The chimeric EWSR1::FLI1 transcription factor is the main oncogenic event in Ewing sarcoma. Recently, it has been proposed that EWSR1::FLI1 levels can fluctuate in Ewing sarcoma cells, giving rise to ...two cell populations. EWSR1::FLI1
cells present a migratory and invasive phenotype, while EWSR1::FLI1
cells are more proliferative. In this work, we described how the CD44 standard isoform (CD44s), a transmembrane protein involved in cell adhesion and migration, is overexpressed in the EWSR1::FLI1
phenotype. The functional characterization of CD44s (proliferation, clonogenicity, migration, and invasion ability) was performed in three doxycycline-inducible Ewing sarcoma cell models (A673, MHH-ES1, and CADO-ES1). As a result, CD44s expression reduced cell proliferation in all the cell lines tested without affecting clonogenicity. Additionally, CD44s increased cell migration in A673 and MHH-ES1, without effects in CADO-ES1. As hyaluronan is the main ligand of CD44s, its effect on migration ability was also assessed, showing that high molecular weight hyaluronic acid (HMW-HA) blocked cell migration while low molecular weight hyaluronic acid (LMW-HA) increased it. Invasion ability was correlated with CD44 expression in A673 and MHH-ES1 cell lines. CD44s, upregulated upon EWSR1::FLI1 knockdown, regulates cell migration and invasion in Ewing sarcoma cells.
Ewing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 promotes a specific ...cellular transcriptional program. Therefore, the study of EWSR1-FLI1 target genes is important to identify critical pathways involved in Ewing sarcoma tumorigenesis. In this work, we focused on the transcription factors regulated by EWSR1-FLI1 in Ewing sarcoma. Transcriptomic analysis of the Ewing sarcoma cell line A673 indicated that one of the genes more strongly upregulated by EWSR1-FLI1 was FEZF1 (FEZ family zinc finger protein 1), a transcriptional repressor involved in neural cell identity. The functional characterization of FEZF1 was performed in three Ewing sarcoma cell lines (A673, SK-N-MC, SK-ES-1) through an shRNA-directed silencing approach. FEZF1 knockdown inhibited clonogenicity and cell proliferation. Finally, the analysis of the FEZF1-dependent expression profile in A673 cells showed several neural genes regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1. In summary, FEZF1 is transcriptionally regulated by EWSR1-FLI1 in Ewing sarcoma cells and is involved in the regulation of neural-specific genes, which could explain the neural-like phenotype observed in several Ewing sarcoma tumors and cell lines.
Tyr is the mouse gene that encodes tyrosinase, an enzyme that triggers the first and rate‐limiting step in the biosynthesis of melanin. Mutations in Tyr might result in non‐functional Tyr protein ...and, consequently, loss of pigment production. This is a rare genetic condition, known as albinism, described for most animal species and one of the most obvious and simple phenotypes to investigate in model organisms. Mutations in the orthologous human TYR gene are associated with oculocutaneous albinism type 1 (OCA1). Over the last thirty years, the mouse Tyr locus has been studied as a paradigm for how genes and expression domains are organized and regulated in mammalian genomes. This review summarizes the major findings and experimental strategies used, from the production of conventional transgenic mice to the latest CRISPR‐Cas9 genome‐edited animals. The main conclusion inferred from all of these studies, which extends beyond the analysis of the mouse Tyr locus, is the relevance of analyzing non‐coding regulatory DNA elements in their natural chromosomal environment, and not only as randomly inserted transgenes. Further, the identification of evolutionary conserved regulatory sequences might highlight new vulnerable sites in the human TYR gene, whose mutations could also be associated with albinism.
A history of genome editing in mammals Fernández, Almudena; Josa, Santiago; Montoliu, Lluis
Mammalian genome,
08/2017, Letnik:
28, Številka:
7-8
Journal Article
Recenzirano
Genome editing is now a routine procedure in many mammalian genetics laboratories. The ostensibly short but intense history of genome-editing approaches illustrates how a disruptive technology can ...universally colonize a field when this new methodology, conceived to alter mammalian genomes at specific locations, is found to efficiently and robustly deliver results. This review summarizes the early development of genome editing using nucleases, from the pioneering experiments using yeast meganucleases, to the latest prokaryotic nucleases used for precise genome manipulation. Gene-editing nucleases belong to one of three known categories: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) tools. All operate on the same principle; they are all capable of inducing a double-strand break (DSB) at a defined genomic sequence that is subsequently corrected by endogenous DNA repair mechanisms. DSBs can be repaired through non-homologous end joining (NHEJ), resulting in small insertions and/or deletions (INDELs) and, hence, often leading to gene disruption. Alternatively, DSBs can be repaired through homology-driven repair (HDR), in the presence of donor homologous DNA sequences, resulting in gene-editing events.
High-resolution isotropic three-dimensional reconstructions of human brain gray and white matter structures can be characterized to quantify aspects of their shape, volume and topological complexity. ...In particular, methods based on fractal analysis have been applied in neuroimaging studies to quantify the structural complexity of the brain in both healthy and impaired conditions. The usefulness of such measures for characterizing individual differences in brain structure critically depends on their within-subject reproducibility in order to allow the robust detection of between-subject differences. This study analyzes key analytic parameters of three fractal-based methods that rely on the box-counting algorithm with the aim to maximize within-subject reproducibility of the fractal characterizations of different brain objects, including the pial surface, the cortical ribbon volume, the white matter volume and the gray matter/white matter boundary. Two separate datasets originating from different imaging centers were analyzed, comprising 50 subjects with three and 24 subjects with four successive scanning sessions per subject, respectively. The reproducibility of fractal measures was statistically assessed by computing their intra-class correlations. Results reveal differences between different fractal estimators and allow the identification of several parameters that are critical for high reproducibility. Highest reproducibility with intra-class correlations in the range of 0.9–0.95 is achieved with the correlation dimension. Further analyses of the fractal dimensions of parcellated cortical and subcortical gray matter regions suggest robustly estimated and region-specific patterns of individual variability. These results are valuable for defining appropriate parameter configurations when studying changes in fractal descriptors of human brain structure, for instance in studies of neurological diseases that do not allow repeated measurements or for disease-course longitudinal studies.
•The study quantifies the reliability of fractal measures within the human brain.•Parameter settings that ensure high reproducibility are identified.•All results provided were validated in two separate subject cohorts.•Estimates of fractal dimension can be derived with high intra-class correlation.•We found robust and reproducible regional differences in fractal dimension.
Recently, some authors have severely criticised the incorporation of the notion of autonomous components of aggregate demand in demand-led growth theory. We show that these components are present in ...Richard Cantillon’s Essai written in the XVIIth century, and that an implicit demand-led theory of capital accumulation can be also developed based on his writings.
The aim of this paper is to use an integrative approach to identify the main correlates and/or predictors at different levels (personal, psychosocial, occupational and outside the workplace) of the ...burnout dimensions. The sample consists of 813 university professors. Results from statistical analyses show that there are, indeed, both common and specific predictors for the different facets of the syndrome. Specifically, while social support and optimism are selected to confirm all manifestations of burnout, other factors (work hours per week, time in the profession, hardiness, Type A behavioural pattern, life events, daily hassles) do increase the emergence of differential profiles. Lastly, findings are discussed and the main conclusions are presented.
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