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ʻO ka pale koko-lolo a me ka pale koko-lolo e pale aku i nā mea hana biotherapeutic mai ka hiki ʻana i kā lākou mau pahuhopu i loko o ka ʻōnaehana nūnū waena, no laila ke keʻakeʻa nei i ka mālama pono ʻana i nā maʻi neurological. No ka ʻike ʻana i nā mea lawe lolo hou i loko o ka vivo, ua hoʻolauna mākou i kahi waihona T7 phage peptide a hōʻiliʻili pū i ke koko a me ka wai cerebrospinal (CSF) me ka hoʻohana ʻana i kahi kumu hoʻohālike wai nui o nā ʻiole. Ua hoʻonui nui ʻia nā clones phage kikoʻī ma CSF ma hope o ʻehā pōʻai koho. Ua hōʻike ʻia ka hoʻāʻo ʻana i nā peptides moho pākahi ma mua o 1000-fold enrichment ma CSF. Ua hōʻoia ʻia ka bioactivity o ka hāʻawi ʻana i ka peptide-mediated i ka lolo e ka hōʻemi ʻana o 40% i ke kiʻekiʻe o ka amyloid-β i loko o ka wai cerebrospinal me ka hoʻohana ʻana i ka BACE1 peptide inhibitor i hoʻopili ʻia i ka peptide transit novel i ʻike ʻia. Hōʻike kēia mau hopena i nā peptides i ʻike ʻia e nā ʻano koho vivo phage he mau kaʻa pono no ka lawe ʻana i nā macromolecules i ka lolo me kahi hopena therapeutic.
Ua kālele nui ʻia ka noiʻi ʻana i ka lāʻau lapaʻau i hoʻopaʻa ʻia a me nā mea hana e hōʻike ana i nā waiwai CNS-targeting, me ka liʻiliʻi o ka hoʻoikaika ʻana i ka ʻike ʻana i nā hana e hoʻokau i ka lāʻau lapaʻau ikaika i ka lolo. Ke hoʻomaka nei kēia e hoʻololi i kēia manawa e like me ka lawe ʻana i ka lāʻau lapaʻau, ʻoi aku ka nui o nā molekala nui, he ʻāpana koʻikoʻi o ka hoʻomohala lāʻau neuroscience hou. Mālama maikaʻi ʻia ke kaiapuni o ka ʻōnaehana nerve waena e ka ʻōnaehana cerebrovascular barrier, ʻo ia hoʻi ka pale koko-lolo (BBB) ​​​​a me ka pale koko-lolo (BCBB) 1, e paʻakikī ana i ka hāʻawi ʻana i nā lāʻau lapaʻau i ka lolo1,2. Ua manaʻo ʻia ʻaneʻane pau nā lāʻau mole nui a ʻoi aku ma mua o 98% o nā lāʻau mole liʻiliʻi e hoʻopau ʻia mai ka lolo. ʻO ia ke kumu he mea nui loa ka ʻike ʻana i nā ʻōnaehana lawe lolo hou e hāʻawi i ka hāʻawi pono a me ka kikoʻī o nā lāʻau lapaʻau i ka CNS 4,5. Eia nō naʻe, hāʻawi ka BBB a me BCSFB i kahi manawa kūpono no ka lawe ʻana i ka lāʻau i ko lākou komo ʻana a komo i nā hale āpau o ka lolo ma o kāna vasculature nui. No laila, ʻo nā hana i kēia manawa e hoʻohana i nā ʻano hana non-invasive o ka lawe ʻana i ka lolo e hoʻokumu nui ʻia ma ke ʻano o ka receptor-mediated transport (PMT) me ka hoʻohana ʻana i ka endogenous BBB6 receptor. ʻOiai ʻo ka holomua nui o ka hoʻohana ʻana i ka transferrin receptor pathway7,8, pono e hoʻomohala hou i nā ʻōnaehana hoʻopuka hou me nā waiwai i hoʻomaikaʻi ʻia. I kēia hopena, ʻo kā mākou pahuhopu ʻo ia ka ʻike ʻana i nā peptides hiki ke hoʻopili i ka lawe ʻana i ka CSF, no ka mea hiki ke hoʻohana ʻia lākou e hāʻawi i nā macromolecules i ka CNS a i ʻole e wehe i nā alaloa hou. Ma keʻano kūikawā, hiki i nā mea loaʻa kūikawā a me nā mea lawe o ka ʻōnaehana cerebrovascular (BBB a me BSCFB) ke lilo i mau pahuhopu no ka hoʻopuka ʻana a me ka kikoʻī o nā lāʻau lapaʻau biotherapeutic. ʻO ka wai cerebrospinal (CSF) kahi hua huna o ka choroid plexus (CS) a pili pololei me ka wai interstitial o ka lolo ma o ka subarachnoid space a me ventricular space4. I kēia mau lā, ua hōʻike ʻia ke kahe nui ʻana o ka wai cerebrospinal subarachnoid i loko o ka interstitium o ka lolo9. Manaʻo mākou e komo i ka parenchymal space me ka hoʻohana ʻana i kēia subarachnoid inflow tract a i ʻole ma o ka BBB. No ka hoʻokō ʻana i kēia, ua hoʻokō mākou i kahi hoʻolālā koho vivo phage ikaika e hoʻomaopopo pono i nā peptides i lawe ʻia e kekahi o kēia mau ala ʻelua.
Ke wehewehe nei mākou i kahi ʻano hōʻike hōʻike hōʻike vivo phage me ka CSF sampling i hui pū ʻia me ka high throughput sequencing (HTS) e nānā i nā koho koho mua me ka ʻokoʻa o ka waihona puke kiʻekiʻe. Ua hana ʻia ka nānā ʻana ma luna o nā ʻiole ʻike me kahi cannula cisterna nui (CM) i hoʻokomo mau ʻia e pale aku i ke koko. ʻO ka mea nui, koho kēia ala i ka lolo-targeting a me nā peptides me ka hana halihali ma waena o ka pale cerebrovascular. Ua hoʻohana mākou i nā phages T7 ma muli o ko lākou liʻiliʻi liʻiliʻi (~ 60 nm) 10 a manaʻo ʻia he kūpono lākou no ka lawe ʻana i nā vesicle e ʻae ai i ka transcellular crossing o ka endothelial a / a i ʻole epithelial-medulla pa. Ma hope o ʻehā mau pōʻai o ka panning, ua hoʻokaʻawale ʻia nā heluna phage e hōʻike ana i ka ikaika i ka vivo CSF ​​​​enrichment a me ka hui microvessel cerebral. ʻO ka mea nui, ua hiki iā mākou ke hōʻoia i kā mākou ʻike ma ka hōʻike ʻana e hiki i nā peptides koho maikaʻi loa i koho ʻia a me ke kemika e hiki 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 o ka hoʻohui ʻana i kahi peptide transit alakaʻi me kahi mea hoʻopale o ka peptide BACE1. Ma kahi o ka hōʻike ʻana i nā hoʻolālā screening hana i loko o ka vivo hiki ke ʻike i nā peptides ka lawe ʻana i ka lolo hou e like me nā mea lawe ukana protein maikaʻi, manaʻo mākou i nā ala koho hana like e lilo i mea koʻikoʻi i ka ʻike ʻana i nā ala lawe lolo.
Ma muli o nā ʻāpana hana plaque (PFU), ma hope o ka pae ʻana o ka phage, ua hoʻolālā ʻia a hana ʻia kahi waihona o 12-mer linear T7 phage peptides me kahi ʻano like ʻole o 109 (e ʻike i nā Materials and Methods). He mea nui e hoʻomaopopo ua nānā pono mākou i kēia waihona ma mua o ka panning in vivo. PCR amplification o phage waihona laʻana me ka hoʻololi primers hana amplicons i pili pono i HTS (Supplementary Fig. 1a). Ma muli o a) HTS11 sequencing hewa, b) hopena i ka maikaʻi o primers (NNK) 1-12, a me c) ke alo o ka hihiu-type (wt) phage (skeleton inserts) i loko o ka standby hale waihona puke, ua hoʻokō 'ia ke kaʻina hana kānana e extract wale hōʻoia kaʻina 'ike (Hoʻohui Fig. 1b). Pili kēia mau kānana i nā hale waihona puke HTS. No ka waihona maʻamau, ua loaʻa ka huina o 233,868 heluhelu, a ʻo 39% i hala i nā koina kānana a ua hoʻohana ʻia no ka nānā ʻana o ka waihona a me ke koho ʻana no nā pōʻai hope (Supplementary Figure 1c–e). ʻO ka heluhelu ʻana he mau helu nui o 3 mau papa kumu i ka lōʻihi me kahi kiʻekiʻe ma 36 nucleotides (Supplementary Fig. 1c), e hōʻoia ana i ka hoʻolālā hale waihona puke (NNK) 1-12. ʻO ka mea nui, ma kahi o 11% o nā lālā waihona he 12-dimensional wild-type (wt) backbone PAGISRELVDKL hoʻokomo, a kokoke i ka hapalua o nā kaʻina (49%) i loaʻa nā hoʻokomo a holoi ʻia paha. Ua hōʻoia ka HTS o ka hale waihona puke i ka ʻokoʻa kiʻekiʻe o nā peptides i loko o ka waihona: ʻoi aku ma mua o 81% o nā kaʻina peptide i loaʻa hoʻokahi wale nō a ʻo 1.5% wale nō i loaʻa i nā kope ≥4 (Supplementary Fig. 2a). ʻO nā alapine o nā waikawa amino (aa) ma nā kūlana 12 a pau i loko o ka repertoire i hoʻopili maikaʻi ʻia me nā alapine i manaʻo ʻia no ka helu o nā codon i hana ʻia e ka repertoire NKK degenerate (Supplementary Fig. 2b). ʻO ke alapine iʻikeʻia o nā koena aa i hoʻopaʻaʻia e kēia mau mea hoʻokomo i hoʻopili maikaʻiʻia me ke alapine i heluʻia (r = 0.893) (Hoʻohui Fig. 2c). ʻO ka hoʻomākaukau ʻana i nā hale waihona puke phage no ka injection e pili ana i nā ʻanuʻu o ka amplification a me ka wehe ʻana o ka endotoxin. Ua hōʻike mua ʻia kēia i hiki ke hōʻemi i ka ʻokoʻa o nā hale waihona puke phage12,13. No laila, ua hoʻonohonoho mākou i kahi waihona phage i hoʻonui ʻia i ka pā i hoʻopau ʻia i ka endotoxin a hoʻohālikelike ʻia me ka waihona kumu e hoʻohālikelike ai i ke alapine o AA. Uaʻikeʻia kahi pilina ikaika (r = 0.995) ma waena o ka punawai kumu a me ka wai i hoʻonuiʻia a hoʻomaʻemaʻeʻia (Supplementary Fig. 2d), e hōʻike ana i ka hoʻokūkū ma waena o nā clones i hoʻonuiʻia ma nā papa me ka hoʻohanaʻana i ka T7 phageʻaʻole i kumu nui. Hoʻokumu ʻia kēia hoʻohālikelike ma luna o ke alapine o nā kumu tripeptide i kēlā me kēia waihona, ʻoiai ʻaʻole hiki ke hoʻopaʻa ʻia ka ʻokoʻa o nā hale waihona puke (~109) me ka HTS. ʻO ka loiloi pinepine o ka aa ma kēlā me kēia kūlana i hōʻike ʻia i kahi kūlana liʻiliʻi i hilinaʻi ʻia ma nā kūlana hope ʻekolu o ka repertoire i komo (Supplementary Fig. 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 o ka ʻokoʻa ma muli o ka hoʻonui a me ka hoʻomākaukau ʻana i nā hale waihona puke phage ma waena o nā pōʻai koho.
Hiki ke hana 'ia ka la'ana o ka wai cerebrospinal ma ka ho'okomo 'ana i ka cannula i loko o ka CM o nā 'iole 'ike e ho'oma'ama'a i ka 'ike 'ana i ka T7 phage i ho'okomo 'ia ma ke kino (iv) ma o ka BBB a/a i 'ole BCSFB (Fig. 1a-b). Ua hoʻohana mākou i ʻelua mau lima koho kūʻokoʻa (nā lima A a me B) i nā pōʻai mua ʻekolu o ke koho in vivo (Fig. 1c). 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 pōʻai mua ʻekolu o ke koho. No ka hā o ka panning, ua hui mākou i nā laʻana mai nā lālā A a me B a hana mākou i ʻekolu mau koho kūʻokoʻa. No ke aʻo ʻana i nā waiwai in vivo o nā ʻāpana phage T7 i kēia kŘkohu, ua hoʻokomo ʻia ka phage ʻano hihiu (PAGISRELVDKL master insert) i loko o nā ʻiole ma o ka huelo. ʻO ka hoʻihoʻi ʻana o nā phages mai ka wai cerebrospinal a me ke koko i nā manawa manawa like ʻole i hōʻike ʻia he liʻiliʻi liʻiliʻi ʻo T7 icosahedral phages i kahi wikiwiki hoʻomaʻemaʻe mua mai ke keʻena koko (Supplementary Fig. 3). Ma muli o nā titers i lawelawe ʻia a me ka nui o ke koko o nā ʻiole, ua helu mākou ma kahi o 1% wt wale nō. Ua ʻike ʻia ka phage mai ke ʻano i hoʻopaʻa ʻia i loko o ke koko 10 mau minuke ma hope o ka hoʻokele intravenous. Ma hope o kēia hāʻule wikiwiki mua, ua ana ʻia kahi hoʻomaʻemaʻe lohi me ka hapalua o ke ola o 27.7 mau minuke. ʻO ka mea nui, he kakaikahi wale nā ​​phages i kiʻi ʻia mai ka CSF compartment, e hōʻike ana i kahi haʻahaʻa haʻahaʻa no ka neʻe ʻana o ka phage wild-type i loko o ka CSF compartment (Supplementary Fig. 3). Ma ka awelika, aia wale nō ma kahi o 1 x 10-3% titers o T7 phage i loko o ke koko a me 4 x 10-8% o nā phages i hoʻomaka mua ʻia i ʻike ʻia i loko o ka wai cerebrospinal i ka manawa hoʻohālike holoʻokoʻa (0-250 min). ʻO ka mea nui, ʻo ka hapalua o ke ola (25.7 min) o ka phage wild-type i loko o ka wai cerebrospinal e like me ka mea i ʻike ʻia i ke koko. Hōʻike kēia mau ʻikepili i ka paʻa ʻana o ka pale e hoʻokaʻawale ana i ka ʻāpana CSF mai ke koko i loko o nā ʻiole CM-cannulated, e ʻae ana i ke koho ʻana i nā hale waihona puke phage e ʻike i nā clones i lawe koke ʻia mai ke koko i loko o ke keʻena CSF.
(a) Hoʻonohonoho ʻana i kahi ala no ka hoʻomaʻamaʻa hou ʻana i ka wai cerebrospinal (CSF) mai kahi wai nui. (b) Ke kiʻi e hōʻike ana i ka wahi kelepona o ka ʻōnaehana nūnū waena (CNS) a me ka hoʻolālā koho i hoʻohana ʻia no ka ʻike ʻana i nā peptides e hele i ka pale koko-lolo (BBB) ​​​​a me ka pale koko-lolo. (c) Ma ka vivo phage hōʻike i ka palapala hoʻoheheʻe. I kēlā me kēia pōʻai koho, ua hoʻokomo ʻia nā phages (nā mea hōʻike holoholona i loko o nā pua). Mālama ʻia ʻelua mau lālā ʻokoʻa kūʻokoʻa (A, B) a hiki i ka pōʻai 4 o ke koho. No nā pōʻai koho 3 a me 4, ua hoʻonohonoho lima ʻia kēlā me kēia clone phage mai CSF. (d) Kinetics o ka phage i hoʻokaʻawale ʻia mai ke koko (nā pōʻai ʻulaʻula) a me ka wai cerebrospinal (nā triangles ʻōmaʻomaʻo) i ka wā o ka pōʻai mua o ke koho ʻana i nā ʻiole cannulated ʻelua ma hope o ka hoʻokomo ʻia ʻana o ka waihona T7 peptide (2 x 1012 phages / holoholona). Hōʻike nā ʻāpana ʻulaʻula i ka awelika kumu mua o ka phage i loko o ke koko, i helu ʻia mai ka nui o ka phage injected, e noʻonoʻo ana i ka nui o ke koko. Hōʻike nā ʻāpana ʻeleʻele i ke kiko o ke kuʻekuʻe ʻana o ka laina y i hoʻonui ʻia mai ka hoʻopaʻa ʻana o ka phage koko. (e,f) Hōʻike i ke alapine pili a me ka puʻunaue ʻana o nā kumu tripeptide e hoʻopili ʻia i loaʻa i loko o ka peptide. Hōʻike ʻia ka helu o nā kumu i loaʻa ma 1000 heluhelu. Hōʻailona ʻia nā kumu waiwai nui (p <0.001) me nā kiko ʻulaʻula. (e) Correlation scatterplot e hoohalike ana i ka pili pinepine o ka tripeptide motif o ka hale waihona puke injected me ka phage i loaa mai ke koko mai na holoholona #1.1 a me #1.2. (f) Correlation scatterplot hoʻohālikelike i nā alapine pili o nā motif phage tripeptide 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) versus injected hale waihona puke a me ka phage i hoʻonui ʻia i CSF (h) versus koko ma hope o ke koho ʻana i loko o vivo i nā holoholona ʻelua. ʻO ka nui o ka helu leka hoʻokahi e hōʻike i ka nui o ka loaʻa ʻana o ka waikawa amino ma kēlā kūlana. ʻOmaomao = polar, poni = neutral, uliuli = kumu, ʻulaʻula = ʻakika a ʻeleʻele = hydrophobic amino acids. Ua hoʻolālā ʻia a hana ʻia ke kiʻi 1a, b e Eduard Urich.
Hoʻokomo mākou i kahi waihona peptide phage i loko o ʻelua mau ʻiole mea kani CM (clades A a me B) a hoʻokaʻawale i ka phage mai ka wai cerebrospinal a me ke koko (Figure 1d). ʻO ka hoʻomaʻemaʻe wikiwiki mua o ka hale waihona puke ʻoi aku ka liʻiliʻi i ka hoʻohālikelike ʻia me ka phage wild-type. ʻO ka hapalua o ke ola o ka hale waihona puke i loko o nā holoholona ʻelua he 24.8 mau minuke i ke koko, e like me ka phage ʻano hihiu, a me 38.5 mau minuke ma CSF. ʻO ke koko a me ka cerebrospinal fluid phage samples mai kēlā me kēia holoholona i hoʻopaʻa ʻia i ka HTS a ua ʻike ʻia nā peptides i ʻike ʻia no ka loaʻa ʻana o kahi kumu tripeptide pōkole. Ua koho ʻia nā kumu Tripeptide no ka mea hāʻawi lākou i kahi kumu liʻiliʻi no ka hoʻokumu ʻana o ka hale a me nā pilina peptide-protein14,15. Ua loaʻa iā mākou kahi pilina maikaʻi i ka hāʻawi ʻana i nā kumu ma waena o ka waihona phage injected a me nā clones i lawe ʻia mai ke koko o nā holoholona ʻelua (Fig. 1e). Hōʻike ka ʻikepili i ka hoʻohui ʻia o ka hale waihona puke i hoʻonui ʻia i loko o ke koko koko. Hoʻopili hou ʻia nā alapine amino acid a me nā kaʻina consensus i kēlā me kēia kūlana me ka hoʻohana ʻana i kahi hoʻololi o ka polokalamu Weblogo16. ʻO ka mea hoihoi, ua loaʻa iā mākou kahi hoʻonui ikaika i nā koena glycine koko (Fig. 1g). Ke hoʻohālikelike ʻia ke koko me nā clones i koho ʻia mai CSF, ua ʻike ʻia ke koho ikaika a me ka wehe ʻana o nā kumu (Fig. 1f), a ʻoi aku ka maikaʻi o kekahi mau amino acid i nā kūlana i koho mua ʻia i ka lālā 12 (Fig. 1h). ʻO ka mea nui, ʻokoʻa loa nā holoholona pākahi i ka wai cerebrospinal, ʻoiai ua ʻike ʻia ka hoʻonui ʻana i ka glycine koko ma nā holoholona ʻelua (Supplementary Fig. 4a-j). Ma hope o ka kānana paʻa ʻana o ka ʻikepili kaʻina i loko o ka wai cerebrospinal o nā holoholona #1.1 a me #1.2, ua loaʻa ka huina o 964 a me 420 ʻokoʻa 12-mer peptides (Supplementary Fig. 1d-e). Ua hoʻonui ʻia nā clones phage i hoʻokaʻawale ʻia a kau ʻia i kahi pōʻai lua o ke koho in vivo. ʻO ka Phage i unuhi ʻia mai ka pōʻai ʻelua o ke koho ʻana i hoʻopaʻa ʻia i ka HTS i kēlā me kēia holoholona a ua hoʻohana ʻia nā peptides i ʻike ʻia ma ke ʻano he hoʻokomo i kahi papahana ʻike kumu no ka nānā ʻana i ka hanana o nā kumu tripeptide (Fig. 2a, b, ef). Ke hoʻohālikelike ʻia i ka pōʻai mua o ka phage i hoʻihoʻi ʻia mai CSF, ua ʻike mākou i ke koho ʻana a me ka wehe ʻana o nā kumu he nui i CSF ma nā lālā A a me B (Fig. 2). Ua hoʻohana ʻia kahi algorithm ʻike pūnaewele e hoʻoholo inā he mau ʻano like ʻole o ke kaʻina kūlike. Ua ʻike ʻia kahi ʻano like ʻole ma waena o nā kaʻina 12-dimensional i hoʻihoʻi ʻia e CSF ma nā clade ʻokoʻa A (Fig. 2c, d) a me clade B (Fig. 2g, h). Ua hōʻike ʻia ka loiloi pooled i kēlā me kēia lālā i nā koho koho like ʻole no 12-mer peptides (Supplementary Fig. 5c, d) a me ka hoʻonui ʻana i ka CSF / blood titer ratio i ka manawa no nā clones pooled ma hope o ka lua o ke koho ʻana i hoʻohālikelike ʻia i ka pōʻai mua o ke koho (Supplementary Fig. 5e). ).
Hoʻonui i nā kumu a me nā peptides i loko o ka wai cerebrospinal e ʻelua mau pōʻai o ka vivo functional phage hōʻike koho.
ʻO nā phages wai cerebrospinal a pau i loaʻa mai ka pōʻai mua o kēlā me kēia holoholona (holoholona #1.1 a me #1.2) ua hui ʻia, hoʻonui ʻia, HT-sequenced a hoʻohui hou ʻia (2 x 1010 phages / holoholona) 2 SM cannulated iole (#1.1 → #). 2.1 a me 2.2, 1.2 → 2.3 a me 2.4). (a, b, e, f) Hoʻohālikelike ʻia nā scatterplots pili i ke alapine pili o nā kumu tripeptide o nā phages i loaʻa i ka CSF i nā koho koho mua a me ka lua. ʻO ka pinepine pili a me ka hāʻawi ʻana i nā kumu e hōʻike ana i nā tripeptides hiki ke hoʻopili ʻia i loaʻa i nā peptides ma nā ʻaoʻao ʻelua. Hōʻike ʻia ka helu o nā kumu i loaʻa ma 1000 heluhelu. ʻO nā kumu i koho nui ʻia (p <0.001) i koho ʻia a i ʻole i hoʻokomo ʻia i loko o kekahi o nā hale waihona puke i hoʻohālikelike ʻia me nā kiko ʻulaʻula. (c, d, g, h) Ka hōʻailona hōʻailona o ka CSF waiwai 12 amino acid lōʻihi ma muli o nā pōʻai 2 a me 1 o ke koho in vivo. ʻO ka nui o ka helu leka hoʻokahi e hōʻike i ka nui o ka loaʻa ʻana o ka waikawa amino ma kēlā kūlana. No ka hōʻike ʻana i ka hōʻailona, ​​ua hoʻohālikelike ʻia ka alapine o nā kaʻina CSF i unuhi ʻia mai kēlā me kēia holoholona ma waena o nā pōʻai koho ʻelua a hōʻike ʻia nā kaʻina waiwai 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. ʻO nā ʻakika amino i hoʻonui ʻia ma ke kūlana i hāʻawi ʻia i (c, d) holoholona no. 2.1 a ʻaʻole. 2.2 a i ʻole (g, h) i nā holoholona no. 2.3 a ʻaʻole. Hōʻike ʻia ka 2.4 i ke kala. ʻOmaomao = polar, poni = neutral, uliuli = kumu, ʻulaʻula = ʻakika a ʻeleʻele = hydrophobic amino acids.
Ma hope o ke kolu o ke koho, ua ʻike mākou he 124 mau ʻano peptide kūikawā (#3.1 a me #3.2) mai 332 CSF-reconstituted phage clones i hoʻokaʻawale ʻia mai nā holoholona ʻelua (Supplementary Fig. 6a). ʻO ke kaʻina LGSVS (18.7%) i loaʻa i ka ʻāpana pili kiʻekiʻe loa, a ukali ʻia e nā mea hoʻokomo ʻano ʻāhiu PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), a me SARGSWREIVSLS (2.2%). I ka pōʻai ʻehā hope loa, ua hui mākou i ʻelua mau lālā i koho ʻia mai ʻekolu mau holoholona ʻokoʻa (Fig. 1c). ʻO ka 925 sequenced phage clones i loaʻa mai CSF, i ka hā o ka pōʻai ua loaʻa iā mākou he 64 mau ʻano peptide kūʻokoʻa (Supplementary Fig. 6b), ma waena o ka hāʻule ʻana o ka pili o ka phage wild-type i 0.8%. ʻO nā clones CSF maʻamau i ka hā o ka puni 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 ka hoʻokomo ʻana/ka holoi ʻana o ka nucleotide a i ʻole nā ​​codons hoʻopau mua ʻana i ka waihona waihona i ka wā e hoʻohana ai i nā codon degenerate no ka hoʻolālā waihona NNK. Hoʻopuka nā codons hoʻopau mua i nā peptides pōkole a koho ʻia no ka mea aia lākou i ke kumu aa maikaʻi. Hiki ke loaʻa nā peptide lōʻihi ma muli o ka hoʻokomo ʻana/ka holoi ʻana i nā kumu mua o nā waihona synthetic. Hoʻonoho kēia i ka codon hoʻomaha i hoʻolālā ʻia ma waho o ke kiʻi a heluhelu ia a hiki i ka puka ʻana o kahi codon pani hou i 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 puka hāpana. No ka pōʻai mua o ka nānā ʻana, ua hoʻohana mākou i nā titers phage ʻano ʻāhiu ma ke ʻano he ʻōlelo kikoʻī ʻole. ʻO ka mea e mahalo ai, ua ikaika loa ke koho ʻana i ka phage maikaʻi ʻole i ka pōʻai CSF mua, akā ʻaʻole i ke koko (Fig. 3a), ma muli paha o ka haʻahaʻa haʻahaʻa o ka diffusion passive o ka hapa nui o nā lālā o ka waihona peptide i loko o ka CSF compartment a i ʻole nā ​​​​mea pili phages e ʻoi aku ka maikaʻi o ka mālama ʻana a wehe ʻia mai ke kahe koko ma mua o ka bacteriophages. Eia nō naʻe, i ka lua o ka pōʻai o ka panning, ua ʻike ʻia ke koho ikaika o nā phages i CSF ma nā papa ʻelua, e hōʻike ana ua hoʻonui ʻia ka pōʻai mua i nā phages e hōʻike ana i nā peptides e hoʻoikaika i ka CSF uptake (Fig. 3a). Eia hou, me ka waiwai ʻole o ke koko. Ma ke kolu a me ka hā o nā pōʻai, ua hoʻonui nui ʻia nā clones phage i CSF. I ka hoʻohālikelike ʻana i ka alapine pili o kēlā me kēia kaʻina peptide kūʻokoʻa ma waena o nā pōʻai hope ʻelua o ke koho ʻana, ua ʻike mākou ua ʻoi aku ka waiwai o nā kaʻina i ka hā o ke koho (Fig. 3b). ʻO ka huina o 931 tripeptide motifs i unuhi ʻia mai nā kaʻina peptide kūʻokoʻa 64 a pau me ka hoʻohana ʻana i nā ʻano peptide ʻelua. ʻO nā kumu i hoʻonaʻauao ʻia i ka pōʻai ʻehā i nānā pono ʻia no kā lākou hoʻonui waiwai ma nā pōʻai āpau i hoʻohālikelike ʻia me ka waihona i hoʻopaʻa ʻia (ʻoki ʻia: 10% enrichment) (Supplementary Fig. 6c). Ua hōʻike ʻia nā hiʻohiʻona maʻamau o ke koho ʻana ua hoʻonui ʻia ka hapa nui o nā kumu i aʻo ʻia i nā pōʻai mua o nā lālā koho ʻelua. Eia nō naʻe, ʻo kekahi mau kumu (e laʻa me SGL, VSG, LGS GSV) no ka papa ʻokoʻa ʻē aʻe, a ʻo nā mea ʻē aʻe (e laʻa me FGW, RTN, WGF, NTR) i hoʻonui ʻia i ka papa B.
ʻO ka hōʻoia o ka lawe ʻana i ka CSF o nā peptides i hōʻike ʻia i ka phage a me nā peptides alakaʻi biotinylated i hoʻohui ʻia i nā uku streptavidin.
(a) Ua helu ʻia nā lakio hoʻonui i nā pōʻai ʻehā (R1-R4) ma muli o nā titers injected (input = I) phage (PFU) a me nā titers phage CSF i hoʻoholo ʻia (output = O). Ua helu ʻia nā kumu hoʻonui no nā pōʻai hope ʻekolu (R2-R4) ma ka hoʻohālikelike ʻana me ka pōʻai mua a me ka puni mua (R1) me ka ʻikepili kaumaha. ʻO nā pā hāmama he wai cerebrospinal, ʻo nā pahu pale he plasma. (***p<0.001, ma muli o ka ho'āʻo-t a ka Haumāna). (b) Ka papa inoa o nā peptides phage nui loa, i hoʻonohonoho ʻia e like me ko lākou pili pili i nā phages āpau i hōʻiliʻili ʻia ma CSF ma hope o ka puni 4 o ke koho. Hōʻike ʻia nā clones phage ʻeono i ka waihoʻoluʻu, helu ʻia a me kā lākou mea waiwai ma waena o nā pōʻai 3 a me 4 o ke koho ʻana (insets). (c, d) ʻO nā clones phage ʻeono i hoʻonui nui ʻia, nā hale waihona puke phage phage makua a me nā hale waihona puke phage peptide mai ka puni 4 i hoʻopaʻa ʻia i loko o kahi kumu hoʻohālike CSF. Ua hōʻiliʻili ʻia nā CSF a me nā laʻana koko i nā manawa i hōʻike ʻia. (c) Ka huina like o 6 moho phage clones (2 x 1010 phages/animals), kaawale phages (#1779) (2 x 1010 phages/animals) a me stock phage peptide libraries (2 x 1012 phages/animals). Hōʻike ʻia ka pharmacokinetics CSF o kēlā me kēia phage clone a me ka waihona phage peptide i ka manawa. (d) hōʻike i ka lakene CSF/koko awelika no nā phages/mL i hoʻihoʻi hou ʻia i ka manawa hoʻohālike. (e) ʻEhā peptides alakaʻi synthetic a me hoʻokahi mana scrambled i hoʻopili ʻia me ka biotin i streptavidin ma o kā lākou N-terminus (tetramer display) a ukali ʻia e ka injection (tail vein iv, 10 mg streptavidin/kg). ʻEkolu mau ʻiole i hoʻokomo ʻia (N = 3). ). Ua hōʻiliʻili ʻia nā hōʻailona CSF i nā manawa manawa i hōʻike ʻia a ua ana ʻia nā manaʻo streptavidin e CSF anti-streptavidin ELISA (nd = ʻaʻole i ʻike ʻia). (* p<0.05, **p<0.01, ***p<0.001, ma muli o ka ho'āʻo ANOVA). (f) Ka hoʻohālikelike o ke kaʻina amino acid o ka phage peptide clone nui loa #2002 (purple) me nā clones phage peptide i koho ʻia mai ka pōʻai 4th o ke koho. ʻO nā ʻāpana amino acid like a like me ka waihoʻoluʻu.
ʻO nā phages i hoʻonui ʻia i ka pōʻai ʻehā (Fig. 3b), ua koho ʻia ʻeono clones moho no ka nānā ʻana i kēlā me kēia kanaka i ke kumu hoʻohālike CSF. Ua hoʻokomo ʻia nā waihona like o ʻeono moho phage, phage hakahaka (ʻaʻohe hoʻokomo) a me nā hale waihona puke prophage peptide i ʻekolu mau holoholona CM cannulated, a ua hoʻoholo ʻia nā pharmacokinetics ma CSF (Fig. 3c) a me ke koko (Supplementary Fig. 7) assays. Ua hoʻāʻo ʻia nā clones phage āpau i ka CSF compartment ma kahi pae 10-1000 mau manawa kiʻekiʻe ma mua o ka phage mana ʻole (#1779). No ka laʻana, loaʻa nā clones #2020 a me #2077 ma kahi o 1000 mau manawa kiʻekiʻe CSF titers ma mua o ka control phage. He ʻokoʻa ka ʻōlelo pharmacokinetic o kēlā me kēia peptide i koho ʻia, akā loaʻa iā lākou a pau ka mana homing CSF kiʻekiʻe. Ua ʻike mākou i ka emi mau ʻana o ka manawa no nā clones #1903 a me #2011, ʻoiai no nā clones #2077, #2002 a me #2009 hiki ke piʻi i loko o nā minuke 10 mua e hōʻike ana i ka lawe ʻana akā pono e hōʻoia ʻia. Hoʻopaʻa ʻia nā Clones #2020, #2002, a me #2077 ma nā pae kiʻekiʻe, ʻoiai ke emi mālie nei ka CSF o ka clone #2009 ma hope o ka piʻi mua ʻana. A laila hoʻohālikelike mākou i ke alapine pili o kēlā me kēia moho CSF ​​me kona ʻano koko (Fig. 3d). Ua hōʻike ʻia ka hoʻopili ʻana o ka mean titer o kēlā me kēia moho CSF ​​me kāna titer koko i nā manawa sampling āpau i hōʻike ʻia ʻekolu o nā moho ʻeono i waiwai nui i ke koko CSF. ʻO ka mea e mahalo ai, ua hōʻike ʻo clone #2077 i ke kūpaʻa koko kiʻekiʻe (Ke Kiʻi 7). No ka hōʻoia ʻana i hiki i nā peptides ke lawe ikaika i nā ukana ʻē aʻe ma mua o nā ʻāpana phage i loko o ke keʻena CSF, ua hoʻohui mākou i ʻehā mau alakaʻi peptides derivatized me ka biotin ma ka N-terminus kahi e hoʻopili ai nā peptides i ka phage particle. ʻO nā peptides biotinylated (nos. 2002, 2009, 2020 a me 2077) i hui pū ʻia me streptavidin (SA) e kiʻi i nā ʻano multimeric e like me ka mimicking phage geometry. Ua ʻae kēia ʻano iā mākou e ana i ka ʻike SA i ke koko a me ka wai cerebrospinal e like me nā peptides protein lawe ukana. ʻO ka mea nui, hiki ke hoʻopuka pinepine ʻia ka ʻikepili phage i ka wā i lawelawe ʻia ai nā peptides synthetic ma kēia ʻano SA-conjugated (Fig. 3e). ʻAʻole liʻiliʻi ka ʻike mua ʻana o nā peptides scrambled a ʻoi aku ka wikiwiki o ka CSF me nā pae ʻike ʻole ʻia i loko o 48 mau hola. No ka loaʻa ʻana o ka ʻike i nā ala hoʻopuka o kēia mau peptide phage clones i loko o ka CSF space, ua loiloi mākou i ka localization o kēlā me kēia phage peptide hits me ka immunohistochemistry (IHC) e ʻike pololei i nā ʻāpana phage 1 hola ma hope o ka injection intravenous in vivo. ʻIke ʻia, hiki ke ʻike ʻia nā clones #2002, #2077, a me #2009 e ka ʻili ikaika ʻana i nā capillaries lolo, aʻo ka control phage (#1779) a me ka clone #2020 ʻaʻole i ʻike ʻia (Supplementary Figure 8). Hōʻike kēia i ka hāʻawi ʻana o kēia mau peptides i ka hopena ma ka lolo ma o ka hele ʻana i ka BBB. Pono ka ʻike kikoʻī hou aku e hoʻāʻo i kēia kuhiakau, no ka mea, pili pū paha ke ala BSCFB. I ka hoʻohālikelike ʻana i ke kaʻina amino acid o ka clone i hoʻonui ʻia (#2002) me nā peptides i koho ʻia, ua ʻike ʻia aia kekahi o lākou i nā hoʻonui like amino acid, kahi e hōʻike ai i kahi mīkini lawe like (Fig. 3f).
Ma muli o kāna kūlana plasma kūikawā a me ka piʻi nui ʻana o ka CSF i ka manawa, ua ʻimi hou ʻia ka phage clone #2077 i kahi manawa lōʻihi 48-hola a hiki ke hana hou i ka piʻi wikiwiki o ka CSF i ʻike ʻia i ka hui ʻana me nā pae SA mau (Fig. 4a). E pili ana i nā clones phage ʻē aʻe i ʻike ʻia, #2077 i hoʻopaʻa ikaika ʻia no nā capillaries lolo a hōʻike i ka colocalization koʻikoʻi me ka lectin marker capillary ke nānā ʻia ma ka hoʻonā kiʻekiʻe a me ke ʻano o ka ʻili i ka wahi parenchymal (Figure 4b). No ka noiʻi inā hiki ke loaʻa nā hopena pharmacological peptide-mediated i loko o ka CNS, ua hana mākou i kahi hoʻokolohua kahi biotinylated versions o i) ka #2077 transit peptide a me ii) ka BACE1 inhibitor peptide i hui pū ʻia me SA ma ʻelua mau ratio like ʻole. No hoʻokahi hui ʻana mākou i hoʻohana wale i ka BACE1 peptide inhibitor a no ka mea ʻē aʻe ua hoʻohana mākou i ka ratio 1:3 o ka BACE1 peptide inhibitor i #2077 peptide. Hāʻawi ʻia nā ʻāpana ʻelua i ka intravenously a ua ana ʻia nā pae koko a me ka cerebrospinal wai o ka beta-amyloid peptide 40 (Abeta40) i ka manawa. Ua ana ʻia ʻo Abeta40 ma CSF e hōʻike ana i ka pale ʻana o BACE1 i ka parenchyma lolo. E like me ka mea i manaʻo ʻia, ua hoʻemi nui nā paʻakikī ʻelua i nā pae koko o Abeta40 (Fig. 4c, d). Eia naʻe, ʻo nā laʻana wale nō i loaʻa kahi hui ʻana o ka peptide no. ʻO 2077 a me kahi mea paʻa o ka peptide BACE1 i hoʻohui ʻia i SA ua hoʻemi nui i ka Abeta40 i ka wai cerebrospinal (Fig. 4c). Hōʻike ka ʻikepili i ka peptide no. Hiki iā 2077 ke lawe i ka protein 60 kDa SA i loko o ka CNS a hoʻoulu pū i nā hopena pharmacological me nā mea hoʻopiʻi SA-conjugated o ka peptide BACE1.
(a) Clonal injection (2 × 10 phages / holoholona) o T7 phage e hōʻike ana i nā profile pharmacokinetic lōʻihi o CSF ​​peptide #2077 (RLSSVDSDLSGC) a me ka phage mana uninjected (#1779) i loko o ʻekolu mau ʻiole CM-intubated. (b) Confocal microscopic kiʻi o represent cortical microvessels i loko o phage-injected iole (2 × 10 10 phages / holoholona) e hōʻike ana counterstaining o peptide #2077 a me nā moku (lectin). Hāʻawi ʻia kēia mau clones phage i 3 mau ʻiole a ʻae ʻia e kaapuni no 1 hola ma mua o ka hoʻoulu ʻana. Ua hoʻokaʻawale ʻia nā lolo me nā antibodies polyclonal FITC-labeled e kūʻē i ka T7 phage capsid. He ʻumi mau minuke ma mua o ka hoʻoheheʻe ʻana a me ka hoʻoponopono hou ʻana, ua lawelawe ʻia ʻo DyLight594-labeled lectin. ʻO nā kiʻi fluorescent e hōʻike ana i ka ʻili lectin (ʻulaʻula) o ka ʻaoʻao luminal o nā microvessels a me nā phages ('ōmaʻomaʻo) i ka lumen o nā capillaries a me ka ʻiʻo o ka lolo perivascular. He 10 µm ka pae pālākiō. (c, d) ʻO Biotinylated BACE1 inhibitory peptide wale nō a i hui pū ʻia me ka biotinylated transit peptide #2077 i hui pū ʻia me streptavidin a ukali ʻia e ka intravenous injection o ʻekolu cannulated CM iole (10 mg streptavidin / kg). ʻO ka BACE1 peptide inhibitor-mediated reduction ma Aβ40 ua ana ʻia e Aβ1-40 ELISA i loko o ke koko (ʻulaʻula) a me ka wai cerebrospinal (alani) i nā manawa i hōʻike ʻia. No ka akaka maikaʻi, ua kaha ʻia kahi laina kiko ma ka pakuhi ma ka pālākiō o 100%. (c) Ka emi pākēneka o ka Aβ40 i loko o ke koko (nā triangle ʻulaʻula) a me ka wai cerebrospinal (nā triangle ʻalani) i nā ʻiole i mālama ʻia me streptavidin i hoʻohui ʻia i ka peptide transit #2077 a me BACE1 inhibitory peptide ma kahi 3: 1 ratio. (d) Ka emi pākēneka o ke koko Aβ40 (nā pōʻai ʻulaʻula) a me ka wai cerebrospinal (nā pōʻai ʻalani) o nā ʻiole i mālama ʻia me streptavidin i hui pū ʻia me kahi peptide inhibitory BACE1 wale nō. ʻO ka manaʻo o Aβ i ka mana he 420 pg / ml (ka hoʻololi maʻamau = 101 pg / ml).
Ua hoʻohana maikaʻi ʻia ka hōʻike Phage ma kekahi mau wahi o ka noiʻi biomedical17. Ua hoʻohana ʻia kēia ʻano no nā haʻawina ʻokoʻa vivo vascular18,19 a me nā haʻawina e kuhikuhi ana i nā moku cerebral20,21,22,23,24,25,26. Ma kēia noiʻi ʻana, ua hoʻonui mākou i ka noi ʻana o kēia ʻano koho ʻaʻole wale i ka ʻike pololei ʻana o nā peptides e kuhikuhi ana i nā moku cerebral, akā i ka loaʻa ʻana o nā moho me nā waiwai lawe ikaika e hele i ka pale koko-lolo. Ke wehewehe nei mākou i ka hoʻomohala ʻana i kahi kaʻina koho in vivo i nā ʻiole intubated CM a hōʻike i kona hiki ke ʻike i nā peptides me nā waiwai homing CSF. Ke hoʻohana nei i ka T7 phage e hōʻike ana i kahi waihona o 12-mer random peptides, ua hiki iā mākou ke hōʻike i ka liʻiliʻi o ka T7 phage (ma kahi o 60 nm ke anawaena) 10 e hoʻololi ʻia i ka pale koko-lolo, a laila e hele pololei i ka pale koko-lolo a i ʻole choroid plexus. Ua ʻike mākou ʻo ka hōʻiliʻili ʻana o ka CSF mai nā ʻiole CM cannulated he mea hoʻokele maikaʻi ʻia i loko o ka vivo functional screening method, a ʻaʻole i hoʻopaʻa ʻia ka phage i lawe ʻia i ka vasculature akā ua hana pū kekahi ma ke ʻano he transporter ma waena o ka pale koko-lolo. Eia kekahi, ma ka hōʻiliʻili ʻana i ke koko a me ka hoʻopili ʻana i ka HTS i ka CSF ​​a me nā phages i loaʻa i ke koko, ua hōʻoia mākou ʻaʻole i hoʻopili ʻia kā mākou koho ʻana i ka CSF e ka hoʻonui koko a i ʻole ke kūpono no ka hoʻonui ʻana ma waena o nā pōʻai koho. Eia nō naʻe, ʻo ka ʻāpana koko kekahi ʻāpana o ke kaʻina koho, no ka mea, ʻo nā phages hiki ke hiki i ke keʻena CSF pono ke ola a kaʻa i ke kahe koko e hoʻonui ai iā lākou iho i ka lolo. I mea e unuhi ai i ka ʻike pili pono mai ka ʻikepili HTS maka, ua hoʻokō mākou i nā kānana i hoʻololi ʻia i nā hewa hoʻonohonoho kikoʻī o ka paepae ma ke kahe hana loiloi. Ma ka hoʻokomo ʻana i nā ʻāpana kinetic i ke ʻano screening, ua hōʻoia mākou i ka pharmacokinetics wikiwiki o nā phages T7 wild-type (t½ ~ 28 min) i ke koko24, 27, 28 a hoʻoholo pū i ko lākou hapalua o ke ola i ka wai cerebrospinal (t½ ~ 26 min) i kēlā me kēia minuke). ʻOiai ke ʻano o ka pharmacokinetic profiles i loko o ke koko a me ka CSF, ʻo 0.001% wale nō o ke kahe koko o ka phage i hiki ke ʻike ʻia ma CSF, e hōʻike ana i ka neʻe haʻahaʻa haʻahaʻa o ka T7 phage ʻano hihiu 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 kahe ʻana, ʻoiai he liʻiliʻi nā clones i hiki ke hiki i ke keʻena CNS. No laila, i ka pōʻai mua, ua nui loa ka hōʻemi ʻana i ka ʻokoʻa o ka waihona, no ka mea, he helu palena wale nō o nā clones i hōʻiliʻili ʻia ma kēia kumu hoʻohālike CSF koʻikoʻi. ʻO kēia hoʻolālā in vivo panning i loko o nā ʻanuʻu koho e like me ka hōʻiliʻili ʻana i loko o ke keʻena CSF, ke ola clone i loko o ke keʻena koko, a me ka wehe wikiwiki ʻana o nā clones T7 phage mai ke koko i loko o nā minuke 10 mua (Fig. 1d a me Supplementary Fig. 4M). ). No laila, ma hope o ka pōʻai mua, ua ʻike ʻia nā clones phage ʻē aʻe ma CSF, ʻoiai ua hoʻohana ʻia ka loko wai mua no kēlā me kēia holoholona. Hōʻike kēia i nā ʻanuʻu koho koʻikoʻi he nui no nā hale waihona puke me ka nui o nā lālā waihona e hopena i kahi hōʻemi nui o ka ʻokoʻa. No laila, e lilo nā hanana hanana i mea nui o ke kaʻina koho mua, e hoʻoikaika nui i ka hopena. Ua like paha ka nui o nā clones i loko o ka waihona kumu i like loa i ka CSF enrichment propensity. Eia nō naʻe, ma lalo o nā kūlana hoʻokolohua like, ʻokoʻa paha nā hopena koho ma muli o ka liʻiliʻi o kēlā me kēia clone i loko o ka wai mua.
ʻOkoʻa nā kumu i hoʻonui ʻia i ka CSF mai nā mea i loko o ke koko. ʻO ka mea mahalo, ua ʻike mākou i ka neʻe mua ʻana i nā peptides waiwai glycine i ke koko o kēlā me kēia holoholona. (Fig. 1g, Hoʻohui Fig. 4e, 4f). ʻOi aku ka paʻa o ka Phage i loko o nā peptides glycine a ʻaʻole hiki ke lawe ʻia i waho o ke kahe. Eia naʻe, ʻaʻole i ʻike ʻia kēia mau peptides waiwai nui o ka glycine i loko o nā laʻana o ka wai cerebrospinal, e hōʻike ana ua hele nā ​​hale waihona puke curated i ʻelua mau ʻanuʻu koho: hoʻokahi i loko o ke koko a ʻae ʻia kekahi e hōʻiliʻili i ka wai cerebrospinal. Ua hoʻāʻo nui ʻia nā clones i hoʻonui ʻia e CSF mai ka hā o ke koho. Ma kahi kokoke i nā clones i hoʻāʻo ʻia i hoʻopaʻa ʻia i hoʻonui ʻia i ka CSF i hoʻohālikelike ʻia me ka blank control phage. Hoʻokahi peptide hit (#2077) i nānā ʻia i nā kikoʻī hou aku. Ua hōʻike ʻia ka hapalua o ke ola plasma lōʻihi i hoʻohālikelike ʻia i nā hits ʻē aʻe (Figure 3d a me Supplementary Figure 7), a me ka hoihoi, aia kēia peptide i kahi koena cysteine ​​​​ma ka C-terminus. Ua hōʻike koke ʻia ʻo ka hoʻohui ʻana o ka cysteine ​​​​i nā peptides hiki 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 a pono e aʻo hou. Ua hōʻike kekahi mau peptides i ka hilinaʻi valence i ka hoʻonui ʻana i ka CSF (ʻaʻole i hōʻike ʻia), pili paha i ka geometry ʻili i hōʻike ʻia o ka T7 capsid. Ua hōʻike ʻia ka ʻōnaehana T7 a mākou i hoʻohana ai i nā kope 5-15 o kēlā me kēia peptide no kēlā me kēia phage particle. Ua hana ʻia ʻo IHC ma luna o nā clones lead phage i hoʻokomo ʻia i loko o ka cerebral cortex o nā ʻiole (Supplementary Fig. 8). Ua hōʻike ka ʻikepili i ʻekolu mau clones (No. 2002, No. 2009 a me No. 2077) i hui pū me ka BBB. Ke hoʻoholo nei inā loaʻa kēia pilina BBB i ka hōʻiliʻili ʻana o CSF ​​a i ʻole ka neʻe ʻana o kēia mau clones i ka BCSFB. ʻO ka mea nui, hōʻike mākou i ka mālama ʻana o nā peptides i koho ʻia i ko lākou hiki ke lawe CSF i ka wā synthesized a hoʻopaʻa ʻia i ka ukana protein. ʻO ka hoʻopaʻa ʻana i nā peptides biotinylated N-terminal i 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 (Fig. 3e). ʻO ka mea hope loa, hōʻike mākou i ka hiki i ka peptide alakaʻi #2077 ke hoʻoikaika i ka hana lolo o kahi mea hoʻopiʻi peptide biotinylated o BACE1 i hoʻohui ʻia i SA, e hoʻopuka ana i nā hopena pharmacodynamic i ka CNS ma o ka hoʻohaʻahaʻa nui ʻana i nā pae Abeta40 ma CSF (Fig. 4). ʻAʻole hiki iā mākou ke ʻike i nā homologues i loko o ka waihona ma o ka hana ʻana i kahi peptide sequence homology hulina o nā hits āpau. He mea nui e hoʻomaopopo i ka nui o ka waihona T7 ma kahi o 109, aʻo ka nui o ka waihona puke no 12-mers he 4 x 1015. No laila, koho wale mākou i kahi hapa liʻiliʻi o ka ʻokoʻa like ʻole o ka waihona peptide 12-mer, ʻo ia paha ke ʻano he hiki ke ʻike ʻia nā peptides i ʻoi aku ka maikaʻi ma o ka loiloi ʻana i nā wahi pili pili. ʻO Hypothetically, ʻo kekahi o nā kumu i ʻike ʻole ai mākou i nā homologues kūlohelohe o kēia mau peptides hiki ke hoʻopau ʻia i ka wā evolution e pale ai i ke komo ʻole ʻana o kekahi mau kumu peptide i loko o ka lolo.
Hoʻohui pū ʻia, hāʻawi kā mākou hopena i kumu no ka hana e hiki mai ana e ʻike a ʻike i nā ʻōnaehana lawe o ka cerebrovascular barrier in vivo i nā kikoʻī hou aku. Hoʻokumu ʻia ka hoʻonohonoho kumu o kēia ʻano hana ma kahi hoʻolālā koho hana ʻaʻole wale e ʻike i nā clones me nā waiwai paʻa cerebral vascular, akā ua komo pū kekahi i kahi hana koʻikoʻi i loaʻa i nā clones kūleʻa nā hana intrinsic e hele i nā pale olaola i loko o ka keʻena CNS. ʻO ia ka wehewehe ʻana i ke ʻano o ka lawe ʻana i kēia mau peptides a me kā lākou makemake no ka hoʻopaʻa ʻana i ka microvasculature kikoʻī i ka ʻāina lolo. Hiki i kēia ke alakaʻi i ka loaʻa ʻana o nā ala hou no ka lawe ʻana i ka BBB a me nā mea loaʻa. Manaʻo mākou e hiki i nā peptides i ʻike ʻia ke hoʻopaʻa pololei i nā mea hoʻokipa cerebrovascular a i ʻole nā ​​​​ligands e holo ana ma o ka BBB a i ʻole BCSFB. E noiʻi hou ʻia nā peptide vectors me ka hana lawe CSF i ʻike ʻia ma kēia hana. Ke noiʻi nei mākou i ka kikoʻī o ka lolo o kēia mau peptides no ko lākou hiki ke hele i ka BBB a / a i ʻole BCSFB. ʻO kēia mau peptides hou e lilo i mea waiwai nui no ka loaʻa ʻana o nā mea loaʻa hou a i ʻole nā ​​alahele a no ka hoʻomohala ʻana i nā paepae hou loa no ka lawe ʻana i nā macromolecules, e like me ka biologics, i ka lolo.
E hoʻopili i ka lua nui (CM) me ka hoʻololi ʻana i ke ʻano i wehewehe mua ʻia. Ua kau ʻia nā ʻiole Wistar Anesthetized (200-350 g) ma luna o kahi stereotaxic apparatus a ua hana ʻia kahi ʻoki waena ma luna o ke poʻo i kahi ʻia a hoʻomākaukau aseptically e hōʻike i ka iwi poʻo. E wili i ʻelua puka ma ka ʻāpana o ke kāʻei luna a hoʻopaʻa i nā wili paʻa 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 ho'ōla kiʻi a me ka paʻakikī sima, ua pani ʻia ka ʻili me ka 4/0 supramid suture. Hoʻopaʻa ʻia ka hoʻokomo pono ʻana o ka cannula e ka leaka ʻana o ka wai cerebrospinal (CSF). Wehe i ka ʻiole mai ka stereotaxic apparatus, e loaʻa i ka mālama postoperative kūpono a me ka mālama ʻana i ka ʻeha, a e ʻae iā ia e ola hou no hoʻokahi pule a ʻike ʻia nā hōʻailona o ke koko i loko o ka wai cerebrospinal. Ua loaʻa nā ʻiole Wistar (Crl:WI/Han) mai ka muliwai ʻo Charles (Palani). Mālama ʻia nā ʻiole a pau ma lalo o nā kūlana pathogen-free. Ua ʻae ʻia nā hoʻokolohua holoholona āpau e ka Veterinary Office o ke kūlanakauhale ʻo Basel, Switzerland, a ua hana ʻia e like me ka Laikini holoholona No. 2474 (Assessment of Active Brain Transport by Measuring Levels of Therapeutic Candidates in the Cerebrospinal Fluid and Brain of Rat).
E mālama mālie i ka ʻiole me ka CM cannula ma ka lima. Wehe iā Datura mai ka cannula a hōʻiliʻili i 10 µl o ka wai cerebrospinal kahe wale. Ma muli o ka hoʻopaʻapaʻa ʻana o ka cannula, ua hoʻokomo ʻia i loko o kēia noiʻi ʻana i nā ʻano wai cerebrospinal akaka me ka ʻole o ka hoʻohaumia ʻana o ke koko. Ma ka like, ma kahi o 10-20 μl o ke koko i lawe ʻia mai kahi ʻoki liʻiliʻi ma ka piko o ka huelo i loko o nā paipu me ka heparin (Sigma-Aldrich). Ua hōʻiliʻili ʻia ʻo CSF ​​a me ke koko i nā manawa like ʻole ma hope o ka hoʻokele intravenous o T7 phage. Ma kahi o 5-10 μl o ka wai i hoʻolei ʻia ma mua o ka hōʻiliʻili ʻia ʻana o kēlā me kēia CSF sample, e pili ana i ka nui make o ka catheter.
Ua hana ʻia nā hale waihona puke me ka hoʻohana ʻana i ka vector T7Select 10-3b e like me ka mea i wehewehe ʻia ma ka manual system T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). ʻO ka pōkole, ua hoʻokomo ʻia kahi hoʻokomo DNA 12-mer ma ke ʻano penei:
Ua hoʻohana ʻia ka codon NNK no ka pale ʻana i nā codons kū ʻelua a me ka overexpression amino acid i loko o ka mea hoʻokomo. ʻO N ka lākiō equimolar hui lima o kēlā me kēia nucleotide, a ʻo K ka lākiō equimolar hui lima o nā nucleotides adenine a me cytosine. Ua hoʻololi ʻia nā ʻāpana stranded hoʻokahi i ka DNA stranded pālua ma o ka incubation hou me dNTP (Novagen) a me Klenow enzyme (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 stranded ʻelua e ka ua EtOH. ʻO ka hopena o ka DNA i hoʻokahe ʻia me nā enzyme restriction EcoRI a me HindIII (mai Roche ʻelua). ʻO ka hoʻokomo ʻia a hoʻomaʻemaʻe ʻia (QIAquick, Qiagen) hoʻokomo (T4 ligase, New England Biolabs) i hoʻopaʻa ʻia i loko o ke kiʻi i loko o kahi vector T7 pre-cleaved ma hope o ka amino acid 348 o ka 10B capsid gene. Hoʻokomo ʻia nā hopena ligation ma 16 ° C. no 18 mau hola ma mua o ka hoʻopili ʻana i loko o ka vitro. Hana ʻia ʻo Phage packaging in vitro e like me nā ʻōlelo aʻoaʻo i hāʻawi ʻia me ka T7Select 10-3b cloning kit (Novagen) a ua hoʻonui ʻia ka solution packaging i hoʻokahi manawa e lysis me ka hoʻohana ʻana iā Escherichia coli (BLT5615, Novagen). Ua centrifuged nā lysates, titrated a maloʻo ma -80 ° C. ma ke ʻano he waihona waiwai o ka glycerol.
Hoʻonui ʻia ka PCR pololei o nā ʻāpana ʻāpana phage i hoʻonui ʻia i ka broth a i ʻole ka pā me ka hoʻohana ʻana i nā kumu mua fusion 454/Roche-amplicon. Aia i loko o ka mua fusion primer nā kaʻina e pili ana i ka ʻāpana ʻokoʻa (NNK) 12 (kahi kikoʻī), GS FLX Titanium Adapter A, a me kahi kaʻina kī waihona waihona ʻehā (TCAG) (Ke Kiʻi Hoʻohui 1a):
Aia i loko o ka reverse fusion primer ka biotin i ho'opili 'ia no ka hopu 'ana i nā pele a me ka GS FLX Titanium Adapter B pono no ka ho'onui clonal i ka wā emulsion PCR.
Hoʻokomo ʻia nā amplicons i ka 454/Roche pyrosequencing e like me ka protocol 454 GS-FLX Titanium. No ke kaʻina hana Sanger manual (Applied Biosystems Hitachi 3730 xl DNA Analyzer), ua hoʻonui ʻia ka T7 phage DNA e ka PCR a ua hoʻonohonoho ʻia me nā hui mua:
Hoʻokomo ʻia nā mea hoʻokomo mai nā plaque pākahi i ka PCR amplification me ka Roche Fast Start DNA Polymerase Kit (e like me nā ʻōlelo a ka mea hana). Hana i kahi hoʻomaka wela (10 min ma 95 °C) a me 35 mau pōʻaiapili hoʻoikaika (50 s ma 95 °C, 1 min ma 50 °C, a me 1 min ma 72 °C).
ʻO Phage mai nā hale waihona puke, phage ʻano hihiu, phage i hoʻopakele ʻia mai CSF a me ke koko, a i ʻole nā ​​clones pākahi i hoʻonui ʻia ma Escherichia coli BL5615 i ka broth TB (Sigma Aldrich) a i ʻole ma 500 cm2 kīʻaha (Thermo Scientific) no 4 mau hola ma 37 ° C. Wehe ʻia ʻo Phage mai nā papa ma ka holoi ʻana i nā pā me Tris-EDTA buffer (Fluka Analytical) a i ʻole ma ka ʻohi ʻana i nā pā me nā ʻōlelo aʻoaʻo pipette sterile. Ua hoʻokaʻawale ʻia ʻo Phage mai ka moʻomeheu supernatant a i ʻole ka mea hoʻoheheʻe ʻokiʻoki me hoʻokahi pōʻai o ka polyethylene glycol (PEG 8000) precipitation (Promega) a hoʻopaneʻe hou ʻia ma Tris-EDTA buffer.
Ua hoʻokau ʻia ka phage i hoʻonui ʻia i ka 2-3 puni o ka wehe ʻana i ka endotoxin me ka hoʻohana ʻana i nā peʻa hoʻohemo endotoxin (Miltenyi Biotec) ma mua o ka hoʻokele intravenous (IV) (500 μl / holoholona). I ka pōʻai mua, ua hoʻokomo ʻia nā phages 2 × 1012; i ka lua, 2×1010 phages; i ke kolu a me ka ha koho puni, 2×109 phages no ka holoholona. ʻO ka maʻiʻo Phage ma CSF a me nā laʻana koko i hōʻiliʻili ʻia i nā manawa i hōʻike ʻia ua hoʻoholo ʻia e ka helu plaque e like me nā kuhikuhi a ka mea hana (T7Select system manual). Ua hana ʻia ke koho ʻana o Phage e ka hoʻokomo ʻana o nā hale waihona puke i hoʻomaʻemaʻe ʻia i loko o ka huelo a i ʻole ka hoʻihoʻi hou ʻana o ka phage i unuhi ʻia mai ka CSF mai ka pōʻai koho mua, a ua hana ʻia nā hōʻiliʻili ma hope ma 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, a me 240 mau minuke koko. He ʻehā mau pōʻai o in vivo panning i mālama ʻia a mālama ʻia nā lālā ʻelua i koho ʻia a hoʻopaʻa ʻia i loko o nā pōʻai koho mua ʻekolu. ʻO nā mea hoʻokomo phage āpau i unuhi ʻia mai ka CSF mai nā pōʻai mua ʻelua o ke koho ʻana i hoʻopaʻa ʻia i ka 454/Roche pyrosequencing, aʻo nā clones āpau i unuhi ʻia mai CSF mai nā pōʻai hope ʻelua o ke koho ʻana i hoʻonohonoho lima ʻia. ʻO nā phages koko a pau mai ka pōʻai mua o ke koho ʻana i kau ʻia i ka 454/Roche pyrosequencing. No ka hoʻokomo ʻana i nā clones phage, ua hoʻonui ʻia nā phages i koho ʻia ma E. coli (BL5615) ma nā papa 500 cm2 ma 37 ° C no 4 mau hola. Ua hoʻolaha ʻia nā clones i koho ʻia a me ka lima i hoʻolaha ʻia ma ke ʻano TB. Ma hope o ka unuhi ʻana o ka phage, hoʻomaʻemaʻe a me ka wehe ʻana o ka endotoxin (e like me ka mea i hōʻike ʻia ma luna nei), 2 × 1010 phages / holoholona i 300 μl i hoʻokomo ʻia i loko o ka vein huelo hoʻokahi.
Preprocessing a me qualitative kānana o ka ʻikepili kaʻina. Ua hoʻololi ʻia ka ʻikepili Raw 454/Roche mai kahi ʻano palapala palapala kahawai maʻamau (sff) i kahi palapala hiki ke heluhelu ʻia e ke kanaka Pearson (fasta) me ka hoʻohana ʻana i ka lako polokalamu mea kūʻai aku. Hana ʻia ka hana hou ʻana o ke kaʻina nucleotide me ka hoʻohana ʻana i nā polokalamu C proprietary a me nā palapala (puke polokalamu i hoʻokuʻu ʻole ʻia) e like me ka mea i wehewehe ʻia ma lalo nei. Hoʻopili ʻia ka ʻikepili o ka ʻikepili kumu i nā kaʻina hana kānana nui. No ka kānana ʻana i ka heluhelu ʻaʻole i loaʻa i ka 12mer insert DNA sequentially, ua hoʻonohonoho ʻia nā heluhelu e hoʻomaka i ka lepili (GTGATGTCGGGGATCCGAATTCT), ka lepili hoʻomaha (TAAGCTTGCGGCCGCACTCGAGTA) a me ka hoʻokomo hope (CCCTGCAGGGATATCCCGGGAGCTCGTCGAC) me ka hoʻohana ʻana i ka honua Needleman-Wunsch test. alignment hiki i ka 2 inconsistencies no ka alignment31. No laila, ʻaʻole i hoʻomaka a hoʻōki i nā hōʻailona a me nā heluhelu e loaʻa ana nā mea hoʻokomo hope, ʻo ia hoʻi, ua wehe ʻia nā alignment i ʻoi aku ma mua o ka helu i ʻae ʻia o nā mismatches. No nā heluhelu i koe, ʻo ka N-mer DNA sequence mai ka hoʻomaka ʻana a me ka pau ʻana ma mua o ka pau ʻana o ka māka i hoʻopau ʻia mai ke kaʻina heluhelu mua a hoʻoponopono hou ʻia (ma hope i kapa ʻia ʻo "hoʻokomo"). Ma hope o ka unuhi ʻana o ka mea hoʻokomo, wehe ʻia ka ʻāpana ma hope o ka codon pani mua ma ka hopena 5′ o ka mea 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 wehe ʻana i nā mea hoʻokomo i loaʻa nā kaʻina hope wale nō, ua wehe ʻia nā mea hoʻokomo i unuhi ʻia e hoʻomaka ana me ke kumu amino acid "PAG". Ua wehe ʻia nā peptide me ka lōʻihi ma hope o ka unuhi ʻana ma lalo o 3 mau waikawa amino mai ka waihona. ʻO ka hope, e wehe i ka redundancy i loko o ka wai hoʻokomo a hoʻoholo i ke alapine o kēlā me kēia hoʻokomo kū hoʻokahi. ʻO nā hopena o kēia hoʻopaʻa ʻana i loaʻa i kahi papa inoa o nā kaʻina nucleotide (hoʻokomo) a me kā lākou (heluhelu) alapine (Supplementary Figures 1c a me 2).
Hoʻokomo pū ʻia ka N-mer DNA ma ke kaʻina like: No ka hoʻopau ʻana i nā hewa kikoʻī kikoʻī 454/Roche (e like me nā pilikia me ka hoʻopili ʻana i nā homopolymer homopolymer) a wehe i nā redundancies liʻiliʻi nui, ua kānana mua ʻia nā mea hoʻokomo o ke kaʻina N-mer DNA (inserts) e like me ka like. hoʻokomo ʻia (a hiki i 2 mau kumu like ʻole i ʻae ʻia) me ka hoʻohana ʻana i ka algorithm iterative i wehewehe ʻia penei: hoʻopili mua ʻia nā hoʻokomo e ko lākou alapine (kiʻekiʻe a haʻahaʻa), a inā like lākou, ma ko lākou ʻano lua ma ka lōʻihi (lōʻihi a pōkole) ). No laila,ʻo nā hoʻokomo pinepine a lōʻihi loa e wehewehe i ka "hui" mua. Hoʻonohonoho ʻia ka alapine hui i ke alapine kī. A laila, ua ho'āʻo ʻia kēlā me kēia hoʻokomo i koe i ka papa inoa i hoʻohui ʻia i ka hui ma ka hoʻohālikelike ʻana ʻo Needleman-Wunsch. Inā ʻaʻole ʻoi aku ka nui o nā ʻokoʻa, hoʻokomo, a holoi ʻia paha i ka paepae o 2, hoʻohui ʻia kahi hoʻokomo i ka pūʻulu, a hoʻonui ʻia ka pinepine o ka hui e ka manawa i hoʻohui ʻia ai ka hoʻokomo. Hōʻailona ʻia nā mea hoʻokomo i hoʻohui ʻia i kahi hui i hoʻohana ʻia a kāpae ʻia mai ka hana hou ʻana. Inā ʻaʻole hiki ke hoʻohui ʻia ke kaʻina hoʻokomo i kahi hui i loaʻa, hoʻohana ʻia ke kaʻina hoʻokomo e hana i kahi hui hou me ke alapine hoʻokomo kūpono a kaha ʻia e like me ka hoʻohana ʻia. Hoʻopau ka ʻike 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 loaʻa. Ma hope o nā mea a pau, ua unuhi ʻia nā mea hoʻokomo i hui pū ʻia me nā nucleotides i nā kaʻina peptide (nā hale waihona puke peptide). ʻO ka hopena o kēia hōʻuluʻulu ʻana he pūʻulu o nā mea hoʻokomo a me kā lākou mau alapine kūpono e hana ai i ka helu o nā heluhelu ʻana (Supplementary Fig. 2).
Motif Generation: Ma muli o ka papa inoa o nā peptides kū hoʻokahi, ua hana ʻia kahi waihona i loaʻa nā ʻano amino acid āpau (aa) e like me ka mea i hōʻike ʻia ma lalo nei. Ua unuhi ʻia kēlā me kēia hiʻohiʻona o ka lōʻihi 3 mai ka peptide a ua hoʻohui ʻia kona ʻano hoʻohālikelike me kahi waihona motif maʻamau i loaʻa nā ʻano āpau (tripeptides). Ua hoʻokaʻawale ʻia nā hale waihona puke o nā kumu hana hou a hoʻopau ʻia. A laila, no kēlā me kēia tripeptide i loko o ka waihona motif, ua nānā mākou i kona noho ʻana i ka waihona me ka hoʻohana ʻana i nā mea hana helu. I kēia hihia, hoʻohui ʻia ka pinepine o ka peptide i loaʻa ka tripeptide kumu i ʻike ʻia a hāʻawi ʻia i ke kumu i loko o ka waihona kumu ("helu o nā kumu"). ʻO ka hopena o ka motif generation, he ʻano ʻelua ʻāpana i loaʻa nā hanana a pau o nā tripeptides (motifs) a me ko lākou mau waiwai ponoʻī, ʻo ia ka helu o ka heluhelu ʻana i ka hopena i ke kumu kūpono ke kānana ʻia nā heluhelu, hui ʻia, a unuhi ʻia. Nā ana e like me ka mea i wehewehe ʻia ma luna.
ʻO ka maʻamau o ka helu o nā kumu a me nā scatterplots e pili ana: Ua hoʻohālikelike ʻia ka helu o nā kumu no kēlā me kēia laʻana.
kahi ni ka helu o nā heluhelu i loaʻa ke kumuhana i. No laila, hōʻike ʻia ka vi i ka pakeneka pinepine o nā heluhelu (a i ʻole peptides) i loaʻa ke kumu i loko o ka hāpana. Ua helu ʻia nā waiwai P no ka helu kumu ʻole i hoʻohana ʻia me ka hoʻāʻo pololei a Fisher. E pili ana i nā correlograms o ka helu o nā kumu, ua helu ʻia nā correlations a Spearman me ka hoʻohana ʻana i ka helu maʻamau o nā kumu me R.
No ka ʻike ʻana i ka ʻike o nā waikawa amino i kēlā me kēia kūlana i ka waihona peptide, ua hana ʻia nā logograms 32, 33 (http://weblogo.threeplusone.com). ʻO ka mea mua, mālama ʻia ka ʻike o nā waikawa amino i kēlā me kēia kūlana o ka peptide 12-mer i loko o kahi matrix 20 × 12. A laila, hoʻokumu ʻia kahi pūʻulu o 1000 peptides i loaʻa i ka maʻiʻo amino acid like ma kēlā me kēia kūlana ma ke ʻano wikiwiki-sequence a hāʻawi ʻia ma ke ʻano he hoʻokomo i ka pūnaewele-logo 3, e hana ana i kahi kiʻi kiʻi o ka maʻiʻo amino acid pili i kēlā me kēia kūlana. no kahi waihona peptide i hāʻawi ʻia. No ka ʻike ʻana i nā ʻikepili multidimensional, ua hana ʻia nā palapala wela me ka hoʻohana ʻana i kahi mea hana i kūkulu 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 hoʻohuihui hierarchical a Ward me ka metric mamao Euclidean. No ka helu helu helu o ka ʻikepili helu kumu, helu ʻia nā helu P no ka helu 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 t-test a ka haumāna a i ʻole ANOVA.
ʻO nā clones phage i koho ʻia a me nā phages me ka ʻole o nā mea hoʻokomo i hoʻokomo ʻia i loko o ka vein huelo (2 × 1010 phages / holoholona i 300 μl PBS). He ʻumi mau minuke ma mua o ka perfusion a me ka hoʻoponopono hou ʻana, ua hoʻokomo ʻia nā holoholona like me ka 100 μl o ka lectin i kapa ʻia ʻo DyLight594 (Vector Laboratories Inc., DL-1177). 60 mau minuke ma hope o ka hoʻoheheʻe ʻana i ka phage, ua honi ʻia nā ʻiole ma o ka puʻuwai me 50 ml PBS a ukali ʻia e 50 ml 4% PFA/PBS. Ua hoʻopaʻa ʻia nā laʻana lolo i ka pō ma 4% PFA/PBS a hoʻomoʻi ʻia i 30% sucrose i ka pō ma 4°C. Hoʻopaʻa ʻia nā laʻana ma ka hui ʻOCT. Hana ʻia ka loiloi immunohistochemical o nā laʻana hau hau ma ka lumi wela ma 30 µm cryosections i ālai ʻia me 1% BSA a hoʻokomo ʻia me ka polyclonal FITC-labeled antibodies kūʻē i ka T7 phage (Novus NB 600-376A) ma 4 °C. Incubate i ka pō. ʻO ka hope, holoi ʻia nā ʻāpana i nā manawa 3 me ka PBS a nānā ʻia me kahi microscope laser confocal (Leica TCS SP5).
ʻO nā peptides a pau me ka maʻemaʻe liʻiliʻi o 98% i synthesized e GenScript USA, biotinylated a lyophilized. Hoʻopaʻa ʻia ʻo Biotin ma o kahi spacer triple glycine hou ma ka N-terminus. E nānā i nā peptide a pau me ka hoʻohana ʻana i ka spectrometry.
Ua hui pū ʻia ʻo Streptavidin (Sigma S0677) me kahi 5-fold equimolar excess of biotinylated peptide, biotinylated BACE1 inhibitory peptide, a i ʻole kahi hui (3: 1 ratio) o ka biotinylated BACE1 inhibitory peptide a me BACE1 inhibitory peptide ma 5-10% DMSO/incubated i loko o PBSSO/incubated. 1 hola ma ka lumi wela ma mua o ka hoʻokomo ʻana. ʻO nā peptides i hoʻohui ʻia ʻo Streptavidin i hoʻopaʻa ʻia ma ke ʻano o 10 mg / kg i loko o kekahi o nā veins huelo o nā ʻiole me kahi lua cerebral.
Ua loiloi ʻia ka ʻike o ka streptavidin-peptide complexes e ELISA. Ua uhi ʻia nā papa microtiter Nunc Maxisorp (Sigma) i ka pō ma 4 ° C me 1.5 μg / ml mouse anti-streptavidin antibody (Thermo, MA1-20011). Ma hope o ka hoʻopaʻa ʻana (paʻa paʻa: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) ma ka lumi wela no 2 mau hola, holoi i ka pā me 0.05% Tween-20/PBS (holoi buffer) no 3 Second, CSF a me nā plasma buffers i hoʻohui ʻia. 1:10,000, CSF 1:115). Hoʻopili ʻia ka pā i ka pō ma 4 ° C me ka antibody detection (1 μg / ml, anti-streptavidin-HRP, Novus NB120-7239). Ma hope o ʻekolu holoi ʻana, ʻike ʻia ka streptavidin e ka incubation i loko o ka TMB substrate solution (Roche) a hiki i 20 min. Ma hope o ka ho'ōki 'ana i ka ho'omohala kala me 1M H2SO4, e ana i ka absorbance ma 450 nm.
ʻO ka hana o ka streptavidin-peptide-BACE1 inhibitor complex i loiloi ʻia e Aβ(1-40) ELISA e like me ka protocol o ka mea hana (Wako, 294-64701). ʻO ka pōkole, ua hoʻoheheʻe ʻia nā laʻana CSF i ka diluent maʻamau (1:23) a hoʻomoʻi ʻia i ka pō ma 4 ° C i loko o nā papa 96-well i uhi ʻia me ka BNT77 hopu antibody. Ma hope o ʻelima holoi ʻana, ua hoʻohui ʻia ka antibody BA27 i hoʻohui ʻia e HRP a hoʻomoʻi ʻia no 2 mau hola ma 4 ° C., a ukali ʻia e ʻelima holoi holoi. Ua ʻike ʻia ʻo Aβ (1-40) e ka incubation i loko o ka hopena TMB no 30 mau minuke ma ka lumi wela. Ma hope o ka pau ʻana o ka hoʻomohala kala ʻana me ka hopena hoʻomaha, e ana i ka absorbance ma 450 nm. Hoʻokomo ʻia nā laʻana plasma i ka unuhi ʻana i ka pae paʻa ma mua o Aβ(1-40) ELISA. Hoʻohui ʻia ka Plasma i ka 0.2% DEA (Sigma) i nā pā 96-well a hoʻokomo ʻia i ka lumi lumi no 30 mau minuke. Ma hope o ka holoi ʻana i nā pā SPE (Oasis, 186000679) me ka wai a me 100% methanol, ua hoʻohui ʻia nā laʻana plasma i nā pā SPE a ua wehe ʻia nā wai āpau. Holoi ʻia nā laʻana (mua me 5% methanol a laila 30% methanol) a eluted me 2% NH4OH/90% methanol. Ma hope o ka hoʻomaloʻo ʻana i ka ʻeluate ma 55 ° C no 99 min i ka manawa N2 mau, ua hoʻemi ʻia nā laʻana i nā diluents maʻamau a ua ana ʻia ʻo Aβ (1-40) e like me ka mea i hōʻike ʻia ma luna.
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