Single stranded fully Modified-Phosphorothioate oligonucleotides can induce structured nuclear inclusions, alter nuclear protein localization and disturb the transcriptome In Vitro

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects

An accelerated closed universe

We study a model in which a closed universe with dust and quintessence matter components may look like an accelerated flat Friedmann-Robertson-Walker (FRW) universe at low redshifts. Several quantities relevant to the model are expressed in terms of observed density parameters, $\Omega_M$ and $\Omega_{\Lambda}$, and of the associated density parameter $\Omega_Q$ related to the quintessence scalar field $Q$.Comment: 11 pages. For a festschrift honoring Alberto Garcia. To appear in Gen. Rel. Gra

Is the evidence for dark energy secure?

Several kinds of astronomical observations, interpreted in the framework of the standard Friedmann-Robertson-Walker cosmology, have indicated that our universe is dominated by a Cosmological Constant. The dimming of distant Type Ia supernovae suggests that the expansion rate is accelerating, as if driven by vacuum energy, and this has been indirectly substantiated through studies of angular anisotropies in the cosmic microwave background (CMB) and of spatial correlations in the large-scale structure (LSS) of galaxies. However there is no compelling direct evidence yet for (the dynamical effects of) dark energy. The precision CMB data can be equally well fitted without dark energy if the spectrum of primordial density fluctuations is not quite scale-free and if the Hubble constant is lower globally than its locally measured value. The LSS data can also be satisfactorily fitted if there is a small component of hot dark matter, as would be provided by neutrinos of mass 0.5 eV. Although such an Einstein-de Sitter model cannot explain the SNe Ia Hubble diagram or the position of the `baryon acoustic oscillation' peak in the autocorrelation function of galaxies, it may be possible to do so e.g. in an inhomogeneous Lemaitre-Tolman-Bondi cosmology where we are located in a void which is expanding faster than the average. Such alternatives may seem contrived but this must be weighed against our lack of any fundamental understanding of the inferred tiny energy scale of the dark energy. It may well be an artifact of an oversimplified cosmological model, rather than having physical reality.Comment: 12 pages, 5 figures; to appear in a special issue of General Relativity and Gravitation, eds. G.F.R. Ellis et al; Changes: references reformatted in journal style - text unchange

Detection of IMBHs from microlensing in globular clusters

Globular clusters have been alternatively predicted to host intermediate-mass black holes (IMBHs) or nearly impossible to form and retain them in their centres. Over the last decade enough theoretical and observational evidence have accumulated to believe that many galactic globular clusters may host IMBHs in their centres, just like galaxies do. The well-established correlations between the supermassive black holes and their host galaxies do suggest that, in extrapolation, globular clusters (GCs) follow the same relations. Most of the attempts in search of the central black holes (BHs) are not direct and present enormous observational difficulties due to the crowding of stars in the GC cores. Here we propose a new method of detection of the central BH -- the microlensing of the cluster stars by the central BH. If the core of the cluster is resolved, the direct determination of the lensing curve and lensing system parameters are possible; if unresolved, the differential imaging technique can be applied. We calculate the optical depth to central BH microlensing for a selected list of Galactic GCs and estimate the average time duration of the events. We present the observational strategy and discuss the detectability of microlensing events using a 2-m class telescope.Comment: 10 pages, 11 figures, accepted in New Astronom

Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major

Background The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. Objectives In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. Methods Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. Results Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. Conclusions These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications

Paraspeckle subnuclear bodies depend on dynamic heterodimerisation of DBHS RNA-binding proteins via their structured domains

RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONOSFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches.Pei Wen Lee, Andrew C. Marshall, Gavin J. Knott, Simon Kobelke, Luciano Martelotto, Ellie Cho, Paul J. McMillan, Mihwa Lee, Charles S. Bond, and Archa H. Fo

Last Glacial Period Cryptotephra Deposits in an Eastern North Atlantic Marine Sequence: Exploring Linkages to the Greenland Ice-Cores

The establishment of a tephra framework for the Greenland ice-cores spanning the last glacial period, particularly between 25 and 45 ka b2k, provides strong potential for precisely correlating other palaeoclimatic records to these key archives. Tephra-based synchronisation allows the relative timing of past climatic changes recorded within different depositional environments and potential causal mechanisms to be assessed. Recent studies of North Atlantic marine records have demonstrated the potential of tracing cryptotephra horizons in these sequences and the development of protocols now allows a careful assessment of the isochronous nature of such horizons. Here we report on tephrochronological investigations of a marine sequence retrieved from the Goban Spur, Eastern North Atlantic, covering ?25–60 ka b2k. Density and magnetic separation techniques and an assessment of potential transport and depositional mechanisms have identified three previously unknown isochronous tephra horizons along with deposits of the widespread North Atlantic Ash Zone II and Faroe Marine Ash Zone III. Correlations between the new horizons and the Greenland ice-core tephra framework are explored and despite no tie-lines being identified the key roles that high-resolution climatostratigraphy and shard-specific trace element analysis can play within the assessment of correlations is demonstrated. The previously unknown horizons are new additions to the overall North Atlantic tephra framework for the last glacial period and could be key horizons for future correlations

Inflation Physics from the Cosmic Microwave Background and Large Scale Structure

Fluctuations in the intensity and polarization of the cosmic microwave background (CMB) and the large-scale distribution of matter in the universe each contain clues about the nature of the earliest moments of time. The next generation of CMB and large-scale structure (LSS) experiments are poised to test the leading paradigm for these earliest moments---the theory of cosmic inflation---and to detect the imprints of the inflationary epoch, thereby dramatically increasing our understanding of fundamental physics and the early universe. A future CMB experiment with sufficient angular resolution and frequency coverage that surveys at least 1 of the sky to a depth of 1 uK-arcmin can deliver a constraint on the tensor-to-scalar ratio that will either result in a 5-sigma measurement of the energy scale of inflation or rule out all large-field inflation models, even in the presence of foregrounds and the gravitational lensing B-mode signal. LSS experiments, particularly spectroscopic surveys such as the Dark Energy Spectroscopic Instrument, will complement the CMB effort by improving current constraints on running of the spectral index by up to a factor of four, improving constraints on curvature by a factor of ten, and providing non-Gaussianity constraints that are competitive with the current CMB bounds

Evidence for Shock Acceleration and Intergalactic Magnetic Fields in a Large-Scale Filament of Galaxies ZwCl 2341.1+0000

We report the discovery of large-scale diffuse radio emission from what appears to be a large-scale filamentary network of galaxies in the region of cluster ZwCl 2341.1+0000, and stretching over an area of at least $6 h^{-1}_{50}$ Mpc in diameter. Multicolour CCD observations yield photometric redshifts indicating that a significant fraction of the optical galaxies in this region is at a redshift of z=0.3. This is supported by spectroscopic measurements of 4 galaxies in the SDSS survey at a mean z=0.27. We present VLA images at 20 cm (NVSS) and 90 cm wavelengths, showing the detailed radio structure of the filaments. Comparison with the VLA high resolution FIRST radio survey shows that the diffuse emission is not due to known individual point sources. The diffuse radio-emission has a spectral index $\alpha \lesssim -0.5$, and is most likely synchrotron emission from relativistic charged particles in an inter-galactic magnetic field. Furthermore, this optical/radio structure is detected in X-rays by the ROSAT all-sky survey. It has a 0.1--2.4 keV luminosity of about $10^{44}$ erg s$^{-1}$ and shows an extended highly non-relaxed morphology. These observations suggest that ZwCl 2341.1+0000 is possibly a proto-cluster of galaxies in which we are witnessing the process of structure formation. We show (both analytically and by numerical simulations) that the energetics of accretion shocks generated in forming large-scale structures are sufficient to produce enough high energy cosmic-ray (CR) electrons required to explain the observed radio emission, provided a magnetic field of strength of about 1 micro Gauss is present there.Thus it is the first evidence of cosmic-ray particle acceleration and magnetic fields occuring on a super-cluster scale. (Abridged)Comment: Replaced with the published version. The published paper can be accessed from http://www.elsevier.com/gej-ng/10/33/29/71/56/53/article.htm

Cold Plus Hot Dark Matter Cosmology in the Light of Solar and Atmospheric Neutrino Oscillations

We explore the implications of possible neutrino oscillations, as indicated by the solar and atmospheric neutrino experiments, for the cold plus hot dark matter scenario of large scale structure formation. We find that there are essentially three distinct schemes that can accommodate the oscillation data and which also allow for dark matter neutrinos. These include (i) three nearly degenerate (in mass) neutrinos, (ii) non-degenerate masses with $\nu_\tau$ in the eV range, and (iii) nearly degenerate $\nu_\mu-\nu_\tau$ pair (in the eV range), with the additional possibility that the electron neutrino is cosmologically significant. The last two schemes invoke a `sterile' neutrino which is light (< or ~ eV). We discuss the implications of these schemes for $\bar{\nu}_\mu - \bar{\nu}_e$ and $\nu_\mu - \nu_\tau$ oscillation, and find that scheme (ii) in particular, predicts them to be in the observable range. As far as structure formation is concerned, we compare the one neutrino flavor case with a variety of other possibilities, including two and three degenerate neutrino flavors. We show, both analytically and numerically, the effects of these neutrino mass scenarios on the amplitude of cosmological density fluctuations. With a Hubble constant of 50 km s$^{-1}$ Mpc$^{-1}$, a spectral index of unity, and $\Omega_{baryon} = 0.05$, the two and three flavor scenarios fit the observational data marginally better than the single flavor scheme. However, taking account of the uncertainties in these parameters, we show that it is premature to pick a clear winner.Comment: 1 LaTEX file plus 1 uuencoded Z-compressed tar file with 3 postscript figure