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Salces-Ortiz J.,INIA | Gonzalez C.,INIA | Bolado-Carrancio A.,Systems Biology Ireland | Rodriguez-Rey J.C.,University of Cantabria | And 2 more authors.
Cell Stress and Chaperones | Year: 2015

When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extrated from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account. © 2015, Cell Stress Society International. Source

Kilinc D.,University College Dublin | Kilinc D.,UCD Conway Institute | Schwab J.,University College Dublin | Rampini S.,University College Dublin | And 10 more authors.
Integrative Biology (United Kingdom) | Year: 2016

We present a microfluidic chip that generates linear concentration gradients of multiple solutes that are orthogonally-aligned to each other. The kinetics of gradient formation was characterized using a fluorescent tracer matching the molecular weight of small inhibitory drugs. Live-cell signalling and motility experiments were conducted to demonstrate the potential uses and advantages of the device. A431 epidermoid carcinoma cells, where EGF induces apoptosis in a concentration-dependent manner, were simultaneously exposed to gradients of MEK inhibitor and EGF receptor (EGFR) inhibitor. By monitoring live caspase activation in the entire chip, we were able to quickly assess the combinatorial interaction between MEK and EGFR pathways, which otherwise would require costly and time consuming titration experiments. We also characterized the motility and morphology of MDA-MB-231 breast cancer cells exposed to orthogonal gradients of EGF and EGFR inhibitor. The microfluidic chip not only permitted the quantitative analysis of a population of cells exposed to drug combinations, but also enabled the morphological characterization of individual cells. In summary, our microfluidic device, capable of establishing concentration gradients of multiple compounds over a group of cells, facilitates and accelerates in vitro cell biology experiments, such as those required for cell-based drug combination assays. © 2016 The Royal Society of Chemistry. Source

News Article
Site: http://phys.org/biology-news/

Accountants everywhere would cry as their books ceased to balance. You might find yourself pausing at the supermarket wondering if you could save money by buying your milk before your toothpaste. Eurovision fans may value a 'douze points' early in the voting more than one later on. Fortunately, there is no new evidence to suggest that order matters in addition. However, researchers at UCD have recently found that order matters a surprising amount for sequence alignment, an important part of modern genetic analyses. Sequence alignment is used to understand similarities and differences between proteins found in different species. Proteins are the building blocks of life and carry out most of the functions in our cells. Consequently understanding proteins and their function is a key part of biology. We can use pairwise sequence alignment to identify which parts of a specific protein are identical between a pair of species (e.g. humans and chimps). Using multiple sequence alignment, we can identify those parts of a protein that are conserved in all mammals or in even larger groups of species. Very large sequence alignments help us understand which parts of proteins are important; if part of a protein is identical in all mammals then it's probably important and also gives us some insight into the three dimensional structure of proteins (as parts of a protein that are close together in 3D space tend to change together across species). PhD student Kieran Boyce, Dr Fabian Sieviers and Professor Des Higgins in UCD Conway Institute & Systems Biology Ireland found that for large protein sequence alignments the order in which sequences are compared matters, i.e. the alignment that you get out of a sequence alignment programme depends on the order that you input your sequences into the programme. This finding is surprising, as people have been performing sequence alignments for decades without knowing how dependent the results are on the input order. It is important because it suggests that most scientific publications that make use of large multiple sequence alignments probably have not provided sufficient information to reproduce their results. Reproducibility is an important part of science. Consequently, when performing sequence alignment, most scientists will provide details of the sequence alignment programme and the settings they used. The findings from Boyce et al suggest that the order is also an important setting that may need to be provided from now on. A question left open by the paper is how we can make sequence alignment programmes ignore order, or how we can choose the best possible ordering. Explore further: Relationships in rank and file: Better sequence searches of genes and proteins More information: Kieran Boyce et al. Instability in progressive multiple sequence alignment algorithms, Algorithms for Molecular Biology (2015). DOI: 10.1186/s13015-015-0057-1

Kilinc D.,University College Dublin | Lesniak A.,University College Dublin | Rashdan S.A.,University College Dublin | Rashdan S.A.,University of Bahrain | And 8 more authors.
Advanced Healthcare Materials | Year: 2015

Multifunctional nanoparticles that actively target-specific tissues are studied for cancer diagnosis and treatment. Magnetically and optically active particles are of particular interest because they enable multiple imaging modalities and physically modulated therapies, such as magnetic hyperthermia. Fe-Au nanorods are synthesized that have a long iron segment, coated with polyethylene glycol, and a short gold tip functionalized with heregulin (HRG), a known ligand of ErbB family of receptors. HRG-nanorods preferentially target MCF7 cells relative to MDA-MB-231 cells, as demonstrated in a novel microfluidics device. Targeting rates of these classical breast cancer cells correlate with their differential expression of ErbB2/3 receptors. HRG-nanorod binding stimulates the extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in MCF7 cells. The increase in ERK phosphorylation is linked to "active zones," dynamic regions in the cell periphery, which exhibit higher rates of particle binding than the rest of the cell. Periodically stretching cells using magnetic tweezers further activates ERK, which leads to cell death in cells co-treated with B-Raf inhibitors, through ERK hyperactivation. Although to a lesser extent, cell death is also achieved through magnetic hyperthermia. These results demonstrate nanoscale targeting and localized mechanochemical treatment of specific cancer cell lines based on their receptor expression using multifunctional nanoparticles. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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