76 research outputs found
Translational masking via a translation regulatory particle (TRP) isolated from Xenopus laevis oocytes
No full textGeometric and computational models of chromatin fibre folding for human embryonic stem cells
No full textIn this study we analyze the chromatin state of human pluripotent stem cells by geometric and computational modelling of fibre conformation. The model takes into account local structure of chromatin organized into euchromatin, permissive for gene activation, and heterochromatin, transcriptionally silenced. Euchromatin was modelled using linear DNA while heterochromatin by means of a solenoid structure in which DNA winds onto six nucleosome spools per turn. Two geometric models are presented and are compared in terms of geometric quantities. The models are tested using in vivo data generated from chromatin human immunoprecipitation from embryonic stem cells. This study provides insight for identifying the relationships between chromosome geometry and epigenomic processes associated with chromatin remodeling, cellular reprograming and maintenance of cellular pluripotency
Modeling chromatin fibre folding for human embryonic stem cells
Get PDFIn this study we analyze the chromatin state of human pluripotent stem cells by geometric modelling of fibre conformation. The model takes into account local structure of chromatin organized into euchromatin, permissive for gene activation, and heterochromatin, transcriptionally silenced. Euchromatin modelled using linear DNA while heterochromatin by means of a solenoid structure in which DNA winds onto six nucleosome spools per turn. Two geometric models are presented and are compared in terms of geometric quantities. The models are tested using in vivo data generated from chromatin immunoprecipitation human from embryonic stem cells. This study provides insight into and tools for identifying the relationships between chromosome geometry and epigenomic processes associated with chromatin remodeling, cellular reprograming and maintenance of cellular pluripotency
GABA-B1 Receptor-Null Schwann Cells Exhibit Compromised In Vitro Myelination
Get PDFGABA-B receptors are important for Schwann cell (SC) commitment to a non-myelinating phenotype during development. However, the P0-GABA-B1fl/fl conditional knockout mice, lacking the GABA-B1 receptor specifically in SCs, also presented axon modifications, suggesting SC non-autonomous effects through the neuronal compartment. In this in vitro study, we evaluated whether the specific deletion of the GABA-B1 receptor in SCs may induce autonomous or non-autonomous cross-changes in sensory dorsal root ganglia (DRG) neurons. To this end, we performed an in vitro biomolecular and transcriptomic analysis of SC and DRG neuron primary cultures from P0-GABA-B1fl/fl mice. We found that cells from conditional P0-GABA-B1fl/fl mice exhibited proliferative, migratory and myelinating alterations. Moreover, we found transcriptomic changes in novel molecules that are involved in peripheral neuron-SC interaction.</p
Occurrence of L1M Elements in Chromosomal Rearrangements Associated to Chronic Myeloid Leukemia (CML): Insights from Patient-Specific Breakpoints Characterization
Get PDFChronic myeloid leukemia (CML) is a rare myeloproliferative disorder caused by the reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem cells (HSCs). This chromosomal translocation results in the formation of an extra-short chromosome 22, called a Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene responsible for the expression of a constitutively active tyrosine kinase that causes uncontrolled growth and replication of leukemic cells. Mechanisms behind the formation of this chromosomal rearrangement are not well known, even if, as observed in tumors, repetitive DNA may be involved as core elements in chromosomal rearrangements. We have participated in the explorative investigations of the PhilosoPhi34 study to evaluate residual Ph+ cells in patients with negative FISH analysis on CD34+/lin- cells with gDNA qPCR. Using targeted next-generation deep sequencing strategies, we analyzed the genomic region around the t(9;22) translocations of 82 CML patients and one CML cell line and assessed the relevance of interspersed repeat elements at breakpoints (BP). We found a statistically higher presence of LINE elements, in particular belonging to the subfamily L1M, in BP cluster regions of both chromosome 22 and 9 compared to the whole human genome. These data suggest that L1M elements could be potential drivers of t(9;22) translocation leading to the generation of the BCR-ABL1 chimeric gene and the expression of the active BCR-ABL1-controlled tyrosine kinase chimeric protein responsible for CML
RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy
No full textCellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262–1269, 2017. © 2016 Wiley Periodicals, Inc
Three-dimensional cultures for collecting CNS and PNS glial secreted factors, vesicles and scaffolds free of animal serum components
Get PDFGlial cells provide physical and chemical support and protection for neurons. Secreted neurotrophic factors, scaffolds and vesicles regulate in addition to normal homeostasis, repair following neural tissue injury or degeneration. We previously demonstrated that human CNS glia have revascularization capacity by inducing endothelial cell sprouting and microchanneling driven by glial cell secreted components. The clinical potential of using secreted factors and vesicles generated by glial cells in conditioned media was shown by their re-normalization of pathological blood vessels and 3D tissue wound healing and remodeling capacity. To identify secreted components of glial cells and how CNS and PNS glial cells differentially regulate neural tissue homeostasis, disease and injury response, we developed a novel culture method for various types of glial cells that allow for isolating and characterizing secreted glial components, free of animal factors and contaminants normally associated with serum-based culture conditions. Our culture method will enable to identify secreted components using ultrasensitive RNA, glycoconjugate and protein analysis technologies. The characterization of glial secreted factors involved microchanneling may help in development of new clinical therapies for glial and neural cells or axons to migrate and reform connections
Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels
Get PDFDuring embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis
A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation
No full textThe cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal-alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level
Wear Improvements Induced by Thermally Grown Oxide Layers and by Nitrogen Ion Implantation
No full textMild steels which have been either thermally oxidized near 300°C or implanted with nitrogen ions have been wear tested using a modified Falex Lubricant Testing machine. Both these treatments cause similar, much reduced wear rates which are about an order of magnitude better than in untreated samples. Specimens worn after treatments by both methods have been examined in detail using scanning electron microscopy and Auger spectroscopy. It is concluded that the wear induced by oxidation is the same as the wear induced by nitrogen ion implantation. Both of these treatments acted to initiate favorable sustained oxide wear and confirm the Initiator/Sustainer model. Additional studies have been done on oxidized samples using a variety of sample processing techniques and sequences including oxide layer stripping, wear recycling, and annealing. These studies clearly show that favorable wear results were caused by the grown oxide layer and not by the heat treatment. Also, it has been found that the favorable oxide wear can be stopped and started numerous times since it is able to reinitiate and sustain itself. However, if a worn oxide layer is stripped from a sample just prior to the wear run, favorable wear does not occur unless the sample was oxidized or favorably worn in the last few weeks. This occurs because wear favorable chemistry at the oxide/metal interface anneals at room temperature. Other discussions of the wear initiating and sustaining processes are presented
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