Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.We acknowledge funding from an EPSRC Platform grant which supported McMurray and a Wellcome Trust project grant which supported Wann and McMurray. Wann is now supported on an ARUK project grant. Thompson was funded by a BBSRC PhD studentshi

Id1 Interacts and Stabilizes the Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) in Nasopharyngeal Epithelial Cells

The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells

Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered

Quantitative Proteome Profiling of C. burnetii under Tetracycline Stress Conditions

The recommended antibiotic regimen against Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever

Toxicogenomic analysis of exposure to TCDD, PCB126 and PCB153: identification of genomic biomarkers of exposure to AhR ligands

<p>Abstract</p> <p>Background</p> <p>Two year cancer bioassays conducted by the National Toxicology Program have shown chronic exposure to dioxin-like compounds (DLCs) to lead to the development of both neoplastic and non-neoplastic lesions in the hepatic tissue of female Sprague Dawley rats. Most, if not all, of the hepatotoxic effects induced by DLC's are believed to involve the binding and activation of the transcription factor, the aryl hydrocarbon receptor (AhR). Toxicogenomics was implemented to identify genomic responses that may be contributing to the development of hepatotoxicity in rats.</p> <p>Results</p> <p>Through comparative analysis of time-course microarray data, unique hepatic gene expression signatures were identified for the DLCs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (100 ng/kg/day) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) (1000 ng/kg/day) and the non-DLC 2,2',4,4',5,5',-hexachlorobiphenyl (PCB153) (1000 μg/kg/day). A common time independent signature of 41 AhR genomic biomarkers was identified which exhibited at least a 2-fold change in expression following subchronic (13-wk) and chronic (52-wk) p.o. exposure to TCDD and PCB126, but not the non DLC, PCB153. Real time qPCR analysis validated that 30 of these genes also exhibited at least a 2-fold change in hepatic expression at 24 hr following a single exposure to TCDD (5 μg/kg, po). Phenotypic anchoring was conducted which identified forty-six genes that were differently expressed both following chronic p.o. exposure to DLCs and in previously reported studies of cholangiocarcinoma or hepatocellular adenoma.</p> <p>Conclusions</p> <p>Together these analyses provide a comprehensive description of the genomic responses which occur in rat hepatic tissue with exposure to AhR ligands and will help to isolate those genomic responses which are contributing to the hepatotoxicity observed with exposure to DLCs. In addition, the time independent gene expression signature of the AhR ligands may assist in identifying other agents with the potential to elicit dioxin-like hepatotoxic responses.</p

Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation

Most tissue cells grown in sparse cultures on linearly elastic substrates typically display a small, round phenotype on soft substrates and become increasingly spread as the modulus of the substrate increases until their spread area reaches a maximum value. As cell density increases, individual cells retain the same stiffness-dependent differences unless they are very close or in molecular contact. On nonlinear strain-stiffening fibrin gels, the same cell types become maximally spread even when the low strain elastic modulus would predict a round morphology, and cells are influenced by the presence of neighbors hundreds of microns away. Time lapse microscopy reveals that fibroblasts and human mesenchymal stem cells on fibrin deform the substrate by several microns up to five cell lengths away from their plasma membrane through a force limited mechanism. Atomic force microscopy and rheology confirm that these strains locally and globally stiffen the gel, depending on cell density, and this effect leads to long distance cell-cell communication and alignment. Thus cells are acutely responsive to the nonlinear elasticity of their substrates and can manipulate this rheological property to induce patterning

Investigating Population Genetic Structure in a Highly Mobile Marine Organism: The Minke Whale Balaenoptera acutorostrata acutorostrata in the North East Atlantic

Inferring the number of genetically distinct populations and their levels of connectivity is of key importance for the sustainable management and conservation of wildlife. This represents an extra challenge in the marine environment where there are few physical barriers to gene-flow, and populations may overlap in time and space. Several studies have investigated the population genetic structure within the North Atlantic minke whale with contrasting results. In order to address this issue, we analyzed ten microsatellite loci and 331 bp of the mitochondrial D-loop on 2990 whales sampled in the North East Atlantic in the period 2004 and 2007–2011. The primary findings were: (1) No spatial or temporal genetic differentiations were observed for either class of genetic marker. (2) mtDNA identified three distinct mitochondrial lineages without any underlying geographical pattern. (3) Nuclear markers showed evidence of a single panmictic population in the NE Atlantic according STRUCTURE's highest average likelihood found at K = 1. (4) When K = 2 was accepted, based on the Evanno's test, whales were divided into two more or less equally sized groups that showed significant genetic differentiation between them but without any sign of underlying geographic pattern. However, mtDNA for these individuals did not corroborate the differentiation. (5) In order to further evaluate the potential for cryptic structuring, a set of 100 in silico generated panmictic populations was examined using the same procedures as above showing genetic differentiation between two artificially divided groups, similar to the aforementioned observations. This demonstrates that clustering methods may spuriously reveal cryptic genetic structure. Based upon these data, we find no evidence to support the existence of spatial or cryptic population genetic structure of minke whales within the NE Atlantic. However, in order to conclusively evaluate population structure within this highly mobile species, more markers will be required