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Umqobo wegazi-ubuchopho kunye nomqobo wegazi-ubuchopho zithintela ii-arhente zonyango lwebhayoloji ekufikeleleni kwiinjongo zazo kwinkqubo yemithambo-luvo ephakathi, ngaloo ndlela zithintela unyango olusebenzayo lwezifo ze-neurological. Ukuze sifumane abathuthi bobuchopho abatsha kwi-vivo, sazisa ithala leencwadi le-T7 phage peptide kunye negazi eliqokelelwe ngokulandelelana kunye nolwelo lwe-cerebrospinal (CSF) sisebenzisa imodeli ye-cannulated conscious large pool yeempuku. Ii-phage clones ezithile zityebile kakhulu kwi-CSF emva kweendlela ezine zokukhetha. Uvavanyo lwee-peptides ezizodwa lubonise ukutyebisa okungaphezulu kwe-1000 kwi-CSF. Ukusebenza kwe-bioactivity yokuhanjiswa kwe-peptide ebuchosheni kuqinisekiswe kukunciphisa kwe-40% kwinqanaba le-amyloid-β kulwelo lwe-cerebrospinal kusetyenziswa i-BACE1 peptide inhibitor edityaniswe ne-transit peptide entsha echongiweyo. Ezi ziphumo zibonisa ukuba ii-peptides ezichongiweyo ziindlela zokukhetha i-phage kwi-vivo zinokuba zizithuthi eziluncedo zokuhambisa ii-macromolecules ebuchosheni ngesiphumo sonyango.
Uphando lonyango olujoliswe kwinkqubo ye-nervous system (CNS) lugxile kakhulu ekuboneni amayeza aphuculweyo kunye neearhente ezibonisa iimpawu zokujolisa kwi-CNS, ngaphandle komzamo omncinci wokufumanisa iindlela eziqhuba ukuhanjiswa kwamayeza asebenzayo engqondweni. Oku kuqala ukutshintsha ngoku njengoko ukuhanjiswa kwamayeza, ingakumbi iimolekyuli ezinkulu, kuyinxalenye ebalulekileyo yophuhliso lwamayeza e-neuroscience yanamhlanje. Indawo engqongileyo yenkqubo ye-nervous system ikhuselwe kakuhle yinkqubo ye-cerebrovascular barrier, equka i-blood-brain barrier (BBB) ​​​​kunye ne-blood-brain barrier (BCBB)1, okwenza kube nzima ukuhambisa amayeza engqondweni1,2. Kuqikelelwa ukuba phantse zonke iziyobisi ezinkulu ze-molecule kunye ne-98% yamayeza amancinci e-molecule asuswa ebuchotsheni3. Yingakho kubaluleke kakhulu ukuchonga iinkqubo ezintsha zokuhambisa ubuchopho ezibonelela ngokuhanjiswa ngokufanelekileyo nangokuthe ngqo kwamayeza onyango kwi-CNS 4,5. Nangona kunjalo, i-BBB kunye ne-BCSFB nazo zibonelela ngethuba elihle lokuhanjiswa kwamayeza njengoko zingena kwaye zingena kuzo zonke izakhiwo zobuchopho ngokusebenzisa imithambo yegazi ebanzi. Ngoko ke, imizamo ekhoyo ngoku yokusebenzisa iindlela ezingangenisi ntsholongwane zokuhambisa ingqondo isekelwe kakhulu kwindlela yokuhambisa i-receptor-mediated transport (PMT) kusetyenziswa i-endogenous BBB6 receptor. Nangona kukho inkqubela phambili yakutshanje kusetyenziswa indlela ye-transferrin receptor7,8, kufuneka uphuhliso olongezelelekileyo lweenkqubo ezintsha zokuhambisa ezineempawu eziphuculweyo. Ngenxa yoku, injongo yethu yayikukuchonga iipeptides ezikwaziyo ukulawula ukuthutha i-CSF, njengoko ngokusisiseko zinokusetyenziselwa ukuhambisa ii-macromolecules kwi-CNS okanye ukuvula iindlela ezintsha ze-receptor. Ngokukodwa, ii-receptors ezithile kunye nabathuthi benkqubo ye-cerebrovascular (BBB kunye ne-BSCFB) zinokusebenza njengeethagethi ezinokubakho zokuhambisa amayeza e-biotherapeutic asebenzayo nakhethekileyo. I-Cerebrospinal fluid (CSF) yimveliso eyimfihlo ye-choroid plexus (CS) kwaye inxibelelana ngokuthe ngqo nolwelo lwe-interstitial lobuchopho ngesithuba se-subarachnoid kunye nesithuba se-ventricular4. Kutshanje kuboniswe ukuba ulwelo lwe-subarachnoid cerebrospinal lusasazeka kakhulu kwi-interstitium yobuchopho9. Sinethemba lokufikelela kwindawo ye-parenchymal sisebenzisa le ndlela yokungena kwe-subarachnoid okanye ngokuthe ngqo nge-BBB. Ukuze sifezekise oku, sisebenzise icebo eliqinileyo lokukhetha i-in vivo phage elichonga ngokufanelekileyo iipeptides ezithuthwa ngenye yezi ndlela zimbini zahlukileyo.
Ngoku sichaza indlela yokuvavanya i-in vivo phage display elandelelanayo kunye ne-CSF sampling edityaniswe ne-high throughput sequencing (HTS) ukujonga imijikelo yokuqala yokukhetha enobuninzi bethala leencwadi. Ukuhlolwa kwenziwe kwiigundane eziqondayo nge-cannula enkulu ye-cisterna (CM) efakwe ngokusisigxina ukuze kuthintelwe ukungcoliswa kwegazi. Okubalulekileyo, le ndlela ikhetha zombini i-brain-targeting kunye nee-peptides ezine-transport activity ngaphesheya komqobo we-cerebrovascular. Sisebenzise ii-T7 phages ngenxa yobukhulu bazo obuncinci (~60 nm)10 kwaye sacebisa ukuba zifanelekile ukuthuthwa kwee-vesicles ezivumela ukuwela kwe-transcellular yomqobo we-endothelial kunye/okanye we-epithelial-medulla. Emva kwemijikelo emine ye-panning, ii-phages populations zahlulwa zibonisa ukutyeba kwe-CSF enamandla kwi-vivo kunye nokudibana kwe-cerebral microvessel. Okubalulekileyo, sikwazile ukuqinisekisa iziphumo zethu ngokubonisa ukuba ii-peptides ezikhethiweyo nezilungiselelwe ngokweekhemikhali ziyakwazi ukuthutha umthwalo weproteni ukuya kwi-cerebrospinal fluid. Okokuqala, iziphumo ze-pharmacodynamic ze-CNS zasekwa ngokudibanisa i-transit peptide ehamba phambili kunye ne-inhibitor ye-BACE1 peptide. Ukongeza ekuboniseni ukuba amaqhinga okuhlola ukusebenza kwe-in vivo anokuchonga iipeptides ezintsha zokuthutha ubuchopho njengabathwali beproteni abasebenzayo, silindele ukuba iindlela ezifanayo zokukhetha ukusebenza nazo zibe zibalulekile ekuchongeni iindlela ezintsha zokuthutha ubuchopho.
Ngokusekelwe kwiiyunithi zokwenza i-plaque (PFU), emva kwenyathelo lokupakisha i-phage, ilayibrari yee-peptides ze-phage ze-12-mer ezi-linear T7 ezingafaniyo ezineentlobo ngeentlobo ezimalunga ne-109 yadalwa (jonga izixhobo kunye neendlela). Kubalulekile ukuqaphela ukuba siyihlalutyile ngononophelo le layibrari ngaphambi kokuba i-in vivo panning. Ukwandiswa kwe-PCR kweesampulu zelayibrari ye-phage kusetyenziswa ii-primers ezilungisiweyo kwavelisa ii-amplicons ezazisebenza ngqo kwi-HTS (Umzobo oNcedisayo 1a). Ngenxa ye-a) iimpazamo zokulandelelana kwe-HTS11, b) impembelelo kumgangatho wee-primers (NNK)1-12, kunye ne-c) ubukho be-phage yohlobo lwe-wild-type (wt) (i-skeleton inserts) kwilayibrari yokulinda, inkqubo yokucoca ulandelelwano yaqaliswa ukukhupha ulwazi oluqinisekisiweyo lolandelelwano (Umzobo oNcedisayo 1b). La manyathelo okucoca asebenza kuzo zonke iilayibrari zokulandelelana kwe-HTS. Kwithala leencwadi eliqhelekileyo, kufunyenwe ukufundwa okungu-233,868, apho ama-39% aphumelele kwiikhrayitheriya zokucoca kwaye asetyenziselwa uhlalutyo lwethala leencwadi kunye nokukhetha imijikelo elandelayo (Umfanekiso oNcedisayo 1c–e). Ukufundwa bekuyi-multiples yezibini ezisisiseko ezi-3 ubude kunye nencopho kwi-36 nucleotides (Umfanekiso oNcedisayo 1c), okuqinisekisa uyilo lwethala leencwadi (NNK) 1-12. Okuphawulekayo kukuba, malunga ne-11% yamalungu ethala leencwadi aqulethe i-12-dimensional wild-type (wt) backbone PAGISRELVDKL insert, kwaye phantse isiqingatha se-sequences (49%) siqulethe ukufakwa okanye ukususwa. I-HTS yethala leencwadi iqinisekisile ukwahluka okuphezulu kweepeptides kwithala leencwadi: ngaphezulu kwe-81% ye-peptide sequences ifunyenwe kube kanye kuphela kwaye yi-1.5% kuphela eyenzekileyo kwiikopi ezi-≥4 (Umfanekiso oNcedisayo 2a). Iifrequencies ze-amino acids (aa) kuzo zonke izikhundla ezili-12 kwirepertoire zidibene kakuhle neefrequencies ezilindelweyo kwinani lee-codons eziveliswa yi-repertoire ye-NKK ewohlokileyo (Umzobo oNcedisayo 2b). Iifrequencies ezibonweyo zee-aa residues ezifakwe kwezi zifakelo zihambelana kakuhle nefrequencies ebaliweyo (r = 0.893) (Umzobo oNcedisayo 2c). Ukulungiswa kweelayibrari ze-phage ukuze zifakwe inaliti kubandakanya amanyathelo okukhulisa nokususa i-endotoxin. Oku kuboniswe ngaphambili ukuba kunokunciphisa ukwahlukana kweelayibrari ze-phage12,13. Ke ngoko, silandelelanise ilayibrari ye-phage e-plate-amplified eyayisuswe i-endotoxin kwaye sayithelekisa nelayibrari yokuqala ukuze siqikelele iifrequencies ze-AA. Ulwalamano oluqinileyo (r = 0.995) lubonwe phakathi kwedama lokuqala kunye nedama elikhuliswe nelicociweyo (Umzobo oNcedisayo 2d), nto leyo ebonisa ukuba ukhuphiswano phakathi kwee-clones ezikhuliswe kwiiplates ezisebenzisa i-T7 phage aluzange lubangele ukuthambekela okukhulu. Olu thelekiso lusekelwe kwinani lee-motifs ze-tripeptide kwithala leencwadi ngalinye, kuba ulwahluko lweelayibrari (~109) alunakuqondwa ngokupheleleyo nokuba usebenzisa i-HTS. Uhlalutyo lwenani le-aa kwindawo nganye lubonise ukuba kukho ucalucalulo oluncinci oluxhomekeke kwindawo kwiindawo ezintathu zokugqibela ze-repertoire engenisiweyo (Umzobo oNcedisayo 2e). Ukuqukumbela, sigqibe kwelokuba umgangatho kunye nolwahluko lwethala leencwadi lwamkelekile kwaye kuphela utshintsho oluncinci kulwahluko olubonwe ngenxa yokwandiswa kunye nokulungiswa kweethala leencwadi ze-phage phakathi kweendlela ezahlukeneyo zokukhetha.
Ukuthathwa kwesampulu yolwelo lwe-cerebrospinal serial kungenziwa ngokufakelwa i-cannula kwi-CM yeempuku eziqondayo ukuze kube lula ukuchonga i-T7 phage efakwe ngaphakathi kwegazi (iv) nge-BBB kunye/okanye i-BCSFB (Umzobo 1a-b). Sisebenzise iingalo ezimbini zokukhetha ezizimeleyo (iingalo A kunye no-B) kwimijikelo emithathu yokuqala yokukhetha kwi-vivo (Umzobo 1c). Siye sandisa kancinci kancinci ukuqina kokukhetha ngokunciphisa inani lilonke le-phage elingeniswe kwimijikelo emithathu yokuqala yokukhetha. Kumjikelo wesine we-panning, sidibanise iisampuli ezivela kumasebe u-A kunye no-B kwaye senza ukhetho oluthathu olongezelelweyo oluzimeleyo. Ukuze sifunde iipropati ze-in vivo zee-T7 phage particles kule modeli, i-wild-type phage (PAGISRELVDKL master insert) ifakwe kwiimpuku nge-tail vein. Ukubuyiselwa kwe-phages kwi-cerebrospinal fluid kunye negazi ngamaxesha ahlukeneyo kubonise ukuba ii-T7 icosahedral phages ezincinci zinesigaba sokuqala sokususwa ngokukhawuleza ukusuka kwindawo yegazi (Umzobo ongezelelweyo 3). Ngokusekelwe kwii-titers ezinikweyo kunye nomthamo wegazi weempuku, sibale ukuba malunga ne-1% ye-wt. phage evela kwidosi enikweyo ifunyenwe egazini emva kwemizuzu eli-10 emva kokufakwa nge-intravenous. Emva koku kwehla okukhawulezileyo kokuqala, ukususwa okucothayo kwe-primary kwalinganiswa nge-half-life yemizuzu engama-27.7. Okubalulekileyo kukuba, zimbalwa kakhulu ii-phage ezifunyenweyo kwi-CSF compartment, nto leyo ebonisa ukuba imvelaphi iphantsi yokufuduka kwe-wild-type phage iye kwi-CSF compartment (Umfanekiso Ongezelelweyo 3). Ngokomyinge, malunga ne-1 x 10-3% yee-titers ze-T7 phage egazini kunye ne-4 x 10-8% yee-phages ezifakwe ekuqaleni zifunyenwe kwi-cerebrospinal fluid kulo lonke ixesha lokuthathwa kwesampulu (0-250 min). Okuphawulekayo kukuba, i-half-life (25.7 min) ye-wild-type phage kwi-cerebrospinal fluid yayifana neyo ebonwe egazini. Ezi datha zibonisa ukuba umqobo owahlula i-CSF compartment egazini awukho kwiimpuku ezifakwe i-CM-cannulated, nto leyo evumela ukuba kukhethwe ii-phage libraries ezifumaneka emzimbeni ukuze kuchongwe ii-clones ezithuthwa ngokulula zisuka egazini zisiya kwi-CSF compartment.
(a) Ukuseta indlela yokuphinda uthabathe iisampulu ze-cerebrospinal fluid (CSF) kwidama elikhulu. (b) Umzobo obonisa indawo yeseli yomqobo we-central nervous system (CNS) kunye necebo lokukhetha elisetyenziselwa ukuchonga iipeptides eziwela umqobo wegazi-ubuchopho (BBB) ​​​​kunye nomqobo wegazi-ubuchopho. (c) Itshati yokujonga i-in vivo phage display. Kumjikelo ngamnye wokukhetha, ii-phages (izihlonzi zezilwanyana ngaphakathi kweentolo) zifakwa nge-intravenously. Amasebe amabini azimeleyo (A, B) agcinwa ngokwahlukeneyo kude kube ngumjikelo wesi-4 wokukhetha. Kwimijikelo yokukhetha 3 kunye nowesi-4, i-phage clone nganye ekhutshwe kwi-CSF yacwangciswa ngesandla. (d) I-Kinetics ye-phage ehlukanisiweyo egazini (izangqa ezibomvu) kunye ne-cerebrospinal fluid (iitriangles eziluhlaza) ngexesha lomjikelo wokuqala wokukhetha kwiigundane ezimbini ezifakwe kwi-cannulated emva kokufakwa kwi-intravenous kwithala leencwadi le-T7 peptide (2 x 1012 phages/isilwanyana). Izikwere eziluhlaza okwesibhakabhaka zibonisa umyinge wokuqala woxinzelelo lwe-phage egazini, olubalwa ukusuka kubungakanani be-phage efakwe kwi-blood phage, kuthathelwa ingqalelo umthamo wegazi uwonke. Izikwere ezimnyama zibonisa indawo apho kudityaniswe khona umgca we-y othathwe kwiingxinano ze-phage yegazi. (e,f) Bonisa ubuninzi kunye nokusasazwa kwazo zonke ii-motifs ze-tripeptide ezidlulayo ezifumaneka kwi-peptide. Inani lee-motifs ezifunyenwe kwiifundo ezili-1000 libonisiwe. Okubalulekileyo (p < 0.001) ii-motifs ezicebileyo ziphawulwe ngamachaphaza abomvu. (e) I-scatterplot yokuhambelana ethelekisa ubuninzi be-motif ye-tripeptide yethala leencwadi elifakwe kwi-phage ephuma kwigazi evela kwizilwanyana #1.1 kunye #1.2. (f) I-scatterplot yokuhambelana ethelekisa ubuninzi be-motifs ze-tripeptide ze-phage yezilwanyana #1.1 kunye #1.2 ezihlukaniswe kwigazi nakwi-cerebrospinal fluid. (g, h) Umzobo we-Sequence ID we-phage etyebileyo kwigazi (g) ngokuchasene neelayibrari ezifakwe kwi-phage kunye ne-phage etyebileyo kwi-CSF (h) ngokuchasene negazi emva komjikelo wokukhethwa kwe-in vivo kuzo zombini izilwanyana. Ubungakanani bekhowudi enonobumba omnye bubonisa ukuba loo amino acid ifumaneka kangaphi kuloo ndawo. Iluhlaza = i-polar, imfusa = ingathathi cala, iluhlaza okwesibhakabhaka = isiseko, ibomvu = i-acidic kwaye imnyama = i-hydrophobic amino acids. Umfanekiso 1a, b wenziwe kwaye waveliswa nguEduard Urich.
Sifake ilayibrari ye-phage peptide kwiigundane ezimbini ze-CM instrument (clades A kunye no-B) kunye ne-phage eyahlukileyo kwi-cerebrospinal fluid kunye negazi (Umfanekiso 1d). Ukususwa ngokukhawuleza kokuqala kwelayibrari bekungabonakali kangako xa kuthelekiswa ne-wild-type phage. Isiqingatha sobomi belayibrari efakwe kwizilwanyana zombini yayiyimizuzu engama-24.8 egazini, efana ne-wild-type phage, kunye nemizuzu engama-38.5 kwi-CSF. Iisampulu zegazi kunye ne-cerebrospinal fluid phage ezivela kwisilwanyana ngasinye zafakwa kwi-HTS kwaye zonke iipeptides ezichongiweyo zahlalutywa ukuba zikhona i-tripeptide motif emfutshane. Ii-Tripeptide motifs zakhethwa kuba zibonelela ngesiseko esincinci sokwakheka kwesakhiwo kunye nokusebenzisana kwe-peptide-protein14,15. Sifumene ulwalamano oluhle ekusasazweni kwee-motifs phakathi kwelayibrari ye-phage efakwe kwi-injection kunye nee-clones ezikhutshwe egazini lezilwanyana zombini (Umzobo 1e). Idatha ibonisa ukuba ukwakheka kwelayibrari kutyetyiswe kancinci kuphela kwindawo yegazi. Ii-Amino acid frequency kunye ne-consensus sequences zahlalutywa ngakumbi kwindawo nganye kusetyenziswa i-adaptation yesoftware ye-Weblogo16. Okunomdla kukuba, sifumene ukutyeba okunamandla kwiintsalela ze-glycine egazini (Umzobo 1g). Xa igazi lithelekiswa nee-clones ezikhethwe kwi-CSF, kwabonwa ukhetho oluqinileyo kunye nokususwa kwezinye ii-motifs (Umzobo 1f), kwaye ii-amino acids ezithile zazikho ngokukhethekileyo kwiindawo ezimiselweyo kwi-12-member (Umzobo 1h). Okuphawulekayo kukuba, izilwanyana ezahlukeneyo zahluke kakhulu kulwelo lwe-cerebrospinal, ngelixa ukutyeba kwe-glycine egazini kwabonwa kwizilwanyana zombini (Umzobo oNcedisayo 4a-j). Emva kokuhluzwa okungqongqo kwedatha yolandelelwano kulwelo lwe-cerebrospinal lwezilwanyana #1.1 kunye #1.2, kwafunyanwa ii-peptides eziyi-964 kunye ne-420 ezikhethekileyo ze-12-mer (Umzobo oNcedisayo 1d-e). Ii-clones ze-phage ezizimeleyo zandiswa kwaye zafakwa kumjikelo wesibini wokukhethwa kwe-in vivo. I-Phage ekhutshwe kumjikelo wesibini wokukhetha ibekwe kwi-HTS kwisilwanyana ngasinye kwaye zonke iipeptides ezichongiweyo zisetyenziswe njengegalelo kwinkqubo yokuqaphela i-motif ukuhlalutya ukwenzeka kwe-motifs ze-tripeptide (Umzobo 2a, b, ef). Xa kuthelekiswa nomjikelo wokuqala we-phage efunyenwe kwi-CSF, siqaphele ukukhethwa okungakumbi kunye nokususwa kokukhethwa kwe-motifs ezininzi kwi-CSF kumasebe A kunye no-B (Umzobo 2). I-algorithm yokuchongwa kwenethiwekhi isetyenzisiwe ukumisela ukuba imele iipatheni ezahlukeneyo zolandelelwano oluhambelanayo. Ukufana okucacileyo kubonwe phakathi kwe-12-dimensional sequences ezifunyenwe yi-CSF kwi-alternative clade A (Umzobo 2c, d) kunye ne-clade B (Umzobo 2g, h). Uhlalutyo oluhlanganisiweyo kwisebe ngalinye lutyhile iiprofayili ezahlukeneyo zokukhetha iipeptides ze-12-mer (Umzobo oNcedisayo 5c, d) kunye nokwanda komlinganiselo we-CSF/blood titer ngokuhamba kwexesha kwii-clones ezihlanganisiweyo emva komjikelo wesibini wokukhetha xa kuthelekiswa nomjikelo wokuqala wokukhetha (Umzobo oNcedisayo 5e).).
Ukwandiswa kwee-motifs kunye neepeptides kulwelo lwe-cerebrospinal ngemijikelezo emibini elandelelanayo yokukhethwa kokubonisa i-phage esebenzayo kwi-vivo.
Zonke ii-phages ze-cerebrospinal fluid ezifunyenwe kumjikelo wokuqala wesilwanyana ngasinye (izilwanyana #1.1 kunye #1.2) zadityaniswa, zandiswa, zalandelelaniswa nge-HT zaza zafakwa kwakhona kunye (2 x 1010 phages/isilwanyana) Iimpuku ezi-2 ze-SM cannulated (#1.1 → #). 2.1 kunye no-2.2, 1.2 → 2.3 kunye no-2.4). (a,b,e,f) Ii-scatterplots zokudibanisa ukuhambelana ezithelekisa i-frequency ye-tripeptide motifs yazo zonke ii-phages ezivela kwi-CSF kumjikelo wokuqala nowesibini wokukhetha. I-frequency ehambelanayo kunye nokusasazwa kwee-motifs ezimele zonke ii-tripeptides ezinokuthi zigqubane ezifumaneka kwii-peptides kuzo zombini iindlela. Inani lee-motifs ezifunyenwe kwi-1000 readings libonisiwe. Ii-motifs ezikhethwe kakhulu (p < 0.001) okanye ezingabandakanywanga kwenye yeelayibrari ezithelekiswayo zigqanyisiwe ngamachaphaza abomvu. (c, d, g, h) Ukubonakaliswa kwelogo yolandelelwano lwazo zonke ii-amino acid ezili-12 ezityebileyo ze-CSF ngokusekelwe kwimijikelo yesi-2 neyesi-1 yokukhethwa kwe-in vivo. Ubungakanani bekhowudi enobumba omnye bubonisa ukuba loo amino acid ifumaneka kangaphi kuloo ndawo. Ukumela ilogo, ukuphindaphinda kwe-CSF sequences ezikhutshwe kwizilwanyana ngazinye phakathi kwemijikelo emibini yokukhetha kuyathelekiswa kwaye ukulandelelana okutyebileyo kumjikelo wesibini kuyaboniswa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 kunye (h) #1.2–#2.4. Ii-amino acid ezityebileyo kakhulu kwindawo ethile kwizilwanyana (c, d) inombolo 2.1 kunye nenombolo 2.2 okanye (g, h) kwizilwanyana inombolo 2.3 kunye nenombolo 2.4 ziboniswa ngombala. Oluhlaza = i-polar, i-purple = i-neutral, i-blue = isiseko, ebomvu = i-acidic kunye ne-black = i-hydrophobic amino acids.
Emva komjikelo wesithathu wokukhetha, sichonge ulandelelwano lwe-peptide olukhethekileyo oluyi-124 (#3.1 kunye no-#3.2) oluvela kwi-332 CSF-reconstituted phage clones ezahlulwe kwizilwanyana ezibini (Umzobo oNcedisayo 6a). Uluhlu lwe-LGSVS (18.7%) lwalunenani eliphezulu kakhulu, lulandelwa yi-wild-type inserts PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), kunye ne-SARGSWREIVSLS (2.2%). Kumjikelo wesine wokugqibela, sidibanise amasebe amabini akhethwe ngokuzimeleyo kwizilwanyana ezintathu ezahlukeneyo (Umzobo 1c). Kwii-925 sequenced phage clones ezifunyenwe kwi-CSF, kumjikelo wesine sifumene i-64 unique peptide sequences (Umzobo oNcedisayo 6b), phakathi kwazo inani elihambelanayo le-wild-type phage lehla laya kwi-0.8%. Ii-clones ze-CSF eziqhelekileyo kumjikelo wesine yayiyi-LYVLHSRGLWGFKLAAALE (18%), i-LGSVS (17%), i-GFVRFRLSNTR (14%), i-KVAWRVFSLFWK (7%), i-SVHGV (5%), i-GRPQKINGARVC (3.6%) kunye ne-RLSSVDSDLSGC (3, 2%). %)). Uluhlu lobude beepeptides ezikhethiweyo lubangelwa kukufakwa/ukususwa kweenucleotide okanye ii-codons zokumisa ngaphambi kwexesha kwi-primers zelayibrari xa kusetyenziswa ii-codons eziwohlokileyo kuyilo lwelayibrari ye-NNK. Ii-codons zokumisa ngaphambi kwexesha zivelisa ii-peptides ezimfutshane kwaye zikhethwa kuba ziqulathe i-aa motif efanelekileyo. Ii-peptides ezinde zinokubangelwa kukufakwa/ukususwa kwi-primers zeelayibrari zokwenziwa. Oku kubeka i-stop codon eyilwe ngaphandle kwesakhelo kwaye kuyifunde de i-stop codon entsha ivele ngezantsi. Ngokubanzi, sibale izinto zokuphucula kuzo zonke ii-select rounds ezine ngokuthelekisa idatha yokufaka kunye nedatha yesiphumo sesampuli. Kwisigaba sokuqala sovavanyo, sisebenzise ii-phage titers ze-wild-type njengereferensi yemvelaphi engangqalanga. Okubangel’ umdla kukuba, ukhetho lwe-phage olungalunganga lwalunamandla kakhulu kumjikelo wokuqala we-CSF, kodwa kungekhona egazini (Umzobo 3a), okunokubangelwa kukungafumaneki okuncinci kokusasazwa okungenamsebenzi kwamalungu amaninzi elayibrari ye-peptide kwi-CSF compartment okanye ii-phage ezizalanayo zihlala zigcinwa okanye zisuswe egazini ngokufanelekileyo kunee-bacteriophages. Nangona kunjalo, kumjikelo wesibini we-panning, ukhetho oluqinileyo lwee-phages kwi-CSF lwabonwa kuzo zombini ii-clades, nto leyo ebonisa ukuba umjikelo wangaphambili wawutyebile kwii-phages ezibonisa ii-peptides ezikhuthaza ukuthathwa kwe-CSF (Umzobo 3a). Kwakhona, ngaphandle kokutyebiswa kwegazi okubalulekileyo. Kwakhona kumjikelo wesithathu nowesine, ii-phage clones zatyebiswa kakhulu kwi-CSF. Ukuthelekisa ukuphindaphinda kolandelelwano ngalunye lwe-peptide olukhethekileyo phakathi kwezigaba ezimbini zokugqibela zokukhetha, sifumanise ukuba ulandelelwano lwalutyebiswe ngakumbi kumjikelo wesine wokukhetha (Umzobo 3b). Iimotif ze-tripeptide ezingama-931 zizonke zikhutshwe kuzo zonke ii-peptide sequences ezingama-64 ezikhethekileyo kusetyenziswa zombini ii-peptide orientations. Iimotifs ezicebileyo kakhulu kumjikelo wesine zihlolwe ngokusondeleyo kwiiprofayili zazo zokutyebisa kuzo zonke ii-rounds xa kuthelekiswa nelayibrari efakiweyo (ukusika: i-10% yokutyebisa) (Umzobo oNcedisayo 6c). Iipateni zokukhetha ngokubanzi zibonise ukuba uninzi lwezizathu ezifundwayo zityebiswe kuzo zonke ii-rounds zangaphambili zamasebe okukhetha omabini. Nangona kunjalo, ezinye iimotifs (umz. i-SGL, i-VSG, i-LGS GSV) zazivela kakhulu kwi-alternative clade A, ngelixa ezinye (umz. i-FGW, i-RTN, i-WGF, i-NTR) zityebiswe kwi-alternative clade B.
Ukuqinisekiswa kokuhanjiswa kwe-CSF kwee-peptides eziboniswe nge-CSF ezityebiswe nge-phage kunye nee-peptides eziphambili ze-biotinylated ezidityaniswe nemithwalo yomthwalo ye-streptavidin.
(a) Umlinganiselo wobutyebi obalwe kuzo zonke iiraundi ezine (R1-R4) ngokusekelwe kwiititer ze-phage (PFU) ezijojoweyo (input = I) kunye neetiter ze-phage ze-CSF ezichongiweyo (output = O). Izinto zobutyebi kwiraundi ezintathu zokugqibela (R2-R4) zibalwe ngokuthelekisa nomjikelo odlulileyo kunye nomjikelo wokuqala (R1) kunye nedatha yobunzima. Iibha ezivulekileyo zilulwelo lwe-cerebrospinal, iibha ezinombala ziyi-plasma. (***p<0.001, ngokusekelwe kuvavanyo lwe-t lomfundi). (b) Uluhlu lwee-peptides ze-phage ezininzi kakhulu, zibekwe ngokwemilinganiselo yazo ehambelana nazo zonke ii-phage eziqokelelwe kwi-CSF emva komjikelo wesi-4 wokukhetha. Ii-clones ze-phage ezintandathu eziqhelekileyo zigqanyisiwe ngombala, zibhalwe kwaye izinto zazo zobutyebi zigqanyisiwe phakathi kweraundi yesi-3 neyesi-4 yokukhetha (ii-insets). (c,d) Ii-clones ze-phage ezintandathu ezityebileyo kakhulu, iilayibrari ze-phage ezingenanto kunye neelayibrari ze-phage peptide zomzali ezivela kumjikelo wesi-4 zihlalutywe ngokwahlukeneyo kwimodeli yesampulu ye-CSF. Iisampulu ze-CSF kunye negazi ziqokelelwe ngamaxesha abonisiweyo. (c) Inani elilinganayo lee-clones ze-phage ezi-6 ezikhethiweyo (ii-phages/izilwanyana ezi-2 x 1010), ii-phages/izilwanyana ezingenanto (#1779) (ii-phages/izilwanyana ezi-2 x 1010) kunye neelayibrari ze-stock phage peptide (ii-phages/izilwanyana ezi-2 x 1012) Faka ubuncinane i-3 CM kwisilwanyana esifakwe kwi-cannulated ngokwahlukeneyo nge-tail vein. I-CSF pharmacokinetics ye-phage clone nganye ejoyiweyo kunye ne-phage peptide library ngokuhamba kwexesha iyaboniswa. (d) ibonisa umlinganiselo ophakathi we-CSF/igazi kuzo zonke ii-phages/mL ezifunyenweyo ngexesha lokuthathwa kwesampulu. (e) Iipeptides ezine ze-synthetic leader kunye nolawulo olunye oluqhekekileyo zidibene ne-biotin kwi-streptavidin nge-N-terminus yazo (tetramer display) elandelwa yi-injection (tail vein iv, 10 mg streptavidin/kg). Ubuncinane iigundane ezintathu ezifakwe kwi-intubated (N = 3).). Iisampulu ze-CSF ziqokelelwe ngamaxesha abonisiwe kwaye ubuninzi be-streptavidin bulinganiswe yi-CSF anti-streptavidin ELISA (nd = ayifunyaniswanga). (*p<0.05, **p<0.01, ***p<0.001, ngokusekelwe kuvavanyo lwe-ANOVA). (f) Uthelekiso lwe-amino acid sequence ye-most rich phage peptide clone #2002 (purple) kunye nezinye ii-phage peptide clones ezikhethiweyo ezivela kumjikelo we-4 wokukhetha. Iziqwenga ze-amino acid ezifanayo nezifanayo zinemibala.
Kuzo zonke ii-phages ezityebileyo kumjikelo wesine (Umzobo 3b), ii-clones ezintandathu ezikhethiweyo zikhethwe ukuze kuhlalutywe ngakumbi umntu ngamnye kwimodeli yesampulu ye-CSF. Inani elilinganayo le-phage ezintandathu ezikhethiweyo, i-phage engenanto (engenazo i-insert) kunye neelayibrari ze-prophage peptide zifakwe kwizilwanyana ezintathu ze-CM ezifakwe kwi-cannulated, kwaye i-pharmacokinetics yamiselwa kwi-CSF (Umzobo 3c) kunye negazi (Umzobo oNcedisayo 7). Zonke ii-clones ze-phage ezivavanyiweyo zijolise kwigumbi le-CSF kwinqanaba eliphindwe ka-10-1000 kunelo le-phage engenanto (#1779). Umzekelo, ii-clones #2020 kunye ne-#2077 zazinee-CSF titers ezingaphezulu ka-1000 kune-phage yokulawula. Iprofayili ye-pharmacokinetic ye-peptide nganye ekhethiweyo yahlukile, kodwa zonke zinamandla aphezulu e-CSF homing. Sibone ukwehla okuqhubekayo ngokuhamba kwexesha kwii-clones #1903 kunye ne-#2011, ngelixa kwii-clones #2077, #2002 kunye ne-#2009 ukwanda ngexesha lemizuzu eli-10 yokuqala kunokubonisa ukuthuthwa okusebenzayo kodwa kufuneka kuqinisekiswe. Ii-Clones #2020, #2002, kunye ne-#2077 zizinzile kumanqanaba aphezulu, ngelixa uxinzelelo lwe-CSF lwe-clone #2009 lwehla kancinci emva kokunyuka kokuqala. Emva koko sithelekise imvamisa yomviwa ngamnye we-CSF kunye noxinzelelo lwakhe lwegazi (Umzobo 3d). Ulwalamano lwe-titer ephakathi yomviwa ngamnye we-CSF kunye ne-titer yakhe yegazi ngamaxesha onke okuthatha iisampulu lubonise ukuba abathathu kwabathandathu batyunjwe kakhulu kwi-CSF yegazi. Okunomdla kukuba, i-clone #2077 ibonise uzinzo oluphezulu lwegazi (Umfanekiso ongezelelweyo 7). Ukuqinisekisa ukuba ii-peptides ngokwazo ziyakwazi ukuthutha imithwalo ngaphandle kwee-phage particles ukuya kwi-CSF compartment, senze ii-peptides ezine eziphambili ezithathwe kwi-biotin kwi-N-terminus apho ii-peptides zinamathela kwi-phage particle. Iipeptides zeBiotinylated (nos. 2002, 2009, 2020 kunye no-2077) zadityaniswa ne-streptavidin (SA) ukuze kufunyanwe iifom ze-multimeric ezilinganisa i-phage geometry. Le fomathi ikwasivumele ukuba silinganise ukuvezwa kwe-SA egazini nakwi-cerebrospinal fluid njengeepeptides zeprotheyini ezithutha impahla. Okubalulekileyo kukuba, idatha ye-phage yayinokuphinda iveliswe xa iipeptides zokwenziwa zinikwa kule fomathi ye-SA-conjugated (Umzobo 3e). Iipeptides eziqhekekileyo zazinokuvezwa okuncinci kokuqala kwaye zazingenakususwa ngokukhawuleza kwe-CSF ngamanqanaba angafumanekiyo kwiiyure ezingama-48. Ukuze sifumane ulwazi malunga neendlela zokuhanjiswa kwezi peptide phage clones kwindawo ye-CSF, sihlalutye indawo ye-phage peptide hits nganye sisebenzisa i-immunohistochemistry (IHC) ukuze sifumane ngokuthe ngqo ii-phage particles emva kweyure e-1 emva kokufakwa kwi-intravenous in vivo. Okuphawulekayo kukuba, ii-clones #2002, #2077, kunye ne-#2009 zinokufunyanwa ngokudaya okunamandla kwi-brain capillaries, ngelixa i-control phage (#1779) kunye ne-clone #2020 azizange zifunyanwe (Umfanekiso oNcedisayo 8). Oku kubonisa ukuba ezi peptides zinegalelo kwisiphumo engqondweni ngokuchanekileyo ngokuwela i-BBB. Uhlalutyo oluneenkcukacha oluthe vetshe luyafuneka ukuvavanya le ngcamango, njengoko indlela ye-BSCFB nayo isenokubandakanyeka. Xa kuthelekiswa ulandelelwano lwe-amino acid ye-clone etyebileyo kakhulu (#2002) kunye nezinye iipeptides ezikhethiweyo, kwaphawulwa ukuba ezinye zazo zine-amino acid extensions ezifanayo, ezinokubonisa indlela efanayo yokuthutha (Umzobo 3f).
Ngenxa yeprofayili yayo yeplasma eyahlukileyo kunye nokwanda okukhulu kwi-CSF ngokuhamba kwexesha, i-phage display clone #2077 yaphononongwa ngakumbi kwixesha elide leeyure ezingama-48 kwaye yakwazi ukuphinda ivelise ukwanda okukhawulezileyo kwi-CSF okubonwe ngokunxulumene namanqanaba e-SA aqhubekayo (Umzobo 4a). Ngokuphathelele ezinye ii-phage clones ezichongiweyo, i-#2077 yanombala oqinileyo kwi-capillaries yobuchopho kwaye yabonisa i-colocalization ebalulekileyo nge-capillary marker lectin xa ijongwa kwisisombululo esiphezulu kwaye mhlawumbi imbala ethile kwindawo ye-parenchymal (Umfanekiso 4b). Ukuphanda ukuba iziphumo ze-pharmacological ezilawulwa yi-peptide zinokufunyanwa kwi-CNS, senze uvavanyo apho iinguqulelo ze-biotinylated ze-i) i-#2077 transit peptide kunye nee-ii) i-BACE1 inhibitor peptide zaxutywa ne-SA kwii-ratio ezimbini ezahlukeneyo. Kwindibaniselwano enye sisebenzise i-BACE1 peptide inhibitor kuphela kwaye kwenye sisebenzise umlinganiselo we-1:3 we-BACE1 peptide inhibitor kwi-#2077 peptide. Zombini iisampulu zinikwe ngemithambo yegazi kunye namanqanaba olwelo lwe-cerebrospinal lwe-beta-amyloid peptide 40 (Abeta40) alinganiswa ngokuhamba kwexesha. I-Abeta40 ilinganiswe kwi-CSF njengoko ibonisa ukuthintelwa kwe-BACE1 kwi-brain parenchyma. Njengoko bekulindelekile, zombini ezi complexes zinciphise kakhulu amanqanaba egazi le-Abeta40 (Umzobo 4c, d). Nangona kunjalo, kuphela iisampulu eziqulethe umxube we-peptide no. 2077 kunye ne-inhibitor ye-BACE1 peptide edityaniswe ne-SA zibangele ukwehla okukhulu kwe-Abeta40 kulwelo lwe-cerebrospinal (Umzobo 4c). Idatha ibonisa ukuba i-peptide no. 2077 iyakwazi ukuhambisa iproteni ye-60 kDa SA kwi-CNS kwaye ikwabangela iziphumo ze-pharmacological nge-SA-conjugated inhibitors ye-BACE1 peptide.
(a) Inaliti yeClonal (2 × 10 phages/isilwanyana) ye-T7 phage ebonisa iiprofayili ze-pharmacokinetic zexesha elide ze-CSF peptide #2077 (RLSSVDSDLSGC) kunye ne-uninjected control phage (#1779) ubuncinane kwiigundane ezintathu ezifakwe kwi-CM. (b) Umfanekiso we-Confocal microscopic we-cortical microvessels emeleyo kwiimpuku ezifakwe kwi-phage (2 × 10 10 phages/isilwanyana) ebonisa ukuphikisana kwe-peptide #2077 kunye nemithambo yegazi (lectin). Ezi clones ze-phage zanikwa iimpuku ezi-3 kwaye zavunyelwa ukuba zijikeleze iyure e-1 ngaphambi kokuphuma kwegazi. Ubuchopho bahlulwahlulwa baza bafakwa i-polyclonal FITC-labeled antibodies ngokuchasene ne-T7 phage capsid. Imizuzu elishumi ngaphambi kokuphuma kwegazi kunye nokulungiswa okulandelayo, i-DyLight594-labeled lectin yanikwa nge-intravenously. Imifanekiso ekhanyayo ebonisa ukudaywa kwe-lectin (ebomvu) kwicala le-luminal le-microvessels kunye ne-phages (eluhlaza) kwi-lumen ye-capillaries kunye ne-perivascular brain tissue. I-scale bar ihambelana ne-10 µm. (c, d) I-Biotinylated BACE1 inhibitory peptide yodwa okanye xa idibene ne-biotinylated transit peptide #2077 yadityaniswa ne-streptavidin yalandelwa kukufakwa kwe-intravenous ubuncinane iigundane ezintathu ze-CM ezifakwe kwi-cannulated (10 mg streptavidin/kg). Ukunciphisa kwe-BACE1 peptide inhibitor-mediated kwi-Aβ40 kwalinganiswa yi-Aβ1-40 ELISA egazini (elibomvu) kunye nolwelo lwe-cerebrospinal (orenji) kwiindawo zexesha ezichaziweyo. Ukuze kucace ngcono, umgca onamachaphaza udwetshwa kwigrafu kwisikali se-100%. (c) Ukwehla kwepesenti kwi-Aβ40 egazini (oonxantathu ababomvu) kunye nolwelo lwe-cerebrospinal (oonxantathu aba-orenji) kwiimpuku ezinyangwe nge-streptavidin edityaniswe ne-transit peptide #2077 kunye ne-BACE1 inhibitory peptide kumlinganiselo we-3:1. (d) Ukwehla kwepesenti kwigazi i-Aβ40 (iizangqa ezibomvu) kunye nolwelo lwe-cerebrospinal (izangqa ezi-orenji) kwiimpuku ezinyangwe nge-streptavidin zidibene ne-BACE1 inhibitory peptide kuphela. Uxinzelelo lwe-Aβ kulawulo yayiyi-420 pg/ml (ukuphambuka okuqhelekileyo = 101 pg/ml).
Ukuboniswa kwePhage kusetyenziswe ngempumelelo kwiindawo ezahlukeneyo zophando lwezonyango17. Le ndlela isetyenziselwe izifundo zokwahluka kwemithambo yegazi kwi-vivo18,19 kunye nezifundo ezijolise kwimithambo yegazi kwi-cerebral20,21,22,23,24,25,26. Kolu phononongo, sandise ukusetyenziswa kwale ndlela yokukhetha kungekuphela nje ekuchongeni ngokuthe ngqo iipeptides ezijolise kwimithambo yegazi kwi-cerebral, kodwa nasekufumaneni abaviwa abaneempawu zokuthutha ezisebenzayo zokuwela umqobo wegazi-ubuchopho. Ngoku sichaza uphuhliso lwenkqubo yokukhetha kwi-vivo kwiimpuku ezifakwe kwi-CM kwaye sibonisa amandla ayo okuchonga iipeptides ezineempawu ze-CSF homing. Sisebenzisa i-T7 phage ebonisa ithala leencwadi leepeptides ezingaqhelekanga ze-12-mer, sikwazile ukubonisa ukuba i-T7 phage incinci ngokwaneleyo (malunga ne-60 nm ububanzi)10 ukuze ilungelelaniswe nomqobo wegazi-ubuchopho, ngaloo ndlela iwela ngokuthe ngqo umqobo wegazi-ubuchopho okanye i-choroid plexus. Siqaphele ukuba ukuvunwa kwe-CSF kwiigundane ze-CM ezifakwe kwi-cannulated yindlela yokuhlola esebenza kakuhle emzimbeni, kwaye i-phage ekhutshiweyo ayibophelelanga nje kuphela kwi-vasculature kodwa isebenza njengomthuthi ngaphesheya komqobo wegazi-ubuchopho. Ngaphezu koko, ngokuqokelela igazi ngaxeshanye nokusebenzisa i-HTS kwi-CSF kunye nee-phage ezithathwe egazini, siqinisekisile ukuba ukukhetha kwethu i-CSF akuzange kuchaphazeleke kukutyebiswa kwegazi okanye ukufaneleka kokukhula phakathi kwemijikelo yokukhetha. Nangona kunjalo, indawo yegazi yinxalenye yenkqubo yokukhetha, kuba ii-phage ezikwaziyo ukufikelela kwindawo ye-CSF kufuneka ziphile kwaye zijikeleze egazini ixesha elide ukuze zityebiswe engqondweni. Ukuze sikhuphe ulwazi oluthembekileyo lokulandelelana kwidatha eluhlaza ye-HTS, sisebenzise izihluzi ezilungelelaniswe neempazamo zokulandelelana ezithile kwiplatifomu kumsebenzi wohlalutyo. Ngokufaka iiparameter ze-kinetic kwindlela yokuhlola, siqinisekisile i-pharmacokinetics ekhawulezayo yee-phage ze-T7 zasendle (t½ ~ 28 min) kwigazi24, 27, 28 kwaye sikwamisele nesiqingatha sobomi bazo kwi-cerebrospinal fluid (t½ ~ 26 min) ngomzuzu). Nangona iiprofayili ezifanayo ze-pharmacokinetic egazini nakwi-CSF, yi-0.001% kuphela yoxinaniso lwegazi lwe-phage olunokubonwa kwi-CSF, nto leyo ebonisa ukuhamba okuphantsi kwe-wild-type T7 phage ngaphaya komqobo wegazi-ubuchopho. Lo msebenzi ugxininisa ukubaluleka komjikelo wokuqala wokukhetha xa kusetyenziswa amaqhinga okuguqula i-in vivo, ingakumbi kwiinkqubo ze-phage ezisuswa ngokukhawuleza kumjikelezo wegazi, njengoko ii-clones ezimbalwa zikwazi ukufikelela kwicandelo le-CNS. Ke ngoko, kumjikelo wokuqala, ukuncipha kokwahluka kwethala leencwadi kwakukhulu kakhulu, njengoko kuphela inani elilinganiselweyo lee-clones ezaqokelelwa kule modeli ye-CSF engqongqo kakhulu. Le ndlela yokuguqula i-in vivo ibandakanya amanyathelo aliqela okukhetha afana nokuqokelelwa okusebenzayo kwicandelo le-CSF, ukusinda kwe-clone kwicandelo legazi, kunye nokususwa ngokukhawuleza kwee-clones ze-T7 phage egazini kwimizuzu eli-10 yokuqala (Umzobo 1d kunye nomzobo ongezelelweyo 4M). ). Ke ngoko, emva komjikelo wokuqala, ii-clones ze-phage ezahlukeneyo zachongwa kwi-CSF, nangona ichibi elifanayo lokuqala lasetyenziswa kwizilwanyana ngazinye. Oku kuthetha ukuba amanyathelo amaninzi angqongqo okukhetha amathala eencwadi aqulethe inani elikhulu lamalungu ethala leencwadi abangela ukuncipha okukhulu kokwahluka. Ke ngoko, iziganeko ezingacwangciswanga ziya kuba yinxalenye ebalulekileyo yenkqubo yokuqala yokukhetha, zichaphazele kakhulu iziphumo. Kusenokwenzeka ukuba uninzi lwee-clones kwithala leencwadi lokuqala zazinotyekelo olufanayo kakhulu lokufumisa i-CSF. Nangona kunjalo, naphantsi kweemeko ezifanayo zovavanyo, iziphumo zokukhetha zinokwahluka ngenxa yenani elincinci le-clone nganye kwidama lokuqala.
Iimotifs ezityebileyo kwi-CSF zahlukile kwezo zisegazini. Okubangel’ umdla kukuba, siphawule utshintsho lokuqala oluya kwiipeptides ezityebileyo kwi-glycine egazini lezilwanyana ngazinye. (Umzobo 1g, IiFigs ezongezelelweyo. 4e, 4f). Iipeptides ze-glycine eziqulathe i-phage zinokuba zizinzile kwaye zingabi nakwenzeka ukuba zisuswe ekujikelezeni kwegazi. Nangona kunjalo, ezi peptides zityebileyo kwi-glycine azizange zifunyanwe kwiisampuli ze-cerebrospinal fluid, nto leyo ebonisa ukuba iilayibrari ezikhethiweyo zidlule kumanyathelo amabini ahlukeneyo okukhetha: enye egazini kwaye enye ivunyelwe ukuba iqokelele kwi-cerebrospinal fluid. Ii-clones ezityebileyo kwi-CSF ezivela kumjikelo wesine wokukhetha ziye zavavanywa kakhulu. Phantse zonke ii-clones ezivavanyiweyo ngabanye ziqinisekisiwe ukuba zityebile kwi-CSF xa kuthelekiswa ne-blank control phage. I-peptide hit enye (#2077) ihlolwe ngokweenkcukacha. Ibonise i-plasma half-life ende xa ithelekiswa nezinye ii-hits (Umfanekiso 3d kunye noMfanekiso oNcedisayo 7), kwaye okunomdla kukuba, le peptide yayine-cysteine ​​​​residue kwi-C-terminus. Kutshanje kuye kwaboniswa ukuba ukongezwa kwe-cysteine ​​​​kwiipeptides kunokuphucula iipropati zazo ze-pharmacokinetic ngokubophelela kwi-albumin 29. Oku okwangoku akwaziwa nge-peptide #2077 kwaye kufuna uphando olongezelelekileyo. Ezinye iipeptides zibonise ukuxhomekeka kwe-valence kwi-CSF enrichment (idatha ayiboniswanga), enokuthi inxulumene ne-geometry yomphezulu obonisiweyo we-T7 capsid. Inkqubo ye-T7 esiyisebenzisileyo ibonise iikopi ezi-5-15 ze-peptide nganye nge-phage particle. I-IHC yenziwe kwi-candidate lead phage clones ezifakwe ngaphakathi kwi-cerebral cortex yeempuku (Umzobo oNcedisayo 8). Idatha ibonise ukuba ubuncinci ii-clones ezintathu (No. 2002, No. 2009 kunye no-No. 2077) zidibene ne-BBB. Kusafuneka kumiselwe ukuba olu nxibelelwano lwe-BBB lubangela ukuqokelelwa kwe-CSF okanye ukuhamba kwezi clones ngqo kwi-BCSFB. Okubalulekileyo, sibonisa ukuba ii-peptides ezikhethiweyo zigcina amandla azo okuthutha e-CSF xa zenziwe kwaye zibotshelelwe kwi-protein cargo. Ukubopha iipeptides ze-N-terminal biotinylated kwi-SA ngokusisiseko kuphinda iziphumo ezifunyenweyo ngee-phage clones zazo egazini nakwi-cerebrospinal fluid (Umzobo 3e). Okokugqibela, sibonisa ukuba i-lead peptide #2077 iyakwazi ukukhuthaza isenzo sobuchopho se-biotinylated peptide inhibitor ye-BACE1 edityaniswe ne-SA, nto leyo ebangela iziphumo ze-pharmacodynamic kwi-CNS ngokunciphisa kakhulu amanqanaba e-Abeta40 kwi-CSF (Umzobo 4). Asikwazanga ukuchonga naziphi na ii-homologues kwi-database ngokwenza uphando lwe-peptide sequence homology lwazo zonke ii-hits. Kubalulekile ukuqaphela ukuba ubungakanani belayibrari ye-T7 bumalunga ne-109, ngelixa ubungakanani belayibrari yethiyori ye-12-mers yi-4 x 1015. Ke ngoko, sikhethe iqhezu elincinci lesithuba sokwahluka kwelayibrari ye-12-mer peptide, nto leyo ethetha ukuba iipeptides ezilungisiweyo ngakumbi zinokuchongwa ngokuvavanya indawo ye-sequence ekufutshane yezi hits zichongiweyo. Ngokwengcamango, esinye sezizathu zokuba singafumananga naziphi na ii-homologues zemvelo zezi peptides kukungakhethwa ngexesha lokuvela kwezinto ukuze kuthintelwe ukungena okungalawulekiyo kwee-peptide motifs ezithile engqondweni.
Xa zizonke, iziphumo zethu zibonelela ngesiseko somsebenzi wexesha elizayo wokuchonga nokuchonga iinkqubo zothutho lomqobo we-cerebrovascular in vivo ngokweenkcukacha ezithe vetshe. Useto olusisiseko lwale ndlela lusekelwe kwisicwangciso sokukhetha esisebenzayo esingachongi kuphela ii-clones ezineempawu zokubopha imithambo yegazi yobuchopho, kodwa sikwabandakanya inyathelo elibalulekileyo apho ii-clones eziphumelelayo zinomsebenzi wangaphakathi wokuwela imiqobo yebhayoloji in vivo ziye kwigumbi le-CNS. kukucacisa indlela yokuthutha ezi peptides kunye nokukhetha kwazo ukubopha kwi-microvasculature ethile kwindawo yobuchopho. Oku kunokukhokelela ekufumaneni iindlela ezintsha zokuthutha i-BBB kunye nee-receptors. Silindele ukuba ii-peptides ezichongiweyo zinokubophelela ngokuthe ngqo kwii-receptors ze-cerebrovascular okanye kwii-ligands ezijikelezayo ezithuthwa nge-BBB okanye i-BCSFB. Ii-peptide vectors ezinomsebenzi wokuthutha we-CSF ezifunyenwe kulo msebenzi ziya kuphandwa ngakumbi. Okwangoku siphanda ubunyani bobuchopho bezi peptides ukuze sibone amandla azo okuwela i-BBB kunye/okanye i-BCSFB. Ezi peptides zintsha ziya kuba zizixhobo ezixabisekileyo kakhulu ekufumaneni ii-receptors okanye iindlela ezintsha kunye nophuhliso lwamaqonga amatsha asebenza kakuhle ekuhambiseni ii-macromolecules, ezifana ne-biologics, engqondweni.
Hlamba i-cisterna enkulu (CM) usebenzisa utshintsho lwendlela echazwe ngaphambili. Iigundane ze-Wistar ezifakwe i-anesthesia (200-350 g) zifakwe kwisixhobo se-stereotaxic kwaye kwenziwa umngxuma ophakathi phezu kwentloko echetyiweyo nelungiselelwe i-aseptic ukuze kuvele ukhakhayi. Gqobhoza imingxunya emibini kwindawo yesash ephezulu uze ubophe izikrufu zokulungisa kwimingxunya. Umngxunya owongezelelweyo ugqobhoziwe kwi-lateral occipital crest ukuze kukhokelwe i-stereotactic ye-cannula yentsimbi engagqwaliyo kwi-CM. Faka isamente yamazinyo ejikeleze i-cannula kwaye uyiqinise ngezikrufu. Emva kokulungiswa ngefoto kunye nokuqina kwesamente, inxeba lesikhumba livalwe nge-4/0 supramid suture. Ukubekwa ngokufanelekileyo kwe-cannula kuqinisekiswa ngokuvuza okuzenzekelayo kolwelo lwe-cerebrospinal (CSF). Susa igundane kwisixhobo se-stereotaxic, ufumane unyango olufanelekileyo emva kotyando kunye nolawulo lweentlungu, kwaye uyivumele ukuba iphile ubuncinane iveki enye de kubonakale iimpawu zegazi kwi-cerebrospinal fluid. Iigundane ze-Wistar (Crl:WI/Han) zifunyenwe kwiCharles River (France). Zonke iimpuku zigcinwe phantsi kweemeko ezithile ezingenazo iintsholongwane. Zonke iimvavanyo zezilwanyana zivunyiwe yiOfisi yeZilwanyana yeSixeko saseBasel, eSwitzerland, kwaye zenziwe ngokuhambelana neLayisensi yeZilwanyana Nombolo 2474 (Uvavanyo lweThutho loBuchopho oluSebenzayo ngokulinganisa amanqanaba abaFundi boNyango kwiCerebrospinal Fluid kunye neBrain of Rat).
Gcina igundane lisesichengeni, i-CM cannula isesandleni. Susa iDatura kwi-cannula uze uqokelele i-10 µl yolwelo lwe-cerebrospinal oluhamba ngokwalo. Ekubeni i-patency ye-cannula ekugqibeleni yayichaphazeleka, kuphela iisampulu zolwelo lwe-cerebrospinal ezicacileyo ezingenabungqina bokungcoliswa kwegazi okanye ukutshintsha kombala ezifakiwe kolu phononongo. Kwangaxeshanye, malunga ne-10-20 μl yegazi yathathwa kwi-incision encinci encotsheni yomsila yaya kwiityhubhu ezine-heparin (Sigma-Aldrich). I-CSF kunye negazi zaqokelelwa ngamaxesha ahlukeneyo emva kokufakwa kwi-intravenous ye-T7 phage. Malunga ne-5-10 μl yolwelo yalahlwa ngaphambi kokuba kuqokelelwe isampulu nganye ye-CSF, ehambelana nomthamo ofileyo we-catheter.
Iilayibrari zenziwe kusetyenziswa i-T7Select 10-3b vector njengoko kuchaziwe kwi-T7Select system manual (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). Ngamafutshane, i-DNA insert engacwangciswanga ye-12-mer yenziwe ngale ndlela ilandelayo:
I-NNK codon yasetyenziselwa ukunqanda ii-codons ezima kabini kunye nokwanda kwe-amino acid kwi-insert. I-N yi-equimolar ratio exutywe ngesandla ye-nucleotide nganye, kwaye i-K yi-equimolar ratio exutywe ngesandla ye-adenine kunye ne-cytosine nucleotides. Iindawo ezimabini ezimabini zaguqulwa zaba yi-double stranded DNA ngokufaka i-dNTP (Novagen) kunye ne-Klenow enzyme (New England Biolabs) kwi-Klenow buffer (New England Biolabs) iiyure ezi-3 kwi-37°C. Emva kwe-reaction, i-double-stranded DNA yafunyanwa yi-EtOH precipitation. I-DNA eyaphumayo yagaywa ngee-restriction enzymes EcoRI kunye neHindIII (zombini zivela kwi-Roche). I-clipped and purified (QIAquick, Qiagen) insert (T4 ligase, New England Biolabs) yaza yafakwa kwi-frame kwi-T7 vector e-pre-claved emva kwe-amino acid 348 ye-10B capsid gene. Ii-Ligation reactions zafakwa kwi-16°C kangangeeyure ezili-18 ngaphambi kokupakishwa kwi-vitro. Ukupakishwa kwePhage kwi-vitro kwenziwe ngokwemiyalelo enikwe yi-T7Select 10-3b cloning kit (Novagen) kwaye isisombululo sokupakishwa sandiswa kube kanye ukuze sinyibilike kusetyenziswa i-Escherichia coli (BLT5615, Novagen). Ii-lysates zafakwa kwi-centrifuge, zaza zaqandiswa kwi--80° C. njengesisombululo se-glycerol esitokhweni.
Ukwandiswa kwe-PCR ngokuthe ngqo kweendawo eziguquguqukayo ze-phage ezikhuliswe kwi-broth okanye kwipleyiti kusetyenziswa ii-primer ze-fusion ze-454/Roche-amplicon ezizimeleyo. I-primer ye-fusion yangaphambili iqulethe ulandelelwano olujikeleze i-variable region (NNK) 12 (ethile kwitemplate), i-GS FLX Titanium Adapter A, kunye nolandelelwano lwezitshixo zelayibrari ezine-base (TCAG) (Umfanekiso ongezelelweyo 1a):
I-reverse fusion primer ikwanayo ne-biotin eqhotyoshelweyo kwi-capture beads kunye ne-GS FLX Titanium Adapter B efunekayo kwi-clonal amplification ngexesha le-emulsion PCR:
Emva koko ii-amplicons zafakwa kwi-454/Roche pyrosequencing ngokwe-454 GS-FLX Titanium protocol. Kwi-manual Sanger sequencing (Applied Biosystems Hitachi 3730 xl DNA Analyzer), i-T7 phage DNA yandiswa yi-PCR yaza yalandelelaniswa ngezi zibini zilandelayo ze-primer:
Izinto ezifakwe kwiiplaque ngazinye ziye zafakwa kwi-PCR amplification kusetyenziswa iRoche Fast Start DNA Polymerase Kit (ngokwemiyalelo yomenzi). Yenza i-hot start (imizuzu eli-10 kwi-95 °C) kunye nemijikelo engama-35 yokunyusa (imizuzwana engama-50 kwi-95 °C, umzuzu o-1 kwi-50 °C, kunye nomzuzu o-1 kwi-72 °C).
I-Phage evela kwiilayibrari, i-phage yohlobo lwe-wild-type, i-phage ehlangulwe kwi-CSF kunye negazi, okanye ii-clones zodwa zandiswa kwi-Escherichia coli BL5615 kwi-TB broth (Sigma Aldrich) okanye kwizitya ezingama-500 cm2 (Thermo Scientific) iiyure ezi-4 kwi-37°C. I-Phage yakhutshwa kwiiplate ngokuhlanjwa kweeplate nge-Tris-EDTA buffer (Fluka Analytical) okanye ngokuqokelelwa kweeplaque ngeencam ze-pipette ezingeyongozi. I-Phage yahlulwa kwi-culture supernatant okanye kwi-extraction buffer nge-round enye ye-polyethylene glycol (PEG 8000) precipitation (Promega) yaza yaphinda yaxhonywa kwi-Tris-EDTA buffer.
I-phage ekhulisiweyo yenziwe imijikelo emi-2-3 yokususwa kwe-endotoxin kusetyenziswa ii-endotoxin removal beads (Miltenyi Biotec) ngaphambi kokufakwa kwi-intravenous (IV) injection (500 μl/animal). Kumjikelo wokuqala, kwangeniswa ii-phages ezi-2×1012; kowesibini, ii-phages ezi-2×1010; kumjikelo wesithathu nowesine wokukhetha, ii-phages ezi-2×109 ngesilwanyana ngasinye. Umxholo we-phage kwi-CSF nakwiisampuli zegazi eziqokelelwe ngamaxesha athile umiselwe ngokubalwa kwe-plaque ngokwemiyalelo yomenzi (i-T7Select system manual). Ukukhethwa kwe-phage kwenziwe ngokufaka i-intravenous kwiilayibrari ezicociweyo kumthambo womsila okanye ngokufaka kwakhona i-phage ekhutshwe kwi-CSF kumjikelo wokukhetha wangaphambili, kwaye ukuvuna okulandelayo kwenziwa kwimizuzu eli-10, imizuzu engama-30, imizuzu engama-60, imizuzu engama-90, imizuzu eli-120, imizuzu eli-180, kunye nemizuzu engama-240 ngokulandelelanayo. Kuqhutywe imijikelo emine iyonke yokugalela i-in vivo apho amasebe amabini akhethiweyo agcinwa ngokwahlukeneyo kwaye ahlalutywa ngexesha lemijikelo emithathu yokuqala yokukhetha. Zonke ii-phage inserts ezikhutshwe kwi-CSF kwimijikelo emibini yokuqala yokukhetha zenziwe nge-454/Roche pyrosequencing, ngelixa zonke ii-clones ezikhutshwe kwi-CSF kwimijikelo emibini yokugqibela yokukhetha zenziwe nge-hand sequencing. Zonke ii-blood phages ezivela kumjikelo wokuqala wokukhetha nazo zenziwe nge-454/Roche pyrosequencing. Ukuze kufakwe ii-phage clones, ii-phages ezikhethiweyo zandiswa kwi-E. coli (BL5615) kwiiplate ezingama-500 cm2 kuma-37°C kangangeeyure ezi-4. Ii-clones ezikhethwe ngabanye nezilandelelaniswe ngesandla zasasazeka kwi-TB medium. Emva kokukhupha ii-phage, ukucocwa kunye nokususwa kwe-endotoxin (njengoko kuchaziwe apha ngasentla), ii-phages ezi-2 × 1010/isilwanyana kwi-300 μl zafakwa nge-intravenously kwi-tail vein enye.
Ukulungiswa kwangaphambili kunye nokuhluzwa kweenkcukacha-manani zedatha yolandelelwano. Idatha engavuthwanga ye-454/Roche iguqulwe ukusuka kwifomathi yemephu yomlambo eqhelekileyo ye-binary (sff) ukuya kwifomathi efundekayo yomntu yePearson (fasta) kusetyenziswa isoftware yomthengisi. Ukulungiswa ngakumbi kolandelelwano lwe-nucleotide kwenziwe kusetyenziswa iinkqubo ze-C kunye nezikripthi (iphakheji yesoftware engakhutshwanga) njengoko kuchaziwe ngezantsi. Uhlalutyo lwedatha ephambili luquka iinkqubo ezingqongqo zokucoca izigaba ezininzi. Ukucoca ukufundwa okungaqulathanga ulandelelwano olusebenzayo lwe-12mer insert DNA, ukufundwa kwalungelelaniswa ngokulandelelana kwileyibhile yokuqala (GTGATGTCGGGGATCCGAATTCT), ileyibhile yokumisa (TAAGCTTGCGGGCGCACTCGAGTA) kunye ne-background insert (CCCTGCAGGGATATCCCGGGAGCTCGTCGAC) kusetyenziswa uvavanyo lwe-Needleman-Wunsch lwehlabathi. ukulungelelaniswa okuvumela ukuya kuthi ga kwi-2 ukungangqinelani ngokulungelelaniswa ngakunye31. Ke ngoko, ukufundwa ngaphandle kweethegi zokuqala kunye nokumisa kunye nokufundwa okuqulethe ukufakelwa kwemvelaphi, oko kukuthi, ukulungelelaniswa okugqitha inani elivunyelweyo lokungafani, kususiwe kwithala leencwadi. Ngokuphathelele izinto ezisele, ulandelelwano lwe-N-mer DNA oluqala kuphawu lokuqala luze luphele ngaphambi kokuba uphawu lokumisa lukhutshwe kulandelelwano lokuqala lokufunda kwaye luqhubekeke (emva koko lubizwa ngokuba "yi-insert"). Emva kokuguqulelwa kwe-insert, inxalenye emva kwe-codon yokuqala yokumisa kwisiphelo se-5′ se-primer isuswa kwi-insert. Ukongeza, ii-nucleotides ezikhokelela kwii-codon ezingaphelelanga kwisiphelo se-3′ se-primer nazo zisusiwe. Ukuze kukhutshwe izinto ezifakwe kuphela ulandelelwano lwangasemva, izinto eziguqulelweyo eziqala ngephethini ye-amino acid "PAG" nazo zisusiwe. Ii-peptides ezinobude be-post-translational obungaphantsi kwe-3 amino acids zisusiwe kwithala leencwadi. Okokugqibela, susa i-redundancy kwi-insert pool kwaye umisele i-frequency ye-insert nganye eyahlukileyo. Iziphumo zolu hlalutyo ziquka uluhlu lwe-nucleotide sequences (i-inserts) kunye ne-frequency yazo (i-read) (Imifanekiso eyongezelelweyo 1c kunye no-2).
Ukufakwa kweDNA yeQela le-N-mer ngokufana kolandelelwano: Ukuze kususwe iimpazamo zokulandelelana ezithile ze-454/Roche (ezifana neengxaki zokwandiswa kwe-homopolymer yokulandelelanisa) kunye nokususa ukuphindaphinda okungabalulekanga kangako, ukufakelwa kokulandelelana kwe-N-mer DNA okuhluziweyo ngaphambili (ukufakwa) kuhlelwe ngokufakwa kokufana. (ukuya kuthi ga kwiziseko ezi-2 ezingahambelaniyo ezivunyelweyo) kusetyenziswa i-algorithm ephindaphindayo echazwe ngolu hlobo lulandelayo: ukufakelwa kuhlelwe kuqala ngokwexesha lazo (eliphezulu ukuya kwelona liphantsi), kwaye ukuba ziyafana, ngokohlobo lwazo lwesibini ngobude (elide ukuya kwelona lifutshane) ). Ke ngoko, ukufakelwa okuqhelekileyo nokude kakhulu kuchaza "iqela" lokuqala. Ukuphindaphinda kweqela kusetelwe kwi-frequency yesitshixo. Emva koko, ukufakelwa ngakunye okusele kuluhlu oluhleliweyo kwazanywa ukongezwa kwiqela ngokulungelelaniswa kwe-Needleman-Wunsch ngababini. Ukuba inani lokungafani, ukufakelwa, okanye ukususwa kulungelelwaniso alidluli umda we-2, ukufakelwa kongezwa kwiqela, kwaye ukuphindaphinda kweqela liphela kunyuswa ngokuba ukufakelwa kongezwe kangaphi. Ukufakwa kongezwe kwiqela kuphawulwa njengokusetyenziswa kwaye kungabandakanywa ekuqhubekeni nokuqhubekeka. Ukuba ulandelelwano lokufaka alunakongezwa kwiqela esele likho, ulandelelwano lokufaka lusetyenziselwa ukudala iqela elitsha elinemvamisa efanelekileyo yokufaka kwaye luphawulwe njengolusetyenzisiweyo. Ukuphindaphinda kuphela xa ulandelelwano ngalunye lokufaka lusetyenziselwe ukwenza iqela elitsha okanye lunokufakwa kwiqela esele likho. Emva kwayo yonke loo nto, ukufakelwa kwamaqela okuquka iinucleotides ekugqibeleni kuguqulelwa kwi-peptide sequences (iilayibrari zepeptide). Isiphumo solu hlalutyo siseti yokufakwa kunye nemvamisa yazo ehambelanayo eyenza inani lokufundwa okulandelanayo (Umzobo ongezelelweyo 2).
Ukuveliswa kweMotif: Ngokusekelwe kuluhlu lweepeptides ezizodwa, kwadalwa ilayibrari equlathe zonke iipateni ze-amino acid (aa) ezinokwenzeka njengoko kubonisiwe ngezantsi. Ipateni nganye enokwenzeka yobude obuyi-3 yakhutshwa kwi-peptide kwaye ipateni yayo echaseneyo yongezwa kunye nelayibrari eqhelekileyo yemotif equlathe zonke iipateni (ii-tripeptides). Amathala eencwadi eemotifs eziphindaphindwayo kakhulu alandelelaniswa kwaye kwasuswa ukuphinda-phinda. Emva koko, kwi-tripeptide nganye kwilayibrari yemotif, sijonge ukuba ikhona na kwilayibrari sisebenzisa izixhobo zokubala. Kule meko, ukuphindaphinda kwe-peptide equlathe i-tripeptide yemotif efunyenweyo kongezwa kwaye kwabelwa imotif kwilayibrari yemotif ("inani leemotifs"). Isiphumo sokuveliswa kwemotif luluhlu olunemilinganiselo emibini oluqulathe zonke iziganeko ze-tripeptides (iimotifs) kunye namaxabiso azo, ezilinani lee-sequencing reads eziphumela kwimotif ehambelanayo xa ii-reads zihluzwa, ziqokelelwa, kwaye ziguqulelwa. Iimetrics njengoko kuchaziwe ngokweenkcukacha apha ngasentla.
Ukulungiswa kwenani lee-motifs kunye nee-scatterplots ezifanelekileyo: Inani lee-motifs kwisampulu nganye lilungisiwe kusetyenziswa
apho u-ni linani lee-reads eziqulathe isihloko u-i. Ngoko ke, u-vi umele ipesenti ye-reads (okanye ii-peptides) eziqulathe i-motif u-i kwisampulu. Amaxabiso e-P enani lee-motifs ezingamiselwanga kakuhle abalwe kusetyenziswa uvavanyo oluchanekileyo lukaFisher. Ngokuphathelele ii-correlograms zenani lee-motifs, ii-correlations zikaSpearman zibalwe kusetyenziswa inani lee-motifs ezimiselweyo kunye no-R.
Ukuze kubonwe umxholo wee-amino acids kwindawo nganye kwilayibrari ye-peptide, kudalwe iilogogram zewebhu 32, 33 (http://weblogo.threeplusone.com). Okokuqala, umxholo wee-amino acids kwindawo nganye ye-peptide ye-12-mer ugcinwa kwi-matrix ye-20×12. Emva koko, iseti yeepeptide ezili-1000 eziqulethe umxholo ofanayo we-amino acid kwindawo nganye iveliswa kwifomathi ye-fasta-sequence kwaye inikwe njengegalelo kwi-logo yewebhu 3, evelisa umzobo womxholo we-amino acid ohambelana nendawo nganye. kwilayibrari ye-peptide ethile. Ukuze kubonwe iiseti zedatha ezininzi, iimephu zobushushu zenziwe kusetyenziswa isixhobo esiphuhliswe ngaphakathi kwi-R (i-biosHeatmap, iphakheji ye-R engekakhutshwa). Ii-dendrograms eziboniswe kwiimephu zobushushu zibalwe kusetyenziswa indlela ye-hierarchical clustering kaWard kunye ne-Euclidean distance metric. Uhlalutyo lwezibalo lwedatha yokubeka amanqaku e-motif, amaxabiso e-P okufumana amanqaku angaqhelekanga abalwe kusetyenziswa uvavanyo oluchanekileyo lukaFisher. Amaxabiso e-P kwezinye iiseti zedatha abalwe kwi-R kusetyenziswa uvavanyo lwe-t lomfundi okanye i-ANOVA.
Ii-phage clones ezikhethiweyo kunye nee-phages ezingenazo izinto ezifakiweyo zifakwe ngemithambo yegazi nge-tail vein (2×1010 phages/animal kwi-300 μl PBS). Imizuzu elishumi ngaphambi kokufakwa kwegazi kunye nokufakwa okulandelayo, izilwanyana ezifanayo zifakwe ngemithambo yegazi nge-100 μl ye-DyLight594-labeled lectin (Vector Laboratories Inc., DL-1177). Imizuzu engama-60 emva kokufakwa kwe-phage, iimpuku zifakwe ngemithambo yegazi nge-50 ml PBS kulandele i-50 ml 4% PFA/PBS. Iisampuli zobuchopho zongezwa ngobusuku bonke kwi-4% PFA/PBS kwaye zifakwe kwi-30% sucrose ubusuku bonke kwi-4°C. Iisampuli ziqandisiwe kwi-OCT mix. Uhlalutyo lwe-immunohistochemical lweesampuli ezikhenkcezisiweyo lwenziwe kubushushu begumbi kwi-cryosections ezingama-30 µm ezivalwe nge-1% BSA kwaye zafakwa kwi-antibodies ezine-polyclonal FITC-labeled against T7 phage (Novus NB 600-376A) kwi-4 °C. Zifake kwi-incubation ubusuku bonke. Ekugqibeleni, ezi nxalenye zahlanjwa izihlandlo ezi-3 nge-PBS zaza zahlolwa nge-confocal laser microscope (Leica TCS SP5).
Zonke iipeptides ezinobumsulwa obuncinci be-98% zenziwe yiGenScript USA, zaza zafakwa kwi-biotinylated. I-Biotin ibotshelelwe nge-triple glycine spacer eyongezelelweyo kwi-N-terminus. Jonga zonke iipeptides usebenzisa i-mass spectrometry.
I-Streptavidin (Sigma S0677) ixutywe ne-peptide ye-biotinylated equimolar ephindwe kahlanu, i-peptide ye-BACE1 ethintelayo eyenziwe yi-biotinylated, okanye indibaniselwano (3:1 ratio) ye-peptide ye-BACE1 ethintelayo eyenziwe yi-biotinylated kunye ne-peptide ye-BACE1 ethintelayo kwi-5–10% DMSO/incubated kwi-PBS. Iyure e-1 kubushushu begumbi ngaphambi kokufakwa. Ii-peptides ezidityanisiweyo ze-Streptavidin zifakwe nge-intravenously ngedosi ye-10 mg/kg kwenye yemithambo yomsila yeempuku ezine-cerebral cavity.
Uxinaniso lwee-complexes ze-streptavidin-peptide luhlolwe yi-ELISA. Iiplate ze-Nunc Maxisorp microtiter (Sigma) zigqunywe ubusuku bonke kwi-4°C nge-1.5 μg/ml ye-mouse anti-streptavidin antibody (Thermo, MA1-20011). Emva kokuvala (i-blocking buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) kubushushu begumbi iiyure ezi-2, hlamba ipleyiti nge-0.05% Tween-20/PBS (i-wash buffer) kangangeeyure ezi-3, iisampulu ze-CSF kunye ne-plasma zongezwa kwimithombo exutywe ne-blocking buffer (plasma 1:10,000, CSF 1:115). Ipleyiti emva koko yafakwa kwi-incubated ubusuku bonke kwi-4°C nge-detection antibody (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Emva kwamanyathelo amathathu okuhlamba, i-streptavidin yafunyanwa ngokufaka i-incubation kwisisombululo se-TMB substrate (Roche) ukuya kuthi ga kwimizuzu engama-20. Emva kokumisa uphuhliso lombala nge-1M H2SO4, linganisa ukufunxwa kwi-450 nm.
Umsebenzi we-streptavidin-peptide-BACE1 inhibitor complex uhlolwe yi-Aβ(1-40) ELISA ngokwemigaqo yomenzi (Wako, 294-64701). Ngamafutshane, iisampulu ze-CSF zaxutywa kwi-standard diluent (1:23) zaza zafakwa kwi-incuent ubusuku bonke kwi-4°C kwiiplate ze-96-well plates ezigqunywe yi-BNT77 capture antibody. Emva kwamanyathelo amahlanu okuhlamba, i-antibody ye-BA27 ehlanganiswe ne-HRP yongezwa yaza yafakwa kwi-incubation iiyure ezi-2 kwi-4°C., kulandele amanyathelo amahlanu okuhlamba. I-Aβ(1–40) yafunyanwa ngokufaka kwisisombululo se-TMB imizuzu engama-30 kubushushu begumbi. Emva kokuba uphuhliso lombala luyekisiwe ngesisombululo sokumisa, linganisa ukufunxwa kwi-450 nm. Iisampulu ze-plasma zafakwa kwi-solid phase extraction ngaphambi kwe-Aβ(1–40) ELISA. I-Plasma yongezwa kwi-0.2% DEA (Sigma) kwiiplate ze-96-wells zaza zafakwa kwi-incubation kubushushu begumbi imizuzu engama-30. Emva kokuhlamba iipleyiti ze-SPE ngokulandelelana (Oasis, 186000679) ngamanzi kunye ne-100% methanol, iisampulu zeplasma zongezwa kwiipleyiti ze-SPE kwaye lonke ulwelo lwasuswa. Iisampulu zahlanjwa (kuqala nge-5% methanol emva koko nge-30% methanol) zaza zahluzwa nge-2% NH4OH/90% methanol. Emva kokomisa i-eluate kwi-55°C kangangemizuzu engama-99 kwi-constant N2 current, iisampulu zancitshiswa kwii-diluents ezisemgangathweni kwaye i-Aβ(1–40) yalinganiswa njengoko kuchaziwe apha ngasentla.
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