Nothing is known, however, about; thedevelopmental mechanisms governing butterfly oogenesis, how polarity in theoocyte is established, or which particular maternal effect genes regulateearly embryogenesis. To gain insights into these developmental mechanismsand to identify the conserved and divergent aspects of butterfly oogenesis,we analysed a Pararge aegeria females expressed74.5% of the genes that are known to be essential for D.melanogaster oogenesis. We discuss the genes involved in allaspects of oogenesis, including vitellogenesis and choriogenesis, plus thoseimplicated in hormonal control of oogenesis and transgenerational hormonaleffects in great detail. Compared to other insects, a number of significantdifferences were observed in; the genes involved in stem cell maintenanceand differentiation in the germarium, establishment of oocyte polarity, andin several aspects of maternal regulation of zygotic development.A total of 17306 contigs were annotated, with 30% possibly novel or highlydivergent sequences observed. This study provides valuable resources to investigate a number of divergentaspects of butterfly oogenesis requiring further research. In order to fullyunscramble butterfly oogenesis, we also now also have the resources toinvestigate expression patterns of oogenesis genes under a range ofenvironmental conditions, and to establish their function. Drosophila melanogaster, areinvolved in setting up; 1) the location of the germ plasm and subsequent germ cellline development in the offspring . Th. ThBomby by mex-3. Pararge-3 Table . Furthernslation . All of anogaster,65, presDrosophila melanogaster includes maternal hunchback(hb) transcripts into the egg, the protein of which will form an APgradient during early embryogenesis and cooperate with Bcd to specify theanterior of the embryo, whilst being repressed at the posterior by Nos [hb RNA or protein is transferred to the egg, as well as in thesignificance of the maternal contribution to the Hb gradient for AP patterning,the transcription of hb during oogenesis appears conserved [Schistocerca americana embryo, maternal hbtranscripts appear to be involved in distinguishing embryonic fromextra-embryonic cells along the AP axis, whilst in D. melanogastermaternal and zygotic Hb are redundant for AP patterning of the embryo [B. mori, the hb transcripts detected appear tobe transcribed by the zygote, not the mother [Pararge aegeria also did not express hb duringoogenesis ),which indeed appear to have divided up these functions [B. mori does not have agerm plasm, the location of maternal B. mori nos-O transcripts in theembryo seems to correspond with where the PGCs will form [nos paralogs, with the exception of nos-P,are expressed during oogenesis in both B. mori and P. aegeria,with maternal transcripts detectable in P. aegeria eggs genome [nos sequences showsnos-P to be quite different from the other paralogs  genome  and phylD. melanogaster nos RNA is accomplishedduring oogenesis by proteins encoded by glorund (glo) and in the earlyembryo by smaug (smg) [D. melanogaster oocytes [P. aegeria ortholog of smg was found, which waspresent as RNA in the oocyte, but not of glo  that preventsrapid deadenylation at the posterior pole, establishing nos as aposterior defining gene [osk ortholog [nosparalogs may not being involved in AP axis formation during oogenesis. Indeed,P. aegeria also does not possess an ortholog of osk(Table P. aegeriagenome).Translational repression of ug (smg) . Transcr oocytes . A P. aelo Table . Furtheranogaster. Rapid d nos RNA . AlthougR4 Table . In D.ming gene . DitrysiPectinophora gossypiella[osk, which has been argued to have been co-opted as theessential gene in germ plasm formation in holometabolous insects [Pararge aegeria may not possess an osk ortholog,but it does express two genes, which in D. melanogaster silenceosk translationally during oogenesis; bruno[cup[D.melanogaster , vasa (vas) and valois(vls) [valois (vls) could not be found in the P.aegeria transcriptome  . Only vae Tables .tud, vas and vls , Brother ofYb (BoYb) and Sister of Yb (SoYb) [P.aegeria transcriptome , TDRD7 andspindle E/homeless (TDRD9) [P. aegeria  . Indeed, (TDRD9) ,110 and  with osk. It is l with osk.piwi,aubergine (aub) and argonaute 3 (AGO3) [P. aegeria pathway genes often disrupt the axes of the developingoocyte, through their effects on the microtubule cytoskeleton; for examplemaelstrom (mael), zucchini (zuc) andsquash (squ) affect DV polarity [aub in D.melanogaster in silencing osk translation during oogenesis [armitage (armi)affects axis formation and is involved in osk translational silencingin D. melanogaster[zuc nor squ was found in the P.aegeria transcriptome, but mael and armi were . All thraFigure  and 10. em cells . Furtherpolarity ,115. Theanogaster. NeithereTables  and 10.D. melanogaster these can befound in nurse cells, but also appear to be involved in compartmentalisation ofmRNA decay and translation repression, for example of osk[EDC4/Ge-1 and pacman(pcm), genes that encode the essential components of P-bodies wereexpressed in P. aegeria  are aggregates of translationallyinactive ribonucleoproteins (RNPs). In le of osk,117. Witogenesis . This waD.melanogaster, a number of (late-acting) maternal-effect genes areessential in germline formation during early embryogenesis  and imp[germ cell-less (gcl) and polar granulecomponent (pgc)  is involved in germ cell migration inD. melanogaster embryos  and imp. However) Tables .These gte Table .Drosophila melanogaster uses an elaborate network of genes to patternthe DV axis during embryogenesis on the basis of the oocyte polarity establishedduring oogenesis , nudel (ndl), gastrulationdefective (gd), snake (snk), easter(ea), spn27A, spz, tube (tub)and pelle (pll) (Tables weckle (wek) have yet been found outsideDrosophila, and wek was also not found in P.aegeria , rather than toll (tl). A siminespace . This pria Table . In D.m)Tables  and 13. igration , and DV igration . While Ppathways . As discster toll. It thusPararge aegeria did express cactus (cact) anddorsal (dl)  is essential for subsequent DV patterning in the D. melanogasterembryo. Dorsal protein activates some genes, whilst repressing others along theDV axis [dpp ventrally and the proteinencoded by grainyhead (NTF-1/grh) acts asco-repressor [grh is deposited maternally into the oocyte to betranslated and used ventrally during embryogenesis [dpp by a Dorsal gradient does not, however,occur in T. casteneum[D. melanogaster embryo and its concentration isfurther restricted ventro-laterally by Short gastrulation (Sog), which in D.melanogaster may also be maternally provided [vrille (vri) gene encodes a Bzip transcriptionfactor that interacts in D. melanogaster with Dpp signalling, acting asdominant maternal enhancers of embryonic DV patterning defects caused byea and dpp mutations [eclair (eca) andbaiser (bai) are essential for the activity of maternalTkv, a type I Dpp receptor [Pararge aegeria females did transfer maternal transcriptsof grh, dpp, tkv, eca, bai and vri into the oocyte,but did not express sog maternally  Table . DorsalDV axis ,124. Whiprovided . Rather provided . The vriutations . Two P24receptor . ParargeDrosophila melanogaster females express a group of genes called theyema genes  during oogenesis,with most of them displaying strict maternal expression. This may be ofimportance in the development of the central nervous system of the embryo [yema genes arenot known and there are no orthologs outside Drosophila[P. aegeriatranscriptome ,elav, brainiac (brn), Fmr1, braintumor (brat), mnb, and terribly reduced opticlobes (trol)  biogenesis [torso (tor) encodes areceptor whose ligand is most probably encoded for by trunk(trk). Furthermore, the protein encoded by torsolike(tsl) plays a role upstream of trk in activating the Torreceptor in a localised manner, and is thought to be essential for terminalspecification [tor and tsl are involved in terminalspecification in T. castaneum, different tissues are patterned andTorso signalling plays a role in defining the posterior growth zone duringembryogenesis in this short germband insect [Apis mellifera) has the genetsl, but not tor and trk in its genome [Pararge aegeria does not express clear orthologs of eithertor or trk during oogenesis, but does express tsl(Table Bombyx mori does have a RTKin its genome (BGIBMGA003976), which shows similarity to torso, as wellas to tie-like and Cad96Ca. Pararge aegeria did notexpress tie-like , although it hadgreater similarity to the receptor tyrosine kinase Fps85D than totor. This transcript is present in both P. aegeria oocytesand ovarioles, but its role in oogenesis has not been described in theliterature. It is clear that P. aegeria uses RTK signalling duringoogenesis and that the sequences of its ligands and receptors have diverged fromthose of other insects. However, at present it is unclear in which functionalcontext RTK signalling takes place.The Torso receptor tyrosine kinase (RTK) pathway has been implicated in a numberof different processes during ogenesis  and in pogenesis . The matfication . Althougd insect . Torso ss genome . The hons genome . ParargeslTable . Bombyx D. melanogaster , sex combs onmidleg (scm), and Arginine methyltransferase 1 and8  andsimilar to D. melanogaster polyhomeotic (ph-p) gene. Both playversatile and important roles in D. melanogaster oogenesis,particularly in ovarian follicle formation [Pararge aegeria females did express and transfer orthologsof other PcG genes into the oocyte. These include the polycomb repressivecomplex 1 (PRC1) genes sex combs extra (sce),polycomb (ph), posterior sex combs(psc), the PRC2 genes extra sex combs (esc),Enhancer of zeste (E(z)) and the polycomb related genesEnhancer of polycomb (E(ph)) and additional sexcombs (asx)  important in oocyte production ,matrimony (mtrm), microcephalin (MCPH1)and chiffon (chif) .A large number of genes that regulate mitosis have been studied in a reproductivecontext in ri Table . Among talin MCPHand chifD. melanogaster germarium [bruno is not onlyessential in regulating the translation of a number of genes during oocytedifferentiation, but it also appears to be involved in regulating the silencingof Cdk1 activity in order to achieve primary meiotic arrest [bruno was expressed by P.aegeria females (Table The cell cycle becomes arrested in meiotic prophase I in the majority ofMetazoans oocytes. This is initiated during the first stages of oogenesis inregion 2 of the ermarium . The intc arrest . It shouc arrest . As indies Table .D. melanogaster germarium [D. melanogaster for SCformation and thus possibly chiasmata formation: crossover suppressor on 2of Manheim (c(2)M); crossover suppressor on 3 ofGowen (c(3)G); corona (cona) andnipped-B , corona (cona),orientation disrupter (ord) and mei-S332, for example, is bothessential in D. melanogaster for maternal provisioning of the eggduring vitellogenesis and to ensure secondary meiotic arrest by stage 14 ofoogenesis in metaphase I [P. aegeriafemales did express this gene during oogenesis  interacts with polo kinase(polo) in mitotic regulation particularly during earlyembryogenesis, and is maternally provided  and LpR [yl/VgR RNA into previtellogenicoocytes, thus preparing the oocyte for Vtg uptake [Pararge aegeria females expressed not only Vtg/Vg,apoLp-III, apoLp, their receptors yl/VgRand LpR, but also the genes described in D. melanogastervitellogenic endocytosis , sec5, sec6,garnet (G) and jagunal (jagn), such as Vitellogenin (Vtg/Vg) and Apolipophorins (ApoLPs) ,9. Predoanogaster. Vitelloanogaster. LLTPs a and LpR . Nurse cg uptake . Pararge)Figure .B. mori and released as ecdysteroids during yolk uptake in theembryo as a result of dephosphorylation by ecdysteroid-phosphate phosphatase (EPPase)[Pararge aegeria did express EPPase and Samia cynthia (ESP) had not been found [G. mellonella yolk protein 2 have been discovered inD. plexippus and Plodia interpunctella, whilstESP does show significant sequence similarity with genes encodingthe KK-42 binding proteins in Antheraea moth species [D. melanogaster, in particular lipase-1 and 3(lip-1 and 3) [. Rather interestingly, although not functioningas a yolk protein, lip-1, but not lip-3, is expressed invitellogenic follicles in D. melanogaster[lip-1, and possibly lip-3 , was expressed by P. aegeria, whilst no clearortholog of a minor yolk protein was found . ParargeseTable . Further protein . The genen found . More re species  (Additio1 and 3) . Lepidopanogaster. An orthnd Table .P. aegeria ovarioles is anortholog of the slime mold Physarum polycephalum genespherulin-2A. No transcripts were found for this gene in eggs  Chico precludes vitellogenesis, whilst a sharp increasein 20-hydroxy-ecdysone (20E) relative to juvenile hormone (JH) results inapoptosis of the egg chamber before vitellogenesis is initiated or completed [D. melanogaster[bbx genes are expressed predominantly in the brain, butsome may also be expressed in ovaries [B. mori, possess a large number ofbbx-like genes in their genome [D. plexippus appears tohave only three such genes [bbxA1-like andbbxA3-like) were transcribed in P. aegeria ovarioles,whilst a third partial IRS transcript showed more sequence similarity tochico than to any bbx-like gene (Table insulin-like receptor (InR) was also expressed by P.aegeria during oogenesis  and the lipid storagedroplet proteins 1 and 2 (Lsd1 and Lsd2). Please refer to Table Reproductive output depends on female nutritional status which not only affectsthe rate and duration of oogenesis significantly, but also whetherprevitellogenic egg chambers will enter the vitellogenic stage or apoptose . Two sigompleted ,155. Altanogaster. The bbx ovaries . Moths, r genome , but thech genes . OrtholoApart from nutritional status, environmental factors such as temperature canaffect hormone concentrations, providing a possibility for environmental controlof reproductive output ,26. The methoprene-tolerant (met) [taiman (tai) in D.melanogaster[tai gene was originally discovered as a gene that wasexpressed in follicle cells in the functional context of border cell migrationand was described as an ecdysone co-receptor  can also been found in the genome of D. plexippus[There has been extensive investigation of JH signalling ,26, but nt (met) -160. It anogaster,160. Theplexippus.kruppel-homolog 1 (krh1) has beendescribed as a JH response gene, inhibiting 20E induced broad(br) expression in D. melanogaster, but not in thespecific context of oogenesis [khr1 and br were expressed by P.aegeria females , which regulates polyamine biosynthesisand appears to be essential for vitellogenesis [odc and its antagonist gutfeeling(oda), also a mitotic cell-cycle regulator, were expressed inP. aegeria. Maternal transcripts of odc and odawere found in eggs  and the JH esterases (JHEs) . JHEs fuB. mori and in aJHbp CDSs were identified in P.aegeria ovaries; JHbp, cytosolic JHbp(cJHbp), hemolymph JHbp (hJHbp) and a sequenceshowing strong orthology to takeout (to) identified in D.melanogaster as involved in JH binding  to enableimmobilisation, regulate degradation or enable transport . Four cong Table . Transcr, like P. aegeria, have been argued tobest match the criteria for group 4 [B.mori and D. melanogaster[There is a significant amount of life-history variation among insects andconsequently in the relative importance of 20E and JH on oogenesis , even wi group 4  where JHanogaster,146.Bombyx mori appears to be capable of producing ecdysteroidsin the ovaries [D. melanogaster[Drosophila melanogaster expresses start1 duringoogenesis in significant amounts in nurse cells, most likely in response toecdysone signalling. The cholesterol transporter Start1 may in turn facilitateecdysteroid production from cholesterol-based precursors [D.melanogaster cholesterol conversion in the ovaries is defective inthe avoidance of repellents (dare), which encodes an Adrenodoxinreductase [D. melanogaster the SGT1 protein homologecdysoneless (ecd) and disembodied (dib)have been described as essential for ecdysone, both for functionality and itsproduction in the ovaries [D. melanogaster start1 arehypothesised to be deposited into the egg to facilitate ecdysteroid signallingin the developing embryo [P. aegeria females did not expressdib, but did express ecd, start1, and dare. Weobserved the transfer of transcripts of all three genes into the oocytes and its partner ultraspiracle  in B. mori) in theovaries , thetranscription factor gene E74 and the Broad-Complex gene Br-C[B. mori andtheir expression increases during vitellogenesis [E74, all of these genes wereexpressed in P. aegeria  during choriogenesis [P. aegeria females did not expressESP, but did express the related gene lip-3 is expressed in all follicle cells and is essential for DVpatterning of the embryo in D. melanogaster[D. melanogaster embryo [Pararge aegeria females expressed ndl and as inD. melanogaster, no transcripts were found in the oocyte is formedhalfway through vitellogenesis , for whie oocyte . This al [B. mori, althouganogaster. Ndl is anogaster. As suchr embryo . ParargeteTable . It remaD. melanogaster VMPgenes outside the genus Drosophila. The best-characterised VMPgene in Lepidoptera is VMP30[P. aegeria ovarioles. Once again, notranscripts were found in the oocyte (Table Insect vitelline membrane protein (VMP) genes show tremendous sequence diversity.For example, no clear orthologs can be found for  is VMP30, for white Table .D. melanogaster and clusters of choriongenes are selectively amplified or expressed at very high levels [P. aegeria did not express anortholog of G1/S specific cycE, which in D. melanogaster isessential for chorion gene amplification and endocycling in general  in D. melanogaster[P. aegeria (Table B. morihave multiple cis-regulatory binding sites for CCAAT/enhancer bindingprotein (C/EBP) transcription factors and their expression levels are C/EBPconcentration dependent [D. melanogaster ortholog of C/EBP is slbo,which is also expressed in follicle cells though predominantly involved inborder cell migration , its negative regulator tribbles (trbl) andHMGa  [B. mori[D. plexippus[cf1 and CbZ were transcribed by P.aegeria, with transcripts of the latter rather intriguingly found to bepresent in the oocyte  . Further [B. mori and orthplexippus. HoweverD. melanogaster cp geneswith those identified in Lepidoptera, including P. aegeria, is very lowindeed , which forms part of the eggshell and is produced by the follicle cells [P. aegeriaoogenesis (Tables Chorion protein (cp) genes evolve possibly even faster than vitelline membraneprotein genes  and sequed Table . The infstrength . Furtherfication . Expressfication ,179. As le cells . BmEP80 D.melanogaster and B. mori, with nurse and follicle cellsundergoing apoptosis as oogenesis progresses, while complete egg chambers mayapoptose in response to environmentally induced hormonal signals such asstarvation [D. melanogasterovaries, where the effector caspase Dcp-1 and the inhibitor of apoptosis proteinBIR-superfamily domain protein Bruce regulate both autophagy and starvation-induced cell death [B. mori, and the results of the study by Zhang and co-workersshowed that most of these genes are highly conserved [D. melanogaster , which is required for posterior follicle cell maturation [D. melanogaster oogenesis  and mob as tumor suppressor   . It is essential for regulating tissue size and growth . Hippo sturation . Ortholo1) Table . Merlin/M1 Table , which iM2Table . In D. mlisation , but clei) Table . The latD. melanogaster, for example, Hsp60C is essential inorganising and maintaining cytoskeletal and cell adhesion components and thusfor establishing AP and DV oocyte polarity [D. melanogaster oogenesis(Tables Heat shock proteins (Hsps) provide a possible mechanism for environmental controlof development in ovaries and as maternal effects. The transcription of genesencoding Hsps, or molecular chaperones in general, is not only regulated inresponse to various environmental factors (e.g. temperature), but is alsoessential during many developmental processes, including oogenesis. It isthought that Hsps are important for both developmental buffering anddifferentiation ,186(furtpolarity . A large)Tables .D. melanogaster oogenesisand early embryogenesis  are among the most highly transcribed genes duringMetazoan oogenesis, as large amounts of the translation machinery are neededboth during oogenesis and by the developing embryo . Just li are amonis Table .P. aegeria and present as maternal transcripts in the oocytes, has been implicated in DV axisformation [Orthologs of the majority of the genes identified from the literature as beinginvolved in immune response during oogenesis were also found to be expressed byesTable  and 2. AesTable , these iesTable .Drosophormation .Wolbachia sp. is anendocytosymbiont in many arthropod species affecting oogenesis in a multitude ofways and the Bacterium is maternally transmitted [D. mauritiana, Wolbachia increases egg production byaffecting the maintenance and division of germ-line stem cells [Asobara tabida, Wolbachiaconfers a reproductive advantage to the females by properly regulating apoptosisduring oogenesis via its regulation of iron metabolism and ferritinexpression [D. melanogaster highly infected females sufferfrom a range of oogenesis defects mediated via grk signalling [Pararge aegeria females were also found to be infected withWolbachia, but how this affects oogenesis in this species is atpresent not known. However, we did observe that the gene encoding an ortholog ofthe Ferritin 2 light chain protein (FER2-LCH) was amongst the most highlytranscribed genes during P. aegeria oogenesis M19/wisp)tail elongation of bcd, toll, and tor transcriptsupon egg activation. It is thus important for proper patterning of the embryo [Given that P. aegeria females did not expressbcd and tor, it remains to be investigated whether wisp isof any importance in patterning of the embryo.As discussed elsewhere in this paper, after vitellogenesis both the aphase I ,194. Unlaphase I , egg actme Table ,which mp)Table . In D. mansition . The D. e embryo , but is e embryo . Given tD. melanogaster oogenesis)were transcribed by P. aegeria and transcripts were transferred to theoocytes. As this was an ovarian transcriptome study, the precise functional contextin which these genes were transcribed has not been identified. Differences in thefunctional context in which particular genes are expressed are to be expectedcompared to model organisms such as D. melanogaster and even B.mori. What is perhaps more revealing, however, is the absence of certaintranscripts in the database, in particular where these transcripts concern paradigmsof maternal regulation for various aspects of early insect embryogenesis [Pararge aegeria differed most significantly from D.melanogaster (and quite a number of other insect species), both in terms ofstem cell maintenance or differentiation in the germarium and in establishing (andmaintaining) polarity along AP, DV and at the termini of the oocyte. In particular,although Pararge aegeria females expressed an ortholog of aspi/krn-like EGF ligand and possibly its receptor, manycomponents of the EGF pathway involved in patterning of the axes in D.melanogaster embryos, as well as pipe andmirror, were not expressed. This may either suggest that there is not muchevidence for a significant role of EGF signalling in establishing P.aegeria oocyte polarity, or that its functional role and genes involved isdivergent from other insects. This requires further study, as well as the functionalrole and significance of Dpp and Notch signalling in this context.A large proportion of the genes currently described in the literature as beingessential during insect oogenesis . Although progress has beenmade in investigating B. mori embryonic patterning [P.aegeria may prove an ideal model these future studies.Although the more derived species such as and-like , it is mand-like ,54, as teristics . It is vtterning ,54, how Unfortunately, maternal effect gene expression and regulation have receivedsignificantly less research attention in Lepidoptera compared to vitellogenesis,choriogenesis and reproductive physiology . This isP. aegeria as an eco-evo-devo modelsystem for the study of butterfly oogenesis. In order to fully unscramble butterflyoogenesis, an investigation of the spatio-temporal expression patterns of the genesdiscussed in this study, as well as establishment of their function, is required.Further studies are also required to establish the function and expression patternsof the uncharacterised contigs identified in this study, which make up 30% of thetotal contigs found, and are undoubtedly composed of genes that are of highimportance in butterfly oogenesis.This study has taken a much-needed first step in determining the conserved anddivergent elements of the butterfly oogenesis GRN  and establishes P. aegeria. This population originatedfrom a woodland population from the south of Belgium  and by the time of the experiment, the butterflies had been rearedin the laboratory for 10 generations. Newly hatched larvae were placed on pottedhost plants (4 larvae per plant) of Poa trivialis L. with access toad libitum food and were reared until eclosion in a climate roomunder a regime  that promotes direct development(i.e. no diapause). On the day of eclosion  females from this laboratory stock placed individually in netted cages (0.5m3) along with a potted P. trivialis plant foroviposition and an artificial flower containing a 10% honey solution [As butterflies were used in this study, no ethical approval was required. Eggswere collected from a large outbred laboratory population of solution . Later tEggs from 50 mated 4-day old females were collected within 20 minutes of beinglaid, which is well before the onset of cleavage and thus early embryogenesis inbutterflies . The eggThe homogenate (both of eggs and ovarioles/ovary) was first centrifuged at 13000gfor 10min primarily to remove the yolk, after which the supernatant was vortexedwith 200μl of chloroform. Phases were separated at 13000g for 15min at roomtemperature. The aqueous phase was removed and precipitated in 0.5ml isopropanol . The RNAPararge aegeria egg and ovary RNA was sequenced by Source BioScience using Illumina short read RNA-Seq technology. Both total RNAsamples went through polyA selection, fragmentation and double stranded cDNAconversion to produce two separate libraries (300bp insert size) in accordancewith the Illumina mRNA-seq library preparation protocol . Sequencing was performed on the Illumina Genome Analyzer IIx platform withone flowcell lane allocated to each library. A total of 61,400,070 single-readsof 38 base pairs (bp) in length were obtained from the ovary and egg flowcelllanes which were pooled to produce a de novo assembly in CLC GenomicsWorkbench v4.0  using the default settings for shortread data (automatic word and bubble size) [le size) . The assde novo assembly usingTopHat on the Galaxy server [The RNA-seq data was analysed by FASTQC on the Galaxy platform ,199. Aday server . FPKM vay server  with quade novo assembly  was discarded from each rank, using the next best hit (which may bein a lower rank or group)  used for annotation, includingthose genes annotated manually, were used as input in the BLAST2GO software  and assi°C for 30 min after an initial 5 min denaturation step at70°C. Negative reverse transcription (NRT) controls were run inparallel without both Verso RT enzyme mix and primers. A final heat deactivationat 95°C for 2 min was also implemented to deactivate the RTenhancer. The resulting cDNA was stored at −20°C.For of a subset of 19 genes the expression in the ovarioles and the presence oftranscripts in the oocyte were confirmed further by means of RT-qPCR . Only those primers exhibiting the best stability were selected. Eachprimer pair was tested on a 3-step 5-fold dilution series of the ovary cDNA intriplicate, which enabled the primer pair efficiencies to be determined usingthe CFX Manager software . Primers withadequate efficiency (>65%) were then used for investigating the transcriptabundance in the egg and ovary cDNA  on white 96-well plates in ABsolute Blue qPCR SYBR Green Mastermix with the recommended amount of ROX reference dye was used to reveal whetherany individual transcript was used as a maternal effect gene transcript or wasmerely necessary for oocyte production. Relative transcript abundance in theovaries and eggs were obtained using the relative expression software tool RESTv2.0.13.0 software package , which uP. aegeria transcriptome investigatedby means of qPCR, transcript abundance calculated on the basis of Cq values bymeans of the methods described in [41 = 2.37, P = 0.011;Additional file The number of reads mapping to a transcript of a particular gene in RNA-seq datawas argued to be correlated linearly with the number of transcripts of that gene . Rather ribed in  showed sThe sequence read data reported in this manuscript have been deposited in theNCBI Sequence Read Archive and are available under the accession numbersSRR771147 (ovarian reads) and SRR772253 (oocyte reads). Additional file GRN: Gene Regulatory Network; eco-evo-devo: Ecological evolutionary development; AP:Anterior-posterior; DV: Dorso-ventral; RNA-seq: RNA-sequencing; RNP:Ribonucleoprotein; RTK: Receptor Tyrosine Kinase; CDK: Cyclin-dependent kinase; SC:Synaptonemal Complex; RN: Recombination Nodules; IRS: Insulin Receptor Substrate;20E: 20-hydroxy-ecdysone; JH: Juvenile Hormone; FPKM: Fragments Per Kilobase of exonper Million of fragments mapped; ORF: Open Reading Frame; SS: Similarity Score; GO:Gene Ontology; RT-qPCR: Real-time reverse transcription quantitative polymerasechain reaction; NRT: Negative reverse transcription; NTC: No template controlThe authors declare that they have no competing interests.nanos. JT assisted in manualannotation of the transcriptome. MG and CJB designed and supervised the study,performed the manual annotation of the transcriptome, and co-wrote the manuscript.All authors have provided comments on earlier drafts of the manuscript and approvedthe final version of the manuscript for publication.JMC collected and analysed RT-qPCR data, designed the automatic annotation pipeline,performed bioinformatic analyses, and co-wrote the manuscript. SCB assisted inRT-qPCR study design and data collection. RP and DRFC prepared RNA samples forRNA-seq. AC performed phylogenetic analyses of Oogenesis genes. Contains a tabulated and fully referenced list ofgenes identified from the literature, which have been studied in the contextof insect oogenesis and maternal regulation of early embryogenesis. The vastmajority of papers concern the fruitfly Drosophila melanogaster andthe silkmoth Bombyx mori. Many genes have multiple functions duringoogenesis, but to avoid repetition, and keep the size of the Tablemanageable, each gene has been listed only once in the functional contextfor which it is probably best known. Referencing has been kept to a minimum,highlighting key papers and databases. Hyperlinks have been provided foralmost all of the genes listed, which will provide full database informationon their myriad functions and further references. Presence (Y) or absence(N) of orthologs in the Pararge aegeria combined oocyte andovariole transcriptome are indicated.Click here for fileAnnotation summary of the combined transcriptome of the Pararge aegeria ovarioles and oocytes. Details the results of both automaticand manual annotation of 25266 contigs. Egg and ovary FPKM values aregiven for each contig. Each column contains a pop-up comment box with anexplanation of the column contents.Click here for fileOverview of the primer pair properties and performance in qPCRconditions. Gives an overview of the forward and reverse primersdesigned for qPCR of a set of 19 oogenesis and 3 housekeeping genes.Efficiency and R2 values are provided for each of theprimers.Click here for fileData generated by the CFX96 qPCR experiments. Details themeasurements from a total of 8 96-well white plates. Cq are given foreach gene of interest or reference gene.Click here for fileRelative Abundance Data generated by REST. Gives the results fromusing REST v2.0.13.0 to process Cq measurements and efficiencies inorder to estimate relative transcript abundance, and thus comparerelative transcript abundance between ovaries and eggs.Click here for fileTranscript abundance: Cq - FPKM correlation. Provides the resultsof the correlation analyses between two measures of transcriptabundance: Cq and FPKM-values.Click here for fileMapping of raw RNA-seq reads against egfr and wingless coding sequences as predicted from the draft Pararge aegeria genome. Provides the complete egfr andwingless (wg) CDS fasta information from ourunpublished P. aegeria genome. Furthermore, raw RNA-seq readswere mapped against these sequences and coverage determined.Click here for filePhylogenetic analysis of Nanos. Provides a phylogenetic analysisof insect Nanos protein sequences.Click here for filePhylogenetic analyses of both chorion and minor yolk proteins inLepidoptera. Provides the phylogenetic analyses of both chorionand minor yolk proteins in Lepidoptera.Click here for fileOocyte and ovarian RNA quality. Provides the Agilent BioAnalyzerElectropherograms detailing oocyte and ovarian RNA quality prior to cDNAsynthesis.Click here for fileFiltering of BLAST hits in the automated annotation. Provides avisualisation of the similarity score distribution and thresholdsapplied in the automated annotation of the P. aegeriatranscriptome.Click here for fileAutomated annotation based on different BLAST Strategies.Provides a summary of the automated annotation method, detailing thedifferent queries.Click here for fileDistribution of similarity classes across BLAST sources. Providesdetails regarding the number of Pararge aegeria contigs in eachof the similarity classes, according to the BLAST strategy used in theautomated annotation.Click here for fileThermocycler and qPCR reaction setup. Provides details regardingthe reaction conditions and thermocycler programming parameters forsuccessful qPCR amplification for each qPCR measurement reported in thisstudy.Click here for fileCombined annotated ovarian and oocyte transcriptome of Pararge aegeria. Provides the fasta format sequences of the contigs, which inAdditional file 2 had a YES in the SubmitFlag column (i.e. to besubmitted to NCBI TSA). Suggested annotated names are given on the basisof the BLAST results listed in Additional file 2, and as described inthe main text. The start and end of the open reading frames can be foundin the final two columns of Additional file 2.Click here for file


Objective. To evaluate the effect of eradication of Helicobacter pylori (H. pylori) on the progression of dementia in Alzheimer's disease (AD) patients with peptic ulcer. Methods. Participants with the diagnosis of AD and peptic ulcer were recruited between 2001 and 2008. We examined the association between eradication of H. pylori and the progression of AD using the multiple regression models. Medication shift from Donepezil, Rivastgmine, and Galantamine to Mematine is defined as progression of dementia according to the insurance of National Health Insurance (NHI) under expert review. Results. Among the 30142 AD patients with peptic ulcers, the ratio of medication shift in AD patients with peptic ulcers is 79.95%. There were significant lower incidence comorbidities  in patients with H. pylori eradication as compared with no H. pylori eradication. Eradication of H. pylori was associated with a decreased risk of AD progression (odds ratio [OR] 0.35 [0.23–0.52]) as compared with no H. pylori eradication, which was not modified by comorbidities. Conclusions. Eradication of H. pylori was associated with a decreased progression of dementia as compared to no eradication of H. pylori in AD patients with peptic ulcers. An impressive intervention study has shown positive results that the H. pylori eradication may improve the cognitive functiona outcome within two years, but the sample size of case (28 patients) and controls (16 patients) might be small for application to general population [H. pylori infection may be more cognitively impaired. Roubaud-Baudron et al. found higher CSF cytokine  and significantly positive correlation between H. pylori immunoglobulin level and homocysteine level and they concluded that the impact of H. pylori infection on AD course may be attributed to cerebrovascular lesions and neuroinflammation [H. pylori infection modulating neuroinflammatory process may have a protective role for AD. Given that Taiwan's National Health Insurance Reimbursement Policy requested physicians to perform eradication of H. pylori infection depending on gastrointestinal endoscopy biopsy with or without 13C-urea breath test and neurologists to treat worsening of AD patients with anti-cholinesterase treatment according to repeated neuropsychological assessment and detailed expert review, we were allowed to observe the impact of eradication of H. pylori on the AD course.Alzheimer's disease (AD) is a common neurodegenerative disorder for which causes are diverse, and it involves similar neuroinflammation cascade as prion disease . Cerebraε4 , 4. Seve pylori) –8. As fopulation . Additiopulation –13. A reammation . These oThe database used in this study included one million randomly selected subjects from the 1996–2007 Taiwan National Health Insurance Research Database (NHIRD), which was developed for research purposes. The NHIRD is a research database developed at the National Health Research Institute, with linked data from the demographic and enrollment records, hospital claims, ambulatory care visits, and pharmacy dispensing claims from hospitals, outpatient clinics, and community pharmacies. Our source population comprised all beneficiaries from the Longitudinal Health Insurance Database 2005 who were at least 50 years of age on January 1, 2001. There were no statistically significant differences in age, gender, or average insured payroll-related amount between the sample group and all enrollees.Study Population. From the NHID, AD patients who were collected from outpatient pharmacy database between January 1, 1997, and December 31, 2004, with a primary diagnosis of dementia (Classifications of Diseases-9 codes: 290.xx) and regularly taking anticholinesterase medications  classification system codes provided in Supplemental Table 1 in the supplementary material available online at http://dx.doi.org/10.1155/2013/175729) for more than 3 months. We then selected AD patients with the diagnosis of peptic ulcer . Patients who received H. pylori eradication therapy and those who did not receive H. pylori eradication therapy were classified into two subgroups. Due to Taiwan's National Health Insurance Reimbursement Policy request, worsening of neuropsychological assessment including minimental status exam may shift anticholinesterase medications  to memantine and defined as worsening of dementia; AD patients who shift or did not shift anti-cholinesterase medications were analyzed separately. Comorbidities were defined as diseases diagnosed before the index outpatient clinic visit.H. pylori eradication with triple or quadruple therapy was defined as proton pump inhibitor or H2 receptor blocker, plus clarithromycin or metronidazole, plus amoxicillin or tetracycline, with or without Bismuth  [H. pylori eradication date after index hospitalization.viously) . These dWe used inpatient and outpatient diagnosis files and prescription files during the 12-month period before the index date to ascertain patients' history of hypertension diabetes mellitus, cerebrovascular disease, coronary artery disease, congestive heart failure, and hyperlipidemia ; we also collected patient information on age, sex, and resource utilization  12 months prior to the index date.H. pylori eradication therapy on risk of shifting anticholinesterase medications by comparing the occurrence of shifting medications after the H. pylori eradication therapy among AD patients with peptic ulcers. Multiple regression model was used to calculate the odds ratios (ORs) and their 95% CIs.Baseline characteristics, comorbidities, and medication use were presented. For all cohort members, we computed their person days of followup in each anti-cholinesterase medication category. We examined the effect of H. pylori eradication (n = 675) and without H. pylori eradication (n = 863). Among these two groups enrolled in our study, several baseline characteristics, including diabetes mellitus, hypertension, cerebrovascular disease, congestive heart failure, coronary artery disease, and hyperlipidemia, were higher in frequency in patients without H. pylori eradication (H. pylori was 89.4% to 90.5% [n = 675) had successful eradication of H. pylori. Our results would underestimate the association between eradication therapy of H. pylori and the progression of AD.After excluding subjects who did not meet our study criteria, a total of 30142 AD patients were included in the analysis . A totaldication . Accordito 90.5% . Thus, iH. pylori eradication, H. pylori eradication was associated with a decreased risk of changing anticholinesterase medication with the OR of 0.58 (95% CI 0.46–0.74). The protective association of H. pylori eradication persisted with OR of 0.34 (95% CI 0.23–0.52) after adjustment of comorbidities , were taken into consideration. However, there are several limitations. First, although we analyzed health care records from a national representative dataset of 1 million people, there were still few AD cases to allow us to have a precise estimation. Second, we did not adjust the use of medications that potentially may affect AD risk such as statins, NSAID, antidiabetic agents, calcium channel blockers, and neuroleptic agents. Third, we were not able to assess the genotype of apolipoprotein E allele ε4, which cement the solid relevance in late-onset AD [H. pylori, and AD.The strength of our study is the enrollment of a nationally representative cohort of a large sample size. The information regarding anticholinesterase medications and eradication therapy of onset AD –32. FourH. pylori had a deceased association with AD progression compared with no eradication therapy among AD patients with peptic ulcer. Further long-term follow-up study is needed to confirm the potential beneficial role of antibacterial therapy of H. pylori in AD.We observed that eradication therapy of Table 1. Anatomical therapeutic chemical (ATC) for anticholinesterase medications.Table 2. ICD codes for comorbid diseases.Click here for additional data file.


Extrapulmonary tuberculosis (TB) account for approximately 15–20% of TB cases in immunocompetent patients. The genitourinary system is the third most commonly affected site. We report the case of a 20-year-old man admitted with fever, chills, dry cough, right flank pain, and oliguria who developed renal function loss. The pyelogram evidenced silence of the right kidney, and the abdominal and pelvic magnetic resonance showed significant dilation of the right pyelocaliceal system and proximal ureter. Biopsies of renal cortex and retroperitoneal lymph nodes showed caseous granuloma consistent with TB. Treatment was started with rifampicin, isoniazid, pyrazinamide, and ethambutol, and the patient presented a favorable outcome but with non-dialytic chronic kidney disease. This case illustrates a case of chronic kidney disease secondary to TB in a young, otherwise healthy man. Tuberculin skin test was 8 mm; human immunodeficiency virus (HIV) serology was negative. Urinalysis showed sterile pyuria, but microscopy showed acid fast bacilli. The plain abdominal X-ray film showed silence of the right kidney (2). The genitourinary system is often affected in TB resulting from hematogenous spread from primary pulmonary TB.A 20-year-old man was admitted with fever, chills, dry cough, right flank pain, and oliguria. Physical examination showed a palpable mass in the right flank. Laboratory analysis was notable for a creatinine clearance = 59 mL/min per 1.73 mt kidney . The chet kidney . The abdt kidney . Biopsie


Oryza diversity. However, the mechanisms underlying gene and genome evolution in Oryza polyploids remain largely unknown. The allotetraploid Oryza minuta, which is estimated to have formed less than one million years ago, along with its putative diploid progenitors , are quite suitable for the study of polyploid genome evolution using a comparative genomics approach.Polyploid species contribute to Shattering4 locus in O. minuta, as well as in O. punctata and O. officinalis. Duplicated genomes in O. minuta have maintained the diploid genome organization, except for several structural variations mediated by transposon movement. Tandem duplicated gene clusters are prevalent in the Sh4 region, and segmental duplication followed by random deletion is illustrated to explain the gene gain-and-loss process. Both copies of most duplicated genes still persist in O. minuta. Molecular evolution analysis suggested that these duplicated genes are equally evolved and mostly manipulated by purifying selection. However, cDNA-SSCP analysis revealed that the expression patterns were dramatically altered between duplicated genes: nine of 29 duplicated genes exhibited expression divergence in O. minuta. We further detected one gene silencing event that was attributed to gene structural variation, but most gene silencing could not be related to sequence changes. We identified one case in which DNA methylation differences within promoter regions that were associated with the insertion of one hAT element were probably responsible for gene silencing, suggesting a potential epigenetic gene silencing pathway triggered by TE movement.Here, we performed a comparative study of a large genomic region surrounding the O. minuta.Our study revealed both genetic and epigenetic mechanisms involved in duplicated gene silencing in the allotetraploid  Oryza have been employed as excellent genetic resources for rice breeding and genetic modification gene locus, respectively), to hybridize to high-density filters containing three Oryza genomic BAC libraries. Combined with physical map positions in Finger Printed Contigs (FPC), a total of 98 positive BAC clones were identified. All screened BAC clones were digested with HindIII, size-selected by electrophoresis and transferred onto nylon filters for Southern blot analysis. For diploid genomes, eight additional probes were used to identify the BAC clones, which maximized the orthologous region coverage and minimized the gaps between consecutive BAC clones. To distinguish between the subgenomes of O. minuta, the digested map of each BAC was compared with that of O. punctata and O. officinalis. The tetrapolyploid BAC clones were divided into two groups  based on their HindIII digestion patterns. Ten BAC clones of three Oryza species were sequenced with an ABI 3730 automated sequencer (Table O. brachyantha (FF) were recently generated[BAC clones covering the orthologous regions of the u et al.. Initialr Table . Specifir Table . Ortholoenerated.japonica Sh4 genome annotation as a reference gene model, transposon-related gene models and hypothetical genes without cDNA, ESTs and homologous proteins were excluded. Gene structures were confirmed using full-length japonica cDNA. To annotate non-japonica genomes, four wild rice genomes were initially repeat-masked and predicted using FGENESH (http://linux1.softberry.com). All predicted genes were aligned with japonica cDNA and proteins from rice, Sorghum, Brachypodium and Maize. Gene models without cDNA or protein supports were excluded. All gene models were manually refined based on the AA-BB-BBCC-CC multiple alignment framework. Conserved gene structures were modified based on japonica gene models. RT-PCR was used to detect gene structure if great variation existed between japonica genes and non-japonica genes. Multiple sequence comparisons were performed with CLUSTALW. Structural variation was detected using Artemis Comparison Tool (ACT)[A comparative gene annotation approach was taken to identify gene models in the BB, CC and BBCC genomes. Before using the ol (ACT).http://www.repeatmasker.org/). LTR-type transposons were also predicted with LTR_STRUC[-8 per site per year[To identify transposon elements, RepeatMasker was initially used to annotate the TEs and other repeat sequences, followed by manual analysis . The SSCP results revealed the gene expression patterns of duplicated genes in O. minuta, including both coexpressed and gene silencing. All primer sequences are listed in Additional fileTotal RNA samples were isolated from six different tissues of three O. punctata, O. officinalis and O. minuta leaves, digested with restriction enzymes and treated with sodium bisulfite to convert the unmethylated cytosine residues to uracil. A set of primers was designed to amplify the genomic region of each gene from the end of the first exon to ~2 kb upstream of the gene; the size of each PCR product was approximately 150–300 bp. Primer sequences are listed in Additional fileDNA was extracted from mature Sh4 genome sequences for wild rices can be downloaded from NCBI at http://www.ncbi.nlm.nih.gov/nuccore/HQ827834 (BB), http://www.ncbi.nlm.nih.gov/nuccore/HQ827835 (BC_B), http://www.ncbi.nlm.nih.gov/nuccore/HQ827836 (BC_C) and http://www.ncbi.nlm.nih.gov/nuccore/HQ827837 (CC). O. sativa ssp. japonica sequences can be downloaded from http://rice.plantbiology.msu.edu/. FF genome can be downloaded from http://www.gramene.org/Oryza_brachyantha/Info/Index.The authors declare that they have no competing interests.MSC designed and managed the project. YS and JFS prepared the materials and generated sequence data and performed the cDNA-SSCP analysis. YS and BL analyzed the data and performed epigenetic experiments. BL and MSC wrote the manuscript. All authors read and approved the final manuscript.Table S2. Gene annotation results for three Oryza species compared with japonica models. Table S3. Annotation details of LTR retrotransposon in four Oryza genomes. Table S4. Intact elements of DNA transposons across the Sh4 region. Table S5. Pair-wise relative rate test of duplicated genes between O. minuta and its diploid progenitors. Table S6. Pair-wise estimates of Ka/Ks and divergence time among the BB, CC and BBCC genomes. Table S7. Expression divergence of duplicated genes in allotetraploid O. minuta.Figure S1. Phylogenetic analysis of Gene 29 clusters. Figure S2. Evolutionary analysis of Gene 54 clusters. Figure S3. TE contents in five Oryza species. Figure S4. Large inversion detected in the CC subgenome of O. minuta. Figure S5. Pseudogene annotation. Figure S6. Distributions of Ks density for homoeologous genes between two subgenomes in O. minuta and corresponding diploid genomes. Figure S7. Results of cDNA-SSCP analysis with 34 pairs of duplicated genes.Removed gene models from MSU Rice Genome Annotation Release 7. Click here for filePrimer information in this study.Click here for file


In patients with malignant hematological disorders receiving immunosuppressive therapy, invasive pulmonary infections are serious complications that are associated with high morbidity and mortality. In immunocompromised hosts with impaired cellular immunity, two or more organisms may coexist leading to a wide range of clinical and radiological manifestations.Nocardia asteroides and Aspergillus niger that was complicated by septic shock. The invasive pulmonary infections were successfully treated with trimethoprim-sulphamethoxazole, amikacin and liposomal amphotericin-B (amBisome).Reported here is an old man who was diagnosed to have angioimmunoblastic T-cell lymphoma at King Faisal Specialist Hospital and Research Centre in Riyadh in December 2004. The lymphoma was treated with various immunosuppressive agents including alemtuzumab. In October 2006, the patient was admitted with severe bronchopneumonia caused by  Infections are the most common cause of morbidity and mortality in patients with malignant disorders . DespiteThe spectrum of pulmonary infections depends upon the underlying immunological abnormalities . In canc9/L, Hb: 109 g/L and PLT: 315 × 109/L. Differential cell count (DCC) showed neutrophils of 1.1 and lymphocytes of 0.3. Renal and hepatic profiles were all within normal limits. Blood, urine, stool cultures and CMV antigen test were all negative. Chest radiography showed bronchiectatic changes involving both lower lobes and bilateral nodular infiltration consistent with severe pneumonia. The patient was commenced on IV tazobactam-piperacillin: 4.5 grams thrice daily and gentamicin 2 mg/kg IV twice daily in addition to oxygen via mask at 2–4 Liter/minute and IV fluids at rate of 50–80 cc/hour. Initially the patient had partial response then he started to have higher pyrexia and respiratory distress. On 6/11/2006, the patient was experiencing high grade fever and rigors and his BP dropped to 85/45 mmHg. Septic screens were repeated and the IV antibiotics were replaced by IV meropenem 1 gram thrice daily and IV vancomycin 1 gram twice daily. Later on, BP improved and fever started to subside. A repeat CT scan of the chest showed: bronchiectatic cavities involving the lower lobes of both lungs, bilateral nodular infiltration, areas of segmental consolidation and bilateral pleural thickening (Nocardia asteroides (N. asteroides). Special stains for fungi were positive and the fungus was identified as Aspergillus niger (A. niger). BAL fluid was negative for acid fast bacilli, pneumocystis carinii, viral cytopathy and malignant cells. Meanwhile, the previously taken sputum cultures grew branching gram positive rods identified as N. asteroides. However, sputum cultures were negative for acid fast bacilli, candida and aspergillus. Aspergillus galactomannan test was positive. Consequently, the following management modifications were made: vancomycin was stopped and meropenem was continued, IV liposomal amphotericin-B (amBisome) 5 mg/kg/day was commenced in addition to oral trimethoprim-sulphamethoxazole (TMP/SMZ) 960 mg thrice daily as well as IV amikacin 15 mg/kg/day. Later on, the patient started to improve clinically and radiologically and oxygen requirements decreased gradually. One week later, the patient sustained his hemodynamic stability and his clinical improvement so IV meropenem was discontinued. On 20/11/2006, IV amikacin was stopped and IV amBisome was replaced by oral voriconazole. Two days later; the patient was totally asymptomatic and his physical examination showed few basal crackles with good air entry bilaterally. FBC showed WBC: 2.25 × 109/L with neutrophils of 1.4, Hb: 94 g/L and PLT: 147 × 109/L. Renal and hepatic profiles were normal. He was discharged on: voriconazole 200 mg orally twice daily and TMP/SMZ 960 mg orally thrice daily for 6 weeks in addition to warfarin, omeprazole, prophylactic valganciclovir as well as ventolin and atrovent inhalers. Thereafter, the patient had regular follow up at the hematology outpatient clinic and he remained clinically stable.A 67 years old male, a non-smoker, with history of ischaemic heart disease, status post—coronary artery bypass graft, and bronchiectasis was diagnosed to have angioimmunoblastic T-cell lymphoma (AIBTCL), stage III B at King Faisal Specialist Hospital and Research Centre (KFSH&RC) in Riyadh in late December 2004. He presented with: fever, skin rash, generalized external lymphadenopathy and splenomegaly. The blood counts, blood film and bone marrow biopsy were all normal. The renal and hepatic profiles as well as the immunoglobulin levels were within normal limits. Serological tests for CMV, Epstein-Barr virus (EBV), hepatitis C virus and HIV were negative. Computed tomography (CT) scans of chest, abdomen and pelvis showed numerous small lymph nodes in mediastinal, mesenteric and retroperitoneal areas in addition to splenomegaly with focal splenic lesions. Skin biopsy showed atypical lymphohistiocytic changes. A right axillary lymph node biopsy revealed diffuse infiltration with: immunoblasts, lymphocytes, polymorphs, plasma cells, histiocytes and eosinophils with considerable proliferation of small blood vessels. The immunohistochemical stains showed positive: CD1A, CD3, CD4, CD7, CD8, CD15, CD20, CD21, CD30, CD45, CD68 and S100. The gene rearrangement studies revealed a monoclonal T-cell population. The AIBTCL was treated with: prednisone, mycophenolate mofetil and subcutaneous alemtuzumab: 30 mg/month for 4 months. Thereafter the patient developed: CMV infection treated with IV ganciclovir and pulmonary embolism treated with IV heparin then oral warfarin. On 29/10/06, the patient was readmitted with two week history of: fever, cough productive of yellowish sputum and mild dyspnea. He denied any associated chest or abdominal pain, headache or bleeding from any site. Physical examination revealed an unwell elderly male who was in mild respiratory distress. The temperature was 38.7 °C, blood pressure (BP): 112/68 mmHg, pulse rate: 92/minute, respiratory rate: 20/minute and oxygen saturation was 93% on room air. There was pallor but no cyanosis, leg oedema, jaundice or external lymphadenopathy. The inspiratory volume was decreased and coarse crackles were heard over mid and lower lung fields bilaterally. There was no abdominal distension, tenderness or palpable organomegaly. Cardiovascular and neurological examinations did not reveal any abnormality. Full blood count (FBC) showed: WBC: 1.83 × 10ickening . EchocarAIBTCL, an intermediate grade/aggressive mature peripheral T-cell lymphoma, was first described in the 1970s as a distinct clinical syndrome characterized by: pruritic skin rash, generalized lymphadenopathy, hepatosplenomegaly, hypergammaglobulinemia, anemia and constitutional B symptoms. It occurs in elderly individuals and has no known etiology, although it has associations with: various infections , antibiotics and autoimmune disorders  . Lymph nExpression of CD52 occurs in 40% of patients with peripheral T-cell lymphoma. Although other factors may play a role in the in vivo response to alemtuzumab (Campath 1H), an anti-CD52 monoclonal antibody, the estimation of CD52 expression may provide a rationale for the selection of patients with a higher probability of treatment response . The feaAll neutropenic patients with pyrexia of unknown etiology having normal chest roentgenograms should undergo high resolution CT scanning as these scans have been shown to have 87% sensitivity and 57% specificity in detecting pneumonic infiltrations in febrile neutropenic patients with unknown foci of infection . The favAspergillus species or presence of positive cultures for Aspergillus in a sample obtained from a sterile site by an invasive procedure e.g. lung biopsy (During the past several decades, there has been a steady increase in the frequency of opportunistic fungal infections in immunocompromised individuals . IPA is g biopsy . Recent g biopsy . Optimalg biopsy .For many years, treatment of severe fungal infections had been limited to amphotericin-B and flucytosine. Fortunately, the past few years have brought remarkable advancements in antifungal pharmacotherapy as several new antifungals have been introduced . With thIn case of intolerance to or failure of treatment with caspofungin or voriconazole, one of the new echinocandins e.g. micafungin or triazoles e.g. posaconazole can be used as an alternative salvage antifungal therapy .N. asteroides (Nocardia infections develop in patients with underlying lung pathology e.g. cystic fibrosis, bronchiectasis or chronic obstructive airway disease as well as in immunocompromised individuals e.g. those receiving corticosteroids and other immunosuppressive agents, organ transplant recipients and cancer patients specially those with lymphoreticular neoplasms. The most commonly involved organs are: the lungs, the central nervous system and the skin . Howeverteroides . Pulmonateroides . It commteroides . In immuteroides . The medteroides . Clinicateroides . Recurreteroides . The diateroides . Early dteroides . Susceptteroides . The isoteroides . Despiteteroides . A synerteroides . Delayedteroides .The patient presented was severely immunocompromised and had several predisposing factors for the invasive pulmonary infections encountered including: his advanced age; having multiple medical illnesses including bronchiectasis; having the T-cell lymphoma treated with various immunosuppressive drugs including corticosteroids; having a recent CMV infection and having moderately severe neutropenia prior to and during the latest hospitalization. We believe that the immunosuppressive therapy administered to this patient, particularly alemtuzumab, contributed not only to the development of both invasive lung infections, but also to the reactivation of CMV, particularly as he developed neutropenia and lymphopenia several months following this immununotherapy. He presented with severe bronchopneumonia clinically and diffuse nodular lung infiltration in addition to segmental consolidation radiologically. As he did not have appropriate antimicrobial cover initially, he developed the septic shock. However, the septic episode was successfully managed with meropenem, vancomycin in addition to the other supportive measures taken. Thereafter, the antimicrobial therapy was modified when nocardia and aspergillus were cultured from sputum and BAL fluid. Subsequently, the patient made an excellent clinical and radiological recovery although the treatment was rather prolonged.Even in septic immunocompromised hosts, slowly growing organisms and microorganisms that are difficult to culture or isolate should always be included in the differential diagnosis. Taking enough cultures and septic screens, making thorough and repeated physical assessment and requesting all the necessary investigations including high resolution CT scans are essential in the management of septic episodes and severe infections. At times, invasive diagnostic procedures such as bronchoscopy, BAL and even open lung biopsies may become essential to determine the causative microorganisms of certain pulmonary infections.


Calciodinellum, Calcigonellum, Leonella, Pernambugia, Scrippsiella, and Thoracosphaera). The shell of the coccoid cells consisted either of one  or two matrices (Scrippsiella and its relatives) of unknown element composition, whereas calcite is deposited in the only or the outer layer, respectively. We observed crystal-like particles in cytoplasmic vacuoles in cells of nine of the strains investigated and assume that they are widespread among calcareous dinophytes. However, similar structures are also found outside the Thoracosphaeraceae, and we postulate an evolutionarily old physiological pathway (possibly involved in detoxification) that later was specialized for calcification. We aim to contribute to a deeper knowledge of the biomineralization process in calcareous dinophytes.Biomineralization in calcareous dinophytes  takes place in coccoid cells and is presently poorly understood. Vacuolar crystal-like particles as well as collection sites within the prospective calcareous shell may play a crucial role during this process at the ultrastructural level. Using transmission electron microscopy, we investigated the ultrastructure of coccoid cells at an early developmental stage in fourteen calcareous dinophyte strains (corresponding to at least ten species of  Biomineralization is defined as the fundamental biological process by which living organisms produce minerals, often to harden or stiffen existing tissues or subcellular organic matrices. Mineralized structures have evolved multiple times independently and are taxonomically widely distributed over the tree of life. Subsequently, many similar cellular steps take place in distantly related lineages Ensiculifera Balech, 1967 and Pentapharsodinium Indel. & A.R.Loebl.), the T/Pf-clade (for Thoracosphaera Kamptner and Pfiesteria Steid. & J.M.Burkh.), and Scrippsiella Balech ex A.R.Loebl. sensu lato (s.l.).Many dinophytes develop at least two principally different stages during their life history: a phototrophic, motile cell  and an immotile coccoid cell (commonly termed a ‘cyst’). In the calcareous dinophytes , it is particularly the shell of the coccoid cells that is mineralized by calcitic crystals Since the early studies of dinophyte anatomy using transmission electron microscopy (TEM) Scrippsiella minima X.Gao & J.D.Dodge, in which the initial phase of mineralization takes place, are surrounded by two continuous matrices of unknown compounds that are delineated by an outer, middle, and inner unit membrane Thoracosphaera heimii (Lohmann) Kamptner, a single matrix develops, delineated by an outer and inner unit membrane Th. heimii in more detail and have discovered small and large cytoplasmic vesicles (or vacuoles) likewise containing cylindrical crystals. Such vacuoles may derive from the Golgi apparatus and actively transport seed crystals from the center towards the periphery of the cell. ‘Crystal-like bodies’ similar in appearance have been reported outside the calcareous dinophytes, including other Peridiniales Immature coccoid cells of ThoracosphaeraIn this study, we investigate the ultrastructure of several calcareous dinophytes to document the subcellular structures that may play a role in biomineralization. Elbrächter and colleagues −2 s−1, and a 12∶12 h light: dark photoperiod by using K-Medium without silicate Fourteen calcareous dinophyte strains were collected and isolated from environmental samples . They weCultivated living cells were observed using an Olympus CKX41 inverse microscope, equipped with a Kappa camera DX 20H-FW . For scanning electron microscopy (SEM) preparation, coccoid cells were desalinated in bi-distillate water and air-dried on a glass slide that was fixed on a SEM stub  each for 15 min and post-fixed with 1% osmiumtetroxide  in 0.2 M cacodylate buffer. Following the instructions of the Embedding Medi Kit (Science Services), samples were dehydrated in a graded ethanol (Roth) or acetone (Roth) series , and gradually infiltrated and embedded in Spurr's resin For TEM standard protocols, large thecate and coccoid cells (with a focus on early stages of the encystment) were fixed with 2.5% glutaraldehyde  in 0.2 M cacodylate buffer  and 0.25 M saccharose (Roth) at pH 8.0 for 1.5 h. An alternative protocol fixed the cells directly in cultivation media that were afterwards transferred to 0.2 M cacodylate buffer and 0.25 M saccharose at pH 8.0. After fixation, the cells were washed in 0.2 M CaCOThe largest thecae and cells with a roundish form even as large as coccoid stages were selected for sectioning. Ultrathin sections were prepared with an ultra microtome (Leica EM UC6 Ultramikrotom). Sections were spread with 99% chloroform (Roth) and collected on copper 200 square mesh and 200 single bar grids (Plano) covered with collodium. Grids were stained with 1% aqueous uranylacetate (Plano) for 2 min and lead citrate (Plano) for 4 min Leonella granifera (Fütterer) Janofske & Karwath and Th. heimii (both members of the T/Pf clade) produced mainly coccoid cells dividing vegetatively (thecate cells were rarely found under cultivation conditions). All investigated species of Scrippsiella s.l.  developed golden brown, photosynthetically active thecate cells, which were always abundant under cultivation conditions because of vegetative division. The shape of the thecate cells was spherical to ovoid, with a rounded to conical apex. Early coccoid cells developed after shed of the theca by ecdysis and were darker than the thecate cells from the beginning. A red accumulation body was often already visible at this stage. Coccoid cells appeared first hyaline and darkened towards the brownish-opaque color at maturity after a few minutes.Th. heimii: Calciodinellum aff. operosum Deflandre, 1947: Scrippsiella trochoidea (F.Stein) A.R.Loebl.: Scrippsiella bicarinata Zinssmeister, S.Soehner, S.Meier & Gottschling: C. operosum: Th. heimii  were surrounded by a single organic matrix. Directly under this layer, protrusions similar in appearance to those found in Scrippsiella s.l. were present  increased during encystment  and in cc matrix , 6C,D ofc matrix , 5F. By ranifera  and Th.  present . Mature ranifera . The calCalcigonellum infula Deflandre, 1949 (C. aff. operosum (S. aff. trochoidea (C. operosum (Pernambugia tuberosa (Kamptner) Janofske & Karwath (S. bicarinata (S. trochoidea (S. trochoidea var. aciculifera Montresor (Th. heimii (C. infula (C. aff. operosum (S. aff. trochoidea (In nine of fourteen strains (corresponding to at least eight species), crystal-like particles were detected in encysting cells. They were found within cytoplasmic vacuoles, which were peripherally located in the cell body. Such vacuoles were variable in size and were particularly large in re, 1949 , C. aff.operosum , and S. ochoidea . The cryoperosum  showed l Karwath , S. bicacarinata , S. trocochoidea , S. trocontresor , and Th.. heimii  had an o. infula , C. aff.operosum , and S. ochoidea , the shaP. tuberosa: . trochoidea: S. bicarinata: Th. heimii: S. aff. trochoidea , while all extant species of Scrippsiella . These two types of shell architecture may correlate with molecular phylogenies, in which Scrippsiella s.l. and the T/Pf-clade represent two distinct lineages of the Thoracosphaeraceae Pfiesteria.The number of layers constituting the shell of coccoid cells might have some phylogenetic significance S. minima, Gao and co-workers Calciodinellum and Pernambugia. They have also been documented (but neither described nor discussed) for ThoracosphaeraScrippsiella s.l. is homologous to the single layer in the T/Pf-clade. To the best of our knowledge, protrusions of mucofibrous material as prerequisite for calcification For Thoracosphaera, calcification starts with the deposition of seed crystals in the single matrix present S. minima, in which mineralization takes place In Scrippsiella sweeneyae Balech ex A.R.Loebl. Th. heimiiTyrannodinium edax (A.J.Schill.) Calado ( = Peridinium berolinense Lemmerm. The presence of vacuoles including crystal-like particles has been previously reported from S. trochoideaTh. heimiiGaleidinium Tam. & T.Horig. Peridiniopsis Lemmerm. Analytical chemistry of the crystal-like particles might likewise be indicative of their independent evolutionary origin, although the precise molecular composition based on ultimate analyses is rarely investigated. In the Suessiales, the vacuolar crystals with a characteristic rectangular shape are composed of calcium oxalate Calcicarpinum Deflandre, 1949 and Posoniella Streng, Banasová, Reháková & H.Willems. Such calcareous dinophytes are primarily found in surface sediments at coastal sites as the establishment of a dormancy seedbank Multiple functions of vacuolar crystal-like particles have been discussed. In the Suessiales, the mature elements are characteristically brick-like Compared to calcareous dinophytes, biomineralization in other unicellular organisms, such as the foraminifers and coccolithophores, has been more thoroughly investigated Scrippsiella and its relatives. More research on the life history, ultrastructure, and physiology is necessary, and high-spatial resolution analyses such as NanoSIMS, Raman spectroscopy, soft X-ray microscopy, and atomic force microscopy may be promising approaches in developing a comprehensive scenario for biomineralization in calcareous dinophytes.The assumption that calcareous dinophytes have a similar calcification mechanism as coccolithophores has been postulated by Tangen and colleagues


Mycobacterium tuberculosis (M. tuberculosis). At present, its dual activity as hemolysin and RNA methyltransferase in M. tuberculosis has been indirectly proposed based on in vitro results. There is no evidence however for TlyA relevance in the survival of tubercle bacilli inside host cells or whether both activities are functionally linked. A thorough analysis of structure prediction for this mycobacterial protein in this study shows the need for reevaluating TlyA's function in virulence.The TlyA protein has a controversial function as a virulence factor in tlyA gene among Mycobacterium species. Additionally, the nucleotide substitution rates suggested purifying selection during tlyA gene evolution and the absence of a common ancestor between TlyA proteins and bacterial pore-forming proteins.Bioinformatics analysis of TlyA identified a ribosomal protein binding domain (S4 domain), located between residues 5 and 68 as well as an FtsJ-like methyltranferase domain encompassing residues 62 and 247, all of which have been previously described in translation machinery-associated proteins. Subcellular localization prediction showed that TlyA lacks a signal peptide and its hydrophobicity profile showed no evidence of transmembrane helices. These findings suggested that it may not be attached to the membrane, which is consistent with a cytoplasmic localization. Three-dimensional modeling of TlyA showed a consensus structure, having a common core formed by a six-stranded β-sheet between two α-helix layers, which is consistent with an RNA methyltransferase structure. Phylogenetic analyses showed high conservation of the in silico curation suggested that TlyA is involved in ribosomal biogenesis and that there is a functional annotation error regarding this protein family in several microbial and plant genomes, including the M. tuberculosis genome.Altogether, our manual  Mycobacterium tuberculosis strains (XDR) . Protein and gene sequence similarity was analyzed by BLAST tools [http://www.ncbi.nlm.nih.gov/] and a homology comparison was made using BLASTp for the PDB database. The TlyA-like sequences so obtained were aligned using the CLUSTALW Multiple Sequence Alignment program [http://align.genome.jp/] [http://www.clcbio.com]   and manuome.jp/] . The alibio.com]  to obtaihttp://smart.embl-heidelberg.de] [http://www.sanger.ac.uk/Software/Pfam/] [This protein's domain composition was analyzed using the SMART-Simple Modular Architecture Research Tool [berg.de]  in combie/Pfam/] .M. tuberculosis TlyA (Rv1694) secondary structure consensus was built based on the predictions obtained with SAM [http://www.sbg.bio.ic.ac.uk/phyre/] prediction servers [An asy.org/] and Prot servers ,41; the  program , and theprofunc/],32,43 toprofunc/] which prM. tuberculosis TlyA amino acid sequence. TlyA's subcellular localization was predicted using the TBpred server  using a 2.965 threshold value  by BLAST search  [DB:1EJ0]  showing Click here for fileFigure S2. Ramachandran plot of TlyA three-dimensional modeled structure. Dispersion zones are shown in blue and orange representing proline and glycine favored and allowed regions .Click here for file


AGO gene family has been analyzed in various plant species; however, there is no report about AGOs in the well-known Traditional Chinese Medicine (TCM) plant, Salvia miltiorrhiza.Argonaute (AGO) is the core component of RNA-induced silencing complex. The SmAGO genes in S. miltiorrhiza. Full-length cDNAs of all SmAGOs were subsequently cloned and sequenced. These SmAGOs were characterized using a comprehensive approach. Sequence features, gene structures and conserved domains were analyzed by the comparison of SmAGOs and AtAGOs. Phylogenetic relationships among AGO proteins from S. miltiorrhiza, Arabidopsis and rice were revealed. The expression levels of SmAGO genes in various tissues of S. miltiorrhiza were investigated. The results implied that some SmAGOs, such as SmAGO1, SmAGO2, SmAGO3, SmAGO7 and SmAGO10, probably played similar roles as their counterparts in Arabidopsis; whereas the others could be more species-specialized. It suggests the conservation and diversity of AGOs in plants. Additionally, we identified a total of 24 hairpin structures, representing six miRNA gene families, to be miRNA precursors. Using the modified 5′-RACE method, we confirmed that SmAGO1 and SmAGO2 were targeted by S. miltiorrhiza miR168a/b and miR403, respectively. It suggests the conservation of AGO1-miR168 and AGO2-miR403 regulatory modules in S. miltiorrhiza and Arabidopsis.Through a genome-wide analysis, we identified ten SmAGOs and miRNAs in S. miltiorrhiza. The results provide useful information for further elucidation of gene silencing pathways in S. miltiorrhiza.This is the first attempt to explore  DCL) family and the RNA dependent RNA polymerase (RDR) family, and each pathway appears to be taken part in by different member of a gene family [Small RNAs (sRNAs) involved in various gene silencing pathways play important and diverse roles in the development and differentiation of organisms through regulating gene expression at the transcriptional and post-transcriptional levels , affectie family . To regue family . RISCs re family ,7.AGO genes differs in different organisms [AGO gene, whereas insects, mammals and worms have five, eight, and twenty six AGO genes, respectively. Annotation of the Arabidopsis and rice genomes revealed ten and eighteen AGO genes, respectively [AGO genes have been identified [AGO genes have been found in various plants, the majority were predicted by computational approaches based on sequence similarity. Only a small proportion of known AGOs were identified or confirmed by full-length cDNA cloning. Among them, Arabidopsis AGO1 is the most well-studied plant AGO gene. It encodes the core component of RISCs associated with the action of miRNAs, trans-acting siRNAs (ta-siRNAs) and transgene-derived siRNAs [Arabidopsis plants [Arabidopsis AGO4, AGO6 and AGO9 genes recruit endogenous 24nt sRNAs for DNA methylation, which causes target gene silencing at the transcriptional level [AGO7 is involved in the generation of ta-siRNAs from TAS3 by collaborating with miR390 in Arabidopsis[Arabidopsis AGO10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 [AtAGO1 is regulated by miR168 through direct cleavage of AtAGO1 transcripts [AtAGO2 is regulated by miR403 [AGO genes from plant species other than Arabidopsis is largely unknown.AGOs are ribonucleases with two conserved domains, including PAZ and PIWI . The PAZrganisms . Fissionectively . Additioentified ,13. Althd siRNAs . AGO2 prs plants . Arabidoal level ,17. AGO7abidopsis. ArabidoR165/166 ,20. In anscripts . Similary miR403 . The funS. miltiorrhiza, which produces two major groups of bioactive compounds, lipophilic diterpenoid tanshinones and hydrophilic phenolic acids, is a well-known traditional Chinese medicine (TCM) widely used for treating various human diseases, such as dysmenorrhoea, amenorrhoea and cardiovascular disease, for thousands of years [S. miltiorrhiza genome has been preliminarily decoded and a working draft of the genome is currently available . The interest of gene silencing pathways in S. miltiorrhiza is increasing. With the aim to elucidate the core components of gene silencing pathways, we performed a genome-wide prediction of the S. miltiorrhiza AGO gene family. Molecular cloning of AGO genes was carried out for validation and error correction of computational prediction. The characteristics of S. miltiorrhiza AGOs were revealed by a comprehensive analysis, including comparison with AGOs from other plant species, gene expression profiling, and analysis of posttranscriptional regulation. The results provide useful information for further elucidation of gene silencing pathways in S. miltiorrhiza.of years ,24. It iof years ,26. The S. miltiorrhiza AGO genes at the genome level, we downloaded all of 10 Arabidopsis and 19 rice AGO amino acid sequences from GenBank (http://www.ncbi.nlm.nih.gov/protein). BLAST analysis of Arabidopsis and rice AGOs against the current assembly of the S. miltiorrhiza genome  was then performed using the tBLASTn algorithm [-10 was applied to the homologue recognition. As a result, a total of 10 genomic loci of SmAGO genes were identified. The genomic DNA sequence was retrieved and gene models of 10 SmAGO genes were predicted using Genscan (http://genes.mit.edu/GENSCAN.html) [AGO genes identified from other plant species using the BLASTx algorithm (http://www.ncbi.nlm.nih.gov/BLAST) [AGO genes are named SmAGO1 to SmAGO10, respectively, based on high sequence similarity with corresponding Arabidopsis AGOs.To predict lgorithm . An e-vaAN.html) . The modv/BLAST) . All of S. miltiorrhiza. In order to confirm the results from prediction and correct errors of computation, molecular cloning of full-length SmAGO cDNA was carried out. As a result, all of ten predicted SmAGO genes were experimentally validated. Analysis of sequence features showed that the length of open reading frames (ORFs) of SmAGOs varied from 2,472 (SmAGO5) to 3,195 bp (SmAGO1)  revealed that all SmAGOs contained DUF1785, PAZ, and PIWI domains . Plant miRNA sequences belonging to the 31 families were aligned with the current assembly of the S. miltiorrhiza genome using SOAP2 with two mismatches allowed [SmAGOs and the identified S. miltiorrhiza miRNAs/miRNAs* and calculation of penalty scores as previously described [S. miltiorrhiza miR168:SmAGO1, miR403:SmAGO2 and miR530:SmAGO1 were 2, 0, and 3.5, respectively. However, the scores for miR167*:SmAGO8, miR169*:SmAGO5, miR396*:SmAGO3 and were at least 5.5, 9.5, and 5.0, respectively. High penalty scores for S. miltiorrhiza miRNA*:SmAGOs were due to low conservation among plant miRNAs*. To verify whether these miRNAs can mediate the cleavage of SmAGO transcripts, we isolated RNAs from roots and pooled samples containing flowers, leaves, stem and roots of S. miltiorrhiza and performed the modified 5′-rapid amplification of cDNA ends (RACE) for SmAGOs. The 5′-RACE products revealed that SmAGO1 and SmAGO2 are indeed the targets of S. miltiorrhiza miR168 and miR403, respectively . No 5′-RACE products were obtained for SmAGO8, SmAGO3 and SmAGO1 that were predicted to be targeted by miR167, miR396, and miR530, respectively. In addition, miRNA-specific qRT-PCR [S. miltiorrhiza miR168 and miR403 experimentally validated to target SmAGO1 and SmAGO2 for cleavage. The results showed that miR168 and miR403 were expressed in all tissues analyzed. The levels of miR168 in roots and flowers were about three times the levels in leaves and stems. The level of miR403 in flowers was more than twice the level in other tissues analyzed . Among nineteen rice OsAGO genes, only six, including OsAGO1a, OsAGO1b, OsAGO1c, OsAGO1d, OsAGO7 and OsPNH1, have been cloned (http://www.ricedata.cn/gene). Except for SlAGO1-1, SlAGO1-2 and SlAGO7, twelve of fifteen tomato SlAGO genes have not been experimentally confirmed [SmAGOs using computational approaches, and then cloned the full-length cDNAs of all predicted SmAGOs. The number of identified S. miltiorrhiza AGO genes is comparable with that in Arabidopsis, although it is significantly less than the number in rice and maize, which are nineteen and eighteen, respectively [AGO genes in S. miltiorrhiza and Arabidopsis as compared with rice and maize AGOs, most of which are evolved by duplication events [AGO functions in S. miltiorrhiza and gene model prediction of AGOs in other plant species.Although Argonautes play very important roles in small RNA-mediated gene silencing and a large number of onfirmed ,42. In tectively ,12. It in events ,12. The SmAGOs revealed similar patterns with their Arabidopsis counterparts  transcribed from miRNA loci . Pri-miRectively . It suggS. miltiorrhiza miRNAs and their targets showed that no simple linear correlations existed between miR168 and SmAGO1 and between miR403 and SmAGO2  with whole genome sequences available was grown in a field nursery. Mature flower buds blooming soon, mature and healthy leaves, young stems and roots in about 0.5 cm diameter were collected from 2-year-old plants on Aug. 15, 2012. Samples from three plants were pooled and stored in liquid nitrogen until use.Arabidopsis and rice AGO protein sequences were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/protein) and used to search for homologues against the current assembly of the S. miltiorrhiza genome  using the tBLASTn algorithm [-10 was applied to the homologue recognition. All retrieved sequences were used for gene prediction on the Genscan web server (http://genes.mit.edu/GENSCAN.html) [AGO genes identified from other plant species using the BLASTx algorithm (http://www.ncbi.nlm.nih.gov/BLAST) [lgorithm . An e-vaAN.html) . The prev/BLAST) .S. miltiorrhiza using Trizol reagent (invitrogen). mRNA was purified using the oligotex mRNA mini kit (invitrogen). RNA ligase-mediated rapid amplification of 5′ cDNA ends (5′-RACE) and 3′ cDNA ends (3′-RACE) was performed on mRNA using the GeneRacer kit (Invitrogen). PCR amplifications were performed using the GeneRacer primer and the nesting gene-specific primers  were predicted using the Compute pI/MW tool on the ExPASy server (http://web.expasy.org/compute_pi/). Intron/exon structures were analyzed manually based on genomic DNA sequences and the cloned cDNA sequences. Conserved domains were analyzed by search the deduced amino acid sequence of SmAGOs against the NCBI conserved domain (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The conserved residues were analyzed by alignment of amino acid sequences using DNAMAN and then checked manually. For phylogenetic tree construction, amino acid sequences of AGOs from S. miltiorrhiza, Arabidopsis and rice were first aligned using ClustalW version 1.83 and then constructed using MEGA version 4.0 by the neighbor-joining method with bootstrap to be 1000 replicates [The molecular weight (MW) and theoretical isoelectric point  and then treated with RNase-free DNase (Promega) to remove genomic DNA contamination. RNA integrity was analyzed on a 1% agarose gel. RNA quantity was determined using a NanoDrop 2000C Spectrophotometer (Thermo Scientific). Reverse transcription was performed on 1 μg total RNA by 200 U Superscript III reverse transcriptase (Invitrogen) in a 20 μl volume. The resulting cDNA was diluted to 200 μl with sterile water. qPCR was carried out in triplicates using the BIO-RAD CFX system (Bio-Rad). Gene-specific primers were listed in Additional file escribed . PCR wasescribed . Cq reprS. miltiorrhiza 5.8S rRNA was used as an endogenous reference. Primers used for miRNA quantification were listed in Additional file The levels of miR168a/b and miR403 were analyzed using the miRNA-specific poly(T) adaptor RT-PCR method . S. milthttp://www.mirbase.org/) [SmAGO cDNA sequences using psRNATarget [S. miltiorrhiza genome  using the SOAP2 program with no more than 2 mismatches allowed [S. miltiorrhiza miRNAs.Known plant miRNA sequences were downloaded from miRBase  . Target NATarget . The max allowed . Hairpin allowed . Criteri allowed  were appSmAGO cleavage sites were mapped using the modified RNA ligase-mediated rapid amplification of 5′ cDNAs method as described [S. miltiorrhiza roots and pooled samples containing flowers, leaves, stem and roots using the GeneRacer 5′ primer and the nesting gene-specific primers (see Additional file escribed . PCRs weAGO: Argonaute; DCL: Dicer-like; dsRNA: double-stranded RNA; miRNA: microRNA; Mw: Molecular weight; ORF: Open reading frame; pI: Isoelectric point; pri-miRNA: Primary miRNA; qRT-PCR: quantitative real-time reverse transcription-PCR; RACE: RNA ligase-mediated rapid amplification of cDNA ends; RDR: RNA dependent RNA polymerase; RISC: RNA-induced silencing complex; siRNA: small interfering RNA; sRNA: small RNA; ssRMA: single-stranded RNA; ta-siRNA: trans-acting siRNA; TCM: Traditional Chinese medicine; UTR: Untranslated region.The authors declare that they have no competing interests.FS analyzed the data, performed qRT-PCR and RACE, and participated in writing the manuscript. SL designed the experiment and wrote the manuscript. Both authors have read and approved the version of manuscript.Conserved domains in SmAGO proteins. DUF1785, PAZ and PIWI domains are shown.Click here for fileAlignment of the deduced SmAGO amino acid sequences. DUF1785, PAZ and PIWI domains are indicated by dotted, solid and broken lines. The conserved DDH/H798 or DDD/H798 residues are indicated by arrows.Click here for filePrimers used for 5′-RACE of SmAGOs. Complete set of primers used for 5′-RACE of SmAGOs.Click here for filePrimers used for 3′-RACE of SmAGOs. Complete set of primers used for 3′-RACE of SmAGOs.Click here for filePrimers used for amplification of full-length SmAGOs. Complete set of primers used for amplification of full-length SmAGOs.Click here for filePrimers used for qRT-PCR. Complete set of primers used for qRT-PCR.Click here for filePrimers used for miRNA quantification. Complete set of primers used for miRNA quantification.Click here for filePrimers used for mapping of SmAGO cleavage sites. Complete set of primers used for mapping of SmAGO cleavage sites.Click here for file