Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease.

Little is known about the genetic regulation of medulloblastoma dissemination, but metastatic medulloblastoma is highly associated with poor outcome. We obtained expression profiles of 23 primary medulloblastomas clinically designated as either metastatic (M+) or non-metastatic (M0) and identified 85 genes whose expression differed significantly between classes. Using a class prediction algorithm based on these genes and a leave-one-out approach, we assigned sample class to these tumors (M+ or M0) with 72% accuracy and to four additional independent tumors with 100% accuracy. We also assigned the metastatic medulloblastoma cell line Daoy to the metastatic class. Notably, platelet-derived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway are upregulated in M+ tumors. Immunohistochemical validation on an independent set of tumors shows significant overexpression of PDGFRA in M+ tumors compared to M0 tumors. Using in vitro assays, we show that platelet-derived growth factor alpha (PDGFA) enhances medulloblastoma migration and increases downstream MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK1 (p42 MAPK) and MAPK3 (p44 MAPK) phosphorylation in a dose-dependent manner. Neutralizing antibodies to PDGFRA blocks MAP2K1, MAP2K2 and MAPK1/3 phosphorylation, whereas U0126, a highly specific inhibitor of MAP2K1 and MAP2K2, also blocks MAPK1/3. Both inhibit migration and prevent PDGFA-stimulated migration. These results provide the first insight into the genetic regulation of medulloblastoma metastasis and are the first to suggest a role for PDGFRA and the RAS/MAPK signaling pathway in medulloblastoma metastasis. Inhibitors of PDGFRA and RAS proteins should therefore be considered for investigation as possible novel therapeutic strategies against medulloblastoma.     

钢管相贯口变参数变结构建库与数据提取方法研究

零件的批处理和零件库的建立是计算机辅助设计的重要内容之一，以钢管相贯口为例应用UG的几种建库方法，通过分析电子表格、零件族、GRIP编程在变参变结构的复杂零件建库时存在的问题，提出了一种将零件族和GRIP编程相结合的建库方法来建立变参变结构的复杂零件的零件库。     

Invariant chain influences the immunological recognition of MHC class II molecules.

Recent experiments have implicated intracellular events in the formation of the MHC class II-peptide complexes recognized by CD4-positive T cells. These data raise the possibility that the intracellular association of class II with the non-polymorphic glycoprotein, invariant chain (Ii), may regulate the interaction between processed antigen and MHC class II molecules. To address this possibility, we have generated a series of transfected fibroblast cell lines that express class II with and without Ii. Although the presence of Ii does not seem to affect the ability of the cells to process and present intact antigen, Ii-negative cells express an altered form of class II at the cell surface. This modified conformation of class II in Ii-negative cells is detectable by an increase in the ability to present antigenic peptides to T cells and a decrease in the binding of several class II-specific monoclonal antibodies.     

A transgene containing lacZ inserted into the dystonia locus is expressed in neural tube

The site of integration of transgenes in the host genome can affect levels of expression and occasionally confer ectopic patterns of expression on otherwise tissue-specific genes. We describe here a line of mice in which an hsp68-lacZ transgene is expressed in unstressed developing neural tissue and where the transgene insertion has caused a mutation of a neural tissue-specific gene, dystonia musculorum (dt). This coincidence suggests that expression of the hsp68-lacZ construct may be controlled directly by cis-acting regulatory sequences that normally control the developmental expression of the dt gene. Such constructs may serve as useful tools for identifying new tissue-specific enhancers and their associated genes.     

Acoelomorph flatworms are deuterostomes related to Xenoturbella

Xenoturbellida and Acoelomorpha are marine worms with contentious ancestry. Both were originally associated with the flatworms (Platyhelminthes), but molecular data have revised their phylogenetic positions, generally linking Xenoturbellida to the deuterostomes and positioning the Acoelomorpha as the most basally branching bilaterian group(s). Recent phylogenomic data suggested that Xenoturbellida and Acoelomorpha are sister taxa and together constitute an early branch of Bilateria. Here we assemble three independent data sets-mitochondrial genes, a phylogenomic data set of 38,330 amino-acid positions and new microRNA (miRNA) complements-and show that the position of Acoelomorpha is strongly affected by a long-branch attraction (LBA) artefact. When we minimize LBA we find consistent support for a position of both acoelomorphs and Xenoturbella within the deuterostomes. The most likely phylogeny links Xenoturbella and Acoelomorpha in a clade we call Xenacoelomorpha. The Xenacoelomorpha is the sister group of the Ambulacraria (hemichordates and echinoderms). We show that analyses of miRNA complements have been affected by character loss in the acoels and that both groups possess one miRNA and the gene Rsb66 otherwise specific to deuterostomes. In addition, Xenoturbella shares one miRNA with the ambulacrarians, and two with the acoels. This phylogeny makes sense of the shared characteristics of Xenoturbellida and Acoelomorpha, such as ciliary ultrastructure and diffuse nervous system, and implies the loss of various deuterostome characters in the Xenacoelomorpha including coelomic cavities, through gut and gill slits.     

Replacement of huntingtin exon 1 by trans-splicing

Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a “gain-of-function” mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.