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Isithintelo segazi-ingqondo kunye nesithintelo segazi-ingqondo sithintela i-biotherapeutic agents ekufikeleleni kwiithagethi zabo kwinkqubo ye-nervous central, ngaloo ndlela ithintela unyango olusebenzayo lwezifo ze-neurological.Ukufumana abathuthi bengqondo yenoveli kwi-vivo, sazise ithala leencwadi le-T7 phage peptide kwaye saqokelela igazi ngokulandelelana kunye nolwelo lwe-cerebrospinal (CSF) sisebenzisa imodeli enkulu yeempuku ekwaziyo.Iiclones ezithile zephage ziye zatyetyiswa kakhulu kwi-CSF emva kwemijikelo emine yokukhetha.Uvavanyo lweepeptides zomgqatswa ngamnye luveze ngaphezulu kwe-1000-fold enrichment kwi-CSF.I-bioactivity yokuhanjiswa kwe-peptide-mediated ukuya kwingqondo yaqinisekiswa ngokunciphisa kwe-40% kwinqanaba le-amyloid-β kwi-cerebrospinal fluid usebenzisa i-BACE1 peptide inhibitor edityaniswe ne-novel transit peptide echongiweyo.Ezi ziphumo zibonisa ukuba iipeptide ezichongiweyo ngeendlela zokukhetha i-vivo phage zinokuba zizithuthi eziluncedo zokuhanjiswa kwenkqubo yeemacromolecules kwingqondo enesiphumo sonyango.
Inkqubo ye-nervous central (CNS) uphando lonyango olujoliswe kuyo lujolise kakhulu ekuchongeni amachiza afanelekileyo kunye nee-arhente ezibonisa iipropati ezijoliswe kwi-CNS, kunye nomgudu omncinci wokufumanisa iindlela eziqhuba ukuhanjiswa kweziyobisi ezisebenzayo kwingqondo.Oku kuyaqala ukutshintsha ngoku njengoko ukuhanjiswa kweziyobisi, ngakumbi iimolekyuli ezinkulu, ziyinxalenye yophuhliso lwamachiza lwale mihla lweneuroscience.Imekobume yenkqubo ye-nervous ephakathi ikhuselwe kakuhle yinkqubo yesithintelo se-cerebrovascular, ebandakanya umqobo we-blood-brain barrier (BBB) ​​kunye nomqobo we-blood-brain barrier (BCBB)1, okwenza kube nzima ukuhambisa iziyobisi kwi-brain1,2.Kuqikelelwa ukuba phantse onke amachiza emolekyuli amakhulu kwaye ngaphezulu kwe-98% yamachiza amancinci emolekyuli ayakhutshwa kwingqondo3.Yingakho kubaluleke kakhulu ukuchonga iinkqubo ezintsha zokuthutha ubuchopho ezibonelela ngokufanelekileyo kunye nokuhanjiswa okuthe ngqo kweziyobisi zonyango kwi-CNS 4,5.Nangona kunjalo, i-BBB kunye ne-BCSFB zikwanika ithuba elibalaseleyo lokuhanjiswa kweziyobisi njengoko zingena kwaye zingena kuzo zonke izakhiwo zengqondo nge-vasculature yayo ebanzi.Ngaloo ndlela, iinzame zangoku zokusebenzisa iindlela ezingabonakaliyo zokuhanjiswa kwengqondo zisekelwe kakhulu kwindlela yokuthutha i-receptor-mediated transport (PMT) usebenzisa i-BBB6 receptor engapheliyo.Ngaphandle kwenkqubela phambili engundoqo yamva nje kusetyenziswa i-transferrin receptor pathway7,8, uphuhliso olongezelelweyo lweenkqubo ezintsha zonikezelo ezineepropati eziphuculweyo ziyafuneka.Ukuza kuthi ga ngoku, injongo yethu yayikukuchonga iipeptide ezikwaziyo ukulamla uthutho lweCSF, njengoko zinokuthi ngokwemigaqo zisetyenziswe ukuhambisa i-macromolecules kwi-CNS okanye ukuvula iindlela ezintsha zokwamkela.Ngokukodwa, ii-receptors ezithile kunye nabathuthi benkqubo ye-cerebrovascular (BBB kunye ne-BSCFB) banokusebenza njengeethagethi ezinokubakho zonikezelo olusebenzayo kunye oluthile lwamachiza e-biotherapeutic.I-Cerebrospinal fluid (CSF) yimveliso eyimfihlo ye-choroid plexus (CS) kwaye idibene ngokuthe ngqo ne-interstitial fluid yobuchopho ngesithuba se-subarachnoid kunye ne-ventricular space4.Kutshanje kuye kwaboniswa ukuba i-subarachnoid cerebrospinal fluid isasazeka ngokugqithisileyo kwi-interstitium yobuchopho9.Sinethemba lokufikelela kwisithuba se-parenchymal sisebenzisa le phecana lokungena kwe-subarachnoid okanye ngokuthe ngqo nge-BBB.Ukufezekisa oku, siphumeze isicwangciso esomeleleyo sokukhetha i-vivo phage echonga iipeptides ezithuthwa ngenye yezi ndlela zimbini zahlukileyo.
Ngoku sichaza ulandelelwano kwi-vivo phage ukubonisa indlela yokuhlola kunye ne-CSF isampuli edityaniswe nolandelelwano oluphezulu lwe-throughput (HTS) ukujonga imijikelo yokukhetha yokuqala kunye nokwahlukana kwethala leencwadi eliphezulu.Uvavanyo lwenziwa kwiimpuku ezinolwazi nge-cisterna enkulu efakwe ngokusisigxina (CM) cannula ukuphepha ukungcoliswa kwegazi.Okubalulekileyo, le ndlela ikhetha zombini ukujoliswa kwengqondo kunye neepeptides ngomsebenzi wothutho kuwo wonke umqobo we-cerebrovascular.Sasebenzisa i-T7 phages ngenxa yobukhulu bawo obuncinci (~ 60 nm) kwaye sicebise ukuba zifanelekile ukuthuthwa kwee-vesicles ezivumela ukuwela i-transcellular ye-endothelial kunye / okanye i-epithelial-medulla barrier.Emva kwemijikelo emine ye-panning, abantu be-phage babebodwa bebonisa ukomelela kwi-vivo CSF ​​​​kunye nombutho we-cerebral microvessel.Okubalulekileyo, siye sakwazi ukuqinisekisa iziphumo zethu ngokubonisa ukuba iipeptides ezikhethiweyo kunye neekhemikhali zabaviwa ziyakwazi ukuthutha i-protein cargo kwi-cerebrospinal fluid.Okokuqala, iziphumo ze-pharmacodynamic ze-CNS zasekwa ngokudibanisa i-peptide ehamba phambili ehamba phambili kunye ne-inhibitor ye-BACE1 peptide.Ukongeza ekuboniseni ukuba kwi-vivo ubuchule bokuhlola obusebenzayo bunokuchonga i-peptides yothutho yobuchopho inoveli njengabathwali beprotheyini abasebenzayo, silindele iindlela ezifanayo zokukhetha ezisebenzayo ukuba nazo zibaluleke ekuchongeni iindlela ezintsha zothutho lobuchopho.
Ngokusekelwe kwiiyunithi ze-plaque-forming units (PFU), emva kwesinyathelo sokupakisha i-phage, ilayibrari ye-random 12-mer linear T7 phage peptides eneentlobo ngeentlobo malunga ne-109 yenzelwe kwaye yadalwa (jonga i-Materials and Methods).Kubalulekile ukuqaphela ukuba sihlalutye ngononophelo eli thala leencwadi phambi kwe-vivo panning.Ukwandiswa kwe-PCR yeesampulu zethala leencwadi ze-phage usebenzisa ii-primers ezilungisiweyo ezenziwe nge-amplicon ezazisebenza ngokuthe ngqo kwi-HTS (i-Supplementary Fig. 1a).Ngenxa ye) HTS11 iimpazamo zolandelelwano, b) impembelelo kumgangatho we-primers (NNK) 1-12, kunye c) nobukho be-wild-type (wt) phage (ukufakwa kwamathambo) kwilayibrari yokulinda, inkqubo yokucoca ngokulandelelana yaphunyezwa ukukhupha kuphela ulwazi oluqinisekisiweyo olulandelelanayo (i-Supplementary Fig. 1b).La manyathelo okucoca asebenza kuwo onke amathala eencwadi alandelelanayo e-HTS.Kwithala leencwadi elisemgangathweni, inani lilonke elifundiweyo elingama-233,868 lifunyenwe, apho i-39% iphumelele ikhrayitheriya yokuhluza kwaye yasetyenziselwa uhlalutyo lwethala leencwadi nokukhetha imijikelo elandelayo (uMzobo owoNgezelelweyo 1c–e).Ukufundwa bekuyi-multiples kakhulu ye-3 izibini zesiseko ubude kunye nencopho kwi-nucleotides ye-36 (i-Supplementary Fig. 1c), eqinisekisa ukuyila kwethala leencwadi (NNK) 1-12.Okuqaphelekayo, malunga ne-11% yamalungu ethala leencwadi aqulethe i-12-dimensional wild-type (wt) umqolo we-PAGISRELVDKL, kwaye phantse isiqingatha solandelelwano (49%) luqulathe izinto ezifakelweyo okanye ezicinyiweyo.I-HTS yethala leencwadi iqinisekise iyantlukwano ephezulu yeepeptides kwithala leencwadi: ngaphezu kwe-81% yolandelelwano lwe-peptide lufunyenwe kanye kuphela kwaye yi-1.5% kuphela eyenzeka kwiikopi ≥4 (umzobo owongezelelweyo 2a).I-frequencies of amino acids (aa) kuzo zonke izikhundla ze-12 kwi-repertoire zihambelana kakuhle kunye namaxesha alindelekileyo kwinani leecodon eziveliswa yi-NKK repertoire eyongezelelweyo (i-Supplementary Fig. 2b).I-frequency eqatshelweyo yee-aa eziseleyo ezifakwe ezi zifakelo zihambelana kakuhle kunye ne-frequency ebalwayo (r = 0.893) (i-Supplementary Fig. 2c).Ukulungiswa kwamathala eencwadi e-phage yokujova kubandakanya amanyathelo okukhulisa kunye nokususwa kwe-endotoxin.Oku ngaphambili kubonakaliswe ukuba kunokunciphisa ukwahlukana kwamathala eencwadi eefage12,13.Ke ngoko, silandelelanise ithala leencwadi le-plate-amplified phage eliye lasuswa i-endotoxin kwaye sayithelekisa nethala leencwadi lokuqala ukuqikelela ukuphindaphinda kwe-AA.Ukulungelelaniswa okuqinileyo (r = 0.995) kwabonwa phakathi kwe-pool yasekuqaleni kunye ne-pool ekhulisiwe kunye nehlanjululwe (i-Supplementary Fig. 2d), ebonisa ukuba ukhuphiswano phakathi kwama-clones akhuliswe kwiiplate usebenzisa i-T7 phage ayizange ibangele ukuxhatshazwa okukhulu.Olu thelekiso lusekwe kumaxesha amaninzi eemotifs ze-tripeptide kwithala leencwadi ngalinye, njengoko iyantlukwano yamathala eencwadi (~109) ayinakubanjwa ngokupheleleyo nange-HTS.Uhlalutyo oluqhelekileyo lwe-aa kwindawo nganye lubonise i-bias encinci exhomekeke kwisikhundla kwiindawo ezintathu zokugqibela ze-repertoire efakiweyo (i-Supplementary Fig. 2e).Ukuqukumbela, sigqibe kwelokuba umgangatho kunye nokwahluka kwethala leencwadi kwamkelekile kwaye kuphela utshintsho oluncinci kwiiyantlukwano eziye zabonwa ngenxa yokwandiswa nokulungiswa kwamathala eencwadi ephage phakathi kwemijikelo emininzi yokukhetha.
Isampulu ye-serial cerebrospinal fluid ingenziwa ngotyando lokufakela i-cannula kwi-CM yeegundane eziqaphelayo ukuququzelela ukuchongwa kwe-T7 phage efakwe nge-intravenously (iv) nge-BBB kunye / okanye i-BCSFB (Fig. 1a-b).Sasebenzisa iingalo ezimbini zokukhetha ezizimeleyo (iingalo A kunye ne-B) kwiinqanaba ezintathu zokuqala zokukhetha kwi-vivo (Umfanekiso 1c).Siye sanyusa kancinci ukuqina kokhetho ngokunciphisa inani lilonke lephage elaziswa kwimijikelo emithathu yokuqala yokukhetha.Ngomjikelo wesine we-panning, sidibanise iisampuli ezivela kumasebe A kunye no-B kwaye senza ukhetho oluzimeleyo oluthathu.Ukufunda iimpawu ze-vivo ze-T7 phage particles kule modeli, i-wild-type phage (i-PAGISRELVDKL master insert) ifakwe kwiigundane nge-vein tail.Ukubuyiswa kwe-phages kwi-cerebrospinal fluid kunye negazi ngamaxesha ahlukeneyo abonisa ukuba i-T7 i-icosahedral phages encinci inesigaba sokuqala sokucocwa ngokukhawuleza kwi-compartment yegazi (i-Supplementary Fig. 3).Ngokusekwe kwii-titers ezilawulwayo kunye nomthamo wegazi leempuku, sibale ukuba malunga ne-1% wt kuphela.I-phage evela kwi-dose elawulwayo ifunyenwe kwigazi le-10 imizuzu emva kwesitofu se-intravenous.Emva koku kuncipha ngokukhawuleza kokuqala, ukucocwa kweprayimari okucothayo kwalinganiswa nesiqingatha sobomi bemizuzu engama-27.7.Okubalulekileyo, kuphela i-phages embalwa kakhulu yafunyanwa kwi-compartment ye-CSF, ebonisa imvelaphi ephantsi ye-wild-type phage ukufuduka kwindawo ye-CSF (i-Supplementary Fig. 3).Ngokomyinge, kuphela malunga ne-1 x 10-3% i-titers ye-T7 phage egazini kunye ne-4 x 10-8% yee-phages ezifakwe ekuqaleni zifunyenwe kwi-cerebrospinal fluid kulo lonke ixesha lokusampula (0-250 min).Ngokucacileyo, i-half-life (25.7 min) ye-wild-type phage kwi-cerebrospinal fluid yayifana naleyo ibonwa egazini.Ezi nkcukacha zibonisa ukuba umqobo ohlula i-CSF compartment egazini ihambelana ne-CM-cannulated rats, evumela ukuba i-vivo ikhethwe kwiilayibrari ze-phage ukuchonga ii-clones ezithunyelwa ngokukhawuleza ukusuka egazini ukuya kwindawo ye-CSF.
(a) Ukumisela indlela yokwenziwa kwakhona kwesampulu ye-cerebrospinal fluid (CSF) ukusuka kwidama elikhulu.(b) Umzobo obonisa indawo yeselula yesithintelo se-nervous central (CNS) kunye nesicwangciso sokukhetha esisetyenziselwa ukuchonga iipeptide eziwela umqobo wegazi-ubuchopho (BBB) ​​kunye nomqobo wegazi-ubuchopho.(c) Kwi-vivo phage bonisa itshati yokuhlola.Kumjikelo ngamnye wokhetho, i-phage (izazisi zezilwanyana ngaphakathi kweentolo) zafakwa nge-intravenously.Amasebe amabini azimeleyo azimeleyo (A, B) agcinwa ngokwahlukeneyo kude kube ngumjikelo we-4 wokukhetha.Kwimijikelo yokukhetha i-3 kunye ne-4, i-clone ye-phage nganye ekhutshwe kwi-CSF yacwangciswa ngesandla.(d) I-Kinetics ye-phage ehlukanisiweyo negazi (izangqa ezibomvu) kunye nolwelo lwe-cerebrospinal (onxantathu oluhlaza) ngexesha lomjikelo wokuqala wokukhetha kwiigundane ezimbini ze-cannulated emva kokutofa nge-intravenous kwilayibrari ye-peptide ye-T7 (2 x 1012 phages / isilwanyana).Izikwere ezihlaza okwesibhakabhaka zibonisa umyinge wokuqala wokuxinwa kwe-phage egazini, ebalwa ukusuka kwisixa se-phage esitofu, ngokuqwalasela umthamo wegazi elipheleleyo.Izikwere ezimnyama zibonisa indawo yokudibanisa umgca we-y okhutshwe kwi-concentrations ye-phage yegazi.(e,f) Bonisa ubuninzi bezihlandlo kunye nosasazo lwazo zonke iimotifs ze-tripeptide ezinokubakho ezifumaneka kwipeptide.Inani le-motifs elifunyenwe kwi-1000 yokufunda libonisiwe.Ngokubalulekileyo (p <0.001) iimotifs ezityetyisiweyo ziphawulwe ngamachaphaza abomvu.(e) I-Correlation scatterplot ethelekisa ukuphindaphindwa kwe-tripeptide motif yethala leencwadi elitofiweyo kunye negazi eliphuma kwizilwanyana #1.1 kunye ne-#1.2.(f) I-Correlation scatterplot ethelekisa ukuphindaphinda kwezilwanyana phage tripeptide motifs #1.1 kunye #1.2 eyedwa kwigazi kunye nolwelo lwe-cerebrospinal.(g, h) Ukubonakaliswa kwe-ID yolandelelwano lwe-phage etyetyiswe egazini (g) ngokuchasene namathala eencwadi atofwayo kunye nephage ephuculwe kwi-CSF (h) ngokuchasene negazi emva komjikelo wokukhetha kwi-vivo kuzo zombini izilwanyana.Ubungakanani bekhowudi enobumba omnye bubonisa ukuba i-amino acid ibakho kangaphi kweso sithuba.Uhlaza = ipolar, purple = neutral, blue = basic, red = acidic and black = hydrophobic amino acids.Umzobo 1a, b uyilwe waze waveliswa nguEduard Urich.
Sifake ilayibrari ye-phage peptide kwiigundane ezimbini ze-CM (i-clades A kunye ne-B) kunye ne-phage eyedwa kwi-cerebrospinal fluid kunye negazi (Umfanekiso 1d).Ukucocwa okukhawulezileyo kwethala leencwadi kwakungavakaliswa kangako xa kuthelekiswa nohlobo lwasendle.Isiqingatha sobomi belayibrari ejoyiweyo kuzo zombini izilwanyana yayiyimizuzu ye-24.8 egazini, efana ne-wild-type phage, kunye nemizuzu ye-38.5 kwi-CSF.Iisampuli zegazi kunye ne-cerebrospinal fluid phage kwisilwanyana ngasinye ziphantsi kwe-HTS kwaye zonke iipeptide ezichongiweyo zahlaziywa ubukho be-tripeptide motif emfutshane.Ii-motifs ze-Tripeptide zakhethwa ngenxa yokuba zibonelela ngesiseko esincinci sokwakheka kwesakhiwo kunye nokusebenzisana kwe-peptide-protein14,15.Sifumene ulungelelwaniso oluhle ekusasazeni i-motifs phakathi kwelayibrari ye-phage injected kunye neeclones ezikhutshwe egazini lezilwanyana zombini (umzobo 1e).Idatha ibonisa ukuba ukubunjwa kwethala leencwadi kutyetyiswe kuphela kwindawo yegazi.Ii-amino acid frequencies kunye nokulandelelana kwemvumelwano kwahlalutywa ngakumbi kwindawo nganye kusetyenziswa ukulungiswa kwesoftware yeWeblogo16.Kuyathakazelisa ukuba sifumene ukutyetyiswa okunamandla kwiintsalela zegazi ze-glycine (umzobo 1g).Xa igazi lifaniswa nama-clones akhethwe kwi-CSF, ukhetho olunamandla kunye nokukhethwa kwe-motifs ethile kwabonwa (Umfanekiso we-1f), kwaye i-amino acids ethile yayikhethwa ngokufanelekileyo kwiindawo ezichazwe kwangaphambili kwi-12-ilungu (umzobo 1h).Ngokucacileyo, izilwanyana ezizimeleyo zahluke kakhulu kwi-cerebrospinal fluid, kanti i-blood glycine enrichment yabonwa kuzo zombini izilwanyana (i-Supplementary Fig. 4a-j).Emva kokucoca okungqongqo kwedatha yokulandelelana kwi-cerebrospinal fluid yezilwanyana #1.1 kunye ne-#1.2, inani le-964 kunye ne-420 ekhethekileyo ye-12-mer peptides yafunyanwa (i-Supplementary Fig. 1d-e).Ii-clones ze-phage ezizimeleyo zandiswa kwaye zixhomekeke kumjikelo wesibini wokukhetha kwi-vivo.I-Phage ekhutshwe kumjikelo wesibini wokukhetha yayiphantsi kwe-HTS kwisilwanyana ngasinye kwaye zonke iipeptide ezichongiweyo zisetyenziswe njengegalelo kwinkqubo yokuqaphela i-motif ukuhlalutya ukwenzeka kwe-tripeptide motifs (Umfanekiso 2a, b, ef).Xa kuthelekiswa nomjikelezo wokuqala we-phage efunyenwe kwi-CSF, siye sabona ukhetho olungakumbi kunye nokukhethwa kwezinto ezininzi kwi-CSF kumasebe A kunye no-B (umzobo 2).I-algorithm yokuchonga inethiwekhi isetyenziswe ukumisela ukuba bamele iipateni ezahlukeneyo zolandelelwano oluhambelanayo.Ukufana okucacileyo kuye kwabonwa phakathi kwe-12-dimensional sequences efunyenwe yi-CSF kwenye i-clade A (Fig. 2c, d) kunye ne-clade B (Umfanekiso 2g, h).Uhlalutyo oludityanisiweyo kwisebe ngalinye lubonakalise iiprofayili ezikhethiweyo ezikhethiweyo ze-12-mer peptides (I-Fig. 5c, d) kunye nokunyuka kwe-CSF / i-blood titer ratio ngexesha le-clones edibeneyo emva komjikelo wesibini wokukhetha xa kuthelekiswa nomjikelo wokuqala wokukhetha (i-Supplementary Fig. 5e).).
Ukutyebisa i-motifs kunye neepeptides kwi-cerebrospinal fluid ngemijikelo emibini elandelelanayo ye-vivo functional phage display display.
Zonke i-cerebrospinal fluid phages zafunyanwa kumjikelo wokuqala wesilwanyana ngasinye (izilwanyana #1.1 kunye ne-#1.2) zahlanganiswa, zandiswa, ze-HT-zilandelelaniswe kwaye zaphinda zafakwa kunye (2 x 1010 phages / isilwanyana) 2 SM iigundane ze-cannulated (#1.1 → #).2.1 kunye no-2.2, 1.2 → 2.3 kunye no-2.4).(a,b,e,f) I-Correlation scatterplots ethelekisa i-frequency ehambelanayo ye-tripeptide motifs yazo zonke i-CSF-derived phages kumjikelezo wokuqala kunye nowesibini wokukhetha.Unxulumano lokuphindaphinda kunye nokusasazwa kweemotifs ezimele zonke ii-tripeptides ezithelelanayo ezifumaneka kwiipeptides kuzo zombini ii-orientation.Inani le-motifs elifunyenwe kwi-1000 yokufunda libonisiwe.Ii-Motifs eziye zakhethwa kakhulu (p <0.001) okanye ezingabandakanywanga kwelinye lamathala eencwadi athelekisekayo ziphawulwe ngamachaphaza abomvu.(c, d, g, h) Ulandelelwano lokumelwa kwelogo yazo zonke ii-CSF-rich 12 amino acid ezide ngokulandelelana ngokusekwe kwimijikelo ye-2 kunye ne-1 ye-vivo yokukhetha.Ubungakanani bekhowudi enobumba omnye bubonisa ukuba i-amino acid ibakho kangaphi kweso sithuba.Ukumela i-logo, i-frequency ye-CSF yokulandelelana ekhutshwe kwisilwanyana ngasinye phakathi kwemijikelo emibini yokukhetha iyathelekiswa kwaye ulandelelwano olutyetyisiweyo kumjikelo wesibini lubonisiwe: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 kunye (h) #1.2–#2.4.Eyona amino acid ezityetyisiweyo kwindawo enikiweyo kwi (c, d) izilwanyana no.2.1 kwaye akukho.2.2 okanye (g, h) kwizilwanyana inomb.2.3 kwaye hayi.2.4 ziboniswe ngombala.Uhlaza = ipolar, purple = neutral, blue = basic, red = acidic and black = hydrophobic amino acids.
Emva komjikelo wesithathu wokukhetha, sichonge i-124 yokulandelelana kwe-peptide eyodwa (#3.1 kunye ne-#3.2) ukusuka kwi-332 CSF-reconstituted phage clones eyedwa kwizilwanyana ezimbini (i-Supplementary Fig. 6a).Ulandelelwano lwe-LGSVS (18.7%) lube nowona mlinganiselo uphezulu ozalanayo, ulandelwa luhlobo lwasendle PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), kunye ne-SARGSWREIVSLS ( 2.2%).Kumjikelo wesine wokugqibela, siye sadibanisa amasebe amabini akhethiweyo ngokuzimeleyo kwizilwanyana ezithathu ezahlukeneyo (Umfanekiso 1c).Kwii-clones ze-925 ezilandelelanayo ze-phage ezifunyenwe kwi-CSF, kumjikelo wesine sifumene i-64 yokulandelelana kwe-peptide eyodwa (i-Fig.Iiclones ze-CSF eziqhelekileyo kumjikelo wesine zaziyi-LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) kunye ne-RLSSVDSDLSGC (3, 2%).%)).Uluhlu lobude beepeptide ezikhethiweyo kungenxa yokufakwa/ukucima i-nucleotide okanye iikhodons zokuyeka kwangaphambi kwexesha kwiiprimer zethala leencwadi xa kusetyenziswa iicodons eziwohlokayo kuyilo lwethala leencwadi le-NNK.Iikhodoli zokuyeka kwangaphambi kwexesha zivelisa iipeptide ezimfutshane kwaye zikhethiwe ngenxa yokuba ziqulethe i-aa motif efanelekileyo.Iipeptides ezinde zinokuba nesiphumo sofakelo/ekucinyweni kwiiprayimari zamathala eencwadi okwenziwa.Oku kubeka ikhowudi yokumisa eyiliweyo ngaphandle kwesakhelo kwaye ifunde de kuvele ikhowudi entsha yokumisa ezantsi komsinga.Ngokubanzi, sibale izinto zokutyebisa kuzo zonke ezine ezikhethiweyo zokukhetha ngokuthelekisa idatha yegalelo kunye nedatha yokuphuma kwesampuli.Kumjikelo wokuqala wokuhlolwa, sasebenzisa i-phage titers zasendle njengereferensi engakhankanyiweyo yangasemva.Kuyathakazelisa ukuba ukhetho olubi lwe-phage lwalunamandla kakhulu kumjikelezo wokuqala we-CSF, kodwa kungekhona egazini (umzobo 3a), onokuthi ubangelwa ukungabikho kwe-passive diffusion yamalungu amaninzi elayibrari ye-peptide kwi-compartment ye-CSF okanye i-phages ehambelanayo ivame ukugcinwa ngokufanelekileyo okanye ukususwa kwigazi kune-bacteriophages.Nangona kunjalo, kwinqanaba lesibini le-panning, ukhetho olunamandla lwe-phages kwi-CSF lubonwe kuzo zombini iiclades, ezibonisa ukuba umjikelezo wangaphambili wawutyetyiswe kwi-phages ebonisa i-peptides ekhuthaza ukuthathwa kwe-CSF (Umfanekiso 3a).Kwakhona, ngaphandle kokutyeba okubalulekileyo kwegazi.Kwakhona kumjikelo wesithathu nowesine, iiclones zephage zatyetyiswa kakhulu kwi-CSF.Ukuthelekisa i-frequency ehambelanayo yolandelelwano ngalunye lwe-peptide ekhethekileyo phakathi kwemijikelo emibini yokugqibela yokukhetha, sifumene ukuba ulandelelwano lwaye lwatyetyiswa ngakumbi kumjikelo wesine wokukhetha (Umfanekiso 3b).Itotali ye-931 ye-tripeptide motifs yacatshulwa kuzo zonke ii-64 zolandelelwano olulodwa lwe-peptide kusetyenziswa zombini ii-peptide orientation.Iimotifs ezityetyisiweyo kakhulu kumjikelo wesine ziye zavavanywa ngokusondeleyo kwiiprofayili zazo zokutyebisa kuzo zonke izijikelezo xa kuthelekiswa nethala leencwadi elitofelweyo (ukunqunyulwa: i-10% yokutyebisa) (Fig. 6c eyoNgezelelweyo).Iipateni ngokubanzi zokhetho zibonise ukuba uninzi lweenjongo ezifundiweyo zatyetyiswa kuyo yonke imijikelo yangaphambili yawo omabini amasebe okhetho.Nangona kunjalo, ezinye iimotifs (umz. SGL, VSG, LGS GSV) ubukhulu becala bezisuka kwelinye iclade A, ngelixa ezinye (umz. FGW, RTN, WGF, NTR) zatyetyiswa kwelinye iclade B.
Ukuqinisekiswa kokuthuthwa kwe-CSF ye-CSF-enriched phage-displayed peptides kunye ne-biotinylated leader peptides conjugated to streptavidin payloads.
(a) Imilinganiselo yokutyebisa ibalwe kuyo yonke imijikelo emine (i-R1-R4) ngokusekwe kwi-injected (input = I) phage (PFU) titers kunye ne-CSF phage titers (isiphumo = O).Izinto zokutyebisa kwimijikelezo emithathu yokugqibela (i-R2-R4) zibalwe ngokuthelekisa kunye nomjikelo wangaphambili kunye nomjikelezo wokuqala (R1) kunye nedatha yesisindo.Imivalo evulekileyo yi-cerebrospinal fluid, imivalo enemithunzi yiplasma.(***p<0.001, ngokusekelwe kuvavanyo loMfundi).(b) Uluhlu lwezona phage peptides zininzi, ezibekwe ngokomlinganiselo wazo kuzo zonke iiphage eziqokelelwe kwiCSF emva komjikelo 4 wokhetho.I-clones ye-phage emithandathu ixhaphake kakhulu ibonakaliswe ngombala, amanani kunye nezinto zabo zokutyebisa phakathi komjikelo we-3 kunye no-4 wokukhetha (i-inset).(c,d) Ezona zintandathu zityetyiswe kakhulu ii-phage clones, i-phage engenanto kunye nethala leencwadi le-peptide labazali ukusuka kumjikelo 4 ziye zahlalutywa ngokwahlukeneyo kwimodeli yesampulu ye-CSF.I-CSF kunye neesampuli zegazi zaye zaqokelelwa ngamaxesha achaziweyo.(c) Izixa ezilinganayo zamaphage ama-6 (2 x 1010 phage/izilwanyana), iiphage ezingenanto (#1779) (2 x 1010 phage/izilwanyana) kunye namathala eencwadi e-stock phage peptide (2 x 1012 phage/izilwanyana) Tofa ubuncinane i-3 CM ilawulwa ngokwahlukileyo kwisilwanyana.I-pharmacokinetics ye-CSF ye-phage clone nganye etofwayo kunye nelayibrari ye-peptide ye-phage ekuhambeni kwexesha ibonisiwe.(d) ibonisa umndilili we-CSF / umlinganiselo wegazi kuzo zonke iiphage/mL ezifunyenweyo ngexesha lesampulu.(e) Iipeptide ezine zeenkokeli zokwenziwa kunye nolawulo oluxutyiweyo ludityaniswe ne-biotin ukuya kwi-streptavidin nge-N-terminus (i-tetramer display) elandelwa yinaliti (i-tail vein iv, 10 mg streptavidin/kg).Ubuncinci iigundane ezintathu ezifakwe ngaphakathi (N = 3).).Iisampulu ze-CSF ziqokelelwe kumanqaku exesha elibonisiweyo kunye nokugxininiswa kwe-streptavidin kulinganiswe yi-CSF anti-streptavidin ELISA (nd = ayibonwa).(*p<0.05, **p<0.01, ***p<0.001, ngokusekelwe kuvavanyo lwe-ANOVA).(f) Ukuthelekiswa kwe-amino acid ulandelelwano lwe-phage peptide clone ephuculweyo kakhulu #2002 (mfusa) kunye nezinye ezikhethiweyo ze-phage peptide clones ukusuka kumjikelo we-4 wokukhetha.Amaqhekeza afanayo kunye afana ne-amino acid anekhowudi yombala.
Kuzo zonke izigaba ezityetyisiweyo kumjikelo wesine (umzobo 3b), ii-clones zabaviwa ezintandathu ziye zakhethwa ukuze kuhlalutywe ngakumbi umntu ngamnye kwimodeli yesampulu ye-CSF.Izixa ezilinganayo ze-phage yabaviwa abathandathu, i-phage engenanto (akukho fakelo) kunye neelayibrari ze-peptide ze-prophage zafakwa kwizilwanyana ezintathu ze-CM ezinama-cannulated, kwaye i-pharmacokinetics yagqitywa kwi-CSF (Fig. 3c) kunye negazi (i-Supplementary Fig. 7).Zonke ii-clones ze-phage ezivavanyiweyo zijolise kwi-CSF compartment kwinqanaba le-10-1000 ngamaxesha aphezulu kunelo lolawulo olungenanto (#1779).Umzekelo, iiclones #2020 kunye ne #2077 zazinamaxesha angama-1000 aphezulu e-CSF kunephage yolawulo.Iprofayili ye-pharmacokinetic yepeptide nganye ekhethiweyo yahlukile, kodwa zonke zinamandla aphezulu e-CSF homing.Siqaphele ukuhla okuthe gqolo ekuhambeni kwexesha kwiiclones #1903 kunye no #2011, ngelixa iiclones #2077, #2002 kunye #2009 ukonyuka ngexesha lemizuzu yokuqala eyi-10 kunokubonisa uthutho olusebenzayo kodwa kufuneka luqinisekiswe.IiClones #2020, #2002, kunye ne-#2077 zizinzile kumanqanaba aphezulu, ngelixa i-CSF concentration ye-clone #2009 yehla ngokukhawuleza emva kokunyuka kokuqala.Emva koko siye sathelekisa i-frequency ehambelana nomviwa ngamnye we-CSF kunye noxinzelelo lwegazi (Umfanekiso 3d).Ukulungelelaniswa kwenani eliphakathi komviwa ngamnye we-CSF kunye nenqanaba legazi kuwo onke amaxesha eesampuli kubonise ukuba abathathu kwabathandathu abagqatswa batyetyiswe kakhulu kwi-CSF yegazi.Kuyathakazelisa ukuba i-clone #2077 ibonise ukuzinza okuphezulu kwegazi (umzobo owongezelelweyo 7).Ukuqinisekisa ukuba iipeptides ngokwazo ziyakwazi ukuthutha imithwalo ngaphandle kwamaqhekeza e-phage kwindawo ye-CSF, senze iipeptide ezine ezikhokelayo ezithathwe kwi-biotin kwi-N-terminus apho i-peptides inamathele kwi-particle phage.I-peptides ye-Biotinylated (iinombolo ze-2002, i-2009, i-2020 kunye ne-2077) yahlanganiswa kunye ne-streptavidin (SA) ukufumana iifom ze-multimeric ngandlela-thile zilinganisa i-phage geometry.Le fomati ikwasivumele ukuba silinganise utyhileko lwe-SA egazini kunye nolwelo lwe-cerebrospinal njenge-cargo-transporting protein peptides.Okubalulekileyo, idatha ye-phage inokuphinda iveliswe xa i-peptides yokwenziwa ilawulwa kule fomethi ye-SA-conjugated (Umfanekiso 3e).Iipeptide ezikhutshiweyo zibe nokuvezwa okuncinci kokuqala kunye nokukhutshwa ngokukhawuleza kwe-CSF kunye namanqanaba angabonakaliyo kwiiyure ezingama-48.Ukufumana ingqiqo kwiindlela zokuhanjiswa kwezi clones ze-peptide phage kwisithuba se-CSF, sihlalutye indawo ye-phage peptide hits usebenzisa i-immunohistochemistry (IHC) ukufumanisa ngokuthe ngqo i-phage particles 1 iyure emva kwe-injection ye-intravenous in vivo.Ngokucacileyo, ii-clones #2002, #2077, kunye ne-#2009 zinokubonwa ngokucolwa ngamandla kwi-brain capillaries, ngelixa i-phage yolawulo (#1779) kunye ne-clone #2020 ayifunyaniswanga (umzobo owongezelelweyo 8).Oku kuphakamisa ukuba ezi peptides zinegalelo kwisiphumo kwingqondo ngokuchanekileyo ngokuwela i-BBB.Uhlalutyo olongezelelweyo oluneenkcukacha luyafuneka ukuvavanya le ngcamango, njengoko indlela ye-BSCFB inokubandakanyeka.Xa kuthelekiswa ukulandelelana kwe-amino acid ye-clone ephuculweyo (#2002) kunye nezinye iipeptide ezikhethiweyo, kwaphawulwa ukuba ezinye zazo zinezandiso ze-amino acid ezifanayo, ezinokubonisa indlela yokuthutha efanayo (Umfanekiso 3f).
Ngenxa yeprofayili ye-plasma ekhethekileyo kunye nokunyuka okuphawulekayo kwe-CSF ngokuhamba kwexesha, i-clone yokubonisa i-phage #2077 yaphononongwa ngakumbi kwixesha elide le-48 kwaye yakwazi ukuvelisa ukwanda okukhawulezayo kwe-CSF ebonwe ngokubambisana namanqanaba e-SA azinzileyo (Umfanekiso 4a).Ngokumalunga nezinye iiclones zephage ezichongiweyo, i-#2077 inebala elinamandla kwiicapillaries zobuchopho kwaye yabonisa i-colocalization ebalulekileyo nge-capillary marker lectin xa ijongwe kwisisombululo esiphezulu kwaye mhlawumbi nebala elithile kwindawo ye-parenchymal (Figure 4b).Ukuphanda ukuba ingaba i-peptide-mediated pharmacological effects inokufumaneka na kwi-CNS, senze uvavanyo apho iinguqulelo ze-biotinylated ze-i) i- #2077 transit peptide kunye ii) i-BACE1 inhibitor peptide zaxutywa ne-SA kwireyishini ezimbini ezahlukeneyo.Kwindibaniselwano enye sisebenzise kuphela i-BACE1 peptide inhibitor kwaye kwenye sasebenzisa umlinganiselo we-1:3 we-BACE1 peptide inhibitor ukuya kwi#2077 peptide.Zombini iisampulu zilawulwe nge-intravenously kwaye igazi kunye namanqanaba e-cerebrospinal fluid ye-beta-amyloid peptide 40 (Abeta40) yalinganiswa ngokuhamba kwexesha.I-Abeta40 yalinganiswa kwi-CSF njengoko ibonisa i-BACE1 inhibition kwi-brainchyma yengqondo.Njengoko kulindelwe, zombini ii-complexes zanciphisa kakhulu amanqanaba egazi le-Abeta40 (Umfanekiso 4c, d).Nangona kunjalo, iisampuli kuphela eziqulethe umxube we-peptide no.I-2077 kunye ne-inhibitor ye-BACE1 peptide conjugated to SA ibangele ukuhla okukhulu kwe-Abeta40 kwi-cerebrospinal fluid (Fig. 4c).Idatha ibonisa ukuba i-peptide ayikho.I-2077 iyakwazi ukuthutha iprotheni ye-60 kDa SA kwi-CNS kwaye iphinda ibangele iziphumo ze-pharmacological nge-SA-conjugated inhibitors ye-BACE1 peptide.
(a) I-injection ye-Clonal (i-2 × i-10 phages / isilwanyana) se-T7 phage ebonisa iiprofayili ze-pharmacokinetic zexesha elide ze-peptide ye-CSF #2077 (RLSSVDSDLSGC) kunye ne-phage yokulawula engafakwanga (#1779) ubuncinane kwiigundane ezintathu ze-CM-intubated.(b) Umfanekiso we-Confocal microscopic we-cortical microvessels ezimele kwiimpuku ezifakwe kwi-phage (2 × 10 10 phages / isilwanyana) ebonisa ukubalwa kwe-peptide #2077 kunye nemikhumbi (i-lectin).La ma-clones e-phage alawulwa kwiigundane ze-3 kwaye avunyelwe ukuba ajikeleze iyure eli-1 ngaphambi kokugcoba.Ubuchopho bahlulwe kwaye bangcoliswa nge-polyclonal FITC-ebhalwe izilwa-buhlungu ezichasene ne-T7 phage capsid.Imizuzu elishumi ngaphambi kokuxutywa kunye nokulungiswa okulandelayo, i-lectin ebhalwe ngeDyLight594 yayilawulwa nge-intravenously.Imifanekiso ye-Fluorescent ebonisa i-lectin staining (ebomvu) yecala elikhanyayo le-microvessels kunye ne-phages (eluhlaza) kwi-lumen ye-capillaries kunye ne-perivascular brain tissue.Ibar yesikali ihambelana ne-10 µm.(c, d) I-Biotinylated BACE1 inhibitory peptide yodwa okanye idibene ne-biotinylated transit peptide #2077 yadityaniswa ne-streptavidin elandelwa yinaliti ye-intravenous ubuncinane ezintathu ze-CM ze-cannulated (10 mg streptavidin/kg).I-BACE1 peptide inhibitor-mediated reduction kwi-Aβ40 yalinganiswa ngu-Aβ1-40 ELISA egazini (obomvu) kunye ne-cerebrospinal fluid (i-orange) kwixesha elibonisiweyo.Ukucaca ngcono, umgca wamachaphaza uzotywe kwigrafu kwisikali se-100%.(c) Ipesenti yokunciphisa i-Aβ40 egazini (onxantathu obomvu) kunye ne-cerebrospinal fluid (onxantathu be-orenji) kwiigundane eziphathwe nge-streptavidin conjugated to transit peptide #2077 kunye ne-BACE1 inhibitory peptide kwi-3: 1 ratio.(d) Ipesenti yokunciphisa igazi i-Aβ40 (izangqa ezibomvu) kunye ne-cerebrospinal fluid (izangqa ze-orange) zeegundane eziphathwe nge-streptavidin zidibene ne-BACE1 inhibitory peptide kuphela.I-concentration ye-Aβ kulawulo yayiyi-420 pg / ml (i-standard deviation = 101 pg / ml).
Umboniso wePhage usetyenziswe ngempumelelo kwiindawo ezininzi zophando lwe-biomedical17.Le ndlela isetyenziswe kwi-vivo vascular diversity studies18,19 kunye nezifundo ezijolise kwimikhumbi ye-cerebral20,21,22,23,24,25,26.Kule sifundo, sandisa ukusetyenziswa kwale ndlela yokukhetha kungekhona nje ukuchongwa ngokuthe ngqo kweepeptide ezijoliswe kwiinqanawa ze-cerebral, kodwa kunye nokufumanisa abaviwa abaneempawu zokuthutha ezisebenzayo ukuwela umqobo wegazi-ingqondo.Ngoku sichaza ukuphuhliswa kwenkqubo yokukhethwa kwe-vivo kwi-CM intubated rats kwaye ibonise amandla ayo okuchonga i-peptides eneempawu ze-CSF homing.Ukusebenzisa i-T7 phage ebonisa ilayibrari ye-peptides ye-12-mer random, sakwazi ukubonisa ukuba i-T7 phage incinci ngokwaneleyo (malunga ne-60 nm ububanzi) i-10 ukuba ilungelelaniswe kwisithintelo segazi-ingqondo, ngaloo ndlela iwela ngokuthe ngqo umqobo wegazi-ubuchopho okanye i-choroid plexus.Siye saqaphela ukuba ukuvunwa kwe-CSF kwiigundane ze-CM e-cannulated yayilawulwa kakuhle kwindlela yokuhlola i-vivo, kwaye i-phage ekhutshiweyo ayibophekanga kuphela kwi-vasculature kodwa yayisebenza njengomthuthi kuwo wonke umqobo wegazi-ingqondo.Ngaphaya koko, ngokuqokelela igazi ngaxeshanye kunye nokusebenzisa i-HTS kwi-CSF kunye nee-phages ezenziwe ngegazi, siqinisekisile ukuba ukhetho lwethu lwe-CSF aluphenjelelwanga kukutyetyiswa kwegazi okanye ukulungela ukwanda phakathi kwemijikelo yokukhetha.Nangona kunjalo, i-compartment yegazi yinxalenye yenkqubo yokukhetha, ekubeni i-phages ekwazi ukufikelela kwi-compartment ye-CSF kufuneka iphile kwaye ijikeleze kwigazi elide ngokwaneleyo ukuze zizityebise engqondweni.Ukuze sikhuphe ulwazi oluthembekileyo lokulandelelana kwidatha ye-HTS ekrwada, siphumeze izihluzi ezilungelelaniswe kwiimpazamo ezichanekileyo zokulandelelana kweqonga kuhlalutyo lokuhamba komsebenzi.Ngokubandakanya i-kinetic parameters kwindlela yokuhlola, siqinisekisile i-pharmacokinetics ekhawulezayo ye-wild-type T7 phages (t½ ~ 28 min) kwigazi24, 27, 28 kwaye kwakhona inqume i-half-life in cerebrospinal fluid (t½ ~ 26 min) ngomzuzu).Ngaphandle kweeprofayili ze-pharmacokinetic ezifanayo kwigazi kunye ne-CSF, kuphela i-0.001% ye-concentration yegazi ye-phage inokufunyanwa kwi-CSF, ebonisa ukuhamba okuphantsi kwemvelaphi ye-wild-type T7 phage kuwo wonke umqobo wegazi-ingqondo.Lo msebenzi ubonisa ukubaluleka komjikelo wokuqala wokukhetha xa usebenzisa izicwangciso ze-panning ze-vivo, ngokukodwa kwiinkqubo ze-phage ezicinywa ngokukhawuleza kwi-circulation, njengoko ii-clones ezimbalwa zikwazi ukufikelela kwi-compartment ye-CNS.Ngoko ke, kumjikelo wokuqala, ukuncitshiswa kweyantlukwano yethala leencwadi bekukukhulu kakhulu, njengoko kuphela linani elilinganiselweyo lee-clones elathi ekugqibeleni laqokelelwa kule modeli ingqongqo kakhulu ye-CSF.Esi sicwangciso se-vivo panning sibandakanya amanyathelo amaninzi okukhetha afana nokuqokelela okusebenzayo kwi-CSF compartment, ukusinda kwe-clone kwi-compartment yegazi, kunye nokususwa ngokukhawuleza kwe-T7 phage clones egazini kwimizuzu yokuqala ye-10 (umzobo we-1d kunye ne-Supplementary Fig. 4M).).Ngaloo ndlela, emva komjikelezo wokuqala, ii-clones ze-phage ezahlukeneyo zachongwa kwi-CSF, nangona i-pool yokuqala efanayo yayisetyenziselwa izilwanyana.Oku kuphakamisa ukuba amanyathelo angqongqo okukhetha amathala eencwadi omthombo anamanani amakhulu amalungu ethala leencwadi akhokelela ekuncipheni okubonakalayo kokwahluka.Ke ngoko, iziganeko ezingalindelekanga ziya kuba yinxalenye yenkqubo yokukhetha yokuqala, ibe nefuthe kakhulu kwisiphumo.Kusenokwenzeka ukuba uninzi lweeclones kwithala leencwadi lokuqala zazinotyekelo lokutyebisa olufana nqwa ne-CSF.Nangona kunjalo, naphantsi kweemeko zovavanyo ezifanayo, iziphumo zokukhetha zinokwahluka ngenxa yenani elincinci le-clone nganye kwi-pool yokuqala.
Iimotifs ezityetyisiweyo kwi-CSF ziyahluka kwezo zisegazini.Okubangel 'umdla kukuba, siqaphele utshintsho lokuqala kwi-peptides ene-glycine-rich egazini lesilwanyana ngasinye.(Umzobo 1g, amakhiwane awongezelelweyo. 4e, 4f).I-Phage equkethe i-glycine peptides inokuthi izinzile kwaye ayinakwenzeka ukuba ikhutshwe kwi-circulation.Nangona kunjalo, ezi peptide ezityebileyo ze-glycine azizange zibonwe kwiisampuli ze-cerebrospinal fluid, ezibonisa ukuba iilayibrari ezigciniweyo zihamba ngamanyathelo amabini ahlukeneyo okukhetha: enye egazini kwaye enye ivunyelwe ukuba iqokelele kwi-cerebrospinal fluid.Iiclones ezityetyisiweyo ze-CSF eziphuma kumjikelo wesine wokhetho ziye zavavanywa ngokubanzi.Phantse zonke ii-clones ezivavanyiweyo ngabanye zaqinisekiswa ukuba zityetyiswe kwi-CSF xa kuthelekiswa nephage yokulawula engenanto.Ipeptide enye hit (#2077) yavavanywa ngokweenkcukacha.Ibonise i-plasma ende yesiqingatha-ubomi xa kuthelekiswa nezinye hits (Figure 3d kunye neFigure Supplementary 7), kwaye okuthakazelisayo, le peptide iqulethe i-cysteine ​​​​residue kwi-C-terminus.Kutshanje kuye kwaboniswa ukuba ukongezwa kwe-cysteine ​​​​kwi-peptides kunokuphucula iimpawu zabo ze-pharmacokinetic ngokuzibophelela kwi-albumin 29.Oku okwangoku akwaziwa ngepeptide #2077 kwaye ifuna uphononongo oluthe kratya.Ezinye iipeptide zibonise ukuxhomekeka kwe-valence kwi-CSF enrichment (idatha engaboniswa), enokuthi inxulumene nejometri yomphezulu obonisiweyo we-T7 capsid.Inkqubo ye-T7 esiyisebenzisileyo ibonise iikopi ze-5-15 zepeptide nganye kwi-phage particle.I-IHC yenziwa kwi-clones ye-phage yabaviwa efakwe nge-intravenously kwi-cortex ye-cerebral yeegundane (i-Supplementary Fig. 8).Idatha ibonise ukuba ubuncinane ii-clones ezintathu (No. 2002, No. 2009 kunye neNo. 2077) zisebenzisana ne-BBB.Kuhlala kugqitywa ukuba le ntsebenziswano ye-BBB iphumela ekuqokeleleni kwe-CSF okanye ukuhamba kwezi clones ngqo kwi-BCSFB.Okubalulekileyo, sibonisa ukuba iipeptide ezikhethiweyo zigcina umthamo wazo wezothutho we-CSF xa zidityanisiwe kwaye zibotshelelwe kwi-protein cargo.Ukubophelela kwe-N-terminal biotinylated peptides kwi-SA ngokusisiseko kuphinda iziphumo ezifunyenweyo nge-clones yazo ye-phage egazini nakwi-cerebrospinal fluid (Fig. 3e).Ekugqibeleni, sibonisa ukuba i-peptide ehamba phambili #2077 iyakwazi ukukhuthaza ukusebenza kwengqondo ye-biotinylated peptide inhibitor ye-BACE1 ehlanganiswe ne-SA, ebangela imiphumo ecacileyo ye-pharmacodynamic kwi-CNS ngokunciphisa kakhulu amanqanaba e-Abeta40 kwi-CSF (Fig. 4).Asikwazanga ukuchonga naziphi na iihomologues kwisiseko sedatha ngokwenza uphando lwehomology yolandelelwano lwepeptide yazo zonke hits.Kubalulekile ukuqaphela ukuba ubungakanani bethala leencwadi le-T7 limalunga ne-109, ngelixa ubungakanani bethala leencwadi lethiyori ye-12-mers yi-4 x 1015. Ngoko ke, sikhethe kuphela iqhezu elincinane lendawo yokwahlukana kwethala leencwadi le-12-mer peptide, elinokuthetha ukuba iipeptides eziphuculweyo zinokuchongwa ngokuvavanya indawo ekufutshane yokubetha kwezi ndawo zilandelelanayo.Ngokwenyani, esinye sezizathu zokuba singafumani naziphi na ii-homologue zendalo zezi peptides zinokungakhethi ngexesha lokuzivelela kwemvelo ukuthintela ukungena okungalawulekiyo kwee-peptide motifs ezithile ebuchotsheni.
Kuthathwe kunye, iziphumo zethu zibonelela ngesiseko somsebenzi wexesha elizayo ukuchonga kunye nokubonakalisa iinkqubo zokuthutha umqobo we-cerebrovascular in vivo ngokubanzi.Isiseko esisisiseko sale ndlela sisekelwe kwisicwangciso sokukhetha esisebenzayo esingachongi kuphela ii-clones ezineempawu zokubopha i-cerebral vascular, kodwa zibandakanya inyathelo elibalulekileyo apho ii-clones eziphumeleleyo zinomsebenzi wangaphakathi wokuwela imiqobo yebhayoloji kwi-vivo kwi-CNS compartment.kukucacisa indlela yokuthuthwa kwezi peptides kunye nokukhetha kwabo ukubophelela kwi-microvasculature ethe ngqo kwingingqi yengqondo.Oku kunokukhokelela ekufumaneni iindlela ezintsha zokuthutha i-BBB kunye ne-receptors.Silindele ukuba i-peptides echongiweyo inokubophelela ngokuthe ngqo kwi-cerebrovascular receptors okanye kwii-ligands ezijikelezayo ezithuthwa nge-BBB okanye i-BCSFB.Iivektha ze-peptide ezinomsebenzi wothutho we-CSF ofunyenwe kulo msebenzi ziya kuphandwa ngakumbi.Sithetha nje siphanda ubugocigoci bezi peptides ngokukwazi kwazo ukuwela i-BBB kunye/okanye i-BCSFB.Ezi peptide zintsha ziya kuba zizixhobo ezibaluleke kakhulu zokufunyanwa kwe-receptors emitsha okanye iindlela kunye nokuphuhliswa kwamaqonga amatsha asebenzayo kakhulu ekuhanjisweni kwee-macromolecules, ezifana ne-biologics, kwingqondo.
Cannulate i-cisterna enkulu (CM) usebenzisa ukuguqulwa kwendlela echazwe ngaphambili.Iimpuku ze-Wistar ezine-Anesthetized Wistar (200-350 g) zaxhonywa kwisixhobo se-stereotaxic kwaye isiko eliphakathi lenziwa phezu kwentloko yentloko echetyiweyo yaza yalungiswa ngokungenaziphene ukuze kuvele ukhakhayi.Gqoka imingxuma emibini kwindawo ye-sash ephezulu kwaye uqinise izikrufu zokulungisa emingxunyeni.Umngxuma owongezelelweyo wagrunjwa kwi-lateral occipital crest kwisikhokelo se-stereotactic ye-cannula yensimbi engenasici kwi-CM.Faka isamente yamazinyo ejikeleze i-cannula kwaye ukhuseleke ngezikrufu.Emva kokunyangwa kwefoto kunye nokuqina kwesamente, inxeba lesikhumba livalwe nge-4/0 supramid suture.Ukubekwa ngokufanelekileyo kwe-cannula kuqinisekiswa ngokuvuza okuzenzekelayo kwe-cerebrospinal fluid (CSF).Susa igundane kwisixhobo se-stereotaxic, ufumane ukhathalelo olufanelekileyo lwasemva kokuhlinzwa kunye nokulawulwa kweentlungu, kwaye uvumele ukuba ibuyiselwe ubuncinci iveki enye de kubonwe iimpawu zegazi kwi-cerebrospinal fluid.Iimpuku zeWistar (Crl:WI/Han) zafunyanwa eCharles River (France).Zonke iimpuku zagcinwa phantsi kweemeko ezithile ezingenasifo.Zonke iimvavanyo zezilwanyana zavunywa yi-Ofisi yezilwanyana zeSixeko sase-Basel, eSwitzerland, kwaye zenziwa ngokuhambelana neLayisensi yezilwanyana No.
Ngobunono yigcine impuku inolwazi ngeCM cannula esandleni.Susa iDatura kwicannula kwaye uqokelele i-10 µl yolwelo lwecerebrospinal oluqukuqelayo ngokuzenzekelayo.Ekubeni i-patency ye-cannula ekugqibeleni yaphazamiseka, kuphela iisampulu ezicacileyo ze-cerebrospinal fluid ezingenabo ubungqina bokungcoliswa kwegazi okanye ukuguqulwa kombala zibandakanyiwe kolu phononongo.Ngokunxuseneyo, malunga ne-10-20 μl yegazi yathathwa kwincinci encinci kwincam yomsila kwiityhubhu ezineheparin (Sigma-Aldrich).I-CSF kunye negazi zaqokelelwa ngamaxesha ahlukeneyo emva kwe-injection ye-intravenous ye-T7 phage.Ngokumalunga ne-5-10 μl yolwelo yalahlwa ngaphambi kokuba isampuli nganye ye-CSF iqokelelwe, ehambelana nomthamo ofileyo we-catheter.
Iilayibrari zenziwe kusetyenziswa i-T7Select 10-3b vector njengoko kuchazwe kwi-T7Select system manual (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996).Ngokufutshane, i-12-mer mer DNA efakelweyo yenziwe yadityaniswa ngolu hlobo lulandelayo:
I-codon ye-NNK yayisetyenziselwa ukuphepha i-double stop codons kunye ne-amino acid overexpression kwi-insert.I-N yi-equimolar ratio exutywe ngesandla kwi-nucleotide nganye, kwaye i-K yi-equimolar ratio exutywe ngesandla ye-adenine kunye ne-cytosine nucleotides.Imimandla exingileyo enye yatshintshwa yaba yi-DNA enemisonto ephindwe kabini ngokufukanyelwa ngakumbi nge-dNTP (Novagen) kunye ne-Klenow enzyme (i-New England Biolabs) kwi-Klenow buffer (i-New England Biolabs) iiyure ezi-3 kuma-37°C.Emva kokusabela, i-DNA ephindwe kabini yafunyanwa yimvula ye-EtOH.I-DNA efunyenweyo yagaywa kunye ne-enzymes yokuthintela i-EcoRI kunye ne-HindIII (zombini zisuka e-Roche).I-cleaved kwaye ihlanjululwe (i-QIAquick, i-Qiagen) ifake (i-T4 ligase, i-New England Biolabs) emva koko ifakwe kwi-frame kwi-vector ye-T7 edibeneyo emva kwe-amino acid 348 ye-10B ye-capsid gene.Iimpendulo ze-Ligation zafukanyelwa kwi-16 ° C. kwiiyure ze-18 ngaphambi kokupakishwa kwe-in vitro.Ukupakishwa kwePhage kwi-vitro kwenziwa ngokwemiyalelo enikezelweyo kunye ne-T7Select 10-3b cloning kit (Novagen) kunye nesisombululo sokupakisha sandiswa kanye kwi-lysis usebenzisa i-Escherichia coli (BLT5615, Novagen).I-lysates yayiyi-centrifuged, i-titrated kwaye ifakwe kwi -80 ° C. njengesisombululo sesitokisi se-glycerol.
Ukwandiswa ngokuthe ngqo kwe-PCR yemimandla eguquguqukayo ye-phage eyandisiweyo kumhluzi okanye ipleyiti usebenzisa i-proprietary 454 / Roche-amplicon fusion primers.I-primer fusion eya phambili iqulethe ulandelelwano olujikelezayo kummandla oguquguqukayo (NNK) 12 (itemplate-specific), i-GS FLX i-Adapter ye-Titanium A, kunye nolandelelwano lwezitshixo zethala leencwadi ezine-base-four (TCAG) (Figurementary Figure 1a):
I-reverse fusion primer ikwaqulethe i-biotin encanyathiselwe ukubamba amaso kunye ne-GS FLX Titanium Adapter B efunekayo kwi-clonal amplification ngexesha le-emulsion PCR:
Emva koko ii-amplicons ziphantsi kwe-454 / Roche pyrosequencing ngokwe-454 GS-FLX Titanium protocol.Ulandelelwano lweSanger lwemanuwali (I-Applied Biosystems Hitachi 3730 xl DNA Analyzer), i-T7 phage DNA yandiswa yi-PCR kwaye ilandelelaniswe nezi zibini zokuqala:
Ukufakwa kwi-plaques nganye kuxhomekeke kwi-PCR amplification usebenzisa i-Roche Fast Start DNA Polymerase Kit (ngokwemiyalelo yomenzi).Yenza isiqalo esishushu (i-10 min kwi-95 ° C) kunye nemijikelezo ye-35 yokunyusa (50 s kwi-95 ° C, i-1 min kwi-50 ° C, kunye ne-1 min kwi-72 ° C).
I-Phage esuka kwiilayibrari, i-wild-type phage, i-phage ehlangulwe kwi-CSF kunye negazi, okanye ii-clones zomntu ngamnye zaye zandiswa kwi-Escherichia coli BL5615 kwi-TB broth (Sigma Aldrich) okanye kwi-500 cm2 izitya (i-Thermo Scientific) kwi-4 h kwi-37 ° C.I-Phage ikhutshwe kwiiplate ngokuhlamba iiplate nge-Tris-EDTA buffer (i-Fluka Analytical) okanye ngokuqokelela amacwecwe aneengcebiso ze-pipette eziyinyumba.I-Phage yahlulwe kwi-culture supernatant okanye i-extraction buffer kunye nomjikelo omnye we-polyethylene glycol (PEG 8000) yemvula (i-Promega) kwaye yaphinde yamiswa kwi-Tris-EDTA buffer.
I-phage eyandisiweyo yayiphantsi kwemijikelo ye-2-3 yokususwa kwe-endotoxin kusetyenziswa iintsimbi zokususa i-endotoxin (i-Miltenyi Biotec) phambi kwenaliti ye-intravenous (IV) (500 μl / isilwanyana).Kumjikelo wokuqala, i-2 × 1012 phages yaziswa;okwesibini, 2×1010 izigaba;kumjikelo wesithathu nowesine wokukhetha, i-2 × 109 phage ngesilwanyana ngasinye.Isiqulatho sephage kwi-CSF kunye neesampuli zegazi eziqokelelwe ngamaxesha achaziweyo agqitywe ngokubalwa kwamacwecwe ngokwemiyalelo yomenzi (T7Select system manual).Ukukhethwa kwe-Phage kwenziwa nge-injection ye-intravenous yamathala eencwadi ahlambulukileyo kwi-vein yomsila okanye ngokuphinda kufakwe i-phage ekhutshwe kwi-CSF ukusuka kwinqanaba elikhethiweyo elikhethiweyo, kwaye izivuno ezilandelayo zenziwa kwi-10 min, i-30 min, i-60 min, i-90 min, i-120 min, i-180 min, kunye ne-240 iisampulu zegazi ngokulandelanayo.Iyonke imijikelo emine ye-in vivo panning yaqhutywa apho amasebe amabini akhethiweyo agcinwa ngokwahlukeneyo kwaye ahlalutywa ngexesha lemijikelo emithathu yokuqala yokukhetha.Zonke izinto ezifakwe kwi-phage ezikhutshwe kwi-CSF ukusuka kwimijikelo emibini yokuqala yokukhetha zixhomekeke kwi-454 / i-Roche pyrosequencing, ngelixa zonke ii-clones ezikhutshwe kwi-CSF ukusuka kwimijikelo emibini yokugqibela yokukhetha zilandelwa ngesandla.Zonke iiphage zegazi ezivela kumjikelo wokuqala wokukhetha nazo ziphantsi kwe-454 / Roche pyrosequencing.I-injection ye-phage clones, i-phage ekhethiweyo yandiswa kwi-E. coli (BL5615) kwiiplate ze-500 cm2 kwi-37 ° C ngeeyure ze-4.Iiclones ezikhethwe ngumntu ngamnye kunye nezilandelelaniswe ngesandla ziye zasasazwa ngendlela ye-TB.Emva kokutsalwa kwe-phage, ukucocwa kunye nokususwa kwe-endotoxin (njengoko kuchazwe ngasentla), i-2 × 1010 phages / isilwanyana kwi-300 μl yafakwa nge-intravenously kwi-vein enye yomsila.
Ukulungiswa kwangaphambili kunye nokuhluzwa okusemgangathweni kwedatha yolandelelwano.Idatha ye-454/Roche ekrwada yatshintshwa ukusuka kwifomati yemephu yomsinga womgangatho wokubini (sff) ukuya kwifomati efundekayo ye-Pearson yabantu (i-fasta) kusetyenziswa isoftware yomthengisi.Ukuqhutyelwa phambili kokulandelelana kwe-nucleotide kwenziwa kusetyenziswa iinkqubo ze-C ze-proprietary kunye nezikripthi (iphakheji yesofthiwe engavumelekanga) njengoko kuchazwe ngezantsi.Uhlalutyo lwedatha ephambili lubandakanya iinkqubo ezingqongqo zokucoca izigaba ezininzi.Ukuhluza ngaphandle ukufunda okungaqulathanga ulandelelwano lwe-DNA olusebenzayo lwe-12mer, ufundo lwalulungelelaniswe ngokulandelelana ukuqala ileyibhile (GTGATGTCGGGGATCCGAATTCT), misa ileyibhile (TAAGCTTGCGGCCGCACTCGAGTA) kunye nofakelo lwangasemva (CCCTGCAGGGATATCCCGGAGCTCTCGTschCGAC) kusetyenziswa uvavanyo lwehlabathi jikelele lwe-Next.ulungelelwaniso oluvumela ukuya kuthi ga kwi-2 engahambelaniyo ngolungelelwaniso ngalunye31.Ngoko ke, ukufunda ngaphandle kokuqala kunye nokumisa iithegi kunye nokufunda okuqulethe izinto ezifakwe ngasemva, okt, ulungelelwaniso oludlula inani elivumelekileyo lokungafani, lususiwe kwithala leencwadi.Ngokumalunga nofundo oluseleyo, ulandelelwano lwe-N-mer DNA olusuka kumanqaku okuqalisa luze luphele phambi kokuba uphawu lokuyeka lucatshulwe kulandelelwano lokufunda lokuqala luze luqhubekeke (emva koku kubhekiselwa kulo “njengokufaka”).Emva kokuguqulelwa kokufakela, isahlulo emva kwe-codon yokuqala yokumisa ekupheleni kwe-5 'ye-primer isuswe kwi-insert.Ukongezelela, i-nucleotides ekhokelela kwii-codons ezingaphelelanga kwi-3 "isiphelo se-primer nayo yasuswa.Ukukhuphela ngaphandle ufakelo oluqulathe ulandelelwano lwangasemva kuphela, ufakelo oluguqulelweyo oluqala ngepateni yeamino acid “PAG” nazo zisusiwe.Iipeptide ezinobude obusemva koguqulelo obungaphantsi kwe-3 amino acids zasuswa kwithala leencwadi.Ekugqibeleni, susa ukuphindaphinda kwi-pool yokufaka kwaye unqume ukuphindaphinda kofakelo ngalunye olulodwa.Iziphumo zolu hlalutyo zibandakanya uluhlu lwee-nucleotide ze-nucleotide (ukufakwa) kunye ne-frequencies yabo (ukufunda) (i-Supplementary Figures 1c kunye ne-2).
Iqela le-N-mer DNA ifaka ngokulandelelana ukufana: Ukuphelisa iimpazamo ezilandelelanayo ze-454 / i-Roche-specific (ezifana neengxaki zokulandelelana kwezandiso ze-homopolymer) kunye nokususa ukuphindaphinda okungabalulekanga, ukufakwa kwangaphambili kwe-N-mer DNA yokulandelelana (ukufakwa) kuhlelwe ngokufana.ukufakwa (ukuya kwiziseko ezi-2 ezingahambelaniyo ezivunyelweyo) kusetyenziswa i-algorithm ephindaphindwayo echazwe ngolu hlobo lulandelayo: ukufakwa kuhlelwe kuqala ngokuphindaphindiweyo (kuphezulu ukuya kusezantsi), kwaye ukuba ziyafana, ngohlobo lwazo lwesibini ngobude (olona lude ukuya kolufutshane) ).Ngaloo ndlela, ukufakwa rhoqo kunye nokude kakhulu kuchaza "iqela" lokuqala.I-frequency yeqela isetelwe kwifrikhwensi yesitshixo.Emva koko, ufakelo ngalunye olushiyekileyo kuluhlu oluhleliweyo lwazanywa ukuba longezwe kwiqela ngolungelelwaniso lweNeedleman-Wunsch ngababini.Ukuba inani lokungahambelani, ukufakwa, okanye ukucinywa kulungelelwaniso akudluli kumda we-2, ukufakwa kongezwa kwiqela, kwaye i-frequency yeqela lilonke yandiswa ngokuba kaninzi kangakanani ukufakwa kongezwa.Izinto ezifakwe kwiqela ziphawulwa njengezisetyenzisiweyo kwaye aziquki ekuqhubekeni phambili.Ukuba ulandelelwano lofakelo alinakudityaniswa kwiqela esele likho, ulandelelwano lofakelo lusetyenziselwa ukwenza iqela elitsha elinefrikhwensi yokufaka efanelekileyo kwaye iphawulwe njengesetyenzisiweyo.Uphindaphindo luphela xa ulandelelwano lofakelo ngalunye lusetyenziswe ukwenza iqela elitsha okanye lunokuqukwa kwiqela esele likho.Emva kwayo yonke loo nto, ukufakwa kwamaqela okubandakanya i-nucleotides ekugqibeleni kuguqulelwa kulandelelwano lwepeptide (iilayibrari zepeptide).Isiphumo solu hlalutyo yisethi yokufakwa kunye nee-frequencies zabo ezihambelanayo ezenza inani lokufunda ngokulandelelanayo (i-Supplementary Fig. 2).
Isizukulwana se-Motif: Ngokusekwe kuluhlu lweepeptides ezizodwa, ithala leencwadi lenziwe liqulathe zonke iipateni ze-amino acid ezinokwenzeka (aa) njengoko kubonisiwe ngezantsi.Ipateni nganye enokwenzeka yobude be-3 ikhutshwe kwi-peptide kwaye iphethini yayo eguquguqukayo yongezwa kunye nelayibrari ye-motif eqhelekileyo equlethe zonke iipatheni (i-tripeptides).Amathala eencwadi anemixholo ephindaphindwayo aye alandelelana kwaye ukungafuneki kwasuswa.Emva koko, kwi-tripeptide nganye kwithala leencwadi le-motif, sajonga ubukho bayo kwithala leencwadi sisebenzisa izixhobo zokubala.Kule meko, ukuphindaphinda kwe-peptide equlethe i-motif tripeptide efunyenweyo yongezwa kwaye yabelwe i-motif kwilayibrari ye-motif ("inani le-motifs").Isiphumo sokwenziwa kwe-motif luluhlu olunamacala amabini aqulathe zonke izehlo ze-tripeptides (i-motifs) kunye namaxabiso azo ahlukeneyo, inani lolandelelwano lofundo olukhokelela kwimotif ehambelanayo xa ufundo luhluzwa, ludityaniswe ngokwamaqela, kwaye luguqulelwe.Iimetriki njengoko zichazwe ngokweenkcukacha ngasentla.
Ukuqhelaniswa kwenani leemotifs kunye ne-scatterplots ezihambelanayo: Inani le-motifs kwisampulu nganye yayiqhelekile kusetyenziswa.
apho i-ni linani lofundo oluqulathe isihloko i.Ke, i-vi imele ipesenti yokufunda (okanye iipeptides) equlethe i-motif i kwisampulu.Amaxabiso e-P yenani elingaqhelekanga lemotifs abalwe kusetyenziswa uvavanyo oluchanekileyo lukaFisher.Ngokumalunga nolungelelwaniso lwenani leenjongo, ulungelelwaniso luka-Spearman lwabalwa kusetyenziswa inani eliqhelekileyo leenjongo ezine-R.
Ukubona umxholo wee-amino acids kwindawo nganye kwithala leencwadi le-peptide, iilogo zewebhu ezingama-32, ezingama-33 (http://weblogo.threeplusone.com) zenziwa.Okokuqala, umxholo we-amino acids kwindawo nganye ye-peptide ye-12-mer igcinwe kwi-matrix ye-20 × 12.Emva koko, iseti yeepeptide ezili-1000 eziqulethe umxholo ofanayo we-amino acid kwindawo nganye iveliswa kwifomathi yolandelelwano lwe-fasta kwaye inikezelwe njengegalelo kwilogo yewebhu yesi-3, evelisa umboniso womzobo wesiqulatho se-amino acid kwindawo nganye.kwilayibrari yepeptide enikiweyo.Ukujonga ngeso lengqondo iiseti zedatha ezininzi, iimephu zobushushu zenziwe kusetyenziswa isixhobo esiphuhliswe ngaphakathi kwi-R (biosHeatmap, iphakheji ye-R engekakhutshwa).Iidendrograms ezithiwe thaca kwiimephu zobushushu zibalwe kusetyenziswa indlela yeWadi yokuhlanganisana ngokwemigangatho yomgama we-Euclidean.Kucazululo lwamanani edatha yamanqaku e-motif, amaxabiso e-P okufumana amanqaku angaqhelekanga abalwa kusetyenziswa uvavanyo oluchanekileyo lukaFisher.Amaxabiso e-P kwezinye iiseti zedatha abalwa ngo-R kusetyenziswa uvavanyo lwe-t loMfundi okanye i-ANOVA.
Ii-clones ze-phage ezikhethiweyo kunye ne-phage ngaphandle kokufaka zifakwe nge-intravenously nge-vein yomsila (2 × 1010 phages / isilwanyana kwi-300 μl PBS).Imizuzu elishumi ngaphambi kokuxutywa kunye nokulungiswa okulandelayo, izilwanyana ezifanayo zafakwa emithanjeni nge-100 μl ye-DyLight594 ebhalwe i-lectin (Vector Laboratories Inc., DL-1177).Imizuzu engama-60 emva kwesitofu se-phage, iigundane zixutywe entliziyweni kunye ne-50 ml ye-PBS elandelwa yi-50 ml 4% PFA / PBS.Iisampulu zobuchopho zalungiswa ubusuku bonke kwi-4% PFA/PBS kwaye zifakwe kwi-30% sucrose ngobusuku kwi-4 ° C.Iisampulu ziqhwanyaza zikhenkce kumxube we-OCT.Uhlalutyo lwe-Immunohistochemical lweesampulu ezikhenkcezisiweyo lwenziwa kwiqondo lobushushu begumbi kwi-30 µm cryosections evalwe nge-1% BSA kwaye incubated nge-polyclonal FITC-ezibhalwe izilwa-buhlungu ezichasene ne-T7 phage (Novus NB 600-376A) kwi-4 °C.Fuka ubusuku bonke.Ekugqibeleni, amacandelo ahlanjwe ngamaxesha e-3 nge-PBS kwaye ahlolwe nge-microscope ye-laser confocal (Leica TCS SP5).
Zonke iipeptide ezinobuncinci bokucoceka kwe-98% zenziwe yi-GenScript USA, i-biotinylated kunye ne-lyophilized.I-Biotin ibotshelelwa nge-triple glycine spacer kwi-N-terminus.Jonga zonke iipeptides usebenzisa i-mass spectrometry.
I-Streptavidin (i-Sigma S0677) ixutywe kunye ne-5-fold equimolar excess of biotinylated peptide, biotinylated BACE1 inhibitory peptide, okanye indibaniselwano (3: 1 ratio) ye-biotinylated BACE1 inhibitory peptide kunye ne-BACE1 inhibitory peptide kwi-5-10% ye-DM ekwi-SOO.Iyure eyi-1 kwiqondo lokushisa kwigumbi ngaphambi kokutofa.I-Streptavidin-conjugated peptides yajojowe nge-intravenously kwi-dose ye-10 mg / kg kwenye yemithanjeni yomsila weegundane kunye ne-cerebral cavity.
Uxinzelelo lwe-streptavidin-peptide complexes luhlolwe ngu-ELISA.I-Nunc Maxisorp microtiter plates (i-Sigma) ifakwe ubusuku bonke kwi-4 ° C kunye ne-1.5 μg / ml i-anti-streptavidin antibody (i-Thermo, MA1-20011).Emva kokuthintela (i-blocking buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) kwiqondo lokushisa kweeyure ze-2, hlamba ipleyiti nge-0.05% Tween-20 / PBS (i-buffer yokuhlamba) ye-3 Second, CluSF1 yongezwa kunye ne-plasma ye-plasma edibeneyo kunye ne-plasma ye-0. 0, CSF 1:115).I-plate yabe ifakwe ngobusuku kwi-4 ° C kunye ne-antibody yokufumanisa (1 μg / ml, i-anti-streptavidin-HRP, i-Novus NB120-7239).Emva kwamanyathelo amathathu okuhlamba, i-streptavidin yafunyanwa ngokufaka i-incubation kwisisombululo se-substrate ye-TMB (i-Roche) ukuya kwi-20 min.Emva kokumisa ukuphuhliswa kombala kunye ne-1M H2SO4, ukulinganisa ukuxhamla kwi-450 nm.
Umsebenzi we-streptavidin-peptide-BACE1 inhibitor complex ihlolwe ngu-Aβ (1-40) ELISA ngokweprotocol yomenzi (Wako, 294-64701).Ngokufutshane, iisampulu ze-CSF zaxutywa kwi-diluent esemgangathweni (1:23) kwaye zafakwa ngobusuku kwi-4 ° C kwiipleyiti zequla ezingama-96 ezigqunywe nge-BNT77 yokubamba i-antibody.Emva kwamanyathelo amahlanu okuhlamba, i-HRP-conjugated i-BA27 ye-antibody yongezwa yaza yafukanyelwa iiyure ezi-2 kwi-4° C., ilandelwa ngamanyathelo amahlanu okuhlamba.I-Aβ (1–40) yafunyanwa ngokufukanyelwa kwisisombululo se-TMB imizuzu engama-30 kwiqondo lobushushu begumbi.Emva kokuba uphuhliso lombala luyekisiwe kunye nesisombululo sokumisa, ukulinganisa ukuxhamla kwi-450 nm.Iisampulu zePlasma ziphantsi kokukhutshwa kwesigaba esiqinileyo ngaphambi kwe-Aβ (1-40) ELISA.I-Plasma yongezwa kwi-0.2% DEA (Sigma) kwiiplate ze-96-well kwaye zifakwe kwindawo yokushisa kwe-30 imizuzu.Emva kokuhlamba ngokulandelelana iipleyiti ze-SPE (Oasis, 186000679) ngamanzi kunye ne-100% ye-methanol, iisampulu zeplasma zongezwa kwiipleyiti ze-SPE kwaye lonke ulwelo lwasuswa.Iisampulu zahlanjwa (kuqala nge-5% methanol emva koko i-30% ye-methanol) kwaye yahlanjululwa nge-2% NH4OH/90% methanol.Emva kokumisa i-eluate kwi-55 ° C kwi-99 min kwi-N2 yangoku, iisampuli zancitshiswa kwii-diluents eziqhelekileyo kwaye i-Aβ (1-40) yalinganiswa njengoko kuchazwe ngasentla.
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