Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency.

UNLABELLED: Point mutants of alpha1 -antitrypsin (α1AT) form ordered polymers that are retained as inclusions within the endoplasmic reticulum (ER) of hepatocytes in association with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. These inclusions cause cell damage and predispose to ER stress in the absence of the classical unfolded protein response (UPR). The pathophysiology underlying this ER stress was explored by generating cell models that conditionally express wild-type (WT) α1AT, two mutants that cause polymer-mediated inclusions and liver disease (E342K [the Z allele] and H334D) and a truncated mutant (Null Hong Kong; NHK) that induces classical ER stress and is removed by ER-associated degradation. Expression of the polymeric mutants resulted in gross changes in the ER luminal environment that recapitulated the changes observed in liver sections from individuals with PI*ZZ α1AT deficiency. In contrast, expression of NHK α1AT caused electron lucent dilatation and expansion of the ER throughout the cell. Photobleaching microscopy in live cells demonstrated a decrease in the mobility of soluble luminal proteins in cells that express E342K and H334D α1AT, when compared to those that express WT and NHK α1AT (0.34 ± 0.05, 0.22 ± 0.03, 2.83 ± 0.30, and 2.84 ± 0.55 μm(2) /s, respectively). There was no effect on protein mobility within ER membranes, indicating that cisternal connectivity was not disrupted. Polymer expression alone was insufficient to induce the UPR, but the resulting protein overload rendered cells hypersensitive to ER stress induced by either tunicamycin or glucose depletion. CONCLUSION: Changes in protein diffusion provide an explanation for the cellular consequences of ER protein overload in mutants that cause inclusion body formation and α1AT deficiency

On the stability of the exact solutions of the dual-phase lagging model of heat conduction

The dual-phase lagging (DPL) model has been considered as one of the most promising theoretical approaches to generalize the classical Fourier law for heat conduction involving short time and space scales. Its applicability, potential, equivalences, and possible drawbacks have been discussed in the current literature. In this study, the implications of solving the exact DPL model of heat conduction in a three-dimensional bounded domain solution are explored. Based on the principle of causality, it is shown that the temperature gradient must be always the cause and the heat flux must be the effect in the process of heat transfer under the dual-phase model. This fact establishes explicitly that the single- and DPL models with different physical origins are mathematically equivalent. In addition, taking into account the properties of the Lambert W function and by requiring that the temperature remains stable, in such a way that it does not go to infinity when the time increases, it is shown that the DPL model in its exact form cannot provide a general description of the heat conduction phenomena

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

Serpins are important regulators of proteolytic pathways with an anti-protease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an inter-molecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of a number of pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39, and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of pre-formed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for rational design of ligands that is able to dynamically influence α1-AT polymerisation

<i>Gaia</i> Data Release 1. Summary of the astrometric, photometric, and survey properties

Context. At about 1000 days after the launch of Gaia we present the first Gaia data release, Gaia DR1, consisting of astrometry and photometry for over 1 billion sources brighter than magnitude 20.7. Aims. A summary of Gaia DR1 is presented along with illustrations of the scientific quality of the data, followed by a discussion of the limitations due to the preliminary nature of this release. Methods. The raw data collected by Gaia during the first 14 months of the mission have been processed by the Gaia Data Processing and Analysis Consortium (DPAC) and turned into an astrometric and photometric catalogue. Results. Gaia DR1 consists of three components: a primary astrometric data set which contains the positions, parallaxes, and mean proper motions for about 2 million of the brightest stars in common with the HIPPARCOS and Tycho-2 catalogues – a realisation of the Tycho-Gaia Astrometric Solution (TGAS) – and a secondary astrometric data set containing the positions for an additional 1.1 billion sources. The second component is the photometric data set, consisting of mean G-band magnitudes for all sources. The G-band light curves and the characteristics of ∼3000 Cepheid and RR-Lyrae stars, observed at high cadence around the south ecliptic pole, form the third component. For the primary astrometric data set the typical uncertainty is about 0.3 mas for the positions and parallaxes, and about 1 mas yr−1 for the proper motions. A systematic component of ∼0.3 mas should be added to the parallax uncertainties. For the subset of ∼94 000 HIPPARCOS stars in the primary data set, the proper motions are much more precise at about 0.06 mas yr−1. For the secondary astrometric data set, the typical uncertainty of the positions is ∼10 mas. The median uncertainties on the mean G-band magnitudes range from the mmag level to ∼0.03 mag over the magnitude range 5 to 20.7. Conclusions. Gaia DR1 is an important milestone ahead of the next Gaia data release, which will feature five-parameter astrometry for all sources. Extensive validation shows that Gaia DR1 represents a major advance in the mapping of the heavens and the availability of basic stellar data that underpin observational astrophysics. Nevertheless, the very preliminary nature of this first Gaia data release does lead to a number of important limitations to the data quality which should be carefully considered before drawing conclusions from the data

Increased ERK signalling promotes inflammatory signalling in primary airway epithelial cells expressing Z α1-antitrypsin.

Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung

Gaia Data Release 1: Testing parallaxes with local Cepheids and RR Lyrae stars

Context. Parallaxes for 331 classical Cepheids, 31 Type II Cepheids, and 364 RR Lyrae stars in common between Gaia and the Hipparcos and Tycho-2 catalogues are published in Gaia Data Release 1 (DR1) as part of the Tycho-Gaia Astrometric Solution (TGAS). Aims. In order to test these first parallax measurements of the primary standard candles of the cosmological distance ladder, which involve astrometry collected by Gaia during the initial 14 months of science operation, we compared them with literature estimates and derived new period-luminosity (PL), period-Wesenheit (PW) relations for classical and Type II Cepheids and infrared PL, PL-metallicity (PLZ), and optical luminosity-metallicity (M V -[Fe/H]) relations for the RR Lyrae stars, with zero points based on TGAS. Methods. Classical Cepheids were carefully selected in order to discard known or suspected binary systems. The final sample comprises 102 fundamental mode pulsators with periods ranging from 1.68 to 51.66 days (of which 33 with σ Ω /Ω < 0.5). The Type II Cepheids include a total of 26 W Virginis and BL Herculis stars spanning the period range from 1.16 to 30.00 days (of which only 7 with σ Ω /Ω < 0.5). The RR Lyrae stars include 200 sources with pulsation period ranging from 0.27 to 0.80 days (of which 112 with σ Ω /Ω < 0.5). The new relations were computed using multi-band (V,I,J,K s ) photometry and spectroscopic metal abundances available in the literature, and by applying three alternative approaches: (i) linear least-squares fitting of the absolute magnitudes inferred from direct transformation of the TGAS parallaxes; (ii) adopting astrometry-based luminosities; and (iii) using a Bayesian fitting approach. The last two methods work in parallax space where parallaxes are used directly, thus maintaining symmetrical errors and allowing negative parallaxes to be used. The TGAS-based PL,PW,PLZ, and M V - [Fe/H] relations are discussed by comparing the distance to the Large Magellanic Cloud provided by different types of pulsating stars and alternative fitting methods. Results. Good agreement is found from direct comparison of the parallaxes of RR Lyrae stars for which both TGAS and HST measurements are available. Similarly, very good agreement is found between the TGAS values and the parallaxes inferred from the absolute magnitudes of Cepheids and RR Lyrae stars analysed with the Baade-Wesselink method. TGAS values also compare favourably with the parallaxes inferred by theoretical model fitting of the multi-band light curves for two of the three classical Cepheids and one RR Lyrae star, which were analysed with this technique in our samples. The K-band PL relations show the significant improvement of the TGAS parallaxes for Cepheids and RR Lyrae stars with respect to the Hipparcos measurements. This is particularly true for the RR Lyrae stars for which improvement in quality and statistics is impressive. Conclusions. TGAS parallaxes bring a significant added value to the previous Hipparcos estimates. The relations presented in this paper represent the first Gaia-calibrated relations and form a work-in-progress milestone report in the wait for Gaia-only parallaxes of which a first solution will become available with Gaia Data Release 2 (DR2) in 2018. © ESO, 2017

Analysis of the common genetic component of large-vessel vasculitides through a meta- Immunochip strategy

Giant cell arteritis (GCA) and Takayasu's arteritis (TAK) are major forms of large-vessel vasculitis (LVV) that share clinical features. To evaluate their genetic similarities, we analysed Immunochip genotyping data from 1,434 LVV patients and 3,814 unaffected controls. Genetic pleiotropy was also estimated. The HLA region harboured the main disease-specific associations. GCA was mostly associated with class II genes (HLA-DRB1/HLA-DQA1) whereas TAK was mostly associated with class I genes (HLA-B/MICA). Both the statistical significance and effect size of the HLA signals were considerably reduced in the cross-disease meta-analysis in comparison with the analysis of GCA and TAK separately. Consequently, no significant genetic correlation between these two diseases was observed when HLA variants were tested. Outside the HLA region, only one polymorphism located nearby the IL12B gene surpassed the study-wide significance threshold in the meta-analysis of the discovery datasets (rs755374, P?=?7.54E-07; ORGCA?=?1.19, ORTAK?=?1.50). This marker was confirmed as novel GCA risk factor using four additional cohorts (PGCA?=?5.52E-04, ORGCA?=?1.16). Taken together, our results provide evidence of strong genetic differences between GCA and TAK in the HLA. Outside this region, common susceptibility factors were suggested, especially within the IL12B locus

Gaia Data Release 1: Open cluster astrometry: performance, limitations, and future prospects

Context. The first Gaia Data Release contains the Tycho-Gaia Astrometric Solution (TGAS). This is a subset of about 2 million stars for which, besides the position and photometry, the proper motion and parallax are calculated using Hipparcos and Tycho-2 positions in 1991.25 as prior information.Aims. We investigate the scientific potential and limitations of the TGAS component by means of the astrometric data for open clusters.Methods. Mean cluster parallax and proper motion values are derived taking into account the error correlations within the astrometric solutions for individual stars, an estimate of the internal velocity dispersion in the cluster, and, where relevant, the effects of the depth of the cluster along the line of sight. Internal consistency of the TGAS data is assessed.Results. Values given for standard uncertainties are still inaccurate and may lead to unrealistic unit-weight standard deviations of least squares solutions for cluster parameters. Reconstructed mean cluster parallax and proper motion values are generally in very good agreement with earlier HIPPARCOS-based determination, although the Gaia mean parallax for the Pleiades is a significant exception. We have no current explanation for that discrepancy. Most clusters are observed to extend to nearly 15 pc from the cluster centre, and it will be up to future Gaia releases to establish whether those potential cluster-member stars are still dynamically bound to the clusters.Conclusions. The Gaia DR1 provides the means to examine open clusters far beyond their more easily visible cores, and can provide membership assessments based on proper motions and parallaxes. A combined HR diagram shows the same features as observed before using the HIPPARCOS data, with clearly increased luminosities for older A and F dwarfs

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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