Phos-tag analysis of Rab10 phosphorylation by LRRK2:a powerful assay for assessing kinase function and inhibitors

Autosomal dominant mutations that activate the leucine-rich repeat kinase-2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved Thr/Ser residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen derived B Cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2 phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase inactive LRRK2[D2017A] knock-in MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knock-in mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A, S935A] knock-in MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aide with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

A Spatial and Temporal Investigation of Eleocharis Macrostachya and Orcuttia tenuis

Volume: 55Start Page: 257End Page: 26

Harmonisation of indoor material emission labelling schemes in the EU - VOC Workshop

Harmonisation of indoor material labelling schemes in the EU is an important aspect of the European Commission¿s policy making process in the field of indoor air quality and associated health effects. This paper describes the outcome of recent activities and a roadmap setting out the steps being taken by a preparatory working group led by the EC for establishing an EU wide harmonised framework of labelling schemes and obtaining broad consensus through open consultation.JRC.DDG.I.2-Chemical assessment and testin

Harmonisation of Material Emission Labelling Schemes in the EU

Growing awareness about requirements for healthy indoor air has resulted in a demand for products demonstrated to be safe for use in indoor environments. Emissions from construction products have been identified as a significant source of indoor air pollution since the beginning of the 1980¿s. Different approaches to evaluate construction products have emerged over time, and considerable practical experience has been gained during recent years. In some markets, emissions originating from indoor construction products have been noticeably reduced by developing quality criteria and labelling systems. A detailed review of the existing labelling schemes was compiled in 2005 in the European Collaborative Action, Report 24. This paper reports on ongoing activities concerningthe development of a harmonised evaluation concept that is under elaboration by the Danish ICL, the German AgBB, the Finnish M1 and the JRC/IHCP/PCE.JRC.I.2-Chemical assessment and testin

ECA report no. 27 on “Harmonisation framework for indoor products labelling schemes in the EU”

Harmonisation of indoor products labelling schemes in the EU is an important aspect of the European Commission’s policy making process in the field of indoor air quality and associated health effects. This report describes the outcome of recent activities and a roadmap setting out the steps being taken by a preparatory working group led by the European Commission for establishing an EU wide harmonisation framework for labelling schemes which consists of core and transitional criteria. The harmonization framework proposed in ECA report no. 27 will help the convergence of existing mandatory and voluntary labelling schemes in EU. This report is the final product of an initiative, coordinated by the JRC-IHCP at Ispra and integrated into the EU strategy on indoor air quality led by the EC Directorate General for Health and Consumer Affairs. This initiative started in 2010 with an international workshop “Harmonized framework on indoor material labeling schemes: challenge with a global perspective”. Member States, the industry and the consumers are concerned by it. Labeling of products will help building designers and consumers making informed choices about the products (existing or new) in the market which are used in indoor environments and also removing existing barriers in trade of consumer products used indoors.JRC.I.1-Chemical Assessment and Testin

Influence of Accidental Impurities on the Spectroscopic and Luminescent Properties of ZnWO₄ Crystal

Special techniques for deep purification of ZnO and WO3 have been developed in this work. A ZnWO4 single crystal has been grown by the Czochralski method using purified ZnO and WO3 chemicals, along with the ZnWO4 crystal-etalon, which has been grown at the same conditions using commercially available 5N ZnO and WO3 chemicals. The actual accidental impurities compositions of both the initial chemicals and the grown crystals have been measured by inductively coupled plasma mass-spectrometry. A complex of comparative spectroscopic studies of the crystals has been performed, including optical absorption spectra, photo-, X-ray-, and cathodoluminescence spectra and decay kinetics, as well as the photoluminescence excitation spectra. The revealed differences in the measured properties of the crystals have been analyzed in terms of influence of the accidental impurities on these properties