identification-skeletal-muscle-satellite-cells-immunofluorescence

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

National Institutes of Health

Las c&eacute;lulas sat&eacute;lite (SCs) sostener crecimiento muscular y la autonom&iacute;a de m&uacute;sculo esquel&eacute;tico adulto con vigorosa capacidad regenerativa. Aqu&iacute;, Divulgamos que EZH2, la subunidad enzim&aacute;tica de la Polycomb-represivas complejo 2 (PRC2), se expresa en ambos Pax7 + / Myf5&#x207B; c&eacute;lulas madre y Pax7 + / Myf5 + cometidos mi&oacute;genas precursores y es necesaria para la homeostasis de la piscina para adultos de SC. Ratones con ablaci&oacute;n condicional de Ezh2 en SCs tienen menos m&uacute;sculo postnatales Pax7 + c&eacute;lulas y muscular reducida masa y no regenerar adecuadamente. Estos defectos se asocian con deterioro proliferaci&oacute;n de SC y derepression de genes expresados en linajes celulares de valvas. As&iacute;, EZH2 controla auto renovaci&oacute;n y proliferaci&oacute;n y mantiene un adecuado programa transcripcional en SCs.

Polycomb EZH2 controla la auto-renovaci&oacute;n y protege la identidad transcripcional de c&eacute;lulas de m&uacute;sculo esquel&eacute;tico.

&#x536B;&#x661F;&#x7EC6;&#x80DE; (SCs) &#x7EF4;&#x6301;&#x808C;&#x8089;&#x751F;&#x957F;&#x548C;&#x589E;&#x5F3A;&#x6210;&#x4EBA;&#x9AA8;&#x9ABC;&#x808C;&#x4E0E;&#x6709;&#x529B;&#x7684;&#x518D;&#x751F;&#x80FD;&#x529B;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A; EZH2 Polycomb &#x9547;&#x538B;&#x590D;&#x6742; 2 (PRC2) &#x7684;&#x9176;&#x4E9A;&#x5355;&#x4F4D;&#x8868;&#x793A;&#x5728;&#x8FD9;&#x4E24;&#x4E2A; Pax7 + Myf5&#x207B; &#x5E72;&#x7EC6;&#x80DE;&#x4E0E; Pax7 + Myf5 + &#x72AF;&#x751F;&#x808C;&#x524D;&#x4F53;&#x548C;&#x9700;&#x8981;&#x7684;&#x6210;&#x4EBA;&#x7684; SC &#x6C60;&#x7684;&#x52A8;&#x6001;&#x5E73;&#x8861;&#x3002;&#x4E0E;&#x6761;&#x4EF6;&#x6D88;&#x878D;&#x672F;&#x5728;&#x516C;&#x52A1;&#x5458;&#x4E8B;&#x52A1;&#x5C40;&#x5C40;&#x957F; Ezh2 &#x8001;&#x9F20;&#x6709;&#x8F83;&#x5C11;&#x7684;&#x808C;&#x8089;&#x4EA7;&#x540E; Pax7 + &#x7EC6;&#x80DE;&#x548C;&#x51CF;&#x5C11;&#x7684;&#x808C;&#x8089;&#x8D28;&#x91CF;&#x548C;&#x672A;&#x80FD;&#x9002;&#x5F53;&#x5730;&#x91CD;&#x65B0;&#x751F;&#x6210;&#x3002;&#x8FD9;&#x4E9B;&#x7F3A;&#x9677;&#x662F;&#x4E0E;&#x53D7;&#x635F;&#x7684; SC &#x6269;&#x6563;&#x548C;&#x7CFB;&#x7EDF;&#x7684;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x5728;&#x975E;&#x808C;&#x7EC6;&#x80DE;&#x7EC6;&#x80DE;&#x8C31;&#x7CFB;&#x76F8;&#x5173;&#x8054;&#x3002;&#x56E0;&#x6B64;&#xFF0C;EZH2 &#x63A7;&#x5236;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#x548C;&#x6269;&#x6563;&#xFF0C;&#x5E76;&#x4FDD;&#x6301;&#x9002;&#x5F53;&#x7684;&#x8F6C;&#x5F55;&#x7A0B;&#x5E8F;&#x5728;&#x516C;&#x52A1;&#x5458;&#x4E8B;&#x52A1;&#x5C40;&#x5C40;&#x957F;&#x3002;

Polycomb EZH2 &#x4FDD;&#x969C;&#x7684;&#x9AA8;&#x9ABC;&#x808C;&#x5E72;&#x7EC6;&#x80DE;&#x8F6C;&#x5F55;&#x6807;&#x8BC6;&#x548C;&#x63A7;&#x5236;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#x3002;

Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5â» stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.

Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.

Cellules satellites (SCs) soutenir la croissance musculaire et l&#39;autonomisation des muscle squelettique adulte avec des capacit&eacute;s de r&eacute;g&eacute;n&eacute;ration vigoureuses. Nous rapportons ici que EZH2, la sous-unit&eacute; enzymatique de la Polycomb-r&eacute;pressif complexe 2 (PRC2), est exprim&eacute; dans les deux Pax7 + / Myf5&#x207B; cellules souches et Pax7 + / Myf5 + commis pr&eacute;curseurs myog&egrave;nes et est n&eacute;cessaire pour l&#39;hom&eacute;ostasie de la piscine de SC adulte. Souris avec ablation conditionnelle de Ezh2 dans SCs ont moins de muscle postnatals Pax7 + cellules et r&eacute;duit muscle masse et ne parviennent pas &agrave; se r&eacute;g&eacute;n&eacute;rer de fa&ccedil;on appropri&eacute;e. Ces d&eacute;fauts sont associ&eacute;s &agrave; la prolif&eacute;ration de SC avec facult&eacute;s affaiblies et d&eacute;r&eacute;pression de g&egrave;nes exprim&eacute;s dans des lign&eacute;es de cellules musculaires. Ainsi, EZH2 contr&ocirc;le l&#39;auto-renouvellement et prolif&eacute;ration et maintient un programme appropri&eacute; de transcriptional dans SCs.

Polycomb EZH2 contr&ocirc;le l&#39;auto-renouvellement et pr&eacute;serve l&#39;identit&eacute; de transcriptional des cellules souches musculaires squelettiques.

Cellule satellite (SCs) sostenere la crescita muscolare e potenziare il muscolo scheletrico adulto con vigorosa capacit&agrave; rigenerativa. Qui, segnaliamo che la EZH2, la subunit&agrave; enzimatica del Polycomb-repressivo complesso 2 (PRC2), si esprime in entrambi Pax7 + Myf5&#x207B; cellule staminali e Pax7 + Myf5 + commesso miogena precursori ed &egrave; richiesto per l&#39;omeostasi della piscina adulti SC. Topi con ablazione condizionale di Ezh2 in SCs hanno meno muscolo cellule Pax7 + postnatale e muscolo ridotto di massa e non riescono a rigenerarsi in modo appropriato. Questi difetti sono associati a ridotta proliferazione di SC e derepression di geni espressi in linee cellulari nonmuscle. Cos&igrave;, EZH2 controlli auto-rinnovamento e proliferazione e mantiene un adeguato programma transcriptional in SCs.

Polycomb EZH2 controlli auto-rinnovamento e salvaguarda l&#39;identit&agrave; trascrizionale di cellule staminali del muscolo scheletrico.

&#x885B;&#x661F;&#x7D30;&#x80DE; &#xFF08;SCs&#xFF09; &#x306F;&#x3001;&#x7B4B;&#x8089;&#x306E;&#x6210;&#x9577;&#x3092;&#x7DAD;&#x6301;&#x3057;&#x3001;&#x7A4D;&#x6975;&#x7684;&#x306A;&#x518D;&#x751F;&#x80FD;&#x529B;&#x3092;&#x6301;&#x3064;&#x5927;&#x4EBA;&#x306E;&#x9AA8;&#x683C;&#x7B4B;&#x306B;&#x529B;&#x3092;&#x4E0E;&#x3048;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306E;&#x6291;&#x5727;&#x7684;&#x3001;&#x30DD;&#x30EA;&#x30B3;&#x30FC;&#x30E0;-&#x306A;&#x8907;&#x96D1;&#x306A; 2 &#xFF08;PRC2 &#x306E;&#xFF09;&#x3001;&#x9175;&#x7D20;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8; ezh2 &#x306F;&#x4E21;&#x65B9; Pax7 &#x3067;&#x8868;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x5831;&#x544A; + Myf5&#x207B; &#x5E79;&#x7D30;&#x80DE;&#x30FB; Pax7 + Myf5 + &#x30B3;&#x30DF;&#x30C3;&#x30C8;&#x7B4B;&#x524D;&#x99C6;&#x4F53;&#x3068;&#x5927;&#x4EBA;&#x7528;&#x306E; SC &#x30D7;&#x30FC;&#x30EB;&#x306E;&#x6052;&#x5E38;&#x6027;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x6761;&#x4EF6;&#x4ED8;&#x304D;&#x306E;&#x30A2;&#x30D6;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3; ezh2 &#x306F; SCs &#x3067;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x304C;&#x3042;&#x308B;&#x5C11;&#x306A;&#x3044;&#x7B4B;&#x8089;&#x7523;&#x5F8C; Pax7 + &#x7D30;&#x80DE;&#x3068;&#x7B4B;&#x8CEA;&#x91CF;&#x3057;&#x3001;&#x9069;&#x5207;&#x306B;&#x518D;&#x751F;&#x6210;&#x306B;&#x5931;&#x6557;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x6B20;&#x9665;&#x969C;&#x5BB3;&#x306E; SC &#x5897;&#x6B96;&#x3068;&#x975E;&#x7D30;&#x80DE;&#x7CFB;&#x8B5C;&#x306B;&#x767A;&#x73FE;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6291;&#x5236;&#x304C;&#x89E3;&#x9664;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;ezh2 &#x306F;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x3068;&#x5897;&#x6B96;&#x3092;&#x5236;&#x5FA1;&#x3057;&#x3001;&#x9069;&#x5207;&#x306A;&#x8EE2;&#x5199;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0; SCs &#x3067;&#x7DAD;&#x6301;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30DD;&#x30EA;&#x30B3;&#x30FC;&#x30E0; ezh2 &#x306F;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x3092;&#x5236;&#x5FA1;&#x3057;&#x3001;&#x9AA8;&#x683C;&#x7B4B;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x8EE2;&#x5199;&#x306E; id &#x3092;&#x4FDD;&#x8B77;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC704;&#xC131; &#xC138;&#xD3EC; (SCs) &#xADFC;&#xC721; &#xC131;&#xC7A5;&#xC744; &#xC720;&#xC9C0; &#xD558; &#xACE0; &#xD65C;&#xBC1C; &#xD55C; &#xC7AC;&#xC0DD; &#xB2A5;&#xB825;&#xC744; &#xAC00;&#xC9C4; &#xC131;&#xC778; &#xACE8;&#xACA9; &#xADFC;&#xC721;&#xC758; &#xB2A5;&#xB825;&#xC744; &#xC8FC;&#xC18C; &#xC11C;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xB294; &#xBAA8;&#xB450; Pax7 ezh2&#xB294; Polycomb-&#xC5B5;&#xC555; &#xBCF5;&#xC7A1; &#xD55C; 2 (PRC2)&#xC758; &#xD6A8;&#xC18C; &#xC18C; &#xB2E8;&#xC704; &#xD45C;&#xD604; &#xB41C; &#xBCF4;&#xACE0;&#xC11C; + Myf5&#x207B; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC640; Pax7 + Myf5 + &#xC870;&#xC9C1;&#xC801; &#xC120;&#xAD6C;&#xC790; &#xD22C;&#xC785; &#xBC0F; &#xC131;&#xC778; &#xC0AC;&#xC6B0;&#xC2A4; &#xCE90;&#xB864;&#xB77C;&#xC774;&#xB098; &#xC218;&#xC601;&#xC7A5;&#xC758; &#xD56D;&#xC0C1; &#xC131;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. Scs&#xC5D0;&#xC11C; ezh2&#xC758; &#xC870;&#xAC74;&#xBD80; &#xC808;&#xC81C;&#xC640; &#xC950;&#xAC00; &#xC801;&#xC740; &#xADFC;&#xC721; &#xCD9C;&#xC0DD; &#xD6C4; Pax7 + &#xC140; &#xBC0F; &#xAC10;&#xC18C; &#xADFC;&#xC721; &#xC9C8;&#xB7C9; &#xBC0F; &#xC801;&#xC808; &#xD558; &#xAC8C; &#xB2E4;&#xC2DC; &#xC0DD;&#xC131; &#xD558;&#xB824;&#xBA74; &#xC2E4;&#xD328; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xD568; &#xC7A5;&#xC560;&#xC778;&#xB41C; SC &#xD655;&#xC0B0; &#xBC0F; nonmuscle &#xC138;&#xD3EC; &#xACC4;&#xBCF4;&#xC5D0;&#xC11C; &#xD45C;&#xD604; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790;&#xC758; derepression&#xC640; &#xC5F0;&#xACB0; &#xB429;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, EZH2 &#xC790;&#xAE30; &#xAC31;&#xC2E0; &#xBC0F; &#xD655;&#xC0B0;, &#xC81C;&#xC5B4; &#xD558; &#xACE0; Scs&#xC5D0; &#xC801;&#xC808; &#xD55C; transcriptional &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xC720;&#xC9C0; &#xD569;&#xB2C8;&#xB2E4;.

Polycomb EZH2 &#xC790;&#xAE30; &#xAC31;&#xC2E0;&#xC744; &#xC81C;&#xC5B4; &#xD558; &#xACE0; &#xACE8;&#xACA9; &#xADFC;&#xC721; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; transcriptional &#xC2E0;&#xC6D0; &#xBCF4;&#xD638;.

Enhancers play a central role in cell-type-specific gene expression and are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. However, the methyltransferases responsible for H3K4me1/2 on enhancers remain elusive. Furthermore, how these enzymes function on enhancers to regulate cell-type-specific gene expression is unclear. In this study, we identify MLL4 (KMT2D) as a major mammalian H3K4 mono- and di-methyltransferase with partial functional redundancy with MLL3 (KMT2C). Using adipogenesis and myogenesis as model systems, we show that MLL4 exhibits cell-type- and differentiation-stage-specific genomic binding and is predominantly localized on enhancers. MLL4 co-localizes with lineage-determining transcription factors (TFs) on active enhancers during differentiation. Deletion of Mll4 markedly decreases H3K4me1/2, H3K27ac, Mediator and Polymerase II levels on enhancers and leads to severe defects in cell-type-specific gene expression and cell differentiation. Together, these findings identify MLL4 as a major mammalian H3K4 mono- and di-methyltransferase essential for enhancer activation during cell differentiation. DOI: http://dx.doi.org/10.7554/eLife.01503.001. 

H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation.

The acetyltransferase Gcn5 is critical for embryogenesis and shows partial functional redundancy with its homolog PCAF. However, the tissue- and cell lineage-specific functions of Gcn5 and PCAF are still not well defined. Here we probe the functions of Gcn5 and PCAF in adipogenesis. We found that the double knockout (DKO) of Gcn5/PCAF inhibits expression of the master adipogenic transcription factor gene PPARγ, thereby preventing adipocyte differentiation. The adipogenesis defects in Gcn5/PCAF DKO cells are rescued by ectopic expression of peroxisome proliferator-activated receptor γ (PPARγ), suggesting Gcn5/PCAF act upstream of PPARγ to facilitate adipogenesis. The requirement of Gcn5/PCAF for PPARγ expression was unexpectedly bypassed by prolonged treatment with an adipogenic inducer, 3-isobutyl-1-methylxanthine (IBMX). However, neither PPARγ ectopic expression nor prolonged IBMX treatment rescued defects in Prdm16 expression in DKO cells, indicating that Gcn5/PCAF are essential for normal Prdm16 expression. Gcn5/PCAF regulate PPARγ and Prdm16 expression at different steps in the transcription process, facilitating RNA polymerase II recruitment to Prdm16 and elongation of PPARγ transcripts. Overall, our study reveals that Gcn5/PCAF facilitate adipogenesis through regulation of PPARγ expression and regulate brown adipogenesis by influencing Prdm16 expression. 

Gcn5 and PCAF regulate PPARγ and Prdm16 expression to facilitate brown adipogenesis.

Stem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD(+) levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology. 

The NAD(+)-dependent SIRT1 deacetylase translates a metabolic switch into regulatory epigenetics in skeletal muscle stem cells.

Although the genetic interactions between signaling pathways and transcription factors have been largely decoded, much remains to be learned about the epigenetic regulation of cerebellar development. Here, we report that cerebellar deletion of Ezh2, the methyltransferase subunit of the PRC2 complex, results in reduced H3K27me3 and profound transcriptional dysregulation, including that of a set of transcription factors directly involved in cerebellar neuronal cell-type specification and differentiation. Such transcriptional changes lead to increased GABAergic interneurons and decreased Purkinje cells. Transcriptional changes also inhibit the proliferation of granule precursor cells derived from the rhombic lip. The loss of both cell types ultimately results in cerebellar hypoplasia. These findings indicate Ezh2/PRC2 plays crucial roles in regulating neurogenesis from both cerebellar germinal zones.

Polycomb Ezh2 controls the fate of GABAergic neurons in the embryonic cerebellum.

The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.

Roles of H3K27me2 and H3K27me3 Examined during Fate Specification of Embryonic Stem Cells.

The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK), an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE) in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

Article	Year
Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells. Genes & development\| PubMed ID: 21498568	2011
H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation. eLife\| PubMed ID: 24368734	2013
Gcn5 and PCAF regulate PPARγ and Prdm16 expression to facilitate brown adipogenesis. Molecular and cellular biology\| PubMed ID: 25071153	2014
The NAD(+)-dependent SIRT1 deacetylase translates a metabolic switch into regulatory epigenetics in skeletal muscle stem cells. Cell stem cell\| PubMed ID: 25600643	2015
Polycomb Ezh2 controls the fate of GABAergic neurons in the embryonic cerebellum. Development (Cambridge, England)\| PubMed ID: 27068104	2016
Roles of H3K27me2 and H3K27me3 Examined during Fate Specification of Embryonic Stem Cells. Cell reports\| PubMed ID: 27783950	2016
Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK. EBioMedicine\| PubMed ID: 28396013	2017

Xuesong Feng

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