Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 110 (2013): 8696-8701, doi:10.1073/pnas.1216683110.Organisms that use ammonium as the sole nitrogen source discriminate between [15N] and [14N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (≥0.89 µM), discrimination against the heavy isotope by the WT strain (−19.2‰) can be accounted for by the equilibrium isotope effect for dissociation of NH4+ to NH3 + H+. NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (≤0.18 µM), discrimination reflects an isotope effect for the NH4+ channel AmtB (−14.1‰). By making E. coli dependent on the low-affinity ammonium-assimilatory pathway, we determined that biosynthetic glutamate dehydrogenase has an inverse isotope effect in vivo (+8.8‰). Likewise, by making unmediated diffusion of NH3 across the cytoplasmic membrane rate-limiting for cell growth in a mutant strain lacking AmtB, we could deduce an in vivo isotope effect for transport of NH3 across the membrane (−10.9‰). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them.This work was supported by NIH grant GM38361 to S.K. J.M.H. thanks WHOI for support as an emeritus scientist.2013-11-0

Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability.

Thrombospondins (Thbs) are a family of five secreted matricellular glycoproteins in vertebrates that broadly affect cell-matrix interaction. While Thbs4 is known to protect striated muscle from disease by enhancing sarcolemmal stability through increased integrin and dystroglycan attachment complexes, here we show that Thbs3 antithetically promotes sarcolemmal destabilization by reducing integrin function, augmenting disease-induced decompensation. Deletion of Thbs3 in mice enhances integrin membrane expression and membrane stability, protecting the heart from disease stimuli. Transgene-mediated overexpression of α7β1D integrin in the heart ameliorates the disease predisposing effects of Thbs3 by augmenting sarcolemmal stability. Mechanistically, we show that mutating Thbs3 to contain the conserved RGD integrin binding domain normally found in Thbs4 and Thbs5 now rescues the defective expression of integrins on the sarcolemma. Thus, Thbs proteins mediate the intracellular processing of integrin plasma membrane attachment complexes to regulate the dynamics of cellular remodeling and membrane stability

Almost a spider: a 305-million-year-old fossil arachnid and spider origins

Spiders are an important animal group, with a long history. Details of their origins remain limited, with little knowledge of their stem group, and no insights into the sequence of character acquisition during spider evolution. We describe a new fossil arachnid, Idmonarachne brasieri gen. et sp. nov. from the Late Carboniferous (Stephanian, ca 305–299 Ma) of Montceau-les-Mines, France. It is three-dimensionally preserved within a siderite concretion, allowing both laboratory- and synchrotron-based phase-contrast computed tomography reconstruction. The latter is a first for siderite-hosted fossils and has allowed us to investigate fine anatomical details. Although distinctly spider-like in habitus, this remarkable fossil lacks a key diagnostic character of Araneae: spinnerets on the underside of the opisthosoma. It also lacks a flagelliform telson found in the recently recognized, spider-related, Devonian–Permian Uraraneida. Cladistic analysis resolves our new fossil as sister group to the spiders: the spider stem-group comprises the uraraneids and I. brasieri. While we are unable to demonstrate the presence of spigots in this fossil, the recovered phylogeny suggests the earliest character to evolve on the spider stem-group is the secretion of silk. This would have been followed by the loss of a flagelliform telson, and then the ability to spin silk using spinnerets. This last innovation defines the true spiders, significantly post-dates the origins of silk, and may be a key to the group's success. The Montceau-les-Mines locality has previously yielded a mesothele spider (with spinnerets). Evidently, Late Palaeozoic spiders lived alongside Palaeozoic arachnid grades which approached the spider condition, but did not express the full suite of crown-group autapomorphies

A Cross-national Comparison of Public Project Benefits Management Practices – The Effectiveness of Benefits Management Frameworks in Application

© 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group. Benefits are the principal reason why an organization may seek to enact change through programmes and projects. The discipline of identification, definition, planning, tracking and realization of benefits is recognized to be instrumental in achieving organizational strategy. In this study, we describe the results of a cross-national comparison of public sector benefits management (BM) practices in Australia, Canada, the UK and the USA. It explores ‘BM practices in action’, considering to what extent ‘espoused’ or ‘mandated’ frameworks are actually practised and perceived by their users. Employing qualitative analysis, semi-structured interview data were analysed from 46 participants with experience in sponsoring, managing and/or reviewing government projects. The results expose considerable variation in the adoption and standardization of BM frameworks from inter and intragovernmental perspectives. We evidence a strong focus on benefits identification across the data set, specifically at the outset (the business case stage seeking project approval) and observe deterioration in focus as the project or programme progresses through the authorization (or assurance) approval gates towards close-out and operations. The results further emphasize the prominence of political interest, leadership buy-in, a benefits-driven culture and a transparent benefits reporting mechanism in the implementation of ‘effective’ BM frameworks

Single Assay for Simultaneous Detection and Differential Identification of Human and Avian Influenza Virus Types, Subtypes, and Emergent Variants

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents

Modulation of Cell Adhesion and Migration by the Histone Methyltransferase Subunit mDpy-30 and Its Interacting Proteins

We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT) complex, also localizes at the trans-Golgi network (TGN), where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF) BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of β1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of β1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to β1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as β1 integrin