Ndatenda nekushanyira Nature.com.Uri kushandisa shanduro yebrowser ine tsigiro shoma yeCSS.Kuti uwane ruzivo rwakanyanya, tinokurudzira kuti ushandise browser yakagadziridzwa (kana kudzima Compatibility Mode muInternet Explorer).Pamusoro pezvo, kuti tive nechokwadi chekutsigira kunoenderera, tinoratidza saiti isina masitaera uye JavaScript.
Inoratidza carousel yemasiraidhi matatu panguva imwe chete.Shandisa mabhatani ePashure neAnotevera kufamba nemasiraidhi matatu panguva, kana kushandisa mabhatani anotsveyamisa ari kumagumo kufamba nemasiraidhi matatu panguva.
Iyo yeropa-brain chipingamupinyi uye yeropa-brain chipingamupinyi chinodzivirira biotherapeutic agents kubva pakusvika pazvinangwa zvavo mukati mepakati tsinga system, nekudaro zvichitadzisa kurapa kunoshanda kwezvirwere zvetsinga.Kuti tiwane vatakuri vehuropi muvivo, takaunza raibhurari yeT7 phage peptide uye takaunganidza ropa uye cerebrospinal fluid (CSF) tichishandisa cannulated conscious pool hombe modhi yemakonzo.Yakananga phage clones yakafumiswa zvakanyanya muCSF mushure memakwikwi mana ekusarudzwa.Kuedzwa kwepeptides yemunhu mumwe nemumwe kwakaratidza kuwedzera kwe1000-fold hupfumi muCSF.Iyo bioactivity yepeptide-mediated kuendeswa kuuropi yakasimbiswa ne 40% kuderedzwa kwehuwandu hweamyloid-β mu cerebrospinal fluid uchishandisa BACE1 peptide inhibitor yakabatanidzwa kune yakazivikanwa novel transit peptide.Mhedzisiro iyi inoratidza kuti mapeptides anoonekwa nein vivo phage nzira dzekusarudza anogona kunge ari mota dzinobatsira dzesystemic kuendesa macromolecules kuhuropi zvine kurapa.
Central nerve system (CNS) yakanangana nekurapa tsvakiridzo yakanangana nekuona optimized zvinodhaka uye maajenti anoratidza CNS-yakananga zvivakwa, nekushomeka kwekuedza kuwana nzira dzinofambisa inoshanda zvinodhaka kuendeswa kuuropi.Izvi zvave kutanga kushanduka iko zvino sekuendeswa kwezvinodhaka, kunyanya mamorekuru makuru, chikamu chakakosha chekuvandudzwa kwemishonga yemazuva ano neuroscience.Nzvimbo yepakati tsinga inodzivirirwa zvakanaka necerebrovascular barriers system, inosanganisira yeropa-brain barriers (BBB) ​​uye yeropa-brain barriers (BCBB)1, zvichiita kuti zviome kuendesa mishonga kuuropi1,2.Zvinofungidzirwa kuti anenge ese makuru mamorekuru madhiragi uye anopfuura 98% ediki mamorekuru madhiragi anobviswa kubva muuropi3.Ndicho chikonzero nei zvakakosha kuziva hutsva hutsva hwekutakura huropi hunopa hunobudirira uye hwakajeka kuendesa mishonga yekurapa kuCNS 4,5.Nekudaro, iyo BBB neBCSFB inopawo mukana wakanakisa wekuendesa zvinodhaka sezvavanopinda nekupinda zvese zvimiro zvehuropi kuburikidza neyakakura masculature.Nokudaro, kuedza kwemazuva ano kwekushandisa nzira dzisingasviki dzekuendesa kuuropi dzinonyanya kuenderana nemaitiro ekugamuchira-mediated transport (PMT) achishandisa endogenous BBB6 receptor.Pasinei nekufambira mberi kwakakosha kwekushandisa iyo transferrin receptor pathway7,8, kuenderera mberi kwekusimudzira masisitimu matsva ekutumira ane zvivakwa zvakagadziridzwa zvinodiwa.Kuti izvi zviitike, chinangwa chedu chaive chekuona mapeptides anokwanisa kuyananisira kutakura kweCSF, sezvo aikwanisa kushandiswa kuendesa macromolecules kuCNS kana kuvhura nzira nyowani dzekugamuchira.Kunyanya, mareceptor chaiwo uye vatakuri vecerebrovascular system (BBB neBSCFB) vanogona kushanda sezvingatarisirwa zvekushanda uye kwakananga kuendeswa kwemishonga yebiotherapeutic.Cerebrospinal fluid (CSF) chinhu chakavanzika chechoroid plexus (CS) uye inosangana zvakananga ne interstitial fluid yeuropi kuburikidza ne subbarachnoid space uye ventricular space4.Munguva pfupi yapfuura zvakaratidzwa kuti subarachnoid cerebrospinal fluid inopararira zvakanyanya mu interstitium yeuropi9.Isu tinotarisira kuwana iyo parenchymal nzvimbo tichishandisa iyi subbarachnoid inflow turakiti kana zvakananga kuburikidza neBBB.Kuti tiite izvi, takaita yakasimba mu vivo phage sarudzo yekusarudza iyo inonyatso ratidza peptides inotakurwa neimwe yeidzi nzira mbiri dzakasiyana.
Isu ikozvino tinotsanangura inoteedzana mu vivo phage inoratidzira yekuongorora nzira neCSF sampling yakasanganiswa neyepamusoro throughput sequencing (HTS) yekutarisa yekutanga kusarudzwa kutenderera nemhando yepamusoro yeraibhurari.Kuongororwa kwakaitwa pamakonzo anoziva ane cisterna hombe akaisirwa zvachose (CM) cannula kudzivirira kusvibiswa kweropa.Zvakakosha, iyi nzira inosarudza zvese zvinonangana nehuropi uye peptides nebasa rekufambisa mhiri kwecerebrovascular barrier.Takashandisa T7 phages nekuda kwehukuru hwavo hudiki (~ 60 nm) 10 uye vakaratidza kuti vakakodzera kutakurwa kwemavhesi anobvumira transcellular kuyambuka endothelial uye / kana epithelial-medulla barrier.Mushure mekutenderera mana ekutenderera, phage vanhu vakaparadzaniswa vachiratidza kusimba mu vivo CSF ​​hupfumi uye cerebral microvessel association.Zvakakosha, isu takakwanisa kusimbisa zvatakawana nekuratidza kuti iyo yakasarudzika uye yakagadzirwa nekemikari yepamusoro peptides inokwanisa kutakura maprotein cargo kupinda mucerebrospinal fluid.Kutanga, iyo pharmacodynamic mhedzisiro yeCNS yakavambwa nekubatanidza inotungamira yekufambisa peptide ine inhibitor yeBACE1 peptide.Pamusoro pekuratidza kuti mu vivo inoshanda yekuongorora nzira dzinogona kuona mapeptide matsva ekutakura uropi seanotakura mapuroteni anotakura zvinhu, isu tinotarisira maitiro akafanana ekusarudza maitiro kuti avewo akakosha mukuona nzira itsva dzekufambisa dzehuropi.
Kubva pane plaque-forming units (PFU), mushure meiyo phage yekurongedza nhanho, raibhurari yezvisina kurongeka 12-mer linear T7 phage peptides ine akasiyana-siyana angangoita 109 akagadzirwa uye akagadzirwa (ona Zvishandiso uye Nzira).Izvo zvakakosha kuti ticherechedze kuti isu takanyatso ongorora raibhurari iyi pamberi mu vivo panning.PCR kukwidziridzwa kwemaibhurari ephage samples uchishandisa ma modified primers akagadzira amplicon ainyatso kushanda kuHTS (Supplementary Fig. 1a).Nekuda kwe) HTS11 zvikanganiso zvekutevedzana, b) kukanganisa kwemhando yeprimers (NNK) 1-12, uye c) kuvapo kwemhando yesango (wt) phage (skeleton inserts) muraibhurari yekumira, nzira yekusefa yakatevedzana yakaitwa kuti ibudise ruzivo rwekutevedzana kwakasimbiswa chete (Supplementary Fig. 1b).Aya matanho ekusefa anoshanda kune ese maHTS anoteedzera maraibhurari.Kune raibhurari yakajairika, huwandu hwe233,868 hwakaverengwa hwakawanikwa, iyo makumi matatu nepfumbamwe muzana yakapfuura nzira yekusefa uye yakashandiswa pakuongorora raibhurari uye kusarudzwa kune aitevera kutenderera (Supplementary Figure 1c-e).Kuverenga kwacho kwainyanya kuwanda kwe3 base pairs pakureba nepamusoro pe36 nucleotides (Supplementary Fig. 1c), kusimbisa raibhurari dhizaini (NNK) 1-12.Zvikuru, ingangoita gumi nerimwe% yenhengo dzeraibhurari yaive negumi nemaviri-dimensional wild-type (wt) musana PAGISRELVDKL kuisa, uye ingangoita hafu yekutevedzana (49%) yaive nekuisa kana kudzima.HTS yeraibhurari yakasimbisa kusiyana kukuru kwepeptide muraibhurari: kupfuura 81% yepeptide sequences yakawanikwa kamwe chete uye chete 1.5% yakaitika mu ≥4 makopi (Supplementary Fig. 2a).Mafambiro eamino acids (aa) panzvimbo dzose dze12 mune repertoire yakabatana zvakanaka nemafambisirwo anotarisirwa kwenhamba yekodons inogadzirwa nekuderera kweNKK repertoire (Supplementary Fig. 2b).Iyo yakacherechedzwa kazhinji yeaa masara akaiswa encoded neaya anoisa anoenderana zvakanaka neakaverengerwa frequency (r = 0.893) (Supplementary Fig. 2c).Kugadzirira kwemaraibhurari ephage yejekiseni kunosanganisira matanho ekusimudzira uye kubviswa kwe endotoxin.Izvi zvakamboratidzwa kuti zvinogona kuderedza kusiyanisa kwephage library12,13.Naizvozvo, isu takateedzera ndiro-amplified phage raibhurari iyo yakanga yabviswa endotoxin uye tikaienzanisa neraibhurari yepakutanga kufungidzira kuwanda kweAA.Kubatana kwakasimba (r = 0.995) kwakaonekwa pakati pechidziva chepakutanga uye dziva rakawedzerwa uye rakacheneswa (Supplementary Fig. 2d), zvichiratidza kuti kukwikwidzana pakati pemakoni akawedzerwa pamahwendefa anoshandisa T7 phage haana kukonzera kukanganisa kukuru.Kuenzanisa uku kunobva pane kuwanda kwe tripeptide motifs muraibhurari yega yega, sezvo kusiyana kwemaraibhurari (~ 109) hakugone kutorwa zvizere kunyangwe neHTS.Frequency kuongororwa kwea pane imwe neimwe nzvimbo yakaratidza diki-inotsamira yakarerekera munzvimbo nhatu dzekupedzisira dzeiyo yakapinda repertoire (Supplementary Fig. 2e).Mukupedzisa, takagumisa kuti kunaka uye kusiyana-siyana kweraibhurari kwakagamuchirwa uye kuchinja kuduku chete mukusiyana kwakaonekwa nekuda kwekuwedzera uye kugadzirira kwemaraibhurari ephage pakati pezvikamu zvakasiyana-siyana zvekusarudzwa.
Serial cerebrospinal fluid sampling inogona kuitwa nekuvhiya kuisa cannula muCM yemakonzo anoziva kuti abatsire kuzivikanwa kweT7 phage jekiseni intravenously (iv) kuburikidza neBBB uye / kana BCSFB (Fig. 1a-b).Takashandisa maoko maviri akazvimiririra ekusarudza (maoko A uye B) mumatanho matatu ekutanga emu vivo kusarudzwa (Fig. 1c).Isu takawedzera zvishoma nezvishoma kusimba kwesarudzo nekudzikisa huwandu hwese hwepage hwakaunzwa mumatatu ekutanga esarudzo.Pachikamu chechina chekugadzira, takabatanidza masampuli kubva kumapazi A neB uye tikaita mamwe matatu akazvimirira sarudzo.Kuti udzidze in vivo zvimiro zveT7 phage particles mune iyi modhi, wild-type phage (PAGISRELVDKL master insert) yakaiswa jekiseni mumakonzo kuburikidza nemuswe tsinga.Kudzorerwa kwepageji kubva ku-cerebrospinal fluid uye ropa panguva dzakasiyana-siyana dzakaratidza kuti zvishoma zvishoma T7 icosahedral phages yakanga ine kukurumidza kwekutanga kusunungurwa kubva muchikamu cheropa (Supplementary Fig. 3).Zvichienderana nemazita anopihwa uye huwandu hweropa remakonzo, isu takaverenga kuti ingangoita 1% wt.phage kubva pamushonga wakapihwa yakaonekwa muropa maminitsi gumi mushure mejekiseni remutsinga.Mushure meiyi yekutanga kuderera nekukurumidza, kunonoka kwekutanga kubvumidzwa kwakayerwa nehafu yehupenyu hwe27.7 maminetsi.Zvinonyanya kukosha, zvikamu zvishomanana chete zvakatorwa kubva kuCSF compartment, zvichiratidza mamiriro akaderera emusango-mhando yekufambisa phage muchikamu cheCSF (Supplementary Fig. 3).Paavhareji, chete nezve 1 x 10-3% titers yeT7 phage muropa uye 4 x 10-8% yepakutanga yakaputirwa phages yakaonekwa mu cerebrospinal fluid pamusoro penguva yose yekuenzanisira (0-250 min).Zvinonyanya kukosha, iyo hafu yehupenyu (25.7 min) yemusango-mhando phage mu cerebrospinal fluid yakanga yakafanana neyakaonekwa muropa.Iyi data inoratidza kuti chipingamupinyi chinoparadzanisa chikamu cheCSF kubva muropa chakasimba muCM-cannulated rats, zvichibvumira mu vivo kusarudzwa kwemaraibhurari ephage kuona ma clones anotakurwa nyore nyore kubva muropa achienda mukamuri yeCSF.
(a) Kugadzira nzira yekuongororazve cerebrospinal fluid (CSF) kubva padziva guru.(b) Dhiagiramu inoratidza nharembozha yepakati tsinga sisitimu (CNS) barriers uye sarudzo yekusarudza inoshandiswa kuona peptides inoyambuka ropa-uropi barriers (BBB) ​​uye yeropa-brain barriers.(c) In vivo phage ratidza yekutarisa kuyerera.Muchikamu chega chega chekusarudza, phage (zviziviso zvemhuka mukati memiseve) zvakaiswa jekiseni.Mapazi maviri akazvimirira ega (A, B) anochengetwa akaparadzana kusvika pachikamu chechina chesarudzo.Pakusarudzwa kutenderera 3 uye 4, imwe neimwe phage clone yakatorwa kubva kuCSF yakatevedzana nemaoko.(d) Kinetics ye phage yakaparadzaniswa neropa (tsvuku denderedzwa) uye cerebrospinal fluid (green triangles) panguva yekutanga kusarudzwa mumakonzo maviri akavharidzirwa mushure mejojo ​​ye intravenous yeT7 peptide library (2 x 1012 phages / mhuka).Mavara ebhuruu anoratidza avhareji yekutanga yekusangana kwephage muropa, inoverengerwa kubva kuhuwandu hweiyo jekiseni phage, uchifunga nezve huwandu hweropa.Mavara matema anoratidza poindi yemharadzano yemutsara y wakawedzerwa kubva muhuwandu hweropa phage.(e,f) Ratidza huwandu hwehukama uye kugovera kwese kunogona kupindirana tripeptide motifs inowanikwa mupeptide.Nhamba yemamotifs inowanikwa mu1000 kuverenga inoratidzwa.Zvinokosha (p <0.001) akafumiswa motifs akaiswa madotsi matsvuku.(e) Correlation scatterplot inofananidzira kuwanda kweiyo tripeptide motif yeraibhurari yakabaiwa neropa-rinobva kumhuka kubva kumhuka #1.1 uye #1.2.(f) Correlation scatterplot kuenzanisa mafrequencies emhuka phage tripeptide motifs #1.1 uye #1.2 ari oga muropa uye cerebrospinal fluid.(g, h) Sequence ID inomiririra yephage yakafumiswa muropa (g) maringe nemaraibhurari akaiswa jekiseni uye phage yakafumiswa muCSF (h) maringe neropa mushure mekutenderera kwemu vivo kusarudzwa mumhuka mbiri.Saizi yekodhi yetsamba imwe inoratidza kuti amino acid inoitika kakawanda sei panzvimbo iyoyo.Green = polar, yepepuru = kwazvakarerekera, bhuruu = basic, red = acidic uye dema = hydrophobic amino acids.Mufananidzo 1a, b wakagadzirwa uye wakagadzirwa naEduard Urich.
Isu takabaya fage peptide raibhurari mumakonzo maviri eCM chiridzwa (clades A uye B) uye yakasarudzika phage kubva kucerebrospinal fluid neropa (Mufananidzo 1d).Kusununguka kwekutanga kweraibhurari kwakange kusinganyanyi kutaurwa kana ichienzaniswa nerudzi rwemusango.Iyo inoreva hafu yehupenyu hweraibhurari yakajairwa mumhuka dzese dziri mbiri yaive 24.8 maminetsi muropa, yakafanana nesango-rudzi phage, uye 38.5 maminetsi muCSF.Ropa uye cerebrospinal fluid phage samples kubva kumhuka yega yega yakaiswa pasi peHTS uye ese akaonekwa peptides akaongororwa kuvepo kwepfupi tripeptide motif.Tripeptide motifs yakasarudzwa nekuti inopa hwaro hushoma hwekuumbwa kwechimiro uye peptide-protein kudyidzana14,15.Takawana kuwirirana kwakanaka mukugoverwa kwezvinyorwa pakati peraibhurari yephage injected uye clones yakatorwa muropa remhuka mbiri (Fig. 1e).Iyo data inoratidza kuti kuumbwa kweraibhurari kunongowedzerwa zvishoma muchikamu cheropa.Amino acid frequencies uye kubvumirana kutevedzana kwakaongororwazve pachinzvimbo chega chega pachishandiswa shanduko yeWeblogo16 software.Zvinofadza, takawana hupfumi hwakasimba muropa glycine residues (Fig. 1g).Apo ropa rakafananidzwa nemaclones akasarudzwa kubva kuCSF, kusarudzwa kwakasimba uye kumwe kusarudzwa kwezvinyorwa zvakaonekwa (Fig. 1f), uye mamwe amino acids ainyanya kuvepo panzvimbo dzakafanotemerwa mu12-nhengo (Fig. 1h).Zvinonyanya kukosha, mhuka dzega dzakasiyana zvakanyanya mu cerebrospinal fluid, nepo ropa glycine enrichment yakaonekwa mumhuka mbiri (Supplementary Fig. 4a-j).Mushure mekuomesesa kusefa kwekutevedzana kwedata mucerebrospinal fluid yemhuka #1.1 uye #1.2, yakazara ye964 uye 420 yakasarudzika 12-mer peptides yakawanikwa (Supplementary Fig. 1d-e).Iwo ega ega phage clones akakwidziridzwa uye akaiswa kune yechipiri kutenderera kwemu vivo kusarudzwa.Phage yakatorwa kubva muchikamu chechipiri chekusarudzwa yakaiswa pasi peHTS mumhuka imwe neimwe uye peptides yose yakaonekwa yakashandiswa sechipo kune purogiramu yekuzivikanwa kwechinyorwa chekuongorora kuitika kwe tripeptide motifs (Fig. 2a, b, ef).Kuenzaniswa nekutanga kutenderera kwephage yakadzorerwa kubva kuCSF, takaona kumwe kusarudzwa uye kusarudzwa kwemaitiro akawanda muCSF mumatavi A uye B (Fig. 2).Netiweki identification algorithm yakashandiswa kuona kana vaimiririra akasiyana mapatani ekutevedzana kunoenderana.Kufanana kwakajeka kwakaonekwa pakati pezvikamu zve 12-dimensional zvakadzorerwa neCSF mune imwe nzira clade A (Fig. 2c, d) uye clade B (Fig. 2g, h).Kuongororwa kwakabatanidzwa mubazi rimwe nerimwe kwakaratidza maitiro akasiyana-siyana ekusarudzwa kwe12-mer peptides (Supplementary Fig. 5c, d) uye kuwedzera kweCSF / blood titer ratio pamusoro penguva yezvikwata zvakabatanidzwa mushure mechikamu chechipiri chekusarudzwa kuenzaniswa nekutanga kwekusarudzwa (Supplementary Fig. 5e).)
Kupfumiswa kwemotifs uye peptides mucerebrospinal fluid nematenderedzwa maviri anoteedzana emu vivo inoshanda phage kuratidza sarudzo.
Yese cerebrospinal fluid phages yakadzoreredzwa kubva kuchikamu chekutanga chemhuka yega yega (mhuka #1.1 uye #1.2) dzakabatanidzwa, dzakakwidziridzwa, HT-yakatevedzana uye dzakadzoserwa pamwechete (2 x 1010 phages / mhuka) 2 SM cannulated rats (#1.1 → #).2.1 uye 2.2, 1.2 → 2.3 uye 2.4).(a,b,e,f) Correlation scatterplots achienzanisa hukama hwehuwandu hwe tripeptide motifs yevose CSF-yakatorwa phage mukutanga uye yechipiri sarudzo kutenderera.Relative frequency uye kugovera kwemotifs inomiririra ese anogona kupindirana tripeptides anowanikwa mumapeptides mune ese ari maviri.Nhamba yemamotifs inowanikwa mu1000 kuverenga inoratidzwa.Motifs dzaive dzakanyanya (p <0.001) dzakasarudzwa kana kusabatanidzwa mune imwe yemaraibhurari akafananidzwa anojekeswa nemavara matsvuku.(c, d, g, h) Sequence logo inomiririra yevose CSF-rich 12 amino acid refu sequences zvichibva pamaround 2 uye 1 emu vivo kusarudzwa.Saizi yekodhi yetsamba imwe inoratidza kuti amino acid inoitika kakawanda sei panzvimbo iyoyo.Kumiririra chiratidzo, kuwanda kweiyo CSF ​​sequences yakatorwa kubva kumhuka yega yega pakati pematenderedzwa maviri ekusarudza inofananidzwa uye akafumiswa akatevedzana muchikamu chechipiri anoratidzwa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 uye (h) #1.2–#2.4.Iyo yakanyanya kupfumiswa amino acids pane yakapihwa chinzvimbo mu (c, d) mhuka kwete.2.1 uye kwete.2.2 kana (g, h) mumhuka kwete.2.3 uye kwete.2.4 inoratidzwa muruvara.Green = polar, yepepuru = kwazvakarerekera, bhuruu = basic, red = acidic uye dema = hydrophobic amino acids.
Mushure mechikamu chechitatu chekusarudza, takaona 124 yakasiyana-siyana peptide sequences (#3.1 uye #3.2) kubva ku332 CSF-yakagadziriswa phage clones yakaparadzaniswa kubva kumhuka mbiri (Supplementary Fig. 6a).Iyo inoteedzana LGSVS (18.7%) yaive nechepamusoro-soro chikamu, ichiteverwa nemhando dzesango dzinoisa PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), uye SARGSWREIVSLS (2.2%).Muchikamu chechina chekupedzisira, takabatanidza mapazi maviri akazvimiririra akasarudzwa kubva kumhuka nhatu dzakasiyana (Fig. 1c).Pakati pe925 sequenced phage clones yakadzorerwa kubva kuCSF, munharaunda yechina takawana 64 yakasiyana-siyana yepeptide sequences (Supplementary Fig. 6b), pakati peiyo chikamu chemusango-mhando yephage yakadonha kusvika ku0.8%.Makoroni eCSF ainyanyozivikanwa muchikamu chechina aive LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) uye RLSSVDSDLSGC (3, 2%).%)).Hurefu hwemapeptide akasarudzwa hunokonzerwa nekuiswa/kubvisa nucleotide kana nguva isati yakwana yekumisa macodon mumaraibhurari ekutanga kana uchishandisa macodon akaora eNNK library library.Premature stop codons inogadzira mapfupi peptides uye anosarudzwa nekuti ane yakanaka aa motif.Mapeptide marefu anogona kubva mukuiswa/kubviswa mumaprimers emaraibhurari ekugadzira.Izvi zvinomisa kodononi yakagadziridzwa kunze kwefuremu uye inoiverenga kusvika painomira codon yaonekwa pasi.Kazhinji, isu takaverenga ehupfumi zvinhu kune ese mana esarudzo kutenderera nekuenzanisa iyo data yekupinza neyemuenzaniso yekubuda data.Pakutenderera kwekutanga kwekuongorora, takashandisa mhuka dzesango-mhando dzephage senge isiri-chaiyo yekumashure referensi.Sezvineiwo, kusarudzwa kwepageji kwakashata kwaive kwakasimba zvikuru mukutanga kweCSF, asi kwete muropa (Fig. 3a), iyo inogona kunge yakakonzerwa nekuderera kwekupararira kwevazhinji nhengo dzepeptide raibhurari muchikamu cheCSF kana kuti mapeji ehama anowanzove akachengetwa zvakanyanya kana kubviswa kubva muropa kupfuura bacteriophages.Zvisinei, muchikamu chechipiri chekupisa, kusarudzwa kwakasimba kwema phage muCSF kwakaonekwa muzvikamu zviviri, zvichiratidza kuti denderedzwa rekare rakagadziriswa mumaphage kuratidza peptides inokurudzira CSF kutora (Fig. 3a).Zvakare, pasina kukosha kwekuwedzera kweropa.Zvakare mune yechitatu uye yechina kutenderera, iyo phage clones yakafumiswa zvakanyanya muCSF.Kuenzanisa huwandu hwehuwandu hwehuwandu hwepeptide imwe neimwe yakasiyana-siyana pakati pemashure maviri ekupedzisira ekusarudzwa, takaona kuti kutevedzana kwakatowedzera kupfumiswa mumutambo wechina wekusarudza (Fig. 3b).Huwandu hwe931 tripeptide motifs dzakatorwa kubva kune ese makumi matanhatu nemana akasiyana epeptide sequence uchishandisa ese peptide orientation.Mamiriro akanyanya kupfumiswa muchikamu chechina akatariswa zvakanyanya kune avo ehupfumi profiles kumativi ese akaenzaniswa nejekiseni raibhurari (kuchekwa-kubvisa: 10% kupfumisa) (Supplementary Fig. 6c).Mamiriro ose esarudzo airatidza kuti mazhinji evavariro dzakadzidzwa dzakapfumiswa mumataundi ese apfuura emapazi maviri esarudzo.Zvakadaro, mamwe motifs (semuenzaniso SGL, VSG, LGS GSV) ainyanya kubva kune imwe clade A, nepo mamwe (eg FGW, RTN, WGF, NTR) akapfumiswa mune imwe clade B.
Kusimbiswa kwekutakura kweCSF yeCSF-yakagadziriswa phage-yakaratidza peptides uye biotinylated mutungamiri peptides conjugated kune streptavidin payloads.
(a) Kupfumisa mareshiyo akaverengerwa mumativi mana ese (R1-R4) zvichibva pane jekiseni (input = I) phage (PFU) titers uye yakatemerwa CSF phage titers (output = O).Kufumisa zvinhu zvematatu matatu ekupedzisira (R2-R4) akaverengerwa nekuenzanisa nechekare uye yekutanga (R1) ine uremu data.Mabhawa akavhurika ari cerebrospinal fluid, shaded bars iplasma.(***p<0.001, zvichibva pabvunzo dzeMudzidzi).(b) Rondedzero yeakanyanya kuwanda phage peptides, akaiswa maererano nehukama hwayo kune ese maphage akaunganidzwa muCSF mushure mekutenderera 4 yekusarudzwa.Iwo nhanhatu anonyanya kuzivikanwa phage clones akaiswa muruvara, akaverengerwa uye hupfumi hwavo zvinhu pakati pematenderedzwa 3 uye 4 esarudzo (inset).(c, d) Iwo matanhatu akanyanya kupfumisa phage clones, isina chinhu phage uye evabereki phage peptide maraibhurari kubva kutenderera 4 akaongororwa ega ega mu CSF sampling modhi.CSF uye samples dzeropa dzakaunganidzwa panguva dzakatarwa nguva.(c) Huwandu hwakaenzana hwe6 mumiriri wephage clones (2 x 1010 phage/mhuka), phage dzisina chinhu (#1779) (2 x 1010 phage/mhuka) uye stock phage peptide maraibhurari (2 x 1012 phage/mhuka) Jekiseni kanenge 3 CM inopihwa muswe wakaparadzana negani.Iyo CSF ​​pharmacokinetics yega yega jekiseni phage clone uye phage peptide raibhurari nekufamba kwenguva inoratidzwa.(d) inoratidza avhareji CSF/reshiyo yeropa kune ese akadzoserwa phage/mL panguva yekuenzanisira.(e) mana ekugadzira mutungamiriri peptides uye imwe scrambled control yakabatanidzwa nebiotin kune streptavidin kuburikidza neN-terminus yavo (tetramer kuratidza) inoteverwa nejekiseni (muswe mutsinga iv, 10 mg streptavidin/kg).Anenge matatu makonzo akaiswa (N = 3).)CSF samples dzakaunganidzwa panguva dzakatarwa nguva uye streptavidin concentrations yakayerwa neCSF anti-streptavidin ELISA (nd = haina kuonekwa).(*p<0.05, **p<0.01, ***p<0.001, zvichibva pabvunzo dzeANOVA).(f) Kuenzanisa kweamino acid kutevedzana kweiyo yakanyanya kupfuma phage peptide clone #2002 (yepepuru) nemamwe akasarudzwa phage peptide clones kubva ku4th kutenderera kwesarudzo.Zvakafanana uye zvakafanana amino acid zvimedu zvine mavara-coded.
Pakati pezvikamu zvose zvakafumiswa mumutambo wechina (Fig. 3b), maclone matanhatu emakwikwi akasarudzwa kuti awedzere kuongororwa kwega mu CSF sampling model.Kuenzana kwematanhatu emumiriri phage, isina chinhu phage (hapana kupinza) uye prophage peptide maraibhurari akaiswa mumatatu cannulated CM mhuka, uye pharmacokinetics akatemerwa CSF (Fig. 3c) uye ropa (Supplementary Fig. 7) assays.Yese phage clones yakaedzwa yakanangana neiyo CSF ​​compartment pamwero 10-1000 nguva dzakakwirira kupfuura iyo isina chinhu control phage (#1779).Semuyenzaniso, clones #2020 uye #2077 yaive neka1000 yakakwira CSF titers pane control phage.Iyo pharmacokinetic mbiri yepeptide yega yega yakasarudzwa yakasiyana, asi ese ane yakakwira CSF homing kugona.Isu takaona kuderera kwekufamba kwenguva kwema clones #1903 uye #2011, nepo kune clones #2077, #2002 uye #2009 kuwedzera mukati memaminitsi gumi ekutanga kungaratidza kufambisa kunoshanda asi kunoda kuongororwa.Clones #2020, #2002, uye #2077 yakagadzikana pamazinga akakwirira, ukuwo CSF ​​concentration yeclone #2009 yakadzikira zvishoma nezvishoma mushure mekuwedzera kwekutanga.Isu takazofananidza kuwanda kwemumiriri wega wega weCSF nehuwandu hweropa (Fig. 3d).Iko kuwirirana kwechirevo chemumiriri wega wega weCSF neropa rayo panguva dzese dzesampling yakaratidza kuti vatatu vevatanhatu vavhoti vakafumiswa zvakanyanya muropa CSF.Zvinofadza, clone #2077 yakaratidza kugadzikana kwepamusoro kweropa (Supplementary Figure 7).Kusimbisa kuti mapeptide pachawo anokwanisa kutakura zvinhu kunze kwezvimedu zvepageji kupinda mukamuri yeCSF, takagadzira mapeptides mana emutungamiri akatorwa nebiotin paN-terminus apo mapeptides anonamira pachikamu chefage.Biotinylated peptides (n. 2002, 2009, 2020 uye 2077) yakasanganiswa ne streptavidin (SA) kuti iwane mafomu e-multimeric achitevedzera phage geometry.Iyi fomati zvakare yakatibvumidza kuyera SA kuratidzwa muropa uye cerebrospinal fluid sekutakura-kutakura mapuroteni peptides.Zvinonyanya kukosha, data yephage yaigona kazhinji kudzokororwa apo peptides yakagadzirwa yakashandiswa mune iyi SA-conjugated format (Fig. 3e).Iwo mapeptide akadzvanywa aive nekutanga kuratidzwa uye nekukurumidza CSF kubvumidzwa nemazinga asingaonekwe mukati me48 maawa.Kuti tiwane nzwisiso mumakwara ekuendesa aya peptide phage clones munzvimbo yeCSF, takaongorora kugariswa kwega kwega peptide hits uchishandisa immunohistochemistry (IHC) kuona zvakananga phage particles 1 awa mushure mejekiseni remutsinga mu vivo.Zvinonyanya kukosha, clones #2002, #2077, uye #2009 inogona kuonekwa nekusvibiswa kwakasimba muuropi capillaries, nepo control phage (#1779) uye clone #2020 hazvina kuonekwa (Supplementary Figure 8).Izvi zvinoratidza kuti mapeptide aya anobatsira pakuita kuuropi chaizvo nekuyambuka BBB.Imwezve ongororo yakadzama inodiwa kuti uedze iyi hypothesis, sezvo nzira yeBSCFB inogona kubatanidzwawo.Kana tichienzanisa kutevedzana kweamino acid yeklone yakapfumisa (#2002) nemamwe mapeptide akasarudzwa, zvakaonekwa kuti mamwe acho ane amino acid ekuwedzera, iyo inogona kuratidza nzira yekufambisa yakafanana (Fig. 3f).
Pamusana peiyo yakasiyana-siyana yeplasma profile uye kuwedzera kukuru kweCSF nekufamba kwenguva, phage display clone #2077 yakaramba ichiongororwa kwenguva yakareba ye48-awa uye yakakwanisa kubereka kukurumidza kuwedzera kweCSF yakaonekwa mukubatana neSA mazinga akasimba (Fig. 4a).Nezve mamwe akacherechedzwa epage clones, #2077 yakasvibiswa zvine simba kuuropi capillaries uye yakaratidza yakakosha colocalization ine capillary marker lectin painotariswa pakugadziriswa kwepamusoro uye pamwe kumwe kusviba munzvimbo yeparenchymal (Mufananidzo 4b).Kuti tiongorore kana peptide-mediated pharmacological mhedzisiro inogona kuwanikwa muCNS, takaita ongororo umo biotinylated shanduro i) iyo #2077 transit peptide uye ii) iyo BACE1 inhibitor peptide yakasanganiswa neSA pazvikamu zviviri zvakasiyana.Pamusanganiswa mumwe takashandisa chete BACE1 peptide inhibitor uye kune imwe yacho takashandisa chiyero che1:3 cheBACE1 peptide inhibitor kusvika #2077 peptide.Masampula ese ari maviri akaiswa mukati memutsinga uye ropa uye cerebrospinal fluid mazinga ebeta-amyloid peptide 40 (Abeta40) akayerwa nekufamba kwenguva.Abeta40 yakayerwa muCSF sezvo ichiratidza BACE1 inhibition muuropi parenchyma.Sezvinotarisirwa, zvose zviri zviviri zvakaderedza zvakanyanya mazinga eropa eAbeta40 (Fig. 4c, d).Nekudaro, masampuli chete ane musanganiswa wepeptide kwete.2077 uye inhibitor yeBACE1 peptide conjugated kuSA yakakonzera kuderera kukuru muAbeta40 mu cerebrospinal fluid (Fig. 4c).Iyo data inoratidza kuti peptide kwete.2077 inokwanisa kutakura 60 kDa SA protein ichipinda muCNS uye zvakare inokonzeresa pharmacological mhedzisiro neSA-conjugated inhibitors yeBACE1 peptide.
(a) Clonal jekiseni (2 × 10 phages / mhuka) yeT7 phage inoratidza kwenguva refu pharmacokinetic profiles yeCSF peptide #2077 (RLSSVDSDLSGC) uye isina jekiseni control phage (#1779) mune angangoita matatu CM-intubated rats.(b) Confocal microscopic mufananidzo weanomiririra cortical microvessels mune phage-injected makonzo (2 × 10 10 phages / mhuka) kuratidza counterstaining peptide #2077 uye midziyo (lectin).Iyi phage clones yaishandiswa kumakonzo e3 uye yakabvumirwa kutenderera kweawa ye1 isati yasvibiswa.Uropi hwakaiswa zvikamu uye hwakasvibiswa nepolyclonal FITC-yakanyorwa masoja ekudzivirira chirwere achipesana neT7 phage capsid.Maminitsi gumi pamberi pekunyungudika uye kugadziridza kwakatevera, DyLight594-yakanyorwa lectin yaipihwa intravenously.Mifananidzo yeFluorescent inoratidza lectin staining (tsvuku) yedivi rinopenya remamicrovessels uye phage (girinhi) mu lumen ye capillaries uye perivascular brain tissue.Chikero bhaa chinoenderana ne10 µm.(c, d) Biotinylated BACE1 inhibitory peptide yoga kana pamwe chete ne biotinylated transit peptide #2077 yakabatanidzwa kune streptavidin ichiteverwa nejekiseni remutsinga weanosvika matatu cannulated CM rats (10 mg streptavidin/kg).BACE1 peptide inhibitor-mediated reduction muAβ40 yakayerwa neAβ1-40 ELISA muropa (tsvuku) uye cerebrospinal fluid (orange) panguva yakaratidzwa nguva.Kuti ujekese zviri nani, mutsara une doti unodhirowa pagirafu pachiyero che100%.(c) Kuderedzwa kwezana muzana muAβ40 muropa (matatu matatu) uye cerebrospinal fluid (orange triangles) mumakonzo anobatwa ne streptavidin conjugated to transit peptide #2077 uye BACE1 inhibitory peptide muchikamu che3: 1.(d) Kuderedzwa kwezana muropa Aβ40 (red circles) uye cerebrospinal fluid (orange circles) yemakonzo anobatwa ne streptavidin akabatanidzwa neBACE1 inhibitory peptide chete.Iyo Aβ yepakati mukutonga yaive 420 pg/ml (standard deviation = 101 pg/ml).
Phage kuratidza yakashandiswa zvakabudirira munzvimbo dzinoverengeka dzebiomedical research17.Iyi nzira yakashandiswa mu vivo vascular diversity studies18,19 pamwe chete nezvidzidzo zvinotarisa midziyo yeuropi20,21,22,23,24,25,26.Muchidzidzo ichi, takawedzera kushandiswa kweiyi nzira yekusarudza kwete kungozivikanwa kwakananga kwepeptides kunanga tsinga dzehuropi, asiwo pakuwanikwa kwevamiriri vane hunyanzvi hwekutakura zvinhu kuti vayambuke chipingamupinyi cheropa-uropi.Isu ikozvino tinotsanangura kuvandudzwa kweiyo in vivo yekusarudza maitiro muCM intubated makonzo uye kuratidza kugona kwayo kuona peptides ine CSF homing zvivakwa.Tichishandisa T7 phage inoratidza raibhurari ye12-mer random peptides, takakwanisa kuratidza kuti T7 phage idiki zvakakwana (inenge 60 nm mudhayamita) 10 kuti igadziriswe kune yeropa-brain barriers, nokudaro yakananga kuyambuka ropa-brain barrier kana choroid plexus.Takaona kuti kukohwa kweCSF kubva kune cannulated CM rats yaive yakanyatso kudzorwa mu vivo inoshanda yekuongorora nzira, uye kuti iyo yakabviswa phage haina kungosungwa kune vasculature asi yakashanda semutakuri mhiri kweropa-brain barriers.Uyezve, nekuunganidza ropa panguva imwe chete uye kushandisa HTS kuCSF uye mapeji anobva muropa, takasimbisa kuti sarudzo yedu yeCSF haina kukanganiswa nekuwedzerwa kweropa kana kusimba kwekuwedzera pakati pezvirongwa zvekusarudzwa.Zvisinei, kamuri yeropa chikamu chemaitiro ekusarudza, sezvo phage dzinokwanisa kusvika mukamuri yeCSF dzinofanira kurarama uye kutenderera muropa kwenguva yakareba zvakakwana kuti dzizvipfumise muuropi.Kuti tibvise ruzivo rwakavimbika rwekutevedzana kubva kuHTS data yakasvibira, takashandisa mafirita akachinjirwa kune-chikuva-chaiwo zvikanganiso zvekuteedzera mukuwongorora mafambiro.Nekubatanidza kinetic parameters munzira yekuongorora, isu takasimbisa iyo inokurumidza pharmacokinetics yemusango-mhando T7 phages (t½ ~ 28 min) muropa24, 27, 28 uye zvakare takasarudza hafu yehupenyu hwavo mucerebrospinal fluid (t½ ~ 26 min) paminiti).Pasinei nemafungiro akafanana emishonga muropa uye CSF, chete 0.001% yeropa rekuiswa kwephage inogona kuonekwa muCSF, ichiratidza kuderera kwemashure ekufamba kwesango-T7 phage kudarika ropa-brain barrier.Iri basa rinoratidza kukosha kwekutenderera kwekutanga kwekusarudzwa paunenge uchishandisa mu vivo panning mazano, kunyanya kune masisitimu ephage anokurumidza kucheneswa kubva mukutenderera, sezvo mashoma clones anokwanisa kusvika kune CNS compartment.Nekudaro, muchikamu chekutanga, kudzikiswa kwekusiyana kweraibhurari kwaive kwakakura kwazvo, sezvo chete nhamba shoma yemakoneti yakazounganidzwa mune iyi yakasimba CSF modhi.Iyi in vivo panning strategy yaisanganisira matanho akawanda ekusarudza akadai sekushanda kuunganidza muchikamu cheCSF, kupona kwe clone muchikamu cheropa, uye kukurumidza kubviswa kweT7 phage clones kubva muropa mukati memaminitsi ekutanga e10 (Fig. 1d uye Supplementary Fig. 4M).)Saka, mushure mekutenderera kwekutanga, akasiyana phage clones akaonekwa muCSF, kunyangwe dziva rimwe chete rekutanga raishandiswa kune mhuka imwe neimwe.Izvi zvinoratidza kuti matanho akasimba ekusarudzwa kwemaraibhurari ane nhamba huru yenhengo dzeraibhurari zvinokonzeresa kudzikiswa kukuru kwekusiyana.Naizvozvo, zviitiko zvisina tsarukano zvichava chikamu chakakosha chekutanga kusarudzwa maitiro, zvichipesvedzera zvakanyanya mhedzisiro.Zvingangoita kuti mazhinji emakoni muraibhurari yekutanga aive neyakafanana CSF kusimudzira propensity.Nekudaro, kunyangwe pasi pemamiriro akafanana ekuyedza, sarudzo dzesarudzo dzinogona kusiyana nekuda kwenhamba diki yega yega clone mune yekutanga dziva.
Mamiriro akafumiswa muCSF akasiyana neaya ari muropa.Sezvineiwo, takaona shanduko yekutanga kuenda kune glycine-rich peptides muropa remhuka imwe neimwe.(Fig. 1g, Supplementary Figs. 4e, 4f).Phage ine glycine peptides inogona kunge yakagadzikana uye isinganyanyi kutorwa kunze kwekutenderera.Zvisinei, aya mapeptide ane glycine-rich haana kuonekwa mune cerebrospinal fluid samples, zvichiratidza kuti maraibhurari akavharidzirwa akapinda nematanho maviri akasiyana ekusarudza: imwe muropa uye imwe yakabvumirwa kuunganidza mu cerebrospinal fluid.CSF-yakapfuma clones inokonzerwa nechikamu chechina chesarudzo yakaedzwa zvakanyanya.Anenge ese ega ega akaedzwa clones akasimbiswa kuve akafumiswa muCSF zvichienzaniswa neasina chinhu control phage.Imwe peptide hit (#2077) yakaongororwa zvakadzama.Yakaratidza hurefu hweplasma hafu yeupenyu kana ichienzaniswa nemamwe hits (Mufananidzo 3d uye Supplementary Figure 7), uye zvinofadza, iyi peptide yaive necysteine ​​​​yakasara paC-terminus.Ichangobva kuratidzwa kuti kuwedzerwa kwe cysteine ​​​​kune peptides kunogona kuvandudza yavo pharmacokinetic zvivakwa nekusunga kune albumin 29.Izvi parizvino hazvizivikanwe peptide #2077 uye inoda kumwe kudzidza.Mamwe mapeptide airatidza valence-kutsamira muCSF enrichment (data isina kuratidzwa), iyo inogona kunge ine hukama neinotaridzwa yepamusoro geometry yeT7 capsid.Iyo T7 system yatakashandisa yakaratidza 5-15 makopi epeptide yega yega pageji particle.IHC yakaitwa pamumiriri anotungamira phage clones akaiswa intravenously mucerebral cortex yemakonzo (Supplementary Fig. 8).Iyo data yakaratidza kuti kanenge katatu clones (No. 2002, No. 2009 uye No. 2077) yakabatana neBBB.Izvo zvinoramba zvichizivikanwa kana kupindirana uku kweBBB kuchikonzera kuunganidzwa kweCSF kana kufamba kwemakoneti aya zvakananga kuBCSFB.Zvakakosha, isu tinoratidza kuti mapeptide akasarudzwa anochengeta CSF yavo yekufambisa kana yagadzirwa uye yakasungwa kune mapuroteni anotakura.Kusunga kweN-terminal biotinylated peptides kuSA kunonyanya kudzokorora mhedzisiro inowanikwa neayo phage clones muropa uye cerebrospinal fluid (Fig. 3e).Pakupedzisira, tinoratidza kuti lead peptide #2077 inokwanisa kusimudzira uropi hwekuita kwebiotinylated peptide inhibitor yeBACE1 conjugated to SA, zvichikonzera kutaurwa kwepharmacodynamic mhedzisiro muCNS nekudzikisa zvakanyanya Abeta40 mazinga muCSF (Fig. 4).Hatina kukwanisa kuziva chero mahomologues mudura re data nekuita peptide sequence homology kutsvaga kwese kurova.Zvakakosha kuziva kuti saizi yeT7 library inoda kusvika 109, nepo saizi yeraibhurari ye12-mers iri 4 x 1015. Naizvozvo, isu takangosarudza chikamu chidiki chenzvimbo yekusiyana-siyana ye12-mer peptide raibhurari, izvo zvinogona kureva kuti mamwe mapeptide akakwenenzverwa anogona kuzivikanwa nekuongorora nzvimbo yepedyo yenzvimbo idzi dzakatevedzana dzakatarisana.Hypothetically, chimwe chezvikonzero nei tisina kuwana chero echisikigo homologues yeaya peptides anogona kunge asina kusarudzwa panguva yekushanduka-shanduka kudzivirira kupinda kusingadzoreki kweimwe peptide motifs muuropi.
Kutorwa pamwechete, mhedzisiro yedu inopa hwaro hwebasa remangwana rekuona uye kuratidza masisitimu ekufambisa ecerebrovascular barriers mu vivo mune zvakadzama.Iyo yekutanga setup yeiyi nzira yakavakirwa pane inoshanda sarudzo zano isingango ratidza ma clones ane cerebral vascular binding properties, asi inosanganisirawo danho rakakosha umo ma clone akabudirira ane intrinsic chiitiko chekuyambuka biological barriers in vivo kupinda mu CNS compartment.ndeyekujekesa mafambisirwo ekufambisa kweaya peptide uye zvavanoda kusungirira kune microvasculature yakananga kunharaunda yehuropi.Izvi zvinogona kutungamira mukuwanikwa kwenzira nyowani dzekutakura kweBBB uye receptors.Isu tinotarisira kuti mapeptide akaonekwa anogona kusungira zvakananga kune cerebrovascular receptors kana kutenderera ligands inotakurwa kuburikidza neBBB kana BCSFB.Iwo ma peptide vectors ane CSF yekufambisa chiitiko akawanikwa mubasa iri achaongororwa zvakare.Parizvino tiri kuongorora huropi hwemapeptide aya nekugona kwavo kuyambuka BBB uye/kana BCSFB.Aya mapeptide matsva anozove maturusi akanyanya kukosha ekuwanikwa kwezvitsva zvinogamuchira kana nzira uye nekusimudzira mapuratifomu matsva anoshanda kwazvo ekuunza macromolecules, senge biologics, kuuropi.
Cannulate the big cisterna (CM) uchishandisa gadziridzo yenzira yakambotsanangurwa.Anesthetized Wistar rats (200-350 g) akaiswa pane stereotaxic apparatus uye kuchekwa kwepakati kwakaitwa pamusoro pemusoro wakagerwa uye wakagadzirwa neseptically kuti dehenya ribude pachena.Dhonza maburi maviri munzvimbo yekumusoro sash uye sunga zvigadziriso zvekugadzirisa mumakomba.Rimwe gomba rakaboorwa mu lateral occipital crest kuitira stereotactic kutungamira kwesimbi isina tsvina cannula muCM.Isa simende yemazino yakatenderedza cannula uye chengetedza nema screws.Mushure mekupora-pikicha uye simende kuomesa, ronda reganda rakavharwa ne 4/0 supramid suture.Kuiswa kwakakodzera kwecannula kunosimbiswa nekubuda kwepakarepo kwecerebrospinal fluid (CSF).Bvisa gonzo kubva kune stereotaxic apparatus, gamuchira kwakakodzera postoperative kutarisirwa uye kugadzirisa marwadzo, uye bvumira kuti iwanezve kweinenge vhiki imwe kusvikira zviratidzo zveropa zvaonekwa mucerebrospinal fluid.Wistar rats (Crl: WI/Han) akawanikwa kubva kuCharles River (France).Makonzo ese aichengetwa pasi pemamiriro chaiwo asina pathogen.Zvose zvinongororwa zvemhuka zvakagamuchirwa neVeterinary Office yeGuta reBasel, Switzerland, uye zvakaitwa maererano neAnimal License No. 2474 (Kuongorora kweActive Brain Transport neKuyera Mazinga eVadzidzi Vekurapa muCerebrospinal Fluid uye Brain of Rat).
Zvinyoro chengetedza gonzo richiziva neCM cannula iri muruoko.Bvisa Datura kubva mucannula uye tora 10 µl ye cerebrospinal fluid inoyerera yega.Sezvo patency yecannula yakapedzisira yakanganiswa, chete yakajeka cerebrospinal fluid samples isina humbowo hwekusvibiswa kweropa kana kubuda kwemavara zvakabatanidzwa muchidzidzo ichi.Mukufanana, inenge 10-20 μl yeropa yakatorwa kubva padiki diki pamucheto wemuswe mumachubhu ane heparin (Sigma-Aldrich).CSF neropa zvakaunganidzwa panguva dzakasiyana-siyana mushure mekupinda mutsinga weT7 phage.Inenge 5-10 μl yemvura yakaraswa isati yatorwa imwe neimwe sampu yeCSF, iyo inowirirana nevhoriyamu yakafa yecatheter.
Maraibhurari akagadzirwa achishandisa T7Select 10-3b vector sezvinotsanangurwa muT7Select system manual (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996).Muchidimbu, yakangoerekana yaiswa 12-mer DNA yakaiswa mune inotevera fomati:
Iyo NNK codon yakashandiswa kudzivirira kaviri kumira codons uye amino acid overexpression mune yekuisa.N ireshiyo yakavhenganiswa nemaoko yenucleotide yega yega, uye K ireshiyo nemaoko yakasanganiswa equimolar yeadenine uye cytosine nucleotides.Matunhu mamwe akaganhurwa akashandurwa kuita DNA yakapetwa kaviri nekuwedzera kuvharirwa nedNTP (Novagen) uye Klenow enzyme (New England Biolabs) muKlenow buffer (New England Biolabs) kwemaawa matatu pa37°C.Mushure mekuita, DNA ine tambo mbiri yakadzoserwa neEtOH kunaya.Iyo DNA yakaguma yakagayiwa nekudzora ma enzymes EcoRI uye HindIII (ose kubva kuRoche).Iyo yakatsemurwa uye yakacheneswa (QIAquick, Qiagen) isa (T4 ligase, New England Biolabs) yakabva yabatanidzwa mu-frame mu pre-cleaved T7 vector mushure meamino acid 348 ye10B capsid gene.Ligation reactions yakaiswa pa 16 ° C. kwemaawa gumi nemasere isati yasvika in vitro packaging.Phage packaging in vitro yakaitwa maererano nemirairo yakapihwa neT7Select 10-3b cloning kit (Novagen) uye mhinduro yekurongedza yakakwidziridzwa kamwe chete kuita lysis uchishandisa Escherichia coli (BLT5615, Novagen).Malysates aive centrifuged, titrated uye akaomeswa nechando pa -80 ° C. semushonga wekutengesa weglycerol.
Yakananga PCR kukwidziridzwa kwephage kusiyanisa matunhu akakwidziridzwa mubroth kana ndiro uchishandisa proprietary 454 / Roche-amplicon fusion primers.Iyo yekumberi fusion primer ine inoteedzana inotenderera nharaunda inoshanduka (NNK) 12 (template-yakananga), GS FLX Titanium Adapter A, uye ina-base raibhurari kiyi sequence (TCAG) (Supplementary Figure 1a):
Iyo reverse fusion primer inewo biotin yakasungirirwa kutora mabhero uye GS FLX Titanium Adapter B inodiwa kuti clonal amplification panguva emulsion PCR:
Maamplicon akazoiswa pasi pe454/Roche pyrosequencing maererano ne454 GS-FLX Titanium protocol.Kune bhuku reSanger sequencing (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage DNA yakakwidziridzwa nePCR uye yakatevedzana neanotevera mapeya ekutanga:
Kuiswa kubva kune ega mapepa akaiswa pasi pePCR amplification uchishandisa iyo Roche Fast Start DNA Polymerase Kit (maererano nemirairo yemugadziri).Ita kutanga kunopisa (10 min pa 95 ° C) uye 35 kukurudzira maitiro (50 s pa 95 ° C, 1 min pa 50 ° C, uye 1 min pa 72 ° C).
Phage kubva mumaraibhurari, mhuka dzesango, phage yakanunurwa kubva kuCSF neropa, kana ma clones ega ega akawedzerwa muEscherichia coli BL5615 muTB muto (Sigma Aldrich) kana mu500 cm2 ndiro (Thermo Scientific) ye4 h pa37 ° C.Phage yakatorwa kubva mumahwendefa nekusuka mahwendefa neTris-EDTA buffer (Fluka Analytical) kana kuunganidza mapepa ane sterile pipette mazano.Phage yaive yakaparadzaniswa netsika inoshamisa kana yekubvisa buffer ine imwe denderedzwa yepolyethylene glycol (PEG 8000) kunaya (Promega) uye yakamiswazve muTris-EDTA buffer.
Iyo phage yakasimudzwa yakaiswa pasi pe 2-3 kutenderera kwekubvisa endotoxin uchishandisa endotoxin kubvisa mabhero (Miltenyi Biotec) isati yaiswa intravenous (IV) jekiseni (500 μl/mhuka).Muchikamu chekutanga, 2 × 1012 phages yakaunzwa;mune yechipiri, 2 × 1010 phages;muchikamu chechitatu nechina chekusarudza, 2 × 109 phages pamhuka.Phage zvemukati muCSF uye masampuli eropa akaunganidzwa panguva dzakatarwa dzakatemwa nekuverenga plaque maererano nemirairo yemugadziri (T7Select system manual).Kusarudzwa kwePhage kwakaitwa nejekiseni remutsinga remaraibhurari akacheneswa mutsinga yemuswe kana nejekiseni repage rakatorwa kubva kuCSF kubva padanho rekutanga rekusarudza, uye kukohwa kwakatevera kwakaitwa pa10 min, 30 min, 60 min, 90 min, 120 min, 180 min, uye 240 min uye samples yeropa zvichiteerana.Huwandu hwematenderedzwa mana emu vivo panning akaitwa umo matavi maviri akasarudzwa akachengetwa zvakasiyana uye akaongororwa panguva yekutanga kutenderera kwesarudzo.Yese fage inoiswa yakatorwa kubva kuCSF kubva kumatanho maviri ekutanga esarudzo akaiswa pasi pe454 / Roche pyrosequencing, nepo maclone ese akatorwa kubva kuCSF kubva kumaviri ekupedzisira esarudzo akateedzana nemaoko.Yese phage dzeropa kubva padanho rekutanga resarudzo dzakaiswawo pasi pe454 / Roche pyrosequencing.Nokuda kwejekiseni repage clones, phage dzakasarudzwa dzakawedzerwa muE. coli (BL5615) pamapuleti 500 cm2 pa 37 ° C kwemaawa mana.Makoni akasarudzwa ega uye akateedzana nemaoko akaparadzirwa mukati meTB medium.Mushure mekubviswa kwephage, kucheneswa uye kubviswa kwe endotoxin (sezvinotsanangurwa pamusoro), 2 × 1010 phages / mhuka mu 300 μl yakabayiwa intravenously mune imwe muswe tsinga.
Preprocessing uye yemhando kusefa kwesequence data.Raw 454/Roche data yakashandurwa kubva kubhinari yakajairwa kuyerera kwemepu fomati (sff) kuenda kuPearson yemunhu inoverengeka fomati (fasta) uchishandisa mutengesi software.Kuenderera mberi kwekugadzirisa kweiyo nucleotide kutevedzana kwakaitwa pachishandiswa proprietary C zvirongwa uye zvinyorwa (isina kuburitswa software package) sekutsanangurwa pazasi.Kuongororwa kwedata rekutanga kunosanganisira akasimba akawanda-nhanho kusefa maitiro.Kusefa maverengi anga asina kutevedzana kwe12mer kuisa DNA, kuverenga kwakarongwa zvakatevedzana kutanga label (GTGATGTCGGGGATCCGAATTCT), stop label (TAAGCTTGCGGCCGCACTCGAGTA) uye yekumashure kuisa (CCCTGCAGGGATATCCCGGAGCTCTCGTschCGAC) uchishandisa iyo yepasi rose Need Need.kurongeka kunobvumira kusvika ku2 kusapindirana pakurongeka31.Naizvozvo, kuverenga pasina kutanga uye kumisa ma tag uye kuverenga ane kumashure ekuisa, kureva, marongero anodarika nhamba inobvumidzwa yekusawirirana, akabviswa muraibhurari.Kana zviri zvezviverengero zvasara, iyo N-mer DNA kutevedzana kunobva pachiratidzo chekutanga uye ichipera chiratidzo chekumira chisati chabviswa kubva pakutevedzana kwekuverenga kwekutanga uyezve kugadziridzwa (zvino zvinonzi "isa").Mushure mekushandura kwekuisa, chikamu mushure mekutanga kumira codon pamagumo e5 'yekutanga inobviswa kubva pakuiswa.Mukuwedzera, nucleotides inotungamirira kumakodheni asina kukwana pamagumo e3 'yekutanga akabviswawo.Kusabvisa zvinoiswa zvine nhevedzano yekumashure chete, zvakaturikirwa zvinotanga neamino acid pateni "PAG" zvakabviswawo.Peptides ane hurefu hwepashure-shanduro isingasviki 3 amino acids akabviswa muraibhurari.Chekupedzisira, bvisa redundancy mudziva rekuisa uye uone kuwanda kweyega yega yakasarudzika yekuisa.Migumisiro yekuongorora iyi yaisanganisira runyoro rwe nucleotide sequences (insets) uye yavo (verenga) frequencies (Supplementary Figures 1c uye 2).
Boka reN-mer DNA rinoisa nekutevedzana kufanana: Kubvisa 454/Roche-chaiyo yekutevedzana kukanganisa (sematambudziko nekutevedzana kwehomopolymer extensions) uye kubvisa kusakosha kusakosha, kwakambosefa N-mer DNA sequence inserts (insets) inorongwa nekufanana.kuiswa (kusvika ku2 mabhesi asingaenderane anotenderwa) uchishandisa algorithm inodzokororwa inotsanangurwa sezvizvi: zvinoiswa zvinorongwa kutanga nemafambiro azvo (pamusoro-soro kusvika pasi pasi), uye kana zvakafanana, nerudzi rwechipiri nekureba (kureba kusvika kupfupi) ).Nokudaro, kuiswa kwakawanda uye kwenguva refu kunotsanangura "boka" rokutanga.Iyo frequency yeboka inoiswa kune kiyi frequency.Zvadaro, kuiswa kwega kwega kwasara muhurongwa hwakarongwa kwakayedzwa kuwedzerwa kuboka nekurongeka kweNeedleman-Wunsch.Kana huwandu hwekusapindirana, kuisirwa, kana kudzima mugwara kusingapfuure chikumbaridzo che2, kuiswa kunowedzerwa kuboka, uye kuwanda kweboka rose kunowedzerwa nekuwanda kwaiiswa kwakawedzerwa.Zvinoiswa paboka zvinomakwa sezvakashandiswa uye hazvibatanidzwe pakuenderera mberi nekugadzirisa.Kana nhevedzano yekuisa isingakwanisi kuwedzerwa kuboka ragara riripo, mutsara wekuisa unoshandiswa kugadzira boka idzva rine frequency yakafanira yekuisa uye yakamaka seyakashandiswa.Kudzokorodza kunopera kana kutevedzana kwega kwega kwekuisa kwave kushandiswa kuumba boka idzva kana kuti kunogona kuverengerwa muboka ragara riripo.Mushure mezvose, kuiswa kwakaiswa mumapoka kunosanganisira nucleotides kunozoshandurwa kuita peptide sequences (peptide library).Mhedzisiro yekuongorora uku igadziriro yekuisa uye maitiro avo anowirirana anoita nhamba yekuverenga kwakatevedzana (Supplementary Fig. 2).
Motif Generation: Kubva pane rondedzero yemapeptides akasiyana, raibhurari yakagadzirwa iine ese anobvira amino acid mapatani (aa) sezvakaratidzwa pazasi.Imwe neimwe inogoneka pateni yehurefu 3 yakatorwa kubva peptide uye yayo inverse pateni yakawedzerwa pamwe neyakajairwa motif raibhurari ine ese mapatani (tripeptides).Maraibhurari ezvekudzokorodza zvakanyanya motifs akateedzana uye redundancy yakabviswa.Zvino, kune yega yega tripeptide muraibhurari yemotif, takatarisa kuvepo kwayo muraibhurari tichishandisa maturusi emakomputa.Muchiitiko ichi, kuwanda kwepeptide ine yakawanikwa motif tripeptide inowedzerwa uye inopihwa kune iyo motif mumotif raibhurari ("nhamba yemamotifs").Mhedzisiro yekugadzirwa kwemotif ndeye maviri-dimensional array ine zvese zvinoitika zve tripeptides (motifs) uye hunhu hwazvo, hunova huwandu hwekutevedzana kwekuverenga kunoguma nemotif inoenderana apo kuverenga kunosefa, kuunganidzwa, uye kushandurwa.Metrics sezvinotsanangurwa zvakadzama pamusoro apa.
Normalization yehuwandu hwema motifs uye anoenderana scatterplots: Huwandu hwemamotifs ega ega sampu yakajairwa uchishandisa.
apo ni ndiyo nhamba yezviverengero zvine musoro wenyaya i.Saka, vi inomiririra huwandu hwehuwandu hwekuverenga (kana peptides) ine motif i mumuenzaniso.P-values ​​yeiyo isiri-yakajairwa nhamba yemotifs yakaverengerwa uchishandisa bvunzo yaFisher chaiyo.Nezve macorrelograms ehuwandu hwevavariro, kuwirirana kwaSpearman kwakaverengerwa pachishandiswa nhamba yakajairwa yevavariro neR.
Kuti uone zviri mukati maamino acids panzvimbo yega yega muraibhurari yepeptide, web logograms 32, 33 (http://weblogo.threeplusone.com) yakagadzirwa.Chekutanga, zvirimo zveamino acids panzvimbo yega yega ye12-mer peptide inochengetwa mu20×12 matrix.Zvadaro, seti ye1000 peptides ine imwechete amino acid yemukati pane imwe neimwe chinzvimbo inogadzirwa mufasta-kutevedzana fomati uye yakapihwa sekuisa kune web-logo 3, iyo inoburitsa mufananidzo wakajeka wehukama amino acid zvirimo panzvimbo yega yega.yeraibhurari yepeptide yakapihwa.Kuti uone dhatabheti dzakawanda, mamepu ekupisa akagadzirwa uchishandisa chishandiso chemukati chakagadzirwa muR (biosHeatmap, ichiri kuburitswa R package).Iwo ma dendrogram akaunzwa mumamepu ekupisa akaverengerwa achishandisa Ward's hierarchical clustering nzira neEuclidean distance metric.Kuongororwa kwenhamba yemotif scoring data, P values ​​​​yezvisina kurongeka zvibodzwa zvakaverengerwa uchishandisa bvunzo yaFisher chaiyo.P-values ​​yemamwe madataset akaverengerwa muR achishandisa Mudzidzi's t-bvunzo kana ANOVA.
Akasarudzwa phage clones uye phage asina kuiswa akaiswa jekiseni kuburikidza nemuswe tsinga (2 × 1010 phages / mhuka mu 300 μl PBS).Maminitsi gumi kusati kwaitwa perfusion nekugadzirisa kwakatevera, mhuka dzakafanana dzakaiswa intravenously ne100 μl yeDyLight594-yakanyorwa lectin (Vector Laboratories Inc., DL-1177).60 maminetsi mushure mejojo ​​yephage, makonzo akasvibiswa nemoyo ne 50 ml PBS yakateverwa ne50 ml 4% PFA / PBS.Sampuli dzehuropi dzakagadziriswazve husiku humwe mu4% PFA/PBS uye yakanyudzwa mu30% sucrose husiku humwe pa4 ° C.Samples anopenya akaomeswa nechando mumusanganiswa weOCT.Ongororo yeImmunohistochemical yemasampuli akaomeswa yakaitwa pakamuri tembiricha pa30 µm cryosections yakavharwa ne1% BSA uye yakavharirwa nepolyclonal FITC-yakanyorwa masoja ekudzivirira chirwere anorwisa T7 phage (Novus NB 600-376A) pa4 °C.Vhura usiku hwose.Pakupedzisira, zvikamu zvakashambidzwa 3 nguva nePBS uye yakaongororwa ne confocal laser microscope (Leica TCS SP5).
Mapeptide ese ane hushoma kuchena kwe98% akagadzirwa neGenScript USA, biotinylated uye lyophilized.Biotin inosungwa kuburikidza neyekuwedzera katatu glycine spacer paN-terminus.Tarisa peptides ese uchishandisa mass spectrometry.
Streptavidin (Sigma S0677) yakasanganiswa ne5-fold equimolar yakawandisa yebiotinylated peptide, biotinylated BACE1 inhibitory peptide, kana musanganiswa (3: 1 ratio) yebiotinylated BACE1 inhibitory peptide uye BACE1 inhibitory peptide mu5-10% yePCB yakavharwa muSOO.1 awa pakamuri tembiricha pamberi jekiseni.Streptavidin-conjugated peptides akaiswa intravenously pachiyero che 10 mg / kg mune imwe yemuswe tsinga dzemakonzo ane cerebral cavity.
Kuwanda kwe streptavidin-peptide complexes kwakaongororwa neELISA.Nunc Maxisorp microtiter plates (Sigma) yakaputirwa usiku hwose pa4 ° C ne 1.5 μg / ml mouse anti-streptavidin antibody (Thermo, MA1-20011).Mushure mekuvhara (kuvharira buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) pamhepo tembiricha kwemaawa maviri, geza ndiro ne 0.05% Pakati-20/PBS (wash buffer) ye3 Second, CluSF yakawedzerwa0 neplasma inovhara 0 plasma: 0 tsime replasma: 0, CSF 1:115).Iyo ndiro yakabva yaiswa usiku hwose pa4 ° C neine antibody yekuona (1 μg / ml, anti-streptavidin-HRP, Novus NB120-7239).Mushure mematanho matatu ekugeza, streptavidin yakaonekwa nekuvharirwa muTMB substrate solution (Roche) kusvika ku20 min.Mushure mekumisa kugadzirwa kwemavara ne1M H2SO4, pima kunyura pa450 nm.
Basa re streptavidin-peptide-BACE1 inhibitor complex rakaongororwa neAβ (1-40) ELISA maererano neprotocol yemugadziri (Wako, 294-64701).Nenguva pfupi, sampuli dzeCSF dzakanatswa mune yakajairwa diluent (1:23) uye yakadzikwa husiku hwese pa4 ° C mu96-tsime mahwendefa akaputirwa neBNT77 kubata antibody.Mushure mematanho mashanu ekugeza, HRP-conjugated BA27 antibody yakawedzerwa uye yakavharirwa kwemaawa maviri pa4° C., ichiteverwa nematanho mashanu ekugeza.Aβ(1–40) yakaonekwa nekuvharirwa muTMB mhinduro kwemaminetsi makumi matatu patembiricha yekamuri.Mushure mekugadzirwa kwemavara kwakamiswa nemhinduro yekumisa, kuyera kunyura pa450 nm.Masampula ePlasma akaiswa pasi pechikamu chakasimba chekutorwa pamberi peAβ (1-40) ELISA.Plasma yakawedzerwa ku 0.2% DEA (Sigma) mumapuleti e96-tsime uye yakavharwa pamhepo tembiricha kwemaminitsi makumi matatu.Mushure mekugeza zvakatevedzana mahwendefa eSPE (Oasis, 186000679) nemvura uye 100% methanol, masampuli eplasma akawedzerwa kumahwendefa eSPE uye mvura yose yakabviswa.Samples dzakagezwa (kutanga ne5% methanol kozoti 30% methanol) uye yakaburitswa ne2% NH4OH/90% methanol.Mushure mekuomesa iyo eluate pa 55 ° C ye 99 min pane nguva dzose N2 ikozvino, sampuli dzakaderedzwa mumhando diluents uye Aβ (1-40) yakayerwa sezvinotsanangurwa pamusoro apa.
Matauriro echinyorwa ichi: Urich, E. et al.Cargo kuendeswa kuuropi uchishandisa transit peptides akaonekwa mu vivo.sainzi.5, 14104;doi:10.1038/srep14104 (2015).
Likhota J., Skjorringe T., Thomsen LB naMoos T. Kuendeswa kwemishonga yemacromolecular kuuropi uchishandisa kurapa kwakanangwa.Nyaya yeNeurochemistry 113, 1-13, 10.1111 / j.1471-4159.2009.06544.x (2010).
Brasnjevic, I., Steinbusch, HW, Schmitz, C., uye Martinez-Martinez, P. Kuendeswa kwepeptide uye mapuroteni zvinodhaka mukati meropa-brain barriers.Prog Neurobiol 87, 212–251, 10.1016/j.pneurobio.2008.12.002 (2009).
Pardridge, WM Iyo yeropa-brain barriers: bhodhoro mukuvandudza zvinodhaka muuropi.NeuroRx 2, 3–14, 10.1602/neurorx.2.1.3 (2005).
Johanson, KE, Duncan, JA, Stopa, EG, naByrd, A. Tarisiro yekuvandudzwa kwekushandisa zvinodhaka uye kunangwa kuuropi kuburikidza nechoroid plexus-CSF nzira.Pharmaceutical Research 22, 1011–1037, 10.1007/s11095-005-6039-0 (2005).
Pardridge, WM Modernization yebiopharmaceuticals ine molecular Trojan mabhiza ekuunza huropi.Bioconjug Chem 19, 1327–1338, 10.1021/bc800148t (2008).
Pardridge, WM receptor-mediated peptide kutakura mhiri kweropa-brain barriers.Endocr Rev. 7, 314–330 (1986).
Niewoehner, J. et al.Wedzera kupinda kwehuropi uye kushanda zvakanaka kwemishonga inorwisa mabhakitiriya uchishandisa monovalent molecular shuttles.Neuron 81, 49–60, 10.1016/j.neuron.2013.10.061 (2014).
Bien-Lee, N. et al.Transferrin receptor (TfR) yekufambisa inosarudza kutora kwehuropi hwehukama hwakasiyana hweTfR antibodies.J Exp Med 211, 233–244, 10.1084/jem.20131660 (2014).