Mayar da Taswirorin Taswirar Tantanin halitta azaman Layer fifita fifiko, Ba Jerin Halittu ba
Canjin mafi amfani shine tunani: yi la'akari da fitowar omics azaman matakin fifiko wanda ke zaɓar inda ilmin halitta ya maida hankali, da kuma ajiye shawarwarin ƙirar peptide don bayan an tabbatar da zaɓin. Gina binciken peptide daga sararin samaniya da bayanan tantanin halitta maimakon daga jerin bambance-bambancen-bayani shine abin da ke barin shirin ya yi niyya zuwa yanayin tantanin halitta maimakon matsakaici.. A cikin aikin gano magunguna, Tsarin RNA-cell guda ɗaya ya zama madaidaicin Layer don tantancewa da ba da fifiko ga maƙasudi saboda yana warware bambance-bambancen cewa matsakaicin jeri mai girma ya ragu.. Reviews na aikace-aikace na jerin RNA-cell guda ɗaya a cikin gano magunguna bayyana yadda scRNA-seq ke goyan bayan gano manufa, cell-subtype-takamaiman bincike, kuma, mai zargi, Haɗarin ɓarna da wuri ta hanyar nuna waɗanne sel ke amsawa da kuma yadda allurai ke shafar su. Hanyoyin sararin samaniya sannan ƙara mahallin babban bayanan lalata: adana bayanan sararin samaniya mai tsayi shine abin da ke barin manazarta su gani sababbin nau'in tantanin halitta, hulɗar salula, da tsarin nama wanda ke da ma'ana kawai da zarar an ajiye wurin tantanin halitta.
Don maƙasudin peptide, Yaduddukan da za a iya aiwatarwa sun rushe zuwa ɗan gajeren jeri:
| Abin da za a cire | Me yasa yake da mahimmanci ga peptide | Peptide Synthesis Hanyar da aka saba |
|---|---|---|
| Masu karɓar sararin samaniya sun wadatar a cikin jihar da aka yi niyya da maƙwabta | Masu ɗaure ɗan takara don peptide mai iya samun damar fili | SRNA-seq bambance-bambancen magana da aka tace zuwa farfajiya |
| Ƙuntataccen wuri mai niches mai mai da hankali mai karɓa | Ƙananan sel marasa manufa, mafi girman ƙayyadaddun gida | Ƙididdigar sararin samaniya niche / alamar haɗin gwiwa |
| Ligand-nau'i-nau'i masu karɓa daga sadarwar gida | Yana zaɓar duka axis da mahallinsa | CellPhoneDB / Bayanin CellChat |
| Alamar canji-jihar (pseudotime, yanayi, gudu) | Yana Nufin abin da aka kunna, gajiye, m, ko bambanta yanayi - ba a tsaye nau'in cell | Binciken yanayin yanayi |
A ƙarshen wannan mataki kuna riƙe jerin sunayen 'yan takara da aka fifita, ba hukunci ba. Hukuncin ya fito ne daga matakan tabbatarwa da ke biyo baya.
Gabatar da Target Peptide: Inda Ƙayyadaddun Jiha-Cell Ya Samu Riba
Makasudin da aka keɓance kawai a cikin duka samfurin shine wurin farawa mai rauni ga peptide, saboda ƙimar peptide yawanci tana rayuwa ne a cikin zaɓin sa. Wannan ilhami yana da goyan bayan ƙididdiga. Wani babban bincike na baya-bayan nan na bayanan tantanin halitta guda ɗaya a cikin kyallen jikin mutum ya gano cewa takamaiman nau'in tantanin halitta da ƙayyadaddun ƙwayoyin cuta duka fasalulluka ne waɗanda aka wadatar a tsakanin maƙasudin shiga ci gaban asibiti., kuma ƙayyadaddun nau'in tantanin halitta a cikin nama mai alaƙa da cuta ya kasance ƙwaƙƙwaran tsinkayar ci gaban manufa daga Mataki na I zuwa Mataki na II. Sakamakon yana fitowa da matakin-safa, kuma ya cancanci auna jimla - amma ya yi daidai da gaskiya mai amfani: mai karɓar mai karɓa wanda aka taƙaita ta hanyar rubutu zuwa yanayin tantanin halitta da kuke kula da shi, da kuma inda jihar ke zaune, shine wanda ya fi dacewa ya ba peptide tagar mai tsabta akan manufa.
Don haka fifiko ya kamata ya ba da lada uku lokaci guda:
- Ƙayyadaddun Jiha. Yakamata a wadatar da mai karɓar mai karɓa a cikin yanayin tantanin halitta da ke da alaƙa dangane da maƙwabta da jihohi makamantansu., ba kawai a cikin nama ba.
- Ƙuntatawa sararin samaniya. Ya kamata ya mai da hankali a cikin alkuki wanda zai iya kaiwa ga jiki ta hanyar da aka nufa, wanda a lokaci guda yana rage yawan adadin ƙwayoyin da ba a kai ba wanda ligand zai ci karo da shi.
- Memba na Surfaceome. Saboda peptides suna aiki akan maƙasudi na waje ko samun dama, ba da fifiko ga kwayoyin halitta waɗanda samfuran da aka san su sunadaran sunadaran membrane maimakon intracellular ko nau'in ɓoye.
Tace mai amfani shine sanya maki kowane ɗan takara akan waɗannan gatura guda uku sannan a ci gaba da jerin sunayen da za su share duka. Mai karɓa wanda ke da takamaiman jiha amma binne, ko fili amma a ko'ina, rashin kyawun fare ne na farko komai ban sha'awa da canjin log-fold ɗin sa ya yi kama.
Ƙayyadaddun Jiha-Tanta: Binciken Gaskiyar Matsayin Protein
Anan ne yawancin shirye-shirye suka yi shiru. Ƙayyadaddun yanayin tantanin halitta tantanin halitta hasashe ne game da gaskiyar furotin; ba hujja ba ce. Kwayoyin halitta na iya kasancewa da ƙarfi a cikin yanayin tantanin halitta kuma har yanzu ba ya kasancewa a matsayin wanda ake iya nunawa, furotin a kan saman da peptide ke buƙatar ɗaure. Zaɓin takamaiman peptide na musamman-jihar tantanin halitta yana buƙatar tabbatar da cewa ƙayyadaddun ƙayyadaddun matakin rubutun ya tsira da fassara da fataucin - ba tare da ɗaukan hakan ba..
Littattafai akan injiniyan ligand masu zaɓin tantanin halitta suna ƙididdige dalilin da yasa wannan ke da mahimmanci. Zaɓin zaɓin yawan jama'a shine mafi kyawun ma'anar aiki - azaman rabon ligand da ke ɗaure ga yawan adadin cell akan ligand da aka ɗaure zuwa yawan jama'a - kuma cimma hakan ya dogara da ainihin maƙasudin bayyanar da manufa., ba ƙididdigewa ba. Kuma saboda peptides gabaɗaya dole ne su kai ga epitope na waje, isa ga manufa na iya zama mahimmanci gwargwadon matakin magana; furotin da aka bayyana sosai da aka binne a cikin hadaddun ko makale a cikin salula bai isa ba.
Tarin ingantaccen matakin furotin, a aikace, gajere ne kuma orthogonal:
- Yawo cytometry akan m, Kwayoyin marasa lahani shine duban layin farko cewa mai karɓa yana zaune akan saman waje kuma yana ƙididdige bambancin cell-to-cell.. An kafa nau'ikan nau'ikan kayan aiki da yawa don bayanin martabar tantanin halitta.
- Immunofluorescence ko immunohistochemistry yana tabbatar da ganowar membrane kuma yana bambanta siginar sigina na gaske daga tafkin intracellular.
- Surface proteomics ko niyya taro spectrometry yana ba da shaidar matakin furotin mara son zuciya kuma yana iya bayyana isoforms ko ƴan takara waɗanda ba su da ƙwayoyin rigakafi masu kyau.
- Hanyoyin proteo-genomic irin su CITE-seq-style readouts sun haɗa RNA kowane tantanin halitta tare da furotin ta saman, da guda-cell proteo-genomic reference maps ya wanzu daidai don tabbatar da cewa yanayin tantanin halitta da aka ayyana RNA yana ɗauke da furotin da aka annabta.
Bayanan lokaci guda ɗaya: a cellular thermal motsi assay (KASHE) ana yawan ambaton su tare da waɗannan kayan aikin, amma yana gano daidaitawar furotin da ke haifar da ligand - yana da maƙasudin karantawa wanda ke ɗauka cewa an riga an sami ɗaure.. Yi amfani da shi bayan kuna da ɗan takara, ba a matsayin kofa mai fallasa ba.
Key Takeaway: Bi da furci na banbanta a matsayin abin da zai sa a duba, sannan tabbatar da sunadarin yana saman yanayin yanayin tantanin halitta da aka nufa kafin ku tsara peptide guda ɗaya a kusa da shi. Ƙayyadaddun ƙayyadaddun jihar tantanin halitta wanda ke tsira daga gwajin matakin furotin shine ƙayyadaddun da allon zai iya ganowa.
Zabar Ligand Axis: Tabbatar da Recetor-Ligand Biyu
Da zarar saman-m, takamaiman mai karɓa na jihar ya tsira da inganci, Tambayar ƙira ta zama wacce za ta shiga - kuma a nan jarabar ita ce a amince da fitar da kayan aikin bincike kamar dai wani lamari ne mai ɗaurewa.. Ba haka ba ne.
Kayan aiki kamar CellPhoneDB kuma CellChat curate ligand-receptor databases hulda da kuma sanin abin da sadarwa gatari aka wadatar a kididdiga tsakanin kungiyoyin cell a guda-cell ko sarari bayanai. Suna da kyau don ba da fifiko ga waɗanne nau'i-nau'i-nau'i-nau'i masu karɓa ne suka cancanci ƙoƙarin gwaji, da CellChat musamman samfura na hadaddun subunit, agonists, masu adawa da juna, da kuma masu haɗin gwiwa. Amma axis da aka kwatanta ɗan takara ne, ba ma'amala da aka tabbatar ba. Haɗa peptide akan mai karɓa da kuma ganin ɗaurin baya da kansa ba ya tabbatar da peptide shine ligand mai aiki da tantanin halitta., kuma nau'i-nau'i da aka annabta cikin ƙididdigewa suna buƙatar tabbatarwa na asali kafin su sami kasafin haɗakarwa.
Kunshin tabbatarwa don axis ligand shine:
- Gasa ko shaidar ƙaura, ko kuma wani nau'i na ɗaure na orthogonal, cewa peptide da mai karɓan da ake tsammani suna hulɗa da gaske.
- A counter-allon panel gina da wuri, keɓance nau'ikan nau'ikan masu karɓa masu alaƙa da sel masu maƙasudi. Abokin dangantaka ba zaɓi ba ne; Zaɓan nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i)) ya samo asali ne kawai ta hanyar gwada dan takarar a kan dangin mai karɓa mafi girma, kamar yadda aikin ƙira akan ƙãra mai karɓa-subtype selectivity ya yi bayyane.
- Zane-sanin jiha ga masu karɓa masu ƙarfin hali. Yawancin makasudin peptide irin su GPCRs sun kasance a cikin abubuwan da ba su da aiki da kuma rashin aiki, kuma fifikon ligand na iya dogara da wace jiha ke yawan jama'a. Zayyana da yanayin da ake so maimakon tsari na tsaye zai iya zama bambanci tsakanin agonist wanda ke tafiyar da ilimin halitta da kuke so da wanda baya..
Jarrabawar yanke shawara shine: shine mai karɓa ya wadatar a cikin yanayin da aka yi niyya, shin yana iya samun samuwa a kan sel na asali, kuma akwai shedar kai tsaye peptide yana ɗaure shi da ƙarfi fiye da kusancin homologs da kyallen da aka kashe? A "a'a" akan kowane ɗayan waɗannan dalili ne don sake fasalin axis kafin yin abu.
Kunshin Shaidar Pre-Synthesis: Ƙofar Go/No-Go
A wannan lokacin shirin ya inganta, m mai karɓa da kuma tabbatar da axis ligand. Dabi'ar da mafi yawan ƙungiyoyi suke buƙata shine guda ɗaya, ƙofa bayyananne wanda dole ne a share kafin ƙira mai tsada da kuma kashe kuɗin tantancewa ba a ba da izini ba.
Kunshin shaidar da aka riga aka yi tana ƙarfafa cak ɗin da ke sama cikin lissafin da za a iya dubawa. Gajere ne da gangan, saboda ana tsallake kofar shaida mai nauyi, kuma wanda yake da haske sosai yana barin raunanan 'yan takara ta hanyar:
| Shaida | Yadda aka kafa shi | Yanayin gazawa idan babu |
|---|---|---|
| Maƙasudin dacewa da ilimin halitta a cikin cuta/jihar tantanin halitta | Na sarari + guda-cell kafin, mahallin hanya, bayanan aiki | Babban haɗin kai ga furotin da bai dace ba |
| furotin mai karɓa yana samuwa a saman | Sitometry mai gudana, IF/IHC, surface proteomics, proteo-genomic guda biyu | Peptide wanda aka ƙera akan fatalwar kwafi |
| Mai karɓa-ligand hulɗa Peptides na roba tabbatar | Gasa / ƙaura, orthogonal dauri, knockout/tarewa | Axis wanda ba ya ɗaure a zahiri |
| Alamar farko-wuta Samuwar Peptide | Daure kai tsaye ko gasa → Kd/Ki | Ba za a iya bambanta ainihin abin ɗaure da hayaniya ba |
| Zaɓin salula vs kashe-manufa/ subtypes | Allon fuska a cikin iyali + Kwayoyin lalata | Wani “takamaiman” peptide wanda ba a zahiri zaɓi ba |
| Binciken haɓakawa da wuri | Solubility, tarawa, serum/plasma kwanciyar hankali, proteolysis, kerawa | Buga mai ƙarfi wanda ba za a iya sarrafa ko sake bugawa ba |
Dan takarar da ke share kowane layi ya cancanci haɗawa da tantancewa. Wanda ya gaza ko da jere guda daya yakamata a sake tsara shi, sake samu, ko faduwa maimakon tilastawa. Kasawa masu tsada a cikin binciken peptide ba safai ba ne jerin abubuwan da ba shakka ba su da kyau - jerin ne waɗanda suka yi kama da kyau a kan gaɓa ɗaya kuma ba a taɓa gwada su akan sauran ba har sai bayan haɗawar ta kashe..
Daga Dan Takara Zuwa Material: Shirye-shiryen Tattaunawa da Bincike
Hukuncin ƙarshe shine game da kayan da kansa, kuma yana da daraja a raba shirye-shiryen sinadarai daga shirye-shiryen nazarin halittu don kada su sami rikice-rikice.
Shirye-shiryen sinadarai al'amari ne na ƙayyadaddun ma'auni masu inganci, kuma ma'auni sun bambanta ta mataki. Don binciken ganowa, sandar yarda da bincike na gama gari shine tsabtar chromatographic na aƙalla 95% ta hanyar juyawa-lokaci HPLC ko UHPLC, tare da kusan 98-99% an tanada don kayan aikin asibiti; peptide ainihi da tsarki ana kafa su ta hanyar juyawa-lokaci HPLC-UV da mass spectrometry maimakon kima guda ɗaya.. Saboda ƙa'idodin tsabta da tsammanin ƙazanta sun bambanta sosai tsakanin allon bincike da tsarin da aka tsara, gwajin peptide na nazari da shirin QC yakamata a saita kafin, ba bayan, isarwa ta farko.
Shirye-shiryen nazarin halittu shine inda aikin taswirar tantanin halitta ke biyan kuɗi. peptide wanda ya kai ga nunawa ya kamata ya ɗauki tabbacin da ya samu a sama: takamaiman mai karɓa na ƙasa-m, hulɗar da aka tabbatar, da Kd-farko da adadi mai zaɓi. Ba tare da waɗannan ba, siginar allo mai kyau ba ta da tabbas kuma mara kyau ba ta da labari. Wannan shine dalilin da ya sa mafi girman dabi'a a cikin wannan aikin shine don haɗa haɗin gwiwa a bayan fakitin shaida maimakon gaba da shi..
Wannan kuma shine inda dan takarar da aka ba da fifiko daga taswira ya hadu da gaskiyar al'adar peptide synthesis bench da kuma m peptide ayyuka - tsarin zane, dabarun gyarawa, solubility-sani analog zane, da takaddun matakin HPLC/MS duk sun zama abubuwan yanke shawara waɗanda ke tantance ko kayan da kike gani shine kwayar halittar da aka yi niyya..
Don Tukwici: Yi oda ƙaramin rukunin farko na analogs na kusa - ba jeri ɗaya ba - don haka haɗawar ku ta farko tana ba ku siginar SAR., shinge mai narkewa, da zaɓin karantawa a zagaye ɗaya maimakon da yawa.
Gina Gano Peptide daga Fayil da Bayanan Tantanin halitta Guda Akewaye da Shaida, Ba Sha'awa ba
Abin da ke raba shirin gano peptide wanda ke canza ilimin halitta mai ƙima zuwa motsin 'yan takara daga wanda ke tattara taswira masu ban sha'awa shine ƙin haɗa sigina tare da mafita.. Taswirorin suna zaɓe; tabbatarwa ya zaɓa; kuma Ƙofar shaida ta fayyace lokacin da ɗan takara ya cancanci abu mai tsada.
Tsarin ayyuka yana da mahimmanci fiye da kowane kayan aiki guda ɗaya. Ba da fifiko ga saman-m, takamaiman mai karɓa na jiha. Tabbatar cewa yana kan saman nau'in tantanin halitta da aka nufa. Tabbatar da ligand axis da kuke shirin shiga. Haɗa ɗan gajeren kunshin da za a iya dubawa - dacewa, gaban kasa, tabbatar da hulɗar, Kd, zaɓe, da haɓakawa - sannan kawai ciyarwa akan haɗawa da nunawa.
Lokacin da kwamitin ƴan takarar ku ya share wannan ƙofar, La'akari na gaba shine ko haɗin ku da kuma abokin tarayya na QC na iya sadar da sifa ta asali, Abubuwan da aka rubuta batch wanda shirin tantancewa ke buƙata - nau'in horo wanda ke kiyaye ƙididdiga na auna kwayoyin ku ba hayaniya ba.. Wannan shine inda abokin tarayya kamar MOL Canje-canje yana ba da ƙarshen-zuwa-ƙarshe al'ada peptide kira da Class 100 bakararre masana'antu, gwajin peptide da QC, kuma babban kundin gyare-gyare na iya taimakawa - don haka peptide da allonku ke gani shine wanda taswirarku tayi alkawari.. Daidaita shawarar nazarin halittu tare da aiwatar da kisa daga rana ɗaya shine abin da ke juya taswirar tantanin halitta zuwa shirin ɗan takara wanda a zahiri ke motsawa..
