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Chidziviriro cheropa-uropi uye chidziviriro cheropa-uropi zvinodzivirira mishonga yekurapa kubva muropa kusvika pazvinangwa zvavo muhurongwa hwetsinga dzeropa, nokudaro zvichitadzisa kurapa zvirwere zvetsinga zvinobudirira. Kuti tiwane zvinhu zvitsva zvinotakura uropi mumuviri, takaunza raibhurari yeT7 phage peptide uye takaunganidza ropa ne cerebrospinal fluid (CSF) tichishandisa modhi yemakonzo ine cannulated conscious large pool. Ma specific phage clones akapfuma zvikuru muCSF mushure menguva ina dzekusarudza. Kuedzwa kwema peptides ega ega kwakaratidza kuwedzera kweCSF kanopfuura ka1000. Kushanda kwe peptide-mediated shipping kuuropi kwakasimbiswa nekudzikira kwe40% kwehuwandu hwe amyloid-β mu cerebrospinal fluid tichishandisa BACE1 peptide inhibitor yakabatana ne novel transit peptide yakawanikwa. Izvi zvinoratidza kuti ma peptides akaonekwa nenzira dze in vivo phage select anogona kuva nzira dzinobatsira pakuendesa ma macromolecules kuuropi nemhedzisiro yekurapa.
Ongororo yekurapa yakanangana nepakati petsinga dzeropa (CNS) yakatarisana zvakanyanya nekuona mishonga yakagadziriswa uye zvinhu zvinoratidza hunhu hweCNS-targeting, pasina kushanda nesimba pakuwana nzira dzinoita kuti mishonga iendeswe kuuropi. Izvi zvava kutanga kuchinja ikozvino sezvo kuendeswa kwemishonga, kunyanya mamorekuru makuru, kuri chikamu chakakosha chekuvandudzwa kwemishonga yeneuroscience yemazuva ano. Nzvimbo yepakati petsinga dzeropa yakachengetedzwa zvakanaka ne cerebrovascular barrier system, ine blood-brain barrier (BBB) ​​​​uye blood-brain barrier (BCBB)1, zvichiita kuti zvive zvakaoma kuendesa mishonga kuuropi1,2. Zvinofungidzirwa kuti mishonga yakawanda yemamorekuru uye inopfuura 98% yemishonga midiki yemamorekuru inobviswa muuropi3. Ndosaka zvichikosha kuona masisitimu matsva ekufambisa uropi anopa kuendesa mishonga yekurapa inoshanda uye yakananga kuCNS 4,5. Zvisinei, BBB neBCSFB zvinopawo mukana wakanaka wekutumira mishonga sezvo ichipinda nekupinda muzvikamu zvese zveuropi kuburikidza netsinga dzayo dzakakura. Saka, kuedza kuri kuitwa pari zvino kushandisa nzira dzisingapindi muuropi kunonyanya kuenderana nekushanda kwekutumira kwemasero (PMT) uchishandisa endogenous BBB6 receptor. Pasinei nekufambira mberi kukuru munguva pfupi yapfuura uchishandisa transferrin receptor pathway7,8, kuenderera mberi kwekuvandudza masisitimu matsva ekutumira ane hunhu hwakavandudzwa kunodiwa. Kuti izvi zviitike, chinangwa chedu chaive chekuona mapeptide anokwanisa kufambisa CSF, sezvo anogona kushandiswa kuendesa macromolecules kuCNS kana kuvhura nzira itsva dzemareceptor. Kunyanya, mareceptor chaiwo uye matransporter e cerebrovascular system (BBB neBSCFB) anogona kushanda sezvinangwa zvekuendeswa kwemishonga inoshanda uye chaiyo ye biotherapeutic. Cerebrospinal fluid (CSF) chigadzirwa chinobuda muropa chechoroid plexus (CS) uye chinosangana zvakananga nemvura yemukati meuropi kuburikidza nenzvimbo ye subarachnoid nenzvimbo ye ventricular4. Munguva pfupi yapfuura zvakaratidzwa kuti mvura ye subarachnoid cerebrospinal inopararira zvakanyanya mu interstitium yeuropi9. Tinotarisira kusvika panzvimbo ye parenchymal tichishandisa nzira iyi ye subarachnoid inflow kana zvakananga kuburikidza ne BBB. Kuti tiite izvi, takaisa nzira yakasimba yekutsvaga ma peptides anotakurwa neimwe yenzira mbiri idzi dzakasiyana.
Iye zvino tava kutsanangura nzira yekuongorora ma "in vivo phage display" neCSF sampling pamwe ne "high throughput sequencing" (HTS) kuti titarise kusarudzwa kwekutanga kwe "rally diversity" ine huwandu hwakanyanya hwe "library diversity". Kuongororwa kwakaitwa pamakonzo ane ruzivo ne "large cisterna" (CM) cannula" yakaisirwa zvachose kudzivirira kusvibiswa kweropa. Zvakakosha, nzira iyi inosarudza "brain-targeting" uye "peptides" dzine "transport activity" kuburikidza ne "cerebrovascular barrier". Takashandisa ma "T7 phages" nekuda kwehukuru hwavo hudiki (~60 nm)10 uye takaratidza kuti akakodzera kutakurwa kwema "vesicles" anobvumira transcellular crossing" ye "endothelial" uye / kana "epithelial-medulla barrier". Mushure me "rounds" ina dze "panning", ma "phage populations" akaparadzaniswa achiratidza kupfuma kwakasimba kwe "in vivo CSF" uye kubatana kwe "cerebral microvessel association". Zvakakosha, takakwanisa kusimbisa zvatakawana nekuratidza kuti ma "best candidate peptides" anodiwa uye akagadzirwa nemakemikari anokwanisa kutakura mapuroteni anotakura mu "cerebrospinal fluid". Chekutanga, mhedzisiro yeCNS mumakemikari yakasimbiswa nekubatanidza peptide inotungamira yekufambisa nechinhu chinodzivirira peptide yeBACE1. Pamusoro pekuratidza kuti nzira dzekuongorora mashandiro emuviri dzinogona kuona mapeptide matsva ekufambisa uropi seanotakura mapuroteni anoshanda, tinotarisira kuti nzira dzakafanana dzekusarudza mashandiro dzichave dzakakoshawo mukuona nzira itsva dzekufambisa uropi.
Zvichibva pamayuniti ekugadzira plaque (PFU), mushure medanho rekuisa phage, raibhurari yemapeptide eT7 phage ane random 12-mer linear ane mhando dzakasiyana dze109 dzakagadzirwa uye dzakagadzirwa (ona Zvinhu neNzira). Zvakakosha kuziva kuti takaongorora raibhurari iyi nokungwarira tisati taisa in vivo panning. Kuwedzerwa kwePCR kwemasampuli eraibhurari yephage uchishandisa ma primer akagadziriswa kwakagadzira ma amplicons aishanda zvakananga kuHTS (Supplementary Fig. 1a). Nekuda kwe a) HTS11 sequencing errors, b) kukanganisa mhando yema primers (NNK)1-12, uye c) kuvapo kwe wild-type (wt) phage (skeleton inserts) muraibhurari yakamirira, nzira yekusefa sequence yakashandiswa kuti ibudise ruzivo rwe sequence rwakavimbiswa chete (Supplementary Fig. 1b). Matanho aya e sefa anoshanda kumaraibhurari ese e HTS sequencing. Kune raibhurari yakajairwa, huwandu hwekuverenga hunosvika 233,868 hwakawanikwa, uye 39% yavo vakapasa zvinodiwa zvekusefa uye vakashandiswa pakuongorora raibhurari nekusarudza marounds anotevera (Supplementary Picture 1c–e). Kuverenga kwacho kwaive kwakawanda kwezvikamu zvitatu zve base pairs pakureba zvine peak pa36 nucleotides (Supplementary Fig. 1c), zvichisimbisa dhizaini yeraibhurari (NNK) 1-12. Zvinonyanya kukosha, inenge 11% yenhengo dzeraibhurari yaive ne 12-dimensional wild-type (wt) backbone PAGISRELVDKL insert, uye inenge hafu yezvikamu (49%) yaive nezvinyorwa kana kubviswa. HTS yeraibhurari yeraibhurari yakasimbisa kusiyana kukuru kwemapeptide muraibhurari: anopfuura 81% ezvikamu zvepeptide zvakawanikwa kamwe chete uye 1.5% chete yakaitika mumakopi ≥4 (Supplementary Fig. 2a). Mafrequency emaamino acids (aa) panzvimbo dzese gumi nembiri mu repertoire akabatana zvakanaka nemafrequency anotarisirwa ehuwandu hwema codons anogadzirwa ne repertoire yeNKK yakaora (Supplementary Fig. 2b). Mafrequency akaonekwa e aa residues akanyorwa neaya ma inserts akabatana zvakanaka nefrequency yakaverengerwa (r = 0.893) (Supplementary Fig. 2c). Kugadzirira maraibhurari e phage kuti apinzwe kunosanganisira matanho ekuwedzera nekubvisa endotoxin. Izvi zvakamboratidzwa kuti zvinogona kuderedza kusiyana kwema libraries e phage12,13. Nokudaro, takaronga raibhurari ye phage yakasimudzwa neplate-amplified yakanga yabviswa endotoxin ndokuienzanisa neraibhurari yekutanga kuti tifungidzire mafrequency eAA. Kuwirirana kwakasimba (r = 0.995) kwakaonekwa pakati pedziva rekutanga nedziva rakasimudzwa uye rakacheneswa (Supplementary Fig. 2d), zvichiratidza kuti kukwikwidzana pakati pema clones akasimudzwa pamaplates achishandisa T7 phage hakuna kukonzera kusarura kukuru. Kuenzanisa uku kwakavakirwa pakuwanda kwemamotif etripeptide muraibhurari yega yega, sezvo kusiyana kwemaraibhurari (~109) kusingakwanise kubatwa zvizere kunyangwe neHTS. Kuongororwa kwekuwanda kweaa panzvimbo yega yega kwakaratidza kusarura kudiki kunoenderana nenzvimbo munzvimbo nhatu dzekupedzisira dzerepertoire yakapinda (Mufananidzo Wekuwedzera 2e). Mukupedzisa, takagumisa kuti mhando nekusiyana kweraibhurari kwaigamuchirwa uye shanduko diki chete mukusiyana kwakaonekwa nekuda kwekuwedzera nekugadzirira maraibhurari ephage pakati pematanho akati wandei ekusarudza.
Kuongororwa kwemvura yemusana kunogona kuitwa nekuiswa kwecannula muCM yemakonzo anoona kuti zviite nyore kuona T7 phage yakapinzwa mutsinga (iv) kuburikidza neBBB uye/kana BCSFB (Mufananidzo 1a-b). Takashandisa maoko maviri ekusarudza akazvimirira (maoko A naB) mumarounds matatu ekutanga ekusarudza muhunhu hwemuviri (Mufananidzo 1c). Takawedzera zvishoma nezvishoma kuoma kwekusarudza nekuderedza huwandu hwese hwephage hwakaunzwa mumarounds matatu ekutanga ekusarudza. Paround yechina yekuisa panning, takabatanidza sampuro kubva kumatavi A naB uye takaita sarudzo nhatu dzekuwedzera dzakazvimirira. Kuti tidzidze hunhu hweT7 phage particles muhunhu hwemuviri muhunhu hwemusana mumuenzaniso uyu, wild-type phage (PAGISRELVDKL master insert) yakapinzwa mumakonzo kuburikidza netsinga yemusana. Kudzoserwa kwemaphages kubva mu cerebrospinal fluid neropa panguva dzakasiyana kwakaratidza kuti maphages madiki eT7 icosahedral aive nechikamu chekubvisa chekukurumidza kubva muchikamu cheropa (Mufananidzo Wekuwedzera 3). Zvichibva pahuwandu hwema titers akapihwa uye huwandu hweropa remakonzo, takaverenga kuti inenge 1% wt. phage kubva pamushonga wakapihwa yakaonekwa muropa mushure memaminitsi gumi mushure mekubayiwa ne intravenous. Mushure mekudzikira uku kwekutanga, kubviswa kwe primary kunononoka kwakayerwa nehafu yehupenyu hwemaminitsi 27.7. Zvakakosha, ma phage mashoma chete akatorwa kubva muCSF compartment, zvichiratidza kuti pane mamiriro akaderera ekufamba kwe wild-type phage muCSF compartment (Supplementary Fig. 3). Paavhareji, ma titers 1 x 10-3% chete eT7 phage muropa uye 4 x 10-8% yema phage akatanga kuiswa akaonekwa mu cerebrospinal fluid munguva yese yekutora sampling (0-250 min). Zvinonyanya kukosha kuti hafu yehupenyu (25.7 min) ye wild-type phage mu cerebrospinal fluid yaive yakafanana neyakaonekwa muropa. Ruzivo urwu runoratidza kuti chipingamupinyi chinoparadzanisa CSF compartment kubva muropa chiripo mumakonzo ane CM-cannulated, zvichibvumira kusarudzwa kwemaraibhurari e phage muhupenyu hwemunhu kuti aone ma clones anotakurwa kubva muropa kuenda kuCSF compartment zviri nyore.
(a) Kugadzira nzira yekudzokorora cerebrospinal fluid (CSF) kubva mudziva guru. (b) Dhiagiramu inoratidza nzvimbo yemasero e central nervous system (CNS) barrier uye nzira yekusarudza inoshandiswa kuona ma peptides anoyambuka blood-brain barrier (BBB) ​​​​neblood-brain barrier. (c) Chati yekuongorora mafambiro emukati me phage display. Muchikamu chega chega chekusarudza, ma phages (zviziviso zvemhuka mukati memiseve) aibayiwa netsinga. Matavi maviri akazvimiririra (A, B) anochengetwa akasiyana kusvika pachikamu chechina chekusarudza. Pachikamu chekusarudza 3 ne4, phage clone yega yega yakatorwa kubva kuCSF yakarongwa nemaoko. (d) Kinetics ye phage yakabviswa muropa (madenderedzwa matsvuku) uye cerebrospinal fluid (matriangles egirini) panguva yekutanga yekusarudza mumakonzo maviri akaiswa mu cannulated mushure mekubayiwa ne intravenous T7 peptide library (2 x 1012 phages/mhuka). Makweya ebhuruu anoratidza avhareji yekutanga yehuwandu hwephage muropa, inoverengwa kubva pahuwandu hwephage yakabatwa, tichifunga nezvehuwandu hweropa rose. Makweya matema anoratidza nzvimbo yekusangana kwemutsara we y wakabva muhuwandu hwephage muropa. (e,f) Ratidza huwandu hwehuwandu uye kupararira kwemhando dzese dze tripeptide dzinopindana dzinowanikwa mupeptide. Huwandu hwemhando dzakawanikwa mukuverenga 1000 hunoratidzwa. Zvakakosha (p < 0.001) mhando dzakapfuma dzakanyorwa nemadotsi matsvuku. (e) Kuenzanisa kupatsanurana kunoenzanisa huwandu hwe tripeptide motif yeraibhurari yakabatwa neropa nephage inobva muropa kubva kumhuka #1.1 na #1.2. (f) Kuenzanisa kupatsanurana kunoenzanisa huwandu hwemhando dzephage tripeptide motifs #1.1 na #1.2 dzakaparadzaniswa muropa nemumvura yemusana. (g, h) Sequence ID inomiririra phage yakapfuma muropa (g) zvichienzaniswa nemaraibhurari akabatwa nejekiseni uye phage yakapfuma muCSF (h) zvichienzaniswa neropa mushure mekusarudzwa kwe in vivo mumhuka dzese dziri mbiri. Saizi yekodhi ine bhii rimwe chete inoratidza kuti amino acid iyoyo inowanikwa kangani panzvimbo iyoyo. Girini = polar, purple = neutral, bhuruu = basic, tsvuku = acidic uye dema = hydrophobic amino acids. Mufananidzo 1a, b wakagadzirwa uye wakagadzirwa naEduard Urich.
Takaisa raibhurari yephage peptide mumakonzo maviri eCM instrument (clades A naB) uye yakapatsanurwa phage kubva mu cerebrospinal fluid neropa (Mufananidzo 1d). Kubviswa kwekutanga nekukurumidza kweraibhurari kwaive kushoma kana tichienzanisa ne wild-type phage. Avhareji yehupenyu hweraibhurari yakaisirwa mumhuka dzese yaive maminetsi makumi maviri nemana.8 muropa, zvakafanana ne wild-type phage, uye maminetsi makumi matatu nemasere neshanu muCSF. Samples dzeropa ne cerebrospinal fluid phage kubva kumhuka yega yega dzakaiswa HTS uye mapeptide ese akaonekwa akaongororwa kuti aone kana paine short tripeptide motif. Tripeptide motifs dzakasarudzwa nekuti dzinopa hwaro hushoma hwekuumbwa kwechimiro uye kudyidzana kwepeptide-protein14,15. Takawana hukama hwakanaka mukugoverwa kwe motifs pakati pe injected phage library nema clones akatorwa kubva muropa remhuka dzese (Mufananidzo 1e). Data rinoratidza kuti kuumbwa kweraibhurari kwakapfuma zvishoma muropa. Amino acid frequency uye consensus sequences zvakaongororwazve panzvimbo yega yega uchishandisa adaptation yeWeblogo16 software. Zvinofadza kuti takawana kuwedzera kwakasimba mumasero eglycine muropa (Mufananidzo 1g). Pakaenzaniswa ropa nemaclone akasarudzwa kubva kuCSF, kusarudzwa kwakasimba uye kusasarudzwa kwemotifs kwakaonekwa (Mufananidzo 1f), uye mamwe maamino acids aivepo zvakanyanya panzvimbo dzakatarwa mu 12-members (Mufananidzo 1h). Zvinonyanya kukosha, mhuka imwe neimwe yakasiyana zvakanyanya mu cerebrospinal fluid, nepo kuwedzera kweglycine muropa kwakaonekwa mumhuka dzese dziri mbiri (Mufananidzo 4a-j). Mushure mekusefa kwakasimba kwedata re sequence mu cerebrospinal fluid yemhuka #1.1 ne #1.2, huwandu hwe964 ne420 unique 12-mer peptides dzakawanikwa (Mufananidzo 1d-e). Ma clone ephage akaparadzaniswa akawedzerwa uye akaiswa ku round yechipiri ye in vivo selection. Phage yakatorwa kubva kuchikamu chechipiri chekusarudza yakaiswa HTS mumhuka yega yega uye mapeptide ese akaonekwa akashandiswa senzira yekuongorora motif kuti aongorore kuitika kwe tripeptide motifs (Mufananidzo 2a, b, ef). Zvichienzaniswa nechikamu chekutanga chephage yakatorwa kubva kuCSF, takaona kusarudzwa kwakawedzerwa uye kubviswa kwe motifs dzakawanda muCSF mumapazi A neB (Mufananidzo 2). Algorithm yekuziva network yakashandiswa kuona kana dzaimiririra mapatani akasiyana ekutevera kwakafanana. Kufanana kwakajeka kwakaonekwa pakati pezvikamu gumi nezviviri zvakatorwa neCSF mune imwe nzira clade A (Mufananidzo 2c, d) uye clade B (Mufananidzo 2g, h). Kuongorora kwakabatanidzwa mubazi rega rega kwakaratidza ma profiles akasiyana ekusarudza ma peptide gumi nemaviri (Mufananidzo 5c, d) uye kuwedzera kweCSF/blood titer ratio nekufamba kwenguva kwema clones akaunganidzwa mushure mechikamu chechipiri chekusarudza zvichienzaniswa nechikamu chekutanga chekusarudza (Mufananidzo 5e).).
Kuwedzerwa kwemamotif nemapeptides mu cerebrospinal fluid nekutenderera kaviri kwakatevedzana kwesarudzo yekuratidza functional phage mu vivo.
Ma phage ese e cerebrospinal fluid akatorwa kubva kudenderedzwa rekutanga remhuka yega yega (mhuka #1.1 na #1.2) akaunganidzwa, akawedzerwa, akatevedzana neHT uye akapinzwazve pamwe chete (2 x 1010 phages/mhuka) Makonzo maviri eSM akacheneswa (#1.1 → #). 2.1 na 2.2, 1.2 → 2.3 na 2.4). (a,b,e,f) Ma scatterplots ekubatana achienzanisa frequency ye tripeptide motifs ye CSF-derived phages mudenderedzwa rekutanga nerechipiri rekusarudza. Relative frequency uye kugoverwa kwe motifs dzinomiririra tripeptides dzese dzinogona kupindirana dzinowanikwa muma peptides mumatanho ese ari maviri. Huwandu hwe motifs dzakawanikwa mukuverenga 1000 hunoratidzwa. Motifs dzakasarudzwa zvakanyanya (p < 0.001) kana kusabatanidzwa mune imwe yemaraibhurari akaenzaniswa dzakaratidzwa nemadotsi matsvuku. (c, d, g, h) Kuratidzwa kwechiratidzo chemutsara wezvikamu zvese zvemaamino acid gumi nemaviri ane CSF zvakapfuma zvichibva pazvikamu zvechipiri ne1 zvekusarudza kwemuviri. Saizi yekodhi ine bhii rimwe chete inoratidza kuti amino acid iyoyo inoitika kangani panzvimbo iyoyo. Kuti imiririre logo, kuwanda kwezvikamu zveCSF zvakatorwa kubva kumhuka imwe neimwe pakati pezvikamu zviviri zvekusarudza kunoenzaniswa uye zvikamu zvakapfuma muchikamu chechipiri zvinoratidzwa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 uye (h) #1.2–#2.4. Maamino acid akapfuma zvakanyanya panzvimbo yakatarwa mumhuka nhamba 2.1 uye nhamba 2.2 kana (g, h) mumhuka nhamba 2.3 uye nhamba 2.4 anoratidzwa neruvara. Girini = polar, purple = neutral, bhuruu = basic, tsvuku = acidic uye dema = hydrophobic amino acids.
Mushure mechikamu chechitatu chekusarudza, takaona 124 unique peptide sequences (#3.1 na #3.2) kubva ku332 CSF-reconstituted phage clones dzakaparadzaniswa nemhuka mbiri (Supplementary Fig. 6a). Sequence LGSVS (18.7%) yaive nechikamu chepamusoro chehukama, ichiteverwa ne wild-type inserts PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), uye SARGSWREIVSLS (2.2%). Muchikamu chekupedzisira chechina, takaunganidza matavi maviri akasarudzwa ega kubva kumhuka nhatu dzakasiyana (Fig. 1c). Pa925 sequenced phage clones dzakatorwa kubva kuCSF, muchikamu chechina takawana 64 unique peptide sequences (Supplementary Fig. 6b), pakati payo chikamu che wild-type phage chakaderera kusvika 0.8%. Ma clones eCSF akajairika muchikamu chechina aive LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) uye RLSSVDSDLSGC (3, 2%). %). Hurefu hwema peptides akasarudzwa hunokonzerwa nekuiswa/kubviswa kwe nucleotide kana ma codons ekumira asati asvika mu library primers pakushandisa ma codons akaora pakugadzira library yeNNK. Ma codons ekumira asati asvika anogadzira ma peptides mapfupi uye anosarudzwa nekuti ane motif yakanaka ye aa. Ma peptides marefu anogona kubva mukuiswa/kubviswa mu primers ema library e synthetic. Izvi zvinoisa stop codon yakagadzirwa kunze kwefuremu uye zvinoiverenga kusvika stop codon itsva yaonekwa pasi. Kazhinji, takaverenga zvinhu zvinovaka zvema rounds ese mana ekusarudza nekuenzanisa data rekupinda ne data re sample output. Pakuongororwa kwekutanga, takashandisa ma wild-type phage titers senzira isina kutsanangurwa. Zvinofadza kuti, kusarudzwa kwe negative phage kwaive kwakasimba kwazvo muCSF cycle yekutanga, asi kwete muropa (Mufananidzo 3a), izvo zvinogona kunge zvichikonzerwa nekusakwanisa kupararira kwenhengo zhinji dze peptide library muCSF compartment kana relative phages dzinowanzo chengetwa kana kubviswa muropa zviri nani kupfuura bacteriophages. Zvisinei, muchikamu chechipiri che panning, kusarudzwa kwakasimba kwephages muCSF kwakaonekwa muma clades ese ari maviri, zvichiratidza kuti chikamu chekare chakanga chakapfuma muphages dzinoratidza ma peptide anokurudzira CSF uptake (Mufananidzo 3a). Zvakare, pasina kuwedzerwa ropa kwakawanda. Zvakare muchikamu chechitatu nechechina, ma phage clones akapfuma zvakanyanya muCSF. Tichienzanisa frequency ye peptide sequence yega yega yakasiyana pakati pezvikamu zviviri zvekupedzisira zvekusarudza, takaona kuti zvikamu zvakapfuma zvakanyanya muchikamu chechina chekusarudza (Mufananidzo 3b). Mamotif etripeptide anosvika 931 akatorwa kubva muzvikamu zvese makumi matanhatu nemana zvepeptide zvakasiyana-siyana uchishandisa nzira dzese dzepeptide. Mamotif akanyanya kupfuma muchikamu chechina akaongororwa zvakanyanya kuti awane ruzivo rwakakura sei muzvikamu zvese zvichienzaniswa neraibhurari yakapinzwa (chidimbu: 10% ruzivo rwakakura) (Mufananidzo 6c). Maitiro ese ekusarudza akaratidza kuti zvikonzero zvakawanda zvakadzidzwa zvakapfuma muzvikamu zvese zvekare zvezvikamu zvese zvekusarudza. Zvisinei, zvimwe zvinhu (semuenzaniso SGL, VSG, LGS GSV) zvakanyanya kubva muchikamu cheA, nepo zvimwe (semuenzaniso FGW, RTN, WGF, NTR) zvakapfuma muchikamu cheAlternative B.
Kusimbiswa kwekutakurwa kweCSF kwemapeptides ane CSF-enriched phage-displayed uye biotinylated leader peptides akabatana ne streptavidin payloads.
(a) Zviyero zvekuwedzera zvakaverengerwa mumarounds ese mana (R1-R4) zvichibva pamatiter ephage (input = I) phage (PFU) uye matiter ephage eCSF akasarudzwa (output = O). Zvinhu zvekuwedzera mumarounds matatu ekupedzisira (R2-R4) zvakaverengerwa nekuenzanisa neround yapfuura neround yekutanga (R1) nedata rehuremu. Mabar akavhurika i cerebrospinal fluid, mabar ane shaded i plasma. (***p<0.001, zvichibva pa t-test yeMudzidzi). (b) Rondedzero yema peptide ephage akawandisa, akarongwa zvichienderana nehuwandu hwawo hwema phage ese akaunganidzwa muCSF mushure meround 4 yekusarudzwa. Ma clones matanhatu akajairika ephage anoratidzwa neruvara, nhamba uye zvinhu zvekuwedzera pakati peround 3 ne4 yekusarudzwa (insets). (c,d) Ma clones matanhatu ephage akanyanya kupfuma, empty phage uye parent phage peptide libraries kubva kuround 4 akaongororwa ega ega mu CSF sampling model. CSF nemasamples eropa zvakaunganidzwa panguva dzakatarwa. (c) Huwandu hwakaenzana hwema "candidate phage clones" matanhatu (2 x 1010 phages/mhuka), ma "empty phages/mhuka" (#1779) (2 x 1010 phages/mhuka) uye ma "stock phage peptide libraries" (2 x 1012 phages/mhuka) Jekiseni rinopihwa kanenge 3 CM kumhuka yakakamurwa yoga kuburikidza ne "tail vein". CSF pharmacokinetics ye "injected phage clone" yega yega uye "phage peptide library" nekufamba kwenguva inoratidzwa. (d) inoratidza avhareji yeCSF/blood ratio ye "phages/mL" dzese dzakadzorerwa mukati menguva yekutora sampu. (e) Ma "synthetic leader peptides" mana uye "scrambled control" imwe chete yakabatana ne biotin ku streptavidin kuburikidza neN-terminus yavo (tetramer display) ichiteverwa ne "injection" (tail vein iv, 10 mg streptavidin/kg). Makonzo matatu akaputirwa muchubhu (N = 3).). Samples dzeCSF dzakaunganidzwa panguva dzakatarwa uye huwandu hwe streptavidin hwakayerwa neCSF anti-streptavidin ELISA (nd = haina kuonekwa). (*p<0.05, **p<0.01, ***p<0.001, zvichibva pa ANOVA test). (f) Kuenzanisa amino acid sequence ye most richized phage peptide clone #2002 (yepepuru) nedzimwe saled phage peptide clones kubva ku 4th round yekusarudza. Zvidimbu zveamino acid zvakafanana uye zvakafanana zvine mavara.
Pama phage ese akapfuma muchikamu chechina (Mufananidzo 3b), ma clone matanhatu akasarudzwa kuti awedzere kuongororwa muCSF sampling model. Huwandu hwakaenzana hwema candidate phage matanhatu, empty phage (isina insert) uye prophage peptide libraries zvakapinzwa mumhuka nhatu dzeCM dzakaiswa mu cannulated, uye pharmacokinetics dzakaongororwa muCSF (Mufananidzo 3c) neropa (Supplementary Fig. 7). Ma phage clones ese akaedzwa akananga kuCSF compartment padanho rakakwirira ka10-1000 kupfuura re empty control phage (#1779). Semuenzaniso, ma clones #2020 na #2077 aive neCSF titers yakakwira ka1000 kupfuura control phage. Pharmacokinetic profile ye peptide yega yega yakasarudzwa yakasiyana, asi ese ane CSF homing ability yakakwira. Takaona kudzikira kunogara kuchiitika nekufamba kwenguva kwema clones #1903 na #2011, nepo kuma clones #2077, #2002 na #2009 kuwedzera mukati memaminitsi gumi ekutanga kungaratidza kutakurwa kuri kushanda asi kunofanirwa kuongororwa. Ma clones #2020, #2002, na #2077 akadzikama pamazinga akakwira, nepo huwandu hweCSF hwe clone #2009 hwakaderera zvishoma nezvishoma mushure mekuwedzera kwekutanga. Takazoenzanisa huwandu hwe CSF yega yega nehuwandu hwayo muropa (Mufananidzo 3d). Kubatana kweavhareji ye titer ye CSF yega yega nehuwandu hwayo muropa panguva dzese dzekutora sampuli kwakaratidza kuti vatatu vevatanhatu vakapfuma zvakanyanya mu CSF yeropa. Zvinofadza kuti clone #2077 yakaratidza kugadzikana kweropa kwakanyanya (Mufananidzo Wekuwedzera 7). Kuti tisimbise kuti ma peptides pachawo anokwanisa kutakura zvinhu zvisiri zve phage particles muCSF compartment, takagadzira ma peptide mana e leader anobva mu biotin paN-terminus apo ma peptides anonamira pa phage particle. Mapeptides ane biotinylated (nhamba 2002, 2009, 2020 na2077) akabatanidzwa ne streptavidin (SA) kuti awane mafomu akawanda akafanana ne phage geometry. Iyi format yakatibvumirawo kuyera SA exposure muropa nemu cerebrospinal fluid se protein peptides dzinotakura zvinhu. Zvakakosha, data re phage raigona kudzokororwa kana ma peptides ekugadzira akapihwa mu SA-conjugated format iyi (Mufananidzo 3e). Ma peptides akasunganidzwa aive ne exposure shoma yekutanga uye CSF clearance nekukurumidza nemazinga asingaonekwe mukati memaawa makumi mana nemasere. Kuti tinzwisise nzira dzekuendeswa kwema peptide phage clones aya munzvimbo yeCSF, takaongorora nzvimbo yema "phage peptide hits" ega ega tichishandisa immunohistochemistry (IHC) kuti tione zvakananga ma "phage particles" awa imwe chete mushure mekubayiwa muropa. Zvinonyanya kukosha kuti ma clones #2002, #2077, uye #2009 anogona kuonekwa nemavara akasimba mu brain capillaries, nepo control phage (#1779) uye clone #2020 zvisina kuonekwa (Mufananidzo Wekuwedzera 8). Izvi zvinoratidza kuti ma peptide aya anobatsira mumhedzisiro pauropi nekuyambuka BBB. Ongororo yakadzama inodiwa kuti kuyedze fungidziro iyi, sezvo nzira yeBSCFB inogonawo kubatanidzwa. Pakuenzanisa amino acid sequence ye most riched clone (#2002) nedzimwe peptide dzakasarudzwa, zvakaonekwa kuti mamwe acho ane amino acid extensions dzakafanana, izvo zvinogona kuratidza nzira yakafanana yekufambisa (Mufananidzo 3f).
Nekuda kwechimiro chayo cheplasma chakasiyana uye kuwedzera kukuru muCSF nekufamba kwenguva, phage display clone #2077 yakaongororwazve kwenguva yakareba kwemaawa makumi mana nemasere uye yakakwanisa kudzokorora kuwedzera nekukurumidza kweCSF kwakaonekwa pamwe chete nemazinga eSA anoramba aripo (Mufananidzo 4a). Nezvemamwe ma phage clones akaonekwa, #2077 yakasvibisa zvakanyanya capillaries yeuropi uye yakaratidza colocalization yakakosha ne capillary marker lectin kana yakaonekwa ne resolution yepamusoro uye pamwe kusvibisa munzvimbo yeparenchymal (Mufananidzo 4b). Kuti tiongorore kana mhedzisiro ye peptide-mediated pharmacological inogona kuwanikwa muCNS, takaita kuyedza umo biotinylated versions dze i) #2077 transit peptide uye ii) BACE1 inhibitor peptide dzakasanganiswa neSA pamatanho maviri akasiyana. Pamusanganiswa mumwe chete takashandisa BACE1 peptide inhibitor chete uye kune mumwe takashandisa 1:3 ratio yeBACE1 peptide inhibitor kune #2077 peptide. Mienzaniso yese miviri yakapihwa netsinga uye huwandu hwe beta-amyloid peptide 40 (Abeta40) muropa nemumuzongoza hwakayerwa nekufamba kwenguva. Abeta40 yakayerwa muCSF sezvo ichiratidza kudziviswa kweBACE1 muuropi. Sezvaitarisirwa, ma complexes ese ari maviri akadzikisa zvakanyanya huwandu hweAbeta40 muropa (Mufananidzo 4c, d). Zvisinei, sampuli chete dzine musanganiswa wepeptide nhamba 2077 uye inhibitor yeBACE1 peptide yakabatana neSA dzakakonzera kudzikira kukuru kweAbeta40 mu cerebrospinal fluid (Mufananidzo 4c). Data rinoratidza kuti peptide nhamba 2077 inokwanisa kutakura 60 kDa SA protein kuenda kuCNS uye inokonzerawo mhedzisiro yemishonga neSA-conjugated inhibitors yeBACE1 peptide.
(a) Kuiswa kweClonal (2 × 10 phages/mhuka) yeT7 phage inoratidza ma profiles enguva refu eCSF peptide #2077 (RLSSVDSDLSGC) uye isina jekiseni rekutonga (#1779) mumakonzo matatu ane CM-intubated. (b) Mufananidzo weConfocal microscopic we cortical microvessels inomiririra mumakonzo ane phage-injected (2 × 10 10 phages/mhuka) uchiratidza counterstain yepeptide #2077 nemidziyo yeropa (lectin). Aya ma phage clones akapihwa kumakonzo matatu uye akabvumirwa kutenderera kweawa imwe chete asati aiswa ropa. Uropi hwakakamurwa ndokuzoiswa mavara e antibodies ane polyclonal FITC-labeled antibodies kurwisa T7 phage capsid. Maminitsi gumi vasati vaiswa ropa uye kugadziriswa kwakatevera, DyLight594-labeled lectin yakapihwa nemukati. Mifananidzo inopenya inoratidza kutsvuka kwe lectin (tsvuku) kwerutivi rwe luminal rwe microvessels ne phages (girini) mu lumen ye capillaries ne perivascular brain tissue. Chikero che scale bar chinoenderana ne 10 µm. (c, d) Biotinylated BACE1 inhibitory peptide yega kana pamwe chete ne biotinylated transit peptide #2077 yakabatanidzwa ne streptavidin zvichiteverwa ne injection ye intravenous ye cannulated CM rats nhatu (10 mg streptavidin/kg). Kuderedzwa kwe BACE1 peptide inhibitor-mediated mu Aβ40 kwakayerwa ne Aβ1-40 ELISA muropa (tsvuku) uye cerebrospinal fluid (orenji) panguva dzakatarwa. Kuti zvive pachena, mutsetse une dotted unodhirowa pagirafu pachiyero che 100%. (c) Kuderedzwa kweAβ40 muropa (matriangle matsvuku) uye cerebrospinal fluid (matriangle eorenji) mumakonzo akarapwa nestreptavidin yakabatana netransit peptide #2077 uye BACE1 inhibitory peptide muchikamu che3:1. (d) Kuderedzwa kweperesenti muropa Aβ40 (madenderedzwa matsvuku) uye cerebrospinal fluid (madenderedzwa eorenji) emakonzo akarapwa nestreptavidin zvakabatana neBACE1 inhibitory peptide chete. Kuwanda kweAβ mukutonga kwaive 420 pg/ml (standard deviation = 101 pg/ml).
Kuratidzwa kwePhage kwave kushandiswa zvinobudirira munzvimbo dzakasiyana-siyana dzeongororo yezvekurapa17. Nzira iyi yakashandiswa muzvidzidzo zvekusiyana kweropa muuropi18,19 pamwe nezvidzidzo zvinotarisa tsinga dzeropa20,21,22,23,24,25,26. Muchidzidzo ichi, takawedzera kushandiswa kwenzira iyi yekusarudza kwete chete pakuzivikanwa zvakananga kwemapeptide anotarisa tsinga dzeropa, asiwo pakuwanikwa kwevanhu vane hunhu hwekufambisa hunoshanda kuti vayambuke chipingamupinyi cheropa-uropi. Iye zvino tinotsanangura kugadzirwa kwenzira yekusarudza muuropi mumakonzo ane CM intubated uye tinoratidza kugona kwayo kuziva mapeptide ane hunhu hweCSF homing. Tichishandisa T7 phage inoratidza raibhurari yemapeptides e12-mer random, takakwanisa kuratidza kuti T7 phage diki zvakakwana (inenge 60 nm mudhayamita)10 kuti igadziriswe kune chipingamupinyi cheropa-uropi, nokudaro ichiyambuka zvakananga chipingamupinyi cheropa-uropi kana choroid plexus. Takaona kuti kukohwa CSF kubva kumakonzo eCM akachekwa kwaive nzira yekuyedza inoshanda zvakanaka, uye kuti phage yakabviswa haina kungobatana neropa chete asiwo yaishanda senzira yekufambisa kuburikidza nechivharo cheropa-uropi. Uyezve, nekuunganidza ropa panguva imwe chete uye kushandisa HTS kuCSF uye maphage anobva muropa, takasimbisa kuti sarudzo yedu yeCSF haina kukanganiswa nekuwedzerwa kweropa kana kugona kwekuwedzera pakati pematanho ekusarudza. Zvisinei, chikamu cheropa chikamu chemaitiro ekusarudza, sezvo maphage anokwanisa kusvika kuchikamu cheCSF anofanirwa kurarama uye kutenderera muropa kwenguva yakareba zvakakwana kuti azvipfumise muuropi. Kuti tiwane ruzivo rwekutevera rwakavimbika kubva kudata reHTS raw, takaisa mafirita akagadziriswa kune zvikanganiso zve sequencing specific platform mu analysis workflow. Nekuisa kinetic parameters munzira yekuyedza, takasimbisa pharmacokinetics ye wild-type T7 phages (t½ ~ 28 min) muropa24, 27, 28 uye takaonawo hafu yehupenyu hwavo mu cerebrospinal fluid (t½ ~ 26 min) paminiti). Pasinei nema pharmacokinetic profiles akafanana muropa neCSF, 0.001% chete yehuwandu hwephage muropa ndiyo yaigona kuonekwa muCSF, zvichiratidza kufamba kushoma kwe wild-type T7 phage pamusoro peblood-brain barrier. Basa iri rinoratidza kukosha kwekutanga kwekusarudza pakushandisa nzira dze in vivo panning, kunyanya kune ma phage systems anobviswa nekukurumidza kubva mukutenderera kweropa, sezvo ma clones mashoma anokwanisa kusvika kuCNS compartment. Saka, muround yekutanga, kudzikira kwekusiyana kweraibhurari kwaive kukuru kwazvo, sezvo nhamba shoma yema clones chete yakazounganidzwa mumuenzaniso uyu wakaoma weCSF. Iyi nzira ye in vivo panning yaisanganisira matanho akati wandei ekusarudza akadai sekuunganidza active muCSF compartment, kupona kwema clone muropa compartment, uye kubviswa nekukurumidza kwema T7 phage clones kubva muropa mukati memaminitsi gumi ekutanga (Mufananidzo 1d uye Supplementary Fig. 4M). ). Saka, mushure meround yekutanga, ma phage clones akasiyana akaonekwa muCSF, kunyangwe dziva rekutanga rakafanana rakashandiswa kumhuka imwe neimwe. Izvi zvinoratidza kuti matanho akawanda ekusarudza maraibhurari ekutsvaga mabhuku ane nhengo dzakawanda dzemaraibhurari anokonzera kudzikira kukuru kwekusiyana-siyana. Nokudaro, zviitiko zvisina kurongeka zvichave chikamu chakakosha chemaitiro ekutanga ekusarudza, zvichikanganisa zvakanyanya mhedzisiro yacho. Zvingangodaro kuti akawanda ema clones muraibhurari yekutanga aive netsika yakafanana yekuwedzera CSF. Zvisinei, kunyangwe mumamiriro ezvinhu akafanana ekuyedza, mhedzisiro yekusarudza inogona kusiyana nekuda kwehuwandu hushoma hwe clone imwe neimwe muboka rekutanga.
Mamotifomu akapfuma muCSF akasiyana neeari muropa. Zvinofadza kuti, takaona shanduko yekutanga kuenda kumapeptide ane glycine-rich muropa remhuka imwe neimwe. (Mufananidzo 1g, Supplementary Figs. 4e, 4f). Mapeptide ane phage ane glycine anogona kunge akasimba uye asingakwanise kubviswa muropa. Zvisinei, aya mapeptide ane glycine-rich haana kuonekwa mumasampuli e cerebrospinal fluid, zvichiratidza kuti maraibhurari akarongwa akapfuura nematanho maviri akasiyana ekusarudza: rimwe muropa uye rimwe rakabvumirwa kuungana mu cerebrospinal fluid. Ma clones ane CSF-rich anobva muchikamu chechina chekusarudza akaedzwa zvakanyanya. Anenge ma clones ese akaedzwa ega ega akasimbiswa kuti akapfuma muCSF zvichienzaniswa ne blank control phage. Imwe peptide hit (#2077) yakaongororwa zvakadzama. Yakaratidza hafu yehupenyu hweplasma yakareba zvichienzaniswa nedzimwe hits (Mufananidzo 3d ne Supplementary Mufananidzo 7), uye zvinonakidza, iyi peptide yaive ne cysteine ​​​​residue paC-terminus. Munguva pfupi yapfuura zvakaratidzwa kuti kuwedzerwa kwe cysteine ​​​​kune ma peptides kunogona kuvandudza hunhu hwavo hwe pharmacokinetic nekusunga ku albumin 29. Izvi hazvizivikanwe parizvino nezve peptide #2077 uye zvinoda kuongororwa kwakawanda. Mamwe ma peptides akaratidza kuvimba kwe valence mu CSF enrichment (data harina kuratidzwa), izvo zvingave zvine chekuita ne geometry yepamusoro yakaratidzwa ye T7 capsid. Sisitimu ye T7 yatakashandisa yakaratidza makopi 5-15 e peptide yega yega pa phage particle. IHC yakaitwa pama candidate lead phage clones akaiswa mukati me intravenously mu cerebral cortex yemakonzo (Supplementary Fig. 8). Data rakaratidza kuti ma clones matatu (Nha. 2002, Nha. 2009 uye Nha. 2077) akabatana ne BBB. Zvinoramba zvichionekwa kana kusangana uku kwe BBB kuchikonzera kuunganidzwa kwe CSF kana kufamba kwema clones aya zvakananga ku BCSFB. Zvakakosha, tinoratidza kuti ma peptides akasarudzwa anochengetedza CSF transport capacity yawo kana akagadzirwa uye akabatana ne protein cargo. Kusunga mapeptide eN-terminal biotinylated kuSA kunodzokorora mhedzisiro inowanikwa nema clones avo ephage muropa uye cerebrospinal fluid (Mufananidzo 3e). Pakupedzisira, tinoratidza kuti lead peptide #2077 inokwanisa kukurudzira kushanda kweuropi kwe biotinylated peptide inhibitor yeBACE1 yakabatana neSA, zvichikonzera mhedzisiro ye pharmacodynamic muCNS nekuderedza zvakanyanya mazinga eAbeta40 muCSF (Mufananidzo 4). Hatina kukwanisa kuziva chero homologues mu database nekuita search ye peptide sequence homology ye hits dzese. Zvakakosha kuziva kuti saizi ye library yeT7 inenge 109, nepo saizi ye theoretical library ye 12-mers iri 4 x 1015. Saka, takangosarudza chidimbu chidiki che diversity space ye 12-mer peptide library, zvinogona kureva kuti ma peptides akagadziridzwa anogona kuzivikanwa nekuongorora advanced sequence space ye hits idzi dzakaonekwa. Mukufungidzira, chimwe chezvikonzero nei tisina kuwana chero hukama hwechisikigo hwemapeptide aya chingave kubviswa kwesarudzo panguva yekushanduka-shanduka kudzivirira kupinda kusingadzoreki kwemamwe mapeptide muuropi.
Zvichitorwa pamwe chete, zvatakawana zvinopa hwaro hwebasa remangwana rekuziva nekuratidza masisitimu ekufambisa echivharo chetsinga dzeropa muuropi zvakadzama. Nzira huru yekushandisa iyi nzira yakavakirwa panzira yekusarudza inoshanda iyo isingangoone chete ma clones ane hunhu hwekubatanidza tsinga dzeropa muuropi, asiwo inosanganisira danho rakakosha umo ma clones anobudirira ane basa remukati rekuyambuka zvipingamupinyi zvebhayoloji muuropi kuenda kuCNS compartment. Kujekesa mashandiro ekufambisa ma peptides aya uye zvavanoda pakusunga kune microvasculature chaiyo kunzvimbo yeuropi. Izvi zvinogona kutungamira mukuwanikwa kwenzira itsva dzekutakura BBB nema receptors. Tinotarisira kuti ma peptides akaonekwa anogona kubatana zvakananga kuma cerebrovascular receptors kana kuma ligands anotenderera anotakurwa kuburikidza neBBB kana BCSFB. Ma peptide vectors ane CSF transport activity akawanikwa mubasa iri achaongororwa zvakanyanya. Pari zvino tiri kuferefeta brain specificity yema peptides aya kuti akwanise kuyambuka BBB uye/kana BCSFB. Mapeptide matsva aya achava maturusi akakosha kwazvo pakuwana mareceptor matsva kana nzira uye pakugadzira mapuratifomu matsva anoshanda zvakanyanya ekutumira macromolecules, akadai se biologics, kuuropi.
Kuisa ganda guru reCisna (CM) uchishandisa nzira yakatsanangurwa kare. Makonzo eWistar ane anesthesia (200-350 g) akaiswa pamudziyo we stereotaxic uye kucheka kwepakati kwakaitwa pamusoro peganda remusoro rakachekwa uye rakagadzirwa zvisina utachiona kuti rionekwe. Boora maburi maviri munzvimbo yesash yepamusoro uye sunga masikuru ekugadzirisa mumaburi. Rimwe gomba rakaboorwa muchikamu cheoccipital lateral kuti ritungamirirwe nesimbi isina tsvina muCM. Isa simende yemazino yakatenderedza cannula uye simbisa nesikuruu. Mushure mekupora nekusimbisa simende, ronda reganda rakavharwa ne 4/0 supramid suture. Kuiswa kwe cannula kwakakodzera kunosimbiswa nekubuda kwemvura yecerebrospinal (CSF) pakarepo. Bvisa gonzo kubva pamudziyo we stereotaxic, gamuchira rubatsiro rwakakodzera mushure mekuvhiyiwa uye kurapa marwadzo, uye rega ripore kweinenge vhiki imwe kusvika zviratidzo zveropa zvaonekwa mu cerebrospinal fluid. Makonzo eWistar (Crl:WI/Han) akawanikwa kubva kuCharles River (France). Makonzo ese akachengetwa pasi pemamiriro ezvinhu asina utachiona. Kuedza kwese kwemhuka kwakatenderwa neVeterinary Office yeGuta reBasel, Switzerland, uye kwakaitwa zvichienderana neAnimal License Nhamba 2474 (Kuongororwa kweActive Brain Transport neKuyera Mazinga eVanorapwa muCerebrospinal Fluid neBrain yeRat).
Chengeta gonzo zvinyoro nyoro uine CM cannula muruoko. Bvisa Datura kubva mu cannula wounganidza 10 µl ye cerebrospinal fluid inoyerera yoga. Sezvo patency ye cannula yakazopedzisira yakanganiswa, sampuro dze cerebrospinal fluid dzakajeka chete dzisina humbowo hwekusvibiswa kweropa kana kuchinja kweruvara dzakaiswa muchidzidzo ichi. Panguva imwe chete, inenge 10-20 μl yeropa yakatorwa kubva pakachekwa kadiki pamucheto wemuswe ikaiswa mumachubhu ane heparin (Sigma-Aldrich). CSF neropa zvakatorwa panguva dzakasiyana mushure mekubayiwa T7 phage netsinga. Inenge 5-10 μl yemvura yakaraswa isati yatorwa sampuro yega yega yeCSF, iyo inoenderana nehuwandu hwekufa kwecatheter.
Maraibhurari akagadzirwa achishandisa T7Select 10-3b vector sezvakatsanangurwa mubhuku reT7Select system (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). Muchidimbu, chinhu cheDNA chine 12-mer chakagadziriswa nenzira inotevera:
Kodhoni yeNNK yakashandiswa kudzivirira macodon maviri anomira uye amino acid yakawanda mu insert. N inhamba ye equimolar yakasanganiswa nemaoko ye nucleotide yega yega, uye K inhamba ye equimolar yakasanganiswa nemaoko ye adenine ne cytosine nucleotides. Nzvimbo dzakasungwa chete dzakachinjwa kuita DNA mbiri dzakasungwa nekuwedzera kusanganiswa ne dNTP (Novagen) ne Klenow enzyme (New England Biolabs) mu Klenow buffer (New England Biolabs) kwemaawa matatu pa37°C. Mushure me reaction, DNA mbiri dzakasungwa dzakadzorerwa ne EtOH precipitation. DNA yakabuda yakagayiwa nema restriction enzymes EcoRI ne HindIII (zvose kubva kuRoche). Insert yakacheneswa uye yakacheneswa (QIAquick, Qiagen) (T4 ligase, New England Biolabs) yakazobatanidzwa mu-frame kuita pre-cleaved T7 vector mushure me amino acid 348 ye 10B capsid gene. Ligation reactions dzakaiswa pa16°C kwemaawa gumi nemasere isati yaiswa mu in vitro packaging. Kurongedza kwePhage mu vitro kwakaitwa zvichienderana nemirayiridzo yakapihwa neT7Select 10-3b cloning kit (Novagen) uye mhinduro yekurongedza yakawedzerwa kamwe chete kuti iore uchishandisa Escherichia coli (BLT5615, Novagen). MaLysates akaiswa mukati me centrifuge, akagadziriswa uye akaomeswa nechando pa -80° C. se stock solution ye glycerol.
Kuwedzerwa kwePCR zvakananga kwenzvimbo dzinoshanduka dzephage kwakasimudzirwa mumuto kana mundiro uchishandisa maprimer e454/Roche-amplicon fusion. Iyo forward fusion primer ine ma sequences ari pedyo ne variable region (NNK) 12 (template-specific), GS FLX Titanium Adapter A, uye four-base library key sequence (TCAG) (Supplementary Picture 1a):
Iyo reverse fusion primer inewo biotin yakabatana ne capture beads uye GS FLX Titanium Adapter B inodiwa pakuwedzera clonal panguva ye emulsion PCR:
Ma amplicons akazoitwa 454/Roche pyrosequencing zvichienderana ne454 GS-FLX Titanium protocol. Pakushandisa Sanger sequencing (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage DNA yakawedzerwa nePCR uye yakarongwa nema primer pairs anotevera:
Zvishandiso zvakaiswa kubva mumaplaque ega ega zvakaiswa PCR amplification uchishandisa Roche Fast Start DNA Polymerase Kit (zvichienderana nemirairo yemugadziri). Ita hot start (10 min pa95 °C) uye boost cycles makumi matatu nemashanu (50 s pa95 °C, 1 min pa50 °C, uye 1 min pa72 °C).
Madhaka emumaraibhurari, madhaka emhando yewild-type, madhaka akanunurwa kubva kuCSF neropa, kana ma clones ega ega akawedzerwa muEscherichia coli BL5615 mumuto weTB (Sigma Aldrich) kana mumidziyo ye500 cm2 (Thermo Scientific) kwemaawa mana pa37°C. Madhaka akabviswa mumaplate nekusuka maplate neTris-EDTA buffer (Fluka Analytical) kana nekuunganidza ma plaque ne sterile pipette tips. Madhaka akabviswa kubva ku culture supernatant kana extraction buffer ne one round of polyethylene glycol (PEG 8000) precipitation (Promega) uye akaiswazve muTris-EDTA buffer.
Mushonga we amplified phage wakabviswa endotoxin ka2-3 uchishandisa endotoxin removal beads (Miltenyi Biotec) usati wabayiwa ne intravenous (IV) (500 μl/animal). Mumutambo wekutanga, 2×1012 phages dzakaiswa; mumutsara wechipiri, 2×1010 phages; mumutsara wechitatu newechina, 2×109 phages pamhuka imwe neimwe. Hunhu hwe phage muCSF nemasampuli eropa akaunganidzwa panguva yakatarwa hwakaongororwa nekuverengwa kweplaque zvichienderana nemirairo yemugadziri (T7Select system manual). Kusarudzwa kwe phage kwakaitwa nekuiswa mutsinga yemaraibhurari akacheneswa mutsinga yemuswe kana nekuiswazve phage yakatorwa kubva kuCSF kubva mumutsara wekare, uye kukohwa kwakatevera kwakaitwa pamaminitsi gumi, maminetsi makumi matatu, maminetsi makumi matanhatu, maminetsi makumi mapfumbamwe, maminetsi zana nemakumi maviri, maminetsi zana nemakumi masere, uye maminetsi mazana maviri nemakumi mana zvichiteerana CSF nemasampuli eropa. Mapoinzi mana e "in vivo panning" akaitwa umo matavi maviri akasarudzwa akachengetwa zvakasiyana uye akaongororwa panguva yematanho matatu ekutanga ekusarudza. Ma "phage inserts" ese akatorwa kubva kuCSF kubva kumatanho maviri ekutanga ekusarudza akaiswa pa "454/Roche pyrosequencing", nepo ma "clones" ese akatorwa kubva kuCSF kubva kumatanho maviri ekupedzisira ekusarudza akarongwa nemaoko. Ma "blood phages" ese kubva ku "first round" ekusarudza akaiswawo pa "454/Roche pyrosequencing". Pakubaiwa ma "phage clones", ma "phages" akasarudzwa akawedzerwa muE. coli (BL5615) pamaplates e500 cm2 pa37°C kwemaawa mana. Ma "clones" akasarudzwa ega ega uye akarongwa nemaoko akapararira munzvimbo yeTB. Mushure mekubviswa kwe "phage", kucheneswa uye kubviswa kwe endotoxin (sezvakatsanangurwa pamusoro), ma "phages" e2×1010/mhuka mu300 μl akaiswa mutsinga imwe chete yemuswe.
Kugadzirisa data re sequence ne qualitative filtering. Data re Raw 454/Roche rakashandurwa kubva pa binary standard stream map format (sff) kuenda pa Pearson human reable format (fasta) uchishandisa software ye vendor. Kuenderera mberi nekugadzirisa nucleotide sequence kwakaitwa uchishandisa proprietary C programs and scripts (unreleased software package) sezvakatsanangurwa pazasi. Kuongororwa kwe primary data kunosanganisira maitiro akaomarara ekusefa akawanda. Kuti usefa ma reads asina 12mer insert DNA sequence inoshanda, ma reads akarongwa sequentially kune start label (GTGATGTCGGGGATCCGAATTCT), stop label (TAAGCTTGCGGGCGCACTCGAGTA) uye background insert (CCCTGCAGGGATATCCCGGGAGCTCGTCGAC) uchishandisa global Needleman-Wunsch test. alignment inobvumira kusvika ku2 insistencies pa alignment31. Nokudaro, ma reads asina start and stop tags uye ma reads ane background inserts, kureva, alignments dzinopfuura nhamba inobvumirwa ye mismatches, dzakabviswa mu library. Kana zviri zvekuverengwa kwasara, N-mer DNA sequence inotangira pachiratidzo chekutanga ichipera isati yabviswa kubva pachikamu chekutanga chekuverenga uye yozogadziriswazve (pano inonzi "insert"). Mushure mekushandurwa kwechinhu chakaiswa, chikamu chinotevera chekodhi yekutanga yekumira pamucheto we5′ weprimer chinobviswa kubva pachinhu chakaiswa. Pamusoro pezvo, manucleotide anotungamira kumacodon asina kukwana pamucheto we3′ weprimer akabviswawo. Kuti zvisasanganiswe zvinhu zvakaiswa zvine background sequences chete, zvinhu zvakaiswa zvinoshandurwa zvinotanga neamino acid pattern "PAG" zvakabviswawo. Mapeptide ane urefu hwepost-translational husingasviki 3 amino acids akabviswa muraibhurari. Pakupedzisira, bvisai redundancy mu insert pool uye sarudza frequency yechinhu chimwe nechimwe chakaiswa. Mhedzisiro yekuongorora uku yaisanganisira runyorwa rwe nucleotide sequences (inserts) uye ma frequency avo (read) (Supplementary Figures 1c na 2).
Kuiswa kweDNA muboka reN-mer nekufanana kwezvikamu: Kuti ubvise zvikanganiso zve454/Roche-specific sequencing errors (senge matambudziko ekuwedzera kwehomopolymer sequencing) uye kubvisa zvidimbu zvisinganyanyi kukosha, kuiswa kweN-mer DNA sequence inserts (inserts) kwakamboseferwa kunorongwa nekuisa kwakafanana. (kusvika kumabhesi maviri asingawirirane anobvumirwa) uchishandisa algorithm iterative inotsanangurwa seinotevera: kuiswa kunorongwa kutanga nefrequency yavo (yakakwirira kusvika yakaderera), uye kana yakafanana, nesecond sort yavo nehurefu (yakareba kusvika pfupi) ). Saka, kuiswa kwakawanda uye kwenguva refu kunotsanangura "boka" rekutanga. Frequency yeboka inoiswa ku key frequency. Zvadaro, kuiswa kwega kwega kwasara mune list yakarongwa kwakaedzwa kuwedzerwa kuboka ne pairing Needleman-Wunsch alignment. Kana nhamba ye mismatches, insertions, kana deletions mu alignment isingapfuure threshold ye2, kuiswa kunowedzerwa kuboka, uye frequency yeboka rese inowedzerwa nekuti kuiswa kwakawedzerwa kangani. Kuiswa kwakawedzerwa kuboka kunoiswa sekushandiswa uye hakubviswi mukugadzirisa mberi. Kana kutevedzana kwezvikamu hakugone kuwedzerwa kuboka ragara riripo, kutevedzana kwezvikamu kunoshandiswa kugadzira boka idzva rine frequency yakakodzera yezvikamu uye kwakanyorwa sekushandiswa. Kudzokorora kunopera kana kutevedzana kwezvikamu kwega kwega kwakashandiswa kuumba boka idzva kana kuti kunogona kuiswa muboka ragara riripo. Pamusoro pezvose, zvikamu zvezvikamu zvine nucleotides zvinozopedzisira zvashandurwa kuita peptide sequences (maraibhurari epeptide). Mugumisiro wekuongorora uku i seti yezvikamu uye ma frequency anoenderana nazvo anoumba nhamba yekuverenga kwakatevedzana (Supplementary Fig. 2).
Kugadzirwa kweMotif: Zvichibva pane runyorwa rwemapeptide akasiyana, raibhurari yakagadzirwa ine mapatani ese eamino acid (aa) anogona kuwanikwa sezvakaratidzwa pazasi. Patani imwe neimwe inobvira yehurefu 3 yakatorwa kubva mupeptide uye patani yayo yakapesana yakawedzerwa pamwe chete neraibhurari yakafanana yemotif ine mapatani ese (tripeptides). Maraibhurari emotif dzinodzokororwa zvakanyanya akarongwa uye kudzokorora kwakabviswa. Zvadaro, patripeptide yega yega muraibhurari yemotif, takatarisa kuti iripo here muraibhurari tichishandisa maturusi ekuverenga. Muchiitiko ichi, frequency yepeptide ine found motif tripeptide inowedzerwa uye inopihwa kune motif muraibhurari yemotif ("nhamba yemotifs"). Mhedzisiro yekugadzirwa kwemotif imhando mbiri ine zviitiko zvese zve tripeptides (motifs) uye kukosha kwavo, inova nhamba yekuverenga kuteedzana kunoguma nemotif inoenderana kana kuverenga kwacho kwasefetwa, kurongwa, uye kushandurwa. Metrics sezvakatsanangurwa zvakadzama pamusoro apa.
Kugadzirisa huwandu hwemamotifs nema scatterplots anoenderana nawo: Huwandu hwemamotifs emuenzaniso wega wega hwakagadziriswa uchishandisa
apo ni inhamba yekuverenga ine musoro wenyaya i. Saka, vi inomiririra huwandu hwekuverenga (kana mapeptide) ane motif i mumuenzaniso. P-values ​​​​yenhamba isina kurongeka ye motifs yakaverengerwa uchishandisa bvunzo chaiyo yaFisher. Nezve correlograms yenhamba yezvikonzero, hukama hwaSpearman hwakaverengerwa uchishandisa nhamba yakarongeka yezvikonzero naR.
Kuti tifungidzire zviri mukati memaamino acids panzvimbo yega yega muraibhurari yepeptide, mawebhu logogram 32, 33 (http://weblogo.threeplusone.com) akagadzirwa. Kutanga, zviri mukati memaamino acids panzvimbo yega yega ye12-mer peptide zvinochengetwa mu20×12 matrix. Zvadaro, seti yemapeptide 1000 ane zviri mukati meamino acid zvakafanana panzvimbo yega yega inogadzirwa mufasta-sequence format uye inopihwa sekupinda muwebhu-logo 3, iyo inogadzira mufananidzo wezviri mukati meamino acid panzvimbo yega yega. yeraibhurari yepeptide yakapihwa. Kuti tifungidzire datasets dzakawanda, mamepu ekupisa akagadzirwa uchishandisa chishandiso chakagadzirwa mukati muR (biosHeatmap, iyo isati yaburitswa R package). Madendrogram anoratidzwa mumamepu ekupisa akaverengerwa uchishandisa nzira yeWard's hierarchical clustering neEuclidean distance metric. Kuti uongorore data rekuongorora motif, maP values ​​​​ekuongororwa kusina kurongeka akaverengerwa uchishandisa bvunzo chaiyo yaFisher. MaP-values ​​​​emamwe madata akaverengerwa muR uchishandisa bvunzo yeT-test yeVadzidzi kana ANOVA.
Ma phage clones akasarudzwa uye ma phage asina kuiswa akaiswa mukati meropa kuburikidza nemutsinga wemuswe (2×1010 phages/mhuka mu300 μl PBS). Maminitsi gumi vasati vaisa mvura mumuviri uye vaisa mvura mushure meizvozvo, mhuka idzodzo dzakaiswa 100 μl yeDyLight594-labeled lectin (Vector Laboratories Inc., DL-1177). Maminitsi makumi matanhatu mushure mekuisa phage, makonzo akaisirwa mvura mumoyo ne50 ml PBS ichiteverwa ne50 ml 4% PFA/PBS. Samples dzeuropi dzakagadziriswazve husiku hwese mu4% PFA/PBS uye dzakanyikwa mu30% sucrose husiku hwese pa4°C. Samples dzinoiswa mufiriji mumusanganiswa weOCT. Kuongororwa kwemasampuli akaomeswa nechando kwakaitwa patembiricha yemukamuri pa30 µm cryosections dzakavharirwa ne1% BSA uye dzakaiswa ma antibodies e polyclonal FITC-labeled against T7 phage (Novus NB 600-376A) pa4 °C. Dziisei mudumbu husiku hwese. Pakupedzisira, zvikamu zvakagezwa katatu nePBS uye zvakaongororwa ne confocal laser microscope (Leica TCS SP5).
Mapeptide ese ane kuchena kushoma kwe98% akaumbwa neGenScript USA, biotinylated uye lyophilized. Biotin inosungwa kuburikidza nekuwedzera triple glycine spacer paN-terminus. Tarisa mapeptide ese uchishandisa mass spectrometry.
Streptavidin (Sigma S0677) yakasanganiswa ne peptide inonyungudika ine huwandu hwe 5-fold equimolar, peptide inonyungudika yeBACE1, kana musanganiswa (3:1 ratio) we peptide inonyungudika yeBACE1 uye peptide inonyungudika yeBACE1 mu 5–10% DMSO/incubated muPBS. Awa imwe chete patembiricha yemukamuri isati yaiswa jekiseni. Ma peptide akabatanidzwa eStreptavidin akaiswa jekiseni ne intravenously pachiyero che 10 mg/kg mune imwe yetsinga dzemusana dzemakonzo ane cerebral cavity.
Kuwanda kwe streptavidin-peptide complexes kwakaongororwa ne ELISA. Nunc Maxisorp microtiter plates (Sigma) dzakaputirwa husiku hwese pa4°C ne 1.5 μg/ml mouse anti-streptavidin antibody (Thermo, MA1-20011). Mushure mekuvharira (blocking buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) patembiricha yemukamuri kwemaawa maviri, gezai plate ne 0.05% Tween-20/PBS (wash buffer) kwemasekondi matatu, CSF ne plasma samples zvakawedzerwa mumatsime akasanganiswa ne blocking buffer (plasma 1:10,000, CSF 1:115). plate yakazoiswa mu incubation husiku hwese pa4°C ne detection antibody (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Mushure mematanho matatu ekusuka, streptavidin yakaonekwa nekuiswa muTMB substrate solution (Roche) kwemaminitsi anosvika makumi maviri. Mushure mekumisa kugadzirwa kweruvara ne1M H2SO4, yera kunyudzwa kweruvara pa450 nm.
Basa re streptavidin-peptide-BACE1 inhibitor complex rakaongororwa neAβ(1-40) ELISA zvichienderana neprotocol yemugadziri (Wako, 294-64701). Muchidimbu, sampuli dzeCSF dzakasanganiswa mu standard diluent (1:23) uye dzakaiswa mu incubator husiku hwese pa4°C mumaplates e 96-well akaputirwa neBNT77 capture antibody. Mushure mematanho mashanu ekusuka, HRP-conjugated BA27 antibody yakawedzerwa uye yakaiswa mu incubator kwemaawa maviri pa4°C., zvichiteverwa nematanho mashanu ekusuka. Aβ(1–40) yakaonekwa ne incubation mu TMB solution kwemaminitsi makumi matatu patembiricha yemumba. Mushure mekunge ruvara rwamiswa ne stop solution, yera absorbance pa450 nm. Plasma samples dzakaiswa mu solid phase extraction isati yaitwa Aβ(1–40) ELISA. Plasma yakawedzerwa ku0.2% DEA (Sigma) mumaplates e 96-well uye yakaiswa mu incubator yemumba kwemaminitsi makumi matatu. Mushure mekusuka maSPE plates (Oasis, 186000679) nemvura uye 100% methanol, plasma samples dzakaiswa mumaSPE plates uye mvura yese yakabviswa. Samples dzakasukwa (kutanga ne5% methanol ndokuzobviswa 30% methanol) ndokuzobviswa ne2% NH4OH/90% methanol. Mushure mekuomesa eluate pa55°C kwemaminitsi 99 pa constant N2 current, sampuli dzakaderedzwa mu standard diluents uye Aβ(1–40) yakayerwa sezvakatsanangurwa pamusoro apa.
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