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Ihe mgbochi ụbụrụ nke ọbara na mgbochi ụbụrụ ọbara na-egbochi ndị na-ahụ maka biotherapeutic ka ha rute ebumnuche ha na sistemụ ụjọ nke etiti, si otú a na-egbochi ọgwụgwọ dị irè nke ọrịa akwara ozi.Iji chọpụta ndị na-ebu ụbụrụ ụbụrụ na vivo, anyị webatara ọbá akwụkwọ T7 phage peptide na chịkọta ọbara na mmiri cerebrospinal (CSF) site na iji ụdị oke oke ama ama ama ama.Emebere clones phage nke ukwuu na CSF ka agbachara nhọrọ anọ gasịrị.Nnwale nke peptides n'otu n'otu gosipụtara mmụba karịa 1000-fold na CSF.Ekwenyere bioactivity nke nnyefe mgbasa ozi peptide na ụbụrụ site na mbelata 40% na ọkwa amyloid-β na mmiri cerebrospinal site na iji ihe mgbochi BACE1 peptide jikọtara ya na peptide transit novel mara.Nsonaazụ ndị a na-atụ aro na peptides mara site na ụzọ nhọrọ nhọrọ vivo phage nwere ike ịbụ ụgbọ ala bara uru maka nnyefe sistemu nke macromolecules na ụbụrụ nwere mmetụta ọgwụgwọ.
Usoro nyocha ọgwụgwọ ezubere iche (CNS) lekwasịrị anya n'ụzọ dị ukwuu n'ịchọpụta ọgwụ kachasị mma na ndị nnọchi anya na-egosipụta akụrụngwa na-eche CNS, yana obere mbọ na-achọpụta usoro na-ebuga ọgwụ na-ebuga ụbụrụ.Nke a na-amalite ịgbanwe ugbu a dị ka nnyefe ọgwụ, karịsịa nnukwu ụmụ irighiri ihe, bụ akụkụ dị mkpa nke mmepe ọgwụ neuroscience ọgbara ọhụrụ.A na-echebe gburugburu ebe obibi nke usoro nhụjuanya nke ọma site na usoro mgbochi ụbụrụ cerebrovascular, nke gụnyere ihe mgbochi ụbụrụ ọbara (BBB) ​​na mgbochi ụbụrụ ọbara (BCBB) 1, na-eme ka ọ sie ike ịnye ọgwụ na ụbụrụ1,2.A na-eme atụmatụ na ihe fọrọ nke nta ka ọ bụrụ ọgwụ niile buru ibu na ihe karịrị 98% nke obere ọgwụ molecule na-ekpochapụ n'ụbụrụ3.Ọ bụ ya mere ọ dị ezigbo mkpa iji chọpụta usoro ụgbọ njem ụbụrụ ọhụrụ nke na-enye nnyefe nke ọma na kpọmkwem nke ọgwụ ọgwụgwọ na CNS 4,5.Otú ọ dị, BBB na BCSFB na-enyekwa ohere magburu onwe ya maka nnyefe ọgwụ ka ha na-abanye ma na-abanye na akụkụ niile nke ụbụrụ site na nnukwu vasculature ya.Ya mere, mgbalị ndị a na-eme ugbu a iji ụzọ ndị na-adịghị emerụ ahụ nke nnyefe na ụbụrụ na-adabere n'ụzọ dị ukwuu na usoro nke nnyefe mgbasa ozi na-anabata (PMT) site na iji BBB6 receptor endogenous.N'agbanyeghị ọganihu dị mkpa n'oge na-adịbeghị anya site n'iji ụzọ nnyefe transferrin7,8, a na-achọkwu mmepe nke usoro nnyefe ọhụrụ na ihe ndị dị mma.Iji mezuo nke a, ebumnuche anyị bụ ịchọpụta peptides nwere ike ịgbazite ụgbọ njem CSF, n'ihi na enwere ike iji ha na-ebuga macromolecules na CNS ma ọ bụ mepee ụzọ nnabata ọhụrụ.Karịsịa, ndị na-anabata ya na ndị na-ebufe sistemu cerebrovascular (BBB na BSCFB) nwere ike ịbụ ihe mgbaru ọsọ maka nnyefe na-arụsi ọrụ ike na nke kpọmkwem nke ọgwụ biotherapeutic.Mmiri Cerebrospinal (CSF) bụ ngwaahịa nzuzo nke choroid plexus (CS) ma na-akpakọrịta kpọmkwem na mmiri interstitial nke ụbụrụ site na oghere subarachnoid na ventricular space4.Na nso nso a, egosiri na mmiri nke cerebrospinal subarachnoid na-agbasa nke ukwuu n'ime interstitium nke ụbụrụ9.Anyị na-atụ anya ịnweta oghere parenchymal site na iji traktị inflow subarachnoid ma ọ bụ ozugbo site na BBB.Iji mezuo nke a, anyị mebere atụmatụ siri ike na vivo phage nhọrọ nke na-akọwapụta nke ọma peptides nke ọ bụla n'ime ụzọ abụọ a dị iche na-ebufe.
Ugbu a, anyị na-akọwa usoro nyocha usoro ihe ngosi na vivo phage yana nlele CSF yana usoro mmepụta dị elu (HTS) iji nyochaa agba nhọrọ mbụ nwere ụdị ọba akwụkwọ kachasị elu.Emere nyocha n'elu oke ndị maara nke ọma nwere nnukwu olulu mmiri (CM) etinyere na-adịgide adịgide iji zere mmetọ ọbara.N'ụzọ dị mkpa, ụzọ a na-ahọrọ ma nlebara anya ụbụrụ yana peptides nwere ọrụ njem gafere ihe mgbochi cerebrovascular.Anyị na-eji T7 phages n'ihi obere nha ha (~ 60 nm) 10 ma na-atụ aro na ha dị mma maka njem nke vesicles nke na-enye ohere ịgafe transcellular nke mgbochi endothelial na / ma ọ bụ epithelial-medulla.Mgbe agba anọ nke panning gasịrị, ndị phage nọpụrụ iche na-egosi ike na vivo CSF ​​​​inrichment na cerebral microvessel Association.N'ụzọ dị mkpa, anyị nwere ike ịkwado nchoputa anyị site n'igosipụta na peptides kachasị mma na nke a na-emepụta na kemịkal nwere ike ibufe ibu protein n'ime mmiri cerebrospinal.Nke mbụ, e hiwere mmetụta ọgwụ ike nke CNS site na ijikọta peptide na-eduga na ihe mgbochi nke BACE1 peptide.Na mgbakwunye na igosi na na vivo atụmatụ nyocha ọrụ nwere ike ịchọpụta peptides na-ebufe ụbụrụ ụbụrụ dị ka ndị na-ebu ibu protein dị irè, anyị na-atụ anya ka usoro nhọrọ ọrụ yiri ya ga-adịkwa mkpa n'ịchọpụta ụzọ ụgbọ njem ụbụrụ ọhụrụ.
Dabere na nkeji na-akpụ plaque (PFU), ka emechara usoro nkwakọ ngwaahịa phage, a haziri ma mepụta ọba akwụkwọ nke 12-mer linear T7 phage peptides nwere ụdị dị iche iche nke ihe dịka 109 (lee Ngwa na Ụzọ).Ọ dị mkpa iburu n'obi na anyị nyochachara nke ọma n'ọbá akwụkwọ a tupu in vivo panning.Mmụba PCR nke ihe atụ ọba akwụkwọ phage site na iji primers gbanwetụrụ emepụtara amplicon ndị dabara na HTS (Mgbakwunye Fig. 1a).N'ihi a) HTS11 njehie sequencing, b) mmetụta na àgwà nke primers (NNK) 1-12, na c) ọnụnọ nke anụ ọhịa-ụdị (wt) phage (skeleton inserts) na njikere n'ọbá akwụkwọ, a usoro nzacha usoro e mejuputa atumatu iji wepụta naanị nkwenye usoro ozi (Mgbakwunye Fig. 1b).Usoro nzacha ndị a metụtara ọba akwụkwọ usoro HTS niile.Maka ọbá akwụkwọ ọkọlọtọ, a nwetara ngụkọta nke 233,868 agụ, nke 39% gafere nzacha nzacha ma jiri ya mee nyocha na nhọrọ nke ọbá akwụkwọ maka agba ndị na-esote (Ngbakwunye 1c-e).Ihe gụpụtara bụ nke kachasị ọnụọgụ nke ụzọ abụọ isi 3 n'ogologo nwere ọnụ ọgụgụ kasị elu na 36 nucleotides (Mgbakwunye Fig. 1c), na-akwado nhazi ọba akwụkwọ (NNK) 1-12.N'ụzọ doro anya, ihe dị ka 11% nke ndị otu ụlọ akwụkwọ ahụ nwere ihe ntinye azụ azụ PAGISRELVDKL nwere akụkụ 12 (wt), yana ihe fọrọ nke nta ka ọ bụrụ ọkara nke usoro (49%) nwere ntinye ma ọ bụ nhichapụ.HTS nke ọbá akwụkwọ ọbá akwụkwọ kwadoro ụdị dị iche iche nke peptides dị n'ọbá akwụkwọ: ihe karịrị 81% nke usoro peptide ka a hụrụ naanị otu ugboro na naanị 1.5% mere na ≥4 mbipụta (Mgbakwunye 2a).Ugboro nke amino acid (aa) na ọnọdụ 12 niile dị na repertoire jikọtara nke ọma na ugboro ole a na-atụ anya maka ọnụọgụ codons sitere na NKK repertoire mebiri emebi (Mgbakwunye 2b).Ugboro ole a na-ahụ anya nke ihe fọdụrụ aa nke ntinye ndị a na-etinye na ya jikọtara nke ọma na ugboro agbakọrọ (r = 0.893) (Fig mgbakwunye. 2c).Nkwadebe nke ọba akwụkwọ phage maka ịgba ọgwụ na-agụnye usoro nke mmụba na mwepụ nke endotoxin.Egosiputara nke a na mbụ nwere ike ibelata ụdị ọba akwụkwọ phage12,13.Ya mere, anyị chịkọtara ọba akwụkwọ phage nke e mechiri efere nke ewepụrụ mwepụta endotoxin wee jiri ya tụnyere ọbá akwụkwọ mbụ iji chọpụta ugboro ole AA.Ahụrụ njikọ siri ike (r = 0.995) n'etiti ọdọ mmiri mbụ na ọdọ mmiri agbakwunyere na nke dị ọcha (Mgbakwunye 2d), na-egosi na asọmpi n'etiti clones agbagoro na efere site na iji T7 phage emeghị ka ọ bụrụ nnukwu mkparị.Ntụle a gbadoro ụkwụ na ọnụọgụ tripeptide motifs n'ọbá akwụkwọ ọ bụla, ebe ọ bụ na ụdị ọba akwụkwọ dị iche iche (~ 109) enweghị ike ijide ya nke ọma ọbụlagodi na HTS.Nyocha ugboro ugboro nke aa n'ọnọdụ ọ bụla gosipụtara obere ọnọdụ na-adabere n'ọnọdụ dị n'ọnọdụ atọ ikpeazụ nke ntinye akwụkwọ (Mgbakwunye 2e).N'ikpeazụ, anyị kwubiri na àgwà na ụdị dị iche iche nke ụlọ akwụkwọ ahụ nabatara na ọ bụ naanị obere mgbanwe dị iche iche ka a hụrụ n'ihi mmụba na nkwadebe nke ọba akwụkwọ phage n'etiti ọtụtụ agba nke nhọrọ.
Enwere ike ịme nlele mmiri serial cerebrospinal site na ịwa ahụ ịkụnye cannula n'ime CM nke oke mara mma iji kwado njirimara T7 phage nke a na-agbanye intravenously (iv) site na BBB na / ma ọ bụ BCSFB (Fig. 1a-b).Anyị na-eji ogwe aka nhọrọ abụọ nwere onwe (ogwe aka A na B) na agba atọ mbụ nke nhọrọ vivo (Fig 1c).Anyị ji nwayọọ nwayọọ mụbaa stringent nke nhọrọ site n'ibelata mkpokọta phage ewebata na agba atọ mbụ nke nhọrọ.Maka agba nke anọ nke panning, anyị jikọtara ihe nlele sitere na alaka A na B wee mee nhọrọ atọ ọzọ nwere onwe.Iji mụọ akụrụngwa in vivo nke ụmụ irighiri ihe T7 phage n'ụdị a, a na-etinye ụdị phage anụ ọhịa (PAGISRELVDKL master insert) n'ime oke site na vein ọdụ.Iweghachite nke phages sitere na mmiri cerebrospinal na ọbara n'oge dị iche iche gosiri na obere T7 icosahedral phages nwere usoro nkwụsị ngwa ngwa ngwa ngwa site na ngalaba ọbara (Ngbakwunye 3).Dabere na titers enyere na oke ọbara nke oke, anyị gbakọrọ na naanị ihe dịka 1% wt.Achọpụtara phage site na ọgwụ a na-enye n'ime ọbara nkeji iri mgbe ịgba ọgwụ mgbochi ọrịa gachara.Mgbe mgbada ngwa ngwa nke mbụ a gasịrị, ejiri ọkara ndụ nke nkeji 27.7 tụọ njiri nwayọ nwayọ.N'ụzọ dị mkpa, ọ bụ naanị phages ole na ole ka ewepụtara na ngalaba CSF, na-egosi nzụlite dị ala maka ịkwaga ụdị phage ọhịa n'ime ngalaba CSF (Mgbakwunye Fig. 3).Na nkezi, naanị ihe dịka 1 x 10-3% titers nke T7 phage n'ọbara na 4 x 10-8% nke phages etinyere na mbụ ka achọpụtara na mmiri cerebrospinal n'ime oge nlele niile (0-250 min).N'ụzọ doro anya, ọkara ndụ (25.7 min) nke ụdị anụ ọhịa phage na cerebrospinal fluid yiri nke a hụrụ n'ime ọbara.Ihe data ndị a na-egosi na ihe mgbochi nkewapụ akụkụ CSF na ọbara adịghị na oke CM-cannulated, na-enye ohere na vivo nhọrọ nke ọba akwụkwọ phage iji chọpụta clones ndị a na-ebuga ngwa ngwa site na ọbara n'ime ngalaba CSF.
(a) Ịtọlite ​​​​usoro maka re-sampling cerebrospinal fluid (CSF) site na nnukwu ọdọ mmiri.(b) Eserese na-egosi ọnọdụ cellular nke ihe mgbochi akwara etiti (CNS) na usoro nhọrọ eji achọpụta peptides na-agafe mgbochi ụbụrụ ọbara (BBB) ​​na mgbochi ụbụrụ ọbara.(c) Ngosipụta ngosi ihe ngosi na vivo phage.Na nhọpụta nke ọ bụla, a na-agbaba phages (ihe na-achọpụta anụmanụ n'ime akụ).A na-edobe alaka abụọ nọọrọ onwe ha (A, B) iche iche ruo agba nke anọ nke nhọrọ.Maka okirikiri nhọrọ 3 na 4, ejiri aka hazie clone phage ọ bụla ewepụtara na CSF.(d) Kinetics nke phage dịpụrụ adịpụ site na ọbara ( okirikiri uhie) na mmiri cerebrospinal (triangles green) n'oge agba nke mbụ nke nhọrọ n'ime oke abụọ nwere cannulated mgbe ịgbanye eriri afọ nke ọba akwụkwọ T7 peptide (2 x 1012 phages / anụmanụ).Oghere na-acha anụnụ anụnụ na-egosi nkezi ntinye mbụ nke phage n'ime ọbara, gbakọọ site na ọnụọgụ phage ịgbanye, na-eburu n'uche mkpokọta ọbara.Oghere ojii ndị ahụ na-egosi ebe nkwụsị nke ahịrị y ewepụtara na mkpokọta phage ọbara.(e,f) Wepụta ugboro ole na nkesa nke ụdị trieptide niile nwere ike imechi nke achọtara na peptide.E gosipụtara ọnụ ọgụgụ nke motifs dị na 1000 ọgụgụ.N'ụzọ dị ịrịba ama (p <0.001) ka e ji ntụpọ uhie mee akara motifs bara ụba.(e) Mgbasa mgbasa ozi na-atụnyere ugboro ugboro nke tripeptide motif nke ọbá akwụkwọ ịgbanye na phage sitere na ọbara sitere na anụmanụ #1.1 na #1.2.(f) Mgbasa mgbasa ozi na-atụnyere frequencies nke anụmanụ phage tripeptide motifs #1.1 na #1.2 dịpụrụ adịpụ na ọbara na mmiri cerebrospinal.(g, h) Nnọchite anya NJ usoro nke phage bara ụba na ọbara (g) megide ọba akwụkwọ ịgbanye na phage bara ụba na CSF (h) n'ọbara ka emechara nhọrọ vivo n'ime anụmanụ abụọ ahụ.Nha koodu nke otu mkpụrụedemede na-egosi ugboro ole amino acid na-apụta n'ọnọdụ ahụ.Green = polar, odo odo = na-anọpụ iche, acha anụnụ anụnụ = isi, uhie = acidic na nwa = hydrophobic amino acids.Ọdịiche 1a, b bụ Eduard Urich chepụtara wee mepụta ya.
Anyị gbabara ọbá akwụkwọ phage peptide n'ime oke ngwá ọrụ CM abụọ (clades A na B) yana phage dịpụrụ adịpụ site na mmiri mmiri na ọbara (Onyonyo 1d).Mwepu ngwa ngwa mbụ nke ụlọ akwụkwọ ahụ adịchaghị akpọpụta ma e jiri ya tụnyere ụdị phage ọhịa.Ogologo ndụ ọkara nke ọbá akwụkwọ ịgbanye n'ime anụmanụ abụọ ahụ bụ nkeji 24.8 n'ọbara, yiri ụdị phage ọhịa, yana nkeji 38.5 na CSF.A na-edobere ihe nlele ọbara na cerebrospinal fluid phage nke anụmanụ ọ bụla na HTS ma nyochara peptides niile achọpụtara maka ọnụnọ nke mkpụmkpụ tripeptide.A họọrọ motifs Tripeptide n'ihi na ha na-enye ntakịrị ihe ndabere maka nhazi nhazi na mmekọrịta protein-protein14,15.Anyị chọtara mmekọrịta dị mma na nkesa nke motifs n'etiti ọbá akwụkwọ phage injected na clones ewepụtara n'ọbara nke anụmanụ abụọ ahụ (Fig 1e).Ihe omuma a na-egosi na a na-eme ka ihe mejupụtara ọbá akwụkwọ ahụ dịkwuo ntakịrị n'ime akụkụ ọbara.A na-enyocha ugboro ugboro Amino acid na usoro nkwenye n'ọnọdụ ọ bụla site na iji mmegharị nke ngwa Weblogo16.N'ụzọ na-akpali mmasị, anyị hụrụ mmụba siri ike n'ime ọbara glycine residues (Fig 1g).Mgbe e jiri ọbara tụnyere clones ahọpụtara site na CSF, a na-ahụ nhọrọ siri ike na ụfọdụ deselection nke motifs (Fig 1f), na ụfọdụ amino acid dị mma na ọnọdụ ndị a kara aka na 12-otu (Fig 1h).N'ụzọ doro anya, anụmanụ dị iche iche dị iche iche na mmiri cerebrospinal, ebe a na-ahụ mmụba glycine n'ọbara n'ime anụmanụ abụọ ahụ (Mgbakwunye 4a-j).Mgbe stringent nzacha nke usoro data na cerebrospinal mmiri mmiri nke anụmanụ #1.1 na #1.2, a ngụkọta nke 964 na 420 pụrụ iche 12-mer peptides nwetara (Mgbakwunye Fig. 1d-e).Emebere clones phage dịpụrụ adịpụ ma tinye ya na agba nke abụọ nke nhọrọ vivo.A na-edobere Phage nke ewepụtara site na agba nke abụọ nke HTS na anụmanụ ọ bụla na peptides niile achọpụtara na-eji dị ka ntinye aka na mmemme nnabata motif iji nyochaa ihe omume nke trieptide motifs (Fig. 2a, b, ef).N'iji ya tụnyere nke mbụ okirikiri nke phage nwetara site na CSF, anyị hụrụ n'ihu nhọrọ na deselection nke ọtụtụ motifs na CSF na alaka A na B (Fig. 2).A na-etinye algọridim njirimara netwọkụ iji chọpụta ma ha na-anọchi anya ụkpụrụ dị iche iche nke usoro na-agbanwe agbanwe.Ahụrụ myirịta doro anya n'etiti usoro 12-akụkụ nke CSF nwetara na clade ọzọ A (Fig 2c, d) na clade B (Fig. 2g, h).Nyocha a gbakọtara na alaka ọ bụla gosipụtara profaịlụ nhọrọ dị iche iche maka 12-mer peptides (Mgbakwunye 5c, d) na mmụba nke CSF / titer titer ratio na oge maka clones jikọtara ọnụ mgbe agba nke abụọ nke nhọrọ ma e jiri ya tụnyere nke mbụ nke nhọrọ (Mgbakwunye 5e).).
Ịba ụba nke motifs na peptides na mmiri cerebrospinal site na agba abụọ na-esochi nke in vivo functional phage ngosi nhọrọ.
All cerebrospinal fluid phages gbakere site na agba nke mbụ nke anụmanụ ọ bụla (anụmanụ # 1.1 na # 1.2) gbakọtara, gbasaa, HT-usoro na reinjected ọnụ (2 x 1010 phages / anụmanụ) 2 SM cannulated oke (#1.1 → #).2.1 na 2.2, 1.2 → 2.3 na 2.4).(a,b,e,f) Mgbasa mgbasa ozi na-atụnyere ọnụọgụ nke trieptide motifs nke phages niile sitere na CSF na agba nke mbụ na nke abụọ.Ugboro ugboro na nkesa motifs na-anọchi anya tripeptides niile nwere ike imechi nke a chọtara na peptides n'akụkụ abụọ ahụ.E gosipụtara ọnụ ọgụgụ nke motifs dị na 1000 ọgụgụ.E ji ntụpọ na-acha uhie uhie pụta ìhè Motifs ndị dị mkpa (p <0.001) ma ọ bụ wepụrụ n'otu ọba akwụkwọ atụnyere.(c, d, g, h) Nnọchite anya akara akara nke usoro amino acid niile nwere CSF nwere ogologo usoro iri na abụọ dabere na agba nke 2 na 1 nke in vivo nhọrọ.Nha koodu nke otu mkpụrụedemede na-egosi ugboro ole amino acid na-apụta n'ọnọdụ ahụ.Iji nọchite anya akara ngosi, a na-atụnyere ugboro usoro CSF ​​nke ewepụtara na anụmanụ n'otu n'otu n'etiti okirikiri nhọrọ abụọ wee gosipụta usoro bara ụba na agba nke abụọ: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 na (h) #1.2–#2.4.Amino acid kachasị emejupụta n'ọnọdụ enyere na (c, d) anụmanụ mba.2.1 na mba.2.2 ma ọ bụ (g, h) n'ime anụmanụ mba.2.3 na mba.2.4 ka egosiri na agba.Green = polar, odo odo = na-anọpụ iche, acha anụnụ anụnụ = isi, uhie = acidic na nwa = hydrophobic amino acids.
Mgbe agba nke atọ nke nhọrọ, anyị chọpụtara 124 pụrụ iche peptide usoro (#3.1 na #3.2) si 332 CSF-reconstituted phage clones dịpụrụ adịpụ site na ụmụ anụmanụ abụọ (Mgbakwunye Fig. 6a).Usoro LGSVS (18.7%) nwere oke ikwu kachasị elu, sochiri ụdị ntinye anụ ọhịa PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), na SARGSWREIVSLS (2.2%).Na agba nke anọ ikpeazụ, anyị jikọtara alaka abụọ ahọpụtara onwe ha site na anụmanụ atọ dị iche iche (Fig 1c).N'ime 925 usoro phage clones nwetara site na CSF, na nke anọ anyị hụrụ 64 peptide usoro pụrụ iche (Mgbakwunye Fig. 6b), n'ime nke ndị ikwu nke ụdị phage ọhịa gbadara ruo 0.8%.Ihe mkpuchi CSF kachasị na agba nke anọ bụ LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) na RLSSVDSDLSGC%) (3.%)).Ogologo ogologo nke peptides ahọpụtara bụ n'ihi ntinye / nchapụta nucleotide ma ọ bụ codons nkwụsị akaghi aka n'ime ụlọ akwụkwọ ọbá akwụkwọ mgbe ị na-eji codons mebiri emebi maka imepụta ọba akwụkwọ NNK.Codons nkwụsị akabeghị aka na-emepụta peptides dị mkpụmkpụ ma họrọ ya n'ihi na ha nwere ihe dị mma aa motif.peptides ogologo nwere ike ịpụta site na ntinye/nhichapụ na mbido nke ọba akwụkwọ sịntetik.Nke a na-edobe codon nkwụsị emebere na mpụga etiti ahụ wee gụọ ya ruo mgbe codon nkwụsị ọhụrụ pụtara n'okpuru.N'ozuzu, anyị gbakọrọ ihe ndị na-eme ka ahụ dịkwuo mma maka okirikiri nhọrọ anọ niile site na iji data ntinye tụnyere data mmepụta ihe nlele.Maka nyocha nke mbụ nke mbụ, anyị na-eji ụdị phage titers dị ka ihe ndabere na-abụghị nke akọwapụtara.N'ụzọ na-akpali mmasị, nhọrọ phage na-adịghị mma dị nnọọ ike na nke mbụ CSF okirikiri, ma ọ bụghị na ọbara (Fig. 3a), nke nwere ike ịbụ n'ihi na obere ihe gbasara nke puru omume nke passive mgbasa nke ọtụtụ ndị òtù nke peptide ọbá akwụkwọ n'ime CSF ngalaba ma ọ bụ ndị ikwu phages na-emekarị nke ọma na-ejigide ma ọ bụ wepụ si n'ọbara karịa bacteriophages.Otú ọ dị, na agba nke abụọ nke panning, a na-ahụ nhọrọ siri ike nke phages na CSF na clades abụọ, na-atụ aro na agba nke gara aga na-emewanyewanye na phages na-egosipụta peptides na-akwalite nkwalite CSF (Fig 3a).Ọzọ, na-enweghị nnukwu mmụba ọbara.Ọzọkwa na agba nke atọ na nke anọ, phage clones emelitere nke ọma na CSF.N'ịtụle ọnụọgụ nke usoro peptide ọ bụla pụrụ iche n'etiti agba abụọ ikpeazụ nke nhọrọ, anyị chọpụtara na usoro ndị ahụ na-emewanyewanyewanye na agba nke anọ nke nhọrọ (Fig 3b).Ngụkọta nke 931 tripeptide motifs ka ewepụtara site na usoro peptide 64 pụrụ iche site na iji ụzọ peptide abụọ.Ebumnuche ndị kachasị baa ọgaranya na agba nke anọ ka a na-enyocha nke ọma maka profaịlụ nkwalite ha n'ofe gburugburu niile ma e jiri ya tụnyere ụlọ akwụkwọ a gbanyere mkpọrọgwụ (nkwụsị: 10% enrichment) (Mgbakwunye 6c).Usoro nhọpụta n'ozuzu gosiri na ọtụtụ n'ime ebumnobi ọmụmụ amụbanyere na agba niile gara aga nke ngalaba abụọ nhọrọ.Agbanyeghị, ụfọdụ motifs (dịka SGL, VSG, LGS GSV) sitere na clade ọzọ A, ebe ndị ọzọ (dịka FGW, RTN, WGF, NTR) emelitere na clade B ọzọ.
Nkwado nke njem CSF nke peptides gosipụtara phage nke CSF na peptides onye ndu biotinylated jikọtara na ibu streptavidin.
(a) A na-agbakọ oke mmụba na agba anọ niile (R1-R4) dabere na ntinye (ntinye = I) phage (PFU) titers na mkpebi CSF phage titers (mpụta = O).A na-agbakọ ihe ndị na-eme ka ọ dịkwuo mma maka agba atọ ikpeazụ (R2-R4) site n'iji tụnyere nke gara aga na nke mbụ (R1) na data arọ.Ogwe mepere emepe bụ mmiri cerebrospinal, ogwe ndo bụ plasma.(***p<0.001, dabere na t-ule nwata akwụkwọ).(b) Ndepụta nke peptides phage kachasị ukwuu, nke a họọrọ dịka nha nha ha na phages niile anakọtara na CSF ka agbachara 4 nke nhọrọ.A na-akọwapụta clones phage isii kachasị na agba, agụọ ọnụ yana ihe na-eme ka ha nwekwuo mmụba n'etiti agba 3 na 4 nke nhọrọ (insets).(c,d) A na-enyocha phage clone isii kachasị baa ọgaranya, phage efu na ọba akwụkwọ peptide nke ndị nne na nna sitere na gburugburu 4 n'otu n'otu na ụdị nlele CSF.A nakọtara CSF na nlele ọbara n'oge oge egosipụtara.(c) nha nha nke 6 nwa akwukwo phage clones (2 x 1010 phages / anụmanụ), phages efu (#1779) (2 x 1010 phages / anụmanụ) na ọba akwụkwọ phage peptide (2 x 1012 phages / anụmanụ) ịgbanye ma ọ dịkarịa ala 3 CM ka a na-enye anụ ahụ iche iche.E gosipụtara ọgwụ ọgwụ CSF nke phage clone na ọba akwụkwọ phage peptide n'ime oge ka egosiri.(d) na-egosi nkezi CSF/ọbara ruru maka phages/mL niile enwetara n'oge nlele.(e) A na-ejikọta peptides onye ndu anọ sịntetik na otu njikwa scrambled na biotin na streptavidin site na N-terminus ha (ngosipụta tetramer) na-esochi injection (tail vein iv, 10 mg streptavidin/kg).Opekata mpe oke atọ etinyere (N = 3).).A na-anakọta ihe nlele CSF n'oge oge egosipụtara na ntinye nke streptavidin bụ CSF anti-streptavidin ELISA (nd = achọpụtaghị).(* p <0.05, ** p<0.01, *** p<0.001, dabere na ule ANOVA).(f) Ntụle nke usoro amino acid nke phage peptide clone kachasị emejupụtara #2002 (acha odo odo) yana ndị ọzọ ahọpụtara phage peptide clones sitere na agba nke anọ nke nhọrọ.Iberibe amino acid ndị yiri ya na ndị yiri ya ka agbanyere agba.
N'ime phages niile bara ụba na agba nke anọ (Fig 3b), a na-ahọrọ clones ndị na-akwado isii maka nyocha ọzọ nke onye ọ bụla na ụdị nlele CSF.A na-agbanye nha nha nke phage nke isii, oghere efu (enweghị ntinye) na ụlọ akwụkwọ peptide prophage n'ime anụmanụ CM atọ nwere cannulated, na pharmacokinetics kpebisiri ike na CSF (Fig 3c) na ọbara (Mgbakwunye 7).All phage clones nwalere lekwasịrị anya na ngalaba CSF na ọkwa 10-1000 dị elu karịa nke phage njikwa efu (#1779).Dịka ọmụmaatụ, clones #2020 na #2077 nwere ihe dị ka 1000 ugboro CSF ​​titers karịa akara njikwa.Profaịlụ pharmacokinetic nke peptide ọ bụla ahọpụtara dị iche, mana ha niile nwere ikike nnabata CSF dị elu.Anyị hụrụ mbelata oge niile maka clones #1903 na #2011, ebe maka clones #2077, #2002 na #2009 mmụba n'ime nkeji iri mbụ nwere ike igosi njem na-arụ ọrụ mana ọ dị mkpa ka enyocha ya.Clones #2020, #2002, na #2077 kwụsiri ike na ọkwa dị elu, ebe mkpokọta CSF nke clone #2009 ji nwayọọ nwayọọ belata mgbe mmụba mbụ gasịrị.Anyị na-atụnyere ugboro ugboro nke onye ọ bụla na-aga CSF na ntinye ọbara ya (Fig 3d).Mmekọrịta nke ihe nrịbama nke onye nyocha CSF ọ bụla na ọkwa ọbara ya n'oge nlele niile gosiri na atọ n'ime ndị a na-eme ntuli aka isii nwere ụbara nke ọma na CSF ọbara.N'ụzọ na-akpali mmasị, clone #2077 gosipụtara nkwụsi ike nke ọbara dị elu (Ntinye mgbakwunye 7).Iji gosi na peptides n'onwe ha nwere ike ibuga ibu na-arụsi ọrụ ike na-abụghị phage ahụ n'ime ngalaba CSF, anyị na-emepụta peptides anọ nke ejiri biotin mee ihe na N-terminus ebe peptides na-ejikọta na phage ahụ.A na-ejikọta peptides biotinylated (ọnụọgụ 2002, 2009, 2020 na 2077) na streptavidin (SA) iji nweta ụdị multimeric nke na-eṅomi geometry phage.Usoro a kwekwara ka anyị tụọ mkpughe SA na ọbara yana mmiri cerebrospinal dị ka peptides protein na-ebu ibu.N'ụzọ dị mkpa, enwere ike ịmegharị data phage mgbe mgbe a na-enye peptides synthetic na usoro SA-conjugated (Fig 3e).The peptides scrambled nwere obere mkpughe mbụ yana mkpochapụ CSF ngwa ngwa yana ọkwa a na-achọpụtaghị n'ime awa 48.Iji nweta nghọta n'ime ụzọ nnyefe nke peptide phage clones ndị a na oghere CSF, anyị nyochara nhazi nke phage peptide hits site na iji immunohistochemistry (IHC) iji chọpụta ihe irighiri ihe phage ozugbo 1 elekere gachara ntụtụ intravenous na vivo.N'ụzọ doro anya, enwere ike ịchọpụta clones #2002, #2077, na #2009 site na ntụpọ siri ike na ụbụrụ ụbụrụ, ebe achọpụtaghị akara njikwa (#1779) na clone #2020 (Ngbakwunye 8).Nke a na-egosi na peptides ndị a na-enye aka na mmetụta na ụbụrụ kpọmkwem site na ịgafe BBB.A choro nyocha nke ọma iji nwalee echiche a, dịka ụzọ BCFB nwekwara ike itinye aka.Mgbe a na-atụnyere usoro amino acid nke clone kachasị bara ụba (#2002) na peptides ndị ọzọ ahọpụtara, ekwuru na ụfọdụ n'ime ha nwere ihe mgbakwunye amino acid yiri nke ahụ, nke nwere ike igosi usoro njem njem yiri ya (Fig 3f).
N'ihi profaịlụ plasma ya pụrụ iche na mmụba dị ịrịba ama na CSF ka oge na-aga, phage ngosi clone #2077 na-enyocha ọzọ n'ime ogologo oge 48 ma nwee ike ịmaliteghachi mmụba ngwa ngwa na CSF hụrụ na njikọ na ọkwa SA kwadoro (Fig 4a).Banyere clones phage ndị ọzọ achọpụtara, #2077 nwere ntụpọ siri ike maka ụbụrụ ụbụrụ wee gosipụta nnukwu colocalization na capillary marker lectin mgbe elere ya na mkpebi dị elu yana ikekwe ụfọdụ ntụpọ na oghere parenchymal (Nyocha 4b).Iji nyochaa ma enwere ike nweta mmetụta ọgwụ peptide-mediated na CNS, anyị mere nnwale nke ụdị biotinylated nke i) na #2077 transit peptide na ii) agwakọta peptide BACE1 inhibitor na SA na oke abụọ dị iche iche.Maka otu ngwakọta anyị na-eji naanị BACE1 peptide inhibitor na nke ọzọ anyị ji 1: 3 ratio nke BACE1 peptide inhibitor na #2077 peptide.A na-enye ihe nlele abụọ ahụ n'ime intravenous na ọbara na ọkwa cerebrospinal nke beta-amyloid peptide 40 (Abeta40) ka a tụrụ ka oge na-aga.A tụrụ Abeta40 na CSF ka ọ na-egosipụta mgbochi BACE1 na parenchyma ụbụrụ.Dị ka a tụrụ anya, ogige abụọ ahụ belatara ọkwa ọbara nke Abeta40 nke ukwuu (Fig 4c, d).Agbanyeghị, naanị ihe atụ nwere ngwakọta nke peptide no.2077 na onye na-egbochi BACE1 peptide conjugated na SA mere ka mbelata dị ukwuu na Abeta40 na mmiri cerebrospinal (Fig. 4c).Ihe omuma a gosiputara na peptide no.2077 nwere ike ibufe protein 60 kDa SA n'ime CNS ma na-ebutekwa mmetụta ọgwụ na SA-conjugated inhibitors nke BACE1 peptide.
(a) Clonal injection (2 × 10 phages / anụmanụ) nke T7 phage na-egosi profaịlụ pharmacokinetic ogologo oge nke CSF peptide #2077 (RLSSVDSDLSGC) na phage nchịkwa na-enweghị nchịkwa (#1779) na ọ dịkarịa ala atọ CM-intubated oke.(b) Confocal microscopic oyiyi nke cortical microvessels nnọchite anya na phage-injected oke (2 × 10 10 phages / anụmanụ) na-egosi counterstaining nke peptide #2077 na arịa (lectin).A na-ekenye clones phage ndị a na oke 3 ma hapụ ya ka ọ gbasaa maka elekere 1 tupu nsị.E kewara ụbụrụ ma jiri ọgwụ mgbochi akpọrọ FITC polyclonal megide T7 phage capsid.Nkeji iri tupu ịmịnye ọbara na nhazi na-esote, a na-enye lectin akara DyLight594 n'ime intravenous.Onyonyo na-egbuke egbuke na-egosi ntụpọ lectin (red) nke akụkụ ọkụ nke microvessels na phages (green) na lumen nke capillaries na ụbụrụ ụbụrụ perivascular.Ogwe ọnụ ọgụgụ ahụ dabara na 10µm.(c, d) Biotinylated BACE1 inhibitory peptide naanị ma ọ bụ jikọtara ya na biotinylated transit peptide #2077 jikọtara ya na streptavidin na-esote ya na ntinye intravenous nke opekata mpe oke CM atọ cannulated (10 mg streptavidin/kg).BACE1 peptide inhibitor-mediated mbelata na Aβ40 ka a tụrụ Aβ1-40 ELISA n'ọbara (ọbara ọbara) na mmiri cerebrospinal (oroma) n'oge egosipụtara.Maka idoanya ka mma, a na-eseta ahịrị nwere ntụpọ na eserese na nha 100%.(c) Mbelata pasent nke Aβ40 n'ime ọbara (triangles uhie) na mmiri cerebrospinal (triangles oroma) na oke a na-emeso ya na streptavidin conjugated na peptide transit #2077 na BACE1 inhibitory peptide na nha 3: 1.(d) Mbelata pasentị n'ọbara Aβ40 (okirikiri uhie) na mmiri cerebrospinal (orange okirikiri) nke oke ejiri streptavidin jikọtara yana naanị peptide inhibitory BACE1.Ntụle Aβ dị na njikwa bụ 420 pg / ml (nkewa ọkọlọtọ = 101 pg / ml).
E tinyela ihe ngosi n'ụzọ gara nke ọma n'ọtụtụ ebe nyocha biomedical17.A na-eji usoro a maka ọmụmụ ihe dị iche iche nke vivo vaskụla18,19 yana ọmụmụ ihe ezubere iche maka ụgbọ mmiri ụbụrụ20,21,22,23,24,25,26.N'ime ọmụmụ ihe a, anyị gbatịpụrụ ngwa nke usoro nhọrọ a ọ bụghị naanị na njirimara kpọmkwem nke peptides na-ezubere maka arịa ụbụrụ, kamakwa na nchọpụta nke ndị na-aga ime ihe na-arụsi ọrụ ike na-ebugharị iji gafere ihe mgbochi ụbụrụ ọbara.Ugbu a, anyị na-akọwa mmepe nke usoro nhọrọ in vivo na oke oke CM ma gosipụta ike ya iji chọpụta peptides nwere akụrụngwa ụlọ CSF.N'iji T7 phage na-egosipụta ọbá akwụkwọ nke 12-mer random peptides, anyị nwere ike igosi na T7 phage dị ntakịrị (ihe dị ka 60 nm na dayameta) 10 iji mee ka ọ bụrụ ihe mgbochi ụbụrụ ụbụrụ ọbara, si otú ahụ na-agafe kpọmkwem ihe mgbochi ụbụrụ ọbara ma ọ bụ choroid plexus.Anyị hụrụ na iwewe ihe ubi CSF sitere na oke CM nwere ike ịchịkwa bụ nke a na-achịkwa nke ọma na usoro nyocha ọrụ vivo, yana na phage ewepụtara abụghị naanị jikọtara ya na vasculature kamakwa ọ na-arụkwa ọrụ dị ka onye na-ebugharị n'ofe mgbochi ụbụrụ ọbara.Ọzọkwa, site n'ịchịkọta ọbara n'otu oge na itinye HTS na CSF na phages sitere na ọbara, anyị kwadoro na nhọrọ nke CSF emetụtaghị mmụba ọbara ma ọ bụ ahụike maka mgbasawanye n'etiti nhọrọ nhọrọ.Otú ọ dị, akụkụ ọbara bụ akụkụ nke usoro nhọrọ, ebe ọ bụ na phages nwere ike iru akụkụ CSF ga-adị ndụ ma na-ekesa n'ọbara ogologo oge iji mee onwe ha ọgaranya na ụbụrụ.Iji wepụta ozi usoro a pụrụ ịdabere na ya site na data HTS raw, anyị mejuputa ihe nzacha emebere maka njehie usoro usoro akọwapụtara n'elu ikpo okwu na usoro nyocha.Site na itinye paramita kinetic n'ime usoro nyocha, anyị kwadoro ngwa ngwa pharmacokinetics nke ụdị anụ ọhịa T7 phages (t½ ~ 28 min) na ọbara24, 27, 28 wee kpebie ọkara ndụ ha na mmiri mmiri cerebrospinal (t½ ~ 26 min) kwa nkeji).N'agbanyeghị profaịlụ pharmacokinetic yiri nke ahụ na ọbara na CSF, naanị 0.001% nke ntinye ọbara nke phage ka enwere ike ịchọpụta na CSF, na-egosi obere mmegharị azụ nke ụdị anụ ọhịa T7 phage gafee mgbochi ụbụrụ ọbara.Ọrụ a na-egosipụta mkpa nke agba nke mbụ nke nhọrọ mgbe ị na-eji usoro vivo panning, karịsịa maka usoro phage nke na-ekpochapụ ngwa ngwa site na mgbasa ozi, ebe ọ bụ na clones ole na ole na-enwe ike iru akụkụ CNS.Ya mere, na agba nke mbụ, mbelata nke iche iche n'ọbá akwụkwọ dị nnọọ ukwuu, n'ihi na ọ bụ nanị ọnụ ọgụgụ dị nta nke clones ka emechara na-anakọta na ụdị CSF a siri ike.Nke a na vivo panning atụmatụ gụnyere ọtụtụ usoro nhọrọ dị ka nchịkọta na-arụsi ọrụ ike na ngalaba CSF, ịlanarị clone n'ime ọbara ọbara, na iwepụ ngwa ngwa nke T7 phage clones na ọbara n'ime nkeji 10 mbụ (Fig 1d na Fig. 4M).).Ya mere, mgbe agba nke mbụ gasịrị, a na-achọpụta clones phage dị iche iche na CSF, ọ bụ ezie na a na-eji otu ọdọ mmiri mbụ ahụ maka anụmanụ ọ bụla.Nke a na-egosi na ọtụtụ usoro nhọrọ siri ike maka ọba akwụkwọ ndị nwere ọnụ ọgụgụ buru ibu nke ndị otu ọba akwụkwọ na-ebute mbelata dị ukwuu na ụdị dị iche iche.Ya mere, ihe omume na-enweghị usoro ga-abụ akụkụ dị mkpa nke usoro nhọrọ mbụ, na-emetụta nsonaazụ ya.O yikarịrị ka ọtụtụ n'ime clones dị n'ọbá akwụkwọ mbụ nwere mmasị ịba ụba CSF yiri nke ahụ.Otú ọ dị, ọbụlagodi n'okpuru otu ọnọdụ nnwale ahụ, nsonaazụ nhọrọ nwere ike ịdị iche n'ihi ntakịrị ọnụ ọgụgụ nke clone ọ bụla na ọdọ mmiri mbụ.
Ebumnuche ndị ahụ bara ụba na CSF dị iche na nke dị n'ọbara.N'ụzọ na-akpali mmasị, anyị kwuru na mbụ mgbanwe kwupụta glycine-ọgaranya peptides n'ime ọbara nke onye ọ bụla anụmanụ.(Fig. 1g, Mgbakwunye 4e, 4f).Phage nwere peptides glycine nwere ike kwụsie ike ma ọ dịkwa obere ka a ga-ewepụ ya.Otú ọ dị, a chọpụtaghị peptides ndị a bara ụba na glycine n'ime ihe nlele nke ụbụrụ cerebrospinal, na-atụ aro na ụlọ akwụkwọ ndị a kwadoro na-agafe usoro nhọrọ abụọ dị iche iche: otu n'ime ọbara na nke ọzọ kwere ka ịgbakọta n'ime mmiri cerebrospinal.CSF-ọgaranya clones sitere na agba nke anọ anwalela nke ukwuu.Ihe fọrọ nke nta ka ọ bụrụ clones niile a nwalere n'otu n'otu ka enwetara ọgaranya na CSF ma e jiri ya tụnyere phage njikwa oghere.Otu peptide hit (#2077) ka enyochara nke ọma.Ọ gosipụtara ogologo ndụ ọkara nke plasma ma e jiri ya tụnyere hits ndị ọzọ (Nyocha 3d na ihe mgbakwunye 7), ma na-akpali mmasị, peptide a nwere ihe fọdụrụ na cysteine ​​​​na C-terminus.E gosipụtara n'oge na-adịbeghị anya na mgbakwunye nke cysteine ​​​​na peptides nwere ike imeziwanye ihe ha na-emepụta ọgwụ site na ijikọta albumin 29.Nke a bụ amabeghị ugbu a maka peptide #2077 ma chọọ ọmụmụ ihe ọzọ.Ụfọdụ peptides gosiri valence-ndabere na nkwalite CSF (data egosighi), nke nwere ike jikọta na geometry elu egosipụtara nke T7 capsid.Usoro T7 anyị ji mee ihe gosipụtara 5-15 nke peptide ọ bụla n'otu akụkụ phage.Emere IHC na clones ndu phage nke nwa akwukwo agbaba n'ime intravenously n'ime cerebral cortex nke oke (Mgbakwunye Fig. 8).Ihe omuma a gosiputara na opekata mpe atọ clones (No. 2002, No. 2009 na No. 2077) na-emekọrịta ihe na BBB.A ka ga-ekpebi ma mmekọrịta BBB a na-ebute mkpokọta CSF ma ọ bụ mmegharị nke clones ndị a ozugbo na BCSFB.N'ụzọ dị mkpa, anyị na-egosi na peptides ahọpụtara na-ejigide ikike mbufe CSF ha mgbe ejikọta ya na ibu protein.Njide nke N-terminal biotinylated peptides na SA n'ezie na-ekwughachi nsonaazụ enwetara na clones nke phage ha dị n'ọbara na mmiri cerebrospinal (Fig 3e).N'ikpeazụ, anyị na-egosi na peptide lead #2077 nwere ike ịkwalite ọrụ ụbụrụ nke biotinylated peptide inhibitor nke BACE1 conjugated na SA, na-eme ka a na-akpọ pharmacodynamic mmetụta na CNS site n'ibelata ọkwa Abeta40 na CSF (Fig 4).Anyị enweghị ike ịchọpụta homologues ọ bụla na nchekwa data site n'ime usoro nyocha nke peptide n'usoro homology nke hits niile.Ọ dị mkpa iburu n'obi na size nke T7 n'ọbá akwụkwọ bụ mkpokọta 109, mgbe theoretical Library size maka 12-mers bụ 4 x 1015. Ya mere, anyị na-ahọrọ naanị a obere akụkụ nke diversity ohere nke 12-mer peptide Ọbá akwụkwọ, nke nwere ike ịpụta na ihe kachasị peptides nwere ike mata site n'ịtụle ndị a n'akụkụ ebe a mata usoro.N'eche echiche, otu n'ime ihe kpatara na anyị ahụbeghị homologues eke nke peptides ndị a nwere ike ịbụ deselection n'oge evolushọn iji gbochie ntinye nke ụfọdụ peptide motifs n'ime ụbụrụ na-achịkwaghị achịkwa.
N'ịchịkọta ọnụ, nsonaazụ anyị na-enye ihe ndabere maka ọrụ n'ọdịnihu iji chọpụta ma mara usoro njem nke ihe mgbochi cerebrovascular na vivo n'ụzọ zuru ezu.Ntọala ntọala nke usoro a dabere na atụmatụ nhọrọ na-arụ ọrụ nke na-abụghị naanị na-achọpụta clones nwere ihe jikọrọ ya na cerebral vaskụla, ma gụnyekwara nzọụkwụ dị oke mkpa nke clones na-aga nke ọma nwere ọrụ dị mkpa iji gafere ihe mgbochi ndu na vivo n'ime ngalaba CNS.bụ ịkọwapụta usoro njem nke peptides ndị a na mmasị ha maka ijikọta microvasculature kpọmkwem na mpaghara ụbụrụ.Nke a nwere ike bute nchọpụta ụzọ ọhụrụ maka ibufe BBB na ndị na-anabata ya.Anyị na-atụ anya na peptides achọpụtara nwere ike jikọta ya na ndị na-anabata cerebrovascular ma ọ bụ na-ekesa ligands na-ebufe site na BBB ma ọ bụ BCSFB.A ga-enyochakwu peptide vectors nwere ọrụ njem CSF achọpụtara n'ọrụ a.Anyị na-enyocha nkọwapụta ụbụrụ nke peptides ndị a ugbu a maka ikike ha ịgafe BBB na/ma ọ bụ BCSFB.Ndị peptides ọhụrụ a ga-abụ ngwá ọrụ bara uru nke ukwuu maka nchọpụta nke ndị na-anabata ma ọ bụ ụzọ ọhụrụ na maka ịmepụta usoro ọhụrụ dị mma maka nnyefe nke macromolecules, dị ka ihe ndị dị ndụ, na ụbụrụ.
Megharịa nnukwu olulu mmiri (CM) site na iji mgbanwe nke usoro akọwara na mbụ.Awụkwasịrị oke Wistar aneshetized (200-350 g) n'elu ngwa stereotaxic yana mbepu etiti etiti n'elu isi isi a kpụrụ akpụ na nke edoziri nke ọma iji kpughee okpokoro isi.Gwuo oghere abụọ na mpaghara sash nke elu ma kechie skru na-edozi na oghere.A kụpụrụ oghere ọzọ na mpụta occipital crest maka ntụzịaka stereotactic nke cannula igwe anaghị agba nchara n'ime CM.Tinye ciment eze gburugburu cannula ma chekwaa ya na kposara.Mgbe ọgwụgwọ foto na simenti siri ike, a na-emechi ọnya akpụkpọ ahụ na 4/0 supramid suture.A na-akwado ntinye nke cannula nke ọma site na mpụta mmiri nke cerebrospinal (CSF).Wepụ oke ahụ na ngwa stereotaxic, nweta nlekọta ahụike mgbe ọ gasịrị na nlekọta mgbu, ma kwe ka ọ gbakee maka opekata mpe otu izu ruo mgbe a na-ahụ ihe ịrịba ama nke ọbara na mmiri cerebrospinal.Enwetara oke Wistar (Crl: WI/Han) site na Charles River (France).Edobere oke niile n'okpuru ọnọdụ enweghị nje nje.Ụlọ ọrụ Veterinary nke Obodo Basel, Switzerland kwadoro nyocha anụmanụ niile ma mee ya dịka ikikere anụmanụ nke 2474 (Ntụle nke Ụgbọ njem Brain Na-arụ Ọrụ site na Ịtụ ọkwa nke Ndị Candidates Ọgwụgwọ na Cerebrospinal Fluid na Brain of Rat).
Jiri nwayọọ mee ka oke mara nke ọma na cannula CM dị n'aka.Wepu Datura na cannula wee kpokọta 10 µl nke mmiri cerebrospinal na-eru n'otu oge.Ebe ọ bụ na e mebiela patency nke cannula n'ikpeazụ, ọ bụ naanị ihe nlele cerebrospinal doro anya na-enweghị ihe akaebe nke mmetọ ọbara ma ọ bụ mwepu agbanyere n'ọmụmụ ihe a.N'otu aka ahụ, a na-ewepụta ihe dịka 10-20 μl ọbara site na obere mbepụ n'ọnụ ọdụ n'ime tubes nwere heparin (Sigma-Aldrich).A nakọtara CSF na ọbara n'oge dị iche iche mgbe ịgba ọgwụ mgbochi T7 phage gasịrị.Ihe dị ka 5-10 μl nke mmiri na-atụfu tupu a na-anakọta ihe nlele CSF ọ bụla, nke kwekọrọ na olu nwụrụ anwụ nke catheter.
Ejiri vector T7Select 10-3b mepụta ọba akwụkwọ dịka akọwara na akwụkwọ ntuziaka sistemụ T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996).Na nkenke, etinyere ntinye DNA 12-mer na-enweghị usoro n'ụdị a:
Ejiri koodu NNK mee ihe iji zere codons nkwụsị okpukpu abụọ na oke amino acid na ntinye.N bụ ihe nucleotide ọ bụla agwakọtara na aka, na K bụ oke nhamola agwakọta nke adenine na cytosine nucleotides.A gbanwere mpaghara naanị otu eriri ka ọ bụrụ DNA nwere eriri okpukpu abụọ site na ntinye ọzọ na dNTP (Novagen) na Klenow enzyme (New England Biolabs) na Klenow buffer (New England Biolabs) maka awa 3 na 37°C.Mgbe mmeghachi omume ahụ gasịrị, mmiri EtOH nwetara DNA nwere eriri abụọ.Ejiri enzymes mmachi EcoRI na HindIII gbarie DNA nke si na ya pụta (ha abụọ sitere na Roche).Ihe ntinye nke gbawara agbawa na nke dị ọcha (QIAquick, Qiagen) (T4 ligase, New England Biolabs) bụ nke etinyere na etiti n'ime vector T7 nke agbawara tupu amino acid 348 nke 10B capsid gene.A na-etinye mmeghachi omume ligation na 16 ° C. maka awa 18 tupu nkwakọ ngwaahịa in vitro.A na-eme nkwakọ ngwaahịa na vitro dị ka ntuziaka enyere ya na T7Select 10-3b cloning kit (Novagen) na ngwọta nkwakọ ngwaahịa abawanyela otu ugboro iji lysis iji Escherichia coli (BLT5615, Novagen).The lysates bụ centrifuged, titrated na oyi kpọnwụrụ na -80 Celsius C. dị ka ngwaahịa ngwọta nke glycerol.
Mmụba PCR ozugbo nke mpaghara phage agbanwe agbagoro na broth ma ọ bụ efere site na iji primers 454/Roche-amplicon fusion primers.Ngwakọta ejikọta na-aga n'ihu nwere usoro na-atụgharị mpaghara agbanwe agbanwe (NNK) 12 (ụkpụrụ-kpọmkwem), GS FLX Titanium Adapter A, na usoro igodo ụlọ akwụkwọ nwere isi anọ (TCAG) (Ntinye mgbakwunye 1a):
Ntugharị fusion primer nwekwara biotin jikọtara iji weghara beads na GS FLX Titanium Adapter B chọrọ maka mmụba clonal n'oge emulsion PCR:
A na-edozi amplicon ndị ahụ na 454/Roche pyrosequencing dị ka 454 GS-FLX Titanium protocol.Maka usoro akwụkwọ ntuziaka Sanger (Applied Biosystems Hitachi 3730 xl DNA Analyzer), PCR kwalitere T7 phage DNA wee were ụzọ abụọ primer na-esote usoro:
A na-etinye ihe ntinye sitere na mbadamba ihe n'otu n'otu na nkwalite PCR site na iji Roche Fast Start DNA Polymerase Kit (dị ka ntuziaka onye nrụpụta siri dị).Mee mmalite na-ekpo ọkụ (minti 10 na 95 Celsius) na usoro nkwalite 35 (50 s na 95 Celsius C, 1 min na 50 Celsius C, na 1 min na 72 Celsius C).
Phage sitere na ọba akwụkwọ, ụdị anụ ọhịa phage, phage napụtara n'aka CSF na ọbara, ma ọ bụ clones nke ọ bụla na-abawanye na Escherichia coli BL5615 na broth TB (Sigma Aldrich) ma ọ bụ na efere 500 cm2 (Thermo Scientific) maka 4 h na 37 ° C.A na-ewepụta ngwa ngwa na efere ndị ahụ site na iji Tris-EDTA buffer (Fluka Analytical) sachaa efere ndị ahụ ma ọ bụ site na iji ndụmọdụ pipette na-adịghị ọcha na-anakọta mbadamba nkume ahụ.Ekewapụrụ n'usoro n'ụdị omenaala ma ọ bụ ihe nchekwa mmịpụta nwere otu okirikiri polyethylene glycol (PEG 8000) mmiri ozuzo (Promega) wee malitegharịa na nchekwa Tris-EDTA.
A na-edobere phage a na-amụbawanye na 2-3 nke mwepụ nke endotoxin site na iji beads mwepụ endotoxin (Miltenyi Biotec) tupu ịgba ntụtụ (IV) (500 μl / anụmanụ).Na agba nke mbụ, ewebata 2 × 1012 phages;na nke abụọ, 2 × 1010 phages;na nke atọ na nke anọ nhọrọ agba, 2 × 109 phages kwa anụmanụ.A na-ekpebi ọdịnaya ọkwa dị na CSF yana nlele ọbara anakọtara n'oge egosipụtara site na ịgụta akara dịka ntuziaka onye nrụpụta si dị (T7Select system manual).A na-eme nhọrọ nke phage site n'intravenous nke ụlọ akwụkwọ ndị a na-eme ka ọ dị ọcha na ọdụ ọdụ ma ọ bụ site na ntinyeghachi nke phage nke ewepụtara na CSF site na nhọrọ nke gara aga, a rụpụtara ihe ubi na-esote na 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, na 240 min na ọbara C.SF n'otu n'otu.A na-eduzi ngụkọta nke agba anọ nke in vivo panning nke a na-echekwa alaka abụọ ahụ ahọpụtara iche iche ma nyochaa n'oge agba atọ mbụ nke nhọrọ.Ihe ntinye phage niile ewepụtara na CSF site na agba abụọ mbụ nke nhọrọ ka edobere na 454/Roche pyrosequencing, ebe ejiri aka hazie clones niile ewepụtara na CSF site na agba abụọ ikpeazụ nke nhọrọ.A na-edozikwa phages ọbara niile sitere na agba nke mbụ na 454/Roche pyrosequencing.Maka ịgba ọgwụ mgbochi nke phage clones, phages ahọpụtara ka agbakwunyere na E. coli (BL5615) na efere 500 cm2 na 37 ° C maka awa 4.A na-agbasa clones nke ahọpụtara n'otu n'otu yana ejiri aka mee ya na usoro TB.Mgbe mmịpụta phage gasịrị, ịdị ọcha na mwepụ nke endotoxin (dị ka akọwara n'elu), 2 × 1010 phages / anụmanụ na 300 μl na-agbanye n'ime intravenously n'ime otu ọdụdụ ọdụ.
Na-ahazi na qualitative nzacha nke data usoro.A tụgharịrị data Raw 454/Roche site na usoro maapụ ọkọlọtọ ọnụọgụ abụọ (sff) gaa na usoro ọgụgụ mmadụ nke Pearson (fasta) site na iji ngwa ndị na-ere ere.Emere nhazi ọzọ nke usoro nucleotide site na iji mmemme C na scripts (ngwungwu ngwanrọ na-ewepụtaghị) dị ka akọwara n'okpuru.Nyocha nke data mbụ gụnyere usoro nzacha ọtụtụ ọkwa.Iji yochaa agụ nke na-enweghị usoro ntinye DNA nke 12mer bara uru, ndị agụpụtara na-ahazi n'usoro iji malite akara (GTGATGTCGGGGATCCGAATTCT), labelụ kwụsị (TAAGCTTGCGGCCGCACTCGAGTA) na ntinye ndabere (CCCTGCAGGATATCCCGeedGGAGCTCGTCGAC) site na iji ule ụwa niile.alignment na-enye ohere ruo 2 ekwekọghị n'otu n'otu alignment31.Ya mere, na-agụ na-enweghị mmalite na kwụsị mkpado ma na-agụ nwere ndabere inserts, ya bụ, alignments gafere ọnụ ọgụgụ nke mismatches, ewepụrụ n'ọbá akwụkwọ.N'ihe gbasara agụ nke fọdụrụ, usoro N-mer DNA na-agbatị site na mmalite akara na-agwụ tupu e wepụ akara nkwụsị n'usoro ọgụgụ mbụ wee hazie ya (nke a na-akpọ "ịtinye").Mgbe ntụgharị ntụgharị nke ntinye gasịrị, a na-ewepụ akụkụ ahụ mgbe codon nkwụsị nke mbụ dị na njedebe 5′ nke primer na ntinye.Na mgbakwunye, e wepụrụkwa nucleotides nke na-eduga na codons na-ezughị ezu na njedebe 3′ nke primer.Iji wepu ihe ntinye nwere naanị usoro ndabere, ewepụrụ ihe ntinye atụgharịrị na-amalite na ụkpụrụ amino acid "PAG".E wepụrụ peptide ndị nwere ogologo ntụgharị nke na-erughị 3 amino asịd n'ọbá akwụkwọ.N'ikpeazụ, wepụ redundancy na ọdọ mmiri ntinye ma chọpụta ugboro ole ntinye ọ bụla pụrụ iche.Nsonaazụ nke nyocha a gụnyere ndepụta nke usoro nucleotide (ntinye) na ugboro ha (gụọ) ugboro (Mgbakwunye 1c na 2).
Otu N-mer DNA ntinye site n'usoro myirịta: Iji kpochapụ njehie 454/Roche kpọmkwem (dị ka nsogbu na nhazi nke ndọtị homopolymer) ma wepụ ihe ndị na-adịchaghị mkpa, ntinye N-mer DNA usoro ntinye (ntinye) na-ahazi site na myirịta.ntinye (ihe ruru 2 ntọala anaghị adabara ekwe) site na iji algọridim iterative akọwapụtara dị ka ndị a: a na-ahazi ntinye nke mbụ site na ugboro ole ha (kachasị elu na nke kachasị ala), ma ọ bụrụ na ha bụ otu, site na ụdị nke abụọ ha n'ogologo (ogologo ruo mkpụmkpụ)).Ya mere, ntinye kachasị na nke kachasị ogologo na-akọwa "otu" nke mbụ.A na-ahazi ugboro otu na ugboro igodo.Mgbe ahụ, a nwara ntinye ọ bụla fọdụrụ na ndepụta ahaziri ka ịgbakwunye ya na otu site na nhazi Needleman-Wunsch n'ụzọ abụọ.Ọ bụrụ na ọnụ ọgụgụ ndakọrịta, ntinye, ma ọ bụ ihichapụ na nhazi agafeghị oke nke 2, a na-agbakwunye ntinye n'ime otu ahụ, a na-abawanye ugboro ole otu n'ozuzu ya site na ugboro ole etinyere ntinye.Ihe ntinye agbakwunyere na otu ka ejiri akara ma wepụ ya na nhazi ọzọ.Ọ bụrụ na enweghị ike ịgbakwunye usoro ntinye na otu dị adị, a na-eji usoro ntinye emepụta otu ọhụrụ nwere ugboro ntinye kwesịrị ekwesị ma kaa akara dịka ejiri ya.Ntụgharị ahụ na-akwụsị mgbe ejirila usoro ntinye ọ bụla iji guzobe otu ọhụrụ ma ọ bụ nwee ike itinye ya na otu dị adị.A sị ka e kwuwe, a na-asụgharị ihe ntinye nke nwere nucleotides n'ime usoro peptide (ọbá akwụkwọ peptide).Nsonaazụ nke nyocha a bụ ntinye ntinye na ugboro ole ha kwekọrọ na-eme ka ọnụọgụgụ nke a na-agụ n'usoro (Mgbakwunye Fig. 2).
Ọgbọ Motif: Dabere na ndepụta peptides pụrụ iche, e mepụtara ọbá akwụkwọ nwere usoro amino acid niile nwere ike (aa) dị ka egosiri n'okpuru.A na-ewepụta ụkpụrụ ọ bụla nwere ike ịdị ogologo 3 site na peptide ma tinye ụkpụrụ ya na-atụgharị ya na ọbá akwụkwọ motif nke nwere ụkpụrụ niile (tripeptides).A na-ahazi ụlọ ọba akwụkwọ nke ebumnobi na-emegharị ugboro ugboro ma wepụrụ ya.Mgbe ahụ, maka tripeptide ọ bụla n'ọbá akwụkwọ motif, anyị na-enyocha ọnụnọ ya n'ọbá akwụkwọ site na iji ngwá ọrụ mgbakọ.N'okwu a, a na-agbakwunye ugboro ole peptide nwere motif tripeptide achọtara ma kenye ya na motif n'ọbá akwụkwọ motif ("ọnụọgụ nke motifs").Nsonaazụ nke ọgbọ motif bụ nhazi akụkụ abụọ nwere ihe omume niile nke tripeptides (motifs) na ụkpụrụ ha dị iche iche, nke bụ ọnụ ọgụgụ nke usoro a na-agụ nke na-eme ka ihe ahụ dabara adaba mgbe a na-enyocha, chịkọta, na nsụgharị.Metric dị ka akọwara n'ụzọ zuru ezu n'elu.
Normalization nke ọnụ ọgụgụ nke motifs na ndị na-ekpo ọkụ kwekọrọ na ya: A na-eme ka ọnụ ọgụgụ nke motifs maka nlele ọ bụla na-eme ka ọ dị mma site na iji.
ebe ni bụ ọnụọgụgụ nke nwere isiokwu i.Ya mere, vi na-anọchi anya ọnụọgụgụ ọnụọgụgụ (ma ọ bụ peptides) nwere motif i na nlele.A na-agbakọ ụkpụrụ P- maka ọnụ ọgụgụ ndị na-abụghị nke ezi uche dị na ya site na iji ule Fisher ziri ezi.Banyere correlograms nke ọnụ ọgụgụ ebumnobi, a gbakọrọ mmekọrịta Spearman site na iji ọnụọgụ ebumnobi emebere nke ọma na R.
Iji jiri anya nke uche hụ ọdịnaya nke amino acid n'ọnọdụ ọ bụla n'ọbá akwụkwọ peptide, e mepụtara akara ngosi weebụ 32, 33 (http://weblogo.threeplusone.com).Nke mbụ, a na-echekwa ọdịnaya nke amino acid n'ọnọdụ ọ bụla nke peptide 12-mer na matriks 20 × 12.Mgbe ahụ, a na-emepụta otu peptides 1000 nwere otu ọdịnaya amino acid dị n'ọnọdụ ọ bụla n'ụdị ngwa ngwa wee wepụta ya dị ka ntinye na logo web 3, nke na-ewepụta ihe ngosi eserese nke ọdịnaya amino acid dị n'ọnọdụ ọ bụla.maka ọbá akwụkwọ peptide nyere.Iji hụta setịpụ data ọtụtụ akụkụ, a na-emepụta maapụ okpomọkụ site na iji ngwa arụpụtara n'ime R (biosHeatmap, ngwugwu R nke a ka ga-ewepụta).A gbakọrọ dendrogram ndị e gosipụtara na maapụ okpomọkụ site na iji usoro nchịkọta usoro nhazi nke Ward jiri metric anya Euclidean.Maka nyocha ndekọ ọnụ ọgụgụ nke data akara motif, a na-agbakọ ụkpụrụ P maka akara na-ezighi ezi site na iji ule Fisher ziri ezi.A gbakọrọ ụkpụrụ P-maka ihe ndekọ data ndị ọzọ na R site na iji t-ule nke ụmụ akwụkwọ ma ọ bụ ANOVA.
A na-agbanye clones phage na phages na-enweghị ntinye n'ime intravenously site na eriri ọdụ (2 × 1010 phages / anụmanụ na 300 μl PBS).Nkeji iri tupu mgbawa na nrụzi na-esote, a na-eji 100 μl nke DyLight594 akpọrọ lectin (Vector Laboratories Inc., DL-1177) gbanye otu anụmanụ ahụ.N'ime nkeji 60 ka ịgbanye ọgwụ phage, a na-agbanye oke n'ime obi na 50 ml PBS na 50 ml 4% PFA/PBS sochiri ya.A na-edozikwa ihe nlele ụbụrụ n'otu abalị na 4% PFA/PBS wee tinye ya na 30% sucrose n'abali na 4 Celsius.A na-ekpochapụ ihe nlele na ngwakọta OCT.A na-eme nyocha nke immunohistochemical nke ihe atụ oyi kpọnwụrụ na ụlọ okpomọkụ na 30 µm cryosections kpuchiri 1% BSA ma tinye ya na polyclonal FITC-labeled antibodies megide T7 phage (Novus NB 600-376A) na 4 Celsius C.Tinyegharịa otu abalị.N'ikpeazụ, a na-eji PBS sachaa akụkụ ahụ ugboro 3 wee jiri microscope laser confocal (Leica TCS SP5) nyochaa ya.
Ihe peptides niile nwere opekempe dị ọcha nke 98% bụ GenScript USA mebere, biotinylated na lyophilized.A na-ejikọta Biotin site na oghere glycine ugboro atọ na N-terminus.Lelee peptides niile site na iji spectrometry mass.
Streptavidin (Sigma S0677) jikọtara ya na 5-fold equimolar ngafe nke biotinylated peptide, biotinylated BACE1 inhibitory peptide, ma ọ bụ ngwakọta (3: 1 ratio) nke biotinylated BACE1 inhibitory peptide na BACE1 inhibitory peptide na 5-10% DMSO / incu.1 hour na ụlọ okpomọkụ tupu igba ogwu.A na-agbanye Streptavidin conjugated peptides n'ime intravenously na dose nke 10 mg / kg n'ime otu n'ime ọdụdụ ọdụ nke oke nwere oghere ụbụrụ.
ELISA tụlere mkpokọta streptavidin-peptide.Nunc Maxisorp microtiter plates (Sigma) na-ekpuchi otu abalị na 4 Celsius na 1.5 μg/ml mouse anti-streptavidin antibody (Thermo, MA1-20011).Mgbe igbochi (nchekwa ihe mgbochi: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) na ụlọ okpomọkụ maka awa 2, na-asa efere na 0.05% Tween-20 / PBS (ihe na-asachapụ) maka 3 nke abụọ, CSF na plasma 0 gbakwunyere na 0. SF 1:115).A na-etinye efere ahụ n'otu abalị na 4 Celsius na mgbochi nchọpụta (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239).Mgbe usoro ịsa ahụ atọ gasịrị, ahụrụ streptavidin site na incubation na TMB substrate solution (Roche) maka ihe ruru 20 min.Mgbe ịkwụsị mmepe agba na 1M H2SO4, tụọ nnabata ahụ na 450 nm.
A na-enyocha ọrụ nke streptavidin-peptide-BACE1 inhibitor complex site na Aβ (1-40) ELISA dịka usoro onye nrụpụta (Wako, 294-64701).Na nkenke, a gbajiri ihe nlele CSF n'ụdị ntụ ntụ ọkọlọtọ (1:23) wee tinye ya n'otu abalị na 4 Celsius n'ime efere 96 nke ọma nke ejiri ihe mgbochi BNT77 kpuchie.Mgbe usoro ịsa ahụ ise gasịrị, agbakwunyere mgbochi BA27 nwere njikọ HRP ma tinye ya maka awa 2 na 4 Celsius, usoro ịsa ahụ ise sochiri.Achọpụtara Aβ (1-40) site na ntinye na ngwọta TMB maka nkeji 30 n'ime ụlọ okpomọkụ.Mgbe emechara mmepe agba na ngwọta nkwụsị, tụọ nnabata ahụ na 450 nm.Edebere ihe nlele Plasma na mmịpụta oge siri ike tupu Aβ (1-40) ELISA.Agbakwunyere Plasma na 0.2% DEA (Sigma) na efere 96 nke ọma ma tinye ya na ụlọ okpomọkụ maka nkeji 30.Mgbe ejiri mmiri na methanol 100% sachaa efere SPE (Oasis, 186000679), a na-agbakwunye ihe nlele plasma na efere SPE wee wepụ mmiri niile.A na-asa ihe nlele (nke mbụ na methanol 5% wee 30% methanol) wee jiri 2% NH4OH/90% methanol pụta.Mgbe ihichapụ eluate na 55 ° C maka 99 min na N2 na-adị ugbu a, a na-ebelata ihe nlele ahụ na diluents ọkọlọtọ na Aβ (1-40) tụrụ dịka akọwara n'elu.
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