Differentiated, promoter-specific response of [4Fe-4S] NsrR DNA-binding to reaction with nitric oxide

NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA-binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ~2 NO per cluster, while for the hmpA1 and nsrR promoters, ~4 and ~8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8 – 10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ~2 NO per cluster, along with two further intermediates at ~4 and ~6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA-binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes

Mass spectrometric identification of intermediates in the O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion in FNR

The iron-sulfur cluster containing protein FNR is the master regulator for the switch between anaerobic and aerobic respiration in Escherichia coli and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O2 that leads to conversion to a [2Fe-2S] form with loss of high affinity DNA-binding. Here we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide new insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a novel [3Fe-3S] cluster and persulfide coordinated [2Fe-2S] clusters ([2Fe-2S](S)n, where n = 1 or 2). Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion, and not as a subsequent, secondary reaction, to generate ([2Fe-2S](S)n species. This methodology shows great potential for broad application to studies of protein cofactorsmall molecule interactions

NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR

Long-term determination of energetic electron precipitation into the atmosphere from AARDDVARK subionospheric VLF observations

We analyze observations of subionospherically propagating very low frequency (VLF) radio waves to determine outer radiation belt energetic electron precipitation (EEP) flux magnitudes. The radio wave receiver in Sodankylä, Finland (Sodankylä Geophysical Observatory) observes signals from the transmitter with call sign NAA (Cutler, Maine). The receiver is part of the Antarctic-Arctic Radiation-belt Dynamic Deposition VLF Atmospheric Research Konsortia (AARDDVARK). We use a near-continuous data set spanning November 2004 until December 2013 to determine the long time period EEP variations. We determine quiet day curves over the entire period and use these to identify propagation disturbances caused by EEP. Long Wave Propagation Code radio wave propagation modeling is used to estimate the precipitating electron flux magnitudes from the observed amplitude disturbances, allowing for solar cycle changes in the ambient D region and dynamic variations in the EEP energy spectra. Our method performs well during the summer months when the daylit ionosphere is most stable but fails during the winter. From the summer observations, we have obtained 693 days worth of hourly EEP flux magnitudes over the 2004–2013 period. These AARDDVARK-based fluxes agree well with independent satellite precipitation measurements during high-intensity events. However, our method of EEP detection is 10–50 times more sensitive to low flux levels than the satellite measurements. Our EEP variations also show good agreement with the variation in lower band chorus wave powers, providing some confidence that chorus is the primary driver for the outer belt precipitation we are monitoring

Diagramming social practice theory:An interdisciplinary experiment exploring practices as networks

Achieving a transition to a low-carbon energy system is now widely recognised as a key challenge facing humanity. To date, the vast majority of research addressing this challenge has been conducted within the disciplines of science, engineering and economics utilising quantitative and modelling techniques. However, there is growing awareness that meeting energy challenges requires fundamentally socio-technical solutions and that the social sciences have an important role to play. This is an interdisciplinary challenge but, to date, there remain very few explorations of, or reflections on, interdisciplinary energy research in practice. This paper seeks to change that by reporting on an interdisciplinary experiment to build new models of energy demand on the basis of cutting-edge social science understandings. The process encouraged the social scientists to communicate their ideas more simply, whilst allowing engineers to think critically about the embedded assumptions in their models in relation to society and social change. To do this, the paper uses a particular set of theoretical approaches to energy use behaviour known collectively as social practice theory (SPT) - and explores the potential of more quantitative forms of network analysis to provide a formal framework by means of which to diagram and visualize practices. The aim of this is to gain insight into the relationships between the elements of a practice, so increasing the ultimate understanding of how practices operate. Graphs of practice networks are populated based on new empirical data drawn from a survey of different types (or variants) of laundry practice. The resulting practice networks are analysed to reveal characteristics of elements and variants of practice, such as which elements could be considered core to the practice, or how elements between variants overlap, or can be shared. This promises insights into energy intensity, flexibility and the rootedness of practices (i.e. how entrenched/ established they are) and so opens up new questions and possibilities for intervention. The novelty of this approach is that it allows practice data to be represented graphically using a quantitative format without being overly reductive. Its usefulness is that it is readily applied to large datasets, provides the capacity to interpret social practices in new ways, and serves to open up potential links with energy modeling. More broadly, a significant dimension of novelty has been the interdisciplinary approach, radically different to that normally seen in energy research. This paper is relevant to a broad audience of social scientists and engineers interested in integrating social practices with energy engineering

Superconductivity in metallic twisted bilayer graphene stabilized by WSe₂

Magic-angle twisted bilayer graphene (TBG), with rotational misalignment close to 1.1 degrees, features isolated flat electronic bands that host a rich phase diagram of correlated insulating, superconducting, ferromagnetic and topological phases. Correlated insulators and superconductivity have been previously observed only for angles within 0.1 degree of the magic angle and occur in adjacent or overlapping electron-density ranges; nevertheless, the origins of these states and the relation between them remain unclear, owing to their sensitivity to microscopic details. Beyond twist angle and strain, the dependence of the TBG phase diagram on the alignment and thickness of the insulating hexagonal boron nitride (hBN) used to encapsulate the graphene sheets indicates the importance of the microscopic dielectric environment. Here we show that adding an insulating tungsten diselenide (WSe₂) monolayer between the hBN and the TBG stabilizes superconductivity at twist angles much smaller than the magic angle. For the smallest twist angle of 0.79 degrees, superconductivity is still observed despite the TBG exhibiting metallic behaviour across the whole range of electron densities. Finite-magnetic-field measurements further reveal weak antilocalization signatures as well as breaking of fourfold spin–valley symmetry, consistent with spin–orbit coupling induced in the TBG via its proximity to WSe₂. Our results constrain theoretical explanations for the emergence of superconductivity in TBG and open up avenues towards engineering quantum phases in moiré systems

Iterative focused screening with biological fingerprints identifies selective Asc-1 inhibitors distinct from traditional high throughput screening

N-methyl-d-aspartate receptors (NMDARs) mediate glutamatergic signaling that is critical to cognitive processes in the central nervous system, and NMDAR hypofunction is thought to contribute to cognitive impairment observed in both schizophrenia and Alzheimer’s disease. One approach to enhance the function of NMDAR is to increase the concentration of an NMDAR coagonist, such as glycine or d-serine, in the synaptic cleft. Inhibition of alanine–serine–cysteine transporter-1 (Asc-1), the primary transporter of d-serine, is attractive because the transporter is localized to neurons in brain regions critical to cognitive function, including the hippocampus and cortical layers III and IV, and is colocalized with d-serine and NMDARs. To identify novel Asc-1 inhibitors, two different screening approaches were performed with whole-cell amino acid uptake in heterologous cells stably expressing human Asc-1: (1) a high-throughput screen (HTS) of 3 M compounds measuring 35S l-cysteine uptake into cells attached to scintillation proximity assay beads in a 1536 well format and (2) an iterative focused screen (IFS) of a 45 000 compound diversity set using a 3H d-serine uptake assay with a liquid scintillation plate reader in a 384 well format. Critically important for both screening approaches was the implementation of counter screens to remove nonspecific inhibitors of radioactive amino acid uptake. Furthermore, a 15 000 compound expansion step incorporating both on- and off-target data into chemical and biological fingerprint-based models for selection of additional hits enabled the identification of novel Asc-1-selective chemical matter from the IFS that was not identified in the full-collection HTS

Realising transition pathways for a more electric, low-carbon energy system in the United Kingdom: challenges, insights and opportunities

The United Kingdom has placed itself on a transition towards a low-carbon economy and society, through the imposition of a legally-binding goal aimed at reducing its ‘greenhouse gas’ emissions by 80% by 2050 against a 1990 baseline. A set of three low-carbon, socio-technical transition pathways were developed and analysed via an innovative collaboration between engineers, social scientists and policy analysts. The pathways focus on the power sector, including the potential for increasing use of low-carbon electricity for heating and transport, within the context of critical European Union developments and policies. Their development started from narrative storylines regarding different governance framings, drawing on interviews and workshops with stakeholders and analysis of historical analogies. The quantified UK pathways were named Market Rules, Central Co-ordination and Thousand Flowers; each reflecting a dominant logic of governance arrangements. The aim of the present contribution was to use these pathways to explore what is needed to realise a transition that successfully addresses the so-called energy policy ‘trilemma,’ i.e. the simultaneous delivery of low carbon, secure and affordable energy services. Analytical tools were developed and applied to assess the technical feasibility,social acceptability, and environmental and economic impacts of the pathways. Technological and behavioural developments were examined, alongside appropriate governance structures and regulations for these low-carbon transition pathways, as well as the roles of key energy system ‘actors’ (both large and small). An assessment of the part that could possibly be played by future demand side response was also undertaken in order to understand the factors that drive energy demand and energy-using behaviour, and reflecting growing interest in demand side response for balancing a system with high proportions of renewable generation. A set of interacting and complementary engineering and technoeconomic models or tools were then employed to analyse electricity network infrastructure investment and operational decisions to assist market design and option evaluation. This provided a basis for integrating the analysis within a whole systems framework of electricity system development, together with the evaluation of future economic benefits, costs and uncertainties. Finally, the energy and environmental performance of the different energy mixes were appraised on a‘life-cycle’ basis to determine the greenhouse gas emissions and other ecological or health burdens associated with each of the three transition pathways. Here, the challenges, insights and opportunities that have been identified over the transition towards a low-carbon future in the United Kingdom are described with the purpose of providing a valuable evidence base for developers, policy makers and other stakeholders

A Spectroscopic Survey of a Sample of Active M Dwarfs

A moderate resolution spectroscopic survey of Fleming's sample of 54 X-ray selected M dwarfs with photometric distances less than 25 pc is presented. Radial and rotation velocities have been measured by fits to the H-alpha profiles. Radial velocities have been measured by cross correlation. Artificial broadening of an observed spectrum has produced a relationship between H-alpha FWHM and rotation speed, which we use to infer rotation speeds for the entire sample by measurement of the H-alpha emission line. We find 3 ultra-fast rotators (UFRs, vsini > 100km/s), and 8 stars with 30 < vsini < 100 km/s. The UFRs have variable emission. Cross-correlation velocities measured for ultra-fast rotators (UFRs) are shown to depend on rotation speed and the filtering used. The radial velocity dispersion of the sample is 17 km/s. A new double emission line spectroscopic binary with a period of 3.55 days has been discovered, and another known one is in the sample. Three other objects are suspected spectroscopic binaries, and at least six are visual doubles. The only star in the sample observed to have significant lithium is a known TW Hya Association member, TWA 8A. These results show that there are a number of young (< 10^8 yr) and very young (< 10^7 yr) low mass stars in the immediate solar neighbourhood. The H-alpha activity strength does not depend on rotation speed. Our fast rotators are less luminous than similarly fast rotators in the Pleiades. They are either younger than the Pleiades, or gained angular momentum in a different way.Comment: 38 pages incl. 14 figures and 4 tables, plus 12 pages of table for electronic journal only; LaTeX, aastex.cls. Accepted 07/18/02 for publication in The Astronomical Journa