Detection of long repeat expansions from PCR-free whole-genome sequence data

Identifying large expansions of short tandem repeats (STRs) such as those that cause amyotrophic lateral sclerosis (ALS) and fragile X syndrome is challenging for short-read whole-genome sequencing (WGS) data. A solution to this problem is an important step towards integrating WGS into precision medicine. We have developed a software tool called ExpansionHunter that, using PCR-free WGS short-read data, can genotype repeats at the locus of interest, even if the expanded repeat is larger than the read length. We applied our algorithm to WGS data from 3,001 ALS patients who have been tested for the presence of the C9orf72 repeat expansion with repeat-primed PCR (RP-PCR). Compared against this truth data, ExpansionHunter correctly classified all (212/212, 95% CI [0.98, 1.00]) of the expanded samples as either expansions (208) or potential expansions (4). Additionally, 99.9% (2,786/2,789, 95% CI [0.997, 1.00]) of the wild type samples were correctly classified as wild type by this method with the remaining three samples identified as possible expansions. We further applied our algorithm to a set of 152 samples where every sample had one of eight different pathogenic repeat expansions including those associated with fragile X syndrome, Friedreich's ataxia and Huntington's disease and correctly flagged all but one of the known repeat expansions. Thus, ExpansionHunter can be used to accurately detect known pathogenic repeat expansions and provides researchers with a tool that can be used to identify new pathogenic repeat expansions. The software is licensed under GPL v3.0 and the source code is freely available on GitHub

Accumulation of an alkyl lysophospholipid in tumor cell membranes affects membrane fluidity and tumor cell invasion

Tumor cells grown in the presence of 1-O-alkyl-2-O-methylglycero-3-phosphocholine (AMG-PC) accumulated this ether lipid in their membranes. Depending on the cell type and the dose of the compound, up to 17% of the total phospholipids of the purified plasma membranes consisted of authentic AMG-PC. Extensive incorporation of the agent resulted in a decrease in plasma membrane fluidity and inhibition of tumor cell invasiveness in embryonic chick heart fragments. The extent of AMG-PC incorporation and fluidity change was not strictly correlated with the degree to which tumor cell invasion was inhibited. © 1985 American Oil Chemists' Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Diacylglycerol kinase counteracts protein kinase C-mediated inactivation of the EGF receptor

Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC)signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGK isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGK physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGK attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGK downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGK translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGK attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signalin

Modeling mechanical signals on the surface of microCT and CAD based rapid prototype scaffold models to predict (early stage) tissue development

Item does not contain fulltextIn the field of tissue engineering, mechano-regulation theories have been applied to help predict tissue development in tissue engineering scaffolds in the past. For this, finite element models (FEMs) were used to predict the distribution of strains within a scaffold. However, the strains reported in these studies are volumetric strains of the material or strains developed in the extracellular matrix occupying the pore space. The initial phase of cell attachment and growth on the biomaterial surface has thus far been neglected. In this study, we present a model that determines the magnitude of biomechanical signals on the biomaterial surface, enabling us to predict cell differentiation stimulus values at this initial stage. Results showed that magnitudes of the 2D strain-termed surface strain-were lower when compared to the 3D volumetric strain or the conventional octahedral shear strain as used in current mechano-regulation theories. Results of both microCT and CAD derived FEMs from the same scaffold were compared. Strain and fluid shear stress distributions, and subsequently the cell differentiation stimulus, were highly dependent on the pore shape. CAD models were not able to capture the distributions seen in the microCT FEM. The calculated mechanical stimuli could be combined with current mechanobiological models resulting in a tool to predict cell differentiation in the initial phase of tissue engineering. Although experimental data is still necessary to properly link mechanical signals to cell behavior in this specific setting, this model is an important step towards optimizing scaffold architecture and/or stimulation regimes. Biotechnol. Bioeng. 2014;111: 1864-1875. (c) 2014 Wiley Periodicals, Inc

Increased cell seeding efficiency in bioplotted three-dimensional PEOT/PBT scaffolds

In regenerative medicine studies, cell seeding efficiency is not only optimized by changing the chemistry of the biomaterials used as cell culture substrates, but also by altering scaffold geometry, culture and seeding conditions. In this study, the importance of seeding parameters, such as initial cell number, seeding volume, seeding concentration and seeding condition is shown. Human mesenchymal stem cells (hMSCs) were seeded into cylindrically shaped 4 × 3 mm polymeric scaffolds, fabricated by fused deposition modelling. The initial cell number ranged from 5 × 104 to 8 × 105 cells, in volumes varying from 50 µl to 400 µl. To study the effect of seeding conditions, a dynamic system, by means of an agitation plate, was compared with static culture for both scaffolds placed in a well plate or in a confined agarose moulded well. Cell seeding efficiency decreased when seeded with high initial cell numbers, whereas 2 × 105 cells seemed to be an optimal initial cell number in the scaffolds used here. The influence of seeding volume was shown to be dependent on the initial cell number used. By optimizing seeding parameters for each specific culture system, a more efficient use of donor cells can be achieved

Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts.

Contains fulltext : 50331.pdf (publisher's version ) (Closed access)PURPOSE: Combined modality treatment has improved outcome in various solid tumors. Besides classic anticancer drugs, a new generation of biological response modifiers has emerged that increases the efficacy of radiation. Here, we have investigated whether perifosine, an orally applicable, membrane-targeted alkylphospholipid, enhances the antitumor effect of radiation in vitro and in vivo. EXPERIMENTAL DESIGN: Several long-term and short-term in vitro assays (clonogenic survival, sulforhodamine B cytotoxicity, apoptosis, and cell cycle analysis) were used to assess the cytotoxic effect of perifosine in combination with radiation. In vivo, the response of human KB squamous cell carcinoma xenografts was measured after treatment with perifosine, irradiation, and the combination. Radiolabeled perifosine was used to determine drug disposition in tumor and normal tissues. At various intervals after treatment, tumor specimens were collected to document histopathologic changes. RESULTS: In vitro, perifosine reduced clonogenic survival, enhanced apoptosis, and increased cell cycle arrest after radiation. In vivo, radiation and perifosine alone induced a dose-dependent tumor growth delay. When combining multiple perifosine administrations with single or split doses of radiation, complete and sustained tumor regression was observed. Histopathologic analysis of tumor specimens revealed a prominent apoptotic response after combined treatment with radiation and perifosine. Radiation-enhanced tumor response was observed at clinically relevant plasma perifosine concentrations and accumulating drug disposition of >100 microg/g in tumor tissue. CONCLUSIONS: Perifosine enhances radiation-induced cytotoxicity, as evidenced by reduced clonogenic survival and increased apoptosis induction in vitro and by complete tumor regression in vivo. These data provide strong support for further development of this combination in clinical studies

Biological and Tribological Assessment of Poly(Ethylene Oxide Terephthalate)/Poly(Butylene Terephthalate), Polycaprolactone, and Poly (L\DL) Lactic Acid Plotted Scaffolds for Skeletal Tissue Regeneration

Additive manufactured scaffolds are fabricated from three commonly used biomaterials, polycaprolactone (PCL), poly (L\DL) lactic acid (P(L\DL)LA), and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT). Scaffolds are compared biologically and tribologically. Cell-seeded PEOT/PBT scaffolds cultured in osteogenic and chondrogenic differentiation media show statistical significantly higher alkaline phosphatase (ALP) activity/DNA and glycosaminoglycans (GAG)/DNA ratios, followed by PCL and P(L\DL)LA scaffolds, respectively. The tribological performance is assessed by determining the friction coefficients of the scaffolds at different loads and sliding velocities. With increasing load or decreasing sliding velocity, the friction coefficient value decreases. PEOT/PBT show to have the lowest friction coefficient value, followed by PCL and P(L\DL)LA. The influence of the scaffold architecture is further determined with PEOT/PBT. Reducing of the fiber spacing results in a lower friction coefficient value. The best and the worst performing scaffold architecture are chosen to investigate the effect of cell culture on the friction coefficient. Matrix deposition is low in the cell-seeded scaffolds and the effect is, therefore, undetermined. Taken together, our studies show that PEOT/PBT scaffolds support better skeletal differentiation of seeded stromal cells and lower friction coefficient compared to PCL and P(L/DL)A scaffolds