1,720,973 research outputs found
Ewsa-dependent regulation of Runx2 during zebrafish skeletogenesis
Get PDFEwing’s sarcoma is the second most common malignant bone cancer found in adolescents, and the genetic hallmark of this disease is the presence of the aberrant chimeric fusion protein EWS/FLI1. This fusion is induced by the t(11; 22) chromosomal translocation of EWS and FLI1, and is the most prominent and common characteristic of Ewing sarcoma tumors found in approximately 85-90% of reported cases. EWS/FLI1 has been shown to directly bind to and inhibit the function of endogenous EWS in a dominant manner. In this study, we seek to increase our understanding of the role of endogenous EWS during development and skeletogenesis to gain insight into the pathogenesis of the disease. Previously, we demonstrated the role of a zebrafish EWS homolog Ewsa by analyzing the phenotype of a maternal zygotic homozygous ewsa/ewsa mutant line (MZ ewsa/ewsa) of zebrafish null for Ewsa protein. Prehypertrophic chondrocytes of Meckel’s cartilage in 4dpf MZ ewsa/ewsa mutants fail to completely differentiate into hypertrophic chondrocytes, followed by structural defects in craniofacial bones (dentary and basihyal bones) at the adult stage. Based on these results, we sought to understand EWS’s involvement in skeletogenesis by asking if Ewsa regulates activity of critical transcription factors involved in chondrocyte development. We have previously shown that Ewsa directs chondrocyte differentiation through modulation of chondrogenesis master transcription factor Sox9. Runx2 is also known to play a critical role in differentiation of both chondrocytes and osteoblasts, so we have addressed whether and how Ewsa regulates Runx2 expression and transcriptional activity. We discovered that Runx2 protein expression dramatically increases in craniofacial chondrocytes of 4-6dpf MZ ewsa/ewsa compared to wt/wt embryos. We have also observed premature mineralization in MZ ewsa/ewsa embryos at 6dpf and 10dpf. MZ ewsa/ewsa fish also display a decrease in expression of collagen10a1, a hypertrophic-specific Runx2 target gene. These data together suggests that Ewsa regulates Runx2 expression and transcriptional activity during chondrogenesis and regulates mineralization of these domains. Additionally, our lab has previously discovered that adult MZ ewsa/ewsa fish display aberrant curved spines. Based on these results we asked how Ewsa is involved in the formation of the axial skeleton. Since Collagen2a1 is a critical component of notochord development as both a precursor to intervertebral disc (IVD) formation and as a component of notochord sheath extracellular matrix (ECM) we have previously asked if Ewsa regulates the collagen2a1a gene. Ewsa interacts with the col2a1a gene and col2a1 is upregulated in the notochord MZ ewsa/ewsa fish which suggests that the notochord sheath ECM is misregulated in mutant fish. Additionally, our analysis also revealed that notochord cells have failed to intercalate 5-10dpf and remain in a single layer. Based on our previous data and the data in this report, we hypothesize that Ewsa regulates axis development through regulation of collagen2a1 expression, which in turn allows for normal notochord sheath ECM distribution, which in turn allows for notochord cell intercalation and later IVD and vertebral body formation. This is the first in vivo evidence for regulation of RUNX2 by EWS during chondrogenesis and axial skeleton development
The role of Ewing’s sarcoma protein EWS in endochondral ossification and angiogenesis
Get PDFEwing sarcoma is a bone and cartilage cancer affecting children and adolescents. Up to 90% of Ewing sarcoma patients express a chimeric EWS/FLI1 protein which is formed by a chromosomal translocation, and is known to induce aberrant transcription, bind with endogenous EWS and cause mitotic defects similar to knockdown of EWS. In EWS knockout studies, EWS-/- mice produce smaller pups with shorter, brittle bones compared to wild-type littermates suggesting EWS has a role in skeletal and overall development. Since Ewing’s sarcoma develops in the bone, the role of endogenous EWS is of interest as it may provide an insight into the parthenogenesis of this disease. To investigate this we generated a maternal zygotic (MZ) ewsa null mutant zebrafish, produced by insertional mutagenesis. Chondrocyte maturation begins after mesenchymal condensation at about 3 days past fertilization (dpf), then cells undergo morphological changes by 4dpf to become prehypertrophic chondrocytes with most fully differentiated into hypertrophic chondrocytes by 6dpf. Previously, we reported that Ewsa modulates the mRNA expression of target genes of transcription factor Sox9, the master transcriptional regulator of chondrocyte differentiation. Given the regulatory role of Sox9 in chondrogenesis, an important question is whether Ewsa directly regulates the expression level of Sox9 proteins. Thus, we generated antibodies against Sox9a and Sox9b, and performed immunohistochemistry in 3-6dpf of wt/wt and MZ ewsa/ewsa zebrafish mutants. As a result, Sox9a localized in the nucleus, and the expression levels of Sox9a proteins were unchanged among 3-6dpf wt/wt and MZ ewsa/ewsa zebrafish mutants. This suggests that Sox9a protein is not regulated by Ewsa during chondrocyte maturation. One upregulated Sox9 target gene was connective tissue growth factor (ctgf). Ctgf is known to have a critical role in endochondral ossification, furthermore the overexpression of Ctgf regulation results in skeletal defects and reduced hypertrophy. We suspect that Ews regulates Ctgf during endochonral ossification and aim to confirm this in this study. We discovered that Ctgf is overexpressed in craniofacial cartilage of mutant embryos at 4dpf and 6dpf. Also measurements of cartilage structures show evidence of skeletal defects in these embryos as early as 4dpf. This is evidence that the regulation of Ctgf by Ewsa is essential for normal chondrocyte maturation and skeletal development. In addition, a reduction of ctgfa signal in situ and Ctgf in immunohistochemistry assays was observed in the vascular structures in mutant embryos at 3dpf and 6dpf suggesting Ewsa may regulate ctgf expression in vascular tissue. Which is important since vascular invasion into mature chondrocytes is necessary in skeletal development. In summary, we discovered that Ewsa may regulate stage-specific, and tissue specific expression of Ctgf during embryonic development. Understanding the pathway of Ewsa-dependent skeletogenesis may supply a platform for the molecular pathogenesis of Ewing sarcoma
RNA Helicase 1 interacts with an ABCRNAi Transporter: Genetic Interactions with haf-6
Get PDFThe C. elegans rha-1 gene encodes a conserved helicase with ATP-dependent DEAD/H-box and double-stranded RNA binding domains. rha-1 is orthologous to the Drosophila maleless gene(MLE), an essential component of the dosage compensation machinery that leads to a two-fold increase in transcription of genes located on the single X chromosome of males in comparison to the transcription rate of homologous genes located on an X chromosome in females. The human ortholog, RNA helicase A (RHA), unwinds double-stranded DNA and RNA in a 3' to 5' direction. RHA is a component of several distinct protein complexes, including the RNA-induced silencing complex (RISC), the coding region determinant (CRD)-mediated complex, mRNP granules, and also associates with BRCA1, CREBbp or SMN1 and the RNA polymerase II complex, phosphorylated histones (H2AFX). RHA affects a number of different biological activities, including CRD-mediated mRNA stabilization, RNA splicing, translation, and transcription. It also has been discovered that regulation of RHA is disrupted in many types of cancers. The C. elegans rha-1 gene is expressed in the gonad where it localizes to the nucleus and the cytoplasm and is required for proper development. Defects in rha-1 lead to germline defects, aberrant expression of genes residing in repetitive transgene arrays, and defects in RNAi. ABC transporter proteins have also been implicated to play a role in RNAi in C. elegans. Ten out of the sixty-one ABC transporters are required for efficient RNAi in the germ line; these transporters are called ABCRNAi transporters. All of the ABCRNAi transporter mutants interact genetically with rde-2 (a novel protein) and mut-7 (a protein with homology to RnaseD). One of these ABCRNAi transporter genes is haf-6. The works presented here show that rha-1 mutants also interact genetically with haf-6, with rde-2, and with mut-7 with respect to RNAi defects. By contrast, mutations in rha-1 suppress the transposon mobilization phenotypes observed in haf-6 mutants. It is also observed that an unknown mutation is genetically interacting with rha-1 and haf-6. We are currently investigating roles for rha-1 in proper accumulation of haf-6 and other ABCRNAi transporter mRNAs in an effort to understand the interrelated functions of these proteins in RNAi
Taxotere suppresses breast cancer growth through inducing lincRNA-p21 expression
Get PDFIt has been reported that long non-coding RNA lincRNA-p21 is induced upon radiation and chemotherapy. This induction contributes to DNA-damage repair, cell death and cell cycle regulation. In this study, we focused on Taxotere (TXT) mediated chemotherapy of breast cancer cells and found that lincRNA-p21 is robotically induced upon TXT treatment. Secondly, we observed increased chemoresistance in three different breast cancer cell lines with lincRNA-p21 knockdown. Mechanistically, we found that lincRNA-p21 knockdown lead to decreased cell death during chemotherapy comparing to the negative control. In addition, our work showed that p21 is a downstream target of lincRNA-p21 in human breast cancer cells, and the loss of lincRNA-p21 caused p21 downregulation at both the RNA and protein levels
Ewing's Sarcoma EWS protein regulates skeletogenesis by modulation of SOX9
Get PDFEwing sarcoma is the second most common form of bone cancer in adolescents, characterized by the presence of an aberrant chimeric fusion gene EWS/FLI1. Wildtype EWS has been proposed to play a role in splicing and transcription. Currently, how these functions affect early developmental stages is unknown. To elucidate the function of EWS in early development, we analyzed an ewsa zebrafish mutant line originally isolated from an insertional mutagenesis method. We generated a Maternal Zygotic (MZ) ewsa/ewsa line because ewsa/wt and ewsa/ewsa zebrafish appear to be normal and are fertile. Alizarin Red staining revealed that there are skeletal formation defects in the lower jaw with an aberrant angle and position of the dentary and basihyal bones in adult MZ ewsa/ewsa mutants. Alcian blue staining revealed that the MZ ewsa/ewsa mutation leads to craniofacial defects with higher numbers of smaller cells with disorganized polarization compared to wt/wt zebrafish at four days post fertilization (dpf). In addition, there were reduced intervertebral discs and asymmetrical vertebrae leading to curved spines in MZ ewsa/ewsa mutants. MZ ewsa/ewsa mutants display disorganized alignment of Sox9 expressing neural crest cells at 27hpf. Because both craniofacial skeletons and vertebrae arise from Sox9 expressing cells, we hypothesized that EWS interacts with Sox9 and modulates the transcriptional regulation activity of Sox9. Co-immunoprecipitation (IP) experiment revealed that EWS interacts with SOX9. Furthermore, qPCR analysis identified that known SOX9 target genes are either upregulated (ctgfa, ctgfb, col2a1a, col2a1b) or downregulated (sox5, nog1, nog2, bmp4) in MZ ewsa/ewsa mutants compared to wt/wt zebrafish embryos. This is the first evidence for a tissue specific role of EWS in skeletogenesis and suggests a novel mechanism by which Sox9 transcriptional regulation is modulated as it directs endochondral bone and cartilage development
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
- …
