models-of-murine-vaginal-colonization-by-anaerobically-grown-bacteria

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

University of California

UCSD

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus.

The circadian clock controls daily oscillations of gene expression at the cellular level. We report the development of a high-throughput circadian functional assay system that consists of luminescent reporter cells, screening automation, and a data analysis pipeline. We applied this system to further dissect the molecular mechanisms underlying the mammalian circadian clock using a chemical biology approach. We analyzed the effect of 1,280 pharmacologically active compounds with diverse structures on the circadian period length that is indicative of the core clock mechanism. Our screening paradigm identified many compounds previously known to change the circadian period or phase, demonstrating the validity of the assay system. Furthermore, we found that small molecule inhibitors of glycogen synthase kinase 3 (GSK-3) consistently caused a strong short period phenotype in contrast to the well-known period lengthening by lithium, another presumed GSK-3 inhibitor. siRNA-mediated knockdown of GSK-3beta also caused a short period, confirming the phenotype obtained with the small molecule inhibitors. These results clarify the role of GSK-3beta in the period regulation of the mammalian clockworks and highlight the effectiveness of chemical biology in exploring unidentified mechanisms of the circadian clock.

A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta.

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&#x5316;&#x5B66;&#x751F;&#x7269;&#x5B66;&#x65B9;&#x6CD5;&#x7531;&#x845B;&#x5170;&#x7D20;&#x53F2;&#x514B; 3beta &#x7279;&#x5F02;&#x6027;&#x6291;&#x5236;&#x63ED;&#x793A;&#x4E86;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x751F;&#x7269;&#x949F;&#x5468;&#x671F;&#x7684;&#x7F29;&#x77ED;&#x3002;

L&#39;horloge circadienne contr&ocirc;le des oscillations quotidiennes d&#39;expression de g&egrave;ne au niveau cellulaire. Nous rapportons le d&eacute;veloppement d&#39;un syst&egrave;me de test fonctionnel circadienne de haut d&eacute;bit qui se compose de cellules journaliste luminescentes, l&#39;automatisation de d&eacute;pistage et un pipeline d&#39;analyse de donn&eacute;es. Nous avons appliqu&eacute; ce syst&egrave;me pour davantage de diss&eacute;quer les m&eacute;canismes mol&eacute;culaires qui sous-tendent l&#39;horloge circadienne chez les mammif&egrave;res, &agrave; l&#39;aide d&#39;une approche de la biologie chimique. Nous avons analys&eacute; l&#39;effet de 1 280 compos&eacute;s pharmacologiquement actifs avec diverses structures sur la longueur de la p&eacute;riode circadienne qui indique le m&eacute;canisme d&#39;horloge de noyau. Notre paradigme de d&eacute;pistage a identifi&eacute; de nombreux compos&eacute;s pr&eacute;c&eacute;demment connus pour changer la p&eacute;riode circadienne ou la phase, ce qui d&eacute;montre la validit&eacute; du syst&egrave;me de dosage. En outre, nous avons trouv&eacute; des petites mol&eacute;cules inhibitrices de la glycog&egrave;ne synthase kinase 3 (GSK-3) toujours caus&eacute; un ph&eacute;notype de p&eacute;riode court fort contraste avec la p&eacute;riode connue, allongement de lithium, un autre pr&eacute;sum&eacute; inhibiteur de GSK-3. induite par le siARN knockdown de GSK-3beta a &eacute;galement provoqu&eacute; une courte p&eacute;riode, confirmant le ph&eacute;notype obtenus avec les inhibiteurs de petites mol&eacute;cules. Ces r&eacute;sultats clarifier le r&ocirc;le de GSK-3beta dans la p&eacute;riode r&eacute;gulation de l&#39;horlogerie de mammif&egrave;res et de mettre en &eacute;vidence l&#39;efficacit&eacute; de la biologie chimique dans l&#39;exploration des m&eacute;canismes non identifi&eacute;s de l&#39;horloge circadienne.

Une approche de la biologie chimique r&eacute;v&egrave;le la p&eacute;riode raccourcissement de l&#39;horloge circadienne chez les mammif&egrave;res par inhibition sp&eacute;cifique de GSK-3beta.

Die circadiane Clock steuert t&auml;gliche Schwingungen der Genexpression auf zellul&auml;rer Ebene. Wir berichten &uuml;ber das Entwicklung eines Hochdurchsatz circadian funktionelle Assay, der leuchtende Reporter Zellen, Screening-Automatisierung und eine Daten-Analyse-Pipeline besteht. Wir verwendet dieses System um die molekularen Mechanismen der S&auml;ugetier-circadianen Uhr mit einem chemischen Biologie-Ansatz weiter zu sezieren. Wir analysierten die Wirkung von 1.280 pharmakologisch aktiven Verbindungen mit unterschiedlichen Strukturen auf die circadiane Periodenl&auml;nge, die bezeichnend f&uuml;r das Kern-Uhrwerk ist. Unsere Screening-Paradigma identifiziert viele chemische Verbindungen, die fr&uuml;her den zirkadianen Periode oder Phase, demonstrieren die G&uuml;ltigkeit der Assay-System &auml;ndern. Dar&uuml;ber hinaus fanden wir, dass ein anderes vermutet GSK-3-Hemmer, niedermolekularer Hemmer der Glykogen-Synthase Kinase 3 (GSK-3) konsequent verursacht einen starken kurzen Periode Ph&auml;notyp im Gegensatz zu den bekannten Zeitraum verl&auml;ngern von Lithium. SiRNA-vermittelte Niederschlag von GSK-3beta verursachte auch einen kurzen Zeitraum, Best&auml;tigung der Ph&auml;notyp mit der niedermolekularen-Inhibitoren erzielten. Diese Ergebnisse verdeutlichen die Rolle der GSK-3beta in der Periode der S&auml;ugetier-Uhrwerk-Verordnung und markieren Sie die Wirksamkeit der chemischen Biologie in der Erforschung der unbekannter Mechanismen der circadianen Uhr.

Ein chemische Biologie-Ansatz zeigt Zeitraum Verk&uuml;rzung der S&auml;ugetier-circadian Clock durch spezifische Hemmung der GSK-3beta.

L&#39;orologio circadiano controlla le oscillazioni giornaliere di espressione genica a livello cellulare. Riportiamo lo sviluppo di un sistema high-throughput test funzionali circadiani che consiste di cellule luminescenti reporter, automazione di screening e una pipeline di analisi dei dati. Abbiamo applicato questo sistema per sezionare ulteriormente i meccanismi molecolari sottostanti l&#39;orologio circadiano mammiferi utilizzando un approccio di biologia chimica. Abbiamo analizzato l&#39;effetto di 1.280 composti farmacologicamente attivi con diverse strutture sulla lunghezza di periodo circadiana &egrave; indicativa del meccanismo dell&#39;orologio di nucleo. Il nostro paradigma di screening identificato molti composti precedentemente conosciuti per cambiare il periodo circadiano o fase, dimostrando la validit&agrave; del sistema di dosaggio. Inoltre, abbiamo scoperto che piccole molecole inibitori delle chinasi 3 di glicogeno sintasi (GSK-3) costantemente ha causato un forte fenotipo periodo breve in contrasto con il periodo ben noto allungamento di litio, un altro presunto GSK-3 inibitore. siRNA-mediata atterramento di GSK-3beta anche causato un breve periodo, confermando il fenotipo ottenuto con piccole molecole inibitori. Questi risultati chiarire il ruolo di GSK-3beta nel periodo regolamento i clockworks mammiferi ed evidenziano l&#39;efficacia di biologia chimica nell&#39;esplorazione non identificati meccanismi dell&#39;orologio circadiano.

Un approccio di biologia chimica rivela periodo accorciamento dell&#39;orologio circadiano mammiferi tramite inibizione specifica di GSK-3 &beta;.

&#x6982;&#x65E5;&#x6642;&#x8A08;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x306F;&#x7D30;&#x80DE;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x306E;&#x6BCE;&#x65E5;&#x306E;&#x632F;&#x52D5;&#x3092;&#x5236;&#x5FA1;&#x3057;&#x307E;&#x3059;&#x3002;&#x767A;&#x5149;&#x30EC;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x7D30;&#x80DE;&#x3001;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x306E;&#x81EA;&#x52D5;&#x5316;&#x3001;&#x30C7;&#x30FC;&#x30BF;&#x5206;&#x6790;&#x306E;&#x30D1;&#x30A4;&#x30D7;&#x30E9;&#x30A4;&#x30F3;&#x3067;&#x69CB;&#x6210;&#x3055;&#x308C;&#x308B;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8;&#x6982;&#x6A5F;&#x80FD;&#x30A2;&#x30C3;&#x30BB;&#x30A4; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x958B;&#x767A;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x3055;&#x3089;&#x306B;&#x5316;&#x5B66;&#x751F;&#x7269;&#x5B66;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x54FA;&#x4E73;&#x985E;&#x306E;&#x6982;&#x65E5;&#x6642;&#x8A08;&#x306E;&#x5206;&#x5B50;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x89E3;&#x5256;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x3053;&#x306E;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x9069;&#x7528;&#x3057;&#x305F;.&#x6211;&#x3005; &#x306E;&#x30B3;&#x30A2;&#x306E;&#x30AF;&#x30ED;&#x30C3;&#x30AF; &#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x6982;&#x65E5;&#x30EA;&#x30BA;&#x30E0;&#x306E;&#x671F;&#x9593;&#x306E;&#x9577;&#x3055;&#x306B;&#x53CA;&#x307C;&#x3059; 1,280 &#x85AC;&#x7406;&#x6D3B;&#x6027;&#x5316;&#x5408;&#x7269;&#x306E;&#x591A;&#x69D8;&#x306A;&#x69CB;&#x9020;&#x3092;&#x5206;&#x6790;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306E;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0; &#x30D1;&#x30E9;&#x30C0;&#x30A4;&#x30E0;&#x6982;&#x65E5;&#x5468;&#x671F;&#x307E;&#x305F;&#x306F;&#x30A2;&#x30C3;&#x30BB;&#x30A4; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x6709;&#x52B9;&#x6027;&#x3092;&#x793A;&#x3059;&#x4F4D;&#x76F8;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x4EE5;&#x524D;&#x77E5;&#x3089;&#x308C;&#x3066;&#x591A;&#x304F;&#x306E;&#x5316;&#x5408;&#x7269;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x6211;&#x3005; &#x30B0;&#x30EA;&#x30B3;&#x30FC;&#x30B2;&#x30F3;&#x30B7;&#x30F3;&#x30BF;&#x30FC;&#x30BC;&#x30AD;&#x30CA;&#x30FC;&#x30BC; 3 &#x306E;&#x5C0F;&#x5206;&#x5B50;&#x963B;&#x5BB3;&#x5264; &#xFF08;GSK 3&#xFF09; &#x306F;&#x3001;&#x4E00;&#x8CAB;&#x3057;&#x3066;&#x5F37;&#x3044;&#x77ED;&#x3044;&#x671F;&#x9593;&#x8868;&#x73FE;&#x578B;&#x306B;&#x3088;&#x308B;&#x30EA;&#x30C1;&#x30A6;&#x30E0;&#x3092;&#x9577;&#x304F;&#x6709;&#x540D;&#x306A;&#x671F;&#x9593;&#x3068;&#x306F;&#x5BFE;&#x7167;&#x7684;&#x539F;&#x56E0;&#x3001;&#x5225; GSK 3 &#x963B;&#x5BB3;&#x5264;&#x3068;&#x63A8;&#x5B9A;&#x304C;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x3057;&#x305F;&#x3002;siRNA &#x3092;&#x4ECB;&#x3057;&#x305F;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3; GSK 3 &beta; &#x306E;&#x5C0F;&#x5206;&#x5B50;&#x963B;&#x5BB3;&#x5264;&#x3067;&#x5F97;&#x3089;&#x308C;&#x308B;&#x8868;&#x73FE;&#x578B;&#x78BA;&#x8A8D;&#x3059;&#x308B;&#x77ED;&#x671F;&#x9593;&#x767A;&#x751F;&#x3053;&#x3068;&#x3082;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x54FA;&#x4E73;&#x985E;&#x306E;&#x6642;&#x8A08;&#x4ED5;&#x639B;&#x3051;&#x306E;&#x671F;&#x9593;&#x306E;&#x898F;&#x5247;&#x306B;&#x304A;&#x3051;&#x308B; GSK 3 &beta; &#x306E;&#x5F79;&#x5272;&#x3092;&#x660E;&#x78BA;&#x306B;&#x3001;&#x6982;&#x65E5;&#x6642;&#x8A08;&#x306E;&#x6B63;&#x4F53;&#x4E0D;&#x660E;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x63A2;&#x6C42;&#x306E;&#x30B1;&#x30DF;&#x30AB;&#x30EB;&#x30D0;&#x30A4;&#x30AA;&#x30ED;&#x30B8;&#x30FC;&#x306E;&#x6709;&#x52B9;&#x6027;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;

&#x5316;&#x5B66;&#x751F;&#x7269;&#x5B66;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3067;&#x306F;&#x54FA;&#x4E73;&#x985E;&#x306E;&#x6982;&#x65E5;&#x6642;&#x8A08;&#x306E;&#x5468;&#x671F;&#x77ED;&#x7E2E; GSK 3 &beta; &#x306B;&#x3088;&#x308B;&#x7279;&#x5B9A;&#x306E;&#x963B;&#x5BB3;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;

Circadian &#xD074;&#xB85D;&#xC744; &#xC138;&#xD3EC; &#xC218;&#xC900;&#xC5D0;&#xC11C; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC758; &#xC77C;&#xC77C; &#xC9C4;&#xB3D9; &#xC81C;&#xC5B4;. &#xC6B0;&#xB9AC; &#xBCF4;&#xACE0; &#xBC1C;&#xAD11; &#xAE30;&#xC790; &#xC804;&#xC9C0;&#xC640; &#xC2EC;&#xC0AC; &#xC790;&#xB3D9;&#xD654;, &#xB370;&#xC774;&#xD130; &#xBD84;&#xC11D; &#xD30C;&#xC774;&#xD504;&#xB77C;&#xC778;&#xC73C;&#xB85C; &#xC774;&#xB8E8;&#xC5B4;&#xC838; &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; circadian &#xAE30;&#xB2A5; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xC2DC;&#xC2A4;&#xD15C; &#xAC1C;&#xBC1C;. &#xC6B0;&#xB9AC;&#xB294; &#xB354; &#xD654;&#xD559; &#xC0DD;&#xBB3C;&#xD559; &#xC811;&#xADFC;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xD3EC;&#xC720;&#xB958; circadian &#xC2DC;&#xACC4; &#xB0B4;&#xBD80; &#xBD84;&#xC790; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD574; &#xBD80;&#xC774; &#xC2DC;&#xC2A4;&#xD15C;&#xC744; &#xC801;&#xC6A9;. &#xC6B0;&#xB9AC;&#xB294; &#xCF54;&#xC5B4; &#xD074;&#xB7ED; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; circadian &#xAE30;&#xAC04; &#xAE38;&#xC774;&#xC5D0; &#xB2E4;&#xC591; &#xD55C; &#xAD6C;&#xC870;&#xC640; 1280 pharmacologically &#xD65C;&#xC131; &#xD654;&#xD569;&#xBB3C;&#xC758; &#xD6A8;&#xACFC; &#xBD84;&#xC11D;. &#xC6B0;&#xB9AC;&#xC758; &#xC2EC;&#xC0AC; &#xD328;&#xB7EC;&#xB2E4;&#xC784; circadian &#xAE30;&#xAC04; &#xB610;&#xB294; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xBCF4;&#xC5EC;&#xC8FC;&#xB294; &#xB2E8;&#xACC4; &#xBCC0;&#xACBD; &#xC774;&#xC804;&#xC758; &#xB9CE;&#xC740; &#xD654;&#xD569;&#xBB3C;&#xC744; &#xC2DD;&#xBCC4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xC791;&#xC740; &#xBD84;&#xC790; &#xC5B5;&#xC81C;&#xC81C; glycogen synthase &#xD0A4; &#xB2C8; &#xC544; &#xC81C; 3&#xC758; (GSK-3) &#xC77C;&#xAD00; &#xB418; &#xAC8C; &#xBC1C;&#xC0DD; &#xB9AC;&#xD2AC;&#xC5D0; &#xC758;&#xD574; &#xAE38;&#xC5B4; &#xC798; &#xC54C;&#xB824;&#xC9C4; &#xAE30;&#xAC04; &#xB2EC;&#xB9AC; &#xAC15;&#xD55C; &#xC9E7;&#xC740; &#xAE30;&#xAC04; &#xD615;, GSK 3 &#xC5B5;&#xC81C;&#xC81C;&#xB97C; &#xCD94;&#xC815; &#xB610; &#xB2E4;&#xB978; &#xBC1C;&#xACAC;. GSK-3beta&#xC758; siRNA &#xC911;&#xC7AC; &#xCD5C;&#xC800;&#xC758; &#xD655;&#xC778; &#xC791;&#xC740; &#xBD84;&#xC790; &#xC5B5;&#xC81C;&#xC81C;&#xB85C; &#xC5BB;&#xC740; &#xD615; &#xC9E7;&#xC740; &#xAE30;&#xAC04;&#xC744; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xD3EC;&#xC720;&#xB958; &#xC6CD;&#xC2A4;&#xC758; &#xAE30;&#xAC04; &#xADDC;&#xC815;&#xC5D0; GSK-3beta&#xC758; &#xC5ED;&#xD560;&#xC744; &#xBA85;&#xD655;&#xD788; &#xD558; &#xACE0; &#xD0D0;&#xD5D8; circadian clock&#xC758; &#xC54C; &#xC218; &#xC5C6;&#xB294; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xD654;&#xD559; &#xC0DD;&#xBB3C;&#xD559;&#xC758; &#xD6A8;&#xACFC; &#xAC15;&#xC870;.

&#xD654;&#xD559; &#xC0DD;&#xBB3C;&#xD559; &#xC811;&#xADFC; GSK-3beta&#xC758; &#xD2B9;&#xC815; &#xC800;&#xD574; &#xD558; &#xC5EC; &#xD3EC;&#xC720;&#xB958; circadian clock&#xC758; &#xAE30;&#xAC04; &#xB2E8;&#xCD95;&#xC744; &#xBCF4;&#xC5EC;&#xC900;&#xB2E4;.

El reloj circadiano controla las oscilaciones diarias de la expresi&oacute;n g&eacute;nica a nivel celular. Se presenta el desarrollo de un sistema de an&aacute;lisis funcional circadiana de alto rendimiento que consiste en c&eacute;lulas de reportero luminiscente, automatizaci&oacute;n de proyecci&oacute;n y una tuber&iacute;a de an&aacute;lisis de datos. Aplicamos este sistema para diseccionar a&uacute;n m&aacute;s los mecanismos moleculares subyacentes reloj circadiano mam&iacute;fero utilizando un enfoque de la Biolog&iacute;a qu&iacute;mica. Se analiz&oacute; el efecto de 1.280 compuestos farmacol&oacute;gicamente activos con diversas estructuras en la duraci&oacute;n del periodo circadiano que es indicativo del mecanismo de reloj del n&uacute;cleo. Nuestro paradigma de investigaci&oacute;n identificado muchos compuestos conocidos para cambiar el per&iacute;odo circadiano o fase, demostrando la validez del sistema de ensayo. Adem&aacute;s, hemos encontrado que inhibidores de mol&eacute;cula peque&ntilde;a de la quinasa de gluc&oacute;geno sintasa 3 (GSK-3) constantemente causaron un fuerte fenotipo per&iacute;odo corto en contraste con el per&iacute;odo conocido alargamiento por litio, otro presunto inhibidor de GSK-3. mediada por siRNA ca&iacute;da de GSK-3beta tambi&eacute;n caus&oacute; un breve per&iacute;odo, confirmando el fenotipo obtenido con los inhibidores de la mol&eacute;cula peque&ntilde;a. Estos resultados aclarar el papel de GSK-3beta en la regulaci&oacute;n de la &eacute;poca de los mecanismos mam&iacute;feros y destacan la eficacia de Biolog&iacute;a qu&iacute;mica en explorar mecanismos no identificados del reloj circadiano.

Un enfoque de la Biolog&iacute;a qu&iacute;mica revela acortamiento per&iacute;odo del reloj circadiano mam&iacute;fero por la inhibici&oacute;n espec&iacute;fica de GSK-3beta.

The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ∼120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.

Article	Year
NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus. The Journal of biological chemistry\| PubMed ID: 17646166	2007
A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19104043	2008
High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase. PLoS biology\| PubMed ID: 21179498	2010

Warren G. Lewis

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