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Wan M.,Karolinska Institutet | Godson C.,UCD Conway Institute | Guiry P.J.,University College Dublin | Agerberth B.,Karolinska Institutet | Haeggstrom J.Z.,Karolinska Institutet
FASEB Journal | Year: 2011

In humans, the antimicrobial peptide LL-37 and leukotriene B 4 (LTB 4) are important proinflammatory mediators, whereas lipoxin A 4 (LXA 4) and resolvin E1 (RvE1) possess anti-inflammatory, proresolving properties. Previously, we reported that LTB 4 triggers LL-37 release from human neutrophils (PMNs) and, conversely, that LL-37 promotes LTB 4 production from these cells. Here we show that this effect of LL-37 is mediated via the GPCR FPR2/ALX. LL-37 (5-30 μg/ml) induces intracellular calcium mobilization in a dose-dependent manner, and the signal transduction leading to LTB 4 release involves p38 MAP kinase and phosphorylation of cPLA 2. LXA 4, an endogenous lipid ligand of FPR2/ALX, and a stable LXA 4 analog [benzo-LXA 4] were ineffective as stimuli at the concentrations of 0.1-10 nM for LTB 4 release from PMNs. Likewise, the BLT1 ligand RvE1, a derivative of eicosapentaenoic acid, inhibited LTB 4-induced LL-37 production from PMNs at 1-100 nM, whereas chemerin, a peptide ligand of the RvE1 receptor ChemR23, failed to block LTB 4- induced LL-37 release at the same concentrations. Hence, in human neutrophils, binding of LL-37 to FPR2/ALX promotes LTB 4 production, which can bind to BLT1 and elicit further LL-37 release. This proinflammatory circuit might be inhibited by LXA 4 and RvE 1 acting at FPR2/ALX and BLT1, respectively, leading to dampened mediator release. © FASEB. Source

Geiger T.,Max Planck Institute of Biochemistry | Geiger T.,Tel Aviv University | Madden S.F.,Dublin City University | Gallagher W.M.,UCD Conway Institute | And 2 more authors.
Cancer Research | Year: 2012

Breast cancer is the second leading cause of cancer death for women in the United States. Of the different subtypes, estrogen receptor-negative (ER -) tumors, which are ErbB2+ or triple-negative, carry a relatively poor prognosis. In this study, we used system-wide analysis of breast cancer proteomes to identify proteins that are associated with the progression of ER - tumors. Our two-step approach included an initial deep analysis of cultured cells that were obtained from tumors of defined breast cancer stages, followed by a validation set using human breast tumors. Using high-resolution mass spectrometry and quantification by Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), we identified 8,750 proteins and quantified 7,800 of them. A stagespecific signature was extracted and validated by mass spectrometry and immunohistochemistry on tissue microarrays. Overall, the proteomics signature reflected both a global loss of tissue architecture and a number of metabolic changes in the transformed cells. Proteomic analysis also identified high levels of IDH2 and CRABP2 and low levels of SEC14L2 to be prognostic markers for overall breast cancer survival. Together, our findings suggest that global proteomic analysis provides information about the protein changes specific to ER - breast tumor progression as well as important prognostic information. ©2012 AACR. 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

Borgeson E.,UCD Conway Institute | Docherty N.G.,Trinity College Dublin | Murphy M.,UCD Conway Institute | Rodgers K.,UCD Conway Institute | And 6 more authors.
FASEB Journal | Year: 2011

Unresolved inflammation underlies the development of fibrosis and organ failure. Here, we investigate the potential of the proresolving eicosanoid lipoxinA 4 (LXA 4) and its synthetic analog benzo-LXA 4to prophylactically modulate fibrotic and inflammatory responses in a model of early renal fibrosis, unilateral ureteric obstruction (UUO). Male Wistar rats (Animalia, Chordata, Rattus norvegicus) were injected intravenously with vehicle (0.1% ethanol), LXA 4 (45 μg/250-g rat), or benzo-LXA 4 (15 μg/250-g rat) 15 min prior to surgery and sacrificed 3 d postligation. Renal gene and protein expression, collagen deposition, macrophage infiltration, and apoptosis were analyzed using manipulated kidneys from sham operations as control. Lipoxins (LXs) attenuated collagen deposition and renal apoptosis (P<0.05) and shifted the inflammatory milieu toward resolution, inhibiting TNF-α and IFN-γexpression, while stimulating proresolving IL-10. LXs attenuated UUO-induced activation of MAP kinases, Akt, and Smads (P<0.05) in injured kidneys. We explored whether the underlying mechanism reflected LX-induced modulation of fibroblast activation. Using cultured rat renal NRK-49F fibroblasts, we report that LXA 4 (1 nM) inhibits TGF-β1 (10 ng/ml)-induced activation of Smad2 and MAP-kinases (P<0.05), and furthermore, LXA 4 reduced TGF-β1-stimulated PAI-1 luciferase activation (P<0.05) relative to vehicle-stimulated cells. We propose that LXs may represent a potentially useful and novel therapeutic strategy for consideration in the context of renal fibrosis. © FASEB. Source

Jin Y.,UCD Conway Institute | Amaral A.,New University of Lisbon | Amaral A.,Institute Biologia Experimental e Tecnologica IBET | McCann A.,UCD Conway Institute | Brennan L.,UCD Conway Institute
Neurochemistry International | Year: 2011

Although the neurotoxic effects of homocysteine have been well elucidated, the effects of homocysteine in astrocytes have received little attention until recently. Previously we have demonstrated that elevated levels of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. However, the mechanisms behind such alterations remain unclear. As homocysteine is a key metabolite in one-carbon metabolism the present study examined if the effects of homocysteine on astrocyte function are mediated through an epigenetic mechanism. Following exposure to homocysteine for 72 h, global DNA methylation and H3K9 acetylation were examined using flow cytometric analysis. Total DNA methyltransferase activity and protein levels of DNA methyltransferase 3B were measured. Exposure to homocysteine resulted in global DNA hypomethylation (p < 0.05) and histone hyperacetylation (p < 0.05). Total DNA methyltransferase activity significantly decreased following exposure to homocysteine (from 11.5 ± 3.9 to 6.0 ± 1.7 OD/h/mg protein, p < 0.01) which was accompanied by a significant reduction in protein levels of DNA methyltransferase 3B (p < 0.05). Treatment of astrocytes with the DNA methyltransferase inhibitor, 5-aza-2′- deoxycytidine, mimicked the functional changes induced by homocysteine. In conclusion, the results demonstrate significant epigenetic modifications following exposure to homocysteine in astrocytes and these changes seem to mediate functional alterations. © 2011 Elsevier Ltd. All rights reserved. Source

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