Publications by authors named "Marion Butler"

7 Publications

  • Page 1 of 1

Automated Raman Micro-Spectroscopy of Epithelial Cell Nuclei for High-Throughput Classification.

Cancers (Basel) 2021 Sep 24;13(19). Epub 2021 Sep 24.

Department of Electronic Engineering, Maynooth University, Maynooth, Ireland.

Raman micro-spectroscopy is a powerful technique for the identification and classification of cancer cells and tissues. In recent years, the application of Raman spectroscopy to detect bladder, cervical, and oral cytological samples has been reported to have an accuracy greater than that of standard pathology. However, despite being entirely non-invasive and relatively inexpensive, the slow recording time, and lack of reproducibility have prevented the clinical adoption of the technology. Here, we present an automated Raman cytology system that can facilitate high-throughput screening and improve reproducibility. The proposed system is designed to be integrated directly into the standard pathology clinic, taking into account their methodologies and consumables. The system employs image processing algorithms and integrated hardware/software architectures in order to achieve automation and is tested using the ThinPrep standard, including the use of glass slides, and a number of bladder cancer cell lines. The entire automation process is implemented, using the open source Micro-Manager platform and is made freely available. We believe that this code can be readily integrated into existing commercial Raman micro-spectrometers.
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http://dx.doi.org/10.3390/cancers13194767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507544PMC
September 2021

IRAK1 Limits TLR3/4- and IFNAR-Driven IL-27 Production through a STAT1-Dependent Mechanism.

J Immunol 2018 10 24;201(7):2070-2081. Epub 2018 Aug 24.

Institute of Immunology, Department of Biology, Maynooth University, Maynooth, County Kildare, Ireland;

IL-27 is a cytokine exerting pleiotropic immunomodulatory effects on a broad spectrum of immune cells. Optimal IL-27 production downstream of TLR3/4 ligand stimulation relies on autocrine type I IFN signaling, defining a first and second phase in IL-27 production. This work shows that IL-1 receptor-associated kinase 1 (IRAK1) limits TLR3/4- and IFNAR-induced IL-27 production. At the mechanistic level, we identified IRAK1 as a novel regulator of STAT1, IRF1, and IRF9. We found hyperactivation of STAT1 together with increased nuclear levels of IRF1 and IRF9 in IRAK1-deficient murine macrophages compared with control cells following stimulation with LPS and poly(I:C). IRAK1-deficient human microglial cells showed higher basal levels of STAT1 and STAT2 compared with control cells. Blocking the kinase activity of TBK1/IKKε in IRAK1 knockdown human microglial cells reduced the high basal levels of STAT1/2, uncovering a TBK1/IKKε kinase-dependent mechanism controlling basal levels of STAT1/2. Stimulating IRAK1 knockdown human microglial cells with IFN-β led to increased IL-27p28 expression compared with control cells. In IRAK1-deficient murine macrophages, increased IL-27 levels were detected by ELISA following IFN-β stimulation compared with control macrophages together with increased nuclear levels of p-STAT1, IRF1, and IRF9. Treatment of wild-type and IRAK1-deficient murine macrophages with fludarabine similarly reduced TLR3/4-induced IL-27 cytokine levels. To our knowledge, this work represents the first report placing IRAK1 in the IFNAR pathway and identifies IRAK1 as an important regulator of STAT1, controlling IL-27 production downstream of TLR3/4 and IFNAR signaling pathways.
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http://dx.doi.org/10.4049/jimmunol.1701373DOI Listing
October 2018

A novel IRAK1-IKKε signaling axis limits the activation of TAK1-IKKβ downstream of TLR3.

J Immunol 2013 Mar 8;190(6):2844-56. Epub 2013 Feb 8.

Institute of Immunology, National University of Ireland Maynooth, Maynooth, County Kildare, Ireland.

IRAK1 is involved in the regulation of type I IFN production downstream of TLR3. Previous work indicated that IRAK1 negatively regulates TRIF-mediated activation of IRF3 and IRF7. We report that IRAK1 limits the activation of the TLR3-NF-κB pathway. Following TLR3 stimulation, IRAK1-deficient macrophages produced increased levels of IL-6 and IFN-β compared with wild type macrophages. Pharmacological inhibition of TAK1 reduced this increase in IFN-β, together with the heightened activation of IRF3 and p65 found in TLR3-ligand stimulated IRAK1-deficient macrophages. Recently, IKKε and TANK-binding kinase 1 (TBK1) were reported to limit activation of the NF-κB pathway downstream of IL-1R, TNFR1, and TLRs. We show that TBK1 has a positive role in the TLR3-NF-κB pathway, because we detected reduced levels of IL-6 and reduced activation of p65 in TBK1-deficient macrophages. In contrast, we show that IKKε limits the activation of the TLR3-NF-κB pathway. Furthermore, we show that IRAK1 is required for the activation of IKKε downstream of TLR3. We report impaired activation of ERK1/2 in IRAK1- and IKKε-deficient macrophages, a novel finding for both kinases. Importantly, this work provides novel mechanistic insight into the regulation of the TLR3-signaling pathway, providing strong evidence that an IRAK1-IKKε-signaling axis acts to limit the production of both type I IFNs and proinflammatory cytokines by regulating TAK1 activity.
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http://dx.doi.org/10.4049/jimmunol.1202042DOI Listing
March 2013

Kinase-active interleukin-1 receptor-associated kinases promote polyubiquitination and degradation of the Pellino family: direct evidence for PELLINO proteins being ubiquitin-protein isopeptide ligases.

J Biol Chem 2007 Oct 3;282(41):29729-37. Epub 2007 Aug 3.

Institute of Immunology, National University of Ireland Maynooth, County Kildare, Ireland.

Members of the Pellino family are interleukin-1 receptor-associated kinase (IRAK)-interacting proteins that possess RING-like domains. The presence of these domains led to the suggestion that Pellino proteins are ubiquitin-protein isopeptide ligases (E3). However, no conclusive data currently exist to prove this proposal. This study provides the first direct evidence that Pellino proteins possess E3 activity. Recombinant forms of Pellino1 and Pellino2 and both spliced variants of Pellino3 are shown in an in vitro ubiquitination assay to be E3 ligases that catalyze Lys(63)-linked polyubiquitination, with Pellino3 exhibiting the greatest ligase activity. Whereas the Pellino proteins cause polyubiquitination of IRAK-1, we also show that kinase-active members of the IRAK family (IRAK-1 and IRAK-4) promote reciprocal polyubiquitination of the Pellino proteins and that this is associated with IRAK-induced degradation of the Pellino family. In contrast, IRAK-2 (which lacks a functional kinase domain) and kinase-dead forms of IRAK-1 and IRAK-4 fail to degrade the Pellino proteins. We show that these kinase-inactive IRAK proteins can associate with Pellino proteins, thus excluding the possibility that their inability to regulate Pellino degradation is due to lack of association with the Pellino proteins. The physiological relevance of IRAK-induced degradation of Pellino proteins is confirmed by the demonstration that lipopolysaccharide causes degradation of endogenous forms of Pellino3 in peripheral blood mononuclear cells. In summary, this study not only demonstrates Pellino proteins to be E3 ligases that can catalyze Lys(63)-linked polyubiquitination but also shows bidirectional signaling between the IRAK and Pellino families and highlights a novel function for IRAK kinase activity.
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http://dx.doi.org/10.1074/jbc.M704558200DOI Listing
October 2007

Pellino3 is a novel upstream regulator of p38 MAPK and activates CREB in a p38-dependent manner.

J Biol Chem 2005 Jul 25;280(30):27759-68. Epub 2005 May 25.

Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.

Engagement of the interleukin-1 (IL-1) and Toll-like receptors triggers mitogen-activated protein kinase (MAPK) pathways and activation of transcription factors such as NFkappaB and AP-1. Recent studies have identified members of the Pellino protein family as novel mediators in mediating activation of these pathways. However, no evidence has been presented to date to suggest a role for the Pellino proteins in activation of the p38 MAPK pathway. We demonstrate herein that Pellino3 is a strong activator of p38 MAPK. RNA interference was used to reveal a physiological role for Pellino3 in the IL-1 pathway leading to activation of p38 MAPK. A series of N-terminal truncation and point mutants of Pellino3 were generated and tested for their ability to activate p38 MAPK in an effort to map sites of protein interaction important for p38 MAPK activation. In this way we show that the binding of Pellino3 to IL-1 receptor-associated kinase 1 coincides with its ability to promote p38 MAPK activation. TRAF-6 and transforming growth factor-beta-activating kinase 1 are shown to act as downstream mediators of the activation of p38 MAPK by Pellino3. Finally we confirm the functional consequences of the activation of p38 MAPK by Pellino3 by demonstrating that Pellino3 promotes translocation of the p38 substrate, MAPK-activated protein kinase2, from the nucleus to the cytoplasm and activates the transcription factor CREB in a p38 MAPK-dependent manner. Our study not only identifies Pellino3 as a novel upstream regulator of the p38 MAPK pathway but also probes the mechanistic basis underlying the ability of Pellino3 to promote activation of this pathway.
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http://dx.doi.org/10.1074/jbc.M500756200DOI Listing
July 2005

Methods of detection of the transcription factor NF-kappa B in rat hippocampal slices.

J Neurosci Methods 2002 Sep;119(2):185-90

Department of Physiology, Earlsfort Terrace, University College Dublin, Dublin 2, Ireland.

The hippocampus is one of the most studied sites for understanding the cellular and molecular mechanisms underlying long-term potentiation (LTP) and long-term depression (LTD), mechanisms believed to underlie the formation and storage of memories. The early-phases of LTP and LTD have been most intensively studied and have been shown to involve the activation of several kinases and phosphatases, respectively. The factors involved in the later stages have largely yet to be elucidated. We have focused our attention on the transcription factor NF-kappaB as a possible factor involved in such late-phase processes, and have developed both immunocytochemistry and electrophoretic mobility shift assay (EMSA) to measure the activated forms of this factor. This is important as many of the studies in this area are performed in vitro and to our knowledge quantitative assessment has not previously being deemed feasible in slice work. The pro-inflammatory cytokines TNF-alpha and IL-1beta both led to pronounced nuclear activation of NF-kappaB in the dentate granule cells as demonstrated by immunostaining and EMSA, respectively. Electrophysiological measurements taken from slices treated with TNF-alpha showed that it inhibited LTP (field excitatory post-synaptic potentials (fEPSP) 116+/-10%, n = 9, 60 min post-tetanus compared to control fEPSP 185+/-9%, n = 6; P < 0.001). The neurotransmitter L-glutamate also led to activation of NF-kappaB and electrophysiology recordings showed a small but sustained increase in synaptic transmission (fEPSP 106+/-12%, 30 min post-drug). These methods provide valuable tools to forward our understanding of the role of NF-kappaB in plasticity as well as in many neurological disorders being mimicked by in vitro studies.
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http://dx.doi.org/10.1016/s0165-0270(02)00171-1DOI Listing
September 2002

Alternative translocation breakpoint cluster region 5' to BCL-6 in B-cell non-Hodgkin's lymphoma.

Cancer Res 2002 Jul;62(14):4089-94

Institute for Cancer Genetics, and the Department of Pathology, Columbia University, New York, NY 10032, USA.

Chromosomal translocations involving band 3q27 with various different partner chromosomes represent a recurrent cytogenetic abnormality in B-cell non-Hodgkin's lymphoma. In a fraction of these translocations, the chromosomal breakpoint is located within the 5' noncoding region of the BCL-6 proto-oncogene where the BCL-6 major breakpoint region (MBR) maps. As a result of the translocation, BCL-6 expression is deregulated by promoter substitution. However, between 30 and 50% of lymphomas with cytogenetically detectable translocations affecting band 3q27 retain a germ-line configuration at the BCL-6 locus. To identify possible additional breakpoint clusters within 3q27, we cloned a t(3;14)(q27;q32) lymphoma without MBR rearrangement and found a novel breakpoint site located between 245 and 285 kb 5' to BCL-6. Breakpoints within this newly described region, which we called the alternative breakpoint region (ABR), were found to be recurrent in lymphomas carrying t(3q27) chromosomal translocations but devoid of BCL-6 MBR rearrangements. Comparative analysis of multiple lymphomas carrying rearrangements within the ABR showed that the breakpoints cluster within a 20-kb distance. Translocations involving the ABR may juxtapose BCL-6 to distantly acting, heterologous transcriptional regulatory elements which cause deregulation of the proto-oncogene. The identification of BCL-6 ABR provides new tools for the diagnosis of lymphomas carrying aberrations at 3q27 and deregulated BCL-6 genes.
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July 2002
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