cryoem-status-update-and-innovative-developments

CryoEM Status Update and Innovative Developments

ARTICLES DISCUSSED:
Truong, C. D. et al. Sample preparation using a lipid monolayer method for electron crystallographic studies. Journal of Visualized ...

Massachusetts Institute of Technology

&#x65B0;&#x578B;&#x751F;&#x7269;&#x53EF;&#x964D;&#x89E3;&#x805A; (&#x916F;&#x9170;&#x80FA;) &#x5F39;&#x6027;&#x4F53;&#x7684;&#x5BB6;&#x5EAD;&#x3002;

A novel family of biodegradable poly(ester amide) elastomers.

Une nouvelle famille d&#39;&eacute;lastom&egrave;res biod&eacute;gradables poly (amide ester).

Eine neuartige Familie von biologisch abbaubaren Poly (Ester Amid) Elastomere.

Una famiglia romanzo di elastomeri biodegradabile poly (ammide estere).

&#x751F;&#x5206;&#x89E3;&#x6027;&#x30DD;&#x30EA; &#xFF08;&#x30A2;&#x30DF;&#x30C9; &#x30A8;&#x30B9;&#x30C6;&#x30EB;&#xFF09; &#x30A8;&#x30E9;&#x30B9;&#x30C8;&#x30DE;&#x30FC;&#x306E;&#x65B0;&#x898F;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x3002;

&#xC0DD; &#xBD84;&#xD574;&#xC131; &#xD3F4; &#xB9AC; (&#xC5D0;&#xC2A4;&#xD14C; &#xB974; &#xC544; &#xBBF8;&#xB4DC;) &#xD0C4;&#xC131; &#xCCB4;&#xC758; &#xC0C8;&#xB85C;&#xC6B4; &#xAC00;&#xC871;.

Nueva familia de elast&oacute;meros de polietileno biodegradable (Amida ester).

Gold-Nanopartikel haben weithin in der wissenschaftlichen Forschung aufgrund ihrer einzigartigen physikalischen und chemischen Eigenschaften verwendet wird. In den letzten Jahren ist ihre Verwendung als SiRNA Delivery Agents untersucht worden. Hier Fortschritte mit Goldene-Nanopartikeln f&uuml;r SiRNA Lieferung wird beschrieben und die verschiedenen Strategien werden verglichen.

F&uuml;nf Jahre SiRNA Lieferumfang: Scheinwerfer am gold-Nanopartikeln.

Nanoparticelle d&#39;oro sono diventato ampiamente utilizzate nella ricerca scientifica a causa della loro propriet&agrave; fisiche e chimiche uniche. Negli ultimi anni &egrave; stato studiato il loro uso come agenti di consegna di siRNA. Qui, progressi usando nanoparticelle d&#39;oro per la consegna di siRNA viene descritto e vengono confrontate le diverse strategie impiegate.

Cinque anni di siRNA consegna: riflettori su nanoparticelle d&#39;oro.

&#x91D1;&#x7EB3;&#x7C73;&#x7C92;&#x5B50;&#x5DF2;&#x6210;&#x4E3A;&#x5E7F;&#x6CDB;&#x5E94;&#x7528;&#x4E8E;&#x79D1;&#x5B66;&#x7814;&#x7A76;&#xFF0C;&#x7531;&#x4E8E;&#x5176;&#x72EC;&#x7279;&#x7684;&#x7269;&#x7406;&#x548C;&#x5316;&#x5B66;&#x7279;&#x6027;&#x3002;&#x5728;&#x8FC7;&#x53BB;&#x7684;&#x51E0;&#x5E74;&#x4E2D;&#x4ED6;&#x4EEC;&#x5229;&#x7528; siRNA &#x4EA4;&#x4ED8;&#x4EE3;&#x7406;&#x5546;&#x4F5C;&#x4E3A;&#x5DF2;&#x8FDB;&#x884C;&#x8C03;&#x67E5;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;siRNA &#x4EA4;&#x4ED8;&#x4F7F;&#x7528;&#x91D1;&#x7EB3;&#x7C73;&#x7C92;&#x5B50;&#x7684;&#x8FDB;&#x5C55;&#x63CF;&#x8FF0;&#x548C;&#x91C7;&#x7528;&#x7684;&#x4E0D;&#x540C;&#x6218;&#x7565;&#x8FDB;&#x884C;&#x6BD4;&#x8F83;&#x3002;

&#x4E94;&#x5E74;&#x7684; siRNA &#x4EA4;&#x4ED8;&#xFF1A; &#x805A;&#x7126;&#x4E8E;&#x91D1;&#x7EB3;&#x7C73;&#x7C92;&#x5B50;&#x3002;

Gold nanoparticles have become widely used in scientific research due to their unique physical and chemical properties. In the last several years their use as siRNA delivery agents has been investigated. Here, progress made using gold nanoparticles for siRNA delivery is described and the different strategies employed are compared.

Five years of siRNA delivery: spotlight on gold nanoparticles.

Nanoparticules d&#39;or ont devenu largement utilis&eacute;s dans la recherche scientifique en raison de leurs propri&eacute;t&eacute;s physiques et chimiques uniques. Dans les derni&egrave;res ann&eacute;es, leur utilisation comme agents d&#39;ex&eacute;cution de siARN a &eacute;t&eacute; &eacute;tudi&eacute;e. Ici, on d&eacute;crit les progr&egrave;s r&eacute;alis&eacute;s en utilisant des nanoparticules d&#39;or pour ARNsi et les diff&eacute;rentes strat&eacute;gies employ&eacute;es sont compar&eacute;s.

Cinq ann&eacute;es d&#39;ARNsi : pleins feux sur les nanoparticules d&#39;or.

&#x91D1;&#x30CA;&#x30CE;&#x7C92;&#x5B50;&#x306F;&#x3001;&#x305D;&#x306E;&#x72EC;&#x7279;&#x306E;&#x7269;&#x7406;&#x7684;&#x30FB;&#x5316;&#x5B66;&#x7684;&#x7279;&#x6027;&#x306B;&#x8D77;&#x56E0;&#x306E;&#x79D1;&#x5B66;&#x7684;&#x7814;&#x7A76;&#x3067;&#x5E83;&#x304F;&#x3055;&#x308C;&#x3066;&#x304D;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;SiRNA &#x914D;&#x4FE1;&#x30A8;&#x30FC;&#x30B8;&#x30A7;&#x30F3;&#x30C8;&#x3068;&#x3057;&#x3066;&#x306E;&#x5229;&#x7528;&#x3001;&#x6700;&#x5F8C;&#x306E;&#x6570;&#x5E74;&#x9593;&#x3067;&#x884C;&#x308F;&#x308C;&#x3066;&#x304D;&#x305F;.&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;siRNA &#x30C7;&#x30EA;&#x30D0;&#x30EA;&#x30FC;&#x306E;&#x91D1;&#x30CA;&#x30CE;&#x7C92;&#x5B50;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x9032;&#x884C;&#x72B6;&#x6CC1;&#x3092;&#x8AAC;&#x660E;&#x3057;&#x3001;&#x7570;&#x306A;&#x308B;&#x6226;&#x7565;&#x3092;&#x63A1;&#x7528;&#x304C;&#x6BD4;&#x8F03;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;

SiRNA &#x30C7;&#x30EA;&#x30D0;&#x30EA;&#x30FC;&#x306E; 5 &#x5E74;&#x9593;&#xFF1A; &#x91D1;&#x30CA;&#x30CE;&#x7C92;&#x5B50;&#x4E0A;&#x306E;&#x30B9;&#x30DD;&#x30C3;&#x30C8; &#x30E9;&#x30A4;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;

&#xAE08; &#xB098;&#xB178; &#xC785;&#xC790; &#xADF8;&#xB4E4;&#xC758; &#xB3C5;&#xD2B9;&#xD55C; &#xBB3C;&#xB9AC;&#xC801;, &#xD654;&#xD559;&#xC801; &#xD2B9;&#xC131; &#xB54C;&#xBB38;&#xC5D0; &#xACFC;&#xD559; &#xC5F0;&#xAD6C;&#xC5D0; &#xB110;&#xB9AC; &#xC0AC;&#xC6A9; &#xB418;&#xB294;. &#xC9C0;&#xB09C; &#xBA87; &#xB144; &#xB3D9;&#xC548;&#xC5D0; siRNA &#xC804;&#xB2EC; &#xC5D0;&#xC774;&#xC804;&#xD2B8;&#xB85C; &#xC0AC;&#xC6A9; &#xC870;&#xC0AC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, siRNA &#xB0A9;&#xD488;&#xC744; &#xC704;&#xD55C; &#xAE08; &#xB098;&#xB178; &#xC785;&#xC790;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB9CC;&#xB4E0; &#xC9C4;&#xD589;&#xB960; &#xC124;&#xBA85; &#xBC0F; &#xBE44;&#xAD50; &#xD558;&#xB294; &#xACE0;&#xC6A9; &#xB2E4;&#xC591; &#xD55C; &#xC804;&#xB7B5;.

SiRNA &#xC804;&#xB2EC;&#xC758; 5 &#xB144;: &#xAE08; &#xB098;&#xB178; &#xC785;&#xC790;&#xC5D0; &#xC2A4;&#xD3EC;&#xD2B8; &#xB77C;&#xC774;&#xD2B8;.

Nanopart&iacute;culas de oro han vuelto ampliamente utilizadas en la investigaci&oacute;n cient&iacute;fica debido a sus propiedades f&iacute;sicas y qu&iacute;micas &uacute;nicas. En los &uacute;ltimos a&ntilde;os se ha investigado su uso como agentes de entrega de siRNA. Aqu&iacute; se describen los progresos realizados con nanopart&iacute;culas de oro para la entrega de siRNA y se comparan las diferentes estrategias empleadas.

Cinco a&ntilde;os de siRNA entrega: foco en nanopart&iacute;culas de oro.

&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; (hESCs) &#x4F5C;&#x4E3A;&#x4E00;&#x79CD;&#x8D44;&#x6E90;&#x518D;&#x751F;&#x533B;&#x5B66;&#x7684;&#x5DE8;&#x5927;&#x6F5C;&#x529B;&#x3002;&#x4E4B;&#x524D;&#x5B9E;&#x73B0;&#x6CBB;&#x7597;&#x7684;&#x76F8;&#x5173;&#x6027;&#xFF0C;&#x5FC5;&#x987B;&#x5236;&#x8BA2;&#x65B9;&#x6CD5;&#xFF0C;&#x63A7;&#x5236;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x3002;&#x5F88;&#x663E;&#x7136;&#x5E72;&#x7EC6;&#x80DE;&#x53EF;&#x4EE5;&#x56DE;&#x5E94;&#x9057;&#x4F20;&#x4FE1;&#x53F7;&#xFF0C;&#x5982;&#x90A3;&#x4E9B;&#x4F20;&#x6388;&#x7684;&#x6838;&#x9178;&#xFF0C;&#x4EE5;&#x4FC3;&#x8FDB;&#x7279;&#x5B9A;&#x6CBF;&#x88AD;&#x7684;&#x5206;&#x5316;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x5F00;&#x53D1;&#x4E86; siRNA &#x9001; hESCs 3D &#x6587;&#x5316;&#x77E9;&#x9635;&#x4F7F;&#x7528;&#x8102;&#x72B6;&#x6750;&#x6599;&#x4E2D;&#x6709;&#x6548;&#x7684;&#x7CFB;&#x7EDF;&#x3002;&#x6211;&#x4EEC;&#x5728; 3D &#x652F;&#x67B6;&#x53EF;&#x4EE5;&#x6709;&#x6548;&#x5730;&#x51FB;&#x5012; 90%GFP hESCs &#x7EFF;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x663E;&#x793A;&#xFF0C;&#x975E;&#x75C5;&#x6BD2; siRNA &#x4EA4;&#x4ED8;&#x3002;&#x6211;&#x4EEC;&#x8FDB;&#x4E00;&#x6B65;&#x663E;&#x793A;&#x6B64;&#x7CFB;&#x7EDF;&#x53EF;&#x4EE5;&#x7528;&#x4F5C;&#x4E00;&#x4E2A;&#x5E73;&#x53F0;&#xFF0C;&#x7528;&#x4E8E;&#x6307;&#x5BFC; hESC &#x5206;&#x5316;&#x3002;&#x901A;&#x8FC7; siRNA &#x6C89;&#x9ED8; KDR &#x53D7;&#x4F53;&#x57FA;&#x56E0;&#x7684;&#x6211;&#x4EEC;&#x5728;&#x57FA;&#x56E0;&#x4EE3;&#x8868;&#x7684;&#x5185;&#x80DA;&#x5C42;&#x80DA;&#x5C42;&#x548C;&#x4E2D;&#x80DA;&#x5C42;&#x80DA;&#x5C42; (27-90 &#x500D;&#xFF09; &#x57FA;&#x56E0;&#x4EE3;&#x8868;&#x91CD;&#x5927;&#x4E0A;&#x8C03;&#x5B9E;&#x73B0;&#x5E76;&#x53D1;&#x4E0B;&#x8C03; (60-90%)&#x3002;&#x8FD9;&#x8868;&#x660E;&#x8BE5; siRNA &#x53EF;&#x4EE5;&#x76F4;&#x63A5;&#x901A;&#x8FC7;&#x963B;&#x6B62;&#x57FA;&#x56E0;&#x4EE3;&#x8868;&#x4E00;&#x4E2A;&#x80DA;&#x5C42;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x548C;&#x8FD8;&#x63D0;&#x4F9B;&#x4E86;&#x9694;&#x79BB;&#x5185;&#x80DA;&#x5C42;&#x80DA;&#x5C42;&#x4E2D;&#x80DA;&#x5C42;&#x548C;&#x5916;&#x80DA;&#x5C42;&#x7684;&#x7279;&#x522B;&#x6709;&#x529B;&#x624B;&#x6BB5;&#x3002;&#x8FD9;&#x79CD;&#x80FD;&#x529B;&#x6765;&#x6291;&#x5236;&#x5185;&#x80DA;&#x5C42;&#x80DA;&#x5C42;&#x5206;&#x5316;&#x53EF;&#x80FD;&#x5141;&#x8BB8;&#x6539;&#x8FDB;&#x63A7;&#x5236; hESC &#x5B9A;&#x5411;&#x8BF1;&#x5BFC;&#x5206;&#x5316;&#x4E3A;&#x6240;&#x9700;&#x7684;&#x5355;&#x5143;&#x683C;&#x7C7B;&#x578B;&#x3002;

&#x5728; 3D &#x6587;&#x5316; siRNA &#x975E;&#x75C5;&#x6BD2;&#x6027;&#x4EA4;&#x4ED8;&#x7684;&#x6307;&#x5BFC;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x3002;

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.

Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture.

Les cellules souches embryonnaires humaines (CSEh) rec&egrave;lent un potentiel grand comme une ressource pour la m&eacute;decine r&eacute;g&eacute;n&eacute;rative. Avant d&#39;atteindre la pertinence th&eacute;rapeutique, les m&eacute;thodes doivent &ecirc;tre &eacute;labor&eacute;s pour contr&ocirc;ler la diff&eacute;renciation des cellules souches. Il est clair que les cellules souches peuvent r&eacute;pondre aux signaux g&eacute;n&eacute;tiques, telles que celles transmises par les acides nucl&eacute;iques, pour promouvoir la diff&eacute;renciation de la lign&eacute;e sp&eacute;cifique. Ici, nous avons d&eacute;velopp&eacute; un syst&egrave;me efficace pour livrer des siARN pour CSEh dans une matrice 3D de la culture en utilisant des mat&eacute;riaux de type lipidique. Nous montrons que non-virale ARNsi dans une 3D &eacute;chafaudages can knockdown efficacement 90 % d&#39;expression de la GFP dans GFP-CSEh. En outre, nous montrent que ce syst&egrave;me peut servir comme plate-forme pour orienter la diff&eacute;renciation CSEh. Par le biais de siARN silencing du g&egrave;ne du r&eacute;cepteur KRR, nous parvenir une diminution concomitante (60-90 %) de repr&eacute;sentant des g&egrave;nes de la couche de germe d&#39;endoderme et significative upregulation of repr&eacute;sentant des g&egrave;nes de la couche de germe du m&eacute;soderme (pli 27-90). Ceci d&eacute;montre que les siARN peuvent diriger la diff&eacute;renciation des cellules souches en bloquant les g&egrave;nes repr&eacute;sentatifs d&#39;une couche de germe et fournit &eacute;galement un moyen particuli&egrave;rement puissant pour isoler la couche de germe d&#39;endoderme du m&eacute;soderme et ectoderme. Cette capacit&eacute; d&#39;inhiber la diff&eacute;renciation des couches germinales endoderme pourrait permettre un contr&ocirc;le am&eacute;lior&eacute; au cours de la diff&eacute;renciation des CSEh aux types de la cellule souhait&eacute;e.

Diriger la diff&eacute;renciation de cellules souches embryonnaires humaines par livraison non viraux de siARN dans la culture 3D.

Menschlicher embryonaler Stammzellen (Zellkulturen) halten gro&szlig;es Potenzial als Ressource f&uuml;r die regenerative Medizin. Vor dem therapeutischen Relevanz erlangen, m&uuml;ssen Methoden entwickelt werden, um Stammzellen Differenzierung zu steuern. Es ist klar, dass Stammzellen genetische Signale, z. B. von Nukleins&auml;uren, vermittelt Herkunfts-spezifische Differenzierung F&ouml;rderung reagieren. Hier haben wir ein effizientes System f&uuml;r die Lieferung von SiRNA f&uuml;r Zellkulturen in einer 3D Kultur-Matrix mit Lipid-&auml;hnliche Materialien entwickelt. Wir zeigen, dass nicht-viralen SiRNA-Lieferung in einem 3D Ger&uuml;sten kann effizient Niederschlag 90 % der GFP-Expression in GFP-Zellkulturen. Wir zeigen weiter, dass dieses System als Plattform f&uuml;r die Leitung der hESC Differenzierung verwendet werden kann. Durch SiRNA Unterdr&uuml;ckender im KDR-Rezeptor-Gen, erreichen wir gleichzeitige Heraufregulation (60-90 %) in Gene Vertreter der Endodermis Mikrobe-Schicht und bedeutende Hochregulation Gene Vertreter des Mesoderm Mikrobe-Schicht (Falten, 27-90). Dies zeigt, dass SiRNA kann Stammzellen Differenzierung durch die Blockierung von Genen, die repr&auml;sentativ f&uuml;r eine Mikrobe-Schicht direkt und bietet auch ein besonders wirksames Instrument, die Endodermis Mikrobe-Schicht aus dem Mesoderm und Ektoderm zu isolieren. Diese F&auml;higkeit Endodermis Mikrobe-Schicht Differenzierung zu hemmen kann f&uuml;r verbesserte Kontrolle &uuml;ber hESC Differenzierung zum gew&uuml;nschten Zelltypen erm&ouml;glichen.

Regie Differenzierung von humanen embryonalen Stammzellen durch nicht-viralen Lieferung von SiRNA in 3D Kultur.

Cellule staminali embrionali umane (hESC) tenere il grande potenziale come una risorsa per la medicina rigenerativa. Prima di raggiungere la pertinenza terapeutica, devono essere sviluppati metodi per controllare la differenziazione delle cellule staminali. &Egrave; chiaro che le cellule staminali possono rispondere ai segnali genetici, come quelli impartita dagli acidi nucleici, per promuovere la differenziazione di lineage-specifici. Qui abbiamo sviluppato un sistema efficiente per la consegna di siRNA a hESCs in una matrice di cultura 3D utilizzando materiali lipido-simile. Mostriamo che consegna non-virale siRNA in un 3D ponteggi pu&ograve; efficacemente atterramento 90% dell&#39;espressione di GFP in GFP-hESCs. Mostriamo inoltre che questo sistema pu&ograve; essere utilizzato come una piattaforma per la regia di differenziazione hESC. Attraverso siRNA silenziamento del gene del recettore KDR, raggiungiamo downregulation simultanee (60-90%) nei geni rappresentante dello strato germinale endoderma e significativo sovraregolazione del rappresentante di geni dello strato germinale mesoderma (27-90 piega). Questo dimostra che siRNA possibile indirizzare la differenziazione delle cellule staminali bloccando i geni rappresentativi di uno strato di germe e fornisce anche un mezzo particolarmente potente per isolare lo strato del germe endoderma dal mesoderma e l&#39;ectoderma. Questa capacit&agrave; di inibire la differenziazione di strato di germe endoderma potrebbe consentire per un controllo migliore hESC differenziazione per i tipi di cella desiderata.

Dirigendo la differenziazione delle cellule staminali embrionali umane dalla consegna non-virale di siRNA nella cultura 3D.

&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE; &#xFF08;hESCs&#xFF09; &#x518D;&#x751F;&#x533B;&#x7642;&#x306E;&#x305F;&#x3081;&#x306E;&#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3068;&#x3057;&#x3066;&#x5927;&#x304D;&#x306A;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x4FDD;&#x6301;&#x3057;&#x307E;&#x3059;&#x3002;&#x6CBB;&#x7642;&#x95A2;&#x9023;&#x6027;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x524D;&#x306B;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x958B;&#x767A;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x5E79;&#x7D30;&#x80DE;&#x7CFB;&#x7D71;&#x7279;&#x7570;&#x7684;&#x306A;&#x5206;&#x5316;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x907A;&#x4F1D;&#x7684;&#x306B;&#x306A;&#x3069;&#x306E;&#x4FE1;&#x53F7;&#x305D;&#x308C;&#x3089;&#x6838;&#x9178;&#x306B;&#x3088;&#x3063;&#x3066;&#x4E0E;&#x3048;&#x3089;&#x308C;&#x308B;&#x5FDC;&#x7B54;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x306F;&#x660E;&#x3089;&#x304B;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x6211;&#x3005; &#x306F; siRNA hESCs &#x8102;&#x8CEA;&#x306E;&#x3088;&#x3046;&#x306A;&#x6750;&#x6599;&#x3092;&#x7528;&#x3044;&#x305F;&#x4E09;&#x6B21;&#x5143;&#x57F9;&#x990A;&#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x5185;&#x306B;&#x914D;&#x4FE1;&#x3059;&#x308B;&#x52B9;&#x7387;&#x7684;&#x306A;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x958B;&#x767A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;3 D &#x8DB3;&#x5834;&#x3067;&#x304D;&#x308B;&#x52B9;&#x7387;&#x7684;&#x306B;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3; 90 &#xFF05; GFP hESCs &#x306B;&#x304A;&#x3051;&#x308B; GFP &#x767A;&#x73FE;&#x306E;&#x975E;&#x30A6;&#x30A4;&#x30EB;&#x30B9; siRNA &#x30C7;&#x30EA;&#x30D0;&#x30EA;&#x30FC;&#x3092;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3053;&#x306E;&#x30B7;&#x30B9;&#x30C6;&#x30E0; hESC &#x5206;&#x5316;&#x3092;&#x6307;&#x793A;&#x3059;&#x308B;&#x305F;&#x3081;&#x3001;&#x30D7;&#x30E9;&#x30C3;&#x30C8;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x3068;&#x3057;&#x3066;&#x3092;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;SiRNA KDR &#x53D7;&#x5BB9;&#x4F53;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x3092;&#x901A;&#x3058;&#x3066;&#x3001;&#x6211;&#x3005; &#x4E26;&#x884C;&#x30C0;&#x30A6;&#x30F3;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3; (60-90%) &#x907A;&#x4F1D;&#x5B50;&#x4EE3;&#x8868;&#x5185;&#x80DA;&#x8449;&#x306E;&#x80DA;&#x5C64;&#x306E;&#x6709;&#x610F;&#x306E;&#x30A2;&#x30C3;&#x30D7;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x4E2D;&#x80DA;&#x8449;&#x306E;&#x80DA;&#x5C64; &#xFF08;27 90 &#x500D;&#xFF09; &#x306E;&#x907A;&#x4F1D;&#x5B50;&#x4EE3;&#x8868;&#x306E;&#x9054;&#x6210;&#x3068;&#x3002;&#x3053;&#x308C;&#x306B;&#x306F;&#x3001;&#x305D;&#x306E; siRNA &#x907A;&#x4F1D;&#x5B50;&#x306E; 1 &#x3064;&#x306E;&#x7D30;&#x83CC;&#x5C64;&#x306E;&#x4EE3;&#x8868;&#x3092;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5E79;&#x7D30;&#x80DE;&#x5206;&#x5316;&#x3092;&#x6307;&#x793A;&#x3067;&#x304D;&#x308B;&#x3057;&#x3001;&#x5185;&#x80DA;&#x8449;&#x80DA;&#x8449;&#x3001;&#x4E2D;&#x80DA;&#x8449;&#x3068;&#x5916;&#x80DA;&#x8449;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x7279;&#x306B;&#x5F37;&#x529B;&#x306A;&#x624B;&#x6BB5;&#x3082;&#x63D0;&#x4F9B;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5185;&#x80DA;&#x8449;&#x80DA;&#x8449;&#x5206;&#x5316;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x3053;&#x306E;&#x80FD;&#x529B;&#x6539;&#x5584; hESC &#x5206;&#x5316;&#x76EE;&#x7684;&#x306E;&#x30BB;&#x30EB;&#x306E;&#x7A2E;&#x985E;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

SiRNA &#x4E09;&#x6B21;&#x5143;&#x57F9;&#x990A;&#x3067;&#x306E;&#x30D2;&#x30C8;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x5316;&#x3092;&#x6F14;&#x51FA;&#x975E;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x914D;&#x9054;&#x306B;&#x3088;&#x3063;&#x3066;&#x3002;

&#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (hESCs) &#xC7AC;&#xC0DD; &#xC758;&#xD559;&#xC5D0; &#xB300; &#xD55C; &#xC790;&#xC6D0;&#xC73C;&#xB85C; &#xD070; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xBCF4;&#xC720;. &#xCE58;&#xB8CC; &#xAD00;&#xB828;&#xC131;&#xC744; &#xB2EC;&#xC131; &#xD558;&#xAE30; &#xC804;&#xC5D0; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654; &#xC81C;&#xC5B4; &#xBC29;&#xBC95;&#xC740; &#xAC1C;&#xBC1C; &#xD574;&#xC57C; &#xD569;&#xB2C8;&#xB2E4;. &#xADF8;&#xAC83;&#xC740; &#xBD84;&#xBA85; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xACC4;&#xBCF4; &#xAD00;&#xB828; &#xCC28;&#xBCC4;&#xD654; &#xD64D;&#xBCF4; &#xB4F1; &#xD575; &#xC0B0;&#xC774; &#xC218; &#xC720;&#xC804;&#xC790; &#xC2E0;&#xD638;&#xC5D0; &#xC751;&#xB2F5;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xB294; &#xC9C0;&#xC9C8; &#xAC19;&#xC740; &#xC7AC;&#xB8CC;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; 3D &#xBB38;&#xD654; &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4;&#xC5D0; Hescs&#xB97C; Sirna&#xB97C; &#xC81C;&#xACF5; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xD6A8;&#xC728;&#xC801;&#xC778; &#xC2DC;&#xC2A4;&#xD15C;&#xC744; &#xAC1C;&#xBC1C; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; GFP Hescs&#xC758; GFP &#xC2DD;&#xC758; 3 &#xCC28;&#xC6D0; &#xAC74;&#xC124; &#xAE30;&#xACC4; &#xC784;&#xB300; &#xC218; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xCD5C;&#xC800;&#xC758; 90%&#xC5D0;&#xC11C; &#xADF8; &#xBE44; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; siRNA &#xC804;&#xB2EC;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB354; &#xC774;&#xC0C1;&#xC774; &#xC2DC;&#xC2A4;&#xD15C; hESC &#xCC28;&#xBCC4;&#xD654; &#xC5F0;&#xCD9C;&#xC744; &#xC704;&#xD55C; &#xD50C;&#xB7AB;&#xD3FC;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. KDR &#xC218;&#xC6A9; &#xCCB4; &#xC720;&#xC804;&#xC790;&#xC758; Sirna&#xB97C; &#xC785;&#xC744; &#xD1B5;&#xD574; &#xC6B0;&#xB9AC; endoderm &#xC138;&#xADE0; &#xCE35;&#xC758; &#xB300;&#xD45C; &#xC720;&#xC804;&#xC790;&#xC640; &#xC720;&#xC804;&#xC790; &#xB300;&#xD45C; mesoderm &#xC138;&#xADE0; &#xB808;&#xC774;&#xC5B4; (27-90 &#xBC30;)&#xC758; &#xC0C1;&#xB2F9;&#xD55C; upregulation &#xB3D9;&#xC2DC; downregulation (60-90%)&#xB97C; &#xB2EC;&#xC131;. &#xC774; siRNA &#xC720;&#xC804;&#xC790; &#xD55C; &#xC138;&#xADE0; &#xCE35;&#xC758; &#xB300;&#xD45C;&#xB97C; &#xCC28;&#xB2E8; &#xD558; &#xC5EC; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654;&#xB97C; &#xC548;&#xB0B4;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; mesoderm ectoderm&#xC5D0;&#xC11C; endoderm &#xC138;&#xADE0; &#xACC4;&#xCE35;&#xC744; &#xBD84;&#xB9AC; &#xD558;&#xB294; &#xD2B9;&#xD788; &#xAC15;&#xB825;&#xD55C; &#xC218;&#xB2E8; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. Endoderm &#xC138;&#xADE0; &#xACC4;&#xCE35; &#xBD84;&#xD654;&#xB97C; &#xC5B5;&#xC81C; &#xD558;&#xB294;&#xC774; &#xAE30;&#xB2A5;&#xC740; &#xC6D0;&#xD558;&#xB294; &#xC140; &#xD615;&#xC2DD; hESC &#xCC28;&#xBCC4;&#xD654;&#xB97C; &#xD1B5;&#xD574; &#xD5A5;&#xC0C1; &#xB41C; &#xC81C;&#xC5B4;&#xC5D0; &#xB300; &#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

3 &#xCC28;&#xC6D0; &#xBB38;&#xD654;&#xC5D0; Sirna&#xC758; &#xBE44; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xC804;&#xB2EC; &#xD558; &#xC5EC; &#xC778;&#xAC04; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xD654;&#xB97C; &#xAC10;&#xB3C5;.

C&eacute;lulas madre embrionarias humanas (hESCs) tener gran potencial como un recurso para la medicina regenerativa. Antes de alcanzar la relevancia terap&eacute;utica, se deben desarrollar m&eacute;todos para controlar la diferenciaci&oacute;n de c&eacute;lulas madre. Est&aacute; claro que las c&eacute;lulas madre puede responder a se&ntilde;ales gen&eacute;ticas, como los impartidos por los &aacute;cidos nucleicos, para promover la diferenciaci&oacute;n del linaje espec&iacute;fico. Aqu&iacute; hemos desarrollado un sistema eficaz para la entrega de siRNA a hESCs en una matriz de cultura 3D utilizando materiales de l&iacute;pidos. Mostramos entrega no viral siRNA en andamios 3D puede eficientemente precipitaci&oacute;n 90% de la expresi&oacute;n de GFP en GFP-hESCs. Adem&aacute;s, demostramos que este sistema puede ser utilizado como una plataforma para dirigir la diferenciaci&oacute;n de c&eacute;lulas. A trav&eacute;s de siRNA silenciamiento del gen del receptor KDR, logramos downregulation concurrente (60-90%) en representante de genes de la capa de germen del endodermo y upregulation significativo de representante de genes de la capa del germen del mesodermo (27-90 veces). Esto demuestra siRNA puede dirigir la diferenciaci&oacute;n de c&eacute;lulas madre mediante el bloqueo de genes representativos de una capa de germen y tambi&eacute;n proporciona un medio particularmente poderoso para aislar la capa del germen del endodermo del ectodermo y mesodermo. Esta capacidad de inhibir la diferenciaci&oacute;n de endodermo capa germinal podr&iacute;a permitir mejor control sobre la diferenciaci&oacute;n de c&eacute;lulas a tipos celulares deseados.

Dirigir la diferenciaci&oacute;n de c&eacute;lulas madre embrionarias humanas por la no entrega de siRNA en cultura 3D.

Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t(1/2) ≈ 24.2 min) than the parent siRNA (t(1/2) ≈ 6 min).

Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery.

siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates.

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis. 

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight.

To be legally sold in the United States, all drugs must go through the FDA approval process. This chapter introduces the FDA approval process and describes the clinical trials required for a drug to gain approval. We then look at the different cancer nanotherapeutics and in vivo diagnostics that are currently in clinical trials or have already received approval. These nanotechnologies are catagorized and described based on the delivery vehicle: liposomes, polymer micelles, albumin-bound chemotherapeutics, polymer-bound chemotherapeutics, and inorganic particles.

Cancer nanotherapeutics in clinical trials.

The spotlighted dual functions of pyridine as a denaturant and as a stabilizer for duplex DNA are thoroughly investigated using spherical nucleic acids (SNAs). At neutral pH, pyridine destabilizes the duplex interconnects of assembled SNAs, resulting in a gradual decrease in their melting temperature (T) as a function of the pyridine concentration. This result is in good agreement with the conventional role of pyridine as a powerful denaturant for free duplex DNA. On the contrary, the addition of pyridine dramatically increases the T of hybridized SNAs under acidic conditions, which could be a striking result of pyridine's stabilizing effect for DNA duplex as previously suggested on the basis of the pyridine-nucleobase interactions. After comprehensive and quantitative investigation based on the analysis of the sharp melting transitions of SNAs, however, we report that, in fact, the pH increase induced by pyridine is also an essential parameter accounting for pyridine's DNA-stabilizing effects under acidic conditions. Importantly, we prove that pyridine, particularly at a low concentration, does not increase the T of hybridized SNAs even under acidic conditions, if the pH increase by pyridine is corrected to maintain the same initial pH.

Pyridine: a Denaturant or Stabilizer of Spherical Nucleic Acids?

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) are a promising class of immunotherapeutics that activate innate immunity to increase tumour immunogenicity. However, the efficacy of CDNs is limited by drug delivery barriers, including poor cellular targeting, rapid clearance and inefficient transport to the cytosol where STING is localized. Here, we describe STING-activating nanoparticles (STING-NPs)-rationally designed polymersomes for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). STING-NPs increase the biological potency of cGAMP, enhance STING signalling in the tumour microenvironment and sentinel lymph node, and convert immunosuppressive tumours to immunogenic, tumoricidal microenvironments. This leads to enhanced therapeutic efficacy of cGAMP, inhibition of tumour growth, increased rates of long-term survival, improved response to immune checkpoint blockade and induction of immunological memory that protects against tumour rechallenge. We validate STING-NPs in freshly isolated human melanoma tissue, highlighting their potential to improve clinical outcomes of immunotherapy.

Endosomolytic polymersomes increase the activity of cyclic dinucleotide STING agonists to enhance cancer immunotherapy.

Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells.

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE). These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

Abigail K. R. Lytton-Jean

Koch Institute for Integrative Cancer Research

Massachusetts Institute of Technology

CryoEM Status Update and Innovative Developments

Abigail K. R. Lytton-Jean

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Koch Institute for Integrative Cancer Research

Massachusetts Institute of Technology

CryoEM Status Update and Innovative Developments

Koch Institute for Integrative Cancer Research