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Chotchinga chamagazi-muubongo komanso chotchinga chamagazi-muubongo chimalepheretsa ma biotherapeutic othandizira kuti afikire zomwe akufuna m'katikati mwa mitsempha, potero amalepheretsa chithandizo chamankhwala chamankhwala amitsempha.Kuti tipeze zotengera zaubongo mu vivo, tidayambitsa laibulale ya T7 phage peptide ndikusonkhanitsa magazi ndi cerebrospinal fluid (CSF) pogwiritsa ntchito dziwe lalikulu la makoswe.Ma clones enieni a phage adalemeretsedwa kwambiri mu CSF pambuyo pa maulendo anayi osankhidwa.Kuyesedwa kwa ma peptides omwe amasankhidwa payekha kunavumbulutsa kuchulukitsa kopitilira 1000 mu CSF.Bioactivity ya peptide-mediated delivery to the brain inatsimikiziridwa ndi 40% kuchepetsa mlingo wa amyloid-β mu cerebrospinal fluid pogwiritsa ntchito BACE1 peptide inhibitor yolumikizidwa ndi peptide yodziwika yodutsa.Zotsatirazi zikuwonetsa kuti ma peptides odziwika ndi njira zosankhidwa mu vivo phage amatha kukhala magalimoto othandiza pakubweretsa ma macromolecules ku ubongo ndi chithandizo chamankhwala.
Kafukufuku wamankhwala omwe amayang'aniridwa ndi dongosolo lapakati lamanjenje (CNS) amayang'ana kwambiri pakuzindikira mankhwala okhathamiritsa ndi othandizira omwe amawonetsa zinthu zomwe zimayang'ana pa CNS, osayesetsa kupeza njira zomwe zimayendetsa kuperekera mankhwala ku ubongo.Izi zikuyamba kusintha tsopano popeza kubweretsa mankhwala, makamaka mamolekyu akuluakulu, ndi gawo lofunikira pakukula kwamankhwala amakono a neuroscience.Malo apakati amanjenje amatetezedwa bwino ndi cerebrovascular chotchinga dongosolo, lopangidwa ndi chotchinga chamagazi-muubongo (BBB) ​​​​ndi chotchinga chamagazi muubongo (BCBB)1, zomwe zimapangitsa kuti zikhale zovuta kupereka mankhwala ku ubongo1,2.Akuti pafupifupi mankhwala onse akuluakulu a molekyulu ndi mankhwala opitilira 98% ang'onoang'ono amachotsedwa muubongo3.Ichi ndichifukwa chake ndikofunikira kwambiri kuzindikira njira zatsopano zoyendera muubongo zomwe zimapereka chithandizo choyenera komanso chachindunji chamankhwala ochizira ku CNS 4,5.Komabe, BBB ndi BCSFB zimaperekanso mwayi wabwino kwambiri woperekera mankhwala osokoneza bongo pamene amalowa ndikulowa m'magulu onse a ubongo kupyolera mu mitsempha yake yambiri.Choncho, zoyesayesa zamakono zogwiritsira ntchito njira zosagwiritsidwa ntchito zoperekera ku ubongo makamaka zimachokera ku njira ya receptor-mediated transport (PMT) pogwiritsa ntchito BBB6 receptor receptor.Ngakhale zapita patsogolo kwambiri zaposachedwa pogwiritsa ntchito njira ya transferrin receptor7,8, kukulitsa kwina kwa machitidwe atsopano operekera zinthu okhala ndi zinthu zabwinoko ndikofunikira.Kuti izi zitheke, cholinga chathu chinali kuzindikira ma peptides omwe amatha kuyimira mayendedwe a CSF, chifukwa amatha kugwiritsidwa ntchito popereka ma macromolecules ku CNS kapena kutsegula njira zatsopano zolandirira.Makamaka, zolandilira enieni ndi zonyamula a dongosolo cerebrovascular (BBB ndi BSCFB) akhoza kutumikira monga mipherezero angathe yogwira ndi mwachindunji yobereka mankhwala biotherapeutic.Cerebrospinal fluid (CSF) ndi chinthu chobisika cha choroid plexus (CS) ndipo imagwirizana mwachindunji ndi interstitial fluid ya ubongo kupyolera mu malo a subbarachnoid ndi ventricular space4.Posachedwapa zawonetsedwa kuti subarachnoid cerebrospinal fluid imafalikira kwambiri mu interstitium ya ubongo9.Tikuyembekeza kupeza malo a parenchymal pogwiritsa ntchito thirakiti lolowera la subbarachnoid kapena mwachindunji kudzera mu BBB.Kuti tikwaniritse izi, tidakhazikitsa njira yodalirika yosankha ma vivo phage yomwe imazindikiritsa ma peptides otengedwa ndi imodzi mwa njira ziwiri izi.
Tsopano tikufotokozera njira yotsatirira mu vivo phage yowonera ndi zitsanzo za CSF zophatikizika ndi kutsata kwapamwamba kwambiri (HTS) kuwunika zoyambira zosankhidwa zokhala ndi laibulale yayikulu kwambiri.Kuwunika kunachitika pa makoswe ozindikira omwe ali ndi kansalu kakang'ono kakang'ono kamene kamayikidwa kosatha (CM) kuti apewe kuipitsidwa kwa magazi.Chofunika kwambiri, njirayi imasankha zonse zomwe zimayang'ana muubongo ndi ma peptides okhala ndi zoyendera kudutsa chotchinga chaubongo.Tidagwiritsa ntchito T7 phages chifukwa cha kukula kwawo kochepa (~ 60 nm) 10 ndipo tidati ndi oyenera kunyamula ma vesicles omwe amalola kudutsa kwa endothelial ndi / kapena epithelial-medulla chotchinga.Pambuyo pa maulendo anayi ozungulira, anthu amtundu wa phage adasiyanitsidwa akuwonetsa amphamvu mu vivo CSF ​​​​kulemeretsa ndi cerebral microvessel association.Chofunika kwambiri, tinatha kutsimikizira zomwe tapeza posonyeza kuti ma peptides omwe amakondedwa komanso opangidwa ndi mankhwala amatha kunyamula katundu wa mapuloteni mu cerebrospinal fluid.Choyamba, zotsatira za pharmacodynamic za CNS zidakhazikitsidwa pophatikiza peptide yotsogola yodutsa ndi inhibitor ya BACE1 peptide.Kuphatikiza pakuwonetsa kuti njira zowunikira mu vivo zitha kuzindikira ma peptide atsopano onyamula muubongo ngati onyamula katundu wama protein, tikuyembekeza njira zofananira zosankhidwa kukhala zofunikanso pakuzindikiritsa njira zatsopano zoyendera muubongo.
Kutengera ma plaque-forming units (PFU), pambuyo pa sitepe yoyika phage, laibulale ya 12-mer linear T7 phage peptides yokhala ndi mitundu pafupifupi 109 idapangidwa ndikupangidwa (onani Zida ndi Njira).Ndikofunika kuzindikira kuti tidasanthula mosamala laibulaleyi mu vivo panning.Kukulitsa kwa PCR kwa zitsanzo za laibulale ya phage pogwiritsa ntchito makina osinthidwa opangidwa ndi amplicons omwe anali okhudzana ndi HTS (Supplementary Fig. 1a).Chifukwa cha a) HTS11 zolakwa zotsatizana, b) zotsatira pa khalidwe la primers (NNK) 1-12, ndi c) kukhalapo kwa nyama zakutchire (wt) phage (zoyika mafupa) mu laibulale yoyimilira, ndondomeko yosefera yotsatizana inakhazikitsidwa kuti ipeze chidziwitso chotsimikizika chotsatira (Supplementary Fig. 1b).Izi zosefera zimagwira ntchito pama library onse otsatizana a HTS.Kwa laibulale yokhazikika, chiwerengero cha 233,868 chowerengedwa chinapezedwa, chomwe 39% chinadutsa njira zosefera ndipo zinagwiritsidwa ntchito pofufuza laibulale ndikusankha maulendo otsatila (Supplementary Figure 1c-e).Zowerengerazo zinali zochulukirachulukira za ma 3 awiriawiri oyambira kutalika ndi nsonga ya 36 nucleotides (Supplementary Fig. 1c), kutsimikizira kapangidwe ka laibulale (NNK) 1-12.Makamaka, pafupifupi 11% ya mamembala a library anali ndi 12-dimensional wild-type (wt) backbone PAGISRELVDKL, ndipo pafupifupi theka la zotsatizanazi (49%) zinali ndi zoyikapo kapena zochotsa.HTS ya laibulale ya laibulale inatsimikizira kusiyana kwakukulu kwa ma peptides mu laibulale: kuposa 81% ya ndondomeko ya peptide inapezeka kamodzi kokha ndipo 1.5% yokha inachitika mu ≥4 makope (Supplementary Fig. 2a).Mafupipafupi a amino acid (aa) pa malo onse a 12 mu repertoire amagwirizanitsa bwino ndi maulendo omwe amayembekezeredwa kwa chiwerengero cha ma codon opangidwa ndi NKK repertoire yowonongeka (Supplementary Fig. 2b).Kuwona pafupipafupi kwa zotsalira za aa zomwe zimayikidwa ndi izi zimalumikizana bwino ndi ma frequency owerengeka (r = 0.893) (Supplementary Fig. 2c).Kukonzekera kwa malaibulale a phage jekeseni kumaphatikizapo masitepe akukulitsa ndikuchotsa endotoxin.Izi zawonetsedwa kale kuti zitha kuchepetsa kusiyanasiyana kwa malaibulale a phage12,13.Chifukwa chake, tidasanja laibulale ya phage yokulitsa mbale yomwe idachotsedwapo endotoxin ndikuyiyerekeza ndi laibulale yoyambirira kuti tiyerekeze kuchuluka kwa AA.Kugwirizana kwamphamvu (r = 0.995) kunawonedwa pakati pa dziwe lapachiyambi ndi dziwe lokulitsa ndi loyeretsedwa (Supplementary Fig. 2d), kusonyeza kuti mpikisano pakati pa ma clones okwezedwa pa mbale pogwiritsa ntchito T7 phage sichinapangitse kukondera kwakukulu.Kuyerekezera uku kumatengera kuchuluka kwa ma tripeptide motifs mu library iliyonse, popeza kusiyanasiyana kwa malaibulale (~109) sikungathe kujambulidwa ngakhale ndi HTS.Kusanthula kwafupipafupi kwa aa pa malo aliwonse kunavumbulutsa tsankho laling'ono lodalira malo mu malo atatu omaliza a repertoire yomwe inalowa (Supplementary Fig. 2e).Pomaliza, tinaganiza kuti ubwino ndi zosiyana za laibulale zinali zovomerezeka ndipo kusintha kwakung'ono kokha kwa mitundu yosiyanasiyana kunawonedwa chifukwa cha kukulitsa ndi kukonzekera malaibulale a phage pakati pa maulendo angapo osankhidwa.
Seri cerebrospinal fluid sampling ikhoza kuchitidwa mwa opaleshoni yoika cannula mu CM ya makoswe odziwa kuti athandize kuzindikira T7 phage jekeseni kudzera m'mitsempha (iv) kudzera pa BBB ndi / kapena BCSFB (Mkuyu 1a-b).Tidagwiritsa ntchito zida ziwiri zodziyimira pawokha (mikono A ndi B) m'magawo atatu oyamba osankhidwa mu vivo (mkuyu 1c).Pang'onopang'ono tinawonjezera kukhwima kwa kusankha mwa kuchepetsa chiwerengero cha phage chomwe chinayambika m'magulu atatu oyambirira osankhidwa.Pachigawo chachinayi chowotchera, tidaphatikiza zitsanzo kuchokera kunthambi A ndi B ndikusankha zina zitatu zodziyimira pawokha.Kuti muphunzire mu vivo katundu wa T7 phage particles mu chitsanzo ichi, wild-type phage (PAGISRELVDKL master insert) anabayidwa mu makoswe kudzera m'mitsempha ya mchira.Kuchira kwa phages kuchokera ku cerebrospinal fluid ndi magazi panthawi yosiyana kunasonyeza kuti T7 icosahedral phages yaying'ono inali ndi gawo lofulumira lachilolezo chochokera ku chipinda cha magazi (Supplementary Fig. 3).Kutengera ma titer omwe amaperekedwa komanso kuchuluka kwa magazi a makoswe, tidawerengera kuti pafupifupi 1% wt.phage ku kutumikiridwa mlingo anapezeka mu magazi mphindi 10 pambuyo mtsempha wa magazi jekeseni.Pambuyo pakutsika koyambirira kumeneku, chilolezo chocheperako chinayesedwa ndi theka la moyo wa mphindi 27.7.Chofunika kwambiri, ma phage ochepa okha ndi omwe adatengedwa kuchokera ku chipinda cha CSF, kusonyeza maziko otsika amtundu wa nyama zakutchire kusamukira ku CSF compartment (Supplementary Fig. 3).Pafupifupi, pafupifupi 1 x 10-3% titers a T7 phage m'magazi ndi 4 × 10-8% ya phage zomwe zidalowetsedwa poyamba zidadziwika mu cerebrospinal fluid panthawi yonse yotsatsira (0-250 min).Mwachidziwitso, theka la moyo (25.7 min) wa phage yamtundu wakuthengo mu cerebrospinal fluid inali yofanana ndi yomwe imawonedwa m'magazi.Deta iyi ikuwonetsa kuti chotchinga cholekanitsa chipinda cha CSF ndi magazi chimakhala chokhazikika mu makoswe a CM-cannulated, kulola mu vivo kusankha malaibulale a phage kuti azindikire ma clones omwe amatengedwa mosavuta kuchokera m'magazi kupita kuchipinda cha CSF.
(a) Kukhazikitsa njira yoyesereranso cerebrospinal fluid (CSF) kuchokera padziwe lalikulu.(b) Chithunzi chosonyeza malo otchinga chapakati pa mitsempha (CNS) chotchinga ndi njira yosankhidwa yomwe imagwiritsidwa ntchito kuzindikira ma peptides omwe amadutsa chotchinga chamagazi-muubongo (BBB) ​​​​ndi chotchinga chamagazi-muubongo.(c) Mu vivo phage chiwonetsero chowonera tchati.Pazosankha zilizonse, ma phages (zidziwitso za nyama mkati mwa mivi) adabayidwa kudzera m'mitsempha.Nthambi ziwiri zodziyimira pawokha (A, B) zimasungidwa padera mpaka kuzungulira 4 kosankha.Pazosankha zozungulira 3 ndi 4, chojambula chilichonse chochokera ku CSF chinatsatiridwa pamanja.(d) Kinetics ya phage yolekanitsidwa ndi magazi (zozungulira zofiira) ndi cerebrospinal fluid (makona atatu obiriwira) panthawi yoyamba yosankhidwa mu makoswe awiri otsekemera pambuyo pa jekeseni wa mtsempha wa laibulale ya T7 peptide (2 x 1012 phages / nyama).Mabwalo a buluu amasonyeza kuchuluka kwa phage m'magazi, kuwerengedwa kuchokera ku kuchuluka kwa jekeseni wa phage, poganizira kuchuluka kwa magazi.Mabwalo akuda amawonetsa podutsa mzere wa y wotuluka kuchokera m'magazi a phage.(e,f) Onetsani kuchuluka kwafupipafupi ndi kugawa kwazinthu zonse zomwe zingathe kuphatikizika za tripeptide zomwe zimapezeka mu peptide.Chiwerengero cha motifs chopezeka mu 1000 kuwerenga chikuwonetsedwa.Zofunikira (p <0.001) zolemetsa zolembedwa zimalembedwa ndi madontho ofiira.(e) Correlation scatterplot kufanizitsa pafupipafupi za tripeptide motif ya laibulale yojambulidwa ndi phage yotengedwa ndi magazi kuchokera ku nyama #1.1 ndi #1.2.(f) Correlation scatterplot kuyerekeza ma frequency achibale a nyama phage tripeptide motifs #1.1 ndi #1.2 olekanitsidwa m'magazi ndi cerebrospinal fluid.(g, h) Chiwonetsero cha ID chotsatizana cha phage cholemeretsedwa m'magazi (g) motsutsana ndi malaibulale ojambulidwa ndi phage yolemeretsedwa mu CSF (h) motsutsana ndi magazi pambuyo pakusankhidwa kwa vivo mu nyama zonse ziwiri.Kukula kwa code ya chilembo chimodzi kumasonyeza kuti amino acid imapezeka kangati pamalo amenewo.Green = polar, purple = neutral, blue = basic, red = acidic and black = hydrophobic amino acid.Chithunzi 1a, b chidapangidwa ndikupangidwa ndi Eduard Urich.
Tidabaya laibulale ya phage peptide mu makoswe awiri a CM (clades A ndi B) ndi phage yakutali kuchokera ku cerebrospinal fluid ndi magazi (Chithunzi 1d).Kutulutsidwa koyambirira kwa laibulale kunali kocheperako poyerekeza ndi phage lamtundu wakuthengo.Theka la moyo wa laibulale yojambulidwa mu nyama zonsezo zinali mphindi 24.8 m'magazi, ofanana ndi nyama zakutchire, ndi mphindi 38.5 mu CSF.Zitsanzo za magazi ndi cerebrospinal fluid phage kuchokera ku nyama iliyonse zinagonjetsedwa ndi HTS ndipo ma peptide onse odziwika adawunikidwa kuti apeze mawonekedwe aafupi a tripeptide.Mitundu ya Tripeptide idasankhidwa chifukwa imapereka maziko ochepa opangira mapangidwe ndi ma peptide-protein interactions14,15.Tinapeza mgwirizano wabwino pakugawidwa kwa zithunzithunzi pakati pa laibulale ya jekeseni ya phage ndi ma clones otengedwa m'magazi a nyama zonse ziwiri (mkuyu 1e).Deta imasonyeza kuti kupangidwa kwa laibulale kumangolemetsedwa pang'ono m'chipinda cha magazi.Mafupipafupi a amino acid ndi machitidwe ogwirizana adawunikidwanso pamalo aliwonse pogwiritsa ntchito pulogalamu ya Weblogo16.N'zochititsa chidwi kuti tinapeza amphamvu kulemeretsa magazi glycine zotsalira (mkuyu. 1g).Pamene magazi ankafaniziridwa ndi ma clones osankhidwa kuchokera ku CSF, kusankhidwa kwamphamvu ndi kusankhidwa kwina kwa ma motifs kunawonedwa (mkuyu 1f), ndipo ma amino acid ena anali osankhidwa mwapadera pa malo okonzedweratu mu 12-membala (mkuyu 1h).Mwachidziwitso, nyama zamtundu uliwonse zimasiyana kwambiri mu cerebrospinal fluid, pamene kupindula kwa magazi a glycine kunawonedwa mu nyama zonse ziwiri (Supplementary Fig. 4a-j).Pambuyo pa kusefa mozama kwa deta yotsatizana mu cerebrospinal fluid ya nyama #1.1 ndi #1.2, chiwerengero cha 964 ndi 420 apadera a 12-mer peptides anapezedwa (Supplementary Fig. 1d-e).Ma clones a phage akutali adakulitsidwa ndikuyika gawo lachiwiri la kusankha kwa vivo.Phage yotengedwa kuchokera kuchigawo chachiwiri chosankhidwa chinayikidwa ku HTS mu nyama iliyonse ndipo ma peptide onse odziwika anagwiritsidwa ntchito monga chothandizira pulogalamu yozindikiritsa motif kuti afufuze zochitika za tripeptide motifs (Fig. 2a, b, ef).Poyerekeza ndi kuzungulira koyamba kwa phage yomwe idapezedwa kuchokera ku CSF, tidawonanso kusankhidwa ndi kusankhidwa kwamitundu yambiri mu CSF m'nthambi A ndi B (mkuyu 2).Ma algorithm ozindikiritsa maukonde adagwiritsidwa ntchito kuti adziwe ngati akuyimira mitundu yosiyanasiyana yotsatizana.Kufanana kowoneka bwino kunawonedwa pakati pa machitidwe a 12-dimensional omwe adapezedwa ndi CSF mu clade A (mkuyu 2c, d) ndi clade B (mkuyu 2g, h).Kusanthula kophatikizana munthambi iliyonse kunavumbulutsa zolemba zosiyana zosankhidwa za 12-mer peptides (Supplementary Fig. 5c, d) ndi kuwonjezeka kwa chiwerengero cha CSF / magazi titer m'kupita kwa nthawi kwa ma clones ophatikizana pambuyo pa ulendo wachiwiri wosankhidwa poyerekeza ndi mzere woyamba wosankhidwa (Supplementary Fig. 5e).).
Kulemera kwa ma motifs ndi ma peptides mu cerebrospinal fluid ndi mizere iwiri yotsatizana ya mu vivo functional phage display.
Ma phage onse a cerebrospinal fluid omwe adachira kuchokera pamzere woyamba wa nyama iliyonse (zinyama #1.1 ndi #1.2) zidaphatikizidwa, kukulitsidwa, kutsatiridwa kwa HT ndikuyikidwanso palimodzi (2 x 1010 phages / nyama) makoswe a 2 SM (#1.1 → #).2.1 ndi 2.2, 1.2 → 2.3 ndi 2.4).(a,b,e,f) Kugwirizana kwa scatterplots kufananiza maulendo afupipafupi a maulendo a tripeptide a phage zonse zochokera ku CSF m'magulu oyamba ndi achiwiri osankhidwa.Kuchuluka kwafupipafupi komanso kugawa kwazithunzi zomwe zimayimira ma tripeptide onse omwe apezeka mu peptides mbali zonse ziwiri.Chiwerengero cha motifs chopezeka mu 1000 kuwerenga chikuwonetsedwa.Zolemba zomwe zidasankhidwa kwambiri (p <0.001) zosankhidwa kapena zosaphatikizidwa mu library imodzi yofananizidwa zimawonetsedwa ndi madontho ofiira.(c, d, g, h) Chifaniziro cha logo chamitundu yonse ya CSF-rich 12 amino acid yotengera mozungulira 2 ndi 1 ya kusankha kwa vivo.Kukula kwa code ya chilembo chimodzi kumasonyeza kuti amino acid imapezeka kangati pamalo amenewo.Kuyimira chizindikiro, mafupipafupi a ma CSF omwe amatsatiridwa kuchokera ku nyama imodzi pakati pa maulendo awiri osankhidwa amafananizidwa ndipo zotsatizana zolemeretsedwa pamzere wachiwiri zikuwonetsedwa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 ndi (h) #1.2–#2.4.Ma amino acid omwe amalemeretsedwa kwambiri pamalo opatsidwa (c, d) nyama ayi.2.1 ndi ayi.2.2 kapena (g, h) mu nyama no.2.3 ndi.2.4 amawonetsedwa mumitundu.Green = polar, purple = neutral, blue = basic, red = acidic and black = hydrophobic amino acid.
Pambuyo pa chisankho chachitatu chosankhidwa, tinazindikira ma 124 ma peptide apadera (#3.1 ndi #3.2) kuchokera ku 332 CSF-reconstituted phage clones olekanitsidwa ndi nyama ziwiri (Supplementary Fig. 6a).Mndandanda wa LGSVS (18.7%) unali ndi gawo lalikulu kwambiri, kutsatiridwa ndi zoyika zakutchire PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), ndi SARGSWREIVSLS (2.2%).M’gawo lomaliza lachinayi, tinaphatikiza nthambi ziwiri zosankhidwa mwaokha kuchokera ku nyama zitatu zosiyana (mkuyu 1c).Pazithunzi za 925 zotsatizana za phage zomwe zinapezedwa kuchokera ku CSF, m'gawo lachinayi tinapeza maulendo apadera a 64 a peptide (Supplementary Fig. 6b), pakati pawo chiwerengero cha phage chakutchire chinagwera ku 0.8%.Mitundu yodziwika bwino ya CSF pamzere wachinayi inali LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) ndi RLSSVDSDLSGC (3, 2%).%)).Kutalika kwa ma peptide osankhidwa ndi chifukwa cha kuyika kwa ma nucleotide / kuchotsedwa kapena ma codon oyimitsa msanga m'mabuku oyambira a library mukamagwiritsa ntchito ma codon osokonekera pakupanga laibulale ya NNK.Ma codon oyimitsa msanga amapanga ma peptide amfupi ndipo amasankhidwa chifukwa amakhala ndi aa motif yabwino.Ma peptide ataliatali amatha chifukwa choyika / kufufutidwa m'mabuku oyambira opangira.Izi zimayika kodoni yoyimitsidwa kunja kwa chimango ndikuyiwerenga mpaka kuyimitsidwa kwatsopano koyimitsidwa kukuwonekera pansi.Nthawi zambiri, tidawerengera zinthu zolemeretsa pazosankha zonse zinayi pofanizira zomwe zalowetsedwa ndi data yotulutsa zitsanzo.Pakuwunika koyambirira, tidagwiritsa ntchito ma phage titers zakuthengo ngati mbiri yosadziwika.Chochititsa chidwi n'chakuti, kusankha kolakwika kwa phage kunali kolimba kwambiri mumayendedwe oyambirira a CSF, koma osati m'magazi (mkuyu 3a), zomwe zingakhale chifukwa cha kuchepa kwapang'onopang'ono kwa mamembala ambiri a laibulale ya peptide mu chipinda cha CSF kapena phages wachibale amakonda kusungidwa bwino kwambiri kapena kuchotsedwa m'magazi kuposa bacteriophages.Komabe, mu gawo lachiwiri la panning, kusankha mwamphamvu kwa phages mu CSF kunawonedwa m'magulu onse awiri, kutanthauza kuti kuzungulira kwapitako kunapindula mu phages kusonyeza ma peptides omwe amalimbikitsa CSF kutengeka (Mkuyu 3a).Apanso, popanda kulimbikitsa kwambiri magazi.Komanso pagawo lachitatu ndi lachinayi, ma clones a phage adalemeretsedwa kwambiri mu CSF.Poyerekeza maulendo afupipafupi amtundu uliwonse wapadera wa peptide pakati pa maulendo awiri omaliza osankhidwa, tinapeza kuti zotsatizanazo zinapindula kwambiri pamtundu wachinayi wosankhidwa (mkuyu 3b).Chiwerengero cha 931 tripeptide motifs chinachotsedwa mumayendedwe onse 64 apadera a peptide pogwiritsa ntchito ma peptide onse.Zolemba zolemerera kwambiri pagawo lachinayi zidafufuzidwa mozama za mbiri yawo yolemeretsa kuzungulira konsekonse poyerekeza ndi laibulale yojambulidwa (kudulidwa: 10% kupindula) (Supplementary Fig. 6c).Kusankhidwa kwachindunji kunawonetsa kuti zolinga zambiri zophunziridwa zidalemeretsedwa m'magulu onse am'mbuyomu a nthambi zonse zosankhidwa.Komabe, ma motifs ena (monga SGL, VSG, LGS GSV) anali makamaka ochokera ku clade A, pomwe ena (monga FGW, RTN, WGF, NTR) adalemeretsedwa mu clade B.
Kutsimikizika kwa kayendedwe ka CSF kwa CSF-enriched phage-displayed peptides ndi biotinylated leader peptides conjugated to streptavidin payloads.
(a) Kuchulukitsa kwachuma kumawerengeredwa m'mizere yonse inayi (R1-R4) kutengera jekeseni (input = I) phage (PFU) titers ndi CSF phage titers (zotulutsa = O).Zinthu zolemetsa zamagulu atatu omaliza (R2-R4) zinawerengedwa poyerekeza ndi kuzungulira kwapitako ndi kuzungulira koyamba (R1) ndi deta yolemera.Mipiringidzo yotseguka ndi cerebrospinal fluid, mipiringidzo yamthunzi ndi plasma.(***p<0.001, kutengera mayeso a Wophunzira).(b) Mndandanda wa ma peptide ochuluka kwambiri a phage, omwe amawerengedwa molingana ndi chiwerengero chawo chamagulu onse omwe amasonkhanitsidwa ku CSF pambuyo pa 4 yosankhidwa.Mitundu isanu ndi umodzi yodziwika bwino ya phage imawonetsedwa mumtundu, kuwerengeredwa ndi zomwe zimapindulitsa pakati pa kuzungulira 3 ndi 4 ya kusankha (zoyika).(c, d) Makanema asanu ndi limodzi olemera kwambiri a phage, malaibulale opanda kanthu ndi malaibulale a phage peptide ochokera kuzungulira 4 adawunikidwa payekhapayekha mu chitsanzo cha CSF.CSF ndi zitsanzo za magazi zinasonkhanitsidwa pa nthawi zosonyezedwa.(c) Zofanana ndi 6 zamagulu a phage (2 x 1010 phage/zinyama), phage zopanda kanthu (#1779) (2 x 1010 phage/zinyama) ndi malaibulale a stock phage peptide (2 x 1012 phage/zinyama) Bayikeni osachepera 3 CM imayendetsedwa pagulu kudzera pa ndowe.Ma pharmacokinetics a CSF amtundu uliwonse wa jekeseni wa phage ndi laibulale ya phage peptide pakapita nthawi akuwonetsedwa.(d) ikuwonetsa chiyerekezo cha CSF/mwazi pa ma phages/mL onse omwe adachira panthawi yoyeserera.(e) Ma peptide otsogolera anayi opangidwa ndi owongolera amodzi adalumikizidwa ndi biotin kupita ku streptavidin kudzera mu N-terminus yawo (chiwonetsero cha tetramer) chotsatiridwa ndi jekeseni (mchira wa mchira iv, 10 mg streptavidin/kg).Makoswe osachepera atatu (N = 3).).Zitsanzo za CSF zinasonkhanitsidwa pazigawo zomwe zasonyezedwa ndi streptavidin zowunikira zinayesedwa ndi CSF anti-streptavidin ELISA (nd = osapezeka).(*p<0.05, **p<0.01, ***p<0.001, kutengera mayeso a ANOVA).(f) Kuyerekeza kwa mndandanda wa amino acid wa phage peptide clone #2002 (wofiirira) ndi ma clones ena osankhidwa a phage peptide kuchokera pamzere wa 4 wosankhidwa.Zidutswa zofanana ndi zofanana za amino acid zimakhala ndi mitundu.
Mwa ma phage onse olemetsedwa pamzere wachinayi (mkuyu 3b), ma clones asanu ndi limodzi adasankhidwa kuti afufuzenso pagulu lachitsanzo la CSF.Zofanana ndi zisanu ndi chimodzi za phage, phage yopanda kanthu (palibe kuyika) ndi malaibulale a prophage peptide adalowetsedwa mu zinyama zitatu za CM za cannulated, ndipo pharmacokinetics inatsimikiziridwa mu CSF (Fig. 3c) ndi magazi (Supplementary Fig. 7) mayesero.Mitundu yonse ya phage yoyesedwa imayang'ana chipinda cha CSF pamlingo wa 10-1000 nthawi yayitali kuposa ya phage yopanda kanthu (#1779).Mwachitsanzo, ma clones #2020 ndi #2077 anali ndi ma CSF okwera pafupifupi 1000 kuposa phage yowongolera.Mbiri ya pharmacokinetic ya peptide iliyonse yosankhidwa ndi yosiyana, koma onse ali ndi luso lapamwamba la CSF.Tidawona kuchepa kosalekeza pakapita nthawi kwa ma clones #1903 ndi #2011, pomwe ma clones #2077, #2002 ndi #2009 kuwonjezeka mkati mwa mphindi 10 zoyambirira zitha kuwonetsa zoyendera koma ziyenera kutsimikiziridwa.Ma Clones #2020, #2002, ndi #2077 adakhazikika pamilingo yayikulu, pomwe kuchuluka kwa CSF #2009 kudatsika pang'onopang'ono pambuyo pakuwonjezeka koyambirira.Kenako tidafanizira pafupipafupi kwa munthu aliyense wa CSF yemwe ali ndi magazi ake (mkuyu 3d).Kulumikizana kwa tanthauzo la munthu aliyense wa CSF ndi gawo lake la magazi nthawi zonse zotsatiridwa kukuwonetsa kuti atatu mwa asanu ndi mmodzi omwe adasankhidwa adalemeretsedwa kwambiri mu CSF yamagazi.Chochititsa chidwi, clone #2077 inasonyeza kukhazikika kwa magazi (Supplementary Figure 7).Kuti titsimikizire kuti ma peptides okha amatha kunyamula katundu wina kupatula tinthu tating'onoting'ono m'chipinda cha CSF, tidapanga ma peptides anayi opangidwa ndi biotin pa N-terminus pomwe ma peptides amamatira ku phage particle.Ma peptides a biotinylated (nos. 2002, 2009, 2020 ndi 2077) adalumikizidwa ndi streptavidin (SA) kuti apeze mitundu yambirimbiri yotsanzira phage geometry.Mtunduwu udatilolanso kuyeza kuwonekera kwa SA m'magazi ndi cerebrospinal fluid ngati ma peptides onyamula katundu.Chofunika kwambiri, deta ya phage nthawi zambiri imatha kupangidwanso pamene ma peptides opangidwa amaperekedwa mumtundu uwu wa SA-conjugated (mkuyu 3e).Ma peptides ophwanyidwa anali ndi kuwonekera koyambirira komanso kuvomerezeka kwa CSF mwachangu ndi milingo yosazindikirika mkati mwa maola 48.Kuti tizindikire njira zoperekera ma peptide phage clones mu malo a CSF, tidasanthula momwe kumenyedwa kwa phage peptide pogwiritsa ntchito immunohistochemistry (IHC) kuti tizindikire mwachindunji tinthu ta phage pa ola la 1 pambuyo pa jekeseni wa mtsempha wa vivo.Makamaka, ma clones #2002, #2077, ndi #2009 amatha kuzindikirika ndi madontho amphamvu mu ma capillaries aubongo, pomwe control phage (#1779) ndi clone #2020 sizinazindikirike (Chithunzi chowonjezera 8).Izi zikuwonetsa kuti ma peptides awa amathandizira kukhudza ubongo ndendende podutsa BBB.Kusanthula mwatsatanetsatane ndikofunikira kuyesa lingaliro ili, chifukwa njira ya BSCFB ingakhalenso nayo.Poyerekeza mndandanda wa amino acid wa clone wolemeretsedwa kwambiri (#2002) ndi ma peptide ena osankhidwa, adadziwika kuti ena mwa iwo ali ndi zowonjezera za amino acid, zomwe zingasonyeze njira yoyendera yofanana (mkuyu 3f).
Chifukwa cha mawonekedwe ake apadera a plasma komanso kuwonjezeka kwakukulu kwa CSF pakapita nthawi, phage chiwonetsero chazithunzi #2077 chinafufuzidwanso kwa nthawi yayitali ya 48-ola ndipo chinatha kuberekanso kuwonjezeka kofulumira kwa CSF komwe kumawonedwa mogwirizana ndi mayendedwe okhazikika a SA (Mkuyu 4a).Ponena za ma clones ena odziwika, #2077 adadetsedwa kwambiri ndi ma capillaries a muubongo ndipo adawonetsa kukhazikika kwakukulu ndi capillary marker lectin ikawonedwa pakutha kwapamwamba komanso mwina madontho ena m'malo a parenchymal (Chithunzi 4b).Kuti tifufuze ngati peptide-mediated pharmacological zotsatira zitha kupezeka mu CNS, tidayesa momwe ma biotinylated versions a i) the #2077 transit peptide ndi ii) BACE1 inhibitor peptide adasakanizidwa ndi SA pamitundu iwiri yosiyana.Pakuphatikiza kumodzi tidagwiritsa ntchito BACE1 peptide inhibitor yokha ndipo inayo tidagwiritsa ntchito chiŵerengero cha 1:3 cha BACE1 peptide inhibitor mpaka #2077 peptide.Zitsanzo zonsezi zidaperekedwa kudzera m'mitsempha ndipo milingo yamagazi ndi cerebrospinal ya beta-amyloid peptide 40 (Abeta40) idayesedwa pakapita nthawi.Abeta40 idayezedwa mu CSF popeza ikuwonetsa kuletsa kwa BACE1 mu parenchyma yaubongo.Monga momwe zimayembekezeredwa, zovuta zonsezi zimachepetsa kwambiri magazi a Abeta40 (mkuyu 4c, d).Komabe, zitsanzo zokha zomwe zili ndi chisakanizo cha peptide ayi.2077 ndi inhibitor ya BACE1 peptide conjugated to SA inachititsa kuchepa kwakukulu kwa Abeta40 mu cerebrospinal fluid (mkuyu 4c).Deta ikuwonetsa kuti peptide ayi.2077 imatha kunyamula puloteni ya 60 kDa SA kupita ku CNS komanso imayambitsa zotsatira zamankhwala ndi SA-conjugated inhibitors ya BACE1 peptide.
(a) Jekeseni wa clonal (2 × 10 phages / nyama) ya T7 phage yomwe ikuwonetsa mbiri yayitali ya pharmacokinetic ya CSF peptide #2077 (RLSSVDSDLSGC) ndi uninjection control phage (#1779) mu makoswe osachepera atatu a CM-intubated.(b) Chifaniziro cha microscopic choyimira ma cortical microvessels mu makoswe opangidwa ndi phage (2 × 10 10 phages / nyama) kuwonetsa kutsutsa kwa peptide #2077 ndi ziwiya (lectin).Mitundu ya phageyi idaperekedwa kwa makoswe a 3 ndikuloledwa kuyendayenda kwa ola la 1 musanayambe kusakaniza.Ubongo udagawidwa ndikudetsedwa ndi ma antibodies olembedwa ndi polyclonal FITC motsutsana ndi T7 phage capsid.Mphindi khumi isanayambe kudzoza ndi kukonzanso kotsatira, DyLight594-yolemba lectin inkaperekedwa kudzera m'mitsempha.Zithunzi za fluorescent zowonetsa madontho a lectin (ofiira) a mbali yowala ya ma microvessels ndi phages (wobiriwira) mu lumen ya ma capillaries ndi minofu yaubongo.Sikelo ya bar imafanana ndi 10 µm.(c, d) Biotinylated BACE1 inhibitory peptide yokha kapena kuphatikiza ndi biotinylated transit peptide #2077 anaphatikizidwa ndi streptavidin kutsatiridwa ndi jekeseni wa mtsempha wa makoswe osachepera atatu a CM (10 mg streptavidin/kg).BACE1 peptide inhibitor-mediated reduction mu Aβ40 inayesedwa ndi Aβ1-40 ELISA m'magazi (wofiira) ndi cerebrospinal fluid (lalanje) pa nthawi zosonyezedwa.Kuti mumveke bwino, mzere wamadontho umajambulidwa pa graph pamlingo wa 100%.(c) Kuchepetsa kuchuluka kwa Aβ40 m'magazi (makona atatu ofiira) ndi cerebrospinal fluid (makona atatu a lalanje) mu makoswe omwe amathandizidwa ndi streptavidin conjugated to transit peptide #2077 ndi BACE1 inhibitory peptide mu chiwerengero cha 3: 1.(d) Kuchepetsa kwaperesenti ya magazi Aβ40 (mabwalo ofiira) ndi cerebrospinal fluid (mabwalo alalanje) a makoswe omwe amathandizidwa ndi streptavidin kuphatikizapo BACE1 inhibitory peptide yokha.Kuphatikizika kwa Aβ muulamuliro kunali 420 pg/ml (kusiyana kokhazikika = 101 pg/ml).
Chiwonetsero cha Phage chagwiritsidwa ntchito bwino m'malo angapo a kafukufuku wamankhwala17.Njirayi yakhala ikugwiritsidwa ntchito mu vivo vascular diversity studies18,19 komanso maphunziro okhudza mitsempha ya ubongo20,21,22,23,24,25,26.Mu phunziro ili, tidakulitsa kugwiritsa ntchito njira yosankhidwayi osati kungozindikiritsa mwachindunji ma peptides omwe akulunjika ziwiya zaubongo, komanso kuti tipeze omwe ali ndi zida zonyamulira zoyendera kuti awoloke chotchinga chamagazi-muubongo.Tsopano tikufotokozera zakukula kwa njira yosankha mu vivo mu makoswe opangidwa ndi CM ndikuwonetsa kuthekera kwake kuzindikira ma peptides okhala ndi CSF homing katundu.Pogwiritsa ntchito T7 phage yomwe ikuwonetsa laibulale ya ma peptides a 12-mer mwachisawawa, tinatha kuwonetsa kuti phage ya T7 ndi yaying'ono mokwanira (pafupifupi 60 nm m'mimba mwake) 10 kuti igwirizane ndi chotchinga cha magazi-ubongo, potero kuwoloka chotchinga chamagazi-ubongo kapena choroid plexus.Tinawona kuti kukolola kwa CSF kuchokera ku makoswe a CM osakanizidwa kunali koyendetsedwa bwino mu vivo ntchito yowunikira njira, komanso kuti phage yotulutsidwa sinangomangidwa ndi vasculature komanso inkagwira ntchito ngati chonyamulira kudutsa chotchinga chamagazi-ubongo.Kuphatikiza apo, pakutolera magazi nthawi imodzi ndikugwiritsa ntchito HTS ku CSF ​​ndi ma phages opangidwa ndi magazi, tidatsimikizira kuti kusankha kwathu CSF sikunakhudzidwe ndi kulemetsedwa kwa magazi kapena kulimba mtima pakukulitsa pakati pazosankha.Komabe, chipinda cha magazi ndi gawo la njira yosankhidwa, popeza ma phages omwe amatha kufika ku chipinda cha CSF ayenera kukhala ndi moyo ndikuzungulira m'magazi nthawi yaitali kuti alemere mu ubongo.Kuti tipeze zambiri zamatsatidwe odalirika kuchokera ku data yaiwisi ya HTS, tidayika zosefera zomwe zidasinthidwa kuti zigwirizane ndi zolakwika zotsatizana ndi pulatifomu pakusanthula kachitidwe.Mwa kuphatikiza magawo a kinetic munjira yowunikira, tidatsimikizira ma pharmacokinetics ofulumira amtundu wamtundu wa T7 phages (t½ ~ 28 min) m'magazi24, 27, 28 ndikutsimikizanso theka la moyo wawo mu cerebrospinal fluid (t½ ~ 26 min) pamphindi).Ngakhale mbiri ya pharmacokinetic yofananira m'magazi ndi CSF, 0.001% yokha ya magazi a phage imatha kudziwika mu CSF, kuwonetsa kutsika kwapansi kwamtundu wamtundu wa T7 phage kudutsa chotchinga chamagazi-ubongo.Ntchitoyi ikuwonetsa kufunikira kwa gawo loyamba losankhira mukamagwiritsa ntchito njira zopangira ma vivo, makamaka machitidwe a phage omwe amachotsedwa mwachangu kuchokera kumayendedwe, popeza ma clones ochepa amatha kufikira chipinda cha CNS.Choncho, m'chigawo choyamba, kuchepetsa kusiyana kwa laibulale kunali kwakukulu kwambiri, chifukwa chiwerengero chochepa cha ma clones pamapeto pake chinasonkhanitsidwa mu chitsanzo chokhwima kwambiri cha CSF.Izi mu vivo panning njira zinaphatikizapo njira zingapo zosankhidwa monga kudzikundikira mwakhama mu chipinda cha CSF, kupulumuka kwa clone m'chipinda cha magazi, ndi kuchotsedwa mwamsanga kwa T7 phage clones m'magazi mkati mwa maminiti oyambirira a 10 (Fig. 1d ndi Supplementary Fig. 4M).).Chifukwa chake, pambuyo pa kuzungulira koyamba, ma clones osiyanasiyana a phage adadziwika mu CSF, ngakhale dziwe loyambirira lomwelo lidagwiritsidwa ntchito kwa nyama payokha.Izi zikuwonetsa kuti njira zingapo zosankhidwa mosamalitsa zama library omwe ali ndi mamembala ambiri a library zimapangitsa kuchepa kwakukulu pakusiyanasiyana.Chifukwa chake, zochitika zachisawawa zidzakhala gawo lofunikira pakusankha koyambirira, zomwe zimakhudza kwambiri zotsatira.Zikuoneka kuti ma clones ambiri mulaibulale yoyambirira anali ndi chizolowezi cholemeretsa cha CSF.Komabe, ngakhale pansi pa zoyesera zomwezo, zotsatira zosankhidwa zikhoza kusiyana chifukwa cha chiwerengero chochepa cha clone iliyonse mu dziwe loyamba.
Zolinga zolemeretsedwa mu CSF zimasiyana ndi zomwe zili m'magazi.Chochititsa chidwi, tidawona kusintha koyamba kwa ma peptides olemera a glycine m'magazi a nyama iliyonse.(Mkuyu 1g, Zowonjezera Nkhuyu. 4e, 4f).Phage yokhala ndi ma glycine peptides imatha kukhala yokhazikika komanso yocheperako kuti ichotsedwe.Komabe, ma peptide olemera a glycine awa sanawonekere mu zitsanzo zamadzimadzi a muubongo, kutanthauza kuti malaibulale osungidwa adadutsa njira ziwiri zosankhidwa: imodzi m'magazi ndi ina yololedwa kuwunjikana mu cerebrospinal fluid.Ma clones olemetsedwa a CSF obwera chifukwa cha mgawo wachinayi wosankhidwa ayesedwa mozama.Pafupifupi ma clones onse omwe adayesedwa payekha adatsimikiziridwa kuti alemeretsedwa mu CSF poyerekeza ndi phage yopanda kanthu.Kugunda kwa peptide kumodzi (#2077) kudawunikidwa mwatsatanetsatane.Idawonetsa theka la moyo wautali wa plasma poyerekeza ndi kugunda kwina (Chithunzi 3d ndi Supplementary Chithunzi 7), ndipo chochititsa chidwi, peptide iyi inali ndi zotsalira za cysteine ​​​​pa C-terminus.Zawonetsedwa posachedwa kuti kuwonjezera kwa cysteine ​​​​ku peptides kumatha kupititsa patsogolo katundu wawo wa pharmacokinetic pomanga ku albumin 29.Izi sizikudziwika pa peptide #2077 ndipo zimafunikira kuphunzira kwina.Ma peptides ena adawonetsa kudalira kwa valence pakulemeretsa kwa CSF (deta yosawonetsedwa), yomwe ingakhale yokhudzana ndi mawonekedwe amtundu wa T7 capsid.Dongosolo la T7 lomwe tidagwiritsa ntchito likuwonetsa makope 5-15 a peptide iliyonse pagawo la phage.IHC inkachitidwa pa otsogolera phage clones jekeseni mtsempha mu ubongo kotekisi wa makoswe (Supplementary Fig. 8).Deta inasonyeza kuti ma clones osachepera atatu (No. 2002, No. 2009 ndi No. 2077) adalumikizana ndi BBB.Ziyenera kutsimikiziridwa ngati kuyanjana kwa BBB uku kumapangitsa kuti CSF ikhale yochuluka kapena kuyenda kwa ma cloneswa molunjika ku BCSFB.Chofunika kwambiri, tikuwonetsa kuti ma peptide osankhidwa amasunga mphamvu zawo zoyendera za CSF akapangidwa ndikumangika ku katundu wa mapuloteni.Kumanga kwa N-terminal biotinylated peptides ku SA kwenikweni kubwereza zotsatira zopezedwa ndi phage clones awo m'magazi ndi cerebrospinal fluid (mkuyu 3e).Pomaliza, tikuwonetsa kuti lead peptide #2077 imatha kulimbikitsa ntchito yaubongo ya biotinylated peptide inhibitor ya BACE1 conjugated to SA, zomwe zimapangitsa kutchulidwa kwa pharmacodynamic mu CNS mwa kuchepetsa kwambiri milingo ya Abeta40 mu CSF (Mkuyu 4).Sitinathe kuzindikira ma homologi aliwonse munkhokwe pofufuza ma peptide motsatizana za homology pazomenyedwa zonse.Ndikofunika kuzindikira kuti kukula kwa laibulale ya T7 ndi pafupifupi 109, pamene kukula kwa laibulale ya 12-mers ndi 4 x 1015. Choncho, tinangosankha gawo laling'ono la malo osiyanasiyana a laibulale ya 12-mer peptide, zomwe zingatanthauze kuti ma peptide okonzedwa bwino amatha kudziwika poyesa malo oyandikana nawo omwe adadziwika.Mongopeka, chimodzi mwazifukwa zomwe sitinapeze ma homologues achilengedwe a ma peptidewa akhoza kukhala osasankhidwa panthawi yachisinthiko kuti aletse kulowa kosalamulirika kwa ma peptide ena muubongo.
Kutengedwa palimodzi, zotsatira zathu zimapereka maziko a ntchito yamtsogolo kuti azindikire ndikuwonetsa machitidwe a mayendedwe a cerebrovascular chotchinga mu vivo mwatsatanetsatane.Kukonzekera koyambirira kwa njirayi kumachokera ku njira yosankha ntchito yomwe sikuti imangozindikiritsa ma clones okhala ndi cerebral vascular binding properties, komanso imaphatikizapo sitepe yofunika kwambiri yomwe ma clones opambana amakhala ndi zochitika zamkati kuti awoloke zolepheretsa zamoyo mu vivo kulowa mu CNS compartment.ndikuwunikira njira yonyamulira ma peptide awa ndi zomwe amakonda kumangirira ku ma microvasculature omwe ali kudera laubongo.Izi zitha kupangitsa kuti pakhale njira zatsopano zonyamulira BBB ndi zolandilira.Tikuyembekeza kuti ma peptides odziwika amatha kumangirira mwachindunji ku cerebrovascular receptors kapena kumayendedwe ozungulira omwe amatumizidwa kudzera mu BBB kapena BCSFB.Ma vector a peptide okhala ndi mayendedwe a CSF omwe apezeka mu ntchitoyi afufuzidwanso.Pakali pano tikufufuza zaubongo wa ma peptidewa kuti athe kuwoloka BBB ndi/kapena BCSFB.Ma peptide atsopanowa adzakhala zida zamtengo wapatali kwambiri zopezera zolandilira zatsopano kapena njira komanso kupanga nsanja zatsopano zoperekera ma macromolecules, monga biologics, ku ubongo.
Cannulate chitsime chachikulu (CM) pogwiritsa ntchito kusinthidwa kwa njira yomwe tafotokozayi.Makoswe a Wistar (200-350 g) adayikidwa pa chipangizo cha stereotaxic ndipo kudulidwa kwapakati kunapangidwa pamutu wometedwa komanso wokonzedwa bwino kuti uwonetse chigaza.Gwirani mabowo awiri m'dera la sash yakumtunda ndikumanga zomangira m'mabowo.Bowo lina linabowoleredwa mu lateral occipital crest kuti muwongolere stereotactic ya cannula yachitsulo chosapanga dzimbiri kulowa mu CM.Ikani simenti ya mano mozungulira kanula ndikutchinjiriza ndi zomangira.Pambuyo pokonza chithunzi ndi kuumitsa simenti, chilonda cha khungu chinatsekedwa ndi 4/0 supramid suture.Kuyika bwino kwa cannula kumatsimikiziridwa ndi kutuluka kwamadzimadzi kwa cerebrospinal fluid (CSF).Chotsani makoswe ku zida za stereotaxic, landirani chisamaliro choyenera cha postoperative ndi kasamalidwe ka ululu, ndipo mulole kuti achire kwa osachepera sabata imodzi mpaka zizindikiro za magazi zikuwonekera mu cerebrospinal fluid.Makoswe a Wistar (Crl: WI/Han) adatengedwa kuchokera ku Charles River (France).Makoswe onse amasungidwa m'malo opanda tizilombo toyambitsa matenda.Kuyesera kwa zinyama zonse kunavomerezedwa ndi Veterinary Office ya Mzinda wa Basel, Switzerland, ndipo kunachitika motsatira License ya Zinyama No.
Pang'onopang'ono samalani khosweyo ali ndi CM cannula m'manja.Chotsani Datura ku cannula ndikusonkhanitsa 10 µl yamadzimadzi a muubongo omwe akuyenda.Popeza patency ya cannula pamapeto pake idasokonekera, zitsanzo zomveka bwino zamadzimadzi am'magazi opanda umboni wa kuipitsidwa kwa magazi kapena kusinthika kwamtundu zidaphatikizidwa mu kafukufukuyu.Mofananirako, pafupifupi 10-20 μl ya magazi idatengedwa kuchokera pang'onopang'ono kumapeto kwa mchira kukhala machubu okhala ndi heparin (Sigma-Aldrich).CSF ndi magazi zinasonkhanitsidwa nthawi zosiyanasiyana pambuyo jekeseni mtsempha wa T7 phage.Pafupifupi 5-10 μl yamadzimadzi idatayidwa isanasonkhanitsidwe chitsanzo chilichonse cha CSF, chomwe chimagwirizana ndi kuchuluka kwakufa kwa catheter.
Malaibulale anapangidwa pogwiritsa ntchito vekitala ya T7Select 10-3b monga momwe tafotokozera mu T7Select system manual (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996).Mwachidule, choyikapo mwachisawawa cha 12-mer DNA chidapangidwa motere:
NNK codon idagwiritsidwa ntchito kupewa ma codons oyimitsa kawiri ndi ma amino acid ochulukirapo pakuyikapo.N ndi pamanja osakaniza equimolar chiŵerengero cha nucleotide aliyense, ndipo K ndi pamanja osakaniza equimolar chiŵerengero cha adenine ndi cytosine nucleotides.Madera ang'onoang'ono osakhazikika adasinthidwa kukhala DNA yokhala ndi zingwe ziwiri polumikizidwanso ndi dNTP (Novagen) ndi enzyme ya Klenow (New England Biolabs) ku Klenow buffer (New England Biolabs) kwa maola atatu pa 37°C.Zitachitika izi, DNA yokhala ndi zingwe ziwiri idapezedwanso ndi mpweya wa EtOH.DNA yotsatilayo idagayidwa ndi ma enzymes oletsa EcoRI ndi HindIII (onse ochokera ku Roche).Chong'ambika ndi choyeretsedwa (QIAquick, Qiagen) choyika (T4 ligase, New England Biolabs) chinalumikizidwa mu-frame mu vekitala ya T7 yomwe idadulidwa kale pambuyo pa amino acid 348 ya jini ya 10B capsid.Zochita za ligation zidakulungidwa pa 16 ° C. kwa maola 18 isanafike kulongedza kwa in vitro.Phage ma CD mu m'galasi anachitidwa molingana ndi malangizo operekedwa ndi T7Select 10-3b cloning kit (Novagen) ndi ma CD njira anakulitsidwa kamodzi lysis ntchito Escherichia coli (BLT5615, Novagen).Ma lysates anali centrifuged, titrated ndi kuzizira pa -80 ° C. monga stock solution ya glycerol.
Kukulitsa kwachindunji kwa PCR kwa zigawo za phage zokulirakulira mu msuzi kapena mbale pogwiritsa ntchito 454 / Roche-amplicon fusion primers.The forward fusion primer ili ndi zotsatizana zomwe zikuzungulira dera losinthika (NNK) 12 (mwachidule-mwachindunji), GS FLX Titanium Adapter A, ndi makiyi anayi a library (TCAG) (Supplementary Figure 1a):
Reverse fusion primer ilinso ndi biotin yolumikizidwa kuti igwire mikanda ndi GS FLX Titanium Adapter B yofunikira pakukulitsa kwa clonal pa emulsion PCR:
The amplicons ndiye pansi 454/Roche pyrosequencing malinga ndi 454 GS-FLX Titanium protocol.Pakutsatizana kwa Sanger pamanja (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage DNA idakulitsidwa ndi PCR ndikutsatiridwa ndi mapeyala oyambira awa:
Zoyikapo kuchokera pamiyala payokha zidapangidwa kuti PCR ikulitsidwe pogwiritsa ntchito Roche Fast Start DNA Polymerase Kit (malinga ndi malangizo a wopanga).Chitani zoyambira zotentha (10 min pa 95 ° C) ndi kuzungulira kwa 35 (50 s pa 95 ° C, 1 min pa 50 ° C, ndi 1 min pa 72 ° C).
Phage kuchokera ku malaibulale, nyama zakutchire, phage yopulumutsidwa ku CSF ndi magazi, kapena ma clones aumwini adakulitsidwa mu Escherichia coli BL5615 mu TB msuzi (Sigma Aldrich) kapena mu 500 cm2 mbale (Thermo Scientific) kwa 4 h pa 37 ° C.Phage anachotsedwa m'mbale potsuka mbale ndi Tris-EDTA buffer (Fluka Analytical) kapena kusonkhanitsa zolembera ndi nsonga wosabala pipette.Phage anali olekanitsidwa ndi chikhalidwe cha supernatant kapena m'zigawo zosungiramo zozungulira za polyethylene glycol (PEG 8000) mpweya (Promega) ndikuyimitsidwanso mu Tris-EDTA buffer.
Phage yokwezeka idapangidwa mozungulira 2-3 yochotsa endotoxin pogwiritsa ntchito mikanda yochotsa endotoxin (Miltenyi Biotec) isanachitike jakisoni wa mtsempha (IV) (500 μl/nyama).Pachigawo choyamba, 2 × 1012 phages adayambitsidwa;chachiwiri, 2 × 1010 phages;muzosankha zachitatu ndi zachinayi, 2 × 109 phages pa nyama.Zomwe zili mu CSF ndi zitsanzo za magazi zomwe zasonkhanitsidwa panthawi yomwe zasonyezedwa zimatsimikiziridwa ndi kuwerengera zolembera malinga ndi malangizo a wopanga (T7Select system manual).Kusankhidwa kwa phage kunkachitidwa ndi jekeseni wa m'mitsempha wa malaibulale oyeretsedwa mumchira wa mchira kapena kuponderezedwa kwa phage yotengedwa ku CSF kuchokera kumalo osankhidwa apitalo, ndipo zokolola zotsatizana zinachitidwa pa 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, ndi 240 mins CSF motsatira.Zozungulira zinayi za mu vivo panning zidachitika momwe nthambi ziwiri zosankhidwa zidasungidwa padera ndikuwunikidwa pamizere itatu yoyamba yosankha.Zoyika zonse za phage zomwe zidachotsedwa ku CSF kuchokera pazosankha ziwiri zoyambirira zidasankhidwa ndi 454 / Roche pyrosequencing, pomwe ma clones onse ochotsedwa ku CSF kuchokera pazosankha ziwiri zomaliza adatsatidwa pamanja.Ma phage onse amagazi kuchokera pamzere woyamba wosankhidwa adayesedwanso ndi 454 / Roche pyrosequencing.Pa jakisoni wa ma clones a phage, phage zosankhidwa zidakulitsidwa mu E. coli (BL5615) pa mbale za 500 cm2 pa 37 ° C kwa maola 4.Ma clones osankhidwa payekha komanso otsatiridwa pamanja adafalitsidwa mu sing'anga ya TB.Pambuyo pochotsa phage, kuyeretsedwa ndi kuchotsa endotoxin (monga tafotokozera pamwambapa), 2 × 1010 phages / nyama mu 300 μl anabayidwa kudzera m'mitsempha mumtsempha umodzi wa mchira.
Kukonzekera ndi kusefa koyenera kwa data yotsatizana.Deta ya Raw 454/Roche idasinthidwa kuchoka pamapu a binary standard stream mapu (sff) kupita ku mtundu wowerengeka wa anthu wa Pearson (fasta) pogwiritsa ntchito mapulogalamu ogulitsa.Kukonzekera kwina kwa ndondomeko ya nucleotide kunachitika pogwiritsa ntchito mapulogalamu a C eni ake ndi zolemba (zosatulutsidwa pulogalamu ya pulogalamu) monga momwe tafotokozera pansipa.Kusanthula kwa deta yoyamba kumaphatikizapo ndondomeko zolimba zosefera masitepe ambiri.Kuti musefe zowerengera zomwe zinalibe DNA yolondola ya 12mer, zowerengerazo zidalumikizidwa motsatana kuti tiyambire lebulo (GTGATGTCGGGGATCCGAATTCT), stop label (TAAGCTTGCGGCCGCACTCGAGTA) ndi background insert (CCCTGCAGGGATATCCCGGAGCTCTCGTCGAC) pogwiritsa ntchito global Needs.kuyanjanitsa kulola kusagwirizana kwa 2 pamayendedwe31.Chifukwa chake, ma tag owerengera osayamba ndi kuyimitsa ndikuwerenga okhala ndi zoyika zakumbuyo, mwachitsanzo, ma alignments omwe amapitilira kuchuluka kololedwa kwa zolakwika, adachotsedwa mulaibulale.Ponena za zowerengera zotsala, mndandanda wa N-mer DNA woyambira pachizindikiro choyambira ndikutha chizindikiro choyimitsa chisanachotsedwe kuchokera pakuwerengera koyambirira ndikukonzedwanso (pambuyo pake amatchedwa "insert").Pambuyo pa kumasuliridwa kwa kuyikapo, gawolo pambuyo pa kodoni yoyamba yoyimitsa kumapeto kwa 5 'mapeto a primer imachotsedwapo.Kuonjezera apo, ma nucleotides omwe amatsogolera ku ma codon osakwanira kumapeto kwa 3 'mapeto a primer adachotsedwanso.Kupatula zoikamo zomwe zili ndi zotsatizana zakumbuyo zokha, zomasulira zoyambira ndi mawonekedwe a amino acid "PAG" zidachotsedwanso.Ma peptides okhala ndi kutalika kwapambuyo komasulira osakwana 3 amino acid adachotsedwa mulaibulale.Pomaliza, chotsani redundancy mu dziwe loyikapo ndikuzindikira kuchuluka kwa kuyika kwapadera kulikonse.Zotsatira za kusanthula uku zinaphatikizapo mndandanda wa ma nucleotide sequences (inserts) ndi maulendo awo (kuwerenga) (Supplementary Figures 1c ndi 2).
Gulu la N-mer DNA limayika motsatira kufanana: Kuchotsa zolakwika zotsatizana za 454/Roche (monga mavuto otsatizana owonjezera a homopolymer) ndikuchotsanso zosafunika kwenikweni, zosefedwa kale za N-mer DNA zoyika (zolowetsa) zimasanjidwa molingana.zolowetsa (mpaka 2 maziko osafananiza amaloledwa) pogwiritsa ntchito ndondomeko yobwerezabwereza yomwe imatanthauzidwa motere: zoyikapo zimasanjidwa poyamba ndi mafupipafupi (okwera kwambiri mpaka otsika kwambiri), ndipo ngati ali ofanana, ndi mtundu wawo wachiwiri ndi kutalika (kutalika mpaka kochepa) ).Choncho, zolowetsa kawirikawiri komanso zazitali zimatanthauzira "gulu" loyamba.Mafupipafupi a gulu amayikidwa kufupipafupi kwachinsinsi.Kenako, choyika chilichonse chomwe chatsala pamndandanda wosankhidwa chidayesedwa kuti chiwonjezedwe pagululo polumikizana ndi Needleman-Wunsch.Ngati chiwerengero cha zolakwika, zoyikapo, kapena zochotsa mumgwirizano sichidutsa malire a 2, kulowetsedwa kumawonjezedwa ku gululo, ndipo mafupipafupi a gulu lonse amawonjezedwa ndi momwe kuyikako kunawonjezeredwa.Zowonjezera zomwe zawonjezeredwa ku gulu zimayikidwa chizindikiro kuti zagwiritsidwa ntchito ndipo sizikuphatikizidwanso pakukonzedwanso.Ngati ndondomeko yoyikayo siingawonjezedwe ku gulu lomwe lilipo kale, ndondomeko yoyikapo imagwiritsidwa ntchito kupanga gulu latsopano lokhala ndi mafupipafupi oyenera oyika ndikulembapo ngati agwiritsidwa ntchito.Kubwerezaku kumatha pamene ndondomeko iliyonse yoyikapo yagwiritsidwa ntchito kupanga gulu latsopano kapena ikhoza kuphatikizidwa mu gulu lomwe lilipo kale.Kupatula apo, zoyika m'magulu zomwe zimakhala ndi ma nucleotides pamapeto pake zimasinthidwa kukhala ma peptide (malaibulale a peptide).Chotsatira cha kusanthula uku ndizomwe zimayikidwa ndi maulendo awo omwe amafanana nawo omwe amapanga chiwerengero cha kuwerenga motsatizana (Supplementary Fig. 2).
Motif Generation: Motengera mndandanda wa ma peptide apadera, laibulale idapangidwa yomwe ili ndi ma amino acid (aa) onse omwe angatheke monga momwe zilili pansipa.Njira iliyonse yotheka ya kutalika kwa 3 idachotsedwa ku peptide ndipo mawonekedwe ake osinthika adawonjezedwa pamodzi ndi laibulale yodziwika bwino yokhala ndi mawonekedwe onse (tripeptides).Ma library okhala ndi zobwerezabwereza kwambiri adatsatiridwa ndikuchotsedwanso.Kenako, pa tripeptide iliyonse mulaibulale ya motif, tinkafufuza ngati ilipo mu laibulale pogwiritsa ntchito zida zowerengera.Pachifukwa ichi, mafupipafupi a peptide omwe ali ndi motif tripeptide yopezeka amawonjezedwa ndikuperekedwa kwa motif mu laibulale ya motif ("nambala ya motifs").Chotsatira cha mtundu wa motif ndi mawonekedwe a mbali ziwiri omwe ali ndi zochitika zonse za tripeptides (motifs) ndi zikhulupiriro zawo, zomwe ndi chiwerengero cha mawerengedwe otsatizana omwe amabweretsa chiganizo chofanana pamene zowerengedwazo zisefedwa, kugawidwa, ndi kumasuliridwa.Metrics monga tafotokozera mwatsatanetsatane pamwambapa.
Kukhazikika kwa kuchuluka kwa ma motifs ndi ma scatterplots ofananira: Chiwerengero cha ma motifs pachitsanzo chilichonse chidasinthidwa kugwiritsa ntchito.
pomwe ni ndi chiwerengero cha zowerengedwa zomwe zili ndi mutu i.Chifukwa chake, vi imayimira kuchuluka kwa kuchuluka kwa zowerengera (kapena ma peptides) okhala ndi motif i mu zitsanzo.Makhalidwe a P a chiwerengero chosasinthika cha motifs adawerengedwa pogwiritsa ntchito mayeso enieni a Fisher.Ponena za ma correlogram a kuchuluka kwa zolinga, kulumikizana kwa Spearman kudawerengedwa pogwiritsa ntchito kuchuluka kwazolinga zomwe zili ndi R.
Kuti muwone zomwe zili mu amino acid pamalo aliwonse mulaibulale ya peptide, ma logogram 32, 33 (http://weblogo.threeplusone.com) adapangidwa.Choyamba, zomwe zili mu amino acid pamalo aliwonse a 12-mer peptide zimasungidwa mu 20 × 12 matrix.Kenako, ma peptides 1000 okhala ndi amino acid omwe ali ndi gawo limodzi pagawo lililonse amapangidwa motsatizana ndi fasta ndipo amaperekedwa ngati cholumikizira ku logo 3 ya intaneti, yomwe imapanga chiwonetsero chazithunzi za wachibale wa amino acid pamalo aliwonse.kwa laibulale ya peptide yopatsidwa.Kuti muwonetsetse ma dataset ambiri, mamapu otentha adapangidwa pogwiritsa ntchito chida chopangidwa mkati mwa R (biosHeatmap, phukusi la R lomwe silinatulutsidwebe).Ma dendrogram omwe amaperekedwa pamapu otentha adawerengedwa pogwiritsa ntchito njira yotsatizana ya Ward ndi metric yamtunda wa Euclidean.Pakusanthula kwa ziwerengero za zigoli za motif, mitengo ya P ya zigoli zosavomerezeka idawerengedwa pogwiritsa ntchito mayeso enieni a Fisher.P-values ​​pama dataset ena adawerengedwa mu R pogwiritsa ntchito t-test ya Student kapena ANOVA.
Magulu osankhidwa a phage ndi ma phage popanda kuyika adalowetsedwa kudzera m'mitsempha ya mchira (2 × 1010 phages / nyama mu 300 μl PBS).Mphindi khumi isanayambe kudzoza ndi kukonzanso kotsatira, nyama zomwezo zinalowetsedwa m'mitsempha ndi 100 μl ya DyLight594-yolembedwa lectin (Vector Laboratories Inc., DL-1177).Mphindi 60 pambuyo pa jekeseni wa phage, makoswe adagwiritsidwa ntchito pamtima ndi 50 ml PBS yotsatiridwa ndi 50 ml 4% PFA / PBS.Zitsanzo zaubongo zidasinthidwanso usiku umodzi mu 4% PFA/PBS ndikuviikidwa mu 30% sucrose usiku umodzi pa 4 ° C.Zitsanzo ndizozizira kwambiri mumsanganizo wa OCT.Kusanthula kwa Immunohistochemical kwa zitsanzo zachisanu kunkachitika pa kutentha kwa firiji pa 30 µm cryosections otsekedwa ndi 1% BSA ndi incubated ndi ma antibodies otchedwa polyclonal FITC motsutsana ndi T7 phage (Novus NB 600-376A) pa 4 ° C.Yalirani usiku wonse.Pomaliza, zigawozo zinatsukidwa nthawi za 3 ndi PBS ndikuyesedwa ndi microscope ya confocal laser (Leica TCS SP5).
Ma peptides onse okhala ndi chiyero chochepera 98% adapangidwa ndi GenScript USA, biotinylated ndi lyophilized.Biotin imamangidwa kudzera pa triple glycine spacer pa N-terminus.Yang'anani ma peptides onse pogwiritsa ntchito misa spectrometry.
Streptavidin (Sigma S0677) idasakanizidwa ndi 5-fold equimolar yochulukirapo ya biotinylated peptide, biotinylated BACE1 inhibitory peptide, kapena kuphatikiza (3:1 chiŵerengero) cha biotinylated BACE1 inhibitory peptide ndi BACE1 inhibitory peptide mu 5-10% ya PBS mu SO.1 ola firiji pamaso jekeseni.Streptavidin-conjugated peptides intravenously intravenously pa mlingo wa 10 mg / kg mu imodzi mwa mitsempha ya mchira ya makoswe ndi ubongo.
Kuchuluka kwa streptavidin-peptide complexes kunayesedwa ndi ELISA.Ma mbale a Nunc Maxisorp microtiter (Sigma) adakutidwa usiku wonse pa 4 ° C ndi 1.5 μg/ml mbewa anti-streptavidin antibody (Thermo, MA1-20011).Pambuyo kutsekereza (kutsekereza chotchinga: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) pa firiji kwa maola 2, sambani mbale ndi 0.05% Tween-20/PBS (kutsuka bafa) kwa 3 Second, CluSF100 ndi plasma 0 zotchinga: 0 plasma wosiyana: 0, CSF 1:115).Chophimbacho chinayikidwa usiku wonse pa 4 ° C ndi anti-antibody (1 μg / ml, anti-streptavidin-HRP, Novus NB120-7239).Pambuyo pa masitepe atatu otsuka, streptavidin inazindikirika ndi incubation mu TMB substrate solution (Roche) mpaka 20 min.Mukayimitsa kukula kwamtundu ndi 1M H2SO4, yesani kuyamwa pa 450 nm.
Ntchito ya streptavidin-peptide-BACE1 inhibitor complex inayesedwa ndi Aβ (1-40) ELISA molingana ndi ndondomeko ya wopanga (Wako, 294-64701).Mwachidule, zitsanzo za CSF zidasungunuka mu diluent wamba (1:23) ndikuyikidwa usiku wonse pa 4 ° C m'mbale za 96-zitsime zokutidwa ndi antibody ya BNT77.Pambuyo pa masitepe asanu ochapira, antibody ya HRP-conjugated BA27 inawonjezedwa ndi kukulungidwa kwa maola awiri pa 4° C., kutsatiridwa ndi masitepe asanu ochapira.Aβ(1–40) inazindikiridwa mwa kulowetsedwa mu njira ya TMB kwa mphindi 30 pa kutentha kwapakati.Pambuyo pakukula kwamtundu kuyimitsidwa ndi njira yoyimitsa, yesani kuyamwa pa 450 nm.Zitsanzo za plasma zinayikidwa pazitsulo zolimba za Aβ (1-40) ELISA.Plasma idawonjezeredwa ku 0.2% DEA (Sigma) m'mbale za 96-zitsime ndikuzikulungidwa kutentha kwa mphindi 30.Pambuyo kutsuka motsatizana mbale za SPE (Oasis, 186000679) ndi madzi ndi 100% methanol, zitsanzo za plasma zinawonjezeredwa ku mbale za SPE ndipo madzi onse anachotsedwa.Zitsanzo zidatsukidwa (poyamba ndi 5% methanol kenako 30% methanol) ndikutulutsidwa ndi 2% NH4OH/90% methanol.Pambuyo poyanika zowonongeka pa 55 ° C kwa 99 min pa nthawi zonse N2 panopa, zitsanzo zinachepetsedwa mu diluents wamba ndipo Aβ (1-40) anayesedwa monga tafotokozera pamwambapa.
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