Maita basa nekushanyira nature.com. Shanduro yebrowser yauri kushandisa ine rutsigiro rushoma rweCSS. Kuti uwane ruzivo rwakanaka, tinokurudzira kushandisa shanduro yebrowser yazvino (kana kudzima compatibility mode muInternet Explorer). Pamusoro pezvo, kuti uve nechokwadi chekuti rutsigiro runoramba ruchienderera mberi, saiti iyi ichave isina masitayera uye JavaScript.
Tsandanyama dzemapfupa imhando dzakasiyana-siyana dzinoumbwa zvakanyanya ne myofibrils, iyo muvanhu inowanzoiswa mumhando nhatu: imwe "inononoka" (rudzi 1) uye mbiri "dzinokurumidza" (rudzi 2A ne2X). Zvisinei, kusiyana pakati uye mukati memhando dzemyofibril dzechinyakare kuchiri kusanzwisiswa zvakanaka. Takashandisa nzira dze transcriptomic ne proteomic ku1050 ne1038 myofibrils dzemunhu kubva ku vastus lateralis yevanhu, zvichiteerana. Chidzidzo che proteomic chaisanganisira varume, uye chidzidzo che transcriptomic chaisanganisira varume gumi nevakadzi vaviri. Kuwedzera kune myosin heavy chain isoforms, takaona mapuroteni e metabolic, ribosomal proteins, uye cellular junctional proteins senzvimbo dze multidimensional intermyofibril variability. Uyezve, pasinei nekuzivikanwa kwemapoka e fibers dzinononoka uye dzinokurumidza, data redu rinoratidza kuti fibers dzerudzi rwe2X hadzisiyaniswi nedzimwe fibers dzinokurumidza. Uyezve, kupatsanurwa kwe myosin heavy chain-based hakuna kukwana kutsanangura phenotype ye myofiber mu nemaline myopathies. Zvose, data redu rinoratidza kusiyana kwe multidimensional myofiber, nemasosi ekuchinja anosvika kupfuura myosin heavy chain isoforms.
Kusiyana kwemasero chinhu chiripo mumasisitimu ese ehupenyu, zvichibvumira masero kuti anyatso shanda kuti asangane nezvinodiwa zvakasiyana zvematishu nemasero.1 Maonero echinyakare ekusawirirana kwefiber yemhasuru yeskeletal yave iri ekuti ma neuron emotor anotsanangura rudzi rwefiber mukati meyuniti yemotor, uye kuti rudzi rwefiber (kureva, rudzi 1, rudzi 2A, uye rudzi 2X muvanhu) rwunotariswa nehunhu hwe myosin heavy chain (MYH) isoforms.2 Izvi pakutanga zvakavakirwa pakusagadzikana kwavo kwepH ATPase, 3,4 uye gare gare pakutaura kwavo kwemamorekuru kweMYH.5 Zvisinei, nekuzivikanwa uye kugamuchirwa kwe "mixed fibers" dzinobatanidza MYH dzakawanda muhuwandu hwakasiyana, skeletal tsandanyama fibers dziri kuonekwa zvakanyanya se continuum pane semhando dzakasiyana dzefiber.6 Pasinei neizvi, munda uchiri kuvimba zvakanyanya neMYH senzira huru yekupatsanura myofiber, maonero angangove achikanganiswa nekusawirirana kwakakosha kwezvidzidzo zvepakutanga zvemakonzo ane MYH expression profiles uye mhando dzakasiyana dzefibers dzakasiyana nedzevanhu.2 Mamiriro acho ezvinhu anowedzera kuoma nekuti tsandanyama dzakasiyana dzeskeletal dzevanhu dzinoratidza mhando dzakasiyana dzefiber. mhando.7 Vastus lateralis imhasuru yakasanganiswa ine chimiro chepakati (uye nekudaro inomiririra) cheMYH.7 Uyezve, nyore kwayo kutora sampuro kunoita kuti ive tsandanyama yakadzidziswa zvakanyanya muvanhu.
Saka, kuferefeta kusina rusaruro pamusoro pekusiyana kwemhasuru dzemapfupa uchishandisa zvishandiso zvine simba zve "omics" kwakakosha asi zvakare kwakaoma, chimwe chikamu nekuda kwehunhu hwemhasuru dzemapfupa dzine nucleic dzakawanda. Zvisinei, matekinoroji e transcriptomics8,9 ne proteomics10 akachinja zvakanyanya mukunzwa kwemuviri mumakore achangopfuura nekuda kwekufambira mberi kwakasiyana-siyana kwetekinoroji, zvichibvumira kuongororwa kwemhasuru dzemapfupa pakugadzirisa kwefiber imwe chete. Nekuda kweizvozvo, kufambira mberi kukuru kwakaitwa mukuratidza kusiyana kwefiber imwe chete uye mhinduro yavo kune atrophic stimuli uye kukwegura11,12,13,14,15,16,17,18. Zvakakosha, kufambira mberi uku kwetekinoroji kune mashandisirwo ekiriniki, zvichibvumira kutsanangurwa kwakanyatsojeka uye kwakarurama kwekusagadzikana kwezvirwere. Semuenzaniso, pathophysiology ye nemaline myopathy, imwe yezvirwere zvemhasuru zvakajairika zvakagarwa nhaka (MIM 605355 neMIM 161800), yakaoma uye inovhiringidza.19,20 Saka, kutsanangurwa kuri nani kwekusagadzikana kwemhasuru dzemapfupa kunogona kutungamira mukufambira mberi kukuru mukunzwisisa kwedu chirwere ichi.
Takagadzira nzira dzekuongorora transcriptomic neproteomic ye single skeletal muscle fibers dzakabviswa nemaoko kubva ku biopsy sampuli dzevanhu ndokudzishandisa kuzviuru zve fibers, zvichitibvumira kuongorora kusiyana kwemasero kwe skeletal muscle fibers dzevanhu. Mukati mebasa iri, takaratidza simba re transcriptomic ne proteomic phenotyping ye muscle fibers uye takaona metabolic, ribosomal, uye cellular junctional proteins senzvimbo dzakakosha dzekusiyana kwe interfiber. Uyezve, tichishandisa iyi proteomic workflow, takatsanangura kukosha kwe nematode myopathy mu single skeletal muscle fibers, zvichiratidza kuchinja kwakabatana kuenda kune non-oxidative fibers isina hunhu hwe fiber hwakavakirwa pa MYH.
Kuti tiongorore kusiyana kwemhasuru dzemhasuru dzemapfupa evanhu, takagadzira nzira mbiri dzekushanda nadzo kuti tigone kuongorora transcriptome neproteome yemhasuru imwe chete yemapfupa (Mufananidzo 1A neMufananidzo Wekuwedzera 1A). Takagadzira nekugadzirisa matanho akati wandei ekuita, kubva pakuchengetedza sampuro nekuchengetedza RNA neprotein integrity kusvika pakugadzirisa throughput yenzira yega yega. Pakuongorora transcriptome, izvi zvakawanikwa nekuisa sample-specific molecular barcodes padanho rekutanga re reverse transcription, zvichibvumira 96 fibers kuti dziunganidzwe kuti dzishandiswe zvakanaka pasi. Kutevedzana kwakadzama (±1 miriyoni inoverengwa pafiber imwe neimwe) zvichienzaniswa nenzira dzechinyakare dze single-cell kwakawedzera kusimudzira data re transcriptome. 21 Pamaproteomics, takashandisa short chromatographic gradient (maminetsi makumi maviri nerimwe) yakasanganiswa neDIA-PASEF data acquisition pa timsTOF mass spectrometer kuti tiwedzere kudzika kweproteome uku tichichengetedza throughput yakakwira. 22,23 Kuti tiongorore kusiyana kwefibers dzetsandanyama dzine hutano, takatsanangura transcriptomes dzefibers 1,050 kubva kuvanhu 14 vane hutano uye proteomes dzefibers 1,038 kubva kuvanhu 5 vane hutano (Table Supplementary 1). Mupepa rino, datasets idzi dzinonzi transcriptomes neproteomes 1,000-fiber, zvichiteerana. Maitiro edu akaona huwandu hwezvinyorwa 27,237 uye mapuroteni 2,983 mu1,000-fiber transcriptomic neproteomic analysis (Mufananidzo 1A, Supplementary Datasets 1–2). Mushure mekusefa datasets dzetranscriptomic neproteomic dzemajini akawanikwa anopfuura chiuru uye 50% valid values pa fiber imwe neimwe, bioinformatics ongororo dzakatevera dzakaitwa dzefibers 925 ne974 mutranscriptome neproteome, zvichiteerana. Mushure mekusefa, avhareji yemajini 4257 ± 1557 uye mapuroteni e2015 ± 234 (avhareji ± SD) akaonekwa pafiber imwe neimwe, nekusiyana kushoma pakati pemunhu (Supplementary Figures 1B–C, Supplementary Datasets 3–4). Zvisinei, kusiyana kwemukati memunhu kwakaonekwa zvakanyanya kune vatori vechikamu, zvichida nekuda kwekusiyana kweRNA/protein goho pakati pefibers dzehurefu hwakasiyana nenzvimbo dzakachinjika. Kune mapuroteni mazhinji (>2000), coefficient of variation yaive pasi pe20% (Supplementary Figure 1D). Nzira dzese dziri mbiri dzakabvumira kutora mhando dzakasiyana dzezvinyorwa nemapuroteni ane siginecha dzakanyatsojeka dzakakosha pakubata tsandanyama (semuenzaniso, ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Supplementary Figures 1E–F). Zvizhinji zvezvinhu zvakaonekwa zvaive zvakajairika pakati pedata re transcriptomic neproteomic (Supplementary Picture 1G), uye avhareji yeUMI/LFQ yezviratidzo izvi yaive yakabatana zvakanaka (r = 0.52) (Supplementary Picture 1H).
Kufamba kweTranscriptomics neproteomics (kwakagadzirwa neBioRender.com). BD Dynamic range curves yeMYH7, MYH2, neMYH1, uye yakaverengerwa miganho yekugoverwa kwemhando yefiber. E, F Kugoverwa kweMYH expression kuburikidza nefibers mu transcriptomics neproteomics datasets. G, H Uniform Diversity Approximation and Projection (UMAP) plots dze transcriptomics neproteomics dzakapendwa neMYH-based fiber type. I, J Feature plots dzinoratidza MYH7, MYH2, uye MYH1 expression mu transcriptomics neproteomics datasets.
Pakutanga takatanga kupa mhando yefiber yakavakirwa paMYH kune fiber yega yega tichishandisa nzira yakagadziriswa inoshandisa mukana wehukuru hwekunzwa uye dynamic range yeMYH expression mumadatasets eomics. Zvidzidzo zvekare zvakashandisa miganhu isina kurongeka kuti inyore mafiber semhando 1 yakachena, mhando 2A, mhando 2X, kana yakasanganiswa zvichibva pahuwandu hwakatarwa hwekutaura kweMYHs11,14,24 dzakasiyana. Takashandisa nzira yakasiyana umo kutaura kwefiber yega yega kwakarongwa neMYHs dzatakashandisa kunyora mafibers: MYH7, MYH2, uye MYH1, zvinoenderana nerudzi 1, rudzi 2A, uye mhando 2X fibers, zvichiteerana. Takazoverenga nemasvomhu poindi yakaderera ye curve yega yega yakabuda uye takaishandisa semuganho wekupa mafibers seakanaka (pamusoro pemuganho) kana asina kunaka (pasi pemuganho) kune yega yega MYH (Mufananidzo 1B–D). Data iri rinoratidza kuti MYH7 (Mufananidzo 1B) uye MYH2 (Mufananidzo 1C) dzine ma profiles ekutaura e on/off akasiyana padanho reRNA zvichienzaniswa ne protein level. Zvechokwadi, padanho reprotein, mafiber mashoma kwazvo aisaburitsa MYH7, uye hapana fiber yaiva ne100% MYH2 expression. Takashandisawo miganhu yakatarwa yeexpression kuti tipe MYH-based fiber types kune mafiber ese ari mudataset yega yega. Semuenzaniso, MYH7+/MYH2-/MYH1- fibers dzakapihwa kurudzi rwe1, nepo MYH7-/MYH2+/MYH1+ fibers dzakapihwa kurudzi rwe2A/2X rwakasanganiswa (ona Supplementary Table 2 kuti uwane tsananguro yakazara). Tichibatanidza mafiber ese, takaona kupararira kwakafanana kweMYH-based fiber types pamatanho ese ari maviri eRNA (Mufananidzo 1E) neprotein (Mufananidzo 1F), nepo kuumbwa kwemhando dzefiber dzakavakirwa paMYH kwakasiyana pamunhu, sezvaitarisirwa (Mufananidzo Wekuwedzera 2A). Mafiber mazhinji akarongwa semhando 1 yakachena (34–35%) kana mhando 2A (36–38%), kunyangwe huwandu hwakawanda hwemafiber emhando 2A/2X akasanganiswa hwakaonekwawo (16–19%). Musiyano unoshamisa ndewekuti fiber dzerudzi rwe2X dzakachena dzaingoonekwa chete padanho reRNA, asi kwete padanho reprotein, zvichiratidza kuti kukurumidza kutaura kweMYH kunodzorwa zvishoma mushure mekunyora.
Takasimbisa nzira yedu yekunyora fiber yakavakirwa paproteomics-based MYH tichishandisa antibody-based dot blotting, uye nzira dzese dziri mbiri dzakawana kubvumirana kwe100% mukuona fibers dzerudzi rwekutanga nedzerudzi rwechipiri (ona Mufananidzo Wekuwedzera 2B). Zvisinei, nzira yeproteomics-based yaive yakanyanya kunzwisisika, inoshanda zvakanyanya mukuona fibers dzakasanganiswa, uye kuyera huwandu hwegene rega rega reMYH mufiber yega yega. Data iri rinoratidza kushanda kwekushandisa nzira yakananga, inonyanya kunzwisisika ye omics-based kuratidza mhando dzefiber dzemhasuru dzeskeletal.
Takabva tashandisa ruzivo rwakabatanidzwa rwakapihwa ne transcriptomics ne proteomics kuti tigovere myofibers zvakananga zvichibva pane transcriptome yavo yakazara kana proteome. Tichishandisa nzira ye uniform manifold approximation and projection (UMAP) kuderedza dimensionality kusvika kuzvikamu zvitanhatu zvikuru (Supplementary Figures 3A–B), takakwanisa kuona myofiber variability mu transcriptome (Mufananidzo 1G) ne proteome (Mufananidzo 1H). Zvinonyanya kukosha, myofibers hadzina kurongwa nevatori vechikamu (Supplementary Figures 3C–D) kana mazuva ekuyedza (Supplementary Figure 3E) mu transcriptomics kana proteomics datasets, zvichiratidza kuti variability yemukati me skeletal tsandanyama fibers yakakwira kupfuura variability yepakati pe subject. MuUMAP plot, mapoka maviri akasiyana anomiririra myofibers "inokurumidza" uye "inononoka" yakabuda (Mifananidzo 1G–H). Ma myofibers eMYH7+ (anononoka) akaunganidzwa pamucheto wakanaka weUMAP1, nepo ma myofibers eMYH2+ neMYH1+ (anononoka) akaunganidzwa pamutsetse usina kunaka weUMAP1 (Mifananidzo 1I–J). Zvisinei, hapana musiyano wakaitwa pakati pemhando dzefiber dzinokurumidza kutsvedza (kureva, mhando 2A, mhando 2X, kana 2A/2X yakasanganiswa) zvichibva pakutaura kweMYH, zvichiratidza kuti MYH1 (Mufananidzo 1I–J) kana mamwe ma classical 2X myofiber markers akadai seACTN3 kana MYLK2 (Mifananidzo Yekuwedzera 4A–B) kutaura hakusiyanisi pakati pemhando dzakasiyana dze myofiber pakufunga nezve transcriptome yese kana proteome. Uyezve, zvichienzaniswa neMYH2 neMYH7, zvinyorwa zvishoma kana mapuroteni akabatana zvakanaka neMYH1 (Mifananidzo Yekuwedzera 4C–H), zvichiratidza kuti kuwanda kweMYH1 hakuratidzi zvizere myofiber transcriptome/proteome. Mhedziso dzakafanana dzakawanikwa pakuongorora kusanganiswa kwema isoform matatu eMYH padanho reUMAP (Supplementary Figs. 4I–J). Saka, nepo 2X fibers ichigona kuonekwa padanho re transcript zvichibva pa MYH quantification chete, MYH1+ fibers hadzisiyaniswi nedzimwe fast fibers kana tichitarisa transcriptome yese kana proteome.
Sekutanga kwekuongorora kusiyana kwe fiber kunononoka kupfuura MYH, takaongorora mapuroteni mana akasimbiswa emhando ye fiber inononoka: TPM3, TNNT1, MYL3, uye ATP2A22. Mhando dze fiber dzinononoka dzakaratidza hukama hwakakwira, kunyange husina kukwana, nePearson neMYH7 mune ese transcriptomics (Mufananidzo Wekuwedzera 5A) uye proteomics (Mufananidzo Wekuwedzera 5B). Inenge 25% ne33% ye fiber dzinononoka hadzina kurongwa se fiber dzinononoka dzakachena nemarudzi ese e gene/protein mu transcriptomics (Mufananidzo Wekuwedzera 5C) uye proteomics (Mufananidzo Wekuwedzera 5D), zvichiteerana. Nokudaro, kupatsanurwa kwe fiber kunononoka kwakavakirwa pane akawanda ma gene/protein subtypes kunounza kuoma kwakawedzerwa, kunyangwe kumapuroteni anozivikanwa kuti ane mhando ye fiber. Izvi zvinoratidza kuti kupatsanurwa kwe fiber kwakavakirwa pa isoforms yemhuri ye gene/protein imwe chete hakugone kuratidza zvakakwana kusiyana kwechokwadi kwe fiber dzemhasuru dzeskeletal.
Kuti tiongorore zvakanyanya kusiyana kwemasero emhasuru dzemapfupa evanhu pachiyero chemuenzaniso weomics wese, takaita kudzikiswa kwedata zvisina rusaruro tichishandisa principal component analysis (PCA) (Mufananidzo 2A). Kufanana neUMAP plots, hapana mutori wechikamu kana zuva rekuyedza rakakanganisa kuunganidzwa kwefiber padanho rePCA (Zvifananidzo Zvekuwedzera 6A–C). Mumadatasets ese ari maviri, MYH-based fiber type yakatsanangurwa nePC2, iyo yakaratidza cluster yemafibers emhando ye1 anononoka uye cluster yechipiri ine fast-twitch type 2A, type 2X, uye mixed 2A/2X fibers (Mufananidzo 2A). Mumadatasets ese ari maviri, ma cluster maviri aya akabatanidzwa nenhamba diki yemafibers emhando ye1/2A akasiyana. Sezvaitarisirwa, kuongororwa kwakawandisa kwemadhiraivha makuru ePC kwakasimbisa kuti PC2 yaitungamirirwa ne contractile uye metabolic signatures (Mufananidzo 2B uye Supplementary Figures 6D–E, Supplementary Datasets 5–6). Kazhinji, mhando yefiber yakavakirwa paMYH yakawanikwa yakakwana kutsanangura kusiyana-siyana kuri kuitika paPC2, kunze kweinonzi 2X fibers dzaiparadzirwa mu transcriptome mukati me fast cluster.
A. Mapuratifomu ekuongorora zvikamu zvikuru (PCA) edata re transcriptome neproteome rakapendwa zvichienderana nerudzi rwefiber zvichibva paMYH. B. Kuongororwa kwekuwedzera kwezvinyorwa zvinofambisa mapuroteni muPC2 nePC1. Kuongororwa kwestatistike kwakaitwa uchishandisa clusterProfiler package uye Benjamini-Hochberg adjusted p-values. C, D. Mapuratifomu ePCA akapendwa zvichienderana neintercellular adhesion gene ontology (GO) terms mu transcriptome ne costamere GO terms mu proteome. Miseve inomiririra zvinyorwa zvinofambisa mapuroteni nemirairo yazvo. E, F. Uniform manifold approximation and projection (UMAP) ine mapuratifomu ezviratidzo zvine chekuita nekiriniki zvinoratidza ma gradients ekutaura asina kutsamira pamhando yefiber inononoka/inokurumidza. G, H. Kubatana pakati pePC2 nePC1 drivers mu transcriptomes neproteomes.
Zvisingatarisirwi, mhando yemyofiber yakavakirwa paMYH yakatsanangura dhigirii rechipiri repamusoro rekusiyana (PC2), zvichiratidza kuti zvimwe zvinhu zvehupenyu zvisina chekuita neMYH-based myofiber type (PC1) zvine basa rakakosha mukugadzirisa kusiyana kwefiber yemhasuru yeskeletal. Kuongororwa kwakawandisa kwezvinokonzeresa zvakanyanya muPC1 kwakaratidza kuti kusiyana muPC1 kwainyanya kutsanangurwa nekunamatira kwemasero ne ribosome mu transcriptome, uye costameres nemapuroteni e ribosomal mu proteome (Mufananidzo 2B ne Supplementary Figures 6D–E, Supplementary Data Set 7). Mutsandanyama yeskeletal, costameres inobatanidza Z-disc ne sarcolemma uye inobatanidzwa mukutumira simba uye kusaina. 25 Annotated PCA plots vachishandisa cell-cell adhesion (transcriptome, Mufananidzo 2C) uye costamere (proteome, Mufananidzo 2D) zvakaratidza shanduko yakasimba yekuruboshwe muPC1, zvichiratidza kuti zvinhu izvi zvakapfuma mune mamwe mafiber.
Kuongororwa kwakadzama kwe myofiber clustering paUMAP level kwakaratidza kuti zvinhu zvakawanda zvakaratidza gradient ye MYH-based expression gradient isina mhando ye myofiber pane ye myofiber subcluster-specific. Kuenderera mberi uku kwakaonekwa kune majini akati wandei ane chekuita nemamiriro ezvinhu ezvirwere (Mufananidzo 2E), akadai seCHCHD10 (chirwere cheneuromuscular), SLIT3 (muscle atrophy), CTDNEP1 (chirwere chemhasuru). Kuenderera mberi uku kwakaonekwawo muproteome, kusanganisira mapuroteni ane chekuita nezvirwere zvetsinga (UGDH), insulin signaling (PHIP), uye transcription (HIST1H2AB) (Mufananidzo 2F). Pamwe chete, data iri rinoratidza kuramba kuripo mu fiber-independent slow/fast twitch heterogeneity mu myofibers dzakasiyana.
Zvinofadza kuti majini emutyairi muPC2 akaratidza hukama hwakanaka hwetranscriptome-proteome (r = 0.663) (Mufananidzo 2G), zvichiratidza kuti marudzi efiber anononoka uye anokurumidza kukweshana, uye kunyanya hunhu hwekubata uye metabolic hwefibers dzemhasuru dzeskeletal, anodzorwa netranscription. Zvisinei, majini emutyairi muPC1 haana kuratidza hukama hwetranscriptome-proteome (r = -0.027) (Mufananidzo 2H), zvichiratidza kuti kusiyana kusina chekuita nemarudzi efiber anononoka/anokurumidza kukweshana kunonyanya kudzorwa mushure mekunyora. Nekuti kusiyana muPC1 kwainyanya kutsanangurwa nemashoko eribosomal gene ontology, uye tichifunga kuti ribosomes inoita basa rakakosha uye rakasiyana musero nekutora chikamu mukushandura mapuroteni,31 takatanga kuongorora kusiyana uku kusingatarisirwi kweribosomal.
Takatanga tapenda puroti yekuongorora zvikamu zvikuru zveproteomics zvichienderana nehuwandu hwemapuroteni muGOCC izwi rekuti "cytoplasmic ribosome" (Mufananidzo 3A). Kunyangwe izwi iri rakapfuma kune divi rakanaka rePC1, zvichikonzera gradient diki, mapuroteni eribosomal anotungamira kupatsanurana munzira dzese dziri mbiri dzePC1 (Mufananidzo 3A). Mapuroteni eribosomal akapfuma kune divi risina kunaka rePC1 aisanganisira RPL18, RPS18, uye RPS13 (Mufananidzo 3B), nepo RPL31, RPL35, uye RPL38 (Mufananidzo 3C) ndiwo ainyanya kukonzera divi rakanaka rePC1. Zvinofadza kuti, RPL38 neRPS13 zvakaratidzwa zvakanyanya mutsandanyama dzemapfupa zvichienzaniswa nedzimwe nyama (Mufananidzo 7A). Aya masaini akasiyana eribosomal muPC1 haana kuonekwa mutranscriptome (Mufananidzo 7B), zvichiratidza mutemo wepost-transcriptional.
A. Plot yekuongorora zvikamu zvikuru (PCA) yakapendwa zvichienderana nemashoko e cytoplasmic ribosomal gene ontology (GO) muproteome. Miseve inoratidza kutungamira kwekuchinja kweprotein muPCA plot. Kureba kwemutsetse kunoenderana ne principal component score yeprotein yakapihwa. B, C. PCA inoratidza ma plots eRPS13 neRPL38. D. Kuongororwa kwe hierarchical clustering ongororo yema cytoplasmic ribosomal proteins. E. Structural model ye 80S ribosome (PDB: 4V6X) inoratidza ma ribosomal proteins ane huwandu hwakasiyana mu skeletal tsandanyama fibers. F. Ribosomal proteins ane stoichiometry dzakasiyana dziri pedyo ne mRNA exit channel.
Pfungwa dze ribosomal heterogeneity uye specialization dzakambotaurwa kare, apo kuvapo kwe different ribosome subpopulations (ribosomal heterogeneity) kunogona kukanganisa zvakananga kushandurwa kweprotein mumatishu akasiyana32 nemasero33 kuburikidza neshanduro yakasarudzwa ye specific mRNA transcript pools34 (ribosome specialization). Kuti tione subpopulations dze ribosomal proteins co-expression mu skeletal tsandanyama fibers, takaita unsupervised hierarchical clustering analysis yema ribosomal proteins mu proteome (Mufananidzo 3D, Supplementary Data Set 8). Sezvaitarisirwa, ribosomal proteins hadzina kuungana ne fiber type zvichibva pa MYH. Zvisinei, takaona ma clusters matatu akasiyana ema ribosomal proteins; cluster yekutanga (ribosomal_cluster_1) co-regulated ne RPL38 uye nokudaro ine kuwedzera kwe expression mu fibers ine positive PC1 profile. Cluster yechipiri (ribosomal_cluster_2) co-regulated ne RPS13 uye yakakwira mu fibers ine negative PC1 profile. Chikamu chechitatu (ribosomal_cluster_3) hachiratidzi kuratidzwa kwakasiyana-siyana mumhasuru dzemapfupa uye chinogona kunzi puroteni ye "core" yemhasuru dzemapfupa. Mapoka ese ari maviri eribosomal 1 ne2 ane mapuroteni eribosomal akamboratidzwa kuti anotonga shanduro dzakasiyana (semuenzaniso, RPL10A, RPL38, RPS19, uye RPS25) uye anokanganisa mashandiro ekukura (semuenzaniso, RPL10A, RPL38).34,35,36,37,38 Zvichienderana nemhedzisiro yePCA, kuratidzwa kwakasiyana kwakaonekwa kwemapuroteni aya eribosomal mumhasuru kwakaratidzawo kuenderera mberi (Mufananidzo Wekuwedzera 7C).
Kuti tifungidzire nzvimbo yemapuroteni eribosomal akasiyana mukati meribosome, takashandisa modhi yemaumbirwo emunhu 80S ribosome (Protein Data Bank: 4V6X) (Mufananidzo 3E). Mushure mekuparadzanisa mapuroteni eribosomal ari mumaboka akasiyana eribosomal, nzvimbo dzawo dzakanga dzisina kuenderana zvakanyanya, zvichiratidza kuti nzira yedu yakakundikana kupa kupfuma kwenzvimbo/zvikamu zveribosome. Zvisinei, zvinofadza kuti huwandu hwemapuroteni makuru esubunit muboka 2 hwaive hwakaderera pane mumaboka 1 ne3 (Mufananidzo 7D Wekuwedzera). Takaona kuti mapuroteni ane stoichiometry yakachinjwa mumapfupa emhasuru dzemapfupa ainyanya kuwanikwa panzvimbo yeribosome (Mufananidzo 3E), zvichienderana nekukwanisa kwawo kusangana nezvinhu zvemukati zveribosome entry site (IRES) mumapoka akasiyana emRNA, nokudaro achibatanidza shanduro yakasarudzwa. 40, 41 Uyezve, mapuroteni mazhinji ane stoichiometry yakachinjwa mumapfupa emhasuru dzemapfupa aive pedyo nenzvimbo dzinoshanda dzakadai semRNA exit tunnel (Mufananidzo 3F), iyo inodzora kurebeswa kweshanduro nekusungwa kwepeptides chaiyo. 42 Mukupfupisa, data redu rinoratidza kuti stoichiometry yemapuroteni e ribosomal muscle muscle anoratidza kusiyana, zvichikonzera mutsauko pakati pemafiber emuscle muscle.
Takazotanga kutsvaga masiginecha efaibha anokasira uye anononoka uye kuongorora mashandiro emutemo wavo wekunyora. Tichienzanisa maboka efaibha anokasira uye anononoka anotsanangurwa neUMAP mumaseti maviri edata (Mifananidzo 1G–H na 4A–B), ongororo dzetranscriptomic neproteomic dzakaratidza zvinhu 1366 ne804 zvakasiyana-siyana, zvichiteerana (Mifananidzo 4A–B, Supplementary Datasets 9–12). Takaona mutsauko waitarisirwa mumasiginecha ane chekuita nemasarcomere (semuenzaniso, tropomyosin netroponin), excitation-contraction coupling (SERCA isoforms), uye energy metabolism (semuenzaniso, ALDOA neCKB). Uyezve, matranscript nemapuroteni anodzora protein ubiquitination akaratidzwa zvakasiyana mumafaibha anokasira uye anononoka (semuenzaniso, USP54, SH3RF2, USP28, uye USP48) (Mifananidzo 4A–B). Uyezve, jini remapuroteni ebhakitiriya RP11-451G4.2 (DWORF), iro rakamboratidzwa kuti rinoratidzwa zvakasiyana-siyana mumhando dzefiber yemhasuru yegwayana43 uye rinowedzera basa reSERCA mumhasuru yemwoyo44, rakawedzerwa zvakanyanya mufiber yemhasuru inononoka (Mufananidzo 4A). Saizvozvowo, padanho refiber yega yega, misiyano yakakosha yakaonekwa muzviratidzo zvinozivikanwa zvakaita se lactate dehydrogenase isoforms ine chekuita nemetabolism (LDHA neLDHB, Mufananidzo 4C ne Supplementary Figure 8A)45,46 pamwe chete nezviratidzo zvemhando yefiber zvaisazivikanwa kare (zvakadai seIRX3, USP54, USP28, uye DPYSL3) (Mufananidzo 4C). Pakanga paine kuwirirana kwakakosha kwezviratidzo zvinoratidzwa zvakasiyana pakati pedata re transcriptomic neproteomic (Mufananidzo Wekuwedzera 8B), pamwe nekubatana kwekuchinja kwefold kunokonzerwa nekuratidzwa kwakasiyana kwezviratidzo zvesarcomere (Mufananidzo Wekuwedzera 8C). Zvinonyanya kukosha kuti mamwe masiginecha (semuenzaniso USP28, USP48, GOLGA4, AKAP13) akaratidza kurongeka kwakasimba kwekutevera mushure mekunyora chete padanho reproteomic uye aive nemaitiro ekutaura kwefiber anononoka/anokurumidza (Supplementary Picture 8C).
Mapuratifomu eA neB evolcano achienzanisa mapoka anononoka uye anokurumidza akaonekwa nepuratifomu yeuniform manifold approximation and projection (UMAP) muMifananidzo 1G–H. Madotsi ane mavara anomiririra zvinyorwa kana mapuroteni akasiyana zvakanyanya paFDR < 0.05, uye madotsi matema anomiririra zvinyorwa kana mapuroteni akasiyana zvakanyanya pakuchinja kwelog > 1. Kuongororwa kwenhamba mbiri kwakaitwa uchishandisa bvunzo yeDESeq2 Wald neBenjamini–Hochberg adjusted p values (transcriptomics) kana nzira yeLimma linear model ne empirical Bayesian analysis ichiteverwa neBenjamini–Hochberg adjustment yekuenzanisa kwakawanda (proteomics). C Zviratidzo zveSignature zvemajini kana mapuroteni akasarudzwa akasiyana pakati pemafiber anononoka uye anokurumidza. D Kuongororwa kwekuwedzera kwezvinyorwa nemapuroteni akaratidzwa zvakasiyana zvakanyanya. Kukosha kwakabatana kwakapfuma mudatasets ese ari maviri, kukosha kwetranscriptome kwakapfuma mutranscriptome chete, uye kukosha kweproteome kwakapfuma muproteome chete. Kuongororwa kwenhamba kwakaitwa uchishandisa clusterProfiler package neBenjamini-Hochberg adjusted p-values. E. Zvinhu zvinonyorwa zvemhando yefiber zvakaonekwa neSCENIC zvichibva paSCENIC-derived regulator specificity scores uye differential mRNA expression pakati pemhando dzefiber. F. Kurongeka kwezvinhu zvakasarudzwa zvinonyorwa zvakasiyana pakati pefibers dzinononoka nedzinokurumidza.
Takabva taita ongororo yakawandisa yemajini nemapuroteni anomiririra zvakasiyana (Mufananidzo 4D, Supplemental Data Set 13). Kuwedzerwa kwenzira yezvinhu zvakasiyana pakati pemadata maviri kwakaratidza misiyano inotarisirwa, senge fatty acid β-oxidation uye ketone metabolism processes (slow fibers), myofilament/muscle contraction (fast and slow fibers, zvichiteerana), uye carbohydrate catabolic processes (fast fibers). Serine/threonine protein phosphatase activity yakakwidziridzwawo mufast fibers, zvichikonzerwa nezvinhu zvakaita se regulatory and catalytic phosphatase subunits (PPP3CB, PPP1R3D, uye PPP1R3A), izvo zvinozivikanwa kuti zvinodzora glycogen metabolism (47) (Supplemental Figures 8D–E). Dzimwe nzira dzakapfuma mu fast fibers dzaisanganisira processing (P-) bodies (YTHDF3, TRIM21, LSM2) mu proteome (Supplementary Fig. 8F), zvingangove zvichibatanidzwa mu post-transcriptional regulation (48), uye transcription factor activity (SREBF1, RXRG, RORA) mu transcriptome (Supplementary Fig. 8G). Slow fibers dzakapfuma mu oxidoreductase activity (BDH1, DCXR, TXN2) (Supplementary Fig. 8H), amide binding (CPTP, PFDN2, CRYAB) (Supplementary Fig. 8I), extracellular matrix (CTSD, ADAMTSL4, LAMC1) (Supplementary Fig. 8J), uye receptor-ligand activity (FNDC5, SPX, NENF) (Supplementary Fig. 8K).
Kuti tinzwisise zvakawanda nezve transcriptional regulation iri pasi pehunhu hwetsandanyama dzinononoka/dzinokurumidza, takaita ongororo ye transcription factor enrichment tichishandisa SCENIC49 (Supplementary Data Set 14). Zvinhu zvakawanda zve transcription zvakapfuma zvikuru pakati petsandanyama dzinokurumidza uye dzinononoka (Mufananidzo 4E). Izvi zvaisanganisira zvinhu zve transcription zvakaita seMAFA, iyo yakambobatanidzwa nekukura kwetsandanyama dzinokurumidza,50 pamwe nezvimwe zvinhu zve transcription zvisati zvambobatanidzwa nemapurogiramu emagene etsandanyama dzinoenderana netsandanyama. Pakati peizvi, PITX1, EGR1, uye MYF6 ndizvo zvinhu zve transcription zvakapfuma zvikuru mutsandanyama dzinokurumidza (Mufananidzo 4E). Kusiyana neizvi, ZSCAN30 neEPAS1 (inozivikanwawo seHIF2A) ndizvo zvinhu zve transcription zvakapfuma zvikuru mutsandanyama dzinononoka (Mufananidzo 4E). Zvichienderana neizvi, MAFA yakaratidzwa pamatanho akakwirira munzvimbo yeUMAP inoenderana netsandanyama dzinokurumidza, nepo EPAS1 yaive nepattern yekutaura yakasiyana (Mufananidzo 4F).
Kuwedzera kune majini anozivikanwa ekugadzira mapuroteni, kune akawanda marudzi eRNA asingakodhesi anogona kunge akabatanidzwa mukudzora kukura kwevanhu uye chirwere. 51, 52 Mumadata etranscriptome, maRNA akati wandei asingakodhesi anoratidza hunyanzvi hwemhando yefiber (Mufananidzo 5A neSupplementary Dataset 15), kusanganisira LINC01405, iyo inonyanya kutsanangurwa kune fibers dzinononoka uye inonzi yakaderera mutsandanyama kubva kuvarwere vane mitochondrial myopathy. 53 Kusiyana neizvi, RP11-255P5.3, inoenderana nejini relnc-ERCC5-5 (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, inoratidza hunyanzvi hwemhando yefiber inokurumidza. Dzese LINC01405 (https://tinyurl.com/x5k9wj3h) neRP11-255P5.3 (https://tinyurl.com/29jmzder) dzinoratidza tsandanyama dzemapfupa (Supplementary Figures 9A–B) uye hadzina majini anozivikanwa ekubatira mukati me1 Mb genomic community yavo, zvichiratidza kuti dzinoita basa rakakosha mukutonga mhando dzefiber pane kutonga majini ari pedyo ekubatira. Ma profiles ekuratidzwa kwefiber type-specific eLINC01405 neRP11-255P5.3, zvichiteerana, akasimbiswa uchishandisa RNAscope (Figures 5B–C).
A. Zvinyorwa zveRNA zvisina kukodha zvinodzorwa zvakanyanya mumhasuru dzinofamba zvishoma nezvishoma uye nekukurumidza. B. Mifananidzo yeRNAscope inoratidza kutsanangurwa kwefiber yeLINC01405 neRP11-255P5.3, zvichiteerana. Scale bar = 50 μm. C. Kuyera kweRNA isina kukodha mhando yemyofiber seyakatsanangurwa neRNAscope (n = 3 biopsies kubva kuvanhu vakazvimiririra, kuenzanisa fiber dzemhasuru dzinofamba nekukurumidza uye dziri mukati memunhu wega wega). Kuongororwa kwenhamba kwakaitwa uchishandisa bvunzo yeT-Test yeMudzidzi ine miswe miviri. Mabhokisi anoratidza maquartiles epakati neekutanga neechitatu, nendebvu dzichinongedzera kuhuwandu hushoma uye hwepamusoro. D. De novo microbial protein identification workflow (yakagadzirwa neBioRender.com). E. Puroteni yemicrobial LINC01405_ORF408:17441:17358 inonyatsoratidzwa mumhasuru dzinononoka dzeskeletal (n=5 biopsies kubva kune vatori vechikamu vakazvimiririra, vachienzanisa mhasuru dzinokurumidza uye dzinononoka dziri muvatori vechikamu mumwe nemumwe). Kuongororwa kwenhamba kwakaitwa uchishandisa nzira yeLimm linear model yakabatanidzwa nenzira yeBayesian, ichiteverwa nenzira yeBenjamini-Hochberg yekuenzanisa kwakawanda ne p-value adjustment. Mabhokisi anoratidza ma quartiles epakati, ekutanga neechitatu, nendebvu dzichinongedzera kune maximum/minimum values.
Munguva pfupi yapfuura, zvidzidzo zvakaratidza kuti zvinyorwa zvakawanda zvisina kunyorwa zvinonyora mapuroteni ehutachiona, mamwe acho anotonga mashandiro emhasuru. 44, 55 Kuti tizive mapuroteni ehutachiona ane mhando yefiber, takatsvaga dataset yedu ye1000 fiber proteome tichishandisa faira reFASTA rakagadzirwa rine marongero ezvinyorwa zvisina kunyorwa (n = 305) zvinowanikwa mudataset ye1000 fiber transcriptome (Mufananidzo 5D). Takaona mapuroteni ehutachiona 197 kubva muzvinyorwa makumi maviri nemaviri zvakasiyana, 71 zvacho zvakadzorwa zvakasiyana pakati pefibers dzemhasuru dzinononoka uye dzinokurumidza (Mufananidzo Wekuwedzera 9C neSeti Yekuwedzera Data 16). Kune LINC01405, zvigadzirwa zvitatu zveprotein yehutachiona zvakawanikwa, chimwe chazvo chakaratidza kufanana kwefiber inononoka nechinyorwa chayo (Mufananidzo 5E neMufananidzo Wekuwedzera 9D). Saka, takaona LINC01405 sejini rinonyora protein yehutachiona chaiyo yefibers dzemhasuru dzinononoka.
Takagadzira nzira yakakwana yekuongorora huwandu hukuru hweproteomic hwemhasuru dzega dzega uye takaona vanodzora kusiyana kwefiber muhutano hwakanaka. Takashandisa nzira iyi yekushanda kuti tinzwisise kuti nemaline myopathies inokanganisa sei kusiyana kwemhasuru dzemhasuru dzeskeletal. Nemaline myopathies zvirwere zvemhasuru zvinogara nhaka zvinokonzera kushaya simba kwemhasuru uye, muvana vakabatwa, zvinosangana nematambudziko akasiyana-siyana anosanganisira kufema, scoliosis, uye kufamba kushoma kwemakumbo. 19,20 Kazhinji, mu nemaline myopathies, mhando dzezvirwere mumajini akadai seactin alpha 1 (ACTA1) dzinokonzera kuwanda kwe fiber myofiber inogadzirwa zvishoma nezvishoma, kunyangwe mhedzisiro iyi yakasiyana. Chimwe chinhu chinoshamisa i troponin T1 nemaline myopathy (TNNT1), iyo ine kuwanda kwe fast fibers. Saka, kunzwisisa zviri nani kusiyana kuri pasi pe skeletal muscle fiber dysregulation inoonekwa mu nemaline myopathies kunogona kubatsira kubvisa hukama hwakaoma pakati pezvirwere izvi nerudzi rwe myofiber.
Zvichienzaniswa nevanochengetedza hutano (n=3 paboka), myofibers dzakabviswa kubva kuvarwere vane nemaline myopathy vane mutations mumajini eACTA1 neTNNT1 dzakaratidza myofiber atrophy kana dystrophy (Mufananidzo 6A, Tafura Yekuwedzera 3). Izvi zvakapa matambudziko makuru ehunyanzvi hwekuongorora proteomic nekuda kwehuwandu hushoma hwezvinhu zviripo. Pasinei neizvi, takakwanisa kuona mapuroteni 2485 mu272 skeletal myofibers. Mushure mekusefa mapuroteni angangoita 1000 akayerwa pafiber imwe neimwe, mafiber 250 akaiswa kuongororwa kwebioinformatics kwakatevera. Mushure mekusefa, avhareji yemapuroteni 1573 ± 359 pafiber imwe neimwe akayerwa (Mufananidzo Wekuwedzera 10A, Supplementary Data Sets 17–18). Zvinonyanya kukosha kuti, pasinei nekuderera kukuru kwehukuru hwefiber, kudzika kweproteome kwemasampuli evarwere vane nemaline myopathy kwakaderedzwa zvishoma. Uyezve, kugadzirisa data iri tichishandisa mafaira edu eFASTA (kusanganisira zvinyorwa zvisiri zvekodhi) zvakatibvumira kuziva mapuroteni mashanu ehutachiona mumapfupa emafupa kubva kuvarwere vemaminerari (Supplementary Data Set 19). Dynamic range yeproteome yaive yakakura zvakanyanya, uye mapuroteni ese ari muboka rekudzora akabatana zvakanaka nemigumisiro yeongororo yapfuura ye1000-fiber proteome (Supplementary Fig. 10B–C).
A. Mifananidzo ye microscopic inoratidza fiber atrophy kana dystrophy uye kuwanda kwemhando dzakasiyana dzefiber zvichibva paMYH muACTA1 neTNNT1 nemaline myopathies (NM). Scale bar = 100 μm. Kuti kuve nechokwadi chekuti staining inogona kudzokororwa muvarwere veACTA1 neTNNT1, ma biopsies matatu evarwere akaiswa mavara kaviri kusvika katatu (zvikamu zvina pachiitiko chimwe nechimwe) vasati vasarudza mifananidzo inomiririra. B. Fiber type proportions muvatori vechikamu zvichibva paMYH. C. Principal component analysis (PCA) plot ye skeletal muscle fibers muvarwere vane nemaline myopathies uye controls. D. Skeletal muscle fibers kubva kuvarwere vane nemaline myopathies uye controls zvakaratidzwa paPCA plot zvakatarwa kubva pa1000 fibers dzakaongororwa muMufananidzo 2. Semuenzaniso, volcano plot dzichienzanisa mutsauko pakati pevatori vechikamu vane ACTA1 neTNNT1 nemaline myopathies uye controls, uye pakati pevatori vechikamu vane ACTA1 neTNNT1 nemaline myopathies. Madenderedzwa ane mavara anoratidza mapuroteni akasiyana zvakanyanya pa π < 0.05, uye madotsi matema anoratidza mapuroteni akasiyana zvakanyanya pa FDR < 0.05. Kuongororwa kwenhamba kwakaitwa uchishandisa nzira yeLimma linear model uye nzira dzeBayesian dze empirical, zvichiteverwa ne p-value adjustment yekuenzanisa kwakawanda uchishandisa nzira yeBenjamini-Hochberg. H. Kuongororwa kwekuwedzera kwemapuroteni akabudiswa zvakasiyana zvakanyanya muproteome yese uye mumhando 1 ne2A fibers. Kuongororwa kwenhamba kwakaitwa uchishandisa clusterProfiler package uye Benjamini-Hochberg adjusted p-values. I, J. Principal component analysis (PCA) plots dzakapendwa ne extracellular matrix uye mitochondrial gene ontology (GO) terms.
Nekuti nemaline myopathies inogona kukanganisa huwandu hwemhando dze MYH-expressing myofiber mutsandanyama dzemapfupa, 19,20 takatanga taongorora mhando dze MYH-expressing myofiber muvarwere vane nemaline myopathies uye controls. Takawana mhando ye myofiber tichishandisa nzira isina rusaruro yakambotsanangurwa kare ye 1000 myofiber assay (Supplementary Figs. 10D–E) uye takatadza zvakare kuona pure 2X myofibers (Mufananidzo 6B). Takaona mhedzisiro yakasiyana ye nemaline myopathies pamhando ye myofiber, sezvo varwere vaviri vane ACTA1 mutations vaive nehuwandu hwakawanda hwemhando ye 1 myofibers, nepo varwere vaviri vane TNNT1 nemaline myopathy vaive nehuwandu hwakaderera hwemhando ye 1 myofibers (Mufananidzo 6B). Zvechokwadi, kuratidzwa kweMYH2 uye fast troponin isoforms (TNNC2, TNNI2, uye TNNT3) kwakadzikira muACTA1-nemaline myopathies, nepo kuratidzwa kweMYH7 kwakadzikira muTNNT1-nemaline myopathies (Mufananidzo Wekuwedzera 11A). Izvi zvinoenderana nemishumo yapfuura yekuchinja kwemhando dzakasiyana dzemyofiber munemaline myopathies.19,20 Takasimbisa mhedzisiro iyi ne immunohistochemistry uye takaona kuti varwere vane ACTA1-nemaline myopathy vaive nehuwandu hwemhando 1 myofibers, nepo varwere vane TNNT1-nemaline myopathy vaive nepatani yakapesana (Mufananidzo 6A).
Padanho re single-fiber proteome, skeletal muscle fibers kubva kuvarwere veACTA1 neTNNT1 nemaline myopathy dzakaungana pamwe chete ne majority control fibers, ne TNNT1 nemaline myopathy fibers dziri idzo dzakanyanya kukanganiswa (Mufananidzo 6C). Izvi zvakaonekwa zvakanyanya pakugadzira principal component analysis (PCA) plots dze pseudo-inflated fibers kumurwere wega wega, nevarwere veTNNT1 nemaline myopathy 2 ne3 vachionekwa vari kure zvakanyanya kubva kuma control samples (Supplementary Picture 11B, Supplementary Data Set 20). Kuti tinzwisise zviri nani kuti fibers kubva kuvarwere ve myopathy dzinoenzaniswa sei nefibers dzine hutano, takashandisa ruzivo rwakadzama rwakawanikwa kubva mu proteomic analysis ye 1,000 fibers kubva kuvanhu vakuru vane hutano. Takafungidzira fibers kubva mu myopathy dataset (ACTA1 neTNNT1 nemaline myopathy varwere uye controls) paPCA plot yakawanikwa kubva mu 1000-fiber proteomic analysis (Mufananidzo 6D). Kupararira kwemhando dzefiber dzeMYH pamwe chete nePC2 mufiber dzinodzora kwakafanana nekupararira kwefiber kwakawanikwa kubva muongororo ye1000-fiber proteomic. Zvisinei, fiber zhinji muvarwere venemaline myopathy dzakachinja PC2, dzichipindirana nefiber dzine hutano dzinokurumidza kupwanyika, zvisinei nerudzi rwefiber dzavo dzemuno dzeMYH. Saka, kunyangwe varwere vane ACTA1 nemaline myopathy vakaratidza kuchinja kuenda kune fiber dzerudzi rwe1 pavakaongororwa vachishandisa nzira dzeMYH, ACTA1 nemaline myopathy neTNNT1 nemaline myopathy zvakachinja skeletal muscle fiber proteome kuenda kune fast-twitch fibers.
Takazoenzanisa zvakananga boka rega rega revarwere nevanodzora hutano uye takaona mapuroteni 256 ne552 akabuda zvakasiyana muACTA1 neTNNT1 nemaline myopathies, zvichiteerana (Mufananidzo 6E–G neSupplementary Mufananidzo 11C, Supplementary Data Set 21). Kuongorora kwekuwedzera kwemajini kwakaratidza kudzikira kwakabatana kwemapuroteni emitochondrial (Mufananidzo 6H–I, Supplementary Data Set 22). Zvinoshamisa kuti, pasinei nekusiyana kwemhando dzefiber muACTA1 neTNNT1 nemaline myopathies, kudzikira uku kwaive kwakasiyana zvachose nerudzi rwefiber rwakavakirwa paMYH (Mufananidzo 6H neSupplementary Figures 11D–I, Supplementary Data Set 23). Mapuroteni matatu emicrobial akadzorwawo muACTA1 kana TNNT1 nemaline myopathies. Mapuroteni maviri aya, ENSG00000215483_TR14_ORF67 (anozivikanwawo seLINC00598 kana Lnc-FOXO1) uye ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), akaratidza kuwanda kwakasiyana mumhando 1 myofibers chete. ENSG00000215483_TR14_ORF67 yakambotaurwa kuti ine basa mukudzora kutenderera kwemasero. 56 Kune rumwe rutivi, ENSG00000232046_TR1_ORF437 (inoenderana neLINC01798) yakawedzerwa mumhando 1 nerudzi 2A myofibers muACTA1-nemaline myopathy zvichienzaniswa nehutachiona hwakanaka (Supplementary Figure 12A, Supplementary Data Set 24). Kusiyana neizvi, mapuroteni eribosomal haana kunyanya kukanganiswa ne nemaline myopathy, kunyange hazvo RPS17 yakaderedzwa mu ACTA1 nemaline myopathy (Mufananidzo 6E).
Kuongororwa kwekuwedzera kwakaratidzawo kuwedzera kwemaitiro ekudzivirira muviri muACTA1 neTNNT1 nemaline myopathies, ukuwo kunamatira kwemasero kwakawedzerawo muTNNT1 nemaline myopathy (Mufananidzo 6H). Kuwedzerwa kwezvinhu izvi zvekunze kwakaratidzirwa nemapuroteni ekunze kwekunze anochinja PCA muPC1 nePC2 nenzira isina kunaka (kureva, kuenda kune fibers dzakanyanya kukanganiswa) (Mufananidzo 6J). Mapoka ese ari maviri evarwere akaratidza kuwedzera kwekutaura kwemapuroteni ekunze kwekunze anobatanidzwa mumhinduro dzemuviri uye nzira dzekugadzirisa sarcolemmal, dzakadai se annexins (ANXA1, ANXA2, ANXA5)57,58 uye protein yavo inobatanidza S100A1159 (Zvifananidzo Zvekuwedzera 12B–C). Maitiro aya akambotaurwa kuti anowedzera mu muscular dystrophies60 asi, pakuziva kwedu, haana kumbobvira abatanidzwa ne nemaline myopathies. Kushanda kwakajairika kwemuchina uyu wemamorekuru kunodiwa kuti sarcolemmal igadziriswe mushure mekukuvara uye kuti myocytes itsva dzisanganiswe ne myofibers58,61. Saka, kuwedzera kwebasa iri mumapoka ese evarwere kunoratidza mhinduro yekudzoreredza kukuvara kunokonzerwa nekusagadzikana kwe myofiber.
Mhedzisiro ye nemaline myopathy yega yega yakabatana zvakanaka (r = 0.736) uye yakaratidza kupindirana kwakanaka (Zvifananidzo Zvekuwedzera 11A–B), zvichiratidza kuti ACTA1 ne TNNT1 nemaline myopathy dzine mhedzisiro yakafanana pa proteome. Zvisinei, mamwe mapuroteni akadzorwa chete mu ACTA1 kana TNNT1 nemaline myopathy (Zvifananidzo Zvekuwedzera 11A na C). Protein yeprofibrotic MFAP4 yaive imwe yemapuroteni akanyanya kukwidziridzwa mu TNNT1 nemaline myopathy asi yakaramba isina kuchinja mu ACTA1 nemaline myopathy. SKIC8, chikamu chePAF1C complex ine basa rekugadzirisa HOX gene transcription, yakaderedzwa mu TNNT1 nemaline myopathy asi haina kukanganiswa mu ACTA1 nemaline myopathy (Zvifananidzo Zvekuwedzera 11A). Kuenzanisa zvakananga kweACTA1 neTNNT1 nemaline myopathy kwakaratidza kudzikira kukuru kwemapuroteni emitochondrial uye kuwedzera kwemapuroteni ekudzivirira muviri muTNNT1 nemaline myopathy (Mufananidzo 6G–H uye Supplementary Figures 11C na11H–I). Data iri rinoenderana ne greater atrophy/dystrophy yakaonekwa muTNNT1 nemaline myopathy zvichienzaniswa neTNNT1 nemaline myopathy (Mufananidzo 6A), zvichiratidza kuti TNNT1 nemaline myopathy inomiririra rudzi rwakanyanya rwechirwere.
Kuti tione kana mhedzisiro yakaonekwa ye nemaline myopathy ichiramba iripo padanho rese remhasuru, takaita ongororo ye bulk proteomic ye muscle biopsies kubva kuboka rimwe chete revarwere vane TNNT1 nemaline myopathy uye takaienzanisa ne controls (n=3 paboka) (Supplementary Fig. 13A, Supplementary Data Set 25). Sezvaitarisirwa, controls dzaive dzakabatana zvakanyanya mu principal component analysis, nepo varwere vane TNNT1 nemaline myopathy vakaratidza musiyano wepamusoro we intersample wakafanana newakaonekwa mu single fiber analysis (Supplementary Fig. 13B). Bulk analysis yakaburitsa mapuroteni akasiyana-siyana (Supplementary Fig. 13C, Supplementary Data Set 26) uye maitiro e biological (Supplementary Fig. 13D, Supplementary Data Set 27) akasimbiswa nekuenzanisa fibers yega yega, asi takarasikirwa nekugona kusiyanisa pakati pemhando dzakasiyana dzefiber uye takatadza kufunga nezvemhedzisiro yezvirwere zvakasiyana-siyana mu fibers.
Kana zvabatanidzwa, data iri rinoratidza kuti single-myofiber proteomics inogona kutsanangura zvinhu zvekiriniki zvemuviri zvisingaonekwe nenzira dzakatarwa dzakadai se immunoblotting. Uyezve, data iri rinoratidza zvipingamupinyi zvekushandisa actin fiber typing (MYH) chete kutsanangura phenotypic adaptation. Zvechokwadi, kunyangwe fiber type switching ichisiyana pakati peactin ne troponin nemaline myopathies, ese anemaline myopathies anoderedza MYH fiber typing kubva ku skeletal muscle fiber metabolism kuenda ku proteome yemhasuru inokurumidza uye isina oxidative.
Kusiyana kwemasero kwakakosha kuti nyama dzikwanise kusangana nezvinodiwa zvakasiyana-siyana. Mutsandanyama dzemapfupa, izvi zvinowanzotsanangurwa semhando dzefiber dzinoratidzwa nemazinga akasiyana ekugadzirwa kwesimba uye kuneta. Zvisinei, zviri pachena kuti izvi zvinotsanangura chikamu chidiki chete chekusiyana kwefiber yetsandanyama dzemapfupa, izvo zvinosiyana-siyana, zvakaoma uye zvine mativi akawanda kupfuura zvaifungidzirwa kare. Kufambira mberi kwetekinoroji ikozvino kwajekesa zvinhu zvinodzora fiber yetsandanyama dzemapfupa. Zvechokwadi, data redu rinoratidza kuti fiber yemhando ye2X inogona kunge isiri mhando yefiber yetsandanyama dzemapfupa. Uyezve, takaona mapuroteni emetabolism, mapuroteni eribosomal uye mapuroteni ane chekuita nemasero sezvinhu zvikuru zvinokonzera kusiyana kwefiber yetsandanyama dzemapfupa. Nekushandisa mafambiro edu eproteomic kuvarwere vane nematode myopathy, takaratidza zvakare kuti MYH-based fiber typing hairatidzi zvizere kusiyana kwetsandanyama dzemapfupa, kunyanya kana system yacho yakanganiswa. Zvechokwadi, pasinei nerudzi rwefiber rwakavakirwa paMYH, nematode myopathy inoguma nekuchinja kuenda kune fiber dzinokurumidza uye dzisina oxidative.
Mafiber emhasuru dzemapfupa ave akaiswa muzvikamu kubva muzana remakore rechi19. Ongororo dzechangobva kuitwa dzemaomic dzakatibvumira kutanga kunzwisisa ma profiles ekutaura kwemarudzi akasiyana eMYH fiber uye mhinduro dzawo kune zvinokurudzira zvakasiyana. Sezvakatsanangurwa pano, nzira dzemaomic dzine mukana wekunzwisisa zvakanyanya pakuyera zviratidzo zvemhando yefiber kupfuura nzira dzechinyakare dzinobva kuma antibody, pasina kuvimba nekuyera kwechiratidzo chimwe chete (kana zvishoma) kutsanangura rudzi rwefiber yemhasuru dzemapfupa. Takashandisa ma workflows ekuwedzera e transcriptomic neproteomic uye takabatanidza mhedzisiro kuti tiongorore kurongeka kwekunyora uye mushure mekunyora kwe fiber heterogeneity mufiber yemhasuru dzemapfupa evanhu. Kufamba uku kwakakonzera kukundikana kuona fibers dzemhando ye2X yakachena padanho reprotein mu vastus lateralis yeboka redu remajaya ane hutano. Izvi zvinoenderana nezvidzidzo zvekare zve single fiber zvakawana <1% fibers dze 2X dzakachena mu vastus lateralis dzine hutano, kunyangwe izvi zvichifanira kusimbiswa mune dzimwe tsandanyama mune ramangwana. Kusiyana pakati pekuonekwa kwe fibers dze 2X dzakachena padanho re mRNA uye fibers dze 2A/2X dzakasanganiswa chete padanho reprotein kunonetsa. Kutaurwa kweMYH isoform mRNA hakusi kwecircadian, 67 zvichiratidza kuti hatingambove "takarasikirwa" nechiratidzo chekutanga kweMYH2 mumafibers anoita seakachena e2X padanho reRNA. Imwe tsananguro inogona kuitika, kunyangwe iri yekufungidzira chete, inogona kunge iri misiyano muprotein uye/kana mRNA kugadzikana pakati peMYH isoforms. Zvechokwadi, hapana fast fiber yakachena 100% kune chero MYH isoform, uye hazvizivikanwe kana MYH1 mRNA expression levels iri pakati pe70–90% yaizoguma nehuwandu hwakaenzana hweMYH1 neMYH2 padanho reprotein. Zvisinei, kana tichifunga nezve transcriptome yese kana proteome, cluster analysis inogona kuona nechivimbo ma cluster maviri akasiyana anomiririra skeletal tsandanyama fibers inononoka uye inokurumidza, zvisinei nekuumbwa kwayo chaiko kweMYH. Izvi zvinoenderana nekuongorora uchishandisa nzira dze single-nucleus transcriptomic, idzo dzinowanzoona ma cluster maviri akasiyana e myonuclear. 68, 69, 70 Uyezve, kunyange zvazvo zvidzidzo zvekare zveproteomic zvakaratidza mafiber emhando ye2X, mafiber aya haaunganidzwe zvakasiyana nemamwe mafiber anokurumidza uye anoratidza huwandu hushoma hwemaprotein akawanda zvakasiyana kana tichienzanisa nemamwe marudzi efiber zvichibva paMYH. 14 Mhedzisiro iyi inoratidza kuti tinofanira kudzokera kumaonero ekutanga kwezana remakore rechi20 ekupatsanurwa kwefiber yemhasuru, iyo yakakamura mafiber emhasuru yemapfupa evanhu kwete muzvikamu zvitatu zvakasiyana zvichibva paMYH, asi muzvikamu zviviri zvichibva pane zvavanoita pakugadzirisa muviri uye pakubata. 63
Zvinonyanya kukosha, kusiyana kwe myofiber kunofanirwa kutariswa nenzira dzakasiyana-siyana. Zvidzidzo zvekare zve "omics" zvakaratidza izvi, zvichiratidza kuti mapfupa emhasuru haaumbi mapoka akasiyana asi akarongwa pamwe chete. 11, 13, 14, 64, 71 Pano, tinoratidza kuti, pamusoro pekusiyana kwehunhu hwekubata uye metabolic hwemhasuru dzemapfupa, myofibers dzinogona kusiyaniswa nezviratidzo zvine chekuita nekudyidzana kwemasero nemasero uye nzira dzekushandura. Zvechokwadi, takawana kusiyana kwe ribosome mumapfupa emhasuru dzemapfupa izvo zvinoita kuti pave nekusiyana kwakasiyana pasina mhando dzefiber dzinononoka uye dzinokurumidza. Chikonzero chikuru cheizvi kusiyana kukuru kwe myofiber, pasina mhando yefiber inononoka uye inokurumidza, hachisati chajeka, asi chinogona kuratidza kurongeka kwakasarudzika kwenzvimbo mukati memhasuru fascicles dzinopindura zvakanaka kumasimba chaiwo nemitoro, 72 kutaurirana kwakasarudzika kwemasero kana nhengo chaiyo nemamwe marudzi emasero munzvimbo diki yemhasuru73,74,75 kana kusiyana kwebasa re ribosome mukati me myofibers yega yega. Zvechokwadi, ribosomal heteroplasmy, kungave kuburikidza nekutsiviwa kweRPL3 neRPL3L kana padanho re2′O-methylation yerRNA, zvakaratidzwa kuti zvine chekuita ne skeletal muscle hypertrophy76,77. Mashandisirwo e multi-omic uye spatial pamwe chete nekushanda kwema myofibers ega ega zvichawedzera kufambira mberi kwedu kunzwisisa kwe biology yemhasuru padanho re multi-omic78.
Nekuongorora mapuroteni ema single myofibers kubva kuvarwere vane nemaline myopathies, takaratidzawo kushanda, kushanda, uye kushanda kwe single myofiber proteomics kujekesa pathophysiology yekiriniki yetsandanyama dzemapfupa. Uyezve, nekuenzanisa mashandiro edu nekuongorora kwepasi rose kweproteomic, takakwanisa kuratidza kuti single myofiber proteomics inopa ruzivo rwakafanana nerwe global tissue proteomics uye inowedzera kudzika uku nekutarisa kusiyana kwe interfiber uye myofiber type. Kuwedzera kune mutsauko unotarisirwa (kunyangwe zvichichinja) mu fiber type ratio wakaonekwa muACTA1 neTNNT1 nemaline myopathies zvichienzaniswa ne healthy controls,19 takaonawo oxidative uye extracellular remodeling isina chekuita neMYH-mediated fiber type switching. Fibrosis yakambotaurwa nezvayo muTNNT1 nemaline myopathies.19 Zvisinei, ongororo yedu inovakira pane zvakawanikwa izvi nekuratidzawo huwandu hwakawanda hwemapuroteni ane chekuita nekushushikana anowanikwa kunze kwemuviri, akadai se annexins, anobatanidzwa mukugadzirisa sarcolemmal, mu myofibers kubva kuvarwere vane ACTA1 neTNNT1 nemaline myopathies.57,58,59 Mukupedzisa, huwandu hwakawanda hwe annexin mu myofibers kubva kuvarwere vane nemaline myopathy hunogona kureva mhinduro yemasero yekugadzirisa myofibers dzakaoma zvakanyanya.
Kunyangwe chidzidzo ichi chichiratidza ongororo huru yevanhu ye single-fiber whole-muscle-omics kusvika pari zvino, hachina miganhu. Takaparadzanisa skeletal tsandanyama kubva mu sample diki uye yakafanana yevatori vechikamu uye tsandanyama imwe chete (vastus lateralis). Nokudaro, hazvigoneki kubvisa kuvapo kwehuwandu hwe fiber hwakasiyana mumhando dzetsandanyama uye pa physiology yetsandanyama. Semuenzaniso, hatigone kubvisa mukana wekuti pane subset ye ultrafast fibers (semuenzaniso, 2X fibers yakachena) inobuda mu sprinters vakadzidziswa zvakanyanya uye/kana vatambi vesimba79 kana panguva yekusaita kwetsandanyama66,80. Uyezve, huwandu hushoma hwesamples dzevatori vechikamu hwakatidzivirira kuti tisaongorore kusiyana kwepabonde mu fiber heterogeneity, sezvo fiber type ratios dzichizivikanwa kuti dzakasiyana pakati pevarume nevakadzi. Uyezve, hatina kukwanisa kuita transcriptomic ne proteomic analysis pa fibers dzetsandanyama dzakafanana kana samples kubva kune vatori vechikamu vamwe chete. Sezvo isu nevamwe tichiramba tichivandudza ongororo dzemasero ese nemaseru ese tichishandisa ongororo dzemaomiki kuti tiwane ongororo shoma (sezvakaratidzwa pano muongororo yemaomiki kubva kuvarwere vane mitochondrial myopathy), mukana wekubatanidza nzira dzemaomiki akawanda (uye anoshanda) mukati memaomiki ese emaseru unova pachena.
Zvose, data redu rinoona uye rinotsanangura zvinokonzeresa kusiyana kwetsandanyama dzemapfupa kuburikidza nekunyora uye mushure mekunyora. Kunyanya, tinopa data rinopikisa dzidziso yenguva refu mu physiology yetsandanyama dzemapfupa ine chekuita netsananguro yekare yemhando dzefayibha yakavakirwa paMYH. Tinotarisira kuvandudza gakava uye pakupedzisira kufunga patsva kunzwisisa kwedu kupatsanurwa kwefayibha dzetsandanyama dzemapfupa uye kusiyana kwefayibha.
Vatori vechikamu gumi nevana vechiCaucasian (varume gumi nevaviri nevakadzi vaviri) vakabvuma kutora chikamu muchidzidzo ichi. Chidzidzo ichi chakatenderwa neEthics Committee yeGhent University Hospital (BC-10237), chakatevedzera 2013 Helsinki Declaration, uye chakanyoreswa paClinicalTrials.gov (NCT05131555). Hunhu hwevatori vechikamu hwakaratidzwa muSupplementary Table 1. Mushure mekunge vapihwa mvumo yekutaura nekunyora, vatori vechikamu vakaongororwa nachiremba vasati vapinda muchidzidzo ichi. Vatori vechikamu vaive vadiki (makore 22-42), vaine hutano hwakanaka (vasina zvirwere, vasina nhoroondo yekusvuta), uye vachishanda zvine mwero. Kutorwa kweoxygen kwakanyanya kwakaongororwa uchishandisa step ergometer yekuongorora hutano hwemuviri sezvakatsanangurwa kare. 81
Sampuli dzetsandanyama dzakatorwa panguva yekuzorora uye panguva yekutsanya katatu, mazuva gumi nemana akasiyana. Nekuti sampuro idzi dzakatorwa sechikamu chechidzidzo chikuru, vatori vechikamu vakadya placebo (lactose), H1-receptor antagonist (540 mg fexofenadine), kana H2-receptor antagonist (40 mg famotidine) maminetsi makumi mana biopsy isati yaitwa. Takamboratidza kuti idzi histamine receptor antagonists hadzikanganisi skeletal muscle fitness81, uye hapana kuungana kwakabatana nemamiriro ezvinhu kwakaonekwa muzvirongwa zvedu zvekudzora hunhu (Zvifananidzo Zvekuwedzera 3 ne6). Kudya kwakajairika (41.4 kcal/kg huremu hwemuviri, 5.1 g/kg body weight carbohydrate, 1.4 g/kg body weight protein, uye 1.6 g/kg body weight fat) kwakachengetedzwa kwemaawa makumi mana nemasere zuva rega rega rekuyedza risati rasvika, uye kudya kwemangwanani kwakajairika (1.5 g/kg body weight carbohydrate) kwakadyiwa mangwanani ezuva rekuyedza. Pasi pemushonga wekutindivadza (0.5 ml 1% lidocaine isina epinephrine), tsandanyama dzakatorwa kubva mutsandanyama yevastus lateralis uchishandisa percutaneous Bergström aspiration.82 Sampuli dzetsandanyama dzakaiswa muRNAlater pakarepo ndokuchengetwa pa4°C kusvika dzabviswa nemaoko (kusvika mazuva matatu).
Mabhandi e myofiber achangobva kupatsanurwa akaendeswa kune RNAlater medium itsva mudhishi rekurima. Mabhandi e myofibers ega ega akabva apatsanurwa nemaoko achishandisa stereomicroscope uye tweezers dzakapfava. Mabhandi makumi maviri nemashanu akapatsanurwa kubva mu biopsy yega yega, achinyanya kutarisisa kusarudza mabhandi kubva munzvimbo dzakasiyana dze biopsy. Mushure mekupatsanurwa, bhandi rega rega rakanyudzwa zvinyoro nyoro mu 3 μl ye lysis buffer (SingleShot Cell Lysis Kit, Bio-Rad) ine proteinase K uye DNase enzymes kubvisa mapuroteni asingadiwe neDNA. Kupatsanurwa kwemasero uye kubviswa kweprotein/DNA zvakazotanga nekutenderera zvishoma, kutenderedza mvura mu microcentrifuge, uye kuisirwa mukati mekushisa kwemumba (10 min). Lysate yakazoiswa mu thermal cycler (T100, Bio-Rad) pa 37°C kwemaminitsi mashanu, 75°C kwemaminitsi mashanu, uye ipapo yakachengetwa ipapo pa -80°C kusvika yagadziriswazve.
Maraibhurari eRNA anoenderana neIllumina akagadzirwa kubva ku2 µl ye myofiber lysate uchishandisa QuantSeq-Pool 3′ mRNA-Seq Library Prep Kit (Lexogen). Nzira dzakadzama dzinogona kuwanikwa mubhuku remugadziri. Maitiro aya anotanga nekugadzirwa kwe cDNA yekutanga ne reverse transcription, panguva iyo unique molecular identifiers (UMIs) uye sample-specific i1 barcodes zvinounzwa kuti zvive nechokwadi chekuunganidza sampuro uye kuderedza kusiyana kwehunyanzvi panguva yekugadzirisa. cDNA kubva ku96 myofibers inozobatanidzwa uye kucheneswa nema magnetic beads, mushure mezvo RNA inobviswa uye kugadzirwa kwe second-strand kunoitwa uchishandisa random primers. Raibhurari inocheneswa nema magnetic beads, ma tag e i5/i7 chaiwo anowedzerwa, uye PCR inowedzerwa. Danho rekupedzisira rekuchenesa rinogadzira maraibhurari anoenderana neIllumina. Hunhu hweraibhurari yega yega hwakaongororwa uchishandisa High Sensitivity Small Fragment DNA Analysis Kit (Agilent Technologies, DNF-477-0500).
Zvichibva pakuyera kweQubit, madziva akaunganidzwazve pahuwandu hwe equimolar (2 nM). Dziva rakabuda rakazorongwa pachishandiswa NovaSeq 6000 mu standard mode uchishandisa NovaSeq S2 Reagent Kit (1 × 100 nucleotides) ine 2 nM loading (4% PhiX).
Pombi yedu yakavakirwa papombi yekuongorora data yeQuantSeq Pool yaLexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). Data rakatanga kubviswa muhuwandu hwema data ne bcl2fastq2 (v2.20.0) zvichibva pa i7/i5 index. Read 2 yakabviswa muhuwandu hwema data ne idemux (v0.1.6) zvichibva pa i1 sample barcode uye UMI sequences dzakabviswa ne umi_tools (v1.0.1). Reads dzakazogadziriswa ne cutadapt (v3.4) muma rounds akawanda kubvisa short reads (<20 in length) kana reads dzine adapter sequences chete. Reads dzakazobatanidzwa ne human genome uchishandisa STAR (v2.6.0c) uye BAM files dzakaiswa index ne SAMtools (v1.11). Duplicate reads dzakabviswa uchishandisa umi_tools (v1.0.1). Pakupedzisira, alignment counting yakaitwa uchishandisa featureCounts mu Subread (v2.0.3). Kudzora mhando kwakaitwa uchishandisa FastQC (v0.11.9) pamatanho akati wandei epakati pepombi.
Kugadziriswa kwese kwe bioinformatics uye kuona kwakaitwa muR (v4.2.3), kunyanya uchishandisa nzira yeSeurat (v4.4.0). 83 Saka, ma UMI values ega ega uye metadata matrices zvakachinjwa kuita zvinhu zveSeurat. Majini akaratidzwa muasingasviki 30% yemafiber ese akabviswa. Samples dzisina kunaka dzakabviswa zvichibva pamuganho wepasi we1000 UMI values uye 1000 majini akaonekwa. Pakupedzisira, mafiber 925 akapfuura matanho ese ekudzora hunhu. Ma UMI values akagadziriswa uchishandisa nzira yeSeurat SCTransform v2, 84 kusanganisira maficha ese 7418 akaonekwa, uye misiyano yepakati pevatori vechikamu yakadzoserwa kumashure. Metadata yese yakakodzera inogona kuwanikwa muSupplementary Dataset 28.
Nguva yekutumira: Gunyana-10-2025