The A2A adenosine receptor: its role in suppressing vascular inflammation and its regulation by phosphorylation

Endothelial inflammation leading to vascular dysfunction is a major contributor to the development of atherosclerosis. The release of adenosine at sites of inflammation represents an endogenous mechanism for limiting excessive inflammation and tissue damage. The majority of the anti-inflammatory effects of adenosine are mediated by signalling through the A2AAR and activation of the A2AAR has been shown to be protective in numerous models of inflammatory disease. Little is known about the molecular mechanisms behind these effects. However, in vitro studies using cultured endothelial cells indicate that signalling through the A2AAR can suppress activation of the NF kappa B and JAK/STAT proinflammatory signalling pathways. NF kappa B appears to be primed for activation in atherosclerosis-prone regions of the aorta indicating that suppression of NF kappa B signalling may protect against the development of atherosclerosis. In this study, the role of the A2AAR in regulating NF kappa B and JAK/STAT signalling pathway activation in the aorta was studied using A2AAR-deficient mice subjected to an LPS-induced model of septic shock. In response to LPS treatment, these mice displayed markedly elevated plasma levels of the pro-inflammatory cytokines TNF-alpha, IL-6, IL-1 beta and GMCSF compared to wild-type mice. Consistent with this finding, heightened activation of the NF kappa B and JAK/STAT pathways was detected in aortic protein samples from A2AAR-deficient mice as demonstrated by increased levels of the phosphorylated forms of I kappa B alpha and STAT1. However, expression of the NF kappa B and STAT1-regulated genes ICAM-1, VCAM-1 and TAP-1 was unaffected indicating the involvement of compensatory negative feedback mechanisms. These findings confirm a role for the A2AAR in regulation of pro-inflammatory signalling in the aorta. Further analysis of mechanisms which mediate this response may reveal new targets for therapeutic intervention to suppress inflammation in inflammatory disorders such as atherosclerosis. While the wide range of anti-inflammatory and tissue-protective responses elicited by the A2AAR have been well documented, the molecular regulation of the A2AAR has been less well studied. The presence of several serine and threonine residues in the extended C-terminal tail of the A2AAR suggests that it may be regulated by phosphorylation events occurring in this region. Indeed, the canine A2AAR is phosphorylated in response to PKC activation. Interestingly, several proteins have recently been identified as being able to interact with the C-terminal tail of the A2AAR. However, how these interactions are regulated is not known. One of the aims of this study was to characterise phosphorylation of the human A2AAR and to determine whether this could provide a means for regulating the binding of C-terminal interacting proteins. This was examined using human umbilical vein endothelial cells infected with recombinant adenovirus encoding the human A2AAR. It was found that phosphorylation of the human A2AAR could be induced in HUVECs by treatment with PMA or by stimulation of endogenous histamine H1 receptors. This was due to activation of PKC, as phosphorylation was inhibited by the PKC inhibitor GF109203X and by depletion of PKC following chronic treatment with PMA. Treatment of cells with the calcium-chelating agent BAPTA/AM did not inhibit PMA-induced phosphorylation indicating that a calcium-insensitive isoform of PKC was responsible. Meanwhile an siRNA-mediated gene silencing approach confirmed that PKC epsilon was not responsible indicating the involvement of either PKC delta or PKC theta. Previously reported interactions between the A2AAR and TRAX and 14-3-3 tau were confirmed in vitro by GST pull-down assay. Binding of 14-3-3 tau to the A2AAR appeared to be unaffected by treatment of HUVECs with PMA. However, A2AAR complex formation with TRAX was significantly reduced in samples from PMA-stimulated cells. These findings indicate that PKC-mediated phosphorylation may represent a means of regulating which proteins can interact with the C-terminal tail of the A2AAR. This may allow the A2AAR to initiate distinct signalling pathways depending on the cellular context in order to achieve the appropriate response

Motivating the knowledge worker to perform

Competitive advantage for any organisation relies on the output of knowledge workers. The more motivated the knowledge worker the more likely they are to perform. This research explores the links between motivation and performance and creates a better understanding of which motivational factors would fall within the ambit of the psychological contract. The secondary portion of the research was to clarify if different types of knowledge workers performed in response to motivation in the same way. The research was done through in-depth interviews of knowledge workers in the 4 defined clusters. A total of 26 interviews were conducted. A questionnaire using open ended questions was used to guide the interview. The interviews were recorded, and the key themes in the results were captured, transformed and analysed, after which a ranking process was done on the results. The findings were analysed from various perspectives. The research found that there are differences in the groups studied from a number of perspectives, such as; motivation and performance scores, the motivation factors, the factors that act against performance, the organisational and individual duties as would be ‘held’ in the psychological contract. The research found that most of the factors that motivate are in the psychological contract. The findings strongly recommend that management needs to fully understand and manage the unique motivators of each individual knowledge worker in order to obtain maximum performance and hence competitive advantage.Dissertation (MBA)--University of Pretoria, 2010.Gordon Institute of Business Science (GIBS)unrestricte

Polarizing intestinal epithelial cells electrically through Ror2

© 2014. Published by The Company of Biologists Ltd.Peer reviewedPublisher PD

Low expression of chloride channel accessory 1 predicts a poor prognosis in colorectal cancer

© 2014 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. Funded by Friends of ANCHOR NHS Grampian Endowment Fund. Grant Number: 12/50Peer reviewedPublisher PD

New insights on Laminaria digitata ultrastructure through combined conventional chemical fixation and cryofixation

Acknowledgements The research leading to these results received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No .730984, ASSEMBLE Plus project, supporting access of CK and FCK to the Station Biologique de Roscoff. This work was conducted in conjunction with the European Marine Biological Resource Centre (EMBRC-ERIC), EMBRC-France. French state funds are managed by the ANR within the Investments of the Future program under reference ANR-10-INSB-02. Also, funding from the UK Natural Environment Research Council (NERC) through grants NE/D521522/1, NE/F012705/1, and Oceans 2025 (WP4.5) programs to FCK; the National Science Foundation (CHE-1664657) and the National Oceanic & Atmospheric Administration to CJC and FCK; and the MASTS pooling initiative (Marine Alliance for Science and Technology for Scotland, funded by the Scottish Funding Council and contributing institutions; grant reference HR09011) is gratefully acknowledged. Finally, we would like to acknowledge Susan Loiseaux-de Goër, Bernard Kloareg, Philippe Potin and Akira F. Peters for their hospitality and support to FCK and CK during their visit to RoscoffPeer reviewedPostprin

Transmission Electron Microscopy Data on drusen-like deposits in the retinal degeneration sTg-IRBP : HEL mouse model

We are grateful to the Microscopy and Histology Core Facility of the University of Aberdeen for their assistance with sample processing. This work was generously funded by the charity Saving Sight in Grampian of the University of Aberdeen Development Trust.Peer reviewedPublisher PD

Understanding the mother-infant bond

NoAbridged version of article Milne E, Johnson SE, Waters GM et al (2018) The mother-infant bond: a systematic review of research that includes mothers’ subjective experience of the relationship. Community Practitioner. Accepted for publication

Polarized retinal pigment epithelium generates electrical signals that diminish with age and regulate retinal pathology

Fight for Sight. Grant Numbers: 1712/13, 1361/1362 NHS Grampian Endowments, Friends of ANCHOR, Action Medical Research. Grant Number: GN2299Peer reviewedPublisher PD

The Viscoelastic Properties of the Fungal Cell Wall Allow Traffic of AmBisome as Intact Liposome Vesicles

NARG thanks The Wellcome Trust (080088, 086827, 075470, 099215 & 097377) and MRC Centre for Medical Mycology (MR/N006364/1) and acknowledges financial support from Gilead Sciences for a studentship and grant IX-EU-131-0262. Dr. Linda Soo Hoo and Tark Bunch of Gilead provided expert technical assistance in liposomal sample preparations and GF provided gold labelled test articles. JAM is funded in part from a research grant from Gilead Sciences Inc. ML was supported by the MRC (MR/J008230/1). AC was supported in part by 5R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733. We thank Debbie Wilkinson and Kevin McKenzie at the Imaging Core Facility at the University of Aberdeen for expert assistance with TEM.Peer reviewedPublisher PD

Derivation and Characterization of Induced Pluripotent Stem Cells from Equine Fibroblasts

Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Induced pluripotent stem cells (iPSCs) are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patient-specific manner. In this study, we report the generation of equine iPSCs from skin fibroblasts obtained from a foal and reprogrammed using viral vectors coding for murine Oct4, Sox2, c-Myc, and Klf4 sequences. The reprogrammed cell lines were morphologically similar to iPSCs reported from other species and could be stably maintained over more than 30 passages. Immunostaining and polymerase chain reaction analyses revealed that these cell lines expressed an array of endogenous markers associated with pluripotency, including OCT4, SOX2, NANOG, REX1, LIN28, SSEA1, SSEA4, and TRA1-60. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of ectoderm, mesoderm, and endoderm, and when injected into immunodeficient mice, gave raise to tumors containing differentiated derivatives of the 3 germ layers. Finally, we also reprogrammed fibroblasts from a 2-year-old horse. The reprogrammed cells were similar to iPSCs derived from neonatal fibroblasts in terms of morphology, expression of pluripotency markers, and differentiation ability. The generation of these novel cell lines constitutes an important step toward the understanding of pluripotency in the horse, and paves the way for iPSC technology to potentially become a powerful research and clinical tool in veterinary biomedicine