Dysfunction of contractile proteins in hypertrophic cardiomyopathy

The contractility of human heart samples from patients diagnosed with hypertrophic cardiomyopathy were studied using a quantitative in vitro motility assay. The aim of this work was to investigate the molecular phenotype of thin filament proteins in the HCM heart. Three biopsy samples with thin filament mutations were studied alongside samples acquired from a subset of HCM patients classified with hypertrophic obstructive cardiomyopathy. The primary effect of thin filament mutations was investigated by reconstituting Factin with ACTC E99K into thin filament with donor troponin. The E99K actin filaments had a higher Ca2+-sensitivity then filaments composed of donor F-actin (with no mutation) (EC50 E99K/donor 0.78±0.14, p=0.02). A similar higher Ca2+- sensitivity was found when recombinant TnT K273N was incorporated into donor troponin and compared to native donor troponin (EC50 K273N/donor 0.54±0.17, p=0.006). Troponin was also purified from HOCM heart samples. This troponin did not contain a causative mutation but behaved abnormally in the response of thin filament Ca2+- sensitivity to changes in TnI phosphorylation (EC50 PKA-HOCM/HOCM 1.08±0.25, p=0.3) as mean TnI phosphorylation of PKA-HOCM was 1.56 molsPi/molsTnI and HOCM was 0.29 molsPi/molsTnI. Thus, thin filament Ca2+-sensitivity was uncoupled from TnI phosphorylation in thin filaments with HOCM troponin. When the native TnT subunits were replaced with recombinant TnT this coupling was restored (EC50 HOCM rTnT/HOCM 0.63±0.26, p=0.03). It would appear that the result of HCM-causing mutations are two-fold. The primary effect of the HCM-causing mutations is to increase thin filament Ca2+-sensitivity. However, the contraction machinery appears to be the target of secondary modifications, that occur due to the pathology of the disease. Resulting in further changes, such as changes in protein composition and post-translational modification. One major consequence of these modifications may be to uncouple the relatively labile regulation of thin filament Ca2+-sensitivity by TnI phosphorylation

Searching for Cooling Signatures in Strong Lensing Galaxy Clusters: Evidence Against Baryons Shaping the Matter Distribution in Cluster Cores

The process by which the mass density profile of certain galaxy clusters becomes centrally concentrated enough to produce high strong lensing (SL) cross-sections is not well understood. It has been suggested that the baryonic condensation of the intra-cluster medium (ICM) due to cooling may drag dark matter to the cores and thus steepen the profile. In this work, we search for evidence of ongoing ICM cooling in the first large, well-defined sample of strong lensing selected galaxy clusters in the range 0.1 < z < 0.6. Based on known correlations between the ICM cooling rate and both optical emission line luminosity and star formation, we measure, for a sample of 89 strong lensing clusters, the fraction of clusters that have [OII]3727 emission in their brightest cluster galaxy (BCG). We find that the fraction of line-emitting BCGs is constant as a function of redshift for z > 0.2 and shows no statistically significant deviation from the total cluster population. Specific star formation rates, as traced by the strength of the 4000 angstrom break, D_4000, are also consistent with the general cluster population. Finally, we use optical imaging of the SL clusters to measure the angular separation, R_arc, between the arc and the center of mass of each lensing cluster in our sample and test for evidence of changing [OII] emission and D_4000 as a function of R_arc, a proxy observable for SL cross-sections. D_4000 is constant with all values of R_arc, and the [OII] emission fractions show no dependence on R_arc for R_arc > 10" and only very marginal evidence of increased weak [OII] emission for systems with R_arc < 10". These results argue against the ability of baryonic cooling associated with cool core activity in the cores of galaxy clusters to strongly modify the underlying dark matter potential, leading to an increase in strong lensing cross-sections.Comment: 9 Pages, 5 Figures, 1 Tabl

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod–res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species

Histories of Deposition: Creating Chronologies for the Late Bronze Age-Early Iron Age transition in southern Britain

The Late Bronze Age–Early Iron Age midden sites of Southern Britain are amongst the richest archaeological sites in the country. The organic accumulations contain substantial quantities of animal bone, decorated ceramics, metalwork and other objects; the often deep stratigraphy allows for changes in material culture and depositional practices, food production and consumption, and shifts in social identities, to be traced through time. The well-stratified assemblages also provide useful materials for dating the deposits. This has been problematic, however, as the majority of samples produce unhelpfully broad calibrated radiocarbon dates, due to the effects of the earlier Iron Age plateau in the calibration curve, which spans c. 800–400 BC. Interpretation has relied on current understandings of the associated pottery and metalwork, which placed most midden sites somewhere between the tenth and the seventh/mid-sixth centuries cal BC (c. 1000–600/550 cal BC), but the end-date of these traditions is particularly uncertain. This article addresses this issue by presenting the results of a new dating programme for East Chisenbury in Wiltshire, southern England. Twenty-eight radiocarbon determinations were obtained and combined with the site stratigraphy in a Bayesian chronological model. The results have transformed the chronology of the site, with the end of the occupation sequence being pulled forward some one-hundred years, to the mid-to-late fifth century cal BC. These new chronologies have significant implications for our understanding of the Late Bronze Age–Early Iron Age transition and require a revision of the currently accepted chronology of post-Deverel Rimbury decorated wares in south-central England

NGTS-4b: A sub-Neptune transiting in the desert

We report the discovery of NGTS-4b, a sub-Neptune-sized planet transiting a 13th magnitude K-dwarf in a 1.34 d orbit. NGTS-4b has a mass M = 20.6 ± 3.0 M⊕ and radius R = 3.18 ± 0.26 R⊕, which places it well within the so-called ‘Neptunian Desert’. The mean density of the planet (3.45 ± 0.95 g cm−3) is consistent with a composition of 100  per cent H2O or a rocky core with a volatile envelope. NGTS-4b is likely to suffer significant mass loss due to relatively strong EUV/X-ray irradiation. Its survival in the Neptunian desert may be due to an unusually high-core mass, or it may have avoided the most intense X-ray irradiation by migrating after the initial activity of its host star had subsided. With a transit depth of 0.13 ± 0.02 per cent, NGTS-4b represents the shallowest transiting system ever discovered from the ground, and is the smallest planet discovered in a wide-field ground-based photometric survey

In situ optical measurement of charge transport dynamics in organic photovoltaics.

We present a novel experimental approach which allows extraction of both spatial and temporal information on charge dynamics in organic solar cells. Using the wavelength dependence of the photonic structure in these devices, we monitor the change in spatial overlap between the photogenerated hole distribution and the optical probe profile as a function of time. In a model system we find evidence for a buildup of the photogenerated hole population close to the hole-extracting electrode on a nanosecond time scale and show that this can limit charge transport through space-charge effects under operating conditions.This work was supported by the EPSRC [Grant number EP/ G060738/1].This is the author accepted manuscript. The final published version is available at http://pubs.acs.org/doi/abs/10.1021/nl503687u

Exploring the Effect of G6PC2 Single Nucleotide Polymorphisms on Enzyme Activity and Human Health

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that is highly expressed in pancreatic islet beta cells. Genome wide association studies (GWAS) have shown that single nucleotide polymorphisms (SNPs) in the G6PC2 gene are associated with variations in fasting blood glucose (FBG), a parameter linked with risk for type 2 diabetes (T2D). Studies in mice have complemented these GWAS data by showing that deletion of G6pc2 abolishes islet glucose-6-phosphatase activity and lowers FBG. We hypothesize that G6pc2 forms a substrate cycle with glucokinase that determines the sensitivity of glucose-stimulated insulin secretion (GSIS) to glucose. In support of this hypothesis we have previously shown that deletion of G6pc2 enhances GSIS at sub-maximal glucose concentrations and abolishes glucose cycling in isolated islets. More recently we have demonstrated that deletion of G6pc2 enhances glycolysis in isolated mouse islets, and that high rates of glucose cycling are also detected in human islets. Our broad hypothesis is that the results of these studies will strongly suggest that G6PC2 inhibition should be considered as a novel therapeutic strategy for lowering FBG and thereby preventing T2D. To extend these observations we have developed a novel intact cell assay for G6PC2 activity. This assay relies on the observation that CREB and ChREBP bound to the rat G6PC1 promoter are highly glucose responsive in the rat islet-derived 832/13 cell line and the fact that endogenous G6PC2 is absent. In the presence of catalytically-dead G6PC2, glucose stimulates G6PC1-luciferase fusion gene expression. However, this induction is blunted in the presence of wild type G6PC2. We are using this assay to determine the effect of non-synonymous G6PC2 SNPs on G6PC2 activity and then examining the association between SNPs that markedly affect G6PC2 activity with their effects on human health as assessed using Vanderbilt’s BioVU biobank. These data will reveal whether SNPs in G6PC2 are associated with only altered FBG or whether G6PC2 affects other aspects of human health

Spin-dependent recombination probed through the dielectric polarizability.

Despite residing in an energetically and structurally disordered landscape, the spin degree of freedom remains a robust quantity in organic semiconductor materials due to the weak coupling of spin and orbital states. This enforces spin-selectivity in recombination processes which plays a crucial role in optoelectronic devices, for example, in the spin-dependent recombination of weakly bound electron-hole pairs, or charge-transfer states, which form in a photovoltaic blend. Here, we implement a detection scheme to probe the spin-selective recombination of these states through changes in their dielectric polarizability under magnetic resonance. Using this technique, we access a regime in which the usual mixing of spin-singlet and spin-triplet states due to hyperfine fields is suppressed by microwave driving. We present a quantitative model for this behaviour which allows us to estimate the spin-dependent recombination rate, and draw parallels with the Majorana-Brossel resonances observed in atomic physics experiments.This work was supported by the Engineering and Physical Sciences Research Council [Grants No. EP/G060738/1]. A. D. C. acknowledges support from the E. Oppenheimer Foundation and St Catharine's College, Cambridge. S. L. B. is grateful for support from the EPSRC Supergen SuperSolar Project, the Armourers and Brasiers Gauntlet Trust and Magdalene College, Cambridge.This is the final published version of the article. It was originally published in Nature Communications (Bayliss et. al, Nature Communications 2015, 6, 8534, doi:10.1038/ncomms9534). The final version is available at http://dx.doi.org/10.1038/ncomms953

Spin-dependent recombination probed through the dielectric polarizability.

Despite residing in an energetically and structurally disordered landscape, the spin degree of freedom remains a robust quantity in organic semiconductor materials due to the weak coupling of spin and orbital states. This enforces spin-selectivity in recombination processes which plays a crucial role in optoelectronic devices, for example, in the spin-dependent recombination of weakly bound electron-hole pairs, or charge-transfer states, which form in a photovoltaic blend. Here, we implement a detection scheme to probe the spin-selective recombination of these states through changes in their dielectric polarizability under magnetic resonance. Using this technique, we access a regime in which the usual mixing of spin-singlet and spin-triplet states due to hyperfine fields is suppressed by microwave driving. We present a quantitative model for this behaviour which allows us to estimate the spin-dependent recombination rate, and draw parallels with the Majorana-Brossel resonances observed in atomic physics experiments.This work was supported by the Engineering and Physical Sciences Research Council [Grants No. EP/G060738/1]. A. D. C. acknowledges support from the E. Oppenheimer Foundation and St Catharine's College, Cambridge. S. L. B. is grateful for support from the EPSRC Supergen SuperSolar Project, the Armourers and Brasiers Gauntlet Trust and Magdalene College, Cambridge.This is the final published version of the article. It was originally published in Nature Communications (Bayliss et. al, Nature Communications 2015, 6, 8534, doi:10.1038/ncomms9534). The final version is available at http://dx.doi.org/10.1038/ncomms953

A TPX2 Proteomimetic Has Enhanced Affinity for Aurora-A Due to Hydrocarbon Stapling of a Helix

Inhibition of protein kinases using ATP-competitive compounds is an important strategy in drug discovery. In contrast, the allosteric regulation of kinases through the disruption of protein-protein interactions has not been widely adopted, despite the potential for selective targeting. Aurora-A kinase regulates mitotic entry and mitotic spindle assembly and is a promising target for anticancer therapy. The microtubule-associated protein TPX2 activates Aurora-A through binding to two sites. Aurora-A recognition is mediated by two motifs within the first 43 residues of TPX2, connected by a flexible linker. To characterize the contributions of these three structural elements, we prepared a series of TPX2 proteomimetics and investigated their binding affinity for Aurora-A using isothermal titration calorimetry. A novel stapled TPX2 peptide was developed that has improved binding affinity for Aurora-A and mimics the function of TPX2 in activating Aurora-A's autophosphorylation. We conclude that the helical region of TPX2 folds upon binding Aurora-A, and that stabilization of this helix does not compromise Aurora-A activation. This study demonstrates that the preparation of these proteomimetics using modern synthesis methods is feasible and their biochemical evaluation demonstrates the power of proteomimetics as tool compounds for investigating PPIs involving intrinsically disordered regions of proteins