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Katangar jini-kwakwalwa da kuma katangar jini-kwakwalwa suna hana magungunan biotherapeutic isa ga burinsu a cikin tsarin jijiyoyi na tsakiya, wanda hakan ke hana maganin cututtukan jijiyoyi masu inganci. Domin gano sabbin masu jigilar kwakwalwa a cikin jiki, mun gabatar da wani dakin karatu na peptide na phage na T7 kuma mun tattara jini da ruwan cerebrospinal (CSF) ta amfani da samfurin beraye masu sane da juna. An wadatar da takamaiman ƙwayoyin phage a cikin CSF bayan zagaye huɗu na zaɓi. Gwajin peptides na kowane mutum ya nuna wadatar CSF fiye da ninki 1000. An tabbatar da cewa aikin isar da peptide zuwa kwakwalwa ya ragu da kashi 40% a cikin matakin amyloid-β a cikin ruwan cerebrospinal ta amfani da mai hana peptide na BACE1 wanda ke da alaƙa da sabon peptide na transit da aka gano. Waɗannan sakamakon sun nuna cewa peptides da aka gano ta hanyar hanyoyin zaɓin phage na in vivo na iya zama hanyoyin amfani don isar da macromolecules zuwa kwakwalwa tare da tasirin warkewa.
Binciken da aka yi niyya ga tsarin jijiyoyi na tsakiya (CNS) ya mayar da hankali sosai kan gano ingantattun magunguna da wakilai waɗanda ke nuna halayen CNS, tare da ƙarancin ƙoƙari wajen gano hanyoyin da ke haifar da isar da magunguna masu aiki zuwa kwakwalwa. Wannan ya fara canzawa yanzu yayin da isar da magunguna, musamman manyan ƙwayoyin halitta, muhimmin ɓangare ne na ci gaban magungunan jijiyoyi na zamani. Yanayin tsarin jijiyoyi na tsakiya yana da kariya sosai ta hanyar tsarin shingen cerebrovascular, wanda ya ƙunshi shingen jini-kwakwalwa (BBB) ​​​​da shingen jini-kwakwalwa (BCBB)1, wanda hakan ke sa ya zama ƙalubale a isar da magunguna zuwa kwakwalwa1,2. An kiyasta cewa kusan dukkan manyan magungunan ƙwayoyin halitta da fiye da kashi 98% na ƙananan magungunan ƙwayoyin halitta ana cire su daga kwakwalwa3. Shi ya sa yana da matukar muhimmanci a gano sabbin tsarin jigilar kwakwalwa waɗanda ke samar da ingantaccen isar da magungunan warkewa ga CNS 4,5. Duk da haka, BBB da BCSFB suma suna ba da kyakkyawar dama ga isar da magunguna yayin da suke shiga da shiga dukkan tsarin kwakwalwa ta hanyar jijiyoyin jini masu yawa. Saboda haka, ƙoƙarin da ake yi a yanzu na amfani da hanyoyin isar da sako marasa amfani ga kwakwalwa ya dogara ne akan tsarin jigilar kaya mai karɓar bayanai (PMT) ta amfani da mai karɓar BBB6 na ciki. Duk da ci gaba mai mahimmanci na baya-bayan nan ta amfani da hanyar mai karɓar bayanai ta transferrin7,8, ana buƙatar ƙarin haɓaka sabbin tsarin isar da saƙo tare da ingantattun halaye. Don wannan dalili, burinmu shine gano peptides waɗanda ke da ikon shiga tsakani na jigilar CSF, domin a ƙa'ida ana iya amfani da su don isar da macromolecules ga CNS ko don buɗe sabbin hanyoyin masu karɓar bayanai. Musamman ma, takamaiman masu karɓar bayanai da masu jigilar bayanai na tsarin cerebrovascular (BBB da BSCFB) na iya zama abubuwan da za a iya cimmawa don isar da magunguna masu aiki da takamaiman magunguna. Ruwan cerebrospinal (CSF) samfuri ne na ɓoye na choroid plexus (CS) kuma yana hulɗa kai tsaye da ruwan interstitial na kwakwalwa ta hanyar sararin subarachnoid da sararin ventricular4. Kwanan nan an nuna cewa ruwan cerebrospinal na subarachnoid yana yaɗuwa sosai zuwa cikin interstitium na kwakwalwa9. Muna fatan samun damar shiga sararin parenchymal ta amfani da wannan hanyar shigar subarachnoid ko kuma kai tsaye ta hanyar BBB. Domin cimma wannan, mun aiwatar da dabarun zaɓi na in vivo phage wanda ya fi dacewa ya gano peptides da ɗayan waɗannan hanyoyi guda biyu daban-daban ke ɗauka.
Yanzu mun bayyana hanyar tantancewa ta in vivo phage tare da samfurin CSF tare da jerin abubuwan da za a iya amfani da su (HTS) don sa ido kan zagayen farko na zaɓe tare da mafi girman bambancin ɗakin karatu. An yi gwajin a kan beraye masu hankali tare da babban cannula na cisterna (CM) da aka dasa har abada don guje wa gurɓatar jini. Abu mafi mahimmanci, wannan hanyar tana zaɓar duka abubuwan da ke kai hari ga kwakwalwa da peptides tare da ayyukan sufuri a kan shingen cerebrovascular. Mun yi amfani da phages na T7 saboda ƙaramin girmansu (~ 60 nm)10 kuma mun ba da shawarar cewa sun dace da jigilar vesicles waɗanda ke ba da damar ketare shingen endothelial da/ko epithelial-medulla. Bayan zagaye huɗu na panning, an ware yawan phage suna nuna ƙarfi a cikin jiki na CSF da haɗin microvessel na kwakwalwa. Abu mafi mahimmanci, mun sami damar tabbatar da bincikenmu ta hanyar nuna cewa mafi kyawun peptides masu dacewa da aka haɗa ta hanyar sinadarai suna iya jigilar kayan furotin zuwa cikin ruwan cerebrospinal. Da farko, an kafa tasirin magunguna na CNS ta hanyar haɗa babban peptide na jigilar kaya tare da mai hana peptide na BACE1. Baya ga nuna cewa dabarun tantance aiki a cikin jiki na iya gano sabbin peptides na jigilar kwakwalwa a matsayin masu ɗaukar kaya masu inganci na furotin, muna tsammanin irin waɗannan hanyoyin zaɓin aiki suma za su zama mahimmanci wajen gano sabbin hanyoyin jigilar kwakwalwa.
Dangane da na'urorin da ke samar da plaque (PFU), bayan matakin marufi na phage, an tsara kuma an ƙirƙiri wani ɗakin karatu na peptides na phage T7 linear 12-mer bazuwar tare da bambancin kusan 109 (duba Kayan aiki da Hanyoyi). Yana da mahimmanci a lura cewa mun yi nazari sosai kan wannan ɗakin karatu kafin a yi amfani da shi a cikin vivo panning. An samar da haɓaka PCR na samfuran ɗakin karatu na phage ta amfani da primers da aka gyara waɗanda suka dace kai tsaye ga HTS (Ƙarin Hoto na 1a). Saboda a) kurakuran jerin HTS11, b) tasiri akan ingancin primers (NNK)1-12, da c) kasancewar phage na nau'in daji (wt) (skeleton inserts) a cikin ɗakin karatu na jiran aiki, an aiwatar da tsarin tace jerin don cire bayanan jerin da aka tabbatar kawai (Ƙarin Hoto na 1b). Waɗannan matakan tacewa sun shafi duk ɗakunan karatu na jerin HTS. Ga ɗakin karatu na yau da kullun, an sami jimillar karatu 233,868, wanda kashi 39% ya wuce ka'idojin tacewa kuma an yi amfani da shi don nazarin ɗakin karatu da zaɓi don zagaye na gaba (Hoto na Ƙarin 1c–e). Karatun sun kasance galibi ninki biyu na nau'ikan tushe guda 3 a tsayi tare da kololutin 36 (Hoto na Ƙarin 1c), yana tabbatar da ƙirar ɗakin karatu (NNK) 1-12. Abin lura, kusan kashi 11% na membobin ɗakin karatu sun ƙunshi sakawar PAGISRELVDKL mai girman girma 12, kuma kusan rabin jerin (49%) sun ƙunshi sakawa ko sharewa. HTS na ɗakin karatu ya tabbatar da bambancin peptides a cikin ɗakin karatu: an sami fiye da kashi 81% na jerin peptide sau ɗaya kawai kuma kashi 1.5% ne kawai ya faru a cikin kwafi ≥4 (Hoto na Ƙarin 2a). Mitar amino acid (aa) a dukkan matsayi 12 a cikin jerin sunadaran sun yi daidai da mitar da ake tsammani don adadin codons da aka samar ta hanyar NKK repertoire mai lalacewa (Hoto na Ƙarin 2b). Mitar da aka lura da ragowar aa da waɗannan abubuwan da aka saka sun yi daidai da mitar da aka ƙididdige (r = 0.893) (Hoto na Ƙarin 2c). Shirye-shiryen ɗakunan karatu na phage don allura sun haɗa da matakan haɓakawa da cire endotoxin. An nuna cewa wannan a baya yana iya rage bambancin ɗakunan karatu na phage12,13. Saboda haka, mun jera ɗakin karatu na phage mai haɓaka faranti wanda aka cire endotoxin kuma muka kwatanta shi da ɗakin karatu na asali don kimanta mitar AA. An lura da alaƙa mai ƙarfi (r = 0.995) tsakanin wurin wanka na asali da wurin wanka mai haɓaka da tsarkakewa (Hoto na Ƙarin 2d), yana nuna cewa gasa tsakanin kwafi da aka haɓaka akan faranti ta amfani da phage T7 bai haifar da babban son kai ba. Wannan kwatancen ya dogara ne akan yawan nau'ikan tripeptide a cikin kowane ɗakin karatu, tunda bambancin ɗakunan karatu (~109) ba za a iya kama shi gaba ɗaya ba ko da tare da HTS. Binciken mita na aa a kowane matsayi ya nuna ƙaramin son kai dangane da matsayi a cikin matsayi uku na ƙarshe na kundin da aka shigar (Ƙarin Hoto na 2e). A ƙarshe, mun kammala da cewa inganci da bambancin ɗakin karatu sun kasance abin karɓa kuma ƙananan canje-canje ne kawai aka lura a cikin bambancin saboda haɓakawa da shirya ɗakunan karatu na phage tsakanin zagaye da yawa na zaɓi.
Ana iya yin samfurin ruwan cerebrospinal serial ta hanyar dasa cannula a cikin CM na berayen da suka sani ta hanyar tiyata don sauƙaƙe gano phage na T7 da aka allura ta hanyar jijiya (iv) ta hanyar BBB da/ko BCSFB (Hoto na 1a-b). Mun yi amfani da hannaye biyu masu zaman kansu na zaɓi (hannaye A da B) a cikin zagaye uku na farko na zaɓin in vivo (Hoto na 1c). A hankali mun ƙara ƙarfin zaɓin ta hanyar rage jimlar adadin phage da aka gabatar a zagaye uku na farko na zaɓi. Don zagaye na huɗu na panning, mun haɗa samfura daga rassan A da B kuma mun yi ƙarin zaɓuɓɓuka masu zaman kansu guda uku. Don nazarin halayen in vivo na barbashi na phage na T7 a cikin wannan samfurin, an allurar phage na daji (PAGISRELVDKL babban saka) a cikin beraye ta hanyar jijiyar wutsiya. Farfado da phages daga ruwan cerebrospinal da jini a lokutan lokaci daban-daban ya nuna cewa ƙananan phages na icosahedral na T7 suna da saurin matakin sharewa daga sashin jini (Hoto na 3 na Ƙarin). Dangane da adadin da aka bayar da kuma yawan jinin berayen, mun ƙididdige cewa an gano kusan kashi 1% na maganin da aka bayar daga allurar da aka bayar a cikin jinin mintuna 10 bayan allurar jijiya. Bayan wannan raguwar farko da sauri, an auna raguwar farko a hankali tare da rabin rai na mintuna 27.7. Abu mai mahimmanci, ƙananan ƙwayoyin cuta ne kawai aka samo daga sashin CSF, wanda ke nuna ƙarancin asali don ƙaura zuwa sashin CSF (Hoto na Ƙarin 3). A matsakaici, kusan kashi 1 x 10-3% na ƙwayar T7 a cikin jini da kashi 4 x 10-8% na ƙwayar cuta da aka fara sakawa a cikin ruwan cerebrospinal a tsawon lokacin ɗaukar samfurin (minti 0-250). Abin lura shi ne, rabin rai (minti 25.7) na ƙwayar cuta ta daji a cikin ruwan cerebrospinal yayi kama da wanda aka gani a cikin jini. Waɗannan bayanai sun nuna cewa shingen da ke raba sashin CSF daga jini yana nan a cikin berayen da aka yi wa allurar CM, wanda hakan ke ba da damar zaɓar ɗakunan karatu na phage don gano ƙwayoyin cuta da ake jigilar su cikin sauƙi daga jinin zuwa cikin sashin CSF.
(a) Shirya wata hanya don sake yin samfurin ruwan cerebrospinal (CSF) daga babban tafki. (b) Zane da ke nuna wurin da shingen tsarin jijiyoyi na tsakiya (CNS) yake da kuma dabarun zaɓi da ake amfani da shi don gano peptides waɗanda suka ratsa shingen jini-kwakwalwa (BBB) ​​​​da shingen jini-kwakwalwa. (c) Jadawalin tantance nunin phage na In vivo. A kowane zagaye na zaɓi, an yi allurar phages (masu gano dabbobi a cikin kibiyoyi) ta hanyar jijiya. Ana ajiye rassa biyu masu zaman kansu (A, B) daban har zuwa zagaye na 4 na zaɓi. Don zagaye na zaɓi na 3 da 4, an tsara kowane phage clone da aka cire daga CSF da hannu. (d) Kinetics na phage da aka ware daga jini (da'irori ja) da ruwan cerebrospinal (masu siffar kore) a lokacin zagaye na farko na zaɓi a cikin beraye biyu da aka yi wa allurar jijiya bayan allurar jijiya ta ɗakin karatu na peptide na T7 (2 x 1012 phages/dabba). Murabba'ai masu launin shuɗi suna nuna matsakaicin yawan farko na phage a cikin jini, wanda aka ƙididdige daga adadin allurar phage, idan aka yi la'akari da jimlar yawan jini. Murabba'ai baƙi suna nuna wurin haɗuwar layin y da aka cire daga yawan phage na jini. (e,f) Gabatar da mitar da rarraba duk yiwuwar haɗuwar peptide motifs da aka samu a cikin peptide. An nuna adadin motifs da aka samu a cikin karatu 1000. Mahimmanci (p < 0.001) motifs masu wadataccen alama an yiwa alama da dige ja. (e) Scatterplot mai daidaitawa yana kwatanta mitar da aka kwatanta ta tripeptide motifs na ɗakin karatu da aka yi wa allurar phage da aka samo daga jini daga dabbobi #1.1 da #1.2. (f) Scatterplot mai daidaitawa yana kwatanta mitar da aka danganta ta tripeptide motifs na dabba #1.1 da #1.2 da aka ware a cikin jini da ruwan cerebrospinal. (g, h) Wakiltar ID na jerin phage da aka wadatar a cikin jini (g) idan aka kwatanta da ɗakin karatu da aka yi wa allurar phage da aka wadatar a cikin CSF (h) idan aka kwatanta da jini bayan zagaye na zaɓin in vivo a cikin dabbobi biyu. Girman lambar harafi ɗaya yana nuna sau nawa wannan amino acid ke faruwa a wannan matsayi. Kore = polar, shunayya = tsaka tsaki, shuɗi = asali, ja = acidic da baƙi = hydrophobic amino acid. Eduard Urich ne ya tsara kuma ya samar da Hoto na 1a, b.
Mun yi allurar peptide na phage a cikin berayen kayan aiki guda biyu na CM (clades A da B) da kuma ware phage daga ruwan cerebrospinal da jini (Hoto na 1d). Farkon sharewar ɗakin karatu cikin sauri bai bayyana ba idan aka kwatanta da phage na nau'in daji. Matsakaicin rabin rayuwar ɗakin karatu da aka yi wa allura a cikin dabbobin biyu shine mintuna 24.8 a cikin jini, kama da phage na nau'in daji, da mintuna 38.5 a cikin CSF. An yi wa samfuran phage na jini da ruwan cerebrospinal daga kowace dabba HTS kuma an yi nazarin duk peptides da aka gano don kasancewar ɗan gajeren motif na tripeptide. An zaɓi motif na tripeptide saboda suna ba da ƙaramin tushe don ƙirƙirar tsari da hulɗar peptide-protein14,15. Mun sami kyakkyawar alaƙa a cikin rarraba motifs tsakanin ɗakin karatu na phage da aka yi wa allura da kwalaye da aka cire daga jinin dabbobin biyu (Hoto na 1e). Bayanan sun nuna cewa abun da ke cikin ɗakin karatu yana da wadatarwa kaɗan ne kawai a cikin sashin jini. An ƙara yin nazarin mitoci na amino acid da jerin yarjejeniya a kowane matsayi ta amfani da daidaitawar software na Weblogo16. Abin sha'awa, mun sami ƙaruwa mai ƙarfi a cikin ragowar glycine na jini (Hoto na 1g). Lokacin da aka kwatanta jini da ƙwayoyin cuta da aka zaɓa daga CSF, an lura da zaɓi mai ƙarfi da kuma cire wasu nau'ikan abubuwa (Hoto na 1f), kuma wasu amino acid sun fi dacewa a cikin matsayin da aka ƙayyade a cikin membobi 12 (Hoto na 1h). Abin lura shi ne, dabbobi daban-daban sun bambanta sosai a cikin ruwan cerebrospinal, yayin da aka lura da wadatar glycine na jini a cikin dabbobi biyu (Hoto na Ƙarin 4a–j). Bayan tace bayanai masu tsauri na jerin a cikin ruwan cerebrospinal na dabbobi #1.1 da #1.2, an sami jimillar peptides 12-mer na musamman guda 964 da 420 (Hoto na Ƙarin 1d–e). An ƙara yawan ƙwayoyin cuta na phage da aka ware kuma an ba su zagaye na biyu na zaɓin in vivo. An yi amfani da Phage da aka cire daga zagaye na biyu na zaɓi a cikin kowace dabba kuma an yi amfani da duk peptides da aka gano a matsayin shigarwa ga shirin gane motif don nazarin faruwar motifs na tripeptide (Hoto na 2a, b, ef). Idan aka kwatanta da zagaye na farko na phage da aka dawo da shi daga CSF, mun lura da ƙarin zaɓi da cire motifs da yawa a cikin CSF a cikin rassan A da B (Hoto na 2). An yi amfani da algorithm na gano hanyar sadarwa don tantance ko suna wakiltar alamu daban-daban na jerin daidaito. An lura da kamanceceniya bayyananne tsakanin jerin girma 12 da CSF ta dawo da su a madadin clade A (Hoto na 2c, d) da clade B (Hoto na 2g, h). Binciken da aka tattara a kowane reshe ya nuna bayanan zaɓi daban-daban don peptides 12-mer (Hoto na Ƙarin 5c, d) da ƙaruwa a cikin rabon CSF/jini akan lokaci don kwafi masu tarin bayan zagaye na biyu na zaɓi idan aka kwatanta da zagaye na farko na zaɓi (Hoto na Ƙarin 5e). ).
Ƙara yawan motifs da peptides a cikin ruwan cerebrospinal ta hanyar zagaye biyu a jere na zaɓin nunin phage mai aiki a cikin jiki.
An tattara dukkan ƙwayoyin halittar cerebrospinal da aka samo daga zagaye na farko na kowace dabba (dabbobi #1.1 da #1.2) an haɗa su, an ƙara musu girma, an haɗa su da HT kuma an sake yin allurar su 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) Tsarin rarrabawa na daidaitawa yana kwatanta mitar alaƙar motifs na duk ƙwayoyin halittar da aka samo daga CSF a zagaye na farko da na biyu na zaɓi. Mita da rarraba motifs da ke wakiltar duk yiwuwar haɗuwar tripeptides da aka samu a cikin peptides a cikin duka yanayin. An nuna adadin motifs da aka samu a cikin karatu 1000. Motifs waɗanda aka zaɓa ko aka cire su sosai (p < 0.001) an nuna su da dige ja. (c, d, g, h) Wakiltar tambarin jerin dukkan jerin amino acid 12 masu wadataccen CSF bisa ga zagaye na 2 da 1 na zaɓin in vivo. Girman lambar harafi ɗaya yana nuna sau nawa wannan amino acid ɗin ke faruwa a wannan matsayin. Don wakiltar tambarin, ana kwatanta mitar jerin CSF da aka cire daga dabbobi daban-daban tsakanin zagaye na zaɓi biyu kuma an nuna jerin da aka wadatar 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. Amino acid mafi wadata a matsayi da aka bayar a cikin (c, d) dabbobi masu lamba 2.1 da lamba 2.2 ko (g, h) a cikin dabbobi masu lamba 2.3 da lamba 2.4 an nuna su da launi. Kore = polar, shunayya = tsaka tsaki, shuɗi = asali, ja = acidic da baƙi = amino acid masu kama da hydrophobic.
Bayan zagaye na uku na zaɓe, mun gano jerin peptide na musamman guda 124 (#3.1 da #3.2) daga cikin kwalayen phage guda 332 da aka sake tsarawa daga dabbobi biyu (Ƙarin Hoto na 6a). Jerin LGSVS (18.7%) yana da mafi girman rabon dangi, sai kuma abubuwan da aka saka na nau'in daji PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), da SARGSWREIVSLS (2.2%). A zagaye na ƙarshe na huɗu, mun haɗa rassa biyu da aka zaɓa daban-daban daga dabbobi uku daban-daban (Hoto na 1c). Daga cikin kwalayen phage guda 925 da aka dawo dasu daga CSF, a zagaye na huɗu mun sami jerin peptide na musamman guda 64 (Hoto na Ƙarin Hoto na 6b), daga cikinsu akwai adadin kwalayen daji na phage ya ragu zuwa 0.8%. Kwafi na CSF da aka fi sani a zagaye na huɗu sune LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) da RLSSVDSDLSGC (3, 2%). %). Tsawon tsawon peptides da aka zaɓa ya faru ne saboda saka/sharewar nucleotide ko kuma codons na dakatarwa da wuri a cikin firam ɗin ɗakin karatu lokacin amfani da codons masu lalacewa don ƙirar ɗakin karatu na NNK. Codons na dakatarwa da wuri suna samar da gajerun peptides kuma ana zaɓar su saboda suna ɗauke da motif ɗin aa mai kyau. Peptides masu tsayi na iya faruwa ne daga saka/sharewa a cikin firam ɗin ɗakin karatu na roba. Wannan yana sanya codon na dakatarwa da aka tsara a wajen firam ɗin kuma yana karanta shi har sai sabon codon na dakatarwa ya bayyana a ƙasa. Gabaɗaya, mun ƙididdige abubuwan haɓakawa don duk zagaye huɗu na zaɓi ta hanyar kwatanta bayanan shigarwa tare da bayanan fitarwa na samfurin. A zagaye na farko na tantancewa, mun yi amfani da jerin phage na daji a matsayin ma'aunin asali mara takamaiman takamaiman bayani. Abin sha'awa, zaɓin phage mara kyau ya yi ƙarfi sosai a zagayen CSF na farko, amma ba a cikin jini ba (Hoto na 3a), wanda wataƙila saboda ƙarancin yuwuwar yaɗuwar yawancin membobin ɗakin karatu na peptide zuwa cikin sashin CSF ko phages na dangi galibi ana riƙe su ko cire su daga jini yadda ya kamata fiye da bacteriophages. Duk da haka, a zagaye na biyu na panning, an lura da zaɓin phages mai ƙarfi a cikin CSF a cikin duka clades, yana nuna cewa zagaye na baya ya wadatar da phages waɗanda ke nuna peptides waɗanda ke haɓaka ɗaukar CSF (Hoto na 3a). Kuma, ba tare da isasshen wadatar jini ba. Hakanan a zagaye na uku da na huɗu, clones na phage sun wadatar da CSF sosai. Idan aka kwatanta mitar kowane jerin peptide na musamman tsakanin zagaye biyu na ƙarshe na zaɓi, mun gano cewa jerin sun fi wadatarwa a zagaye na huɗu na zaɓi (Hoto na 3b). An ciro jimillar nau'ikan tripeptide guda 931 daga dukkan jerin peptide guda 64 na musamman ta amfani da hanyoyin peptide guda biyu. An yi nazari sosai kan mafi yawan nau'ikan da aka ƙara wa girma a zagaye na huɗu don bayanan haɓaka su a duk zagaye idan aka kwatanta da ɗakin karatu da aka yi wa allura (ƙayyadewa: 10% haɓaka) (Ƙarin Hoto na 6c). Tsarin zaɓi na gabaɗaya ya nuna cewa yawancin dalilan da aka yi nazari an ƙara wa girma a duk zagayen da suka gabata na rassan zaɓi biyu. Duk da haka, wasu nau'ikan (misali SGL, VSG, LGS GSV) galibi sun fito ne daga madadin nau'in A, yayin da wasu (misali FGW, RTN, WGF, NTR) an ƙara wa girma a madadin nau'in B.
Tabbatar da jigilar CSF na peptides masu wadatarwa na CSF da peptides na jagora masu biotinylated waɗanda aka haɗa zuwa ga kayan aikin streptavidin.
(a) Rabon wadatarwa da aka ƙididdige a cikin dukkan zagaye huɗu (R1-R4) bisa ga allurar titers na phage (PFU) da kuma ƙaddarar titers na phage na CSF (fitarwa = O). An ƙididdige abubuwan da suka ƙara wadatarwa na zagaye uku na ƙarshe (R2-R4) ta hanyar kwatantawa da zagaye na baya da zagaye na farko (R1) tare da bayanan nauyi. Sandunan da aka buɗe ruwan cerebrospinal ne, sandunan da aka yi wa inuwa sune plasma. (***p<0.001, bisa ga gwajin t na ɗalibi). (b) Jerin peptides na phage mafi yawan gaske, waɗanda aka tsara bisa ga rabon su na dangi ga duk phages da aka tattara a cikin CSF bayan zagaye na 4 na zaɓi. An haskaka kwalayen phage guda shida da aka fi sani da launi, an ƙidaya su da abubuwan wadatarwa tsakanin zagaye na 3 da 4 na zaɓi (insets). (c,d) An yi nazarin kwalayen phage guda shida da suka fi wadatarwa, kwalayen phage marasa komai da ɗakunan karatu na peptide na phage na iyaye daga zagaye na 4 daban-daban a cikin samfurin samfurin CSF. An tattara samfuran CSF da jini a lokutan da aka nuna. (c) Daidai adadin ƙwayoyin cuta guda 6 (2 x 1010 phages/dabbobi), ƙwayoyin cuta marasa komai (#1779) (2 x 1010 phages/dabbobi) da ɗakunan karatu na peptide na phage (2 x 1012 phages/dabbobi) A yi allura aƙalla 3 CM ga dabbar da aka yi wa allura daban ta hanyar jijiyar wutsiya. An nuna magungunan CSF na kowane allurar phage clone da phage peptide library akan lokaci. (d) yana nuna matsakaicin rabon CSF/jini ga duk phages/mL da aka dawo dasu a tsawon lokacin ɗaukar samfurin. (e) An haɗa peptides huɗu na roba da kuma sarrafawa ɗaya da aka yi wa rauni da biotin zuwa streptavidin ta hanyar N-terminus ɗinsu (nuni na tetramer) sannan a yi allura (jini na wutsiya iv, 10 mg streptavidin/kg). Aƙalla beraye uku da aka yi wa allura (N = 3).). An tattara samfuran CSF a lokutan da aka nuna kuma an auna yawan streptavidin ta hanyar amfani da CSF anti-streptavidin ELISA (nd = ba a gano ba). (*p<0.05, **p<0.01, ***p<0.001, bisa ga gwajin ANOVA). (f) Kwatanta jerin amino acid na clone peptide na phage mafi wadatar #2002 (shuɗi) tare da wasu clones na peptide na phage daga zagaye na 4 na zaɓi. An yi wa guntun amino acid iri ɗaya da makamantansu alama.
Daga cikin dukkan ƙwayoyin cuta masu wadata a zagaye na huɗu (Hoto na 3b), an zaɓi ƙwayoyin cuta guda shida don ƙarin bincike na mutum ɗaya a cikin samfurin samfurin CSF. An yi allurar daidai gwargwado na ƙwayoyin cuta guda shida, ƙwayoyin cuta marasa komai (babu abin sakawa) da kuma ɗakunan karatu na peptide na prophage a cikin dabbobin CM guda uku da aka yi wa allura, kuma an tantance ƙwayoyin cuta a cikin gwaje-gwajen CSF (Hoto na 3c) da jini (Hoto na Ƙarin 7). Duk ƙwayoyin cuta na phage da aka gwada sun yi niyya ga sashin CSF a matakin 10-1000 fiye da na ƙwayoyin cuta marasa komai (#1779). Misali, ƙwayoyin cuta #2020 da #2077 suna da adadin CSF mafi girma sau 1000 fiye da ƙwayoyin cuta masu sarrafawa. Bayanin pharmacokinetic na kowane peptide da aka zaɓa ya bambanta, amma duk suna da babban ikon homing na CSF. Mun lura da raguwar da ke faruwa akai-akai ga ƙwayoyin cuta #1903 da #2011, yayin da ga ƙwayoyin cuta #2077, #2002 da #2009 ƙaruwa a cikin mintuna 10 na farko na iya nuna jigilar aiki amma yana buƙatar a tabbatar. Kwayoyin cuta #2020, #2002, da #2077 sun daidaita a manyan matakan, yayin da yawan ƙwayoyin cuta na CSF na ƙwayoyin cuta #2009 ya ragu a hankali bayan ƙaruwar farko. Sannan muka kwatanta yawan ƙwayoyin cuta na kowane ɗan takarar CSF da yawan ƙwayoyin cuta na jini (Hoto na 3d). Alaƙar matsakaicin adadin ƙwayoyin cuta na kowane ɗan takarar CSF da yawan ƙwayoyin cuta na jini a duk lokutan ɗaukar samfur ya nuna cewa uku daga cikin 'yan takarar shida sun sami wadataccen CSF na jini sosai. Abin sha'awa, ƙwayoyin cuta #2077 sun nuna ƙarin kwanciyar hankali na jini (Hoto na Ƙarin 7). Don tabbatar da cewa ƙwayoyin cuta da kansu suna da ikon jigilar kaya banda ƙwayoyin cuta na ƙwayoyin cuta zuwa sashin CSF, mun haɗa manyan peptides guda huɗu waɗanda aka samo daga biotin a tashar N-terminus inda peptides ɗin suka haɗu da ƙwayar cuta ta ƙwayoyin cuta. An haɗa peptides masu biotinylated (lambobi. 2002, 2009, 2020 da 2077) tare da streptavidin (SA) don samun siffofi masu yawa waɗanda suka yi kama da yanayin phage. Wannan tsari ya kuma ba mu damar auna fallasar SA a cikin jini da ruwan cerebrospinal a matsayin peptides masu jigilar kaya. Abu mai mahimmanci, ana iya sake buga bayanan phage sau da yawa lokacin da aka ba da peptides na roba a cikin wannan tsarin haɗin SA (Hoto na 3e). Peptides masu narkewa suna da ƙarancin fallasa farko da sauri tare da matakan da ba a iya gano su ba cikin awanni 48. Don samun fahimta game da hanyoyin isar da waɗannan clones na peptide phage zuwa sararin CSF, mun bincika inda aka gano bugun peptide na phage na mutum ɗaya ta amfani da immunohistochemistry (IHC) don gano ƙwayoyin phage kai tsaye awa 1 bayan allurar jijiya a cikin vivo. Abin lura shi ne, ana iya gano ƙwayoyin cuta #2002, #2077, da #2009 ta hanyar tabo mai ƙarfi a cikin ƙwayoyin kwakwalwa, yayin da ba a gano ƙwayoyin cuta masu sarrafawa (#1779) da ƙwayoyin cuta #2020 ba (Hoto na Ƙarin 8). Wannan yana nuna cewa waɗannan ƙwayoyin cuta suna ba da gudummawa ga tasirin da ke kan kwakwalwa daidai ta hanyar ketare BBB. Ana buƙatar ƙarin cikakken bincike don gwada wannan hasashe, domin hanyar BSCFB ma tana iya shiga. Lokacin da aka kwatanta jerin amino acid na ƙwayoyin cuta mafi wadata (#2002) da sauran peptides da aka zaɓa, an lura cewa wasu daga cikinsu suna da irin wannan faɗaɗa amino acid, wanda zai iya nuna irin wannan tsarin sufuri (Hoto na 3f).
Saboda yanayin plasma na musamman da kuma ƙaruwa mai yawa a cikin CSF akan lokaci, an ƙara bincika clone nuni na phage #2077 a cikin tsawon awanni 48 kuma ya sami damar sake haifar da ƙaruwa mai sauri a cikin CSF da aka lura tare da matakan SA masu dorewa (Hoto na 4a). Dangane da sauran clones na phage da aka gano, #2077 ya yi wa capillaries na kwakwalwa fenti sosai kuma ya nuna babban haɗin gwiwa tare da lectin mai alamar capillary lokacin da aka duba shi a mafi girman ƙuduri da wataƙila wasu tabo a cikin sararin parenchymal (Hoto na 4b). Don bincika ko za a iya samun tasirin magunguna na peptide a cikin CNS, mun yi gwaji inda aka haɗa nau'ikan biotinylated na i) peptide na transit #2077 da ii) peptide mai hana BACE1 tare da SA a rabo biyu daban-daban. Don haɗuwa ɗaya mun yi amfani da mai hana peptide na BACE1 kawai kuma ga ɗayan mun yi amfani da rabo 1:3 na mai hana peptide na BACE1 zuwa peptide na #2077. An yi amfani da dukkan samfuran ta hanyar jijiya kuma an auna matakan beta-amyloid peptide 40 (Abeta40) a cikin jini da ruwan cerebrospinal akan lokaci. An auna Abeta40 a cikin CSF saboda yana nuna hana BACE1 a cikin parenchyma na kwakwalwa. Kamar yadda aka zata, duka hadaddun sun rage matakan jini na Abeta40 sosai (Hoto na 4c, d). Duk da haka, samfuran da ke ɗauke da cakuda peptide mai lamba 2077 da kuma mai hana BACE1 peptide da aka haɗa da SA sun haifar da raguwa mai yawa a cikin Abeta40 a cikin ruwan cerebrospinal (Hoto na 4c). Bayanan sun nuna cewa peptide mai lamba 2077 yana iya jigilar furotin 60 kDa SA zuwa CNS kuma yana haifar da tasirin magunguna tare da masu hana SA-conjugated na peptide BACE1.
(a) Allurar clonal (2 × 10 phages/dabba) ta T7 phage da ke nuna bayanan magunguna na dogon lokaci na peptide CSF #2077 (RLSSVDSDLSGC) da phage mai sarrafa ba tare da allura ba (#1779) a cikin aƙalla beraye uku da aka saka a cikin CM. (b) Hoton microscopic na confocal na ƙananan ƙwayoyin cuta na cortical a cikin beraye da aka yi wa allurar phage (2 × 10 10 phages/dabba) wanda ke nuna rashin tabo na peptide #2077 da tasoshin jini (lectin). An ba wa beraye 3 waɗannan phage clones kuma an bar su su zagaya na tsawon awa 1 kafin a yi musu allurar. An raba kwakwalwa kuma an yi musu fenti da ƙwayoyin rigakafi masu lakabin polyclonal FITC akan capsid na phage T7. Minti 10 kafin a yi musu allurar da kuma gyarawa daga baya, an ba wa lectin mai lakabin DyLight594 ta hanyar jijiya. Hotunan haske da ke nuna tabon lectin (ja) na gefen haske na ƙwayoyin cuta da phages (kore) a cikin lumen na capillaries da ƙwayoyin kwakwalwa na perivascular. Sandar sikelin ta yi daidai da 10 µm. (c, d) An haɗa peptide mai hana BACE1 na Biotinylated shi kaɗai ko tare da haɗin biotinylated transit peptide #2077 da streptavidin sannan aka yi allurar jijiya ta aƙalla beraye uku na cannulated CM (10 mg streptavidin/kg). An auna raguwar da BACE1 peptide inhibitor ya haifar a Aβ40 ta hanyar Aβ1-40 ELISA a cikin jini (ja) da ruwan cerebrospinal (lemu) a lokutan da aka nuna. Don ƙarin haske, an zana layi mai dige-dige akan jadawalin a sikelin 100%. (c) Rage kashi na Aβ40 a cikin jini (ja triangles) da ruwan cerebrospinal (orange triangles) a cikin beraye da aka yi wa magani da streptavidin wanda aka haɗa zuwa transit peptide #2077 da BACE1 inhibitory peptide a cikin rabo na 3: 1. (d) Rage kashi na Aβ40 a cikin jini (ja da'irori ja) da ruwan cerebrospinal (orange fluid) na beraye da aka yi wa magani da streptavidin wanda aka haɗa zuwa peptide mai hana BACE1 kawai. Yawan Aβ a cikin kulawa shine 420 pg/ml (daidaitaccen karkacewa = 101 pg/ml).
An yi amfani da wannan hanyar wajen yin bincike a fannoni da dama na binciken likitanci17. An yi amfani da wannan hanyar don nazarin bambancin jijiyoyin jini18,19 da kuma nazarin da ya shafi jijiyoyin kwakwalwa20,21,22,23,24,25,26. A cikin wannan binciken, mun faɗaɗa amfani da wannan hanyar zaɓi ba wai kawai ga gano peptides kai tsaye da ke kai hari ga tasoshin kwakwalwa ba, har ma ga gano 'yan takara masu halayen sufuri masu aiki don ketare shingen jini-kwakwalwa. Yanzu mun bayyana ci gaban hanyar zaɓi a cikin beraye da aka sanya wa CM kuma mun nuna yuwuwar gano peptides tare da halayen homing na CSF. Ta amfani da phage na T7 wanda ke nuna ɗakin karatu na peptides marasa tsari na 12-mer, mun sami damar nuna cewa phage na T7 ƙarami ne (kimanin diamita 60 nm)10 don a daidaita shi da shingen jini-kwakwalwa, ta haka kai tsaye keta shingen jini-kwakwalwa ko choroid plexus. Mun lura cewa girbe CSF daga berayen CM da aka yi wa allurar rigakafi hanya ce ta tantancewa mai kyau a cikin jiki, kuma phage da aka cire ba wai kawai yana ɗaure da jijiyoyin jini ba ne, har ma yana aiki a matsayin mai jigilar jini a kan shingen jini-kwakwalwa. Bugu da ƙari, ta hanyar tattara jini a lokaci guda da kuma amfani da HTS zuwa CSF ​​da phages da aka samo daga jini, mun tabbatar da cewa zaɓin CSF ɗinmu bai shafi wadatar jini ko dacewa da faɗaɗawa tsakanin zagaye na zaɓi ba. Duk da haka, sashin jini wani ɓangare ne na tsarin zaɓi, tunda phages da ke da ikon isa ga sashin CSF dole ne su rayu kuma su zagaya a cikin jini tsawon lokaci don wadatar da kansu a cikin kwakwalwa. Domin cire ingantattun bayanai na jerin bayanai daga bayanan HTS da ba a sarrafa ba, mun aiwatar da matattara waɗanda suka dace da kurakuran jerin abubuwa na musamman a cikin aikin bincike. Ta hanyar haɗa sigogin motsi a cikin hanyar tantancewa, mun tabbatar da saurin pharmacokinetics na phages T7 na daji (t½ ~ minti 28) a cikin jini24, 27, 28 kuma mun ƙayyade rabin rayuwarsu a cikin ruwan cerebrospinal (t½ ~ minti 26) a minti ɗaya). Duk da irin waɗannan bayanan pharmacokinetic a cikin jini da CSF, kashi 0.001% ne kawai na yawan phage a cikin jini aka iya gano a cikin CSF, wanda ke nuna ƙarancin motsi na baya na phage T7 na daji a kan shingen jini-kwakwalwa. Wannan aikin yana nuna mahimmancin zagaye na farko na zaɓi lokacin amfani da dabarun panning in vivo, musamman ga tsarin phage waɗanda aka share cikin sauri daga zagayawar jini, saboda ƙwayoyin cuta kaɗan ne ke iya isa ga sashin CNS. Don haka, a zagaye na farko, raguwar bambancin ɗakin karatu ya yi yawa sosai, domin an tattara adadi kaɗan na ƙwayoyin cuta a ƙarshe a cikin wannan ƙirar CSF mai tsauri. Wannan dabarar panning in vivo ta haɗa da matakai da yawa na zaɓi kamar tarin aiki a cikin sashin CSF, tsira da clone a cikin sashin jini, da kuma cire ƙwayoyin T7 cikin sauri daga jini a cikin mintuna 10 na farko (Hoto na 1d da Ƙarin Hoto na 4M). ). Don haka, bayan zagaye na farko, an gano ƙwayoyin phage daban-daban a cikin CSF, kodayake an yi amfani da wannan wurin farko ga dabbobi daban-daban. Wannan yana nuna cewa matakai da yawa na zaɓe masu tsauri ga ɗakunan karatu masu yawan membobin ɗakin karatu suna haifar da raguwar bambancin ra'ayi sosai. Saboda haka, abubuwan da suka faru bazuwar za su zama muhimmin ɓangare na tsarin zaɓen farko, wanda ke tasiri sosai ga sakamakon. Wataƙila yawancin kwafi a cikin ɗakin karatu na asali suna da irin wannan yanayin haɓaka CSF. Duk da haka, ko da a ƙarƙashin yanayi iri ɗaya na gwaji, sakamakon zaɓe na iya bambanta saboda ƙarancin adadin kowane kwafi na musamman a cikin rukunin farko.
Abubuwan da aka wadatar a cikin CSF sun bambanta da waɗanda ke cikin jini. Abin sha'awa, mun lura da canjin farko zuwa ga peptides masu arzikin glycine a cikin jinin dabbobi daban-daban. (Hoto na 1g, Ƙarin Hoto na 4e, 4f). Phage dauke da peptides na glycine na iya zama mafi karko kuma ba za a iya cire su daga zagayawa ba. Duk da haka, ba a gano waɗannan peptides masu arzikin glycine a cikin samfuran ruwan cerebrospinal ba, yana nuna cewa ɗakunan karatu da aka shirya sun bi matakai biyu daban-daban na zaɓi: ɗaya a cikin jini ɗayan kuma an yarda ya taru a cikin ruwan cerebrospinal. An gwada clones masu wadatar CSF waɗanda suka fito daga zagaye na huɗu na zaɓi sosai. An tabbatar da cewa kusan duk clones da aka gwada daban-daban suna da wadatar CSF idan aka kwatanta da phage mai sarrafa blank. An binciki bugun peptide ɗaya (#2077) dalla-dalla. Ya nuna tsawon rabin rai na plasma idan aka kwatanta da sauran hits (Hoto na 3d da Ƙarin Hoto na 7), kuma abin sha'awa, wannan peptide ya ƙunshi ragowar cysteine ​​​​a C-terminus. Kwanan nan an nuna cewa ƙara cysteine ​​​​zuwa ga peptides na iya inganta halayen pharmacokinetic ɗinsu ta hanyar ɗaurewa da albumin 29. Wannan ba a san shi ba a peptide #2077 a halin yanzu kuma yana buƙatar ƙarin bincike. Wasu peptides sun nuna dogaro da valence a cikin wadatar CSF (bayanai ba a nuna ba), wanda ƙila yana da alaƙa da yanayin saman da aka nuna na T7 capsid. Tsarin T7 da muka yi amfani da shi ya nuna kwafi 5-15 na kowane peptide a kowane barbashi na phage. An yi IHC akan kwafi na gubar phage na ɗan takarar da aka allura ta cikin jijiya a cikin kwakwalwar beraye (Ƙarin Hoto na 8). Bayanan sun nuna cewa aƙalla kwafi uku (Lambar 2002, Lamba 2009 da Lamba 2077) sun yi hulɗa da BBB. Har yanzu ba a tantance ko wannan hulɗar BBB ta haifar da tarin CSF ko motsi na waɗannan kwafi kai tsaye zuwa BCSFB ba. Abu mafi mahimmanci, mun nuna cewa peptides da aka zaɓa suna riƙe da ƙarfin jigilar CSF lokacin da aka haɗa su kuma aka ɗaure su zuwa kayan furotin. Haɗa peptides na N-terminal biotinylated zuwa SA a zahiri yana maimaita sakamakon da aka samu tare da ƙwayoyin phage ɗinsu a cikin jini da ruwan cerebrospinal (Hoto na 3e). A ƙarshe, mun nuna cewa peptide na lead #2077 yana iya haɓaka aikin kwakwalwa na mai hana peptide na biotinylated na BACE1 wanda aka haɗa zuwa SA, yana haifar da tasirin pharmacodynamic a cikin CNS ta hanyar rage matakan Abeta40 sosai a cikin CSF (Hoto na 4). Ba mu iya gano duk wani homologues a cikin bayanan ba ta hanyar yin binciken homology na jerin peptide na duk bugun. 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 ƙaramin yanki ne kawai na sararin bambancin ɗakin karatu na peptide 12-mer, wanda ke nufin cewa za a iya gano peptides mafi kyau ta hanyar kimanta sararin jerin da ke kusa da waɗannan bugun da aka gano. A zato, ɗaya daga cikin dalilan da ya sa ba mu sami wani nau'in homologue na halitta na waɗannan peptides ba shine rashin zaɓe yayin juyin halitta don hana shigar wasu nau'ikan peptide cikin kwakwalwa ba tare da kulawa ba.
Idan aka haɗa su wuri ɗaya, sakamakonmu ya samar da tushe don aikin nan gaba don gano da kuma bayyana tsarin sufuri na shingen cerebrovascular a cikin jiki dalla-dalla. Tsarin asali na wannan hanyar ya dogara ne akan dabarun zaɓi na aiki wanda ba wai kawai yana gano clones tare da halayen ɗaure jijiyoyin kwakwalwa ba, har ma ya haɗa da muhimmin mataki inda clones masu nasara ke da aikin ciki don ketare shingayen halittu a cikin jiki zuwa cikin sashin CNS. shine don bayyana hanyar jigilar waɗannan peptides da kuma fifikonsu na ɗaurewa 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 ga masu karɓar cerebrovascular ko kuma zuwa ligands masu yawo da aka ɗauka ta hanyar BBB ko BCSFB. Za a ƙara bincika vectors na peptide tare da ayyukan jigilar CSF da aka gano a cikin wannan aikin. A halin yanzu muna binciken takamaiman kwakwalwa na waɗannan peptides don ikon su na ƙetare BBB da/ko BCSFB. Waɗannan sabbin peptides za su zama kayan aiki masu matuƙar amfani don gano sabbin masu karɓa ko hanyoyin da za a bi da kuma haɓaka sabbin dandamali masu inganci don isar da ƙwayoyin cuta, kamar su ilimin halittu, zuwa kwakwalwa.
A shafa babban cisterna (CM) ta amfani da gyaran hanyar da aka bayyana a baya. An sanya berayen Wistar masu sa maye (200-350 g) a kan na'urar stereotaxic kuma an yi tsaka-tsaki a kan fatar kai da aka aske kuma aka shirya ta hanyar septic don fallasa kwanyar. A haƙa ramuka biyu a yankin saman sash ɗin kuma a ɗaure sukurori masu gyara a cikin ramukan. An haƙa ƙarin rami a cikin gefen occipital crest don jagorantar stereotactic na cannula na bakin ƙarfe zuwa cikin CM. A shafa simintin haƙori a kusa da cannula kuma a ɗaure shi da sukurori. Bayan an shafa hoto da tauri na siminti, an rufe raunin fata da suture na supramid 4/0. Ana tabbatar da sanya cannula daidai ta hanyar zubewar ruwan cerebrospinal (CSF) ba zato ba tsammani. A cire bera daga na'urar stereotaxic, a sami kulawar da ta dace bayan tiyata da kuma kula da ciwo, sannan a bar shi ya warke na akalla mako guda har sai an ga alamun jini a cikin ruwan cerebrospinal. An samo berayen Wistar (Crl:WI/Han) daga Charles River (Faransa). An ajiye dukkan berayen a ƙarƙashin takamaiman yanayi marasa ƙwayoyin cuta. Ofishin Kula da Dabbobi na Birnin Basel, Switzerland ya amince da duk gwaje-gwajen dabbobi, kuma an yi su ne bisa ga Lasisin Dabbobi Mai Lamba 2474 (Kimanta Sufurin Kwakwalwa Mai Aiki ta hanyar auna Matakan Masu Neman Magani a cikin Ruwan Cerebrospinal da Kwakwalwar Bera).
A hankali a hankali a riƙe bera da CM cannula a hannu. Cire Datura daga cannula sannan a tattara 10 µl na ruwan cerebrospinal da ke gudana ba zato ba tsammani. Tunda ƙarfin cannula ya lalace, an haɗa samfuran ruwan cerebrospinal bayyanannu kawai ba tare da wata shaida ta gurɓatar jini ko canza launi ba a cikin wannan binciken. A lokaci guda, an ɗauki kimanin 10-20 μl na jini daga ƙaramin yankewa a ƙarshen wutsiya zuwa cikin bututu tare da heparin (Sigma-Aldrich). An tattara CSF da jini a wurare daban-daban bayan allurar T7 phage ta jijiyoyi. An jefar da kimanin 5-10 μl na ruwa kafin a tattara kowane samfurin CSF, wanda ya yi daidai da mataccen adadin catheter.
An samar da ɗakunan karatu ta amfani da na'urar T7Select 10-3b kamar yadda aka bayyana a cikin littafin jagorar tsarin T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). A takaice, an haɗa wani nau'in DNA mai nau'in 12-mer bazuwar a cikin tsarin da ke ƙasa:
An yi amfani da lambar NNK don guje wa codons masu tsayawa biyu da kuma yawan bayyanar amino acid a cikin abin da aka saka. N rabo ne na haɗin equimolar na kowane nucleotide da hannu, kuma K rabo ne na equimolar na adenine da cytosine nucleotides da hannu. An canza yankuna guda ɗaya da aka ɗaure zuwa DNA mai ɗaure biyu ta hanyar ƙara haɗawa da dNTP (Novagen) da enzyme Klenow (New England Biolabs) a cikin Klenow buffer (New England Biolabs) na tsawon awanni 3 a zafin jiki na 37°C. Bayan amsawar, an dawo da DNA mai ɗaure biyu ta hanyar ruwan EtOH. An narke DNA ɗin da ya haifar da shi tare da enzymes masu ƙuntatawa EcoRI da HindIII (dukansu daga Roche). Sannan an haɗa sashin da aka yanke kuma aka tsarkake (QIAquick, Qiagen) (T4 ligase, New England Biolabs) a cikin firam zuwa vector T7 da aka riga aka ɗaure bayan amino acid 348 na kwayar halittar capsid 10B. An haɗa halayen ligation a zafin jiki na 16°C na tsawon awanni 18 kafin a saka a cikin vitro. An yi amfani da marufin Phage in vitro bisa ga umarnin da aka bayar tare da kayan aikin cloning na T7Select 10-3b (Novagen) kuma an ƙara yawan marufin sau ɗaya zuwa lysis ta amfani da Escherichia coli (BLT5615, Novagen). An sanya lysates ɗin a cikin centrifuge, an sanya su a titrated sannan aka daskare su a -80°C. a matsayin maganin glycerol.
Ƙara PCR kai tsaye na yankunan da ke canza yanayin phage da aka ƙara a cikin ruwan 'ya'yan itace ko faranti ta amfani da firam ɗin haɗin 454/Roche-amplicon na musamman. Firam ɗin haɗin gaba ya ƙunshi jerin da ke kewaye da yankin da ke canzawa (NNK) 12 (takamaiman samfuri), GS FLX Titanium Adapter A, da jerin maɓallan ɗakin karatu mai tushe huɗu (TCAG) (Karin Hoto na 1a):
Reverse fusion primer kuma ya ƙunshi biotin da aka haɗa don kama beads da kuma GS FLX Titanium Adapter B da ake buƙata don haɓaka clonal yayin emulsion PCR:
An yi amfani da tsarin pyrosequen na 454/Roche bisa ga ka'idar Titanium ta 454 GS-FLX. Don tsarin Sanger da hannu (Applied Biosystems Hitachi 3730 xl DNA Analyzer), an ƙara girman DNA na phage na T7 ta hanyar PCR kuma an haɗa shi da waɗannan nau'ikan primers:
An yi amfani da kayan da aka saka daga kowane farantin don ƙara ƙarfin PCR ta amfani da Roche Fast Start DNA Polymerase Kit (bisa ga umarnin masana'anta). Yi fara zafi (minti 10 a 95 °C) da kuma zagayowar haɓakawa 35 (daki 50 a 95 °C, minti 1 a 50 °C, da minti 1 a 72 °C).
An ƙara yawan sinadarin Phage daga ɗakunan karatu, nau'in phage na daji, phage da aka ceto daga CSF da jini, ko kuma ƙwayoyin cuta daban-daban a cikin Escherichia coli BL5615 a cikin ruwan TB (Sigma Aldrich) ko a cikin kwano mai girman cm2 500 (Thermo Scientific) na tsawon awanni 4 a zafin jiki na 37°C. An cire Phage daga faranti ta hanyar wanke faranti da Tris-EDTA buffer (Fluka Analytical) ko kuma ta hanyar tattara allunan da aka yi da bututun da ba a tsaftace ba. An ware Phage daga ruwan da ke fitowa daga culture supernatant ko extraction buffer tare da zagaye ɗaya na polyethylene glycol (PEG 8000) precision (Promega) sannan aka sake dakatar da shi a cikin Tris-EDTA buffer.
An yi wa phage mai ƙarfi zagaye 2-3 na cire endotoxin ta amfani da beads na cire endotoxin (Miltenyi Biotec) kafin a yi allurar ta jijiya (IV) (500 μl/dabba). A zagaye na farko, an gabatar da phages 2×1012; a zagaye na biyu, phages 2×1010; a zagaye na uku da na huɗu na zaɓi, phages 2×109 ga kowace dabba. An ƙayyade yawan phage a cikin CSF da samfuran jini da aka tattara a lokutan da aka ƙayyade ta hanyar ƙidaya plaque bisa ga umarnin masana'anta (littafin tsarin T7Select). An yi zaɓin phage ta hanyar allurar ta jijiya ta ɗakunan karatu masu tsabta a cikin jijiyar wutsiya ko kuma ta hanyar sake allurar phage da aka cire daga CSF daga zagaye na zaɓin da ya gabata, kuma an yi girbin da ya biyo baya a minti 10, minti 30, minti 60, minti 90, minti 120, minti 180, da minti 240 bi da bi CSF da samfuran jini. An gudanar da zagaye huɗu na panning a cikin jiki inda aka adana rassan biyu da aka zaɓa daban-daban kuma aka yi nazari a kansu a lokacin zagaye uku na farko na zaɓe. Duk abubuwan da aka saka a cikin phage daga CSF daga zagaye biyu na farko na zaɓe an yi musu 454/Roche pyrosequencing, yayin da duk clones da aka cire daga CSF daga zagaye biyu na ƙarshe na zaɓe an yi musu 454/Roche pyrosequencing da hannu. Duk phages na jini daga zagaye na farko na zaɓe an kuma yi musu 454/Roche pyrosequencing. Don allurar clones na phage, an ƙara phages da aka zaɓa a cikin E. coli (BL5615) akan faranti 500 cm2 a zafin jiki na 37°C na tsawon awanni 4. An yaɗa clones da aka zaɓa daban-daban kuma aka yi musu jadawali da hannu a cikin maganin TB. Bayan cire phage, tsarkakewa da cire endotoxin (kamar yadda aka bayyana a sama), an yi musu phages 2×1010/dabba a cikin 300 μl ta hanyar jijiya ɗaya.
Tsarin sarrafawa da tace bayanai na jerin bayanai kafin lokaci. An canza bayanan 454/Roche marasa inganci daga tsarin taswirar rafi na binary (sff) zuwa tsarin Pearson na ɗan adam mai iya karantawa (fasta) ta amfani da software na mai siyarwa. An yi ƙarin sarrafa jerin nucleotide ta amfani da shirye-shiryen C na mallaka da rubutun (fakitin software mara fitarwa) kamar yadda aka bayyana a ƙasa. Binciken bayanan farko ya haɗa da tsauraran hanyoyin tacewa masu matakai da yawa. Don tace karatun da ba su ƙunshi jerin DNA mai inganci na 12mer ba, an daidaita karatun zuwa lakabin farawa (GTGATGTCGGGGATCCGGAATTCT), lakabin tsayawa (TAAGCTTGCGGCCGCACTCGAGTA) da shigarwar bango (CCCTGCAGGGATATCCCCGGGAGCTCGTCGAC) ta amfani da gwajin Needleman-Wunsch na duniya. daidaitawa wanda ke ba da damar har zuwa rashin daidaito 2 a kowane daidaitawa31. Saboda haka, an cire karantawa ba tare da alamun farawa da tsayawa ba kuma an cire karatun da ke ɗauke da shigarwar bango, watau, daidaitawa waɗanda suka wuce adadin rashin daidaito da aka yarda, daga ɗakin karatu. Dangane da sauran karatun, an cire jerin DNA na N-mer wanda ya tashi daga alamar farko zuwa ƙarshe kafin alamar tsayawa daga jerin karatun asali kuma an ƙara sarrafa shi (wanda daga nan ake kira "saka"). Bayan fassarar shigarwar, an cire ɓangaren bayan lambar farko da ke tsayawa a ƙarshen 5′ na farkon daga cikin shigarwar. Bugu da ƙari, an kuma cire nucleotides waɗanda ke haifar da rashin kammala codons a ƙarshen 3′ na farkon. Don cire shigarwar da ke ɗauke da jerin bayanan baya kawai, an kuma cire shigarwar da aka fassara waɗanda suka fara da tsarin amino acid "PAG". An cire peptides masu tsawon bayan fassarar ƙasa da amino acid 3 daga ɗakin karatu. A ƙarshe, cire ƙarin aiki a cikin wurin shigarwar kuma a tantance mitar kowane shigarwar ta musamman. Sakamakon wannan binciken ya haɗa da jerin jerin nucleotides (saka) da mitar su (karantawa) (Karin Figures 1c da 2).
Shigar da DNA na Rukunin N-mer ta hanyar kamanceceniya: Don kawar da kurakuran jerin 454/Roche na musamman (kamar matsalolin tsawaita jerin homopolymer) da kuma cire canje-canje marasa mahimmanci, ana rarraba shigarwar jerin DNA na N-mer da aka tace a baya (saka) ta hanyar kamanceceniya. shigarwar (har zuwa tushe 2 marasa daidaituwa da aka yarda) ta amfani da tsarin maimaitawa wanda aka bayyana kamar haka: ana tsara shigarwar da farko ta mitar su (mafi girma zuwa mafi ƙanƙanta), kuma idan sun kasance iri ɗaya, ta hanyar nau'in sakandare ta tsawon (mafi tsawo zuwa ga mafi guntu)). Don haka, shigarwar da ta fi yawa kuma mafi tsayi tana bayyana "rukunin" na farko. Ana saita mitar rukuni zuwa mitar maɓalli. Sannan, kowane shigarwa da ya rage a cikin jerin da aka tsara an yi ƙoƙarin ƙara shi zuwa rukunin ta hanyar daidaitawar Needleman-Wunsch ta hanyar biyu. Idan adadin rashin daidaito, sakawa, ko gogewa a cikin daidaitawa bai wuce iyaka ta 2 ba, ana ƙara sakawa zuwa rukunin, kuma jimlar mitar rukuni ana ƙaruwa ta hanyar sau nawa aka ƙara sakawa. An yiwa shigarwar da aka ƙara zuwa rukuni alama a matsayin an yi amfani da ita kuma an cire ta daga ƙarin sarrafawa. Idan ba za a iya ƙara jerin shigarwar zuwa ga rukunin da ke akwai ba, ana amfani da jerin shigarwar don ƙirƙirar sabon rukuni tare da mitar shigarwar da ta dace kuma an yi masa alama kamar an yi amfani da shi. Juyawan yana ƙarewa lokacin da aka yi amfani da kowace jerin shigarwa don ƙirƙirar sabuwar ƙungiya ko kuma za a iya haɗa shi cikin rukunin da ke akwai. Bayan haka, za a fassara shigarwar rukuni waɗanda suka ƙunshi nucleotides zuwa jerin peptide (ɗakunan karatu na peptide). Sakamakon wannan bincike shine saitin shigarwar da mitoci masu dacewa waɗanda suka ƙunshi adadin karantawa a jere (Ƙarin Hoto na 2).
Samar da Motif: Dangane da jerin peptides na musamman, an ƙirƙiri wani ɗakin karatu wanda ke ɗauke da dukkan samfuran amino acid (aa) kamar yadda aka nuna a ƙasa. An cire kowane tsari mai yuwuwa na tsawon 3 daga peptide kuma an ƙara tsarinsa na juzu'i tare da ɗakin karatu na motif na gama gari wanda ke ɗauke da dukkan alamu (tripeptides). An jera ɗakunan karatu na motif masu maimaitawa sosai kuma an cire ƙarin aiki. Sannan, ga kowane tripeptide a cikin ɗakin karatu na motif, mun duba kasancewarsa a cikin ɗakin karatu ta amfani da kayan aikin lissafi. A wannan yanayin, ana ƙara mitar peptide ɗin da ke ɗauke da motif ɗin da aka samo kuma an sanya shi ga motif ɗin a cikin ɗakin karatu na motif ("adadin motifs"). Sakamakon samar da motif shine jerin girma biyu wanda ya ƙunshi duk abubuwan da suka faru na tripeptides (motifs) da ƙimar su, waɗanda sune adadin karatun jerin da ke haifar da motif ɗin da ya dace lokacin da aka tace karatun, aka haɗa su, kuma aka fassara su. Ma'auni kamar yadda aka bayyana dalla-dalla a sama.
Daidaita adadin motifs da kuma shirye-shiryen warwatse masu dacewa: An daidaita adadin motifs na kowane samfurin ta amfani da
inda ni shine adadin karatun da ke ɗauke da batu i. Don haka, vi yana wakiltar kashi na yawan karantawa (ko peptides) waɗanda ke ɗauke da motif i a cikin samfurin. An ƙididdige ƙimar P don adadin motifs marasa daidaituwa ta amfani da gwajin Fisher daidai. Dangane da correlograms na adadin dalilai, an ƙididdige alaƙar Spearman ta amfani da adadin dalilai da aka daidaita tare da R.
Don hango 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). Da farko, ana adana abubuwan da ke cikin amino acid a kowane matsayi na peptide 12-mer a cikin matrix 20 × 12. Sannan, ana samar da saitin peptides 1000 waɗanda ke ɗauke da irin wannan abun ciki na amino acid a kowane matsayi a cikin tsarin sauri kuma ana ba su azaman shigarwa zuwa tambarin yanar gizo 3, wanda ke samar da wakilcin zane na abun ciki na amino acid a kowane matsayi. don ɗakin karatu na peptide da aka bayar. Don hango bayanai masu girma dabam-dabam, an ƙirƙiri taswirar zafi ta amfani da kayan aiki da aka haɓaka a ciki a cikin R (biosHeatmap, kunshin R da ba a sake shi ba tukuna). An ƙididdige dendrograms da aka gabatar a cikin taswirar zafi ta amfani da hanyar rukuni na Ward tare da ma'aunin nisa na Euclidean. Don nazarin ƙididdiga na bayanan maki motif, an ƙididdige ƙimar P don maki mara daidaituwa ta amfani da gwajin Fisher na ainihi. An ƙididdige ƙimar P don wasu bayanan bayanai a cikin R ta amfani da gwajin t-test na ɗalibi ko ANOVA.
An yi wa ƙwayoyin cuta da aka zaɓa da kuma ƙwayoyin cuta ba tare da an saka su ba allura ta jijiya ta hanyar jijiya ta wutsiya (2×1010 phages/dabba a cikin 300 μl PBS). Minti 10 kafin a yi perfusion da kuma bayan an gyara su, an yi wa waɗannan dabbobin allurar ta jijiya da 100 μl na lectin mai lakabin DyLight594 (Vector Laboratories Inc., DL-1177). Minti 60 bayan allurar phage, an zuba beraye a cikin zuciya da 50 ml na PBS sannan sai 50 ml na 4% na PFA/PBS. An kuma gyara samfuran kwakwalwa cikin dare ɗaya a cikin 4% na PFA/PBS sannan aka jiƙa su cikin 30% na sucrose a cikin dare ɗaya a zafin jiki na 4°C. An daskare samfuran a cikin cakuda OCT. An gudanar da nazarin kwayoyin halitta na samfuran daskararre a zafin ɗaki a kan sassan daskararru na 30 µm waɗanda aka toshe da 1% BSA kuma aka haɗa su da ƙwayoyin rigakafi masu lakabin polyclonal FITC akan T7 phage (Novus NB 600-376A) a zafin jiki na 4 °C. A saka su cikin duhu. A ƙarshe, an wanke sassan sau 3 da PBS kuma an duba su da na'urar hangen nesa ta laser confocal (Leica TCS SP5).
An haɗa dukkan peptides masu ƙarancin tsarki na kashi 98% ta hanyar GenScript USA, an yi musu biotinylated kuma an yi musu lyophilized. Ana ɗaure Biotin ta hanyar ƙarin glycine spacer sau uku a N-terminus. Duba duk peptides ta amfani da mass spectrometry.
An haɗa Streptavidin (Sigma S0677) da yawan peptide mai kama da biotinylated sau 5, peptide mai hana BACE1 biotinylated, ko kuma haɗuwa (rabo 3:1) na peptide mai hana BACE1 biotinylated da peptide mai hana BACE1 a cikin 5-10% DMSO/an saka a cikin PBS. Awa 1 a zafin ɗaki kafin allura. An yi allurar peptides masu haɗin Streptavidin ta jijiya a cikin allurar 10 mg/kg a cikin ɗaya daga cikin jijiyoyin wutsiya na beraye masu ramin kwakwalwa.
An tantance yawan ƙwayoyin streptavidin-peptide ta hanyar ELISA. An shafa faranti na Nunc Maxisorp microtiter (Sigma) cikin dare a 4°C da maganin rigakafi na linzamin kwamfuta na 1.5 μg/ml (Thermo, MA1-20011). Bayan toshewa (buffer mai toshewa: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) a zafin ɗaki na tsawon awanni 2, a wanke farantin da 0.05% Tween-20/PBS (wanke buffer) na tsawon awanni 3, an ƙara samfuran CSF da plasma a cikin rijiyoyin da aka narkar da buffer mai toshewa (plasma 1:10,000, CSF 1:115). Sannan an sanya farantin a cikin dare a 4°C tare da maganin rigakafi na ganowa (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Bayan matakai uku na wankewa, an gano streptavidin ta hanyar saka shi a cikin maganin TMB substrate (Roche) na tsawon mintuna 20. Bayan dakatar da haɓakar launi da 1M H2SO4, a auna yadda yake sha a 450 nm.
An tantance aikin hadadden maganin hana streptavidin-peptide-BACE1 ta hanyar amfani da tsarin masana'anta na Aβ(1-40) ELISA. A takaice, an narkar da samfuran CSF a cikin maganin diluent na yau da kullun (1:23) kuma an sanya su cikin dare a 4°C a cikin faranti 96 da aka lulluɓe da maganin hana BNT77. Bayan matakai biyar na wankewa, an ƙara maganin hana BA27 mai haɗin HRP kuma an sanya su cikin kwantena na tsawon awanni 2 a 4°C, sannan aka biyo baya da matakai biyar na wankewa. An gano Aβ(1–40) ta hanyar sanya su cikin maganin TMB na tsawon mintuna 30 a zafin ɗaki. Bayan an dakatar da haɓakar launi da maganin tsayawa, a auna shan su a 450 nm. An yi wa samfuran plasma cirewa mai ƙarfi kafin Aβ(1–40) ELISA. An ƙara jini zuwa 0.2% DEA (Sigma) a cikin faranti 96 kuma an sanya su a zafin ɗaki na tsawon mintuna 30. Bayan an wanke faranti na SPE (Oasis, 186000679) a jere da ruwa da kuma 100% methanol, an ƙara samfuran plasma a cikin faranti na SPE kuma an cire duk wani ruwa. An wanke samfuran (da farko da 5% methanol sannan 30% methanol) sannan aka fitar da 2% NH4OH/90% methanol. Bayan an busar da eluate a 55°C na tsawon mintuna 99 a kan wutar lantarki ta N2 mai ɗorewa, an rage samfuran a cikin daidaitattun abubuwan narkewa kuma an auna Aβ(1–40) kamar yadda aka bayyana a sama.
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