Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Rutgers University

&#x81EA;&#x566C;&#xFF0C;&#x5BFC;&#x81F4;&#x9000;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x6210;&#x5206;&#xFF0C;&#x771F;&#x6838;&#x7EC6;&#x80DE;&#x6D3B;&#x52A8;&#x4F5C;&#x4E3A;&#x6253;&#x51FB;&#x517C;&#x6027;&#x7EC6;&#x80DE;&#x5185;&#x7684;&#x7EC6;&#x83CC;&#xFF0C;&#x4EE5;&#x53CA;&#x9884;&#x7559;&#x80DE;&#x5185;&#x7EC6;&#x83CC;&#x8D1D;&#x6C0F;&#x67EF;&#x514B;&#x65AF;&#x88AB;&#x5355;&#x4E3A;&#x589E;&#x957F;&#x5229;&#x57FA;&#x7684;&#x9632;&#x5FA1;&#x673A;&#x5236;&#x3002;&#x6211;&#x4EEC;&#x5728;&#x8FD9;&#x91CC;&#x8868;&#x73B0;&#x51FA;&#x9884;&#x7559;&#x80DE;&#x5185;&#x7EC6;&#x83CC;&#x75C5;&#x539F;&#x4F53;&#x8863;&#x539F;&#x4F53;&#x6C99;&#x773C;&#x6DCB;&#x5DF4;&#x8089;&#x82BD;&#x80BF; venereum &#x5F3A;&#x70C8;&#x8BF1;&#x5BFC;&#x8863;&#x539F;&#x4F53;&#x7684;&#x53D1;&#x80B2;&#x5468;&#x671F; (&#x611F;&#x67D3;&#x540E; 24 h)&#xFF0C;&#x5728;&#x81EA;&#x566C;&#x4E0E;&#x8863;&#x539F;&#x4F53;&#x7684;&#x590D;&#x5236;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x7684;&#x65E9;&#x671F;&#x9636;&#x6BB5;&#x4E0E;&#x4F4E;&#x603B;&#x4F53;&#x8863;&#x539F;&#x4F53;&#x4EE3;&#x8C22; (&#x524D; 8 h) &#x7684;&#x6700;&#x5927;&#x7EA7;&#x522B;&#x7684;&#x65F6;&#x95F4;&#x70B9;&#x3002;&#x6CA1;&#x6709;&#x81EA;&#x566C;&#x8BF1;&#x5BFC;&#x663E;&#x7136;&#x66B4;&#x9732;&#x5230;&#x70ED;&#x548C;&#x7D2B;&#x5916;&#x7EBF;&#x706D;&#x6D3B;&#x5C0F;&#x5B66;&#x673A;&#x6784; &#xFF08;EBs&#x3001; &#x8863;&#x539F;&#x4F53;&#x611F;&#x67D3;&#x5F62;&#x5F0F;&#xFF09; &#x6216;&#x4ECE;&#x4E2D; EBs &#x5DF2;&#x5220;&#x9664;&#x4E4B;&#x524D;&#x63A5;&#x79CD;&#x83CC;&#x5242;&#x7684;&#x5355;&#x5143;&#x683C;&#x4E2D;&#x3002;&#x963B;&#x6B62;&#x4E0E;&#x6C2F;&#x9709;&#x7D20;&#x6C99;&#x773C;&#x8863;&#x539F;&#x4F53;&#x53D1;&#x5C55;&#x8FD8;&#x963B;&#x6B62;&#x611F;&#x67D3;&#x4F20;&#x67D3;&#x6027; EBs &#x7EC6;&#x80DE;&#x81EA;&#x566C;&#x8BF1;&#x5BFC;&#x3002;&#x5B83;&#x5C06;&#x663E;&#x793A;&#x8BE5;&#x81EA;&#x566C;&#x6FC0;&#x6D3B;&#x4E3B;&#x8981;&#x5728;&#x4EE3;&#x8C22;&#x538B;&#x529B;&#x76F8;&#x5E94;&#x7684;&#x8863;&#x539F;&#x4F53;&#x590D;&#x5236;&#x54CD;&#x5E94;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x81EA;&#x566C;&#x7F3A;&#x9677; ATG5(-/-) &#x7EC6;&#x80DE;&#x81EA;&#x566C;&#x7CBE;&#x901A; ATG5(+/+) &#x5355;&#x5143;&#x683C;&#x4F5C;&#x4E3A;&#x6709;&#x6548;&#x5730;&#x652F;&#x6301;&#x8863;&#x539F;&#x4F53;&#x6027;&#x53D1;&#x5C55;&#x3002;

&#x751F;&#x4EA7;&#x6027;&#x6C99;&#x773C;&#x8863;&#x539F;&#x4F53;&#x6DCB;&#x5DF4;&#x8089;&#x82BD;&#x80BF; venereum 434 &#x611F;&#x67D3;&#x7EC6;&#x80DE;&#x589E;&#x5F3A;&#x6216;&#x706D;&#x6D3B;&#x81EA;&#x566C;&#x6027;&#x6D3B;&#x52A8;&#x3002;

Autophagy, a eukaryotic cellular activity leading to the degradation of cellular components, serves as a defense mechanism against facultative intracellular bacteria as well as a growth niche for the obligate intracellular bacterium Coxiella burnetii. We here demonstrate that the obligate intracellular bacterial pathogen Chlamydia trachomatis lymphogranuloma venereum strongly induced autophagy in the middle of the chlamydial developmental cycle (24 h after infection), a time point with maximal level of chlamydial replication, but not during the early stages with low overall chlamydial metabolism (before 8 h). No autophagy induction was evident in cells exposed to heat- and UV-inactivated elementary bodies (EBs, the infectious form of Chlamydia) or to inocula from which EBs had been removed before inoculation. Blocking chlamydial development with chloramphenicol also prevented autophagy induction in cells infected with infectious EBs. It appears that autophagy is activated primarily in response to the metabolic stress consequent to chlamydial replication. However, autophagy-defective ATG5(-/-) cells supported chlamydial development as efficiently as autophagy-proficient ATG5(+/+) cells.

Productive Chlamydia trachomatis lymphogranuloma venereum 434 infection in cells with augmented or inactivated autophagic activities.

Autophagie, une activit&eacute; cellulaire eucaryote, conduisant &agrave; la d&eacute;gradation des composants cellulaires, constitue un m&eacute;canisme de d&eacute;fense contre les bact&eacute;ries intracellulaires facultatives, mais aussi une niche de croissance de la bact&eacute;rie intracellulaire obligatoire Coxiella burnetii. Nous avons ici d&eacute;montrent que l&#39;obligatoires intracellulaire bact&eacute;rie pathog&egrave;ne Chlamydia trachomatis lymphogranulome v&eacute;n&eacute;rien fortement induite autophagie au milieu du cycle de d&eacute;veloppement Chlamydia (24 h apr&egrave;s l&#39;infection), un point de temps avec un niveau maximal de r&eacute;plication chlamydiale, mais pas durant les premiers stades avec faible m&eacute;tabolisme global de Chlamydia (avant 8 h). Aucune induction autophagie n&#39;&eacute;tait &eacute;vidente dans les cellules expos&eacute;es &agrave; des corps &eacute;l&eacute;mentaires et UV-inactiv&eacute;s par la chaleur (EBs, la forme infectieuse du Chlamydia) ou &agrave; inoculums qui EBs avaient &eacute;t&eacute; enlev&eacute;s avant l&#39;inoculation. Bloquant le d&eacute;veloppement Chlamydia avec chloramph&eacute;nicol pr&eacute;venue aussi l&#39;induction de l&#39;autophagie dans les cellules atteintes infectieuses EBs. Il semble que l&#39;autophagie est activ&eacute; principalement en r&eacute;ponse &agrave; la cons&eacute;quente de stress m&eacute;tabolique &agrave; la r&eacute;plication de Chlamydia trachomatie. Cependant, les cellules d&eacute;fectueuses autophagie ATG5(-/-) soutenu d&eacute;veloppement Chlamydia aussi efficacement que les cellules ATG5(+/+) d&#39;autophagie-comp&eacute;tent.

Infection 434 &agrave; productive Chlamydia trachomatis lymphogranulome v&eacute;n&eacute;rien en cellules avec augment&eacute;e ou inactiv&eacute; autophagiques activit&eacute;s.

Autophagy, einer eukaryotischen Zellaktivit&auml;t f&uuml;hrt zu einer Verschlechterung von Zellbestandteilen, dient als Abwehrmechanismus gegen fakultativ intrazellul&auml;re Bakterien sowie ein Wachstum-Nische f&uuml;r die obligat intrazellul&auml;res Bakterium Coxiella Burnetii. Zeigen wir hier, dass die obligat intrazellul&auml;re bakterielle Erreger Chlamydia Trachomatis venerische Lymphknotenentz&uuml;ndung stark Autophagy in der Mitte der Chlamydien Entwicklungs-Zyklus (24 h nach der Infektion), induziert einen Zeitpunkt mit maximalen Ebene der Chlamydien-Replikation, aber nicht w&auml;hrend der fr&uuml;hen Phasen mit niedrigen insgesamt Chlamydien Stoffwechsel (vor 8 Std.). Keine Induktion Autophagy zeigte sich in den Zellen ausgesetzt, bei Hitze und UV-inaktiviert elementare Einrichtungen (EBs, die infekti&ouml;se Form der Chlamydien) und Inokula von denen EBs vor Inokulation entfernt worden war. Chlamydien-Entwicklung mit Chloramphenicol blockieren verhindert auch Autophagy Induktion in Zellen, die mit infekti&ouml;sen EBs infiziert. Es scheint, dass diese Autophagy in erster Linie als Reaktion auf die metabolische Belastung Folge&auml;nderung aufgrund der Chlamydien Replikation aktiviert wird. Jedoch unterst&uuml;tzt Autophagy-Genen ATG5(-/-) Zellen Chlamydien Entwicklung m&ouml;glichst effizient Autophagy-profizienten ATG5(+/+) Zellen.

Produktive Chlamydia Trachomatis Lymphogranuloma Venereum 434 Infektion in den Zellen mit erg&auml;nzt oder inaktiviert autophagic Aktivit&auml;ten.

Autophagy, un&#39;attivit&agrave; cellulare eucariotica che portano alla degradazione dei componenti cellulari, serve come meccanismo di difesa contro i batteri intracellulari facoltativi cos&igrave; come una nicchia di crescita per il batterio intracellulare obligate Coxiella burnetii. Abbiamo qui dimostrare che il patogeno batterico intracellulare obligate Chlamydia trachomatis Linfogranuloma venereo fortemente indotta autophagy met&agrave; del ciclo di sviluppo da clamidia (24 ore dopo l&#39;infezione), un punto di tempo con il massimo livello di replica da clamidia, ma non durante le prime fasi con basso metabolismo generale da clamidia (prima di 8 h). Nessuna induzione autophagy era evidente in cellule esposte a corpi elementari (EBs, la forma infettiva della clamidia) inattivato da calore e raggi UV o a inoculi da cui EBs era stato rimosso prima dell&#39;inoculazione. Bloccando lo sviluppo da clamidia con cloramfenicolo anche impedito ad induzione autophagy in cellule infettate con EBs infettive. Sembra che autophagy &egrave; attiva principalmente in risposta alla conseguente stress metabolico di replica da clamidia. Tuttavia, le cellule ATG5(-/-) autophagy-difettoso sostegno sviluppo da clamidia nel modo pi&ugrave; efficiente le cellule ATG5(+/+) autophagy-competente.

Produttivo Chlamydia trachomatis Linfogranuloma venereo 434 infezione nelle cellule con aumentata o inattivato autophagic delle attivit&agrave;.

&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x7D30;&#x80DE;&#x6210;&#x5206;&#x306E;&#x4F4E;&#x4E0B;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x771F;&#x6838;&#x751F;&#x7269;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x6D3B;&#x52D5;&#x306F;&#x3001;&#x6210;&#x9577;&#x306E;&#x30CB;&#x30C3;&#x30C1;&#x3092;&#x7FA9;&#x52D9;&#x3065;&#x3051;&#x308B;&#x7D30;&#x80DE;&#x5185;&#x7D30;&#x83CC;&#x30B3;&#x30AF;&#x30B7;&#x30A8;&#x30E9;&#x9855;&#x8457;&#x3068;&#x540C;&#x69D8;&#x306B;&#x901A;&#x6027;&#x7D30;&#x80DE;&#x5185;&#x7D30;&#x83CC;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x9632;&#x5FA1;&#x6A5F;&#x69CB;&#x3068;&#x3057;&#x3066;&#x6A5F;&#x80FD;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x3053;&#x3053; &#xFF05;30 &#x306E;&#x7D30;&#x80DE;&#x5185;&#x7D30;&#x83CC;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x306E;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2; &#x2010; &#x30C8;&#x30E9;&#x30B3;&#x30DE;&#x30C6;&#x30A3;&#x30B9;&#x9F21;&#x5F84;&#x30EA;&#x30F3;&#x30D1;&#x8089;&#x816B;&#x3092;&#x5F37;&#x304F;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2;&#x767A;&#x9054;&#x30B5;&#x30A4;&#x30AF;&#x30EB; &#xFF08;&#x611F;&#x67D3;&#x5F8C; 24 h&#xFF09; &#x306E;&#x9014;&#x4E2D;&#x3067;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x8A98;&#x5C0E;&#x3092;&#x793A;&#x3059;&#x6642;&#x9593;&#x70B9;&#x3068;&#x6700;&#x5927;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x3059;&#x304C;&#x4F4E;&#x306E;&#x5168;&#x4F53;&#x7684;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2;&#x4EE3;&#x8B1D; &#xFF08;8 h&#xFF09; &#x306E;&#x524D;&#x306B;&#x521D;&#x671F;&#x6BB5;&#x968E;&#x306A;&#x3044;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2; &#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x3002;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x8A98;&#x5C0E;&#x71B1;&#x3068; UV &#x4E0D;&#x6D3B;&#x5316;&#x3057;&#x305F;&#x5C0F;&#x5B66;&#x6821;&#x4F53; (EBs&#x3001;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2;&#x306E;&#x4F1D;&#x67D3;&#x6027;&#x5F62;&#x5F0F;)&#x3001;&#x307E;&#x305F;&#x306F;&#x63A5;&#x7A2E;&#x304B;&#x3089; EBs &#x63A5;&#x7A2E;&#x524D;&#x306B;&#x524A;&#x9664;&#x3055;&#x308C;&#x3066;&#x3044;&#x305F;&#x7D30;&#x80DE;&#x3067;&#x660E;&#x3089;&#x304B;&#x3067;&#x306F;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;&#x30AF;&#x30ED;&#x30E9;&#x30E0;&#x30D5;&#x30A7;&#x30CB; &#x30B3;&#x30FC;&#x30EB;&#x3068;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2;&#x958B;&#x767A;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3082;&#x611F;&#x67D3; EBs &#x3068;&#x611F;&#x67D3;&#x7D30;&#x80DE;&#x3067;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x8A98;&#x5C0E;&#x3092;&#x9632;&#x6B62;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x305D;&#x308C;&#x306F;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x4EE3;&#x8B1D;&#x30B9;&#x30C8;&#x30EC;&#x30B9;&#x5E30;&#x7D50;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2; &#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3078;&#x306E;&#x5FDC;&#x7B54;&#x3067;&#x4E3B;&#x306B;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x304C;&#x8868;&#x793A;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x6B20;&#x9665; ATG5(-/-) &#x7D30;&#x80DE;&#x306F;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x719F;&#x7DF4; ATG5(+/+) &#x7D30;&#x80DE;&#x3068;&#x3057;&#x3066;&#x52B9;&#x7387;&#x7684;&#x306B;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2;&#x306E;&#x958B;&#x767A;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;

&#x751F;&#x7523;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x30AF;&#x30E9;&#x30DF;&#x30B8;&#x30A2; &#x2010; &#x30C8;&#x30E9;&#x30B3;&#x30DE;&#x30C6;&#x30A3;&#x30B9;&#x9F21;&#x5F84;&#x30EA;&#x30F3;&#x30D1;&#x8089;&#x816B; 434 &#x611F;&#x67D3;&#x7D30;&#x80DE;&#x3067;&#x306F;&#x3001;&#x62E1;&#x5F35;&#x73FE;&#x5B9F;&#x611F;&#x307E;&#x305F;&#x306F; autophagic &#x6D3B;&#x52D5;&#x4E0D;&#x6D3B;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;

Autophagy, &#xC9C4; &#xD575; &#xC138;&#xD3EC; &#xD65C;&#xB3D9; &#xC138;&#xD3EC;&#xC9C8; &#xBD84;&#xC758; &#xC800;&#xD558;&#xB85C; &#xC774;&#xC5B4;&#xC9C0;&#xB294; &#xC758;&#xC0AC; &#xC138;&#xD3EC;&#xB0B4; &#xC138;&#xADE0;&#xC73C;&#xB85C; &#xC758;&#xBB34; &#xC138;&#xD3EC;&#xB0B4; &#xBC15;&#xD14C;&#xB9AC;&#xC544; Coxiella burnetii&#xC5D0; &#xB300; &#xD55C; &#xC131;&#xC7A5; &#xD2C8;&#xC0C8; &#xC2DC;&#xC7A5;&#xC5D0; &#xB300; &#xD55C; &#xBC29;&#xC5B4; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xC5EC;&#xAE30;&#xC5D0; &#xC785;&#xC99D; &#xC758;&#xBB34; &#xC138;&#xD3EC;&#xB0B4; &#xC138;&#xADE0;&#xC131; &#xBCD1;&#xC6D0; &#xCCB4; &#xD074; &#xB77C; &#xBBF8;&#xB514; &#xC544;&#xC758; trachomatis lymphogranuloma venereum &#xAC15;&#xB825; &#xD558; &#xAC8C; autophagy chlamydial &#xBC1C;&#xB2EC; &#xC8FC;&#xAE30; (&#xAC10;&#xC5FC; &#xD6C4; 24 h), &#xC911;&#xAC04; &#xC720;&#xB3C4; chlamydial &#xBCF5;&#xC81C; &#xD558;&#xC9C0;&#xB9CC; (8 h) &#xC804;&#xC5D0; &#xB0AE;&#xC740; &#xC804;&#xBC18;&#xC801;&#xC778; chlamydial &#xC2E0;&#xC9C4; &#xB300;&#xC0AC;&#xC640; &#xCD08;&#xAE30; &#xB2E8;&#xACC4; &#xD558;&#xC9C0; &#xCD5C;&#xB300; &#xC218;&#xC900;&#xC73C;&#xB85C; &#xC2DC;&#xAC04; &#xD3EC;&#xC778;&#xD2B8;. &#xC544;&#xB2C8; autophagy &#xC720;&#xB3C4; &#xC5F4; &#xBC0F; UV inactivated &#xCD08;&#xB4F1;&#xD559;&#xAD50; &#xC2DC;&#xCCB4; (EBs, &#xD074; &#xB77C; &#xBBF8;&#xB514; &#xC544; &#xAC10;&#xC5FC; &#xD615;&#xD0DC;) &#xB610;&#xB294; inocula &#xC811;&#xC885; &#xC804;&#xC5D0; &#xC81C;&#xAC70; &#xD588;&#xB2E4; Ebs&#xC5D0;&#xC11C; &#xB178;&#xCD9C; &#xD558;&#xB294; &#xC140;&#xC5D0; &#xBD84;&#xBA85; &#xD588;&#xB2E4;. &#xC138;&#xD3EC;&#xC5D0; &#xAC10;&#xC5FC; &#xB41C; &#xC804;&#xC5FC; &#xC131; Ebs&#xC5D0;&#xC11C; autophagy &#xC720;&#xB3C4; &#xBC29;&#xD574;&#xB3C4; &#xD398;&#xB2C8;&#xC640; chlamydial &#xAC1C;&#xBC1C;&#xC744; &#xCC28;&#xB2E8;. &#xADF8;&#xAC83;&#xC740; &#xADF8; autophagy chlamydial &#xBCF5;&#xC81C; &#xB300;&#xC0AC; &#xC2A4;&#xD2B8;&#xB808;&#xC2A4; &#xACB0;&#xACFC;&#xC5D0; &#xB300; &#xD55C; &#xC751;&#xB2F5;&#xC5D0; &#xC8FC;&#xB85C; &#xD65C;&#xC131;&#xD654; &#xB418;&#xC5B4; &#xB098;&#xD0C0;&#xB0A9;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; autophagy &#xACB0;&#xD568; ATG5(-/-) &#xC140; chlamydial &#xAC1C;&#xBC1C; autophagy &#xC2E4;&#xB825; ATG5(+/+) &#xC140; &#xD6A8;&#xC728;&#xC801; &#xC9C0;&#xC6D0;.

&#xC0DD;&#xC0B0;&#xC801;&#xC778; &#xD074; &#xB77C; &#xBBF8;&#xB514; &#xC544; trachomatis lymphogranuloma venereum 434 &#xAC10;&#xC5FC; &#xC140;&#xC5D0; &#xC99D;&#xAC15; &#xB610;&#xB294; autophagic &#xD65C;&#xB3D9;&#xC744; &#xBE44;&#xD65C;&#xC131;&#xD654; &#xD569;&#xB2C8;&#xB2E4;.

Autofagia, una actividad celular eucariota conduce a la degradaci&oacute;n de componentes celulares, sirve como un mecanismo de defensa contra las bacterias intracelulares facultativas como un nicho de crecimiento de la bacteria intracelular obligan Coxiella burnetii. Aqu&iacute; demostramos que la obligue pat&oacute;geno bacteriano intracelular Chlamydia trachomatis Linfogranuloma ven&eacute;reo fuertemente inducida por autofagia en medio del ciclo de desarrollo por clamidia (24 h despu&eacute;s de la infecci&oacute;n), un punto de tiempo con el m&aacute;ximo nivel de replicaci&oacute;n por clamidia, pero no durante las primeras etapas con bajo metabolismo general por clamidia (antes de las 8 h). Inducci&oacute;n autofagia no era evidente en las c&eacute;lulas expuestas a inactivado con calor y UV cuerpos elementales (EBs, la forma infecciosa de Chlamydia) o in&oacute;culos de que EBs hab&iacute;a eliminado antes de la inoculaci&oacute;n. Bloqueo por desarrollo con cloranfenicol tambi&eacute;n prevenir la inducci&oacute;n de la autofagia en las c&eacute;lulas infectadas con EBs infecciosas. Parece que autofagia se activa principalmente en respuesta a la consecuente estr&eacute;s metab&oacute;lico para replicaci&oacute;n por clamidia. Sin embargo, c&eacute;lulas ATG5(-/-) con defectos autofagia apoya desarrollo clamidi&aacute;cea tan eficientemente como las c&eacute;lulas ATG5(+/+) de dominio autofagia.

Productivo Chlamydia trachomatis Linfogranuloma ven&eacute;reo 434 infecci&oacute;n en c&eacute;lulas con aumentada o hab&iacute;a inactivado actividades autofagia.

2008 &#x5E74;&#x6211;&#x4EEC;&#x53D1;&#x8868;&#x4E86;&#x7B2C;&#x4E00;&#x5957;&#x6307;&#x5F15;&#xFF0C;&#x89C4;&#x8303;&#x81EA;&#x566C;&#x7684;&#x7814;&#x7A76;&#x3002;&#x81EA;&#x90A3;&#x65F6;&#x4EE5;&#x6765;&#xFF0C;&#x5173;&#x4E8E;&#x8FD9;&#x4E00;&#x4E3B;&#x9898;&#x7684;&#x7814;&#x7A76;&#x5DF2;&#x7EE7;&#x7EED;&#x52A0;&#x901F;&#xFF0C;&#x548C;&#x8BB8;&#x591A;&#x65B0;&#x79D1;&#x5B66;&#x5BB6;&#x5DF2;&#x8F93;&#x5165;&#x8BE5;&#x5B57;&#x6BB5;&#x3002;&#x6B64;&#x5916;&#x6269;&#x5927;&#x6211;&#x4EEC;&#x7684;&#x77E5;&#x8BC6;&#x5E93;&#x548C;&#x76F8;&#x5173;&#x65B0;&#x6280;&#x672F;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x5B83;&#x662F;&#x91CD;&#x8981;&#x7684;&#x662F;&#x8981;&#x66F4;&#x65B0;&#x8FD9;&#x4E9B;&#x51C6;&#x5219;&#xFF0C;&#x4EE5;&#x4FBF;&#x76D1;&#x6D4B;&#x4E0D;&#x540C;&#x751F;&#x7269;&#x4F53;&#x4E2D;&#x7684;&#x81EA;&#x566C;&#x3002;&#x5404;&#x9879;&#x5BA1;&#x67E5;&#x6240;&#x63CF;&#x8FF0;&#x5DF2;&#x7528;&#x4E8E;&#x6B64;&#x76EE;&#x7684;&#x7684;&#x68C0;&#x6D4B;&#x7684;&#x8303;&#x56F4;&#x3002;&#x5C3D;&#x7BA1;&#x5982;&#x6B64;&#xFF0C;&#x7EE7;&#x7EED;&#x5173;&#x4E8E;&#x53EF;&#x63A5;&#x53D7;&#x7684;&#x65B9;&#x6CD5;&#x6765;&#x8861;&#x91CF;&#x81EA;&#x566C;&#xFF0C;&#x5C24;&#x5176;&#x662F;&#x5728;&#x591A;&#x7EC6;&#x80DE;&#x771F;&#x6838;&#x751F;&#x7269;&#x7684;&#x6DF7;&#x4E71;&#x3002;&#x9700;&#x8981;&#x5F3A;&#x8C03;&#x6307;&#x51FA;&#x7684;&#x5173;&#x952E;&#x70B9;&#x5C31;&#x662F;&#x76D1;&#x6D4B;&#x6570;&#x5B57;&#x6216;&#x5377;&#x7684;&#x81EA;&#x566C;&#x6027;&#x5143;&#x7D20; &#xFF08;&#x4F8B;&#x5982;&#xFF0C;autophagosomes &#x6216; autolysosomes&#xFF09; &#x7684;&#x6D4B;&#x91CF;&#x7ED3;&#x679C;&#x4E4B;&#x95F4;&#x7684;&#x5DEE;&#x5F02;&#x4E0E;&#x90A3;&#x4E9B;&#x81EA;&#x566C;&#x8FC7;&#x7A0B;&#x7684;&#x4EFB;&#x4F55;&#x9636;&#x6BB5;&#xFF0C;&#x68C0;&#x6D4B;&#x6D41;&#x91CF;&#x901A;&#x8FC7;&#x81EA;&#x566C;&#x9014;&#x5F84; &#xFF08;&#x5373;&#xFF0C;&#x5B8C;&#x6574;&#x7684;&#x8FDB;&#x7A0B;&#xFF09; &#xFF1B;&#x56E0;&#x6B64;&#xFF0C;macroautophagy autophagosome &#x79EF;&#x7D2F;&#x7684;&#x7ED3;&#x679C;&#x9700;&#x8981;&#x52A0;&#x4EE5;&#x533A;&#x5206;&#x4ECE;&#x81EA;&#x566C;&#x6027;&#x6D3B;&#x52A8;&#x589E;&#x52A0;&#xFF0C;&#x4F1A;&#x5BFC;&#x81F4;&#x7684;&#x523A;&#x6FC0;&#x4E2D;&#x7684;&#x5757;&#x5B9A;&#x4E49;&#x4E3A;&#x589E;&#x52A0;&#x81EA;&#x566C;&#x8BF1;&#x5BFC;&#x52A0;&#x4E0A;&#x589E;&#x52A0;&#x7684;&#x4EA4;&#x4ED8;&#x548C;&#x6EB6;&#x9176;&#x4F53; &#xFF08;&#x5728;&#x5927;&#x591A;&#x6570;&#x9AD8;&#x7B49;&#x771F;&#x6838;&#x751F;&#x7269;&#x548C;&#x76D8;&#x57FA;&#x7F51;&#x67C4;&#x7B49;&#x4E00;&#x4E9B;&#x751F;&#x7269;&#x4F53;&#xFF09; &#x6216; &#xFF08;&#x5728;&#x690D;&#x7269;&#x548C;&#x771F;&#x83CC;&#xFF09; &#x6CE1;&#x5185;&#x7684;&#x964D;&#x89E3;&#x3002;&#x6362;&#x53E5;&#x8BDD;&#x8BF4;&#xFF0C;&#x5B83;&#x662F;&#x5C24;&#x5176;&#x91CD;&#x8981;&#x7684;&#x662F;&#x65B0;&#x7684;&#x9886;&#x57DF;&#x7684;&#x8C03;&#x67E5;&#x4EBA;&#x5458;&#x4E86;&#x89E3;&#x66F4;&#x591A; autophagosomes &#x7684;&#x5916;&#x89C2;&#x4E0D;&#x4E00;&#x5B9A;&#x7B49;&#x540C;&#x66F4;&#x591A;&#x81EA;&#x566C;&#x3002;&#x4E8B;&#x5B9E;&#x4E0A;&#xFF0C;&#x5728;&#x8BB8;&#x591A;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;autophagosomes &#x79EF;&#x7D2F;&#x7531;&#x4E8E;&#x8D29;&#x8FD0;&#x5230;&#x6EB6;&#x9176;&#x4F53;&#x4E0D;&#x968F;&#x4E4B;&#x6539;&#x53D8;&#x7684;&#x751F;&#x7269;&#x53D1;&#x751F; autophagosome&#xFF0C;&#x4E00;&#x4E2A;&#x5757;&#x800C;&#x589E;&#x52A0; autolysosomes &#x53EF;&#x964D;&#x89E3;&#x6D3B;&#x52A8;&#x4E2D;&#x53CD;&#x6620;&#x51CF;&#x5C11;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x63D0;&#x51FA;&#x4E00;&#x5957;&#x51C6;&#x5219;&#x7684;&#x9009;&#x62E9;&#x548C;&#x4F7F;&#x7528;&#x65B9;&#x6CD5;&#x7684;&#x89E3;&#x91CA;&#x7531;&#x8C03;&#x67E5;&#x4EBA;&#x5458;&#xFF0C;&#x4ED6;&#x4EEC;&#x7684;&#x76EE;&#x7684;&#x662F;&#x68C0;&#x67E5; macroautophagy &#x548C;&#x76F8;&#x5173;&#x7684;&#x8FDB;&#x7A0B;&#xFF0C;&#x4EE5;&#x53CA;&#x5BA1;&#x9605;&#x8005;&#x9700;&#x8981;&#x63D0;&#x4F9B;&#x8BBA;&#x6587;&#x4FA7;&#x91CD;&#x4E8E;&#x8FD9;&#x4E9B;&#x8FDB;&#x7A0B;&#x7684;&#x73B0;&#x5B9E;&#x548C;&#x5408;&#x7406;&#x7684;&#x6279;&#x8BC4;&#x3002;&#x8FD9;&#x4E9B;&#x51C6;&#x5219;&#x4E0D;&#x662F;&#x8981;&#x5957;&#x516C;&#x5F0F;&#x5316;&#x7684;&#x89C4;&#x5219;&#xFF0C;&#x56E0;&#x4E3A;&#x9002;&#x5F53;&#x7684;&#x68C0;&#x6D4B;&#x90E8;&#x5206;&#x53D6;&#x51B3;&#x4E8E;&#x7684;&#x8D28;&#x8BE2;&#x548C;&#x6B63;&#x5728;&#x4F7F;&#x7528;&#x7684;&#x7CFB;&#x7EDF;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x5F3A;&#x8C03;&#x4FDD;&#x8BC1;&#x6CA1;&#x6709;&#x4E2A;&#x522B;&#x6D4B;&#x5B9A;&#x90FD;&#x662F;&#x5728;&#x6BCF;&#x4E2A;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;&#x4E00;&#x4E2A;&#x6700;&#x5408;&#x9002;&#xFF0C;&#x6211;&#x4EEC;&#x5F3A;&#x70C8;&#x5EFA;&#x8BAE;&#x4F7F;&#x7528;&#x591A;&#x4E2A;&#x5316;&#x9A8C;&#x76D1;&#x6D4B;&#x81EA;&#x566C;&#x3002;&#x5728;&#x672C;&#x6307;&#x5357;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x8003;&#x8651;&#x8FD9;&#x4E9B;&#x4E0D;&#x540C;&#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x8BC4;&#x4F30;&#x81EA;&#x566C;&#x548C;&#x54EA;&#x4E9B;&#x4FE1;&#x606F;&#x53EF;&#x4EE5;&#xFF0C;&#x6216;&#x4E0D;&#x80FD;&#xFF0C;&#x4ECE;&#x5B83;&#x4EEC;&#x83B7;&#x5F97;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x901A;&#x8FC7;&#x8BA8;&#x8BBA;&#x6848;&#x60C5;&#x548C;&#x7279;&#x5B9A;&#x7684;&#x81EA;&#x566C;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x7684;&#x9650;&#x5236;&#xFF0C;&#x6211;&#x4EEC;&#x5E0C;&#x671B;&#x9F13;&#x52B1;&#x6280;&#x672F;&#x521B;&#x65B0;&#x7684;&#x9886;&#x57DF;&#x3002;

&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x76D1;&#x6D4B;&#x81EA;&#x566C;&#x7684;&#x89E3;&#x91CA;&#x548C;&#x4F7F;&#x7528;&#x7684;&#x6307;&#x5357;&#x3002;

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy.

En 2008, nous avons publi&eacute; le premier ensemble de lignes directrices pour la normalisation des recherches dans l&#39;autophagie. Depuis lors, les recherches sur ce sujet a continu&eacute; d&#39;acc&eacute;l&eacute;rer, et beaucoup de scientifiques nouveaux entr&eacute;s dans le champ. Notre base de connaissances et les nouvelles technologies ont &eacute;galement &eacute;tendu. En cons&eacute;quence, il est important de mettre &agrave; jour ces lignes directrices pour la surveillance de l&#39;autophagie dans diff&eacute;rents organismes. Diff&eacute;rents examens ont d&eacute;crit les divers essais qui ont &eacute;t&eacute; utilis&eacute;s &agrave; cet effet. N&eacute;anmoins, il continue d&#39;y avoir confusion au sujet des m&eacute;thodes acceptables pour mesurer l&#39;autophagie, surtout chez les eucaryotes multicellulaires. Un point essentiel qui doit &ecirc;tre soulign&eacute;, c&#39;est qu&#39;il y a une diff&eacute;rence entre les diff&eacute;rentes mesures qui surveillent les chiffres ou le volume des &eacute;l&eacute;ments autophagiques (autophagosomes ou autolysosomes) &agrave; tout stade du processus autophagique vs ceux ce flux de mesure par la voie de l&#39;autophagie (c.-&agrave;-d. le processus complet) ; ainsi, un bloc en macroautophagy, ce qui entra&icirc;ne l&#39;accumulation autophagosome doit &ecirc;tre diff&eacute;renci&eacute;s des stimuli qui r&eacute;sultent dans l&#39;augmentation de l&#39;activit&eacute; autophagique, d&eacute;finie comme augmentation autophagie induction coupl&eacute;e avec livraison accrue &agrave; et d&eacute;gradation dans les lysosomes (dans la plupart des eucaryotes sup&eacute;rieurs et certains protistes comme Dictyostelium) ou la vacuole (chez les plantes et les champignons). En d&#39;autres termes, il est particuli&egrave;rement important que les chercheurs &agrave; nouveau dans le domaine comprennent que l&#39;apparence des autophagosomes plus ne correspond pas n&eacute;cessairement avec autophagie plus. En effet, dans de nombreux cas, autophagosomes s&#39;accumulent en raison d&#39;un bloc dans le trafic vers les lysosomes sans changement concomitant autophagosome biogen&egrave;se, alors qu&#39;une augmentation d&#39;autolysosomes peut refl&eacute;ter une r&eacute;duction dans l&#39;activit&eacute; de d&eacute;gradation. Nous pr&eacute;sentons ici une s&eacute;rie de lignes directrices pour la s&eacute;lection et l&#39;interpr&eacute;tation des m&eacute;thodes pour l&#39;utilisation par les enqu&ecirc;teurs, qui visent &agrave; examiner les macroautophagy et les processus connexes, ainsi que pour les examinateurs qui doivent fournir des critiques r&eacute;alistes et raisonnables des papiers qui mettent l&#39;accent sur ces processus. Ces lignes directrices ne visent pas &agrave; &ecirc;tre un ensemble de formule de r&egrave;gles, parce que les dosages appropri&eacute;s d&eacute;pendent en partie de la question pos&eacute;e et le syst&egrave;me utilis&eacute;. En outre, nous soulignons qu&#39;aucun dosage individuel n&#39;est garanti d&#39;&ecirc;tre le plus appropri&eacute; dans chaque situation, et nous recommandons fortement l&#39;utilisation de multiples essais pour surveiller l&#39;autophagie. Dans ces lignes directrices, nous consid&eacute;rons ces diverses m&eacute;thodes d&#39;&eacute;valuation d&#39;autophagie et quels renseignements peuvent, ou ne peut &ecirc;tre obtenu d&#39;eux. Enfin, en discutant les avantages et les limites des analyses particuli&egrave;res autophagie, nous esp&eacute;rons encourager l&#39;innovation technique dans le domaine.

Lignes directrices pour l&#39;utilisation et l&#39;interpr&eacute;tation d&#39;essais pour la surveillance d&#39;autophagie.

Im Jahr 2008 haben wir den ersten Satz von Richtlinien f&uuml;r die Standardisierung der Forschung in Autophagy ver&ouml;ffentlicht. Seitdem Forschung zu diesem Thema hat sich weiter beschleunigen, und viele neue Wissenschaftler haben das Spielfeld betreten. Unsere Wissensdatenbank und relevante neue Technologien haben auch erweitert. Dementsprechend ist es wichtig, diese Leitlinien f&uuml;r die &Uuml;berwachung Autophagy in verschiedenen Organismen zu aktualisieren. Verschiedene Meinungen haben die Proben beschrieben, die f&uuml;r diesen Zweck verwendet wurden. Dennoch weiterhin besteht Verwirrung in Bezug auf akzeptierte, Autophagy, vor allem bei mehrzelligen Eukaryoten zu messen. Ein wesentlicher Punkt, der betont werden muss ist, dass es ein Unterschied zwischen Messungen, die &Uuml;berwachung der Zahlen oder Volume autophagic Elemente (z. B. Autophagosomes oder Autolysosomes) in jedem Stadium des Prozesses gegen die autophagic dieser Ma&szlig;nahme-Fluss durch den Autophagy Pathway (d. h. der vollst&auml;ndige Proze&szlig;); so ein Block in Macroautophagy, Ergebnisse in Autophagosomen Anh&auml;ufung von Reize unterschieden werden, die erh&ouml;hte autophagic Aktivit&auml;t, f&uuml;hren die definiert als erh&ouml;hte Autophagy Induktion gepaart mit erh&ouml;hter Lieferung an und Verschlechterung in Lysosomen (in den meisten h&ouml;heren Eukaryotes und einigen Einzellern wie Dictyostelium) oder die Vakuole (in Pflanzen und Pilzen). Mit anderen Worten, ist es besonders wichtig, dass die Ermittler in das Feld neue verstehen, dass die Darstellung des mehr Autophagosomes nicht unbedingt mit mehr Autophagy gleichzusetzen ist. In der Tat sammeln in vielen F&auml;llen Autophagosomes wegen einen Block in den Handel zu Lysosomen ohne eine damit einhergehende &Auml;nderung in Autophagosomen Biogenese, w&auml;hrend eine Zunahme der Autolysosomes eine Verringerung in Degradations-Aktivit&auml;t widerspiegeln kann. Hier pr&auml;sentieren wir eine Reihe von Leitlinien f&uuml;r die Auswahl und Interpretation der Methoden f&uuml;r die Verwendung von Ermittlern, die Macroautophagy und die damit verbundenen Prozesse untersuchen wollen, sowie f&uuml;r Pr&uuml;fer, die realistische und vern&uuml;nftige Kritiken von Papier bereitstellen, die auf diese Prozesse gerichtet werden m&uuml;ssen. Diese Richtlinien sollen kein formelhaften Satz von Regeln, weil die entsprechenden Assays teilweise auf die Frage und das System verwendet wird. Dar&uuml;ber hinaus betonen wir, dass keine einzelne Assay ist garantiert das passende Produkt in jeder Situation, und wir empfehlen die Verwendung von mehreren Proben Autophagy &uuml;berwachen. In diesen Leitlinien halten wir diese verschiedenen Methoden zur Bewertung Autophagy und welche Informationen kann, oder nicht, von ihnen abgerufen werden. Abschlie&szlig;end diskutieren die Vorz&uuml;ge und Grenzen von bestimmten Autophagy Assays, hoffen wir auf technische Innovationen im Bereich zu f&ouml;rdern.

Richtlinien f&uuml;r die Verwendung und Interpretation von Assays zur &Uuml;berwachung Autophagy in Verbindung.

Nel 2008 abbiamo pubblicato la prima serie di linee guida per la standardizzazione della ricerca in autophagy. Da allora, ricerca su questo argomento ha continuato ad accelerare, e molti nuovi scienziati hanno entrato in campo. Hanno anche ampliato la nostra knowledge base e le nuove tecnologie pertinenti. Di conseguenza, &egrave; importante aggiornare queste linee guida per il monitoraggio autophagy in diversi organismi. Varie recensioni hanno descritto la gamma di dosaggi che sono stati utilizzati per questo scopo. Tuttavia, ci continua a essere confusione per quanto riguarda i metodi accettabili per misurare autophagy, specialmente negli eucarioti pluricellulari. Un punto chiave che deve essere sottolineato &egrave; che c&#39;&egrave; una differenza tra le misure che monitorare il volume di autophagic elementi (ad esempio, autophagosomes o autolysosomes) o numeri in qualsiasi fase del processo autophagic vs quelli che misura flusso attraverso il pathway autophagy (cio&egrave;, il processo completo); cos&igrave;, un blocco in macroautophagy che risultati in accumulo di autophagosome deve essere differenziato da stimoli che si traducono in maggiore attivit&agrave; autophagic, definita come aumento autophagy induzione accoppiato con consegna aumentata e la degradazione nei lisosomi (nella maggior parte dei eucarioti superiori e alcuni protisti come Dictyostelium) o il vacuolo (in piante e funghi). In altre parole, &egrave; particolarmente importante che gli investigatori di nuovi al campo capiscano che l&#39;aspetto di pi&ugrave; autophagosomes non necessariamente identificare con pi&ugrave; autophagy. Infatti, in molti casi, autophagosomes si accumulano a causa di un blocco nel traffico ai lisosomi senza un cambiamento concomitante di autophagosome biogenesi, considerando che un aumento di autolysosomes pu&ograve; riflettere una riduzione nell&#39;attivit&agrave; degradanti. Qui, vi presentiamo una serie di linee guida per la selezione e l&#39;interpretazione dei metodi per l&#39;uso dagli investigatori che mirano a esaminare macroautophagy e dei processi correlati, nonch&eacute; per i revisori che hanno bisogno di fornire critiche realistiche e ragionevole delle carte che sono focalizzate su questi processi. Queste linee guida non sono destinate ad essere una serie stereotipata di regole, perch&eacute; i dosaggi appropriati dipendono in parte la domanda e il sistema in uso. Inoltre, sottolineiamo che nessun dosaggio individuale &egrave; garantito per essere quello pi&ugrave; appropriato in ogni situazione, e si consiglia vivamente l&#39;uso di dosaggi multipli di monitorare autophagy. In queste linee guida, consideriamo questi vari metodi di valutazione autophagy e quali informazioni possono, o non pu&ograve; essere ottenuto da loro. Infine, da discutere i meriti e i limiti delle analisi particolare autophagy, speriamo di incoraggiare l&#39;innovazione tecnica nel campo.

Linee guida per l&#39;uso e l&#39;interpretazione delle analisi per il monitoraggio autophagy.

2008 &#x5E74;&#x306B;&#x6700;&#x521D;&#x306E;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x306E;&#x7814;&#x7A76;&#x6A19;&#x6E96;&#x5316;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x306E;&#x30BB;&#x30C3;&#x30C8;&#x3092;&#x516C;&#x958B;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x5F8C;&#x3001;&#x3053;&#x306E;&#x30C8;&#x30D4;&#x30C3;&#x30AF;&#x306B;&#x95A2;&#x3059;&#x308B;&#x7814;&#x7A76;&#x3092;&#x52A0;&#x901F;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x7D9A;&#x3051;&#x3066;&#x3044;&#x308B;&#x3001;&#x591A;&#x304F;&#x306E;&#x65B0;&#x3057;&#x3044;&#x79D1;&#x5B66;&#x8005;&#x53C2;&#x5165;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x79C1;&#x9054;&#x306E;&#x77E5;&#x8B58;&#x30D9;&#x30FC;&#x30B9;&#x3068;&#x95A2;&#x9023;&#x3059;&#x308B;&#x65B0;&#x3057;&#x3044;&#x30C6;&#x30AF;&#x30CE;&#x30ED;&#x30B8;&#x3082;&#x62E1;&#x5927;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x7570;&#x306A;&#x308B;&#x751F;&#x7269;&#x306B;&#x304A;&#x3051;&#x308B;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x66F4;&#x65B0;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x69D8;&#x3005; &#x306A;&#x30EC;&#x30D3;&#x30E5;&#x30FC;&#x306F;&#x3053;&#x306E;&#x76EE;&#x7684;&#x306E;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x7BC4;&#x56F2;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x305D;&#x308C;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x3001;&#x7279;&#x306B;&#x591A;&#x7D30;&#x80DE;&#x771F;&#x6838;&#x751F;&#x7269;&#x306B;&#x304A;&#x3051;&#x308B;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x6E2C;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x53D7;&#x3051;&#x5165;&#x308C;&#x65B9;&#x6CD5;&#x306B;&#x95A2;&#x3059;&#x308B;&#x6DF7;&#x4E71;&#x7D9A;&#x3051;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x91CD;&#x8981;&#x306A;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x3092;&#x5F37;&#x8ABF;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x756A;&#x53F7;&#x307E;&#x305F;&#x306F;&#x30DC;&#x30EA;&#x30E5;&#x30FC;&#x30E0; autophagic &#x8981;&#x7D20; &#xFF08;&#x4F8B;&#x3048;&#x3070;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x307E;&#x305F;&#x306F; autolysosomes) &#x306E;&#x76E3;&#x8996;&#x6E2C;&#x5B9A;&#x306E;&#x9055;&#x3044;&#x3082;&#x306E;&#x5BFE; autophagic &#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x3069;&#x306E;&#x6BB5;&#x968E;&#x3067;&#x3082;&#x305D;&#x306E;&#x30E1;&#x30B8;&#x30E3;&#x30FC; &#x30D5;&#x30E9;&#x30C3;&#x30AF;&#x30B9; &#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x7D4C;&#x8DEF; (&#x3059;&#x306A;&#x308F;&#x3061;&#x3001;&#x5B8C;&#x4E86;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;); &#x3092;&#x4ECB;&#x3057;&#x3066;&#x3067;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x84C4;&#x7A4D;&#x306E;&#x7D50;&#x679C;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308B;&#x5897;&#x52A0;&#x306E; autophagic &#x6D3B;&#x52D5;&#x306E;&#x7D50;&#x679C;&#x523A;&#x6FC0;&#x304B;&#x3089;&#x533A;&#x5225;&#x3055;&#x308C;&#x308B;&#x305F;&#x3081;&#x306B;&#x3067;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x5897;&#x52A0;&#x306E;&#x914D;&#x4FE1;&#x3068;&#x52A3;&#x5316;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0; &#xFF08;&#x306E;&#x307B;&#x3068;&#x3093;&#x3069;&#x9AD8;&#x7B49;&#x771F;&#x6838;&#x751F;&#x7269;&#x3068;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x539F;&#x751F;&#x751F;&#x7269;&#x7D30;&#x80DE;&#x6027;&#x7C98;&#x83CC;&#x306A;&#x3069;&#xFF09; &#x307E;&#x305F;&#x306F; &#xFF08;&#x306E;&#x690D;&#x7269;&#x3068;&#x83CC;&#x985E;&#xFF09; &#x6DB2;&#x80DE;&#x5185;&#x7D50;&#x5408;&#x5897;&#x52A0;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x8A98;&#x5C0E;&#x3068;&#x3057;&#x3066;&#x3002;&#x4ED6;&#x306E;&#x8A00;&#x8449;&#x3067;&#x306F;&#x3001;&#x635C;&#x67FB;&#x5B98;&#x306F;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x306B;&#x65B0;&#x3057;&#x3044;&#x8A73;&#x7D30;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x5916;&#x89B3;&#x306F;&#x3001;&#x5FC5;&#x305A;&#x3057;&#x3082;&#x3088;&#x308A;&#x591A;&#x304F;&#x306E;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3068;&#x3092;&#x4E00;&#x81F4;&#x3057;&#x306A;&#x3044;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x7279;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x5B9F;&#x969B;&#x306B;&#x306F;&#x3001;autolysosomes &#x306E;&#x5897;&#x52A0;&#x5206;&#x89E3;&#x6D3B;&#x6027;&#x306E;&#x6E1B;&#x5C11;&#x3092;&#x53CD;&#x6620;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x306B;&#x5BFE;&#x3057;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0;&#x4F75;&#x7528;&#x5909;&#x5316;&#x306A;&#x3057;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x5F62;&#x6210;&#x306B;&#x304A;&#x3051;&#x308B;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3067;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x306E;&#x305F;&#x3081;&#x591A;&#x304F;&#x306E;&#x5834;&#x5408;&#x304C;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x84C4;&#x7A4D;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x4E00;&#x9023;&#x306E;&#x9078;&#x629E;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3068;&#x5229;&#x7528;&#x65B9;&#x6CD5;&#x306E;&#x89E3;&#x91C8;&#x3068;&#x95A2;&#x9023;&#x3059;&#x308B;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3092;&#x691C;&#x8A0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x76EE;&#x6307;&#x3057;&#x3066;&#x8ABF;&#x67FB;&#x5B98;&#x306B;&#x3088;&#x3063;&#x3066;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x6821;&#x95B2;&#x8005;&#x304C;&#x3053;&#x308C;&#x3089;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x7126;&#x70B9;&#x3092;&#x5F53;&#x3066;&#x3066;&#x3044;&#x308B;&#x66F8;&#x985E;&#x306E;&#x73FE;&#x5B9F;&#x7684;&#x304B;&#x3064;&#x5408;&#x7406;&#x7684;&#x306A;&#x6279;&#x5224;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308B;&#x305F;&#x3081;&#x672C;.&#x9069;&#x5207;&#x306A;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x4E00;&#x90E8;&#x3001;&#x8CEA;&#x554F;&#x3068;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x308B;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x308B;&#x305F;&#x3081;&#x306B;&#x5B9A;&#x578B;&#x7684;&#x306A;&#x4E00;&#x9023;&#x306E;&#x30EB;&#x30FC;&#x30EB;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x306F;&#x610F;&#x5473;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x3059;&#x3079;&#x3066;&#x306E;&#x72B6;&#x6CC1;&#x3067;&#x6700;&#x3082;&#x9069;&#x5207;&#x306A; 1 &#x3064;&#x306B;&#x500B;&#x3005; &#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x4FDD;&#x8A3C;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x304A;&#x3088;&#x3073;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x8907;&#x6570;&#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x4F7F;&#x7528;&#x3092;&#x304A;&#x52E7;&#x3081;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3067;&#x306F;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3068;&#x3069;&#x306E;&#x3088;&#x3046;&#x306A;&#x60C5;&#x5831;&#x3092;&#x3067;&#x304D;&#x308B;&#x3001;&#x8A55;&#x4FA1;&#x306E;&#x3053;&#x308C;&#x3089;&#x306E;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x65B9;&#x6CD5;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x307E;&#x305F;&#x306F;&#x305D;&#x308C;&#x3089;&#x304B;&#x3089;&#x53D6;&#x5F97;&#x3059;&#x308B;&#x3053;&#x3068;&#x306F;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x30E1;&#x30EA;&#x30C3;&#x30C8;&#x3068;&#x7279;&#x5B9A;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x9650;&#x754C;&#x3092;&#x8AD6;&#x8B70;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x3067;&#x306E;&#x6280;&#x8853;&#x9769;&#x65B0;&#x3092;&#x5968;&#x52B1;&#x3059;&#x308B;&#x9858;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x4F7F;&#x7528;&#x3068;&#x89E3;&#x91C8;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3002;

2008 &#xB144; &#xC6B0;&#xB9AC; &#xCD9C;&#xD310; &#xC5F0;&#xAD6C; autophagy&#xC758; &#xD45C;&#xC900;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;&#xC758; &#xCCAB; &#xC138;&#xD2B8;. &#xADF8; &#xC774;&#xD6C4;&#xB85C;, &#xAC00;&#xC18D;,&#xC774; &#xC8FC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC5F0;&#xAD6C;&#xB294; &#xACC4;&#xC18D; &#xD558; &#xACE0; &#xB9CE;&#xC740; &#xC0C8;&#xB85C;&#xC6B4; &#xACFC;&#xD559;&#xC790; &#xD544;&#xB4DC;&#xB97C; &#xC785;&#xB825; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xAE30;&#xC220; &#xC790;&#xB8CC; &#xBC0F; &#xAD00;&#xB828; &#xC2E0;&#xAE30;&#xC220;&#xB3C4; &#xD655;&#xB300; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xB530;&#xB77C;, &#xADF8;&#xAC83;&#xC740; &#xB2E4;&#xB978; &#xC720;&#xAE30; &#xCCB4;&#xC5D0;&#xC11C; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;&#xC5D0; &#xB300; &#xD55C;&#xC774; &#xC9C0;&#xCE68;&#xC744; &#xC5C5;&#xB370;&#xC774;&#xD2B8; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC911;&#xC694;&#xC785;&#xB2C8;&#xB2E4;. &#xB2E4;&#xC591; &#xD55C; &#xB9AC;&#xBDF0;&#xB294;&#xC774; &#xBAA9;&#xC801;&#xC744; &#xC704;&#xD574; &#xC0AC;&#xC6A9; &#xB41C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xBC94;&#xC704;&#xB97C; &#xC124;&#xBA85; &#xD588;&#xB2E4;. &#xADF8;&#xB7FC;&#xC5D0;&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0;, &#xAC70;&#xAE30;&#xC5D0; &#xD5C8;&#xC6A9; &#xD2B9;&#xD788; &#xB2E4;&#xC138;&#xD3EC; &#xC9C4;&#xD575;&#xC0DD;&#xBB3C;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; autophagy &#xCE21;&#xC815; &#xBC29;&#xBC95;&#xC5D0; &#xAD00;&#xD55C; &#xD63C;&#xB780; &#xB418; &#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xAC15;&#xC870; &#xD558;&#xB294; &#xD575;&#xC2EC; &#xD3EC;&#xC778;&#xD2B8;&#xAC00;&#xC785;&#xB2C8;&#xB2E4; &#xC22B;&#xC790; &#xB610;&#xB294; autophagic &#xC694;&#xC18C; (&#xC608;: autophagosomes &#xB610;&#xB294; autolysosomes)&#xC758; &#xBCFC;&#xB968;&#xC744; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xCE21;&#xC815;&#xC758; &#xCC28;&#xC774; &#xADF8; &#xB300; autophagic &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xBAA8;&#xB4E0; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; autophagy &#xD1B5;&#xB85C; (&#xC989;, &#xC804;&#xCCB4; &#xD504;&#xB85C;&#xC138;&#xC2A4;);&#xB97C; &#xD1B5;&#xD574; &#xADF8; &#xCE21;&#xC815; &#xC720;&#xB7C9; &#xB530;&#xB77C;&#xC11C;, &#xACB0;&#xACFC;: autophagosome &#xCD95;&#xC801; &#xC99D;&#xAC00; autophagic &#xD65C;&#xB3D9;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC790;&#xADF9;&#xC5D0;&#xC11C; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; macroautophagy&#xC5D0;&#xC11C; &#xBE14;&#xB85D; &#xC99D;&#xAC00; autophagy &#xC720;&#xB3C4;, &#xC99D;&#xAC00; &#xC804;&#xB2EC;&#xACFC; &#xB9AC;&#xC18C;&#xC880; (&#xB300;&#xBD80;&#xBD84;&#xC758; &#xB192;&#xC740; &#xC9C4;&#xD575;&#xC0DD;&#xBB3C;&#xC5D0; Dictyostelium &#xAC19;&#xC740; &#xC77C;&#xBD80; protists) &#xB610;&#xB294; (&#xC2DD;&#xBB3C;&#xACFC; &#xACF0; &#xD321;&#xC774;)&#xC5D0; &#xACF5;&#xD3EC; &#xB0B4; &#xC800;&#xD558;&#xC640; &#xACB0;&#xD569;&#xC73C;&#xB85C; &#xC815;&#xC758; &#xB429;&#xB2C8;&#xB2E4;. &#xB2E4;&#xB978; &#xB9D0;&#xB85C; &#xD558;&#xBA74;, &#xADF8;&#xAC83;&#xC740; &#xD2B9;&#xD788; &#xC911;&#xC694; &#xD55C; &#xC218;&#xC0AC; &#xBD84;&#xC57C;&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xB354; &#xB9CE;&#xC740; autophagosomes&#xC758; &#xC678;&#xAD00;&#xC744; &#xBC18;&#xB4DC;&#xC2DC; &#xB354; autophagy&#xC640; &#xB3D9;&#xC77C;&#xC2DC; &#xD558;&#xC9C0; &#xC54A;&#xB294;&#xB2E4; &#xC774;&#xD574;. &#xC0AC;&#xC2E4;, &#xB300;&#xBD80;&#xBD84;&#xC758; &#xACBD;&#xC6B0;&#xC5D0;&#xC11C; autophagosomes &#xCD95;&#xC801; autophagosome &#xC18D;&#xC5D0; &#xC218; &#xBC18;&#xD558;&#xB294; &#xBCC0;&#xD654; &#xC5C6;&#xC774; &#xB9AC;&#xC18C;&#xC880;&#xC744; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xBE14;&#xB85D; &#xB54C;&#xBB38;&#xC5D0; &#xBC18;&#xBA74;&#xC5D0; autolysosomes &#xC99D;&#xAC00; degradative &#xD65C;&#xB3D9;&#xC758; &#xAC10;&#xC18C;&#xB97C; &#xBC18;&#xC601; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xD604;&#xC7AC; &#xC120;&#xD0DD; &#xC601;&#xC5ED;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;&#xC758; &#xC124;&#xC815; &#xBC0F; &#xC0AC;&#xC6A9; &#xBC29;&#xBC95;&#xC758; &#xD574;&#xC11D; &#xC774;&#xB7EC;&#xD55C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xC9D1;&#xC911; &#xB418;&#xB294; &#xB17C;&#xBB38;&#xC758; &#xD604;&#xC2E4;&#xC801;&#xC774; &#xACE0; &#xD569;&#xB9AC;&#xC801;&#xC778; &#xBE44;&#xD310;&#xC744; &#xC81C;&#xACF5; &#xD574;&#xC57C; &#xD558;&#xB294; &#xAC80;&#xD1A0;&#xC790;&#xC5D0; &#xB300; &#xD55C; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; macroautophagy &#xBC0F; &#xAD00;&#xB828;&#xB41C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; &#xAC80;&#xC0AC; &#xD558;&#xB294; &#xAC83;&#xC744; &#xBAA9;&#xD45C;&#xB85C; &#xD558;&#xB294; &#xC870;&#xC0AC;&#xC5D0; &#xC758;&#xD574;. &#xC801;&#xC808; &#xD55C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8; &#xBD80;&#xBD84;&#xC5D0;&#xC11C; &#xC694;&#xCCAD;&#xC744; &#xBC1B;&#xACE0; &#xC9C8;&#xBB38;&#xC744; &#xD558; &#xACE0; &#xC0AC;&#xC6A9; &#xC911;&#xC778; &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0; &#xC758;&#xC874; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xC774;&#xB7EC;&#xD55C; &#xC9C0;&#xCE68; &#xADDC;&#xCE59;, formulaic &#xC9D1;&#xD569;&#xC73C;&#xB85C; &#xC758;&#xBBF8;&#xAC00; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xC544;&#xBB34; &#xAC1C;&#xBCC4; &#xBD84;&#xC11D; &#xACB0;&#xACFC;, &#xBAA8;&#xB4E0; &#xC0C1;&#xD669;&#xC5D0;&#xC11C; &#xAC00;&#xC7A5; &#xC801;&#xD569; &#xD55C; &#xBCF4;&#xC7A5; &#xD558; &#xACE0; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;&#xD560; &#xC5EC;&#xB7EC; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xC0AC;&#xC6A9;&#xC744; &#xAC15;&#xC870;. &#xC774;&#xB7EC;&#xD55C; &#xC9C0;&#xCE68;&#xC5D0; &#xC6B0;&#xB9AC; autophagy&#xC640; &#xC815;&#xBCF4; &#xC218; &#xC788;&#xB294; &#xD3C9;&#xAC00;&#xC758; &#xC774;&#xB7EC;&#xD55C; &#xB2E4;&#xC591; &#xD55C; &#xBC29;&#xBC95;&#xC744; &#xACE0;&#xB824; &#xADF8;&#xB4E4; &#xB85C;&#xBD80;&#xD130; &#xC5BB;&#xC744; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;, &#xB610;&#xB294;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC7A5;&#xC810; &#xBC0F; &#xD2B9;&#xC815; autophagy &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xD55C;&#xACC4;&#xB97C; &#xB17C;&#xC758; &#xD558; &#xC5EC; &#xD604;&#xC7A5;&#xC5D0;&#xC11C; &#xAE30;&#xC220; &#xD601;&#xC2E0;&#xC744; &#xC7A5;&#xB824; &#xD558;&#xACA0;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC0AC;&#xC6A9; &#xBC0F; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xD574;&#xC11D;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;.

En 2008 public&oacute; el primer conjunto de pautas para estandarizar la investigaci&oacute;n en la autofagia. Desde entonces, ha continuado la investigaci&oacute;n sobre este tema acelerar, y muchos nuevos cient&iacute;ficos han entrado en el campo. Tambi&eacute;n han estado ampliando nuestra base de conocimientos y tecnolog&iacute;as relevantes. Por consiguiente, es importante actualizar estas directrices para la supervisi&oacute;n de autofagia en diferentes organismos. Varios comentarios han descrito la gama de ensayos que se han utilizado para este prop&oacute;sito. Sin embargo, contin&uacute;an confusi&oacute;n con respecto a los m&eacute;todos aceptables para medir la autofagia, especialmente en eucariotas pluricelulares. Un punto clave que debe destacarse es que hay una diferencia entre las medidas que monitorean los n&uacute;meros o volumen de autofagia elementos (por ejemplo, autophagosomes o autolysosomes) en cualquier etapa del proceso de autofagia vs los ese flujo medida a trav&eacute;s de la v&iacute;a de la autofagia (es decir, el proceso completo); as&iacute;, un bloque en macroautophagy que resulta en acumulaci&oacute;n de autophagosome debe ser distinguido de est&iacute;mulos que resultan en un aumento de la actividad autofagia, definido como inducci&oacute;n autofagia creciente juntada con mayor entrega y degradaci&oacute;n dentro, lisosomas (en la mayor&iacute;a de eucariotas superiores y algunos protistas como Dictyostelium) o la vacuola (en plantas y hongos). En otras palabras, es especialmente importante que los investigadores de nuevos al campo entiendan que la aparici&oacute;n de m&aacute;s autophagosomes no corresponde necesariamente con autofagia m&aacute;s. De hecho, en muchos casos, autophagosomes se acumulan debido a un bloque en el tr&aacute;fico a los lisosomas sin un cambio concomitante en la biog&eacute;nesis de autophagosome, mientras que un aumento en la autolysosomes puede reflejar una reducci&oacute;n en la actividad de degradaci&oacute;n. Aqu&iacute;, presentamos un conjunto de directrices para la selecci&oacute;n e interpretaci&oacute;n de los m&eacute;todos para su uso por los investigadores que tienen como objetivo examinar macroautophagy y procesos relacionados, as&iacute; como para los revisores que deban proporcionar cr&iacute;ticas realistas y razonables de papeles que se centran en estos procesos. Estas directrices no pretenden ser un formulista conjunto de reglas, porque los ensayos adecuados dependen en parte de la pregunta y el sistema utilizado. Adem&aacute;s, hacemos hincapi&eacute; en que ning&uacute;n ensayo individual est&aacute; garantizada para ser el m&aacute;s adecuado en cada situaci&oacute;n, y le recomendamos el uso de m&uacute;ltiples ensayos para monitorear la autofagia. En estas directrices, consideramos estos diversos m&eacute;todos de evaluaci&oacute;n de autofagia y qu&eacute; informaci&oacute;n puede, o no puede obtenerse de ellos. Finalmente, discutiendo los m&eacute;ritos y los l&iacute;mites de an&aacute;lisis particular de la autofagia, queremos fomentar la innovaci&oacute;n t&eacute;cnica en el campo.

Directrices para el uso e interpretaci&oacute;n de ensayos para el control de la autofagia.

The bacterial RNA polymerase holoenzyme consists of a catalytic core enzyme in complex with a σ factor that is required for promoter-specific transcription initiation. Primary, or housekeeping, σ factors are responsible for most of the gene expression that occurs during the exponential phase of growth. Primary σ factors share four regions of conserved sequence, regions 1-4, which have been further subdivided. Many primary σ factors also contain a nonconserved region (NCR) located between subregions 1.2 and 2.1, which can vary widely in length. Interactions between the NCR of the primary σ factor of Escherichia coli, σ(70), and the β' subunit of the E. coli core enzyme have been shown to influence gene expression, suggesting that the NCR of primary σ factors represents a potential target for transcription regulation. Here, we report the identification and characterization of a previously undocumented Chlamydia trachomatis transcription factor, designated GrgA (general regulator of genes A). We demonstrate in vitro that GrgA is a DNA-binding protein that can stimulate transcription from a range of σ(66)-dependent promoters. We further show that GrgA activates transcription by contacting the NCR of the primary σ factor of C. trachomatis, σ(66). Our findings suggest GrgA serves as an important regulator of σ(66)-dependent transcription in C. trachomatis. Furthermore, because GrgA is present only in chlamydiae, our findings highlight how nonconserved regions of the bacterial RNA polymerase can be targets of regulatory factors that are unique to particular organisms.

Chlamydia trachomatis protein GrgA activates transcription by contacting the nonconserved region of σ66.

Chlamydia is a common bacterial pathogen responsible for many diseases. Methods for transforming this important organism using a β-lactamase as a selection marker have been developed very recently. However, the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules do not permit transformation experiments with β-lactamase gene-containing vectors for certain human chlamydial pathogens. Therefore, a different selection marker is urgently needed for transformation of those chlamydiae.

Chloramphenicol acetyltransferase as a selection marker for chlamydial transformation.

Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value. 

Benzylidene acylhydrazides inhibit chlamydial growth in a type III secretion- and iron chelation-independent manner.

Lactobacillus species dominate the microbiome in the lower genital tract of most reproductive-age women. Producing lactic acid and H2O2, lactobacilli are believed to play an important role in prevention of colonization by and growth of pathogens. However, to date, there have been no reported studies characterizing how lactobacilli interact with Chlamydia trachomatis, a leading sexually transmitted bacterium. In this report, we demonstrate inactivation of C. trachomatis infectivity by culture media conditioned by Lactobacillus crispatus, L. gasseri and L. jensenii, known to be dominating organisms in the human vaginal microbiome. Lactobacillus still cultures produced lactic acid, leading to time- and concentration-dependent killing of C. trachomatis. Neutralization of the acidic media completely reversed chlamydia killing. Addition of lactic acid into Lactobacillus-unconditioned growth medium recapitulated the chlamydiacidal activity of conditioned media. The H2O2 concentrations in the still cultures were found to be comparable to those reported for the cervicovaginal fluid, but insufficient to inactivate chlamydiae. Aeration of Lactobacillus cultures by shaking markedly induced H2O2 production, but strongly inhibited Lactobacillus growth and lactic acid production, and thus severely affected acidification, leading to significantly reduced chlamydiacidal efficiency. These observations indicate lactobacilli inactivate chlamydiae primarily through maintaining acidity in a relatively hypoxic environment in the vaginal lumen with limited H2O2, which is consistent with the notion that women with higher vaginal pH are more prone to sexually transmitted C. trachomatis infection. In addition to lactic acid, formic acid and acetic acid also exhibited potent chlamydiacidal activities. Taken together, our findings imply that lowering the vaginal pH through engineering of the vaginal microbiome and other means will make women less susceptible to C. trachomatis infection. 

Lactobacilli inactivate Chlamydia trachomatis through lactic acid but not H2O2.

Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. 

Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

Chlamydiae are widely distributed pathogens of human populations, which can lead to serious reproductive and other health problems. In our search for novel antichlamydial metabolites from marine derived-microorganisms, one new () and two known () dimeric indole derivatives were isolated from the sponge-derived actinomycete . The chemical structures of these metabolites were elucidated by NMR spectroscopic data as well as CD calculations. All three metabolites suppressed chlamydial growth in a concentration-dependent manner. Among them, compound  exhibited the most effective antichlamydial activity with IC values of 46.6 ~ 96.4 µM in the production of infectious progeny. Compounds appeared to target the mid-stage of the chlamydial developmental cycle by interfering with reticular body replication, but not directly inactivating the infectious elementary body.

Antichlamydial Dimeric Indole Derivatives from Marine Actinomycete Rubrobacter radiotolerans.

The First Chinese Chlamydia Research Meeting was held in Lanzhou, China in May 2017, 60 years after the disclosure of reproducible isolation of Chlamydia trachomatis by (Fei-fan Tang). We report current state of the Chlamydia research community in China, and briefly review recent progress in Chlamydia vaccinology. The meeting represents a new milestone for Chlamydia research in the country. The Chinese Chlamydia Research Society (CCRS) was formed during the meeting. Future meetings will be held biennially and should facilitate collaboration of Chinese researchers with their domestic and international colleagues.

2017: beginning of a new era for Chlamydia research in China and the rest of the world.

The obligate intracellular bacterium  is a widespread human pathogen that causes serious problems, including (but not limited to) infertility and blindness. Our search for novel antichlamydial metabolites from marine-derived microorganisms led to the isolation of pyocyanin, a small compound from  Pyocyanin is an effective antichlamydial for all three  spp. tested. It has a 50% inhibitory concentration (IC) of 0.019 to 0.028 μM, which is comparable to the IC of tetracycline. At concentrations as low as 0.0039 μM, pyocyanin disables infectivity of the chlamydial elementary body (EB). At 0.5 μM or higher concentrations, the continuous presence of pyocyanin also inhibits chlamydial growth in the inclusion during later stages of the developmental cycle. Oxidative stress, a major known antimicrobial mechanism of pyocyanin, appears to be responsible only for the inhibition of bacterial growth and not for the disinfection of EBs. Pyocyanin is well-tolerated by probiotic vaginal  spp. Our findings suggest that pyocyanin is of therapeutic value for chlamydial infections and can serve as a valuable chemical probe for studying chlamydial biology.

Pyocyanin Inhibits Chlamydia Infection by Disabling Infectivity of the Elementary Body and Disrupting Intracellular Growth.

The obligate intracellular bacterial pathogen  has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary  sigma factor, σ, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor, σ In prior work, we identified GrgA as a specific transcription factor that activates σ-dependent transcription by binding DNA and interacting with a nonconserved region (NCR) of σ Here, we extend these findings by showing GrgA can also activate σ-dependent transcription through direct interaction with σ We measure the binding affinity of GrgA for both σ and σ, and we identify regions of GrgA important for σ-dependent transcription. Similar to results obtained with σ, we find that GrgA's interaction with σ involves an NCR located upstream of conserved region 2 of σ Our findings suggest that GrgA is an important regulator of both σ- and σ-dependent transcription in  and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms. is the number one sexually transmitted bacterial pathogen worldwide. A substantial proportion of -infected women develop infertility, pelvic inflammatory syndrome, and other serious complications.  is also a leading infectious cause of blindness in underdeveloped countries. The pathogen has a unique developmental cycle that is transcriptionally regulated. The discovery of an expanded role for the specific transcription factor GrgA helps us understand the progression of the chlamydial developmental cycle.

Role for GrgA in Regulation of σ-Dependent Transcription in the Obligate Intracellular Bacterial Pathogen Chlamydia trachomatis.

Chlamydia is a common pathogen that can causes serious complications in the reproductive system and eyes. Lack of vaccine and other effective prophylactic measures coupled with the largely asymptomatic nature and unrare clinical treatment failure calls for development of new antichlamydials, particularly selective antichlamydials without adverse effects on humans and the beneficial microbiota. We previously reported that benzal-N-acylhydrazones (BAH) can inhibit chlamydiae without detectable adverse effects on host cells and beneficial lactobacilli that dominate the human vaginal microbiota among reproductive-age women. However, the antichlamydial mechanism of BAH is not known. Whereas 4 single nucleotide polymorphisms (i.e., SNP1-4) were identified in a rare Chlamydia variant with a low level of BAH resistance, termed MCR, previous studies failed to establish a causal effect of any particular SNP(s). In the present work, we performed recombination to segregate the four SNPs. Susceptibility tests indicate that the R51G GrgA allele is both necessary and sufficient for the low level of BAH resistance. Thus, the Chlamydia-specific transcription factor GrgA either is a direct target of BAH or regulates BAH susceptibility. We further confirm an extremely low rate of BAH resistance in Chlamydia. Our findings warrant exploration of GrgA as a therapeutic and prophylactic target for chlamydial infections.

Huizhou Fan

Department of Pharmacology,
Robert Wood Johnson Medical School,
Graduate Program in Physiology and Integrative Biology,
School of Graduate Studies,
Department of Pharmacology, Robert Wood Johnson Medical School,
Graduate Program in Physiology and Integrative Biology, School of Graduate Studies

Rutgers University

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription