Confocal laser endomicroscopy is a new imaging modality for recognition of intramucosal bacteria in inflammatory bowel disease in vivo

Interaction of bacteria with the immune system within the intestinal mucosa plays a key role in the pathogenesis of inflammatory bowel disease (IBD). The aim of the current study was to develop a fluorescein-aided confocal laser endomicroscopy (CLE) method to visualise intramucosal enteric bacteria in vivo and to determine the involved mucosal area in the colon and ileum in patients with ulcerative colitis (UC) and Crohn's disease (CD)

Phosphorylation Modulates the Subcellular Localization of SOX11

SOX11 is a key Transcription Factor (TF) in the regulation of embryonic and adult neurogenesis, whose mutation has recently been linked to an intellectual disability syndrome in humans. SOX11’s transient activity during neurogenesis is critical to ensure the precise execution of the neurogenic program. Here, we report that SOX11 displays differential subcellular localizations during the course of neurogenesis. Western-Blot analysis of embryonic mouse brain lysates indicated that SOX11 is post-translationally modified by phosphorylation. Using Mass Spectrometry, we found 10 serine residues in the SOX11 protein that are putatively phosphorylated. Systematic analysis of phospho-mutant SOX11 resulted in the identification of the S30 residue, whose phosphorylation promotes nuclear over cytoplasmic localization of SOX11. Collectively, these findings uncover phosphorylation as a novel layer of regulation of the intellectual disability gene Sox11

Harmonisation framework for health based evaluation of indoor emissions from construction products in the European Union using the EU-LCI concept

This report describes a harmonised procedure for establishing a list of compounds and their associated LCI (Lowest Concentration of Interest) values for the evaluation of emissions from construction products taking into account existing procedures used in some Member States (i.e. ANSES in France and AgBB in Germany). It provides an appropriate health‐protective, science-based and transparent yet pragmatic approach with a flexible framework that enables review of the procedure to take into account new knowledge (e.g. data resulting from the REACH implementation process) for future revision of the EU-LCI master list in terms of both the compounds listed and their EU-LCI values.JRC.I.1-Chemical Assessment and Testin

Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD

A homologue of the Parkinson's disease-associated protein LRRK2 undergoes a monomer-dimer transition during GTP turnover.

Mutations in LRRK2 are a common cause of genetic Parkinson's disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2

CpG-Methylation Regulates a Class of Epstein-Barr Virus Promoters

DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to ‘read’ DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRASG13D

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.This work was supported by European Union FP7 Grant No. 278568 “PRIMES” and Science Foundation Ireland Investigator Program Grant 14/IA/2395 to W.K. B.K. is supported by SmartNanoTox (Grant no. 686098), NanoCommons (Grant no. 731032), O.R. by MSCA-IF-2016 SAMNets (Grant no. 750688). D.M. is supported by Science Foundation Ireland Career Development award 15-CDA-3495. I.J. is supported by the Canada Research Chair Program (CRC #225404), Krembil Foundation, Ontario Research Fund (GL2-01-030 and #34876), Natural Sciences Research Council (NSERC #203475), Canada Foundation for Innovation (CFI #225404, #30865), and IBM. O.S. is supported by ERC investigator Award ColonCan 311301 and CRUK. I.S. is supported by the Canadian Cancer Society Research Institute (#703889), Genome Canada via Ontario Genomics (#9427 & #9428), Ontario Research fund (ORF/ DIG-501411 & RE08-009), Consortium Québécois sur la Découverte du Médicament (CQDM Quantum Leap) & Brain Canada (Quantum Leap), and CQDM Explore and OCE (#23929). T.C. was supported by a Teagasc Walsh Fellowshi

LRRK2 Biology from structure to dysfunction: research progresses, but the themes remain the same

Since the discovery of leucine-rich repeat kinase 2 (LRRK2) as a protein that is likely central to the aetiology of Parkinson's disease, a considerable amount of work has gone into uncovering its basic cellular function. This effort has led to the implication of LRRK2 in a bewildering range of cell biological processes and pathways, and probable roles in a number of seemingly unrelated medical conditions. In this review we summarise current knowledge of the basic biochemistry and cellular function of LRRK2. Topics covered include the identification of phosphorylation substrates of LRRK2 kinase activity, in particular Rab proteins, and advances in understanding the activation of LRRK2 kinase activity via dimerisation and association with membranes, especially via interaction with Rab29. We also discuss biochemical studies that shed light on the complex LRRK2 GTPase activity, evidence of roles for LRRK2 in a range of cell signalling pathways that are likely cell type specific, and studies linking LRRK2 to the cell biology of organelles. The latter includes the involvement of LRRK2 in autophagy, endocytosis, and processes at the trans-Golgi network, the endoplasmic reticulum and also key microtubule-based cellular structures. We further propose a mechanism linking LRRK2 dimerisation, GTPase function and membrane recruitment with LRRK2 kinase activation by Rab29. Together these data paint a picture of a research field that in many ways is moving forward with great momentum, but in other ways has not changed fundamentally. Many key advances have been made, but very often they seem to lead back to the same places

Experimental and Numerical Investigation of the Outer Ring Cooling Concept in a Hybrid and in an All-Steel Ball Bearing Used in Aero-Engines by the Introduction of a Helical Duct

Rolling element bearings for aero engine applications have to withstand very challenging operating conditions because of the high thermal impact due to elevated rotational speeds and loads. The high rate of heat generation in the bearing has to be sustained by the materials, and in the absence of lubrication these will fail within seconds. For this reason, aero engine bearings have to be lubricated and cooled by a continuous oil stream. When the oil has reached the outer ring it has already been heated up, thus its capability to remove extra heat from the outer ring is considerably reduced. Increasing the mass flow of oil to the bearing is not a solution since excess oil quantity would cause high parasitic losses (churning) in the bearing chamber and also increase the demands in the oil system for oil storage, scavenging, cooling, hardware weight, etc. A method has been developed for actively cooling the outer ring of the bearing. The idea behind the outer ring cooling concept was adopted from fins that are used for cooling electronic devices. A spiral groove engraved in the outer ring material of the bearing would function as a fin body with oil instead of air as the cooling medium. The method was first evaluated in an all steel ball bearing and the results were a 50% reduction in the lubricating oil flow with an additional reduction in heat generation by more than 25%. It was then applied on a Hybrid ball bearing of the same size and the former results were reconfirmed. Hybrid bearings are a combination of steel made parts, like the outer ring, the inner ring, and the cage and of ceramic rolling elements. This paper describes the work done to-date as a follow up of the work described in, and demonstrates the potential of the outer ring cooling for a bearing. Friction loss coefficient, Nusselt number, and efficiency correlations have been developed on the basis of the test results and have been compared to correlations from other authors. Computational Fluid Dynamics (CFD) analysis with ANSYS CFX has been used to verify test results and also for parametric studies