apoptosis in R-N2c-infected cells was not associated with a lower level of infection,as 70to 85%of Jurkat rtTA and SK-N-SH cells were infected by R-N2c and SN-10in these experiments.Altogether,these data indicate that only the removal of the G protein gene from the proapoptotic RV strain is suf ficient to inhibit the proapoptotic property of this RV strain.Further-more,the N,P,M,and L proteins of the nonpathogenic viral strains (i.e.,ERA and SN-10)on their own cannot counteract this loss of apoptosis.Altogether,these data show that the G protein is a key element in apoptosis induction.Whether the G protein acts on its own or in combination with other viral and/or cellular proteins remains to be elucidated.The NC and G protein distributions are driven by the origin of the G protein gene.As described above,the G protein and NC of proapoptotic RV strains have distributions in infected cells different from those of nonapoptotic RV strains.Further-more,large amounts of NC accumulate in ERA-and CVS-infected SK-N-SH cells without the induction of apoptosis,andFIG.4.Inducible expression of RV proteins in Jurkat T cells.Theexpression of virus proteins in transgenic Jurkat rtTA cells (JrtTA)containing the gene encoding N-ERA (A),N-CVS (B),G-ERA (C),or G-CVS (D)was analyzed after 18h in the presence of doxycycline by flow cytometry with a MAb speci fic for the G or N protein (bold lines).Thin lines show Ab reactivity with cells that did not express the protein (cells without doxycycline).The numbers show the percentages of doxycycline-treated cells that expressed a fluorescence signal that was higher than fluorescence channel 5.These graphs are representative of four experiments.10542PRE´HAUD ET AL.J.V IROL .on June 2, 2012 by GUANGXI UNIVERSITY/Downloaded from。
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∞
(1.1)
L=
k=1
Lk ,
where L1 has dimension 2, Lk has dimension 1 for k > 1, and Lk+1 = [Lk , L1 ] for all k ≥ 1. Shalev’s construction starts from certain finite-dimensional simple modular Lie algebras, originally introduced by Albert and Frank [AF55], and applies a loop algebra construction (strictly speaking, taking the positive part of a twisted loop algebra, see Subsection 5.1 for details). As a consequence, the resulting graded Lie algebras of maximal class, despite being insoluble, have a kind of periodic structure, in a precise sense. Therefore, one could still hope that each of them is uniquely determined by a suitable finite-dimensional quotient, as is the case with pro-p groups of finite coclass (because each p-group of finite coclass r and class large enough, depending on p and r, is a quotient of a unique infinite pro-p group of coclass r). The investigation carried out in [CMN97] showed that this is not the case. In fact, it turned out that most graded Lie algebras of maximal class are not periodic. Nevertheless, Shalev’s algebras occupy a unique place in the description of the graded Lie algebras of maximal class over fields of odd characteristic, which have been completely classified in [CN00]. An analogous classification over fields of characteristic two will soon appear in [Jur05]. After the coclass conjectures for pro-p groups were proved, other invariants have been suggested, which may possibly lead to a finer classification of p-groups than the rough one provided by the coclass theorems. We do not need the precise definitions of these invariants, which are called width, obliquity and rank, and we refer the interested reader to Chapter 12 of the book [LGM02] by Leedham-Green and McKay. The simplest nontrivial case in terms of these invariants consists of the pro-p groups of width two and obliquity zero. These groups, which do not have finite coclass in general, have also been given the name thin in [Bra88, BCS92] (originally in the finite case) because of a narrowness characterization in terms of their lattice of (closed) normal subgroups. Some of our terminology, like the diamonds introduced in the next paragraph, originates from that point of view. An approach to thin groups via the associated graded Lie algebra was taken in [CMNS96]. The lower central factors in a thin group are elementary abelian of rank at most two. Those of rank two, in the group or in its associated graded Lie algebra, are called diamonds. There is of course a diamond G/γ2 (G) on top of a thin group G, and if this is the only diamond then G has maximal class. Otherwise, it follows from the theory of p-groups of maximal class that in a thin group the second diamond occurs in class at most p. In [CMNS96] we proved that the associated Lie