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Katangar kwakwalwar jini da shingen kwakwalwar jini suna hana magungunan biotherapeutic kaiwa ga burinsu a cikin tsarin juyayi na tsakiya, don haka hana ingantaccen magani na cututtukan jijiya.Don gano sabbin masu jigilar kwakwalwa a cikin vivo, mun gabatar da ɗakin karatu na T7 phage peptide da jerin jini da aka tattara jini da ruwan cerebrospinal (CSF) ta amfani da babban ƙirar berayen sane.An inganta ƙayyadaddun ƙayyadaddun ƙa'idodin phage a cikin CSF bayan zagaye huɗu na zaɓi.Gwajin peptides na ɗan takara ɗaya ya nuna haɓaka fiye da ninki 1000 a cikin CSF.An tabbatar da bioactivity na isar da tsaka-tsakin peptide zuwa kwakwalwa ta hanyar raguwar 40% a matakin amyloid-β a cikin ruwan cerebrospinal ta amfani da mai hana BACE1 peptide wanda ke da alaƙa da gano peptide na sabon labari.Waɗannan sakamakon suna ba da shawarar cewa peptides da aka gano ta hanyoyin zaɓi na vivo phage na iya zama ababen hawa masu amfani don isar da tsarin macromolecules zuwa kwakwalwa tare da tasirin warkewa.
Binciken da aka yi niyya na tsarin jijiya na tsakiya (CNS) ya fi mayar da hankali kan gano ingantattun magunguna da wakilai waɗanda ke nuna kaddarorin CNS-targeting, tare da ƙarancin ƙoƙarin gano hanyoyin da ke fitar da isar da magunguna zuwa kwakwalwa.Wannan ya fara canzawa yanzu yayin da isar da magunguna, musamman manyan kwayoyin halitta, wani bangare ne na ci gaban magungunan neuroscience na zamani.Yanayin tsarin jijiya na tsakiya yana da kariya da kyau ta hanyar tsarin shinge na cerebrovascular, wanda ya ƙunshi shingen kwakwalwar jini (BBB) ​​da kuma shingen kwakwalwar jini (BCBB) 1, yana sa ya zama kalubale don isar da kwayoyi zuwa kwakwalwa1,2.An kiyasta cewa kusan dukkanin manyan magungunan ƙwayoyin cuta da fiye da kashi 98% na ƙananan ƙwayoyin ƙwayoyin cuta an kawar da su daga kwakwalwa3.Wannan shine dalilin da ya sa yana da matukar mahimmanci don gano sababbin tsarin jigilar kwakwalwa wanda ke ba da ingantaccen kuma takamaiman isar da magungunan warkewa zuwa CNS 4,5.Koyaya, BBB da BCSFB suma suna ba da kyakkyawar dama don isar da ƙwayoyi yayin da suke shiga kuma suna shiga duk sassan kwakwalwa ta hanyar ɗimbin jijiyoyi.Don haka, yunƙurin da ake yi na amfani da hanyoyin da ba sa ɓarna ba na isarwa zuwa kwakwalwa sun fi dogara ne akan tsarin jigilar mai karɓa (PMT) ta amfani da mai karɓar BBB6 na ƙarshe.Duk da mahimman ci gaba na kwanan nan ta amfani da hanyar mai karɓa na transferrin7,8, ana buƙatar ƙarin haɓaka sabbin tsarin bayarwa tare da ingantattun kaddarorin.Don wannan karshen, manufarmu ita ce gano peptides masu iya yin sulhu da sufuri na CSF, kamar yadda za a iya amfani da su bisa manufa don isar da macromolecules zuwa CNS ko don buɗe sababbin hanyoyin masu karɓa.Musamman, masu karɓa na musamman da masu sufuri na tsarin cerebrovascular (BBB da BSCFB) na iya zama maƙasudin maƙasudi don aiki da takamaiman isar da magungunan biotherapeutic.Ruwan Cerebrospinal (CSF) samfur ne na sirri na choroid plexus (CS) kuma yana cikin hulɗar kai tsaye tare da ruwan tsaka-tsakin kwakwalwa ta hanyar sararin subarachnoid da sararin samaniya4.Kwanan nan an nuna cewa ruwan subarachnoid cerebrospinal ruwa yana yaduwa da yawa a cikin interstitium na kwakwalwa9.Muna fatan samun damar sararin samaniya ta hanyar amfani da wannan sashin shigar subarachnoid ko kai tsaye ta cikin BBB.Don cimma wannan, mun aiwatar da dabarun zaɓin vivo phage mai ƙarfi wanda ya dace da gano peptides da aka ɗauka ta ɗayan waɗannan hanyoyi guda biyu.
Yanzu mun bayyana hanyar nunawa a cikin vivo phage mai lamba tare da samfurin CSF haɗe tare da babban tsarin kayan aiki (HTS) don saka idanu kan zagayen zaɓi na farko tare da mafi girman bambancin ɗakin karatu.An gudanar da bincike akan berayen da suka sane tare da dasa babban rijiyar (CM) cannula na dindindin don guje wa gurɓatar jini.Mahimmanci, wannan hanyar tana zaɓar duka-ƙwaƙwal-ƙira da peptides tare da ayyukan jigilar kayayyaki a cikin shingen cerebrovascular.Mun yi amfani da T7 phages saboda ƙananan girman su (~ 60 nm) 10 kuma sun ba da shawarar cewa sun dace da jigilar vesicles waɗanda ke ba da izinin hayewa ta hanyar salula na shingen endothelial da / ko epithelial-medulla.Bayan zagaye huɗu na panning, yawan phage sun keɓance suna nuna ƙarfi a cikin haɓakawar CSF da ƙungiyar microvessel na cerebral.Mahimmanci, mun sami damar tabbatar da bincikenmu ta hanyar nuna cewa mafi kyawun ɗan takara da aka haɗa da sinadarai peptides suna iya jigilar kayan furotin a cikin ruwan cerebrospinal.Na farko, an kafa tasirin magunguna na CNS ta hanyar haɗa manyan peptide na wucewa tare da mai hana BACE1 peptide.Baya ga nuna cewa a cikin dabarun tantance aikin vivo na iya gano sabbin peptides na jigilar kwakwalwa a matsayin ingantattun masu jigilar furotin, muna sa ran hanyoyin zaɓe iri ɗaya kuma su zama masu mahimmanci wajen gano sabbin hanyoyin jigilar kwakwalwa.
Dangane da raka'o'in ƙirƙira plaque (PFU), bayan matakin marufin phage, an ƙirƙira da ƙirƙira wani ɗakin karatu na bazuwar 12-mer linear T7 phage peptides tare da bambancin kusan 109 (duba Kayayyaki da Hanyoyi).Yana da mahimmanci a lura cewa mun yi nazarin wannan ɗakin karatu a hankali kafin in vivo panning.PCR na haɓaka samfuran ɗakin karatu na phage ta amfani da gyare-gyaren gyare-gyare sun haifar da amplicons waɗanda suka dace da HTS kai tsaye (Ƙarin Hoto 1a).Saboda a) HTS11 kurakurai na jerin, b) tasiri a kan ingancin na'urorin (NNK) 1-12, da kuma c) kasancewar nau'in daji (wt) phage (skeleton sakawa) a cikin ɗakin karatu na jiran aiki, an aiwatar da tsarin tacewa don cire kawai bayanan jeri da aka tabbatar (Ƙarin Fig. 1b).Waɗannan matakan tacewa sun shafi duk dakunan karatu na HTS.Don daidaitaccen ɗakin karatu, an sami jimlar karatun 233,868, waɗanda 39% suka wuce ƙa'idodin tacewa kuma an yi amfani da su don nazarin ɗakin karatu da zaɓi don zagaye na gaba (Ƙarin Hoto 1c-e).Abubuwan da aka karanta sun fi yawa na 3 tushe nau'i-nau'i a tsayi tare da kololuwa a 36 nucleotides (Ƙarin Hoton 1c), yana tabbatar da ƙirar ɗakin karatu (NNK) 1-12.Musamman, kusan kashi 11% na membobin ɗakin karatu sun ƙunshi nau'in daji mai girma 12 (wt) kashin baya PAGISRELVDKL, kuma kusan rabin jerin (49%) sun ƙunshi shigarwa ko gogewa.HTS na ɗakin karatu na ɗakin karatu ya tabbatar da babban bambancin peptides a cikin ɗakin karatu: fiye da 81% na peptide jerin an samo sau ɗaya kawai kuma kawai 1.5% ya faru a cikin ≥4 kofe (Ƙarin Hoto 2a).Mitar amino acid (aa) a duk matsayi 12 a cikin repertoire sun dace sosai tare da mitocin da ake tsammanin adadin codons da aka samu ta hanyar lalatar NKK repertoire (Ƙarin Hoto 2b).Mitar da aka lura na ragowar aa da waɗannan abubuwan da aka saka suka yi daidai da mitar ƙididdigewa (r = 0.893) (Ƙarin Hoton 2c).Shirye-shiryen dakunan karatu na phage don allura sun haɗa da matakan haɓakawa da cirewar endotoxin.An nuna wannan a baya yana iya rage bambance-bambancen ɗakunan karatu na phage12,13.Sabili da haka, mun tsara ɗakin karatu na farantin karfe wanda aka yi wa cirewar endotoxin kuma muka kwatanta shi da ɗakin karatu na asali don kimanta mitar AA.An lura da alaƙa mai ƙarfi (r = 0.995) tsakanin tafkin asali da kuma wurin da aka haɓaka da kuma tsarkakewa (Ƙarin Hoton 2d), yana nuna cewa gasa tsakanin clones da aka haɓaka akan faranti ta amfani da T7 phage ba ta haifar da babban bambanci ba.Wannan kwatancen ya dogara ne akan yawan abubuwan motsa jiki na tripeptide a cikin kowane ɗakin karatu, tun da bambancin ɗakunan karatu (~ 109) ba za a iya kama su gaba ɗaya ko da tare da HTS ba.Binciken akai-akai na aa a kowane matsayi ya nuna ƙananan ra'ayi na dogara ga matsayi a cikin matsayi uku na ƙarshe na rubutun da aka shigar (Ƙarin Hoton 2e).A ƙarshe, mun yanke shawarar cewa inganci da bambance-bambancen ɗakin karatu sun kasance abin karɓa kuma ƙananan canje-canje a cikin bambance-bambancen an lura da su saboda haɓakawa da shirye-shiryen ɗakunan karatu na phage tsakanin zagaye da yawa na zaɓi.
Serial cerebrospinal ruwa samfurin za a iya yi ta hanyar tiyata dasa cannula a cikin CM na berayen sane don sauƙaƙe ganewar T7 phage da aka allura ta cikin jini (iv) ta hanyar BBB da / ko BCSFB (Fig. 1a-b).Mun yi amfani da makamai masu zaman kansu guda biyu (makamai A da B) a cikin zagaye uku na farko na zaɓin vivo (Fig. 1c).A hankali mun ƙara daɗaɗɗen zaɓi ta hanyar rage jimillar adadin phage da aka gabatar a zagaye uku na farko na zaɓi.Don zagaye na huɗu na panning, mun haɗa samfurori daga rassan A da B kuma mun yi ƙarin zaɓuɓɓuka masu zaman kansu guda uku.Don nazarin in vivo kaddarorin T7 phage barbashi a cikin wannan samfurin, nau'in phage na daji (PAGISRELVDKL babban sakawa) an allura a cikin berayen ta hanyar jijiyar wutsiya.Farfadowa na phages daga ruwan cerebrospinal da jini a wurare daban-daban na lokaci ya nuna cewa ƙananan ƙananan T7 icosahedral phages suna da saurin cirewa na farko daga sashin jini (Ƙarin Hoton 3).Dangane da titers da aka gudanar da adadin jinin berayen, mun ƙididdige cewa kusan 1% kawai wt.An gano phage daga maganin da aka yi amfani da shi a cikin jini mintuna 10 bayan allurar ta jijiya.Bayan wannan raguwar saurin farko na farko, an auna matakin farko a hankali tare da rabin rayuwa na mintuna 27.7.Mahimmanci, ƙananan phages ne kawai aka dawo dasu daga sashin CSF, yana nuna ƙarancin baya don ƙaura na nau'in phage na daji zuwa sashin CSF (Ƙarin Hoton 3).A matsakaita, kawai game da 1 x 10-3% titers na T7 phage a cikin jini da 4 x 10-8% na farkon infused phages an gano su a cikin ruwan cerebrospinal a duk tsawon lokacin samfur (0-250 min).Musamman ma, rabin rayuwa (minti 25.7) na nau'in phage na daji a cikin ruwan cerebrospinal yayi kama da wanda aka gani a cikin jini.Waɗannan bayanan sun nuna cewa shingen da ke raba sashin CSF daga jini yana nan a cikin berayen CM-cannulated, yana ba da damar zaɓin ɗakin karatu na phage a cikin vivo don gano clones waɗanda ake ɗauka da sauri daga jini zuwa sashin CSF.
(a) Ƙirƙiri hanyar sake yin samfurin ruwa na cerebrospinal (CSF) daga babban tafkin.(b) Zane yana nuna wurin salon salula na shinge na tsakiya (CNS) da tsarin zaɓin da aka yi amfani da shi don gano peptides da ke ƙetare shingen kwakwalwar jini (BBB) ​​da kuma shingen kwakwalwar jini.(c) A cikin vivo phage nunin taswirar nuni.A kowane zagaye na zaɓi, an yi allurar phages (masu gano dabbobi a cikin kibiyoyi) ta cikin jini.Sauran rassa biyu masu zaman kansu (A, B) ana kiyaye su daban har zuwa zagaye na 4 na zaɓi.Don zagaye na 3 da 4, kowane phage clone da aka samo daga CSF an jera shi da hannu.(d) Kinetics na phage ware daga jini (ja da'ira) da kuma cerebrospinal ruwa (kore triangles) a lokacin zagaye na farko na zabi a cikin biyu cannulated berayen bayan jijiya allura na T7 peptide library (2 x 1012 phages / dabba).murabba'ai masu shuɗi suna nuna matsakaicin matsakaicin matakin farko na phage a cikin jini, ƙididdiga daga adadin phage ɗin allura, la'akari da jimlar adadin jini.Baƙaƙen murabba'i suna nuna madaidaicin layin y wanda aka cire daga ma'aunin phage na jini.e,fAn nuna adadin motifs da aka samo a cikin karatun 1000.Mahimmanci (p <0.001) ingantattun motifs ana yiwa alama da ɗigo ja.(e) Rarrabuwar daidaitawa da kwatanta mitar dangi na motsin tripeptide na ɗakin karatu na allura tare da phage da aka samo daga jini daga dabbobi #1.1 da #1.2.(f) Tsarin watsawa na daidaitawa yana kwatanta mitocin dangi na dabbobin phage tripeptide motifs # 1.1 da # 1.2 ware a cikin jini da ruwan cerebrospinal.(g, h) Jerin jerin wakilcin phage da aka wadatar da jini (g) tare da alluran dakunan karatu da phage da aka wadatar a CSF (h) da jini bayan zagaye na in vivo a cikin dabbobin biyu.Girman lambar harafi ɗaya yana nuna sau nawa amino acid ke faruwa a wannan matsayi.Green = iyakacin duniya, purple = tsaka tsaki, blue = asali, ja = acidic da baki = hydrophobic amino acid.Hoto na 1a, b Eduard Urich ne ya tsara shi kuma ya samar da shi.
Mun allura ɗakin karatu na phage peptide cikin berayen kayan aikin CM guda biyu (clades A da B) da keɓaɓɓen phage daga ruwan cerebrospinal da jini (Hoto 1d).Farkon saurin share ɗakin ɗakin karatu bai kasance mai faɗi sosai ba idan aka kwatanta da nau'in phage na daji.Matsakaicin rabin rayuwar ɗakin karatun allura a cikin dabbobin biyu shine mintuna 24.8 cikin jini, kama da nau'in phage na daji, da mintuna 38.5 a cikin CSF.Samfuran phage na jini da na cerebrospinal daga kowace dabba an yi su zuwa HTS kuma an bincika duk peptides da aka gano don kasancewar ɗan gajeren motsin tripeptide.An zaɓi motifs na Tripeptide saboda suna samar da ƙaramin tushe don ƙirƙirar tsari da hulɗar peptide-protein14,15.Mun sami kyakkyawar alaƙa a cikin rarraba motifs tsakanin injected phage library da clones da aka samo daga jinin dabbobin biyu (Fig. 1e).Bayanan sun nuna cewa abubuwan da ke cikin ɗakin karatu an wadatar da su ne kawai a cikin sashin jini.An kara nazarin mitocin Amino acid da jerin yarjejeniya a kowane matsayi ta amfani da daidaitawar software na Weblogo16.Abin sha'awa, mun sami wadata mai ƙarfi a cikin ragowar glycine na jini (Fig. 1g).Lokacin da aka kwatanta jini tare da clones da aka zaɓa daga CSF, an lura da zaɓi mai ƙarfi da wasu zaɓe na motifs (Fig. 1f), kuma wasu amino acid sun fi dacewa a wurare da aka ƙayyade a cikin 12-memba (Fig. 1h).Musamman ma, kowane ɗayan dabbobi ya bambanta sosai a cikin ruwan cerebrospinal, yayin da an lura da wadatar jini na glycine a cikin dabbobin biyu (Ƙarin Hoton 4a-j).Bayan stringent tace bayanan jeri a cikin ruwan cerebrospinal na dabbobi #1.1 da #1.2, an sami jimlar 964 da 420 peptides na musamman na 12-mer (Ƙarin Hoton 1d-e).An haɓaka keɓaɓɓen clones na phage kuma an ƙaddamar da zagaye na biyu na zaɓi na vivo.Phage da aka samo daga zagaye na biyu na zaɓi an yi amfani da HTS a cikin kowane dabba kuma an yi amfani da duk peptides da aka gano a matsayin shigarwa zuwa tsarin ƙaddamar da ƙaddamarwa don nazarin abubuwan da suka faru na tripeptide motifs (Fig. 2a, b, ef).Idan aka kwatanta da sake zagayowar farko na phage da aka dawo daga CSF, mun lura da ƙarin zaɓi da zaɓe na yawancin motifs a cikin CSF a cikin rassan A da B (Fig. 2).An yi amfani da algorithm na gano hanyar sadarwa don tantance ko suna wakiltar alamu daban-daban na daidaitattun jeri.An lura da kamanni mai kama da juna tsakanin nau'ikan nau'ikan nau'ikan 12 da CSF ta dawo dasu a madadin clade A (Fig. 2c, d) da clade B (Fig. 2g, h).Binciken da aka tattara a cikin kowane reshe ya bayyana bayanan zaɓuɓɓuka daban-daban don 12-mer peptides (Ƙarin Hotuna 5c, d) da karuwa a cikin CSF / jini na jini a kan lokaci don clones da aka tattara bayan zagaye na biyu na zaɓi idan aka kwatanta da zagaye na farko na zaɓi (Ƙarin 5e).).
Haɓaka motifs da peptides a cikin ruwan cerebrospinal ta zagaye biyu jere na in vivo aikin nunin phage.
All cerebrospinal fluid phages dawo dasu daga zagaye na farko na kowace dabba (dabbobi #1.1 da #1.2) an tattara su, an ƙara su, HT-sequenced da sake yin allura tare (2 x 1010 phages / dabba) 2 SM cannulated berayen (#1.1 → #).2.1 da 2.2, 1.2 → 2.3 da 2.4).(a,b,e,f) Matsakaicin rarrabuwar kawuna kwatankwacin dangin mitar abubuwan motsa jiki na duk phages da aka samu na CSF a zagaye na farko da na biyu.Dangantakar mitar da rarraba motifs masu wakiltar duk yuwuwar haɗuwar tripeptides da aka samu a cikin peptides a cikin bangarorin biyu.An nuna adadin motifs da aka samo a cikin karatun 1000.Motifs waɗanda aka zaɓa masu mahimmanci (p <0.001) waɗanda aka zaɓa ko aka ware su a ɗayan ɗakunan karatu waɗanda aka kwatanta ana haskaka su da ɗigo ja.(c, d, g, h) Tambarin jeri na wakilcin duk dogon jerin amino acid 12 masu arzikin CSF dangane da zagaye na 2 da 1 na zabin vivo.Girman lambar harafi ɗaya yana nuna sau nawa amino acid ke faruwa a wannan matsayi.Don wakiltar tambarin, ana kwatanta adadin jerin CSF da aka samo daga kowane ɗayan dabbobi tsakanin zaɓe biyu kuma ana nuna jerin wadatattun abubuwa a zagaye na biyu: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 da (h) #1.2–#2.4.Mafi wadatar amino acid a wani matsayi a cikin (c, d) dabbobi a'a.2.1 kuma babu.2.2 ko (g, h) a cikin dabbobi no.2.3 kuma ba.2.4 ana nunawa a launi.Green = iyakacin duniya, purple = tsaka tsaki, blue = asali, ja = acidic da baki = hydrophobic amino acid.
Bayan zagaye na uku na zaɓi, mun gano 124 na musamman peptide jerin (# 3.1 da # 3.2) daga 332 CSF-reconstituted phage clones ware daga dabbobi biyu (Ƙarin Hoto 6a).Jerin LGSVS (18.7%) yana da mafi girman girman dangi, sannan kuma abubuwan shigar da nau'in daji PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), da SARGSWREIVSLS (2.2%).A zagaye na huɗu na ƙarshe, mun haɗu da rassa guda biyu waɗanda aka zaɓa daga dabbobi daban-daban (Fig. 1c).Daga cikin 925 jerin phage clones da aka dawo dasu daga CSF, a cikin zagaye na hudu mun sami nau'ikan peptide na musamman na 64 (Ƙarin Hoton 6b), wanda adadin dangi na nau'in phage na daji ya ragu zuwa 0.8%.Abubuwan da aka fi sani da CSF a zagaye na huɗu sune LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) da RLSSVDS DLSGC%) (3,%)).Tsawon kewayon peptides ɗin da aka zaɓa ya kasance saboda shigarwa / gogewa na nucleotide ko dodon tasha da wuri a cikin ma'ajin ɗakin karatu lokacin amfani da codons masu lalacewa don ƙirar ɗakin karatu na NNK.Codons na tsayawa da wuri suna haifar da gajerun peptides kuma an zaɓa saboda sun ƙunshi ingantaccen aa motif.Dogayen peptides na iya haifar da sakawa/sharewa a cikin madaidaitan ɗakunan karatu na roba.Wannan yana sanya codon tasha da aka ƙera a waje da firam kuma yana karanta shi har sai sabon codon tasha ya bayyana a ƙasa.Gabaɗaya, mun ƙididdige abubuwan haɓakawa don duk zagaye huɗun zaɓi ta hanyar kwatanta bayanan shigarwa tare da bayanan fitar da samfur.Don zagaye na farko na nunawa, mun yi amfani da nau'in phage titers a matsayin bayanan baya da ba takamaiman ba.Abin sha'awa, zaɓin phage mara kyau ya kasance mai ƙarfi sosai a cikin sake zagayowar CSF na farko, amma ba a cikin jini ba (Fig. 3a), wanda zai iya kasancewa saboda ƙarancin yuwuwar watsawa mara kyau na yawancin mambobi na ɗakin karatu na peptide a cikin ɗakin CSF ko phages dangi sun fi dacewa da kiyayewa ko cire su daga cikin jini fiye da bacteriophages.Duk da haka, a cikin zagaye na biyu na panning, an lura da zaɓi mai karfi na phages a cikin CSF a cikin nau'i biyu, yana nuna cewa zagaye na baya ya wadata a cikin phages da ke nuna peptides wanda ke inganta haɓakar CSF (Fig. 3a).Bugu da ƙari, ba tare da haɓakar jini mai mahimmanci ba.Hakanan a cikin zagaye na uku da na huɗu, phage clones sun inganta sosai a cikin CSF.Idan aka kwatanta mitar dangi na kowane nau'in peptide na musamman tsakanin zagaye biyu na ƙarshe na zaɓi, mun gano cewa jerin sun fi wadatar a zagaye na huɗu na zaɓi (Fig. 3b).An fitar da jimlar 931 tripeptide motifs daga duk nau'ikan peptide na musamman guda 64 ta amfani da duka hanyoyin peptide.Abubuwan da suka fi dacewa a zagaye na huɗu an yi nazari sosai don bayanan haɓakar su a duk zagaye idan aka kwatanta da ɗakin karatu na allura (yankewa: 10% haɓakawa) (Ƙarin Hoto 6c).Tsarin zaɓi na gabaɗaya ya nuna cewa yawancin dalilan da aka yi nazari sun wadatar a duk zagayen da suka gabata na rassan zaɓin biyu.Koyaya, wasu motifs (misali SGL, VSG, LGS GSV) galibi sun kasance daga madadin clade A, yayin da wasu (misali FGW, RTN, WGF, NTR) aka wadatar a madadin clade B.
Tabbatar da jigilar CSF na peptides masu nuna wadatar phage da CSF da peptides jagoran biotinylated waɗanda aka haɗa zuwa streptavidin payloads.
(a) Matsakaicin wadatar da aka ƙididdige su a cikin duk zagaye huɗu (R1-R4) dangane da allura (shigarwar = I) titers phage (PFU) da ƙayyadaddun titers na CSF phage (fitarwa = O).Abubuwan haɓakawa na zagaye uku na ƙarshe (R2-R4) an ƙididdige su ta hanyar kwatanta da zagaye na baya da zagaye na farko (R1) tare da bayanan nauyi.Buɗe sanduna ruwan cerebrospinal ne, sanduna masu inuwa sune plasma.(***p<0.001, bisa ga t-gwajin ɗalibi).(b) Jerin mafi yawan phage peptides, wanda aka jera bisa ga danginsu zuwa duk phages da aka tattara a cikin CSF bayan zagaye na 4 na zaɓi.An ba da fifikon nau'ikan clones na phage guda shida a cikin launi, ƙididdiga da abubuwan haɓaka su tsakanin zagaye na 3 da 4 na zaɓi (insets).(c,d) Mafi yawan wadatattun phage clones guda shida, phage mara komai da ɗakunan karatu na peptide na iyaye daga zagaye na 4 an yi nazari akai-akai a cikin samfurin samfurin CSF.An tattara samfuran CSF da jini a wuraren da aka nuna.(c) Daidaitaccen adadin 6 ɗan takarar phage clones (2 x 1010 phages / dabbobi), phages mara kyau (#1779) (2 x 1010 phages / dabbobi) da ɗakunan karatu na peptide na phage (2 x 1012 phages / dabbobi) Allurar aƙalla 3 CM ana gudanar da shi ta hanyar dabbobi daban-daban.Ana nuna magunguna na CSF na kowane allurar phage clone da ɗakin karatu na peptide na tsawon lokaci.(d) yana nuna matsakaicin adadin CSF/jini don duk phages/ml da aka dawo dasu akan lokacin samfur.(e) peptides jagoran roba guda hudu da kuma sarrafawa guda ɗaya an haɗa su tare da biotin zuwa streptavidin ta hanyar N-terminus (nuni na tetramer) tare da allura (wutsiya iv, 10 mg streptavidin / kg).Aƙalla berayen da ke cikin ciki (N = 3).).An tattara samfuran CSF a wuraren da aka nuna kuma an auna matakan streptavidin ta CSF anti-streptavidin ELISA (nd = ba a gano ba).(* p <0.05, ** p <0.01, *** p<0.001, dangane da gwajin ANOVA).(f) Kwatanta jerin amino acid na mafi wadatar phage peptide clone #2002 (purple) tare da sauran zaɓaɓɓun phage peptide clones daga zagaye na 4 na zaɓi.Iri ɗaya da makamantansu gutsuttsun amino acid suna da launi.
Daga cikin dukkanin abubuwan da aka wadatar a cikin zagaye na hudu (Fig. 3b), an zaɓi clones 'yan takara shida don ƙarin bincike na mutum a cikin samfurin CSF.Daidaitaccen adadin phage na ɗan takara shida, phage mara kyau (babu sakawa) da ɗakunan karatu na peptide prophage an allura su cikin dabbobin CM guda uku masu gwangwani, kuma an ƙaddara pharmacokinetics a cikin CSF (Fig. 3c) da jini (Ƙarin Fig. 7).Duk phage clones da aka gwada sun yi niyya ga sashin CSF a matakin 10-1000 mafi girma fiye da na phage mara komai (#1779).Misali, clones #2020 da #2077 suna da kusan sau 1000 mafi girma CSF titers fiye da sarrafa phage.Bayanan pharmacokinetic na kowane peptide da aka zaɓa ya bambanta, amma dukkansu suna da babban ƙarfin homing na CSF.Mun lura da raguwa akai-akai akan lokaci don clones #1903 da #2011, yayin da clones #2077, #2002 da #2009 karuwa a cikin mintuna 10 na farko na iya nuna jigilar aiki amma yana buƙatar tabbatarwa.Clones #2020, #2002, da #2077 sun daidaita a manyan matakan, yayin da CSF maida hankali na clone #2009 a hankali ya ragu bayan haɓakar farko.Sa'an nan kuma muka kwatanta mitar dangi na kowane ɗan takarar CSF tare da ƙaddamarwar jini (Fig. 3d).Matsakaicin ma'anar ma'anar kowane ɗan takarar CSF tare da ma'aunin jini a duk lokutan samfur ya nuna cewa uku daga cikin 'yan takara shida sun sami wadata sosai a cikin CSF na jini.Abin sha'awa, clone #2077 ya nuna kwanciyar hankali na jini (Ƙarin Hoto 7).Don tabbatar da cewa peptides da kansu suna da ikon yin jigilar kaya ban da phage barbashi a cikin rukunin CSF, mun haɗa peptides jagororin guda huɗu waɗanda aka samo su tare da biotin a cikin N-terminus inda peptides ke haɗe zuwa ƙwayar phage.Biotinylated peptides (las. 2002, 2009, 2020 da 2077) an haɗa su tare da streptavidin (SA) don samun nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan juzu'i da ɗan kwaikwayi phage geometry.Wannan tsarin kuma ya ba mu damar auna bayyanar SA a cikin jini da ruwan cerebrospinal azaman peptides furotin masu jigilar kaya.Mahimmanci, bayanan phage sau da yawa ana iya sake yin su lokacin da aka yi amfani da peptides na roba a cikin wannan tsarin SA-conjugated (Fig. 3e).Abubuwan peptides da aka ruɗe suna da ƙarancin bayyanar farko da saurin izinin CSF tare da matakan da ba a iya ganowa a cikin sa'o'i 48.Don samun haske cikin hanyoyin isar da waɗannan peptide phage clones a cikin sararin CSF, mun bincikar ganowar kowane phage peptide hits ta amfani da immunohistochemistry (IHC) don gano ƙwayoyin phage kai tsaye 1 sa'a bayan allurar intravenous a cikin vivo.Musamman, ana iya gano clones #2002, #2077, da #2009 ta hanyar ƙaƙƙarfan tabo a cikin capillaries na kwakwalwa, yayin da ba a gano phage (#1779) da clone #2020 ba (Ƙarin Hoto 8).Wannan yana nuna cewa waɗannan peptides suna ba da gudummawa ga tasiri akan kwakwalwa daidai ta hanyar ketare BBB.Ana buƙatar ƙarin cikakken bincike don gwada wannan hasashe, kamar yadda hanyar BCFB kuma za ta iya shiga.Lokacin kwatanta jerin amino acid na clone mafi wadatarwa (#2002) tare da wasu zaɓaɓɓun peptides, an lura cewa wasu daga cikinsu suna da haɓakar amino acid iri ɗaya, wanda zai iya nuna irin tsarin jigilar kayayyaki (Fig. 3f).
Saboda bayanin martaba na musamman na plasma da kuma karuwa mai yawa a cikin CSF a tsawon lokaci, an sake bincika clone na phage # 2077 a cikin tsawon sa'o'i 48 kuma ya iya sake haifar da karuwa mai sauri a CSF da aka lura tare da ci gaba da matakan SA (Fig. 4a).Game da wasu nau'ikan clones na phage da aka gano, #2077 ya yi kyau sosai don capillaries na kwakwalwa kuma ya nuna babban colocalization tare da lectin mai alamar capillary lokacin da aka duba shi a mafi girman ƙuduri kuma wataƙila wasu tabo a cikin sararin samaniya (Hoto 4b).Don bincika ko za a iya samun tasirin maganin magungunan peptide a cikin CNS, mun yi gwaji wanda nau'ikan biotinylated na i) #2077 transit peptide da ii) peptide mai hana BACE1 an gauraye shi da SA a cikin rabo biyu daban-daban.Don haɗuwa ɗaya mun yi amfani da mai hana BACE1 peptide kawai kuma ga ɗayan mun yi amfani da 1: 3 rabo na BACE1 peptide inhibitor zuwa #2077 peptide.Duk samfuran biyu an gudanar da su ta cikin hanji kuma an auna jini da matakan ruwa na cerebrospinal na beta-amyloid peptide 40 (Abeta40) akan lokaci.An auna Abeta40 a cikin CSF yayin da yake nuna hana BACE1 a cikin kwakwalwar kwakwalwa.Kamar yadda aka zata, duka rukunin biyu sun rage matakan jini na Abeta40 sosai (Fig. 4c, d).Koyaya, kawai samfuran da ke ɗauke da cakuda peptide no.2077 da mai hanawa na BACE1 peptide conjugated zuwa SA ya haifar da raguwa mai yawa a Abeta40 a cikin ruwa na cerebrospinal (Fig. 4c).Bayanan sun nuna cewa peptide no.2077 yana iya ɗaukar furotin 60 kDa SA a cikin CNS kuma yana haifar da tasirin magunguna tare da masu hana SA-conjugated na peptide BACE1.
(a) allurar Clonal (2 × 10 phages / dabba) na T7 phage yana nuna bayanan martaba na pharmacokinetic na dogon lokaci na CSF peptide #2077 (RLSSVDSDLSGC) da phage mai sarrafawa mara allura (#1779) a cikin aƙalla uku CM-intubated berayen.(b) Hoton microscopic na microvessels na wakilai na cortical microvessels a cikin berayen da aka yi wa allurar phage (2 × 10 10 phages / dabba) suna nuna rashin daidaituwa na peptide #2077 da tasoshin (lectin).An gudanar da waɗannan clones na phage zuwa berayen 3 kuma an ba su izinin yawo na awa 1 kafin turare.An raba sassan kwakwalwa kuma an lalata su da polyclonal FITC antibodies a kan T7 phage capsid.Minti 10 kafin turawa da gyarawa na gaba, DyLight594-labeled lectin an gudanar da shi ta cikin jini.Hotunan masu walƙiya suna nuna alamar lectin (ja) na gefen haske na microvessels da phages (kore) a cikin lumen na capillaries da ƙwayoyin kwakwalwa na perivascular.Ma'aunin ma'auni yayi daidai da 10µm.(c, d) Biotinylated BACE1 inhibitory peptide kadai ko a hade tare da biotinylated transit peptide #2077 an haɗe shi zuwa streptavidin wanda ya biyo bayan allurar a cikin jini na akalla berayen CM guda uku cannulated (10 mg streptavidin/kg).BACE1 peptide inhibitor-matsakaicin raguwa a cikin Aβ40 an auna ta Aβ1-40 ELISA a cikin jini (ja) da ruwan cerebrospinal (orange) a wuraren da aka nuna.Don ƙarin haske, ana zana layi mai dige-dige akan jadawali a sikelin 100%.(c) Rage yawan kashi a cikin Aβ40 a cikin jini (jajayen triangles) da ruwa na cerebrospinal (triangles orange) a cikin berayen da aka yi tare da streptavidin da aka haɗa zuwa peptide na wucewa #2077 da BACE1 inhibitory peptide a cikin rabo na 3: 1.(d) Rage kashi cikin jini Aβ40 (janye da'ira) da ruwan cerebrospinal (orange da'irar) na berayen da aka yi da streptavidin tare da peptide mai hana BACE1 kawai.Matsakaicin Aβ a cikin kulawa shine 420 pg / ml (daidaituwar daidaitaccen = 101 pg / ml).
An yi nasarar amfani da nunin faifai a fagage da dama na binciken nazarin halittu17.An yi amfani da wannan hanyar don nazarin bambancin bambancin jini na jijiyoyi18,19 da kuma nazarin da ake nufi da tasoshin kwakwalwa20,21,22,23,24,25,26.A cikin wannan binciken, mun tsawaita aikace-aikacen wannan hanyar zaɓi ba kawai don gano kai tsaye na peptides da ke niyya tasoshin kwakwalwa ba, har ma da gano 'yan takarar da ke da kayan sufuri masu aiki don ketare shingen kwakwalwar jini.Yanzu mun bayyana haɓakar hanyar zaɓin in vivo a cikin berayen da ke cikin CM kuma muna nuna yuwuwar sa don gano peptides tare da kaddarorin homing na CSF.Yin amfani da T7 phage yana nuna ɗakin karatu na peptides bazuwar 12-mer, mun sami damar nuna cewa T7 phage yana da ƙananan isa (kimanin 60 nm a diamita) 10 don daidaitawa zuwa shingen kwakwalwar jini, ta haka kai tsaye ke ƙetare shingen kwakwalwar jini ko choroid plexus.Mun lura cewa girbin CSF daga berayen CM mai gwangwani shine ingantaccen sarrafawa a cikin hanyar tantance aikin vivo, kuma cewa phage ɗin da aka fitar ba wai kawai an ɗaure shi da vasculature ba amma yana aiki azaman mai jigilar kaya a kan shingen kwakwalwar jini.Bugu da ƙari, ta hanyar tattara jini lokaci guda da kuma amfani da HTS zuwa CSF da phages da aka samo daga jini, mun tabbatar da cewa zaɓin mu na CSF bai tasiri ta hanyar wadatar jini ko dacewa don faɗaɗa tsakanin zagaye na zaɓi ba.Koyaya, sashin jini yana cikin tsarin zaɓin, tunda phages masu iya isa sashin CSF dole ne su rayu kuma suyi yawo cikin jini har tsawon lokaci don wadatar da kansu a cikin kwakwalwa.Domin fitar da ingantaccen bayanin jeri daga danyen bayanan HTS, mun aiwatar da matattarar da suka dace da takamaiman kurakuran tsarin dandamali a cikin aikin bincike.Ta hanyar haɗa sigogin motsi a cikin hanyar nunawa, mun tabbatar da saurin pharmacokinetics na nau'in nau'in nau'in T7 na daji (t½ ~ 28 min) a cikin jini24, 27, 28 kuma sun ƙaddara rabin rayuwarsu a cikin ruwan cerebrospinal (t½ ~ 26 min) a minti daya).Duk da irin bayanan pharmacokinetic iri ɗaya a cikin jini da CSF, kawai 0.001% na maida hankali na jini na phage za a iya gano shi a cikin CSF, yana nuna ƙarancin motsi na phage na nau'in daji na T7 a cikin shingen kwakwalwar jini.Wannan aikin yana nuna mahimmancin zaɓin zagaye na farko lokacin amfani da dabarun panning vivo, musamman ga tsarin phage waɗanda ke saurin kawar da su daga wurare dabam dabam, yayin da ƙananan clones ke iya isa sashin CNS.Don haka, a zagaye na farko, raguwar bambance-bambancen ɗakin karatu ya yi yawa sosai, domin kawai an tattara iyakataccen adadin clones a ƙarshe a cikin wannan ƙirar CSF mai tsananin gaske.Wannan in vivo dabarun panning ya haɗa da matakai masu yawa na zaɓi kamar tarawa mai aiki a cikin sashin CSF, tsirar clone a cikin sashin jini, da saurin cire T7 phage clones daga jini a cikin mintuna na 10 na farko (Fig. 1d da Ƙarin Hoto 4M).).Don haka, bayan zagaye na farko, an gano nau'ikan nau'ikan nau'ikan phage daban-daban a cikin CSF, kodayake an yi amfani da tafkin farko ɗaya don kowane dabbobi.Wannan yana ba da shawarar matakan zaɓi masu yawa don ɗakunan karatu na tushen tare da adadi mai yawa na membobin ɗakin karatu suna haifar da raguwa mai yawa a cikin bambancin.Sabili da haka, abubuwan da bazuwar za su zama wani ɓangare na tsarin zaɓi na farko, suna tasiri sosai ga sakamakon.Wataƙila yawancin clones a cikin ɗakin karatu na asali suna da kusancin haɓakar CSF iri ɗaya.Duk da haka, ko da a ƙarƙashin yanayin gwaji iri ɗaya, sakamakon zaɓi na iya bambanta saboda ƙananan adadin kowane nau'i na musamman a cikin tafkin farko.
Abubuwan da aka wadatar a cikin CSF sun bambanta da waɗanda ke cikin jini.Abin sha'awa, mun lura da motsi na farko zuwa ga peptides mai arzikin glycine a cikin jinin kowane dabba.(Fig. 1g, Ƙarin Hotuna 4e, 4f).Fage mai ƙunshe da peptides glycine na iya zama mafi kwanciyar hankali kuma ba za a iya fitar da shi daga wurare dabam dabam ba.Duk da haka, waɗannan peptides masu arziki na glycine ba a gano su a cikin samfurori na ruwa na cerebrospinal ba, suna nuna cewa ɗakunan karatu da aka yi amfani da su sun wuce ta hanyoyi guda biyu daban-daban: daya a cikin jini kuma wani ya ba da izinin tarawa a cikin ruwa na cerebrospinal.CSF-inrich clones wanda ya haifar da zagaye na huɗu na zaɓi an gwada su sosai.Kusan dukkanin clones ɗin da aka gwada daidaiku an tabbatar sun wadatar da su a cikin CSF idan aka kwatanta da phage na sarari.An bincika bugun peptide guda ɗaya (#2077) daki-daki.Ya nuna tsawon rabin rayuwar plasma idan aka kwatanta da sauran hits (Hoto 3d da Ƙarin Hoto 7), kuma abin sha'awa, wannan peptide ya ƙunshi ragowar cysteine ​​a C-terminus.Kwanan nan an nuna cewa ƙari na cysteine ​​​​to peptides na iya inganta kayan aikin su na pharmacokinetic ta hanyar ɗaure zuwa albumin 29.Wannan a halin yanzu ba a san shi don peptide #2077 ba kuma yana buƙatar ƙarin nazari.Wasu peptides sun nuna valence-dogara a cikin wadatar CSF (bayanan da ba a nuna ba), wanda ƙila yana da alaƙa da nunin joometry na saman T7 capsid.Tsarin T7 da muka yi amfani da shi ya nuna kwafin 5-15 na kowane peptide kowane ɓangarorin phage.An yi IHC a kan ɗan takarar phage clones da aka yi wa allurar cikin jijiya cikin ƙwayar berayen kwakwalwa (Ƙarin Hoto 8).Bayanan sun nuna cewa aƙalla clones uku (No. 2002, No. 2009 da No. 2077) sun yi hulɗa tare da BBB.Ya rage don tantance ko wannan hulɗar BBB ta haifar da tarawar CSF ko motsi na waɗannan clones kai tsaye zuwa BCSFB.Mahimmanci, muna nuna cewa peptides da aka zaɓa suna riƙe ƙarfin jigilar su na CSF lokacin da aka haɗa su kuma an ɗaure su da kayan furotin.Daure na N-terminal biotinylated peptides zuwa SA da gaske yana maimaita sakamakon da aka samu tare da clones na phage na su a cikin jini da ruwa na cerebrospinal (Fig. 3e).A ƙarshe, mun nuna cewa peptide gubar # 2077 yana iya haɓaka aikin kwakwalwa na mai hana peptide biotinylated na BACE1 da aka haɗa zuwa SA, yana haifar da tasirin pharmacodynamic a cikin CNS ta hanyar rage matakan Abeta40 a cikin CSF (Fig. 4).Ba mu iya gano kowane mahaluki a cikin ma'ajin bayanai ta hanyar yin binciken peptide jerin homology na duk hits.Yana da mahimmanci a lura cewa girman ɗakin karatu na T7 yana da kusan 109, yayin da girman ɗakin karatu na ka'idar don 12-mers shine 4 x 1015. Saboda haka, mun zaɓi ɗan ƙaramin juzu'i na bambancin sararin samaniya na ɗakin karatu na 12-mer peptide, wanda na iya nufin cewa ana iya gano ƙarin peptides da aka inganta ta hanyar kimanta jerin abubuwan da ke kusa da su.A hasashe, daya daga cikin dalilan da ya sa ba mu sami wani mahaluki na dabi'a na wadannan peptides ba na iya zama ɓata lokacin juyin halitta don hana shigar da wasu abubuwan peptide cikin kwakwalwa mara sarrafa su.
A hade tare, sakamakonmu yana ba da tushe don aikin nan gaba don ganowa da kuma kwatanta tsarin sufuri na shinge na cerebrovascular a cikin vivo daki-daki.Saitin asali na wannan hanyar yana dogara ne akan dabarun zaɓin aiki wanda ba wai kawai ya gano clones tare da kaddarorin ɗaurin ƙwayar cuta ba, har ma ya haɗa da muhimmin mataki wanda clones masu nasara suna da ayyuka na zahiri don ketare shingen nazarin halittu a cikin vivo cikin sashin CNS.shine don bayyana tsarin jigilar waɗannan peptides da fifikon su don ɗaure ga microvasculature musamman ga yankin kwakwalwa.Wannan na iya haifar da gano sabbin hanyoyi don jigilar BBB da masu karɓa.Muna tsammanin cewa peptides da aka gano za su iya ɗaure kai tsaye zuwa masu karɓa na cerebrovascular ko kuma zuwa ga ligands masu rarraba ta hanyar BBB ko BCSFB.Za a ci gaba da bincikar peptide vectors tare da ayyukan sufuri na CSF da aka gano a cikin wannan aikin.A halin yanzu muna binciken ƙayyadaddun kwakwalwa na waɗannan peptides don ikonsu na ketare BBB da/ko BCSFB.Wadannan sababbin peptides za su kasance kayan aiki masu mahimmanci masu mahimmanci don yuwuwar gano sabbin masu karɓa ko hanyoyi da kuma haɓaka sabbin dandamali masu inganci don isar da macromolecules, kamar ilimin halittu, zuwa kwakwalwa.
Cannute babban rijiyar (CM) ta amfani da gyara hanyar da aka bayyana a baya.An ɗora berayen Wistar (200-350 g) a kan na'urar stereotaxic kuma an yi tsaka-tsakin tsaka-tsaki a kan wanda aka aske kuma aka shirya kan kai don fallasa kwanyar.Hana ramuka biyu a cikin yankin sash na sama kuma a ɗaure sukurori a cikin ramukan.An haƙa wani ƙarin rami a cikin ɓangarorin occipital na gefe don jagorar stereotactic na cannula bakin karfe a cikin CM.Aiwatar da siminti na hakori a kusa da cannula kuma aminta da sukurori.Bayan gyaran hoto da taurin siminti, an rufe raunin fata tare da suturar supramid 4/0.Ana tabbatar da wurin da ya dace na cannula ta hanyar zubar da ruwa na cerebrospinal (CSF).Cire bera daga na'urar stereotaxic, karɓar kulawar da ta dace bayan tiyata da kula da jin zafi, kuma ba shi damar murmurewa na akalla mako guda har sai an ga alamun jini a cikin ruwan cerebrospinal.An samo berayen Wistar (Crl: WI/Han) daga kogin Charles (Faransa).Duk berayen an kiyaye su a ƙarƙashin takamaiman yanayi marasa ƙwayoyin cuta.Dukkan gwaje-gwajen dabba sun yarda da Ofishin Kula da Dabbobin Dabbobi na birnin Basel, Switzerland, kuma an yi su daidai da Lasisi na Dabbobi 2474 (Kimanin Jirgin Jirgin Kwakwalwa Mai Aiki ta Ma'auni na Matakan Candidatewar warkewa a cikin Ruwan Cerebrospinal da Brain of Rat).
A hankali ka kiyaye bera a hankali tare da CM cannula a hannu.Cire Datura daga cannula kuma tattara 10 µl na ruwan cerebrospinal mai gudana kwatsam.Tun da patency na cannula a ƙarshe ya lalace, kawai bayyanannun samfuran ruwa na cerebrospinal ba tare da wata shaida na gurɓataccen jini ko canza launi ba a cikin wannan binciken.A cikin layi daya, an ɗauki kusan 10-20 μl na jini daga ƙaramin yanki a ƙarshen wutsiya cikin bututu tare da heparin (Sigma-Aldrich).An tattara CSF da jini a wurare daban-daban bayan allurar T7 phage ta cikin jijiya.An yi watsi da kusan 5-10 μl na ruwa kafin a tattara kowane samfurin CSF, wanda yayi daidai da mataccen adadin catheter.
An samar da ɗakunan karatu ta hanyar amfani da T7Select 10-3b vector kamar yadda aka bayyana a cikin littafin tsarin T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996).A taƙaice, an haɗa abin da aka saka DNA mai lamba 12 bazuwar a cikin tsari mai zuwa:
An yi amfani da code ɗin NNK don guje wa codons na tsayawa sau biyu da kuma wuce gona da iri na amino acid a cikin abin da aka saka.N shine haɗe-haɗe daidai gwargwado da hannu na kowane nucleotide, kuma K shine haɗe-haɗe da hannu na adenine da cytosine nucleotides.An canza yankuna guda ɗaya zuwa DNA mai ɗaure biyu ta hanyar haɓakawa tare da dNTP (Novagen) da Klenow enzyme (New England Biolabs) a cikin Klenow buffer (New England Biolabs) na awanni 3 a 37°C.Bayan abin da ya faru, an gano DNA mai madauri biyu ta hazo EtOH.Sakamakon DNA an narkar da shi tare da ƙuntatawa enzymes EcoRI da HindIII (dukansu daga Roche).Sa'an nan an haɗa abin da aka tsinke da tsarkakewa (QIAquick, Qiagen) saka (T4 ligase, New England Biolabs) a cikin firam ɗin cikin firam ɗin T7 da aka rigaya bayan amino acid 348 na 10B capsid gene.An ƙaddamar da halayen ligation a 16 ° C. na 18 hours kafin in vitro marufi.An yi fakitin fakiti a cikin vitro bisa ga umarnin da aka bayar tare da T7Select 10-3b cloning kit (Novagen) kuma an haɓaka maganin marufi sau ɗaya zuwa lysis ta amfani da Escherichia coli (BLT5615, Novagen).An yi amfani da lysates centrifuged, titrated kuma daskararre a -80 ° C. a matsayin maganin jari na glycerol.
PCR kai tsaye haɓakawa na yankuna masu canji na phage waɗanda aka haɓaka a cikin broth ko faranti ta amfani da kayan aikin haɗin gwiwar 454/Roche-amplicon.Fusion na gaba yana ƙunshe da jeri-jeri mai faɗin yanki mai canzawa (NNK) 12 (takamammen samfuri), GS FLX Titanium Adapter A, da jerin maɓallin ɗakin karatu mai tushe huɗu (TCAG) (Ƙarin Hoto 1a):
Juya fusion primer shima ya ƙunshi biotin da aka haɗe don kama beads da GS FLX Titanium Adapter B da ake buƙata don haɓaka clonal yayin emulsion PCR:
An yi amfani da amplicon ɗin zuwa 454/Roche pyrosequencing bisa ga ka'idar 454 GS-FLX Titanium.Don jerin Sanger na hannu (Aikace-aikacen Tsarin Halittu Hitachi 3730 xl DNA Analyzer), T7 phage DNA an haɓaka ta PCR kuma an jera shi tare da nau'i-nau'i masu zuwa:
Abubuwan da aka shigar daga allunan guda ɗaya an sa su zuwa haɓaka PCR ta amfani da Roche Fast Start DNA Polymerase Kit (bisa ga umarnin masana'anta).Yi farawa mai zafi (minti 10 a 95 ° C) da hawan haɓaka 35 (50 s a 95 ° C, 1 min a 50 ° C, da 1 min a 72 ° C).
Phage daga ɗakunan karatu, nau'in nau'in daji, phage da aka ceto daga CSF da jini, ko clones guda ɗaya an haɓaka su a cikin Escherichia coli BL5615 a cikin broth TB (Sigma Aldrich) ko a cikin jita-jita na 500 cm2 (Thermo Scientific) don 4 h a 37 ° C.An fitar da fage daga faranti ta hanyar kurkura faranti tare da buffer Tris-EDTA (Fluka Analytical) ko kuma ta hanyar tattara allunan tare da bakararre pipette.An keɓe matakin daga babban al'ada ko buffer cirewa tare da zagaye ɗaya na hazo polyethylene glycol (PEG 8000) (Promega) kuma an sake dakatar da shi a cikin buffer Tris-EDTA.
An ƙaddamar da haɓakar phage ɗin zuwa zagaye na 2-3 na cirewar endotoxin ta amfani da beads cirewar endotoxin (Miltenyi Biotec) kafin allurar jini (IV) (500 μl / dabba).A zagaye na farko, an gabatar da 2×1012 phages;a cikin na biyu, 2 × 1010 phages;a cikin zagaye na uku da na huɗu, 2 × 109 phages kowace dabba.Abubuwan da ke cikin kashi a cikin CSF da samfuran jini da aka tattara a wuraren da aka nuna an ƙaddara su ta hanyar kirga plaque bisa ga umarnin masana'anta (T7Select system manual).An yi zaɓin zaɓin ta hanyar allurar da aka tsarkake ta cikin jijiyar wutsiya ko kuma ta sake yin allura na phage da aka samo daga CSF daga zagaye na zaɓin da ya gabata, kuma an yi girbi na gaba a 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, da 240 da samfuran jini bi da bi.An gudanar da jimlar zagaye huɗu na in vivo panning inda aka adana rassan biyu da aka zaɓa daban tare da tantance su yayin zagaye uku na farko na zaɓin.Duk abubuwan da aka saka na phage da aka samo daga CSF daga zagaye biyu na farko na zaɓi an yi su zuwa 454/Roche pyrosequencing, yayin da duk clones da aka samo daga CSF daga zagaye biyu na zaɓi na ƙarshe da hannu aka jera su.Dukkanin matakan jini daga zagaye na farko na zaɓin an kuma yiwa 454/Roche pyrosequencing.Don allurar clones na phage, an haɓaka phages da aka zaɓa a cikin E. coli (BL5615) akan faranti 500 cm2 a 37 ° C don 4 hours.An yaɗa clones ɗin da aka zaɓa da hannu ɗaya a cikin matsakaicin tarin fuka.Bayan hakar phage, tsarkakewa da kuma kawar da endotoxin (kamar yadda aka bayyana a sama), 2 × 1010 phages / dabba a cikin 300 μl an yi musu allura ta hanyar jini a cikin jijiya wutsiya daya.
Preprocessing da qualitative tace bayanan jeri.An canza bayanan Raw 454/Roche daga tsarin taswirar daidaitaccen rafi na binary (sff) zuwa tsarin karantawa na ɗan adam na Pearson (fasta) ta amfani da software mai siyarwa.An gudanar da ƙarin sarrafa tsarin nucleotide ta amfani da shirye-shiryen C na mallakar mallaka da rubutun (kunshin software wanda ba a saki ba) kamar yadda aka bayyana a ƙasa.Binciken bayanan farko ya haɗa da tsauraran matakan tace matakai masu yawa.Don tace abubuwan karantawa waɗanda basu ƙunshi ingantacciyar hanyar saka DNA ta 12mer ba, an daidaita karatun a jere don fara lakabin (GTGATGTCGGGGATCCGAATTCT), lakabin tsayawa (TAAGCTTGCGGCCGCACTCGAGTA) da bayanan bayanan (CCCTGCAGGATATCCCGeedGGAGCTCGTCGAC) ta amfani da gwajin duniyaalignment yana ba da damar har zuwa 2 rashin daidaituwa a kowane alignment31.Don haka, karantawa ba tare da farawa ba kuma dakatar da tags da karantawa mai ɗauke da bayanan bayanan, watau, daidaitawa waɗanda suka wuce adadin da ba a yarda da su ba, an cire su daga ɗakin karatu.Dangane da sauran karatun, jerin DNA na N-mer wanda ya tashi daga alamar farawa kuma yana ƙarewa kafin a cire alamar tasha daga jerin karatun na asali kuma a ci gaba da sarrafa su (nan gaba ana kiranta "saka").Bayan fassarar abin da aka saka, ana cire ɓangaren bayan codon tasha ta farko a ƙarshen 5′ na farko daga abin da aka saka.Bugu da ƙari, an cire nucleotides da ke haifar da codons da ba su cika ba a ƙarshen 3′ na farko.Don keɓance abubuwan da ke ɗauke da jerin bayanan baya kawai, an cire abubuwan da aka fassara waɗanda suka fara da tsarin amino acid “PAG” kuma an cire su.Peptides tare da tsayin bayan fassarorin ƙasa da amino acid 3 an cire su daga ɗakin karatu.A ƙarshe, cire sakewa a cikin wurin da aka saka kuma ƙayyade yawan kowane abin sakawa na musamman.Sakamakon wannan bincike ya haɗa da jerin jerin nucleotide (saƙawa) da mitocin su (karanta) (Ƙarin Figures 1c da 2).
Rukunin N-mer DNA suna shigar ta hanyar kamanceceniya: Don kawar da kurakuran jeri na musamman na 454/Roche (kamar matsaloli tare da jerin abubuwan kari na homopolymer) da kuma cire ƙarin abubuwan da ba su da mahimmanci, abubuwan da aka tace N-mer DNA jerin abubuwan sawa (sakewa) a baya ana jerawa ta kamanni.shigarwa (har zuwa 2 ba daidaitattun tushe ba a yarda) ta yin amfani da algorithm na jujjuya da aka ayyana kamar haka: ana rarraba abubuwan da aka fara ta hanyar mitar su (mafi girma zuwa mafi ƙasƙanci), kuma idan sun kasance iri ɗaya, ta nau'in na biyu da tsayi (mafi tsayi zuwa mafi gajarta)).Don haka, mafi yawan lokuta kuma mafi tsayin shigarwa suna bayyana "ƙungiyar" ta farko.An saita mitar ƙungiyar zuwa mitar maɓalli.Sa'an nan, kowane shigarwa da ya rage a cikin jerawa an yi ƙoƙarin ƙara shi zuwa ƙungiyar ta hanyar daidaitawar Needleman-Wunsch.Idan adadin rashin daidaituwa, shigarwa, ko gogewa a cikin jeri bai wuce madaidaicin 2 ba, ana ƙara sakawa cikin ƙungiyar, kuma ana ƙara yawan mitar rukunin gabaɗaya ta sau nawa aka ƙara.Abubuwan da aka ƙara zuwa rukuni ana yiwa alama alama azaman amfani kuma an cire su daga ci gaba da sarrafawa.Idan ba za a iya ƙara jerin abubuwan da aka saka zuwa ƙungiyar da ta riga ta kasance ba, ana amfani da jerin sa don ƙirƙirar sabuwar ƙungiya tare da mitar shigar da ta dace kuma a yi alama kamar yadda aka yi amfani da ita.Ƙaddamarwa tana ƙarewa lokacin da kowane jerin shigarwa ko dai an yi amfani da shi don kafa sabuwar ƙungiya ko za a iya haɗa shi cikin ƙungiyar da ta riga ta kasance.Bayan haka, abubuwan da aka haɗa da suka ƙunshi nucleotides ana fassara su zuwa jerin peptide (dakunan karatu na peptide).Sakamakon wannan bincike shine saitin shigarwa da kuma mitoci masu kama da juna waɗanda ke yin adadin karantawa a jere (Ƙarin Hoto 2).
Motif Generation: Dangane da jerin peptides na musamman, an ƙirƙiri ɗakin karatu mai ɗauke da duk yuwuwar tsarin amino acid (aa) kamar yadda aka nuna a ƙasa.Kowane tsari mai yuwuwar tsayin 3 an fitar da shi daga peptide kuma an ƙara ƙirar sa tare da ɗakin karatu na yau da kullun wanda ya ƙunshi duk alamu (tripeptides).An jera dakunan karatu na dalilai masu yawan maimaitawa kuma an cire su.Sannan, ga kowane tripeptide a cikin ɗakin karatu na motif, mun bincika kasancewarsa a ɗakin karatu ta amfani da kayan aikin lissafi.A wannan yanayin, ana ƙara yawan adadin peptide da ke ɗauke da motif tripeptide da aka samo kuma an sanya shi zuwa motif a cikin ɗakin karatu na motif ("yawan motifs").Sakamakon tsara tsararru shine tsararru mai girma biyu mai ɗauke da duk abubuwan da suka faru na tripeptides (motifs) da ƙimar su, wanda shine adadin jerin abubuwan karantawa waɗanda ke haifar da madaidaicin abin da aka karanta lokacin da aka tace, haɗawa, da fassara.Ma'auni kamar yadda aka bayyana dalla-dalla a sama.
Matsakaicin adadin ƙididdiga da madaidaicin rarrabuwa: An daidaita adadin ƙididdiga ga kowane samfurin ta amfani da
inda ni ne adadin karantawa dauke da topic i.Don haka, vi yana wakiltar yawan adadin karantawa (ko peptides) mai ɗauke da motif i a cikin samfurin.An ƙididdige ƙimar P-darajar adadin abubuwan da ba a daidaita su ta amfani da ainihin gwajin Fisher.Game da ƙididdiga na adadin dalilai, an ƙididdige alaƙar Spearman ta amfani da daidaitattun adadin dalilai tare da R.
Don ganin abubuwan da ke cikin amino acid a kowane matsayi a cikin ɗakin karatu na peptide, an ƙirƙiri tambarin yanar gizo 32, 33 (http://weblogo.threeplusone.com).Na farko, abun ciki na amino acid a kowane matsayi na 12-mer peptide ana adana shi a cikin matrix 20 × 12.Sa'an nan, saitin peptides 1000 wanda ke ɗauke da abun ciki na amino acid iri ɗaya a kowane matsayi ana ƙirƙira shi a cikin tsarin fasta-jerin kuma an samar da shi azaman shigarwa zuwa tambarin yanar gizo 3, wanda ke haifar da hoto mai hoto na abun ciki na amino acid dangi a kowane matsayi.don ɗakin karatu na peptide.Don ganin manyan bayanai masu girma dabam, an ƙirƙiri taswirorin zafi ta amfani da kayan aikin da aka haɓaka a ciki a cikin R (biosHeatmap, fakitin R da ba a fito ba tukuna).Dendrograms da aka gabatar a cikin taswirorin zafi an ƙididdige su ta amfani da hanyar tari na Ward tare da ma'aunin nisa na Euclidean.Don ƙididdigar ƙididdiga na bayanan ƙididdiga na motif, ƙimar P don ƙididdige ƙididdiga marasa daidaituwa an ƙididdige su ta amfani da ainihin gwajin Fisher.An ƙididdige ƙimar P-darajar sauran bayanan bayanai a cikin R ta amfani da t-test Student ko ANOVA.
Zaɓuɓɓukan phage clones da phages ba tare da sakawa ba an yi musu allura ta hanyar jijiyar wutsiya (2 × 1010 phages / dabba a cikin 300 μl PBS).Minti 10 kafin turawa da gyarawa na gaba, an yi wa dabbobi iri ɗaya allura ta cikin jijiya tare da 100 μl na lectin mai alamar DyLight594 (Vector Laboratories Inc., DL-1177).Mintuna 60 bayan allurar phage, an zubar da berayen a cikin zuciya tare da 50 ml PBS sannan 50 ml 4% PFA/PBS.Samfurin kwakwalwa an kuma gyara su cikin dare a cikin 4% PFA/PBS kuma an jika shi cikin 30% sucrose na dare a 4°C.Samfurori suna walƙiya a cikin cakuda OCT.An gudanar da bincike na immunohistochemical na samfurori daskararre a dakin da zafin jiki akan 30 µm cryosections da aka katange tare da 1% BSA kuma an sanya su tare da polyclonal FITC-labeled antibodies a kan T7 phage (Novus NB 600-376A) a 4 ° C.Shiga cikin dare.A ƙarshe, an wanke sassan sau 3 tare da PBS kuma an gwada su tare da microscope na laser confocal (Leica TCS SP5).
Duk peptides tare da mafi ƙarancin tsabta na 98% an haɗa su ta GenScript USA, biotinylated da lyophilized.Ana ɗaure Biotin ta ƙarin sararin glycine sau uku a N-terminus.Bincika duk peptides ta amfani da ma'aunin spectrometry.
Streptavidin (Sigma S0677) an haɗe shi da 5-fold equimolar wuce haddi na biotinylated peptide, biotinylated BACE1 inhibitory peptide, ko hade (3: 1 rabo) na biotinylated BACE1 inhibitory peptide da BACE1 inhibitory peptide a cikin 5-10% DMSO / incu.Sa'a 1 a zafin jiki kafin allura.Streptavidin-conjugated peptides an yi musu allura ta hanyar jini a cikin kashi na 10 mg/kg a cikin ɗayan wutsiya na berayen tare da rami na cerebral.
ELISA ta tantance yawan hadaddun streptavidin-peptide.Nunc Maxisorp microtiter faranti (Sigma) an shafe dare a 4 ° C tare da 1.5 μg / ml linzamin kwamfuta anti-streptavidin antibody (Thermo, MA1-20011).Bayan toshewa (buffer toshe: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) a dakin da zafin jiki na 2 hours, wanke farantin tare da 0.05% Tween-20 / PBS (wash buffer) na 3 Na biyu, CSF da 0 samfurin plasma, an ƙara 0 tare da 0. SF 1:115).An sanya farantin a cikin dare a 4 ° C tare da gano antibody (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239).Bayan matakai uku na wankewa, an gano streptavidin ta hanyar shiryawa a cikin TMB substrate solution (Roche) har zuwa 20 min.Bayan dakatar da ci gaban launi tare da 1M H2SO4, auna abin sha a 450 nm.
Ayyukan streptavidin-peptide-BACE1 inhibitor hadaddun an kimanta ta Aβ (1-40) ELISA bisa ga ka'idar masana'anta (Wako, 294-64701).A taƙaice, samfuran CSF an diluted a daidaitaccen diluent (1:23) kuma an sanya su cikin dare a 4 ° C a cikin faranti 96-riji mai rufi da BNT77 kama antibody.Bayan matakai biyar na wankewa, an ƙara HRP-conjugated BA27 antibody kuma a sanya shi na tsawon awanni 2 a 4° C., sannan matakan wankewa biyar.An gano Aβ (1-40) ta hanyar shiryawa a cikin maganin TMB na mintuna 30 a zafin jiki.Bayan an dakatar da ci gaban launi tare da maganin dakatarwa, auna abin sha a 450 nm.Samfurori na Plasma sun kasance sun kasance masu tsauri mai tsauri kafin Aβ (1-40) ELISA.An ƙara Plasma zuwa 0.2% DEA (Sigma) a cikin faranti 96-rijiya kuma an sanya shi a cikin zafin jiki na minti 30.Bayan an gama wanke faranti na SPE (Oasis, 186000679) tare da ruwa da 100% methanol, an ƙara samfuran plasma zuwa faranti na SPE kuma an cire duk ruwa.An wanke samfurori (na farko tare da 5% methanol sannan 30% methanol) kuma an cire shi da 2% NH4OH/90% methanol.Bayan bushewa da eluate a 55 ° C na 99 min a akai-akai na N2 na yanzu, an rage samfurori a cikin daidaitattun diluents kuma an auna Aβ (1-40) kamar yadda aka bayyana a sama.
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