isolation-culture-dental-epithelial-stem-cells-from-adult-mouse

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

University of California, San Francisco

MicroRNAs (miRNAs) &#xC720;&#xC804;&#xC790; &#xADDC;&#xC815; &#xBD84;&#xC790; (&#xC2EC;&#xD310; 1, 2 &#xAC80;&#xD1A0;)&#xC758; &#xD48D;&#xBD80;&#xD55C; &#xD074;&#xB798;&#xC2A4;&#xB294;. &#xC804;&#xC0B0; &#xC791;&#xC5C5; miRNAs &#xAC1C; &#xC774;&#xC0C1;&#xC758; &#xC778;&#xAC04; &#xC720;&#xC804;&#xC790;&#xC758; 3 &#xC8FC;&#xCCB4;&#xB97C; &#xB098;&#xD0C0;&#xB0B4;&#xC9C0;&#xB9CC;, &#xCC99;&#xCD94; &#xAC1C;&#xBC1C;&#xC5D0; &#xC790;&#xC2E0;&#xC758; &#xC5ED;&#xD560;&#xB9CC; &#xC9C0;&#xAE08; &#xACB0;&#xC815; &#xB418;&#xC5B4;&#xC57C; &#xC2DC;&#xC791; &#xB429;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4; &#xBBF8;&#xB974;-196 &#xD589;&#xC704; hoxb8&#xC640; &#xC18C;&#xB2C9; &#xACE0;&#xC2B4;&#xB3C4;&#xCE58; (&#xC26C;)&#xC758; &#xCEE8;&#xD14D;&#xC2A4;&#xD2B8;&#xC5D0;&#xC11C; vivo &#xC0C1;&#xB958; &#xAC08;&#xAE30;&#xAC08;&#xAE30; &#xAC1C;&#xBC1C;, &#xADF8;&#xB85C; &#xC778;&#xD558;&#xC5EC; &#xC2DD;&#xBCC4; &#xC774;&#xC804;&#xC5D0; &#xAD00;&#xCC30; &#xB41C; &#xC788;&#xC9C0;&#xB9CC; uncharacterized &#xAE08;&#xC9C0;&#xB294; hindlimb&#xC5D0;&#xC11C; &#xAD6C;&#xCCB4;&#xC801;&#xC73C;&#xB85C; &#xC791;&#xB3D9; &#xD558;&#xB294; &#xD65C;&#xB3D9;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130;&#xB294; &#xB9CE;&#xC740; &#xCC99;&#xCD94; miRNAs &#xC8FC;&#xB85C; &#xC544;&#xC774;&#xB514;&#xC5B4;&#xB97C; &#xC9C0; &#xC6D0;&#xD558;&#xB294; transcriptional &#xC218;&#xC900;&#xC5D0;&#xC11C; &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; &#xC2DD; &#xB3C4;&#xBA54;&#xC778;&#xC758; &#xCDA9;&#xC2E4;&#xB3C4; &#xBCF4;&#xC7A5; &#xD558;&#xAE30; &#xC704;&#xD574;&#xC11C; &#xC548;&#xC804; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0;&#xC11C; &#xBBF8;&#xB974;-196 &#xAE30;&#xB2A5; &#xC720;&#xC804;&#xC790; &#xC870;&#xC808;&#xC758; &#xBCF4;&#xC870; &#xC218;&#xC900;&#xC73C;&#xB85C; &#xC791;&#xB3D9; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

MicroRNA &#xBBF8;&#xB974;-196 &#xD589;&#xC704; &#xC0C1;&#xB958; hoxb8&#xC640; &#xC26C;&#xC774; &#xC787;&#xC758; &#xB2E4;&#xB9AC;&#xB97C; &#xAC1C;&#xBC1C;.

(Mirna) &#x5C0F;&#x5206;&#x5B50; rna &#x662F;&#x57FA;&#x56E0;&#x8C03;&#x63A7;&#x5206;&#x5B50; &#xFF08;&#x5BA1;&#x67E5;&#x5728;&#x88C1;&#x5224; 1&#xFF0C;2&#xFF09; &#x4E30;&#x5BCC;&#x7C7B;&#x3002;&#x5C3D;&#x7BA1;&#x8BA1;&#x7B97;&#x5DE5;&#x4F5C;&#x6307;&#x793A; Mirna &#x538B;&#x5236;&#x8D85;&#x8FC7;&#x4E09;&#x5206;&#x4E4B;&#x4E00;&#x7684;&#x4EBA;&#x7C7B;&#x57FA;&#x56E0;&#xFF0C;&#x5B83;&#x4EEC;&#x5728;&#x810A;&#x690E;&#x52A8;&#x7269;&#x53D1;&#x80B2;&#x7684;&#x4F5C;&#x7528;&#x73B0;&#x5728;&#x624D;&#x5F00;&#x59CB;&#x5F85;&#x5B9A;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x8868;&#x660E;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9; 196 &#x884C;&#x4E3A;&#x4E0A;&#x6E38; Hoxb8 &#x548C;&#x58F0;&#x6CE2;&#x523A;&#x732C; (Shh) &#x5728;&#x4F53;&#x5185;&#x7684;&#x80A2;&#x4F53;&#x53D1;&#x5C55;&#xFF0C;&#x4ECE;&#x800C;&#x627E;&#x51FA;&#x4EE5;&#x524D;&#x89C2;&#x5BDF;&#x5230;&#x4F46; uncharacterized &#x6291;&#x5236;&#x8BBE;&#x6709;&#x4E13;&#x95E8;&#x7684;&#x540E;&#x80A2;&#x7684;&#x6D3B;&#x52A8;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x8868;&#x660E;&#x6545;&#x969C;&#x4FDD;&#x62A4;&#x7684;&#x673A;&#x5236;&#xFF0C;&#x4EE5;&#x786E;&#x4FDD;&#x4E3B;&#x8981;&#x53D7;&#x8F6C;&#x5F55;&#x4E00;&#x7EA7;&#xFF0C;&#x652F;&#x6301;&#x8FD9;&#x4E00;&#x60F3;&#x6CD5;&#x7684;&#x8868;&#x8FBE;&#x5F0F;&#x57DF;&#x7684;&#x4FDD;&#x771F;&#x5EA6;&#xFF0C;&#x8BB8;&#x591A;&#x810A;&#x690E;&#x52A8;&#x7269; Mirna &#x5728;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9; 196 &#x51FD;&#x6570;&#x53EF;&#x4EE5;&#x4F5C;&#x4E3A;&#x57FA;&#x56E0;&#x8C03;&#x63A7;&#x7684;&#x4E2D;&#x7B49;&#x6C34;&#x5E73;&#x3002;

MicroRNA &#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9; 196 &#x4E0A;&#x6E38; Hoxb8 &#x548C;&#x884C;&#x4E3A; Shh &#x7684;&#x80A2;&#x4F53;&#x53D1;&#x80B2;&#x3002;

MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.

The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development.

Les microARN (miARN) sont une classe abondante de mol&#xE9;cules r&#xE9;gulatrices de g&#xE8;nes (revue dans les r&#xE9;f&#xE9;rences 1, 2). Bien que le travail de calcul indique que les miRNAs r&#xE9;primer plus d&#x26;#39;un tiers des g&#xE8;nes humains, leurs r&#xF4;les dans le d&#xE9;veloppement des vert&#xE9;br&#xE9;s commencent seulement maintenant &#xE0; &#xEA;tre d&#xE9;termin&#xE9;e. Ici, nous montrons que miR-196 agit en amont de Hoxb8 et Sonic hedgehog (Shh) in vivo dans le contexte du d&#xE9;veloppement des membres, identifiant ainsi une activit&#xE9; observ&#xE9; pr&#xE9;c&#xE9;demment, mais non caract&#xE9;ris&#xE9;s inhibitrice qui fonctionne pr&#xE9;cis&#xE9;ment dans le membre post&#xE9;rieur. Nos donn&#xE9;es indiquent que miR-196 dans les fonctions d&#x26;#39;un m&#xE9;canisme &#xE0; s&#xE9;curit&#xE9; int&#xE9;gr&#xE9;e pour assurer la fid&#xE9;lit&#xE9; de domaines d&#x26;#39;expression qui sont principalement r&#xE9;gul&#xE9;e au niveau transcriptionnel, en soutenant l&#x26;#39;id&#xE9;e que de nombreux miARN vert&#xE9;br&#xE9;s peut fonctionner comme un second niveau de la r&#xE9;gulation des g&#xE8;nes.

Le microARN Mir-196 agit en amont de Hoxb8 et Shh dans le d&#xE9;veloppement des membres

MicroRNAs (MiRNAs) sind eine reichliche gen regulatorische Molek&uuml;le (nachgepr&uuml;ft in Schiedsrichter 1, 2). Obwohl computational Arbeit angibt, dass MiRNAs mehr als ein Drittel der menschlichen Gene unterdr&uuml;cken, beginnen ihre Rollen in vertebrate Entwicklung erst jetzt bestimmt werden. Hier zeigen wir, dass MiR-196 Handlungen oberhalb der Hoxb8 und Sonic Hedgehog (Shh) in-vivo im Rahmen der Entwicklung des K&ouml;rpers, damit zur Identifizierung einer zuvor beobachteten aber uncharacterized hemmende Aktivit&auml;t, die speziell in der Hinterbeine t&auml;tig ist. Unsere Daten zeigen, dass MiR-196-Funktionen in eine Ausfallsicherung versichern der Treue Ausdruck-Dom&auml;nen, die in erster Linie auf die transkriptionelle, unterst&uuml;tzen die Idee geregelt sind, dass viele Wirbeltiere MiRNAs als sekund&auml;re Ebene der Genregulation funktionieren k&ouml;nnen.

Die MicroRNA MiR-196 fungiert stromaufw&auml;rts von Hoxb8 und Shh Gliedma&szlig;en Entwicklung.

MicroRNA (miRNAs) &egrave; una classe di molecole regolatrici del gene (rivisto nel Rif 1, 2) abbondante. Anche se lavoro computazionale indica che i miRNAs reprimere pi&ugrave; di un terzo dei geni umani, loro ruoli nello sviluppo vertebrato solo ora stanno cominciando a essere determinato. Qui vi mostriamo che miR-196 atti a Monte di Hoxb8 e Sonic hedgehog (Shh) in vivo nel contesto dell&#39;arto sviluppo, quindi identificare un precedentemente osservati ma uncharacterized inibitoria attivit&agrave; che opera in particolare l&#39;arto posteriore. I nostri dati indicano che miR-196 funzioni in un meccanismo di fail-safe per assicurare la fedelt&agrave; dei domini di espressione che sono principalmente regolato a livello trascrizionale, sostenendo l&#39;idea che molti vertebrati miRNAs possono funzionare come un livello secondario di regolazione genica.

I microRNA miR-196 atti a Monte di Hoxb8 e Shh nello sviluppo degli arti.

&#x30DE;&#x30A4;&#x30AF;&#x30ED;RNA&#xFF08;miRNA&#xFF09;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x8ABF;&#x7BC0;&#x5206;&#x5B50;&#x306E;&#x8C4A;&#x5BCC;&#x306A;&#x30AF;&#x30E9;&#x30B9;&#xFF08;&#x6587;&#x732E;1&#x3001;2&#x65E5;&#xFF09;&#x3067;&#x3059;&#x3002;&#x8A08;&#x7B97;&#x4F5C;&#x696D;&#x306F;&#x3001;miRNA&#x304C;&#x30D2;&#x30C8;&#x907A;&#x4F1D;&#x5B50;&#x306E;3&#x5206;&#x306E;1&#x4EE5;&#x4E0A;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x304C;&#x3001;&#x810A;&#x690E;&#x52D5;&#x7269;&#x306E;&#x767A;&#x751F;&#x306B;&#x304A;&#x3051;&#x308B;&#x5F79;&#x5272;&#x306F;&#x3001;&#x73FE;&#x5728;&#x6C7A;&#x5B9A;&#x3055;&#x308C;&#x59CB;&#x3081;&#x3066;&#x3044;&#x308B;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x4E0A;&#x6D41;&#x306E;Hoxb8&#x3068;&#x56DB;&#x80A2;&#x306E;&#x958B;&#x767A;&#x306E;&#x6587;&#x8108;&#x306B;&#x304A;&#x3051;&#x308B;in vivo&#x3067;&#x306E;&#x30BD;&#x30CB;&#x30C3;&#x30AF;&#x30D8;&#x30C3;&#x30B8;&#x30DB;&#x30C3;&#x30B0;&#xFF08;Shh&#xFF09;&#x306E;&#x306E;miR-196&#x306E;&#x884C;&#x70BA;&#x306F;&#x3001;&#x305D;&#x308C;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F8C;&#x80A2;&#x306B;&#x7279;&#x7570;&#x7684;&#x306B;&#x52D5;&#x4F5C;&#x3059;&#x308B;&#x4EE5;&#x524D;&#x306B;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x305F;&#x304C;&#x672A;&#x540C;&#x5B9A;&#x306E;&#x200B;&#x200B;&#x963B;&#x5BB3;&#x6D3B;&#x6027;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x6211;&#x3005;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x30D5;&#x30A7;&#x30A4;&#x30EB;&#x30BB;&#x30FC;&#x30D5;&#x6A5F;&#x69CB;&#x306B;miR-196&#x306E;&#x6A5F;&#x80FD;&#x306F;&#x591A;&#x304F;&#x306E;&#x810A;&#x690E;&#x52D5;&#x7269;&#x306E;miRNA&#x304C;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x5236;&#x5FA1;&#x306E;&#x4E8C;&#x6B21;&#x30EC;&#x30D9;&#x30EB;&#x3068;&#x3057;&#x3066;&#x6A5F;&#x80FD;&#x3057;&#x5F97;&#x308B;&#x3068;&#x3044;&#x3046;&#x8003;&#x3048;&#x3092;&#x652F;&#x6301;&#x3059;&#x308B;&#x3001;&#x4E3B;&#x306B;&#x8EE2;&#x5199;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x8ABF;&#x7BC0;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x8868;&#x73FE;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x5FE0;&#x5B9F;&#x5EA6;&#x3092;&#x4FDD;&#x8A3C;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30DE;&#x30A4;&#x30AF;&#x30ED;RNA&#x306E;miR-196&#x30EA;&#x30E0;&#x958B;&#x767A;&#x306B;&#x304A;&#x3051;&#x308B;Hoxb8&#x3068;Shh&#x306E;&#x4E0A;&#x6D41;&#x306E;&#x884C;&#x70BA;

Los microARN (miRNA) son una clase de abundancia de las mol&#xE9;culas reguladoras de genes (revisado en refs 1, 2). Aunque el trabajo de c&#xE1;lculo indica que los miRNAs reprimen m&#xE1;s de un tercio de los genes humanos, su papel en el desarrollo de vertebrados s&#xF3;lo ahora est&#xE1;n comenzando a determinar. Aqu&#xED; nos muestran que miR-196 act&#xFA;a antes de Hoxb8 y Sonic hedgehog (Shh) en vivo en el contexto del desarrollo de las extremidades, con lo que la identificaci&#xF3;n de una actividad ya se observ&#xF3;, pero no caracterizados inhibidor que opera espec&#xED;ficamente en las extremidades inferiores. Nuestros datos indican que el miR-196 funciona en un mecanismo de seguridad para asegurar la fidelidad de los dominios de expresi&#xF3;n que se regula principalmente en el nivel de la transcripci&#xF3;n, el apoyo a la idea de que muchos miRNAs vertebrados puede funcionar como un segundo nivel de la regulaci&#xF3;n g&#xE9;nica.

El microARN miR-196 act&#xFA;a antes de Hoxb8 y Shh en el desarrollo de las extremidades

Runx transcription factors determine cell fate in many lineages. Maintaining balanced levels of Runx proteins is crucial, as deregulated expression leads to cancers and developmental disorders. We conducted a forward genetic screen in zebrafish for positive regulators of runx1 that yielded the cohesin subunit rad21. Zebrafish embryos lacking Rad21, or cohesin subunit Smc3, fail to express runx3 and lose hematopoietic runx1 expression in early embryonic development. Failure to develop differentiated blood cells in rad21 mutants is partially rescued by microinjection of runx1 mRNA. Significantly, monoallelic loss of rad21 caused a reduction in the transcription of runx1 and of the proneural genes ascl1a and ascl1b, indicating that downstream genes are sensitive to Rad21 dose. Changes in gene expression were observed in a reduced cohesin background in which cell division was able to proceed, indicating that cohesin might have a function in transcription that is separable from its mitotic role. Cohesin is a protein complex essential for sister chromatid cohesion and DNA repair that also appears to be essential for normal development through as yet unknown mechanisms. Our findings provide evidence for a novel role for cohesin in development, and indicate potential for monoallelic loss of cohesin subunits to alter gene expression.

Cohesin-dependent regulation of Runx genes.

Myosin is a motor protein associating with actin and ATP. It translates along actin filaments against a force by transduction of free energy liberated with ATP hydrolysis. Various myosin crystal structures define time points during ATPase showing the protein undergoes large conformation change during transduction over a cycle with approximately 10 ms periodicity. The protein conformation trajectory between two intermediates in the cycle is surmised by non-equilibrium Monte Carlo simulation utilizing free-energy minimization. The trajectory shows myosin transduction of free energy to mechanical work giving evidence for: (i) a causal relationship between product release and work production in the native isoform that is correctly disrupted in a chemically modified protein, (ii) the molecular basis of ATP-sensitive tryptophan fluorescence enhancement and acrylamide quenching, (iii) an actin-binding site peptide containing the free-energy barrier to ATPase product release defining the rate limiting step and, (iv) a scenario for actin-activation of myosin ATPase.

Myosin dynamics on the millisecond time scale.

Wnt5a is an important factor patterning many aspects of early development, including the lung. We find pulmonary non-canonical Wnt5a uses Ror2 to control patterning of both distal air and vascular tubulogenesis (alveolarization). Lungs with mis/overexpressed Wnt5a develop with severe pulmonary hypoplasia associated with altered expression patterns of Shh, L-CAM, fibronectin, VEGF and Flk1. This hypoplastic phenotype is rescued by either replacement of the Shh protein or inhibition of fibronectin function. We find that the effect of Wnt5a on vascular patterning is likely to be through fibronectin-mediated VEGF signaling. These results demonstrate the pivotal role of Wnt5a in directing the essential coordinated development of pulmonary airway and vasculature, by affecting fibronectin levels directly, and by affecting the fibronectin pattern of expression through its regulation of Shh. Data herein suggest that Wnt5a functions in mid-pulmonary patterning (during alveolarization), and is distinct from the Wnt canonical pathway which is more important in earlier lung development.

Chick pulmonary Wnt5a directs airway and vascular tubulogenesis.

We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog (Hh) signaling pathway are among the genes identified in the screen, including Gli2, which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo, a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk-condyle separation and provide a molecular framework for future studies of the TMJ.

Temporomandibular joint formation requires two distinct hedgehog-dependent steps.

SNAP-25 (synaptosomal-associated protein of 25 kDa) is an integral part of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), a docking complex for synaptic vesicle exocytosis and neurotransmitter release. Results with SNAP-25 deficient mouse models highly accelerated association studies of SNAP-25 as a candidate gene for psychiatric disorders, such as Attention Deficit Hyperactivity Disorder (ADHD) and Schizophrenia. Candidate gene studies implicate a large number of single nucleotide polymorphisms (SNPs). Among the numerous SNPs, the miR SNPs are novel functional variants affecting the binding of specific microRNA to their target mRNA. According to our in silico studies there are two putative miR SNPs in the 3'untranslated region (3'UTR) of the SNAP-25 gene. If the putative miR SNPs are shown to have a function in vivo their implication in further psychogenetic association studies will have a higher impact.

SNAP-25: a novel candidate gene in psychiatric genetics.

We have taken a synthetic biology approach to the generation and screening of transcription factor binding sites for activity in human cells. All possible 10-mer DNA sequences were printed on microarrays as 100-mers containing 10 repeats of the same sequence in tandem, yielding an oligonucleotide library of 52,429 unique sequences. This library of potential enhancers was introduced into a retroviral vector and screened in multiple cell lines for the ability to activate GFP transcription from a minimal CMV promoter. With this method, we isolated 100 bp synthetic enhancer elements that were as potent at activating transcription as the WT CMV immediate early enhancer. The activity of the recovered elements was strongly dependent on the cell line in which they were recovered. None of the elements were capable of achieving the same levels of transcriptional enhancement across all tested cell lines as the CMV enhancer. A second screen, for enhancers capable of synergizing with the elements from the original screen, yielded compound enhancers that were capable of twofold greater enhancement activity than the CMV enhancer, with higher levels of activity than the original synthetic enhancer across multiple cell lines. These findings suggest that the 10-mer synthetic enhancer space is sufficiently rich to allow the creation of synthetic promoters of all strengths in most, if not all, cell types.

Synthetic design of strong promoters.

Poxvirus DNA replication generates linear concatemers containing many copies of the viral genome with inverted repeat sequences at the junctions between monomers. The inverted repeats refold to generate Holliday junctions, which are cleaved by the virus-encoded resolvase enzyme to form unit-length genomes. Here we report studies of the influence of metal cofactors on the activity and structure of the resolvase of fowlpox virus, which provides a tractable model for in vitro studies. Small-molecule inhibitors of related enzymes bind simultaneously to metal cofactors and nearby surface amino acid residues, so understanding enzyme-cofactor interactions is important for the design of antiviral agents. Analysis of inferred active-site residues (D7, E60, K102, D132, and D135) by mutagenesis and metal rescue experiments specified residues that contribute to binding metal ions and that multiple binding sites are probably involved. Differential electrophoretic analysis was used to map the conformation of the DNA junction when bound by resolvase. For the wild-type complex in the presence of EDTA (ethylenediaminetetraacetic acid) or Ca(2+), migration was consistent with the DNA arms arranged in near-tetrahedral geometry. However, the D7N active-site mutant resolvase held the arms in a more planar arrangement in EDTA, Ca(2+), or Mg(2+) conditions, implicating metal-dependent contacts at the active site in the larger architecture of the complex. These data show how divalent metals dictate the conformation of FPV resolvase-DNA complexes and subsequent DNA cleavage.

Metal cofactors in the structure and activity of the fowlpox resolvase.

&#x810A;&#x690E;&#x52A8;&#x7269;&#x7684;&#x80A2;&#x4F53;&#x662F;&#x7528;&#x4E8E;&#x4E09;&#x7EF4;&#x7ED3;&#x6784;&#x7684;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x8FC7;&#x7A0B;&#x7684;&#x7406;&#x89E3;&#x9635;&#x5217;&#x7684;&#x7ECF;&#x5178;&#x6A21;&#x578B;&#x3002;&#x867D;&#x7136;&#x6570;&#x5341;&#x5E74;&#x7684;&#x7814;&#x7A76;&#x5DF2;&#x9610;&#x660E;&#x7684;&#x7EC4;&#x7EC7;&#x548C;&#x5185;&#x90E8;&#x6240;&#x9700;&#x7684;&#x56FE;&#x6848;&#x53CA;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x7684;&#x80A2;&#x4F53;&#x7684;&#x80A2;&#x82BD;&#x5206;&#x5B50;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#xFF0C;&#x5F62;&#x72B6;&#x672C;&#x8EAB;&#x7684;&#x80DA;&#x80CE;&#x80A2;&#x82BD;&#x7EC6;&#x80DE;&#x548C;&#x5206;&#x5B50;&#x7684;&#x4E8B;&#x4EF6;&#x4ECD;&#x5F88;&#x5927;&#x7A0B;&#x5EA6;&#x4E0A;&#x672A;&#x77E5;&#x3002;

WNT5A/JNK &#x548C; FGF/MAPK &#x901A;&#x8DEF;&#x8C03;&#x8282;&#x5851;&#x9020;&#x810A;&#x690E;&#x52A8;&#x7269;&#x80A2;&#x82BD;&#x7EC6;&#x80DE;&#x4E8B;&#x4EF6;&#x3002;

The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown.

WNT5A/JNK and FGF/MAPK pathways regulate the cellular events shaping the vertebrate limb bud.

Le membre de vert&#xE9;br&#xE9; est un mod&#xE8;le classique pour comprendre la structuration de structures tridimensionnelles au cours du d&#xE9;veloppement embryonnaire. Bien que des d&#xE9;cennies de recherche ont permis d&#x26;#39;&#xE9;lucider le tissu et les interactions mol&#xE9;culaires dans le bourgeon de membre requise pour la mod&#xE9;lisation et la morphogen&#xE8;se du membre, les &#xE9;v&#xE9;nements cellulaires et mol&#xE9;culaires qui d&#xE9;terminent le bourgeon de membre lui-m&#xEA;me sont rest&#xE9;s largement inconnus.

WNT5A/JNK et FGF / MAPK Pathways R&#xE9;glementer les &#xE9;v&#xE9;nements cellulaires qui fa&#xE7;onneront le bourgeon de membre des Vert&#xE9;br&#xE9;s

Das vertebrate Glied ist ein klassisches Modell f&uuml;r Verst&auml;ndnis Musterung von dreidimensionalen Strukturen w&auml;hrend der Embryonalentwicklung. Obwohl jahrzehntelanger Forschung der Gewebe und Molekulare Wechselwirkungen innerhalb der Gliedma&szlig;en Knospe f&uuml;r die Musterbildung und Morphogenese der Extremit&auml;t aufgekl&auml;rt haben, sind die zellul&auml;ren und molekularen Ereignisse, die die Gliedma&szlig;en-Knospe selbst gestalten weitgehend unbekannt geblieben.

WNT5A/JNK und FGF/MAPK-Wege Regeln die zellul&auml;re Ereignisse, die Gestaltung der vertebrate Glied Knospe.

L&#39;arto vertebrato &egrave; un modello classico per il modello di comprensione delle strutture tridimensionali durante lo sviluppo embrionale. Nonostante decenni di ricerche hanno chiarito il tessuto e le interazioni molecolari entro la gemma dell&#39;arto necessaria per la modellatura e la morfogenesi dell&#39;arto, gli eventi cellulari e molecolari che forma il germoglio dell&#39;arto stesso sono rimasti in gran parte sconosciuti.

WNT5A/JNK e percorsi di FGF/MAPK regolano eventi cellulari plasmare il germoglio di vertebrati dell&#39;arto.

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La extremidad de los vertebrados es un modelo cl&#xE1;sico para la comprensi&#xF3;n de los patrones de tres dimensiones de estructuras durante el desarrollo embrionario. A pesar de d&#xE9;cadas de investigaci&#xF3;n han aclarado que el tejido y las interacciones moleculares dentro de la yema de la extremidad se requiere para los patrones y la morfog&#xE9;nesis de la extremidad, los eventos celulares y moleculares que dan forma a la yema de la extremidad se han mantenido en gran parte desconocido.

WNT5A/JNK y v&#xED;as FGF / MAPK Regular los eventos celulares que configuran la yema del miembro de Vertebrados

Synaptosomal-associated protein 25 (SNAP-25) plays a crucial role in exocitosis. Single nucleotide polymorphisms (rs3746544 and rs1051312) in the 3' un-translated region of the SNAP-25 gene have been described to be in association with attention-deficit hyperactivity disorder. As the disease affects millions of children world-wide, understanding the genetic background of attention-deficit hyperactivity disorder is of crucial importance. Efficient and reliable PCR-RFLP protocols were elaborated for the genotyping of the rs3746544 and rs1051312 SNPs employing a high-throughput capillary electrophoresis method for fragment analysis. A novel real-time PCR-based technique was used applying sequence specific TaqMan probes to haplotype the two SNPs, and the G-C haplotype could not be detected in a large Caucasian population (N=1376). These findings have been confirmed by molecular biology tools as well as by the PHASE Bayesian computational approach. In silico analyses have suggested that the two SNPs might alter microRNA binding and thus have an effect on SNAP-25 production. We have demonstrated that this biological information can be revealed only by direct haplotype analysis emphasizing the importance of our novel molecular haplotye analysis protocol. Results of the study of the two SNPs might shed light on the association of SNAP-25 variants and pathological phenotypes at the molecular level.

Haplotyping of putative microRNA-binding sites in the SNAP-25 gene.

Two broad classes of models have been proposed to explain the patterning of the proximal-distal axis of the vertebrate limb (from the shoulder to the digit tips). Differentiating between them, we demonstrate that early limb mesenchyme in the chick is initially maintained in a state capable of generating all limb segments through exposure to a combination of proximal and distal signals. As the limb bud grows, the proximal limb is established through continued exposure to flank-derived signal(s), whereas the developmental program determining the medial and distal segments is initiated in domains that grow beyond proximal influence. In addition, the system we have developed, combining in vitro and in vivo culture, opens the door to a new level of analysis of patterning mechanisms in the limb.

Initiation of proximal-distal patterning in the vertebrate limb by signals and growth.

An analytical method using a liquid chromatograph combined with a simple online column switching sample pre-treatment system was developed for the determination of 1-hydroxypyrene (1-HP) in urine. This compound is the metabolite of pyrene and is used to assess the exposure of workers to polycyclic aromatic hydrocarbons (PAHs). After enzymatic hydrolysis, a urine sample was directly injected into a high-performance liquid chromatograph (HPLC) where it automatically underwent a sample cleanup using a column switching device. The procedure is simpler than previous methods because it uses only one switching valve, one extraction column and one HPLC pump. The analyte was retained on a short extraction column and after interferences were eluted to waste, was subsequently switched onto the analytical column. This allowed a short analysis time of 15 min. The calibration graph was found to be linear within the concentration range of 0.5 to 20 µg/L with a coefficient of determination exceeding r(2) = 0.99. Recoveries were found to be greater than 96% in the range 1 to 10 µg/L with intermediate precision of 2.5 to 5.8% relative standard deviation. This online method was verified by a comparison with an existing manual method by the analysis of 81 urine samples from workers exposed to PAHs and showed that the test results from both methods were in agreement with a probability obtained from the paired Student's t-test of P > 0.76. The proposed online method was found to be simple, fast and suited to routine analyses of 1-HP in urine for the assessment of occupational exposure to PAHs.

The determination of occupational exposure to polycyclic aromatic hydrocarbons by the analysis of 1-hydroxypyrene in urine using a simple automated online column switching device and high-performance liquid chromatography.

Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context.

Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation.

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell-cycle inhibitors p16(Ink4a) and p19(Arf). However, deletion of Ink4a/Arf only partially rescues Bmi1-null phenotypes, indicating that other important targets of BMI1 exist. Here, using the continuously growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.

Article	Year
The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature\| PubMed ID: 16319892	2005
Cohesin-dependent regulation of Runx genes. Development (Cambridge, England)\| PubMed ID: 17567667	2007
Myosin dynamics on the millisecond time scale. Biophysical chemistry\| PubMed ID: 17913331	2007
Chick pulmonary Wnt5a directs airway and vascular tubulogenesis. Development (Cambridge, England)\| PubMed ID: 18305003	2008
Temporomandibular joint formation requires two distinct hedgehog-dependent steps. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19815519	2009
SNAP-25: a novel candidate gene in psychiatric genetics. Neuropsychopharmacologia Hungarica : a Magyar Pszichofarmakológiai Egyesület lapja = official journal of the Hungarian Association of Psychopharmacology\| PubMed ID: 19827316	2009
Synthetic design of strong promoters. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20133776	2010
Metal cofactors in the structure and activity of the fowlpox resolvase. Journal of molecular biology\| PubMed ID: 20380839	2010
WNT5A/JNK and FGF/MAPK pathways regulate the cellular events shaping the vertebrate limb bud. Current biology : CB\| PubMed ID: 21055947	2010
Haplotyping of putative microRNA-binding sites in the SNAP-25 gene. Electrophoresis\| PubMed ID: 21557260	2011
Initiation of proximal-distal patterning in the vertebrate limb by signals and growth. Science (New York, N.Y.)\| PubMed ID: 21617075	2011
The determination of occupational exposure to polycyclic aromatic hydrocarbons by the analysis of 1-hydroxypyrene in urine using a simple automated online column switching device and high-pe... Journal of analytical toxicology\| PubMed ID: 22582268	2012
Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation. Genes & development\| PubMed ID: 22987639	2012
BMI1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor. Nature cell biology\| PubMed ID: 23728424	2013

Jimmy Hu

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