Trevor DeVaney

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA1, LPA2, and LPA3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

Endothelin (ET)-1 and ET-3 promote expression of c-fos and c-jun in human choriocarcinoma via ETB receptor-mediated Gi- and Gq-pathways and MAP kinase activation

Background and purpose: Endothelins (ETs) and their G protein‐coupled receptors exert key physiological functions during normal and aberrant placental development. Trophoblast cells mediate the contact between the embryo and the mother, by establishing a transient organ, the placenta. Choriocarcinoma cells display many of the biochemical and morphological characteristics of in utero invasive trophoblast cells and may therefore be used as a suitable model to study epithelial tumour progression of foetal‐derived cells.

Reflux Symptom Index and Reflux Finding Score in Otolaryngologic Practice

OBJECTIVES To evaluate whether patients with abnormal Reflux Symptom Index (RSI) and Reflux Finding Score (RFS) benefit from proton pump inhibitor (PPI) therapy. STUDY DESIGN Open, multicenter, prospective longitudinal cohort study. METHODS Patients with suspected reflux-associated laryngologic symptoms were evaluated by 40 community practice otolaryngologists using RSI and RFS. Patients were treated with pantoprazole 40-80 mg/d for 8-12 weeks if RSI was greater than 9 and RFS greater than 7. Pre- and posttherapeutic RSI and RFS were compared using Wilcoxon signed rank test and additionally controlled with the symmetry test of Bowker. RESULTS A total of 1044 patients were included over a period of 20 months. Median total score of RSI before therapy was 12 and decreased to 3 (P≪0.001). Median total score of RFS before therapy was 16 and decreased to 6 (P≪0.001). Assessment of the treatment effect by otolaryngologists and patients was judged as being excellent in at least 50%. In 2% of the patients, gastrointestinal side effects were documented. CONCLUSION RSI and RSF are easy to administer in the routine care of patients suspected of having laryngopharyngeal reflux. Patients identified by positive results of these tests have a high likelihood of excellent improvement after 8-12 weeks of PPI treatment. By implementation of RFS and RSI in daily use, most patients may not need time-consuming and cost-intensive examinations in the first-line assessment of LPR. These examinations can be reserved for nonresponders, and uncontrolled prescription of PPIs can be restricted.

The influence of edge detection algorithms on the estimation of the fractal dimension of binary digital images

The boundary of a fractal object, represented in a two-dimensional space, is theoretically a line with an infinitely small width. In digital images this boundary or contour is limited to the pixel resolution of the image and the width of the line commonly depends on the edge detection algorithm used. The Minkowski dimension was evaluated by using three different edge detection algorithms (Sobel, Roberts, and Laplace operator). These three operators were investigated because they are very widely used and because their edge detection result is very distinct concerning the line width. Very common fractals (Sierpinski carpet and Koch islands) were investigated as well as the binary images from a cancer invasion assay taken with a confocal laser scanning microscope. The fractal dimension is directly proportional to the width of the contour line and the fact, that in practice very often the investigated objects are fractals only within a limited resolution range is considered too.

Piezo1 forms mechanosensitive ion channels in the human MCF-7 breast cancer cell line

Mechanical interaction between cells – specifically distortion of tensional homeostasis-emerged as an important aspect of breast cancer genesis and progression. We investigated the biophysical characteristics of mechanosensitive ion channels (MSCs) in the malignant MCF-7 breast cancer cell line. MSCs turned out to be the most abundant ion channel species and could be activated by negative pressure at the outer side of the cell membrane in a saturable manner. Assessing single channel conductance (GΛ) for different monovalent cations revealed an increase in the succession: Li+ < Na+ < K+ ≈Rb+ ≈ Cs+. Divalent cations permeated also with the order: Ca2+ < Ba2+. Comparison of biophysical properties enabled us to identify MSCs in MCF-7 as ion channels formed by the Piezo1 protein. Using patch clamp technique no functional MSCs were observed in the benign MCF-10A mammary epithelial cell line. Blocking of MSCs by GsMTx-4 resulted in decreased motility of MCF-7, but not of MCF-10A cells, underscoring a possible role of Piezo1 in invasion and metastatic propagation. The role of Piezo1 in biology and progression of breast cancer is further substantiated by markedly reduced overall survival in patients with increased Piezo1 mRNA levels in the primary tumor.

How much resolution is enough? Influence of downscaling the pixel resolution of digital images on the generalised dimensions

Fractals are very often illustrated by digital images, this is a consequence of the widespread availability of electronic and computational equipment. These images are always limited by their pixel resolution. Subsequently the determination of dimensions for such images is restricted practically because for their calculation the range of scales is not infinite. Downscaling the pixel resolution of images to get images with reduced resolution worsens the achievable absolute computational results but surprisingly the changes in these values and especially the changes of the courses of the calculated generalised dimensions give very promising results. This is due to the qualitative nature of these changes with respect to pixel downscaling. This opens the possibility of determining the quality of the estimation of the fractal dimensions of any image with any particular pixel resolution. Furthermore the pattern of changes itself caused by downscaling is different between the individual fractals. Several geometrical fractals (Sierpinski gasket, a fern, a Menger gasket and a modified Menger gasket with circles) as well as an example of a measured fractal, the digital images of cancer invasion studies in vitro were investigated to study the influence of downscaling the pixel resolution of digital images on the estimation of fractal dimensions.

Fractal dimension of K1735 mouse melanoma clones and spheroid invasion in vitro.

Abstract. An in vitro tumour-host confrontation method to investigate the invasion behaviour of cancer has been applied to K1735 mouse melanomas. Fluorescently labelled spheroids of cancer cells and host cells were confronted and the temporal course of cancer invasion into the host was investigated using confocal laser scanning microscopy. To improve the quantitative data of this method, the boundary images of the fluorescently labelled confrontation pairs were treated as fractals. The physical and mathematical framework for determination of the fractal capacity dimension is widely used in biology and medicine and has proved to be a very useful tool for describing the cancer invasion process. The fractal capacity dimension determination was carried out by dilation of the binary boundaries of the objects, which were treated as an estimate of the Minkowski-Bouligand dimension. The fractal dimension correlated well with the degree of invasion of the K1735-M2 clone. Control experiments, with host-host confrontations and various K1735 clones with reduced invasiveness, support these results.

Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-JunS73 phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment.

Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line

The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA‐MB‐453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a–e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNAs, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNAs encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer. J. Cell. Biochem. 110: 598–608, 2010.

Carcinoma of the Larynx: Predictors of Diagnostic Delay

Objectives In tumors of the upper aerodigestive tract a dose‐response relationship between tumor stage and its prognosis raises the chance of a reduction in the diagnostic delay. The interval between the first symptom and the start of tumor‐specific therapy and the influencing factors is not well known. The goals of this study were to investigate the diagnostic delay and the influencing factors and to predict the factors that prolong the diagnostic process.