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ʻO ke keʻakeʻa koko-lolo a me ke keʻakeʻa koko-lolo e pale aku i nā mea hana biotherapeutic mai ka hiki ʻana i kā lākou mau pahuhopu i loko o ka ʻōnaehana nerve waena, no laila e pale ana i ka mālama pono ʻana i nā maʻi neurological. No ka ʻike ʻana i nā mea lawe lolo hou i loko o ke kino, ua hoʻolauna mākou i kahi waihona peptide phage T7 a ua hōʻiliʻili ʻia ke koko a me ka wai cerebrospinal (CSF) me ka hoʻohana ʻana i kahi kumu hoʻohālike wai nui i hoʻomaopopo ʻia o nā ʻiole. Ua hoʻonui nui ʻia nā clones phage kikoʻī i loko o ke CSF ma hope o ʻehā mau pōʻai o ke koho ʻana. Ua hōʻike ka hoʻāʻo ʻana o nā peptides moho pākahi i ka hoʻonui ʻana ma mua o 1000-fold i loko o ke CSF. Ua hōʻoia ʻia ka bioactivity o ka lawe ʻana i ka peptide-mediated i ka lolo e ka emi ʻana o 40% i ka pae o ka amyloid-β i loko o ka wai cerebrospinal me ka hoʻohana ʻana i kahi mea hoʻopaʻa peptide BACE1 i hoʻopili ʻia i ka peptide transit hou i ʻike ʻia. Hōʻike kēia mau hopena he mau kaʻa pono nā peptides i ʻike ʻia e nā ʻano koho phage in vivo no ka lawe ʻana i nā macromolecules i loko o ke kino me ka hopena therapeutic.
Ua kālele nui ka noiʻi ʻana i ka lāʻau lapaʻau i hoʻopaʻa ʻia no ka ʻōnaehana nerve waena (CNS) i ka ʻike ʻana i nā lāʻau lapaʻau a me nā mea hana i hoʻomaikaʻi ʻia e hōʻike ana i nā waiwai CNS-targeting, me ka emi ʻana o ka hoʻoikaika ʻana e ʻike i nā ʻano hana e hoʻokele ai i ka lawe ʻana i ka lāʻau lapaʻau i ka lolo. Ke hoʻomaka nei kēia e loli i kēia manawa ʻoiai ʻo ka lawe ʻana i ka lāʻau lapaʻau, ʻoiai nā molekala nui, he ʻāpana koʻikoʻi ia o ka hoʻomohala ʻana i ka lāʻau neuroscience hou. Pale maikaʻi ʻia ke kaiapuni o ka ʻōnaehana nerve waena e ka ʻōnaehana pale cerebrovascular, nona ka pale koko-lolo (BBB) ​​​​a me ka pale koko-lolo (BCBB)1, e paʻakikī ai ka lawe ʻana i nā lāʻau lapaʻau i ka lolo1,2. Ua manaʻo ʻia ua hoʻopau ʻia ka aneane pau nā lāʻau lapaʻau molekala nui a ʻoi aku ma mua o 98% o nā lāʻau lapaʻau molekala liʻiliʻi mai ka lolo3. ʻO ia ke kumu he mea nui loa ka ʻike ʻana i nā ʻōnaehana lawe lolo hou e hāʻawi ana i ka lawe pono a kikoʻī o nā lāʻau lapaʻau i ka CNS 4,5. Eia nō naʻe, hāʻawi pū ka BBB a me BCSFB i kahi manawa kūpono no ka lawe ʻana i ka lāʻau lapaʻau i ko lākou komo ʻana a komo i nā ʻano āpau o ka lolo ma o kona vasculature ākea. No laila, ʻo nā hoʻoikaika o kēia manawa e hoʻohana i nā ʻano hana ʻole invasive o ka lawe ʻana i ka lolo e pili nui ana i ke ʻano o ka lawe ʻana i ka receptor-mediated (PMT) me ka hoʻohana ʻana i ka receptor BBB6 endogenous. ʻOiai nā holomua koʻikoʻi hou e hoʻohana ana i ke ala receptor transferrin7,8, pono ka hoʻomohala hou ʻana o nā ʻōnaehana lawe hou me nā waiwai i hoʻomaikaʻi ʻia. No kēia hopena, ʻo kā mākou pahuhopu ka ʻike ʻana i nā peptides i hiki ke hoʻopili i ka lawe ʻana o ka CSF, no ka mea, hiki ke hoʻohana ʻia ma ke ʻano nui e lawe i nā macromolecules i ka CNS a i ʻole e wehe i nā ala receptor hou. Ma ke ʻano kūikawā, hiki i nā receptors kikoʻī a me nā mea lawe o ka ʻōnaehana cerebrovascular (BBB a me BSCFB) ke lawelawe ma ke ʻano he mau pahuhopu kūpono no ka lawe ʻana i nā lāʻau biotherapeutic. ʻO ka wai Cerebrospinal (CSF) kahi huahana huna o ka choroid plexus (CS) a pili pono me ka wai interstitial o ka lolo ma o ka wahi subarachnoid a me ka wahi ventricular4. Ua hōʻike ʻia i kēia manawa ua hoʻolaha nui ʻia ka wai cerebrospinal subarachnoid i loko o ka interstitium o ka lolo9. Ke manaʻolana nei mākou e komo i ka hakahaka parenchymal me ka hoʻohana ʻana i kēia ala komo subarachnoid a i ʻole pololei ma o ka BBB. No ka hoʻokō ʻana i kēia, ua hoʻokō mākou i kahi hoʻolālā koho phage in vivo ikaika e ʻike pono ana i nā peptides i lawe ʻia e kekahi o kēia mau ala ʻelua.
I kēia manawa, ke wehewehe nei mākou i kahi ʻano hana nānā ʻana i ka hōʻike ʻana o ka phage in vivo me ka laʻana CSF i hui pū ʻia me ka hoʻonohonoho ʻana o ka throughput kiʻekiʻe (HTS) e nānā i nā pōʻai koho mua me ka ʻokoʻa waihona puke kiʻekiʻe loa. Ua hana ʻia ka nānā ʻana ma nā ʻiole ʻike me kahi cannula cisterna nui (CM) i hoʻokomo mau ʻia e pale aku i ka haumia koko. ʻO ka mea nui, koho kēia ʻano hana i ka hoʻopaʻa ʻana i ka lolo a me nā peptides me ka hana lawe ma waena o ka pale cerebrovascular. Ua hoʻohana mākou i nā phages T7 ma muli o ko lākou liʻiliʻi (~60 nm)10 a ua manaʻo ʻia he kūpono lākou no ka lawe ʻana i nā vesicles e ʻae ai i ke keʻa transcellular o ka pale endothelial a/a i ʻole epithelial-medulla. Ma hope o ʻehā pōʻai o ka panning, ua hoʻokaʻawale ʻia nā heluna phage e hōʻike ana i ka hoʻonui ikaika ʻana o ka CSF in vivo a me ka hui ʻana o nā microvessel cerebral. ʻO ka mea nui, ua hiki iā mākou ke hōʻoia i kā mākou mau ʻike ma ka hōʻike ʻana e hiki i nā peptides moho maikaʻi loa i makemake ʻia a synthesized chemically ke lawe i ka ukana protein i loko o ka wai cerebrospinal. ʻO ka mea mua, ua hoʻokumu ʻia nā hopena pharmacodynamic o ka CNS ma ka hoʻohui ʻana i kahi peptide transit alakaʻi me kahi mea hoʻopaʻa o ka peptide BACE1. Ma waho aʻe o ka hōʻike ʻana e hiki i nā hoʻolālā loiloi hana in vivo ke ʻike i nā peptides halihali lolo hou e like me nā mea lawe ukana protein maikaʻi, manaʻo mākou e lilo nā ʻano koho hana like i mea nui i ka ʻike ʻana i nā ala halihali lolo hou.
Ma muli o nā ʻāpana hoʻokumu plaque (PFU), ma hope o ke kaʻina hana hoʻopili phage, ua hoʻolālā ʻia a hana ʻia kahi waihona puke o nā peptides phage T7 linear 12-mer me kahi ʻano like ʻole o kahi 109 (e ʻike i nā Mea Hana a me nā Hana). He mea nui e hoʻomaopopo ua kālailai pono mākou i kēia waihona puke ma mua o ka panning in vivo. ʻO ka hoʻonui ʻana o PCR o nā laʻana waihona puke phage me ka hoʻohana ʻana i nā primers i hoʻololi ʻia ua hana i nā amplicons i pili pono i ka HTS (Kiʻi Hoʻohui 1a). Ma muli o a) nā hewa sequencing HTS11, b) ka hopena ma ke ʻano o nā primers (NNK)1-12, a me c) ke alo o ka phage wild-type (wt) (skeleton inserts) i loko o ka waihona puke standby, ua hoʻokō ʻia kahi kaʻina hana kānana kaʻina e unuhi wale i ka ʻike kaʻina i hōʻoia ʻia (Kiʻi Hoʻohui 1b). Pili kēia mau ʻanuʻu kānana i nā waihona puke sequencing HTS āpau. No ka waihona puke maʻamau, ua loaʻa he 233,868 mau heluhelu, kahi i hala ai ka 39% i nā pae kānana a ua hoʻohana ʻia no ka nānā ʻana i ka waihona puke a me ke koho ʻana no nā pōʻai ma hope (Kiʻi Hoʻohui 1c–e). ʻO ka hapa nui o nā heluhelu he mau hui he 3 mau hui kumu i ka lōʻihi me kahi piko ma 36 mau nucleotides (Kiʻi Hoʻohui 1c), e hōʻoia ana i ka hoʻolālā waihona puke (NNK) 1-12. ʻIke ʻia, ma kahi o 11% o nā lālā waihona puke i loaʻa kahi hoʻokomo PAGISRELVDKL iwi kuamoʻo 12-dimensional (wt), a kokoke i ka hapalua o nā kaʻina (49%) i loaʻa nā hoʻokomo a i ʻole nā ​​holoi ʻana. Ua hōʻoia ka HTS o ka waihona puke i ke ʻano like ʻole o nā peptides i loko o ka waihona puke: ʻoi aku ma mua o 81% o nā kaʻina peptide i loaʻa hoʻokahi wale nō a he 1.5% wale nō i loaʻa ma ≥4 mau kope (Kiʻi Hoʻohui 2a). ʻO nā alapine o nā waikawa amino (aa) ma nā kūlana 12 a pau i loko o ka repertoire ua pili pono me nā alapine i manaʻo ʻia no ka helu o nā codons i hana ʻia e ka repertoire NKK degenerate (Kiʻi Hoʻohui 2b). ʻO ke alapine i ʻike ʻia o nā koena aa i hoʻopili ʻia e kēia mau hoʻokomo ua pili pono me ke alapine i helu ʻia (r = 0.893) (Kiʻi Hoʻohui 2c). ʻO ka hoʻomākaukau ʻana o nā hale waihona puke phage no ka injection e komo pū ana me nā ʻanuʻu o ka hoʻonui ʻana a me ka wehe ʻana o ka endotoxin. Ua hōʻike mua ʻia kēia e hiki ke hōʻemi i ka ʻokoʻa o nā hale waihona puke phage12,13. No laila, ua hoʻonohonoho mākou i kahi hale waihona puke phage i hoʻonui ʻia i ka pā i hala i ka wehe ʻana o ka endotoxin a hoʻohālikelike iā ia me ka hale waihona puke mua e kuhi i ke alapine o AA. Ua ʻike ʻia kahi pilina ikaika (r = 0.995) ma waena o ka waihona mua a me ka waihona i hoʻonui ʻia a hoʻomaʻemaʻe ʻia (Kiʻi Hoʻohui 2d), e hōʻike ana ʻaʻole i hoʻokumu ka hoʻokūkū ma waena o nā clones i hoʻonui ʻia ma nā pā me ka hoʻohana ʻana i ka phage T7 i ka bias nui. Hoʻokumu ʻia kēia hoʻohālikelike ma ke alapine o nā motif tripeptide i kēlā me kēia hale waihona puke, ʻoiai ʻaʻole hiki ke hopu piha ʻia ka ʻokoʻa o nā hale waihona puke (~109) me HTS. Ua hōʻike ka loiloi alapine o aa ma kēlā me kēia kūlana i kahi bias liʻiliʻi e pili ana i ke kūlana ma nā kūlana ʻekolu hope loa o ka repertoire i hoʻokomo ʻia (Kiʻi Hoʻohui 2e). I ka hopena, ua hoʻoholo mākou ua ʻae ʻia ka maikaʻi a me ka ʻokoʻa o ka hale waihona puke a ua ʻike ʻia nā loli liʻiliʻi wale nō i ka ʻokoʻa ma muli o ka hoʻonui ʻana a me ka hoʻomākaukau ʻana o nā hale waihona puke phage ma waena o kekahi mau pōʻai o ke koho ʻana.
Hiki ke hana ʻia ka laʻana wai cerebrospinal serial ma ke ʻoki ʻana i kahi cannula i loko o ka CM o nā ʻiole ʻike e hoʻomaʻamaʻa i ka ʻike ʻana o ka T7 phage i hoʻokomo ʻia i loko o ka intravenous (iv) ma o ka BBB a/a i ʻole BCSFB (Kiʻi 1a-b). Ua hoʻohana mākou i ʻelua mau lima koho kūʻokoʻa (nā lima A a me B) i nā puni ʻekolu mua o ke koho ʻana i loko o ke kino (Kiʻi 1c). Ua hoʻonui mālie mākou i ke koʻikoʻi o ke koho ʻana ma ka hoʻemi ʻana i ka nui o ka phage i hoʻokomo ʻia i nā puni ʻekolu mua o ke koho ʻana. No ka puni ʻehā o ka panning, ua hui pū mākou i nā laʻana mai nā lālā A a me B a ua hana i ʻekolu mau koho kūʻokoʻa hou aʻe. No ke aʻo ʻana i nā waiwai in vivo o nā ʻāpana T7 phage i loko o kēia kumu hoʻohālike, ua hoʻokomo ʻia ka phage wild-type (PAGISRELVDKL master insert) i loko o nā ʻiole ma o ka huelo vein. Ua hōʻike ʻia ka hoʻihoʻi ʻana o nā phages mai ka wai cerebrospinal a me ke koko i nā manawa like ʻole he wikiwiki ka pae hoʻomaʻemaʻe mua o nā phages icosahedral T7 liʻiliʻi mai ka ʻāpana koko (Kiʻi Hoʻohui 3). Ma muli o nā titers i hāʻawi ʻia a me ka nui o ke koko o nā ʻiole, ua helu mākou he 1% wt. phage wale nō mai ka mahele i hāʻawi ʻia i ʻike ʻia i loko o ke koko 10 mau minuke ma hope o ka hoʻokomo ʻana i loko o ke kino. Ma hope o kēia emi wikiwiki mua, ua ana ʻia kahi hoʻomaʻemaʻe mua lohi me ka hapalua ola o 27.7 mau minuke. ʻO ka mea nui, he kakaikahi wale nō nā phages i lawe ʻia mai ka ʻāpana CSF, e hōʻike ana i kahi haʻahaʻa haʻahaʻa no ka neʻe ʻana o ka phage wild-type i loko o ka ʻāpana CSF (Kiʻi Hoʻohui 3). Ma ka awelika, he 1 x 10-3% wale nō nā titers o ka T7 phage i loko o ke koko a me 4 x 10-8% o nā phages i hoʻokomo mua ʻia i ʻike ʻia i loko o ka wai cerebrospinal ma luna o ka manawa laʻana holoʻokoʻa (0-250 min). ʻIke ʻia, ʻo ka hapalua ola (25.7 min) o ka phage wild-type i loko o ka wai cerebrospinal ua like ia me ka mea i ʻike ʻia i loko o ke koko. Hōʻike kēia ʻikepili ua paʻa ka pale e hoʻokaʻawale ana i ka ʻāpana CSF mai ke koko i loko o nā ʻiole i hoʻokomo ʻia i loko o ka CM, e ʻae ana i ke koho ʻana o nā hale waihona puke phage in vivo e ʻike i nā clones i lawe maʻalahi ʻia mai ke koko a i loko o ka ʻāpana CSF.
(a) Ke hoʻonohonoho nei i kahi ʻano hana no ka hoʻihoʻi hou ʻana i ka wai cerebrospinal (CSF) mai kahi loko nui. (b) Kiʻikuhi e hōʻike ana i kahi cellular o ka pale o ka ʻōnaehana nerve waena (CNS) a me ke ʻano koho i hoʻohana ʻia e ʻike i nā peptides e hele ana i ka pale koko-lolo (BBB) ​​​​a me ka pale koko-lolo. (c) Kahe kahe hōʻike phage in vivo. I kēlā me kēia pōʻai o ke koho ʻana, ua hoʻokomo ʻia nā phages (nā mea hōʻike holoholona i loko o nā pua) ma ke ʻano intravenous. ʻElua mau lālā ʻē aʻe kūʻokoʻa (A, B) i mālama ʻia a hiki i ka pōʻai 4 o ke koho ʻana. No nā pōʻai koho 3 a me 4, ua hoʻonohonoho lima ʻia kēlā me kēia clone phage i unuhi ʻia mai CSF. (d) Kinetics o ka phage i hoʻokaʻawale ʻia mai ke koko (nā pōʻai ʻulaʻula) a me ka wai cerebrospinal (nā huinakolu ʻōmaʻomaʻo) i ka wā o ka pōʻai mua o ke koho ʻana i ʻelua mau ʻiole cannulated ma hope o ka hoʻokomo intravenous o ka waihona peptide T7 (2 x 1012 phages/holoholona). Hōʻike nā huinahā polū i ka awelika o ka hoʻohui mua ʻana o ka phage i loko o ke koko, i helu ʻia mai ka nui o ka phage i hoʻokomo ʻia, me ka noʻonoʻo ʻana i ka nui o ke koko. Hōʻike nā huinahā ʻeleʻele i ke kiko o ke kihi o ka laina y i hoʻonui ʻia mai nā hoʻohuihui phage koko. (e,f) Hōʻike i ka alapine pili a me ka hoʻolaha ʻana o nā motif tripeptide āpau i loaʻa i loko o ka peptide. Hōʻike ʻia ka helu o nā motif i loaʻa i nā heluhelu 1000. ʻIke nui ʻia (p < 0.001) nā motif i hoʻonui ʻia me nā kiko ʻulaʻula. (e) ʻO ka scatterplot correlation e hoʻohālikelike ana i ka alapine pili o ka motif tripeptide o ka waihona puke i hoʻokomo ʻia me ka phage i loaʻa mai ke koko mai nā holoholona #1.1 a me #1.2. (f) ʻO ka scatterplot correlation e hoʻohālikelike ana i nā alapine pili o nā motif tripeptide phage holoholona #1.1 a me #1.2 i hoʻokaʻawale ʻia i loko o ke koko a me ka wai cerebrospinal. (g, h) Hōʻike ID Sequence o ka phage i hoʻonui ʻia i ke koko (g) e kūʻē i nā waihona puke i hoʻokomo ʻia a me ka phage i hoʻonui ʻia i CSF (h) e kūʻē i ke koko ma hope o kahi pōʻai o ke koho in vivo i nā holoholona ʻelua. ʻO ka nui o ke code hoʻokahi leka e hōʻike ana i ka pinepine o ka loaʻa ʻana o kēlā waikawa amino ma kēlā kūlana. ʻŌmaʻomaʻo = polar, poni = neutral, polū = basic, ʻulaʻula = acidic a ʻeleʻele = hydrophobic amino acids. Ua hoʻolālā ʻia a hana ʻia ke Kiʻi 1a, b e Eduard Urich.
Ua hoʻokomo mākou i kahi waihona peptide phage i loko o ʻelua mau ʻiole mea hana CM (clades A a me B) a hoʻokaʻawale i ka phage mai ka wai cerebrospinal a me ke koko (Kiʻi 1d). ʻOi aku ka liʻiliʻi o ka hoʻomaʻemaʻe wikiwiki mua o ka waihona i hoʻohālikelike ʻia me ka phage wild-type. ʻO ka hapalua ola awelika o ka waihona i hoʻokomo ʻia i loko o nā holoholona ʻelua he 24.8 mau minuke i ke koko, e like me ka phage wild-type, a me 38.5 mau minuke i ka CSF. Ua hoʻokomo ʻia nā laʻana phage koko a me ka wai cerebrospinal mai kēlā me kēia holoholona i ka HTS a ua kālailai ʻia nā peptides i ʻike ʻia no ke alo o kahi motif tripeptide pōkole. Ua koho ʻia nā motif Tripeptide no ka mea hāʻawi lākou i kahi kumu liʻiliʻi no ka hoʻokumu ʻana i ka hale a me nā pilina peptide-protein14,15. Ua loaʻa iā mākou kahi pilina maikaʻi i ka hoʻolaha ʻana o nā motif ma waena o ka waihona phage i hoʻokomo ʻia a me nā clones i unuhi ʻia mai ke koko o nā holoholona ʻelua (Kiʻi 1e). Hōʻike ka ʻikepili ua hoʻonui iki ʻia ka haku mele ʻana o ka waihona i loko o ka ʻāpana koko. Ua kālailai hou ʻia nā alapine waikawa Amino a me nā kaʻina consensus ma kēlā me kēia kūlana me ka hoʻohana ʻana i kahi hoʻololi o ka polokalamu Weblogo16. ʻO ka mea hoihoi, ua ʻike mākou i kahi hoʻonui ikaika i nā koena glycine koko (Kiʻi 1g). I ka hoʻohālikelike ʻia ʻana o ke koko me nā clones i koho ʻia mai CSF, ua ʻike ʻia ke koho ikaika a me kekahi deselection o nā motifs (Kiʻi 1f), a ua makemake ʻia kekahi mau waikawa amino ma nā kūlana i hoʻoholo mua ʻia ma ka 12-lālā (Kiʻi 1h). ʻO ka mea nui, ua ʻokoʻa nui nā holoholona pākahi i ka wai cerebrospinal, ʻoiai ua ʻike ʻia ka hoʻonui ʻana o ka glycine koko i nā holoholona ʻelua (Kiʻi Hoʻohui 4a-j). Ma hope o ka kānana koʻikoʻi o ka ʻikepili moʻo i loko o ka wai cerebrospinal o nā holoholona #1.1 a me #1.2, ua loaʻa he 964 a me 420 mau peptides 12-mer kū hoʻokahi (Kiʻi Hoʻohui 1d-e). Ua hoʻonui ʻia nā clones phage i hoʻokaʻawale ʻia a ua hoʻokau ʻia i ka lua o ka pōʻai o ke koho in vivo. Ua hoʻokau ʻia nā phage i unuhi ʻia mai ka lua o ka pōʻai o ke koho ʻana i ka HTS i kēlā me kēia holoholona a ua hoʻohana ʻia nā peptides i ʻike ʻia ma ke ʻano he mea hoʻokomo i kahi papahana ʻike motif e kālailai i ka hanana o nā motif tripeptide (Kiʻi 2a, b, ef). I ka hoʻohālikelike ʻia me ke kaʻina hana mua o ka phage i loaʻa mai CSF, ua ʻike mākou i ke koho hou ʻana a me ka wehe ʻana o nā motif he nui ma CSF ma nā lālā A a me B (Kiʻi 2). Ua hoʻopili ʻia kahi algorithm ʻike pūnaewele e hoʻoholo ai inā hōʻike lākou i nā ʻano like ʻole o ke kaʻina kūlike. Ua ʻike ʻia kahi ʻano like maopopo ma waena o nā kaʻina 12-dimensional i loaʻa e CSF ma ke clade ʻē aʻe A (Kiʻi 2c, d) a me ke clade B (Kiʻi 2g, h). Ua hōʻike ka loiloi hui ʻia ma kēlā me kēia lālā i nā ʻano koho like ʻole no nā peptides 12-mer (Kiʻi Hoʻohui 5c, d) a me ka hoʻonui ʻana o ka ratio titer CSF/koko i ka hala ʻana o ka manawa no nā clones hui ʻia ma hope o ka lua o ka puni o ke koho ʻana i hoʻohālikelike ʻia me ka puni mua o ke koho ʻana (Kiʻi Hoʻohui 5e). ).
Ka hoʻonui ʻia ʻana o nā motif a me nā peptides i loko o ka wai cerebrospinal e ʻelua mau pōʻai hope o ke koho hōʻike phage hana in vivo.
Ua hōʻuluʻulu ʻia nā phages wai cerebrospinal āpau i loaʻa mai ka pōʻai mua o kēlā me kēia holoholona (nā holoholona #1.1 a me #1.2), hoʻonui ʻia, hoʻonohonoho ʻia ʻo HT a hoʻokomo hou ʻia (2 x 1010 phages/holoholona) 2 mau ʻiole cannulated SM (#1.1 → #). 2.1 a me 2.2, 1.2 → 2.3 a me 2.4). (a,b,e,f) Nā scatterplots correlation e hoʻohālikelike ana i ka alapine pili o nā motif tripeptide o nā phages i loaʻa mai CSF āpau i nā pōʻai koho mua a me ka lua. ʻO ka alapine pili a me ka hoʻolaha ʻana o nā motif e hōʻike ana i nā tripeptides āpau e pili ana i loaʻa i nā peptides ma nā kuhikuhi ʻelua. Hōʻike ʻia ka helu o nā motif i loaʻa i nā heluhelu 1000. ʻO nā motif i koho nui ʻia (p < 0.001) a i ʻole i hoʻokaʻawale ʻia i loko o kekahi o nā waihona puke i hoʻohālikelike ʻia ua hōʻike ʻia me nā kiko ʻulaʻula. (c, d, g, h) Hōʻike ʻia ka hōʻailona moʻo o nā moʻo lōʻihi 12 waikawa amino waiwai CSF āpau e pili ana i nā pōʻai 2 a me 1 o ke koho in vivo. Hōʻike ka nui o ke code hoʻokahi leka i ka pinepine o ka loaʻa ʻana o kēlā waikawa amino ma kēlā kūlana. No ka hōʻike ʻana i ka logo, ua hoʻohālikelike ʻia ke alapine o nā moʻo CSF ​​i unuhi ʻia mai nā holoholona pākahi ma waena o ʻelua mau pōʻai koho a ua hōʻike ʻia nā moʻo i hoʻonui ʻia i ka pōʻai ʻelua: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 a me (h) #1.2–#2.4. Hōʻike ʻia nā waikawa amino i hoʻonui nui ʻia ma kahi kūlana i hāʻawi ʻia i (c, d) nā holoholona helu 2.1 a me helu 2.2 a i ʻole (g, h) i nā holoholona helu 2.3 a me helu 2.4 i ke kala. ʻŌmaʻomaʻo = polar, poni = neutral, uliuli = basic, ʻulaʻula = acidic a ʻeleʻele = hydrophobic amino acids.
Ma hope o ke kolu o ke koho ʻana, ua ʻike mākou i 124 mau moʻo peptide kū hoʻokahi (#3.1 a me #3.2) mai 332 mau clone phage i hoʻoponopono hou ʻia e CSF i hoʻokaʻawale ʻia mai ʻelua mau holoholona (Kiʻi Hoʻohui 6a). ʻO ka moʻo LGSVS (18.7%) ka mea i loaʻa ka hapa pili kiʻekiʻe loa, ukali ʻia e nā hoʻokomo ʻano hihiu PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), a me SARGSWREIVSLS (2.2%). I ka pōʻai ʻehā hope loa, ua hōʻuluʻulu mākou i ʻelua mau lālā i koho kūʻokoʻa ʻia mai ʻekolu mau holoholona kaʻawale (Kiʻi 1c). No nā 925 mau clone phage i hoʻonohonoho ʻia i loaʻa mai CSF, i ka pōʻai ʻehā ua loaʻa iā mākou he 64 mau moʻo peptide kū hoʻokahi (Kiʻi Hoʻohui 6b), kahi i hāʻule ai ka hapa pili o ka phage ʻano hihiu i 0.8%. ʻO nā clones CSF maʻamau i ka pōʻai ʻehā, ʻo ia ʻo LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) a me RLSSVDSDLSGC (3, 2%). %)). ʻO ka lōʻihi o nā peptides i koho ʻia ma muli o nā hoʻokomo/holoi nucleotide a i ʻole nā ​​codons hoʻōki mua i loko o nā primers waihona puke i ka wā e hoʻohana ai i nā codons degenerate no ka hoʻolālā waihona puke NNK. Hoʻopuka nā codons hoʻōki mua i nā peptides pōkole a ua koho ʻia no ka mea aia iā lākou ke kumu aa maikaʻi. Hiki ke loaʻa nā peptides lōʻihi mai nā hoʻokomo/holoi i loko o nā primers o nā waihona puke synthetic. Hoʻonoho kēia i ka codon hoʻōki i hoʻolālā ʻia ma waho o ka mōlina a heluhelu a hiki i ka wā e ʻike ʻia ai kahi codon hoʻōki hou ma lalo. Ma keʻano laulā, ua helu mākou i nā kumu hoʻonui no nā pōʻai koho ʻehā ma ka hoʻohālikelike ʻana i ka ʻikepili hoʻokomo me ka ʻikepili hoʻopuka laʻana. No ka pōʻai mua o ka nānā ʻana, ua hoʻohana mākou i nā titers phage wild-type ma ke ʻano he kuhikuhi hope ʻole. ʻO ka mea hoihoi, ua ikaika loa ke koho phage maikaʻi ʻole i ka pōʻai CSF mua, akā ʻaʻole i ke koko (Kiʻi 3a), ʻo ia paha ma muli o ka haʻahaʻa o ka hoʻolaha passive o ka hapa nui o nā lālā o ka waihona peptide i loko o ka ʻāpana CSF a i ʻole nā ​​phages pili e mālama pono ʻia a wehe ʻia paha mai ke kahe koko ma mua o nā bacteriophages. Eia naʻe, i ka pōʻai ʻelua o ka panning, ua ʻike ʻia ke koho ikaika o nā phages i CSF ma nā clades ʻelua, e hōʻike ana ua hoʻonui ʻia ka pōʻai ma mua i nā phages e hōʻike ana i nā peptides e hoʻolaha ana i ka lawe ʻana o CSF ​​(Kiʻi 3a). Eia hou, me ka ʻole o ka hoʻonui nui ʻana o ke koko. Eia kekahi i nā pōʻai ʻekolu a me ka hā, ua hoʻonui nui ʻia nā clones phage i CSF. Ke hoʻohālikelike nei i ka alapine pili o kēlā me kēia kaʻina peptide kū hoʻokahi ma waena o nā pōʻai ʻelua hope loa o ke koho ʻana, ua ʻike mākou ua ʻoi aku ka waiwai o nā kaʻina i ka pōʻai ʻehā o ke koho ʻana (Kiʻi 3b). Ua unuhi ʻia he 931 mau motif tripeptide mai nā kaʻina peptide kū hoʻokahi 64 me ka hoʻohana ʻana i nā kuhikuhi peptide ʻelua. Ua nānā pono ʻia nā motif i hoʻonui nui ʻia i ka pōʻai ʻehā no kā lākou mau ʻikepili hoʻonui ma nā pōʻai āpau i hoʻohālikelike ʻia me ka waihona puke i hoʻokomo ʻia (ʻoki: 10% hoʻonui) (Kiʻi Hoʻohui 6c). Ua hōʻike nā ʻano koho maʻamau ua hoʻonui ʻia ka hapa nui o nā motif i aʻo ʻia i nā pōʻai mua a pau o nā lālā koho ʻelua. Eia nō naʻe, ʻo kekahi mau motif (e laʻa me SGL, VSG, LGS GSV) mai ka clade ʻē aʻe A, aʻo nā mea ʻē aʻe (e laʻa me FGW, RTN, WGF, NTR) ua hoʻonui ʻia i ka clade ʻē aʻe B.
Ka hōʻoia ʻana o ka lawe ʻana o ka CSF o nā peptides i hōʻike ʻia e ka phage i hoʻonui ʻia i ka CSF a me nā peptides alakaʻi biotinylated i hoʻohui ʻia i nā ukana streptavidin.
(a) Nā lakio hoʻonui i helu ʻia ma nā puni ʻehā (R1-R4) ma muli o nā titers phage i hoʻokomo ʻia (input = I) (PFU) a me nā titers phage CSF i hoʻoholo ʻia (output = O). Ua helu ʻia nā kumu hoʻonui no nā puni ʻekolu hope loa (R2-R4) ma ka hoʻohālikelike ʻana me ka puni ma mua a me ka puni mua (R1) me ka ʻikepili kaumaha. ʻO nā kaola hāmama he wai cerebrospinal, ʻo nā kaola i hoʻoluʻu ʻia he plasma. (***p<0.001, ma muli o ka hoʻāʻo t a Student). (b) Papa inoa o nā peptides phage nui loa, i hoʻonohonoho ʻia e like me kā lākou ʻāpana pili i nā phages āpau i hōʻiliʻili ʻia ma CSF ma hope o ka puni 4 o ke koho ʻana. ʻO nā clones phage ʻeono maʻamau i hōʻike ʻia i ke kala, helu ʻia a me kā lākou mau kumu hoʻonui ma waena o nā puni 3 a me 4 o ke koho ʻana (nā insets). (c,d) ʻO nā clones phage ʻeono i hoʻonui nui ʻia, nā waihona puke peptide phage hakahaka a me nā mākua mai ka puni 4 i kālailai pākahi ʻia i loko o kahi kumu hoʻohālike laʻana CSF. Ua hōʻiliʻili ʻia nā laʻana CSF a me ke koko ma nā manawa i hōʻike ʻia. (c) Nā nui like o 6 mau clones phage moho (2 x 1010 phages/holoholona), nā phages hakahaka (#1779) (2 x 1010 phages/holoholona) a me nā waihona peptide phage waihona (2 x 1012 phages/holoholona) Hoʻokomo ʻia ma ka liʻiliʻi he 3 CM i ka holoholona cannulated ma o ka huelo vein. Hōʻike ʻia ka pharmacokinetics CSF o kēlā me kēia clone phage i hoʻokomo ʻia a me ka waihona peptide phage i ka hala ʻana o ka manawa. (d) hōʻike i ka awelika CSF/koko no nā phages/mL i hoʻōla ʻia i ka manawa laʻana. (e) Ua hoʻopili ʻia ʻehā mau peptides alakaʻi synthetic a me hoʻokahi mana scrambled me biotin i streptavidin ma o kā lākou N-terminus (hōʻike tetramer) a ukali ʻia e ka injection (tail vein iv, 10 mg streptavidin/kg). Ma ka liʻiliʻi he ʻekolu mau ʻiole intubated (N = 3). ). Ua hōʻiliʻili ʻia nā laʻana CSF ma nā manawa i hōʻike ʻia a ua ana ʻia nā ʻāpana streptavidin e CSF anti-streptavidin ELISA (nd = ʻaʻole i ʻike ʻia). (*p<0.05, **p<0.01, ***p<0.001, ma muli o ka hoʻāʻo ANOVA). (f) Hoʻohālikelike ʻia o ke kaʻina amino acid o ka clone peptide phage waiwai nui loa #2002 (poni) me nā clones peptide phage ʻē aʻe i koho ʻia mai ka pōʻai 4 o ke koho ʻana. Ua hoʻopaʻa ʻia nā ʻāpana amino acid like a like.
ʻO nā phages i hoʻonui ʻia i ka pōʻai ʻehā (Kiʻi 3b), ua koho ʻia ʻeono mau clones moho no ka nānā hou ʻana i kēlā me kēia kanaka i ke kumu hoʻohālike laʻana CSF. Ua hoʻokomo ʻia nā nui like o ʻeono mau hale waihona puke peptide phage moho, phage hakahaka (ʻaʻohe hoʻokomo) a me prophage i loko o ʻekolu mau holoholona CM cannulated, a ua hoʻoholo ʻia nā pharmacokinetics ma CSF (Kiʻi 3c) a me ke koko (Kiʻi Hoʻohui 7) assays. Ua kuhikuhi nā clones phage āpau i hoʻāʻo ʻia i ka ʻāpana CSF ma kahi pae 10-1000 mau manawa kiʻekiʻe ma mua o ka phage kaohi hakahaka (#1779). No ka laʻana, ʻo nā clones #2020 a me #2077 he 1000 mau manawa kiʻekiʻe aʻe nā titers CSF ma mua o ka phage kaohi. He ʻokoʻa ka ʻikepili pharmacokinetic o kēlā me kēia peptide i koho ʻia, akā he hiki ke hoʻokipa CSF kiʻekiʻe ko lākou āpau. Ua ʻike mākou i ka emi mau ʻana i ka hala ʻana o ka manawa no nā clones #1903 a me #2011, ʻoiai no nā clones #2077, #2002 a me #2009, ʻo ka piʻi ʻana i loko o nā minuke 10 mua e hōʻike paha i ka lawe ʻana i ka hana akā pono e hōʻoia ʻia. Ua paʻa nā Clones #2020, #2002, a me #2077 ma nā pae kiʻekiʻe, ʻoiai ua emi mālie ka nui o ka CSF o ka clone #2009 ma hope o ka piʻi mua ʻana. A laila ua hoʻohālikelike mākou i ka alapine pili o kēlā me kēia moho CSF ​​me kona nui koko (Kiʻi 3d). Ua hōʻike ka pilina o ka titer awelika o kēlā me kēia moho CSF ​​me kona titer koko i nā manawa laʻana āpau ua hoʻonui nui ʻia ʻekolu o nā moho ʻeono i ka CSF koko. ʻO ka mea hoihoi, ua hōʻike ka clone #2077 i ke kūpaʻa koko kiʻekiʻe (Kiʻi Hoʻohui 7). No ka hōʻoia ʻana he hiki i nā peptides iā lākou iho ke lawe ikaika i nā ukana ʻē aʻe ma mua o nā ʻāpana phage i loko o ka ʻāpana CSF, ua synthesize mākou i ʻehā mau peptides alakaʻi i loaʻa me ka biotin ma ka N-terminus kahi e hoʻopili ai nā peptides i ka ʻāpana phage. Ua hoʻohui ʻia nā peptides Biotinylated (nos. 2002, 2009, 2020 a me 2077) me streptavidin (SA) e loaʻa ai nā ʻano multimeric e hoʻohālike iki ana i ke ʻano o ka phage. Ua ʻae pū kēia ʻano iā mākou e ana i ka hōʻike ʻana o SA i loko o ke koko a me ka wai cerebrospinal e like me nā peptides protein lawe ukana. ʻO ka mea nui, hiki ke hana hou ʻia nā ʻikepili phage i ka wā i lawelawe ʻia ai nā peptides synthetic i kēia ʻano SA-conjugated (Kiʻi 3e). ʻOi aku ka liʻiliʻi o ka hōʻike mua ʻana o nā peptides i kāwili ʻia a ʻoi aku ka wikiwiki o ka hoʻomaʻemaʻe CSF me nā pae i ʻike ʻole ʻia i loko o 48 mau hola. No ka loaʻa ʻana o ka ʻike i nā ala lawe o kēia mau clones peptide phage i loko o ka hakahaka CSF, ua kālailai mākou i ka localization o nā hits peptide phage pākahi me ka hoʻohana ʻana i ka immunohistochemistry (IHC) e ʻike pololei i nā ʻāpana phage 1 hola ma hope o ka hoʻokomo ʻana i loko o ke kino. ʻIke ʻia, hiki ke ʻike ʻia nā clones #2002, #2077, a me #2009 e ka hoʻoluʻu ikaika ʻana i nā capillaries o ka lolo, ʻoiai ʻaʻole i ʻike ʻia ka phage control (#1779) a me ka clone #2020 (Kiʻi Hoʻohui 8). Hōʻike kēia e hāʻawi kēia mau peptides i ka hopena ma ka lolo ma ke keʻa ʻana i ka BBB. Pono ka loiloi kikoʻī hou aku e hoʻāʻo i kēia kuhiakau, no ka mea, pili pū paha ke ala BSCFB. I ka hoʻohālikelike ʻana i ka moʻo amino acid o ka clone i hoʻonui nui ʻia (#2002) me nā peptides i koho ʻia, ua ʻike ʻia he like kekahi o lākou me nā hoʻonui amino acid like, e hōʻike ana paha i kahi ʻano halihali like (Kiʻi 3f).
Ma muli o kona ʻano plasma kū hoʻokahi a me ka piʻi nui ʻana o ka CSF i ka hala ʻana o ka manawa, ua ʻimi hou ʻia ka clone hōʻike phage #2077 ma luna o kahi manawa lōʻihi he 48 hola a ua hiki ke hana hou i ka piʻi wikiwiki ʻana o ka CSF i ʻike ʻia ma ka pilina me nā pae SA i hoʻomau ʻia (Kiʻi 4a). E pili ana i nā clones phage ʻē aʻe i ʻike ʻia, ua hoʻoluʻu ikaika ʻia ʻo #2077 no nā capillaries o ka lolo a hōʻike i ka colocalization koʻikoʻi me ka capillary marker lectin i ka wā i nānā ʻia ai ma ka hoʻonā kiʻekiʻe a me kekahi mau hoʻoluʻu ʻana i ka hakahaka parenchymal (Kiʻi 4b). No ka noiʻi ʻana inā hiki ke loaʻa nā hopena pharmacological i hoʻopili ʻia e ka peptide i loko o ka CNS, ua hana mākou i kahi hoʻokolohua kahi i hui pū ʻia ai nā mana biotinylated o i) ka peptide transit #2077 a me ii) ka peptide inhibitor BACE1 me SA ma nā lakio ʻelua like ʻole. No ka hui pū ʻana hoʻokahi ua hoʻohana wale mākou i ka inhibitor peptide BACE1 a no kekahi ua hoʻohana mākou i ka lakio 1:3 o ka inhibitor peptide BACE1 i ka peptide #2077. Ua lawelawe ʻia nā hāpana ʻelua ma ke ʻano intravenous a ua ana ʻia nā pae koko a me nā wai cerebrospinal o ka beta-amyloid peptide 40 (Abeta40) i ka hala ʻana o ka manawa. Ua ana ʻia ʻo Abeta40 ma CSF no ka mea e hōʻike ana i ka pale ʻana o BACE1 i loko o ka parenchyma o ka lolo. E like me ka mea i manaʻo ʻia, ua hōʻemi nui nā ʻano paʻakikī ʻelua i nā pae koko o Abeta40 (Kiʻi 4c, d). Eia naʻe, ʻo nā hāpana wale nō i loaʻa ka hui ʻana o ka peptide no. 2077 a me kahi mea hoʻopaʻa o ka peptide BACE1 i hoʻohui ʻia i SA i hoʻoulu ai i ka emi nui ʻana o Abeta40 i loko o ka wai cerebrospinal (Kiʻi 4c). Hōʻike ka ʻikepili hiki i ka peptide no. 2077 ke lawe i ka protein 60 kDa SA i loko o ka CNS a hoʻoulu pū i nā hopena pharmacological me nā mea hoʻopaʻa SA-conjugated o ka peptide BACE1.
(a) ʻO ka inikini Clonal (2 × 10 phages/holoholona) o ka T7 phage e hōʻike ana i nā ʻikepili pharmacokinetic lōʻihi o ka CSF peptide #2077 (RLSSVDSDLSGC) a me ka phage mana i hoʻokomo ʻole ʻia (#1779) ma ka liʻiliʻi he ʻekolu mau ʻiole i hoʻokomo ʻia i loko o ka CM. (b) Kiʻi microscopic confocal o nā microvessels cortical i hōʻike ʻia i loko o nā ʻiole i hoʻokomo ʻia i ka phage (2 × 10 10 phages/holoholona) e hōʻike ana i ka counterstaining o ka peptide #2077 a me nā kīʻaha koko (lectin). Ua hāʻawi ʻia kēia mau clones phage i 3 mau ʻiole a ua ʻae ʻia e kaʻapuni no 1 hola ma mua o ka perfusion. Ua ʻoki ʻia nā lolo a ua hoʻoluʻu ʻia me nā antibodies polyclonal FITC-labeled e kūʻē i ka T7 phage capsid. He ʻumi mau minuke ma mua o ka perfusion a me ka hoʻopaʻa ʻana ma hope, ua hāʻawi ʻia ka lectin DyLight594-labeled intravenously. ʻO nā kiʻi fluorescent e hōʻike ana i ka hoʻoluʻu lectin (ʻulaʻula) o ka ʻaoʻao luminal o nā microvessels a me nā phages (ʻōmaʻomaʻo) i loko o ka lumen o nā capillaries a me ka ʻiʻo lolo perivascular. Pili ka pā unahi i 10 µm. (c, d) Ua hoʻopili ʻia ka peptide inhibitory BACE1 Biotinylated wale nō a i ʻole i hui pū ʻia me ka peptide transit biotinylated #2077 i ka streptavidin a ukali ʻia e ka injection intravenous o ʻekolu mau ʻiole CM cannulated (10 mg streptavidin/kg). Ua ana ʻia ka hōʻemi ʻana o ka peptide inhibitor BACE1 ma Aβ40 e Aβ1-40 ELISA i loko o ke koko (ʻulaʻula) a me ka wai cerebrospinal (ʻalani) ma nā manawa i hōʻike ʻia. No ka maopopo maikaʻi ʻana, ua kahakiʻi ʻia kahi laina kiko ma ka pakuhi ma kahi unahi o 100%. (c) Ka emi ʻana o ka pakeneka o Aβ40 i loko o ke koko (nā huinakolu ʻulaʻula) a me ka wai cerebrospinal (nā huinakolu ʻalani) i loko o nā ʻiole i mālama ʻia me ka streptavidin i hoʻohui ʻia i ka peptide transit #2077 a me ka peptide inhibitory BACE1 ma kahi lakio 3:1. (d) Ka emi ʻana o ka pakeneka o ke koko Aβ40 (nā pōʻai ʻulaʻula) a me ka wai cerebrospinal (nā pōʻai ʻalani) o nā ʻiole i mālama ʻia me ka streptavidin i hoʻohui ʻia i kahi peptide inhibitory BACE1 wale nō. ʻO ka nui o Aβ i loko o ka mana he 420 pg/ml (ʻokoʻa maʻamau = 101 pg/ml).
Ua hoʻopili pono ʻia ka hōʻike ʻana o Phage ma kekahi mau wahi o ka noiʻi biomedical17. Ua hoʻohana ʻia kēia ʻano hana no nā haʻawina ʻokoʻa vascular in vivo18,19 a me nā haʻawina e pili ana i nā kīʻaha lolo20,21,22,23,24,25,26. Ma kēia haʻawina, ua hoʻonui mākou i ka hoʻohana ʻana o kēia ʻano koho ʻaʻole wale i ka ʻike pololei ʻana o nā peptides e pili ana i nā kīʻaha lolo, akā i ka ʻike ʻana i nā moho me nā waiwai lawe ikaika e hele i ke ālai koko-lolo. Ke wehewehe nei mākou i kēia manawa i ka hoʻomohala ʻana o kahi kaʻina koho in vivo i nā ʻiole CM intubated a hōʻike i kona hiki ke ʻike i nā peptides me nā waiwai homing CSF. Me ka hoʻohana ʻana i ka phage T7 e hōʻike ana i kahi waihona puke o nā peptides random 12-mer, ua hiki iā mākou ke hōʻike he liʻiliʻi ka phage T7 (ma kahi o 60 nm ke anawaena)10 e hoʻololi ʻia i ka ālai koko-lolo, no laila e hele pololei ana i ka ālai koko-lolo a i ʻole choroid plexus. Ua ʻike mākou ʻo ka ʻohi ʻana o ka CSF mai nā ʻiole CM cannulated he ʻano hana nānā pono i loko o vivo, a ʻo ka phage i unuhi ʻia ʻaʻole wale i hoʻopaʻa ʻia i ka vasculature akā hana pū kekahi ma ke ʻano he mea lawe ma waena o ka pale koko-lolo. Eia kekahi, ma ka hōʻiliʻili ʻana i ke koko a me ka hoʻopili ʻana iā HTS i ka CSF a me nā phages i loaʻa mai ke koko, ua hōʻoia mākou ʻaʻole i hoʻopilikia ʻia kā mākou koho ʻana o CSF ​​e ka hoʻonui ʻana i ke koko a i ʻole ke kūpono no ka hoʻonui ʻana ma waena o nā pōʻai koho. Eia naʻe, ʻo ka ʻāpana koko kekahi hapa o ke kaʻina koho, ʻoiai ʻo nā phages i hiki ke hiki i ka ʻāpana CSF pono e ola a kaʻapuni i loko o ke kahe koko no ka manawa lōʻihi e hoʻonui ai iā lākou iho i loko o ka lolo. I mea e unuhi ai i ka ʻike kaʻina hilinaʻi mai ka ʻikepili HTS maka, ua hoʻokō mākou i nā kānana i hoʻololi ʻia i nā hewa kaʻina hana kikoʻī i ke kaʻina hana loiloi. Ma ka hoʻohui ʻana i nā palena kinetic i loko o ke ʻano nānā, ua hōʻoia mākou i ka pharmacokinetics wikiwiki o nā phages T7 wild-type (t½ ~ 28 min) i loko o ke koko24, 27, 28 a ua hoʻoholo pū i ko lākou hapalua-ola i loko o ka wai cerebrospinal (t½ ~ 26 min) i kēlā me kēia minuke). ʻOiai nā ʻano pharmacokinetic like i loko o ke koko a me ka CSF, ʻo 0.001% wale nō o ka nui o ke koko o ka phage i hiki ke ʻike ʻia ma CSF, e hōʻike ana i ka neʻe haʻahaʻa o ka hope o ka wild-type T7 phage ma waena o ka pale koko-lolo. Hōʻike kēia hana i ke koʻikoʻi o ka pōʻai mua o ke koho ʻana i ka wā e hoʻohana ai i nā hoʻolālā panning in vivo, ʻoi aku hoʻi no nā ʻōnaehana phage i hoʻomaʻemaʻe koke ʻia mai ke kaʻapuni, ʻoiai he kakaikahi nā clones e hiki ke hōʻea i ka ʻāpana CNS. No laila, i ka pōʻai mua, ua nui loa ka emi ʻana o ka ʻokoʻa o ka waihona puke, ʻoiai he helu palena wale nō o nā clones i hōʻiliʻili ʻia ma kēia kumu hoʻohālike CSF paʻa loa. ʻO kēia hoʻolālā panning in vivo i hoʻokomo i kekahi mau ʻanuʻu koho e like me ka hōʻiliʻili ikaika i loko o ka ʻāpana CSF, ke ola ʻana o ka clone i loko o ka ʻāpana koko, a me ka wehe wikiwiki ʻana o nā clones phage T7 mai ke koko i loko o nā minuke 10 mua (Kiʻi 1d a me ke Kiʻi Hoʻohui 4M). ). No laila, ma hope o ka pōʻai mua, ua ʻike ʻia nā clones phage like ʻole ma CSF, ʻoiai ua hoʻohana ʻia ka waihona mua like no nā holoholona pākahi. Hōʻike kēia he nui nā ʻanuʻu koho koʻikoʻi no nā hale waihona puke kumu me ka nui o nā lālā hale waihona puke e hopena i ka emi nui ʻana o ka ʻokoʻa. No laila, e lilo nā hanana kaulele i ʻāpana koʻikoʻi o ke kaʻina koho mua, e hoʻohuli nui ana i ka hopena. He mea hiki paha i ka nui o nā clones i loko o ka hale waihona puke mua ke ʻano hoʻonui CSF like loa. Eia nō naʻe, ʻoiai ma lalo o nā kūlana hoʻokolohua like, ʻokoʻa paha nā hopena koho ma muli o ka liʻiliʻi o kēlā me kēia clone kikoʻī i loko o ka waihona mua.
ʻOkoʻa nā motif i hoʻonui ʻia i loko o ka CSF mai nā mea i loko o ke koko. ʻO ka mea hoihoi, ua ʻike mākou i ka neʻe mua ʻana i nā peptides waiwai glycine i loko o ke koko o nā holoholona pākahi. (Kiʻi 1g, Nā Kiʻi Hoʻohui. 4e, 4f). ʻOi aku ka paʻa o nā peptides glycine i loaʻa i ka phage a emi iki ka hiki ke lawe ʻia mai ke kaʻapuni. Eia naʻe, ʻaʻole i ʻike ʻia kēia mau peptides waiwai glycine i loko o nā laʻana wai cerebrospinal, e hōʻike ana ua hele nā ​​​​hale waihona puke i mālama ʻia ma o ʻelua mau ʻanuʻu koho like ʻole: hoʻokahi i loko o ke koko a ʻo kekahi i ʻae ʻia e hōʻiliʻili i loko o ka wai cerebrospinal. Ua hoʻāʻo nui ʻia nā clones i hoʻonui ʻia i ka CSF i loaʻa mai ka pōʻai ʻehā o ke koho ʻana. Ua hōʻoia ʻia ua hoʻonui ʻia nā clones i hoʻāʻo pākahi ʻia i ka CSF i hoʻohālikelike ʻia me ka phage mana blank. Ua nānā ʻia kekahi peptide hit (#2077) me nā kikoʻī hou aku. Ua hōʻike ia i kahi hapalua ola plasma lōʻihi i hoʻohālikelike ʻia me nā hits ʻē aʻe (Kiʻi 3d a me ke Kiʻi Hoʻohui 7), a he mea hoihoi, aia i loko o kēia peptide kahi cysteine ​​​​residue ma ka C-terminus. Ua hōʻike ʻia nei ua hiki i ka hoʻohui ʻana o ka cysteine ​​​​i nā peptides ke hoʻomaikaʻi i kā lākou mau waiwai pharmacokinetic ma o ka hoʻopaʻa ʻana i ka albumin 29. ʻAʻole ʻike ʻia kēia no ka peptide #2077 i kēia manawa a pono e noiʻi hou aku. Ua hōʻike kekahi mau peptides i kahi valence-dependency i ka hoʻonui ʻana o ka CSF (ʻaʻole i hōʻike ʻia ka ʻikepili), kahi e pili ana paha i ke ʻano o ka ʻili i hōʻike ʻia o ka capsid T7. Ua hōʻike ka ʻōnaehana T7 a mākou i hoʻohana ai i 5-15 mau kope o kēlā me kēia peptide no kēlā me kēia ʻāpana phage. Ua hana ʻia ʻo IHC ma nā clones phage alakaʻi moho i hoʻokomo ʻia i loko o ka intravenous i loko o ka cerebral cortex o nā ʻiole (Kiʻi Hoʻohui 8). Ua hōʻike ka ʻikepili ma ka liʻiliʻi he ʻekolu mau clones (No. 2002, No. 2009 a me No. 2077) i launa pū me ka BBB. Ke waiho nei e hoʻoholo ʻia inā hopena kēia pilina BBB i ka hōʻiliʻili ʻana o CSF ​​a i ʻole ka neʻe ʻana o kēia mau clones pololei i ka BCSFB. ʻO ka mea nui, hōʻike mākou e mālama nā peptides i koho ʻia i ko lākou hiki ke lawe i ka CSF i ka wā i synthesized a hoʻopaʻa ʻia i ka ukana protein. ʻO ka hoʻopaʻa ʻana o nā peptides biotinylated N-terminal iā SA e hana hou i nā hopena i loaʻa me kā lākou mau clones phage i loko o ke koko a me ka wai cerebrospinal (Kiʻi 3e). ʻO ka mea hope loa, hōʻike mākou e hiki i ka peptide alakaʻi #2077 ke hoʻolaha i ka hana lolo o kahi mea hoʻopaʻa peptide biotinylated o BACE1 i hoʻohui ʻia iā SA, e hana ana i nā hopena pharmacodynamic koʻikoʻi i loko o ka CNS ma ka hoʻemi nui ʻana i nā pae Abeta40 ma CSF (Kiʻi 4). ʻAʻole hiki iā mākou ke ʻike i kekahi homologues i loko o ka waihona ʻikepili ma ka hana ʻana i kahi ʻimi homology peptide o nā hits āpau. He mea nui e hoʻomaopopo ʻo ka nui o ka waihona puke T7 ma kahi o 109, ʻoiai ʻo ka nui o ka waihona puke theoretical no 12-mers he 4 x 1015. No laila, ua koho wale mākou i kahi hapa liʻiliʻi o ka hakahaka diversity o ka waihona puke peptide 12-mer, ʻo ia hoʻi ke ʻano hiki ke ʻike ʻia nā peptides i ʻoi aku ka maikaʻi ma ka loiloi ʻana i ka hakahaka kaʻina pili o kēia mau hits i ʻike ʻia. ʻO ke kuhiakau, ʻo kekahi o nā kumu i loaʻa ʻole ai iā mākou kekahi homologues kūlohelohe o kēia mau peptides ʻo ia ka deselection i ka wā o ka evolution e pale ai i ke komo ʻole ʻia o kekahi mau motif peptide i loko o ka lolo.
I ka hui pū ʻia ʻana, hāʻawi kā mākou mau hopena i kahi kumu no ka hana e hiki mai ana e ʻike a wehewehe i nā ʻōnaehana halihali o ka pale cerebrovascular in vivo me nā kikoʻī hou aku. ʻO ke hoʻonohonoho kumu o kēia ʻano hana e pili ana i kahi hoʻolālā koho hana ʻaʻole wale e ʻike i nā clones me nā waiwai hoʻopaʻa vascular cerebral, akā hoʻokomo pū kekahi i kahi hana koʻikoʻi kahi e loaʻa ai i nā clones holomua ka hana kūloko e hele i nā pale olaola in vivo i loko o ka ʻāpana CNS. ʻo ia ka wehewehe ʻana i ke ʻano o ka halihali ʻana o kēia mau peptides a me ko lākou makemake no ka hoʻopaʻa ʻana i ka microvasculature kikoʻī i ka ʻāpana lolo. Hiki i kēia ke alakaʻi i ka ʻike ʻana i nā ala hou no ka halihali ʻana o ka BBB a me nā mea loaʻa. Manaʻo mākou e hiki i nā peptides i ʻike ʻia ke hoʻopaʻa pololei i nā mea loaʻa cerebrovascular a i ʻole i nā ligands e kaapuni ana i lawe ʻia ma o ka BBB a i ʻole BCSFB. E noiʻi hou ʻia nā vectors peptide me ka hana halihali CSF i ʻike ʻia ma kēia hana. Ke noiʻi nei mākou i kēia manawa i ka ʻike kikoʻī o ka lolo o kēia mau peptides no ko lākou hiki ke hele i ka BBB a/a i ʻole BCSFB. E lilo kēia mau peptides hou i mau mea hana waiwai nui no ka ʻike ʻana i nā mea loaʻa hou a i ʻole nā ​​​​ala hou a no ka hoʻomohala ʻana i nā kahua hou a kūpono loa no ka lawe ʻana i nā macromolecules, e like me nā biologics, i ka lolo.
E hoʻokomo i ka cisterna nui (CM) me ka hoʻohana ʻana i ka hoʻololi ʻana o ke ʻano hana i wehewehe mua ʻia. Ua kau ʻia nā ʻiole Wistar i hoʻopaʻa ʻia (200-350 g) ma luna o kahi mea hana stereotaxic a ua hana ʻia kahi ʻoki waena ma luna o ke poʻo i kahili ʻia a hoʻomākaukau ʻia me ka aseptically e hōʻike i ke poʻo. E wili i ʻelua mau lua ma kahi o ke kāʻei luna a hoʻopaʻa i nā wili hoʻopaʻa i loko o nā lua. Ua wili ʻia kekahi lua hou ma ka ʻaoʻao occipital crest no ke alakaʻi stereotactic o kahi cannula kila kila i loko o ka CM. E hoʻopili i ka sima niho a puni ka cannula a hoʻopaʻa me nā wili. Ma hope o ka photo-curing a me ka paʻakikī ʻana o ka sima, ua pani ʻia ka ʻeha ʻili me ka 4/0 supramid suture. Ua hōʻoia ʻia ke kau kūpono o ka cannula e ka leakage spontaneous o ka wai cerebrospinal (CSF). Wehe i ka ʻiole mai ka mea hana stereotaxic, e loaʻa i ka mālama postoperative kūpono a me ka hoʻokele ʻeha, a e ʻae iā ia e hoʻōla no hoʻokahi pule a hiki i ka ʻike ʻia ʻana o nā hōʻailona o ke koko i loko o ka wai cerebrospinal. Ua loaʻa nā ʻiole Wistar (Crl:WI/Han) mai Charles River (France). Ua mālama ʻia nā ʻiole a pau ma lalo o nā kūlana kūikawā ʻaʻohe pathogen. Ua ʻāpono ʻia nā hoʻokolohua holoholona a pau e ke Keʻena Holoholona o ke kūlanakauhale ʻo Basel, Switzerland, a ua hana ʻia e like me ka Laikini Holoholona Helu 2474 (Loiloi o ka Halihali ʻAna o ka Lolo ma ke Ana ʻana i nā Pae o nā Moho Lapaʻau i loko o ka Wai Cerebrospinal a me ka Lolo o ka ʻIole).
E mālama mālie i ka ʻiole me ka cannula CM ma ka lima. E wehe i ka Datura mai ka cannula a hōʻiliʻili i 10 µl o ka wai cerebrospinal e kahe ana. Ma muli o ka hoʻopilikia ʻia ʻana o ka patency o ka cannula, ʻo nā laʻana wai cerebrospinal maopopo wale nō me ka ʻole o nā hōʻike o ka haumia koko a i ʻole ka hoʻololi ʻana i ke kala i hoʻokomo ʻia i loko o kēia haʻawina. Ma ka manawa like, ua lawe ʻia ma kahi o 10-20 μl o ke koko mai kahi ʻoki liʻiliʻi ma ka piko o ka huelo i loko o nā paipu me ka heparin (Sigma-Aldrich). Ua hōʻiliʻili ʻia ka CSF a me ke koko i nā manawa like ʻole ma hope o ka hoʻokomo ʻana i ka intravenous o ka T7 phage. Ua hoʻolei ʻia ma kahi o 5-10 μl o ka wai ma mua o ka hōʻiliʻili ʻia ʻana o kēlā me kēia laʻana CSF, kahi e kūlike ana i ka nui make o ka catheter.
Ua hana ʻia nā waihona puke me ka hoʻohana ʻana i ka vector T7Select 10-3b e like me ka mea i wehewehe ʻia ma ka manual ʻōnaehana T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). I ka pōkole, ua hoʻohui ʻia kahi hoʻokomo DNA 12-mer ma ke ʻano penei:
Ua hoʻohana ʻia ke codon NNK e pale aku i nā codons pāpālua a me ka hoʻonui ʻia ʻana o ka waikawa amino i loko o ka hoʻokomo. ʻO N kahi lakio equimolar i hui lima ʻia o kēlā me kēia nucleotide, a ʻo K kahi lakio equimolar i hui lima ʻia o ka adenine a me nā cytosine nucleotides. Ua hoʻololi ʻia nā ʻāpana kaula hoʻokahi i DNA kaula pālua ma o ka hoʻoulu hou ʻana me dNTP (Novagen) a me ka enzyme Klenow (New England Biolabs) ma Klenow buffer (New England Biolabs) no 3 mau hola ma 37°C. Ma hope o ka hopena, ua hoʻihoʻi ʻia ka DNA kaula pālua e ka EtOH precipitation. Ua ʻeli ʻia ka DNA hopena me nā enzymes restriction EcoRI a me HindIII (ʻelua mai Roche). A laila ua hoʻopili ʻia ka hoʻokomo i ʻoki ʻia a hoʻomaʻemaʻe ʻia (QIAquick, Qiagen) (T4 ligase, New England Biolabs) i loko o kahi vector T7 i ʻoki mua ʻia ma hope o ka waikawa amino 348 o ka gene 10B capsid. Ua hoʻoulu ʻia nā hopena ligation ma 16°C no 18 mau hola ma mua o ka hoʻopili ʻana i loko o vitro. Ua hana ʻia ka hoʻopili ʻana o ka phage in vitro e like me nā kuhikuhi i hāʻawi ʻia me ka T7Select 10-3b cloning kit (Novagen) a ua hoʻonui ʻia ka hopena hoʻopili i hoʻokahi manawa i ka lysis me ka hoʻohana ʻana iā Escherichia coli (BLT5615, Novagen). Ua centrifuged ʻia nā lysates, titrated a hoʻopaʻahau ʻia ma -80° C. ma ke ʻano he hopena waihona o ka glycerol.
Hoʻonui pololei ʻia ka PCR o nā ʻāpana loli phage i hoʻonui ʻia i loko o ka hupa a i ʻole ka pā me ka hoʻohana ʻana i nā primer fusion 454/Roche-amplicon ponoʻī. Loaʻa i ka primer fusion mua nā moʻo e hoʻopuni ana i ka ʻāpana loli (NNK) 12 (kikoʻī-template), GS FLX Titanium Adapter A, a me kahi moʻo kī waihona puke ʻehā-kumu (TCAG) (Kiʻi Hoʻohui 1a):
Loaʻa pū i ka reverse fusion primer ka biotin i hoʻopili ʻia i nā beads hopu a me ka GS FLX Titanium Adapter B e pono ai no ka clonal amplification i ka wā o ka emulsion PCR:
A laila ua hoʻokomo ʻia nā amplicons i ka 454/Roche pyrosequencing e like me ke kaʻina hana 454 GS-FLX Titanium. No ka hoʻonohonoho lima ʻana o Sanger (Applied Biosystems Hitachi 3730 xl DNA Analyzer), ua hoʻonui ʻia ka T7 phage DNA e ka PCR a ua hoʻonohonoho ʻia me nā hui primer penei:
Ua hoʻokomo ʻia nā mea hoʻokomo mai nā plaque pākahi i ka hoʻonui ʻana o PCR me ka hoʻohana ʻana i ka Roche Fast Start DNA Polymerase Kit (e like me nā kuhikuhi a ka mea hana). Hana i kahi hoʻomaka wela (10 min ma 95 °C) a me 35 mau pōʻaiapuni hoʻonui (50 s ma 95 °C, 1 min ma 50 °C, a me 1 min ma 72 °C).
Ua hoʻonui ʻia nā phage mai nā hale waihona puke, nā phage wild-type, nā phage i hoʻopakele ʻia mai CSF a me ke koko, a i ʻole nā ​​​​clones pākahi i loko o Escherichia coli BL5615 i loko o ka wai TB (Sigma Aldrich) a i ʻole i loko o nā kīʻaha 500 cm2 (Thermo Scientific) no 4 hola ma 37°C. Ua unuhi ʻia nā phage mai nā papa ma ka holoi ʻana i nā papa me ka Tris-EDTA buffer (Fluka Analytical) a i ʻole ma ka hōʻiliʻili ʻana i nā plaques me nā wēlau pipette sterile. Ua hoʻokaʻawale ʻia nā phage mai ka supernatant moʻomeheu a i ʻole ka buffer extraction me hoʻokahi pōʻai o ka polyethylene glycol (PEG 8000) precipitation (Promega) a ua hoʻokuʻu hou ʻia i loko o ka buffer Tris-EDTA.
Ua hoʻokomo ʻia ka phage i hoʻonui ʻia i 2-3 mau puni o ka wehe ʻana o ka endotoxin me ka hoʻohana ʻana i nā beads wehe endotoxin (Miltenyi Biotec) ma mua o ka inikini intravenous (IV) (500 μl/holoholona). I ka puni mua, ua hoʻokomo ʻia nā phages 2 × 1012; i ka lua, 2 × 1010 phages; i nā puni koho ʻekolu a me ka hā, 2 × 109 phages no kēlā me kēia holoholona. Ua hoʻoholo ʻia ka nui o ka phage i loko o ka CSF a me nā laʻana koko i hōʻiliʻili ʻia ma nā manawa i hōʻike ʻia e ka helu ʻana o ka plaque e like me nā kuhikuhi a ka mea hana (manual ʻōnaehana T7Select). Ua hana ʻia ke koho ʻana o ka phage ma ka inikini intravenous o nā waihona puke i hoʻomaʻemaʻe ʻia i loko o ke aʻa huelo a i ʻole ma ka inikini hou ʻana o ka phage i unuhi ʻia mai CSF mai ka puni koho mua, a ua hana ʻia nā ʻohiʻohi ma hope ma 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, a me 240 min pakahi nā laʻana CSF a me ke koko. Ua mālama ʻia he ʻehā mau puni o ka panning in vivo kahi i mālama ʻokoʻa ʻia ai nā lālā ʻelua i koho ʻia a kālailai ʻia i nā puni ʻekolu mua o ke koho ʻana. Ua hoʻokomo ʻia nā hoʻokomo phage āpau i unuhi ʻia mai CSF mai nā puni ʻelua mua o ke koho ʻana i ka 454/Roche pyrosequencing, ʻoiai ua hoʻonohonoho lima ʻia nā clones āpau i unuhi ʻia mai CSF mai nā puni ʻelua hope loa o ke koho ʻana. Ua hoʻokomo pū ʻia nā phages koko āpau mai ka puni mua o ke koho ʻana i ka 454/Roche pyrosequencing. No ka hoʻokomo ʻana i nā clones phage, ua hoʻonui ʻia nā phages i koho ʻia i loko o E. coli (BL5615) ma nā papa 500 cm2 ma 37°C no 4 mau hola. Ua hoʻolaha ʻia nā clones i koho pākahi ʻia a hoʻonohonoho lima ʻia i loko o ka medium TB. Ma hope o ka unuhi ʻana o ka phage, ka hoʻomaʻemaʻe ʻana a me ka wehe ʻana o ka endotoxin (e like me ka mea i wehewehe ʻia ma luna), ua hoʻokomo ʻia he 2 × 1010 phages/holoholona i loko o 300 μl ma ke ʻano intravenous i loko o hoʻokahi vein huelo.
ʻO ka hana mua ʻana a me ka kānana qualitative o ka ʻikepili kaʻina. Ua hoʻololi ʻia ka ʻikepili maka 454/Roche mai kahi ʻano palapala kahawai maʻamau binary (sff) i kahi ʻano heluhelu kanaka Pearson (fasta) me ka hoʻohana ʻana i ka polokalamu mea kūʻai aku. Ua hana hou ʻia ka hana ʻana o ka kaʻina nucleotide me ka hoʻohana ʻana i nā polokalamu C ponoʻī a me nā palapala (pūʻolo polokalamu i hoʻokuʻu ʻole ʻia) e like me ka mea i wehewehe ʻia ma lalo nei. ʻO ka nānā ʻana i ka ʻikepili mua e pili ana i nā kaʻina hana kānana multi-stage koʻikoʻi. No ka kānana ʻana i nā heluhelu ʻaʻole i loaʻa kahi kaʻina DNA hoʻokomo 12mer kūpono, ua hoʻonohonoho ʻia nā heluhelu i ka lepili hoʻomaka (GTGATGTCGGGGATCCGAATTCT), lepili hoʻōki (TAAGCTTGCGGCGCCACTCGAGTA) a me ka hoʻokomo hope (CCCTGCAGGGATATCCCGGGAGCTCGAC) me ka hoʻohana ʻana i ka hoʻāʻo Needleman-Wunsch honua. hoʻonohonoho ʻana e ʻae ana i 2 mau kūlike ʻole no kēlā me kēia hoʻonohonoho31. No laila, ua wehe ʻia nā heluhelu me ka ʻole o nā lepili hoʻomaka a me nā lepili hoʻōki a me nā heluhelu e loaʻa ana nā hoʻokomo hope, ʻo ia hoʻi, nā hoʻonohonoho e ʻoi aku ma mua o ka helu i ʻae ʻia o nā mismatches, mai ka waihona puke. No nā heluhelu i koe, ua ʻoki ʻia ka moʻo DNA N-mer e hoʻolōʻihi ana mai ka māka hoʻomaka a hoʻopau ma mua o ka māka hoʻōki mai ka moʻo heluhelu mua a hana hou ʻia (ma hope aku nei i kapa ʻia ʻo "insert"). Ma hope o ka unuhi ʻana o ka hoʻokomo, ua wehe ʻia ka ʻāpana ma hope o ke codon hoʻōki mua ma ka hopena 5′ o ka primer mai ka hoʻokomo. Eia kekahi, ua wehe ʻia nā nucleotides e alakaʻi ana i nā codons piha ʻole ma ka hopena 3′ o ka primer. No ka hoʻokaʻawale ʻana i nā hoʻokomo e loaʻa ana nā moʻo hope wale nō, ua wehe ʻia nā hoʻokomo i unuhi ʻia e hoʻomaka ana me ke ʻano amino acid "PAG". Ua wehe ʻia nā peptides me ka lōʻihi ma hope o ka unuhi ʻana ma lalo o 3 mau waikawa amino mai ka waihona puke. ʻO ka hope loa, wehe i ka redundancy i loko o ka waihona hoʻokomo a hoʻoholo i ke alapine o kēlā me kēia hoʻokomo kū hoʻokahi. ʻO nā hopena o kēia loiloi i hoʻokomo ʻia kahi papa inoa o nā moʻo nucleotide (inserts) a me kā lākou (heluhelu) alapine (Nā Kiʻi Hoʻohui 1c a me 2).
Nā hoʻokomo DNA hui N-mer ma ke ʻano like o ke kaʻina: No ka hoʻopau ʻana i nā hewa kaʻina kikoʻī 454/Roche (e like me nā pilikia me nā hoʻonui homopolymer kaʻina) a wehe i nā redundancies koʻikoʻi ʻole, ua hoʻokaʻawale ʻia nā hoʻokomo kaʻina DNA N-mer i kānana mua ʻia (nā hoʻokomo) ma ke ʻano like. nā hoʻokomo (a hiki i 2 mau kumu kūlike ʻole i ʻae ʻia) me ka hoʻohana ʻana i kahi algorithm iterative i wehewehe ʻia penei: ua hoʻokaʻawale mua ʻia nā hoʻokomo e ko lākou alapine (kiʻekiʻe a haʻahaʻa), a inā like lākou, e ko lākou ʻano lua ma ka lōʻihi (lōʻihi a pōkole loa) ). No laila, ʻo nā hoʻokomo pinepine a lōʻihi loa e wehewehe i ka "hui" mua. Hoʻonohonoho ʻia ke alapine hui i ke alapine koʻikoʻi. A laila, ua hoʻāʻo ʻia kēlā me kēia hoʻokomo i koe i ka papa inoa i hoʻokaʻawale ʻia e hoʻohui ʻia i ka hui ma o ka hoʻonohonoho ʻana o Needleman-Wunsch pairwise. Inā ʻaʻole i ʻoi aku ka nui o nā mismatches, hoʻokomo, a i ʻole nā ​​​​holoʻi ʻana i kahi hoʻonohonoho ma mua o ka paepae o 2, hoʻohui ʻia kahi hoʻokomo i ka hui, a hoʻonui ʻia ke alapine hui holoʻokoʻa e ka pinepine o ka hoʻohui ʻana o ka hoʻokomo. Ua hōʻailona ʻia nā hoʻokomo i hoʻohui ʻia i kahi hui e like me ka hoʻohana ʻia a ua kāpae ʻia mai ka hana hou aku. Inā ʻaʻole hiki ke hoʻohui ʻia ke kaʻina hoʻokomo i kahi hui i kū mua ʻia, hoʻohana ʻia ke kaʻina hoʻokomo e hana i kahi hui hou me ke alapine hoʻokomo kūpono a hōʻailona ʻia he mea i hoʻohana ʻia. Pau ka hana hou ʻana i ka wā i hoʻohana ʻia ai kēlā me kēia kaʻina hoʻokomo e hana i kahi hui hou a i ʻole hiki ke hoʻokomo ʻia i loko o kahi hui i kū mua ʻia. Ma hope o nā mea a pau, ua unuhi hope ʻia nā hoʻokomo i hui ʻia me nā nucleotides i loko o nā kaʻina peptide (nā hale waihona puke peptide). ʻO ka hopena o kēia loiloi he hoʻonohonoho o nā hoʻokomo a me kā lākou mau alapine e pili ana e hana ana i ka helu o nā heluhelu hope (Kiʻi Hoʻohui 2).
Hana ʻana i nā Motif: Ma muli o kahi papa inoa o nā peptides kū hoʻokahi, ua hana ʻia kahi waihona puke e loaʻa ana nā ʻano amino acid āpau (aa) e like me ka mea i hōʻike ʻia ma lalo nei. Ua unuhi ʻia kēlā me kēia ʻano hiki o ka lōʻihi 3 mai ka peptide a ua hoʻohui ʻia kona ʻano inverse me kahi waihona puke motif maʻamau e loaʻa ana nā ʻano āpau (tripeptides). Ua hoʻonohonoho ʻia nā waihona puke o nā motif i hana hou ʻia a ua wehe ʻia ka redundancy. A laila, no kēlā me kēia tripeptide i loko o ka waihona puke motif, ua nānā mākou no kona alo i loko o ka waihona puke me ka hoʻohana ʻana i nā mea hana computational. I kēia hihia, ua hoʻohui ʻia ke alapine (frequency) o ka peptide e loaʻa ana ka motif tripeptide i loaʻa a ua hāʻawi ʻia i ka motif i loko o ka waihona puke motif ("helu o nā motif"). ʻO ka hopena o ka hana ʻana o nā motif he ʻelua-dimensional e loaʻa ana nā hanana āpau o nā tripeptides (motifs) a me kā lākou mau waiwai, ʻo ia ka helu o nā heluhelu sequencing e hopena i ka motif like ke kānana ʻia, hoʻohui ʻia, a unuhi ʻia nā heluhelu. Nā Metrics e like me ka mea i wehewehe kikoʻī ʻia ma luna.
Ka hoʻonohonoho ʻana o ka helu o nā motif a me nā scatterplots e pili ana: Ua hoʻonohonoho ʻia ka helu o nā motif no kēlā me kēia laʻana me ka hoʻohana ʻana
kahi ʻo ni ka helu o nā heluhelu e loaʻa ana ke kumuhana i. No laila, hōʻike ʻo vi i ka pakeneka pinepine o nā heluhelu (a i ʻole peptides) e loaʻa ana ke motif i i loko o ka laʻana. Ua helu ʻia nā waiwai P no ka helu i hoʻonohonoho ʻole ʻia o nā motif me ka hoʻohana ʻana i ka hoʻāʻo pololei a Fisher. E pili ana i nā correlograms o ka helu o nā motif, ua helu ʻia nā pilina a Spearman me ka hoʻohana ʻana i ka helu i hoʻonohonoho ʻia o nā motif me R.
No ka ʻike ʻana i ka ʻike o nā waikawa amino ma kēlā me kēia kūlana ma ka waihona peptide, ua hana ʻia nā logograms pūnaewele 32, 33 (http://weblogo.threeplusone.com). ʻO ka mea mua, ua mālama ʻia ka ʻike o nā waikawa amino ma kēlā me kēia kūlana o ka peptide 12-mer i loko o kahi matrix 20 × 12. A laila, ua hana ʻia kahi hoʻonohonoho o 1000 peptides e loaʻa ana ka ʻike waikawa amino like ma kēlā me kēia kūlana ma ke ʻano fasta-sequence a hāʻawi ʻia ma ke ʻano he input i ka web-logo 3, kahi e hana ai i kahi hōʻike kiʻi o ka ʻike waikawa amino pili ma kēlā me kēia kūlana. no kahi waihona peptide i hāʻawi ʻia. No ka ʻike ʻana i nā ʻikepili multidimensional, ua hana ʻia nā palapala ʻāina wela me ka hoʻohana ʻana i kahi mea hana i hoʻomohala ʻia i loko o R (biosHeatmap, kahi pūʻolo R i hoʻokuʻu ʻia). Ua helu ʻia nā dendrograms i hōʻike ʻia ma nā palapala ʻāina wela me ka hoʻohana ʻana i ke ʻano clustering hierarchical a Ward me ka metric mamao Euclidean. No ka loiloi helu o ka ʻikepili helu motif, ua helu ʻia nā waiwai P no ka helu ʻana unnormalized me ka hoʻohana ʻana i ka hoʻāʻo pololei a Fisher. Ua helu ʻia nā waiwai P no nā ʻikepili ʻē aʻe ma R me ka hoʻohana ʻana i ka hoʻāʻo t-Student a i ʻole ANOVA.
Ua hoʻokomo ʻia nā clones phage i koho ʻia a me nā phages me nā mea ʻole i hoʻokomo ʻia ma ke ʻano intravenous ma o ke aʻa huelo (2 × 1010 phages / holoholona i loko o 300 μl PBS). He ʻumi mau minuke ma mua o ka perfusion a me ka hoʻopaʻa ʻana ma hope, ua hoʻokomo ʻia nā holoholona like me 100 μl o ka lectin i hōʻailona ʻia ʻo DyLight594 (Vector Laboratories Inc., DL-1177). 60 mau minuke ma hope o ka hoʻokomo ʻana i ka phage, ua hoʻokomo ʻia nā ʻiole ma o ka puʻuwai me 50 ml PBS a ukali ʻia e 50 ml 4% PFA / PBS. Ua hoʻopaʻa pū ʻia nā laʻana lolo i ka pō i loko o 4% PFA / PBS a ua hoʻomoʻa ʻia i loko o 30% sucrose i ka pō ma 4 ° C. Hoʻopaʻa koke ʻia nā laʻana i loko o ka hui OCT. Ua hana ʻia ka loiloi Immunohistochemical o nā hāpana hau i ka mahana o ka lumi ma nā cryosections 30 µm i ālai ʻia me 1% BSA a ua hoʻoulu ʻia me nā antibodies polyclonal FITC-labeled e kūʻē i ka T7 phage (Novus NB 600-376A) ma 4 °C. E hoʻoulu i ka pō. ʻO ka hope loa, ua holoi ʻia nā ʻāpana i 3 mau manawa me PBS a ua nānā ʻia me kahi microscope laser confocal (Leica TCS SP5).
Ua hana ʻia nā peptides āpau me ka maʻemaʻe haʻahaʻa o 98% e GenScript USA, biotinylated a lyophilized. Hoʻopaʻa ʻia ʻo Biotin ma o kahi triple glycine spacer hou ma ka N-terminus. E nānā i nā peptides āpau me ka hoʻohana ʻana i ka mass spectrometry.
Ua hui pū ʻia ʻo Streptavidin (Sigma S0677) me kahi keu equimolar 5-fold o ka peptide biotinylated, ka peptide inhibitory BACE1 biotinylated, a i ʻole kahi hui pū ʻana (3:1 ratio) o ka peptide inhibitory BACE1 biotinylated a me ka peptide inhibitory BACE1 i loko o 5-10% DMSO/incubated i loko o PBS. 1 hola ma ka mahana o ka lumi ma mua o ka hoʻokomo ʻana. Ua hoʻokomo ʻia nā peptides i hoʻohui ʻia me Streptavidin ma ke ʻano intravenous ma kahi mahele o 10 mg/kg i loko o kekahi o nā aa huelo o nā ʻiole me kahi lua lolo.
Ua loiloi ʻia ka nui o nā hui streptavidin-peptide e ELISA. Ua uhi ʻia nā papa microtiter Nunc Maxisorp (Sigma) i ka pō ma 4°C me ka 1.5 μg/ml mouse anti-streptavidin antibody (Thermo, MA1-20011). Ma hope o ka pale ʻana (pale pale: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) ma ka mahana o ka lumi no 2 mau hola, e holoi i ka pā me 0.05% Tween-20/PBS (pale holoi) no 3 kekona, ua hoʻohui ʻia nā laʻana CSF a me plasma i nā lua i hoʻoheheʻe ʻia me ka pale pale (plasma 1:10,000, CSF 1:115). A laila ua hoʻomoʻa ʻia ka pā i ka pō ma 4°C me ka antibody ʻike (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Ma hope o ʻekolu mau ʻanuʻu holoi, ua ʻike ʻia ka streptavidin ma o ka hoʻomoʻa ʻana i loko o ka hopena substrate TMB (Roche) a hiki i 20 min. Ma hope o ka hoʻōki ʻana i ka hoʻomohala ʻana o ke kala me 1M H2SO4, e ana i ka absorbance ma 450 nm.
Ua loiloi ʻia ka hana o ka hui streptavidin-peptide-BACE1 inhibitor e Aβ(1-40) ELISA e like me ke kaʻina hana a ka mea hana (Wako, 294-64701). I ka pōkole, ua hoʻoheheʻe ʻia nā laʻana CSF i loko o ka diluent maʻamau (1:23) a ua hoʻomoʻa ʻia i ka pō ma 4°C i loko o nā pā 96-well i uhi ʻia me ka antibody hopu BNT77. Ma hope o ʻelima mau ʻanuʻu holoi, ua hoʻohui ʻia ka antibody HRP-conjugated BA27 a ua hoʻomoʻa ʻia no 2 mau hola ma 4°C., a ukali ʻia e ʻelima mau ʻanuʻu holoi. Ua ʻike ʻia ʻo Aβ(1–40) ma o ka hoʻomoʻa ʻana i loko o ka hopena TMB no 30 mau minuke ma ka mahana o ka lumi. Ma hope o ka hoʻōki ʻia ʻana o ka hoʻomohala ʻana o ke kala me ka hopena hoʻōki, e ana i ka absorbance ma 450 nm. Ua hoʻokomo ʻia nā laʻana plasma i ka unuhi ʻana o ka pae paʻa ma mua o ka Aβ(1–40) ELISA. Ua hoʻohui ʻia ka plasma i 0.2% DEA (Sigma) i loko o nā pā 96-well a ua hoʻomoʻa ʻia i ka mahana o ka lumi no 30 mau minuke. Ma hope o ka holoi mau ʻana i nā papa SPE (Oasis, 186000679) me ka wai a me 100% methanol, ua hoʻohui ʻia nā laʻana plasma i nā papa SPE a ua wehe ʻia nā wai āpau. Ua holoi ʻia nā laʻana (mua me 5% methanol a laila 30% methanol) a ua eluted ʻia me 2% NH4OH/90% methanol. Ma hope o ka hoʻomaloʻo ʻana i ka eluate ma 55°C no 99 min ma ke au mau o N2, ua hoʻemi ʻia nā laʻana i nā diluents maʻamau a ua ana ʻia ʻo Aβ(1–40) e like me ka mea i wehewehe ʻia ma luna.
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