cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis

The AP-2 adaptor β2 appendage scaffolds alternate cargo endocytosis

The independently folded appendages of the large α and β2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The β2 subunit appendage contains a common binding site for β-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing β2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 α subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the β2 chain with the closely related endogenous β1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both β1 and β2 subunit transcripts recapitulates the strong α subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive β2-yellow fluorescent protein (YFP) expressed in the β1 + β2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the β appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a β2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a β-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with β2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and β-arrestin binding to a site upon the β2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and β-arrestin depend on a privileged β2 appendage site for proper cargo recruitment to clathrin bud sites

Transit timing variation in exoplanet WASP-3b

Photometric follow-ups of transiting exoplanets may lead to discoveries of additional, less massive bodies in extrasolar systems. This is possible by detecting and then analysing variations in transit timing of transiting exoplanets. We present photometric observations gathered in 2009 and 2010 for exoplanet WASP-3b during the dedicated transit-timing-variation campaign. The observed transit timing cannot be explained by a constant period but by a periodic variation in the observations minus calculations diagram. Simplified models assuming the existence of a perturbing planet in the system and reproducing the observed variations of timing residuals were identified by three-body simulations. We found that the configuration with the hypothetical second planet of the mass of about 15 Earth masses, located close to the outer 2:1 mean motion resonance is the most likely scenario reproducing observed transit timing. We emphasize, however, that more observations are required to constrain better the parameters of the hypothetical second planet in WASP-3 system. For final interpretation not only transit timing but also photometric observations of the transit of the predicted second planet and the high precision radial-velocity data are needed.Comment: MNRAS accepte

Soft branes in supersymmetry-breaking backgrounds

We revisit the analysis of effective field theories resulting from non-supersymmetric perturbations to supersymmetric flux compactifications of the type-IIB superstring with an eye towards those resulting from the backreaction of a small number of anti-D3-branes. Independently of the background, we show that the low-energy Lagrangian describing the fluctuations of a stack of probe D3-branes exhibits soft supersymmetry breaking, despite perturbations to marginal operators that were not fully considered in some previous treatments. We take this as an indication that the breaking of supersymmetry by anti-D3-branes or other sources may be spontaneous rather than explicit. In support of this, we consider the action of an anti-D3-brane probing an otherwise supersymmetric configuration and identify a candidate for the corresponding goldstino.Comment: 36+5 pages. References added, minor typos correcte

Evidence for alignment of the rotation and velocity vectors in pulsars

We present strong observational evidence for a relationship between the direction of a pulsar's motion and its rotation axis. We show carefully calibrated polarization data for 25 pulsars, 20 of which display linearly polarized emission from the pulse longitude at closest approach to the magnetic pole. Such data allow determination of the position angle of the linear polarisation which in turn reflects the position angle of the rotation axis. Of these 20 pulsars, 10 show an offset between the velocity vector and the polarisation position angle which is either less than 10\degr or more than 80\degr, a fraction which is very unlikely by random chance. We believe that the bimodal nature of the distribution arises from the presence of orthogonal polarisation modes in the pulsar radio emission. In some cases this orthogonal ambiguity is resolved by observations at other wavelengths so that we conclude that the velocity vector and the rotation axis are aligned at birth. Strengthening the case is the fact that 4 of the 5 pulsars with ages less than 3 Myr show this relationship, including the Vela pulsar. We discuss the implications of these findings in the context of the Spruit & Phinney (1998)\nocite{sp98} model of pulsar birth-kicks. We point out that, contrary to claims in the literature, observations of double neutron star systems do not rule out aligned kick models and describe a possible observational test involving the double pulsar system.Comment: MNRAS, In Pres

Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1β in Cellular and Animal Infection Models

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1 beta. Furthermore, inhibition of LasBmediated IL-1 beta activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen

Natriuretic peptides and integrated risk assessment for cardiovascular disease. an individual-participant-data meta-analysis

BACKGROUND: Guidelines for primary prevention of cardiovascular diseases focus on prediction of coronary heart disease and stroke. We assessed whether or not measurement of N-terminal-pro-B-type natriuretic peptide (NT-proBNP) concentration could enable a more integrated approach than at present by predicting heart failure and enhancing coronary heart disease and stroke risk assessment. METHODS: In this individual-participant-data meta-analysis, we generated and harmonised individual-participant data from relevant prospective studies via both de-novo NT-proBNP concentration measurement of stored samples and collection of data from studies identified through a systematic search of the literature (PubMed, Scientific Citation Index Expanded, and Embase) for articles published up to Sept 4, 2014, using search terms related to natriuretic peptide family members and the primary outcomes, with no language restrictions. We calculated risk ratios and measures of risk discrimination and reclassification across predicted 10 year risk categories (ie, <5%, 5% to <7·5%, and ≥7·5%), adding assessment of NT-proBNP concentration to that of conventional risk factors (ie, age, sex, smoking status, systolic blood pressure, history of diabetes, and total and HDL cholesterol concentrations). Primary outcomes were the combination of coronary heart disease and stroke, and the combination of coronary heart disease, stroke, and heart failure. FINDINGS: We recorded 5500 coronary heart disease, 4002 stroke, and 2212 heart failure outcomes among 95 617 participants without a history of cardiovascular disease in 40 prospective studies. Risk ratios (for a comparison of the top third vs bottom third of NT-proBNP concentrations, adjusted for conventional risk factors) were 1·76 (95% CI 1·56-1·98) for the combination of coronary heart disease and stroke and 2·00 (1·77-2·26) for the combination of coronary heart disease, stroke, and heart failure. Addition of information about NT-proBNP concentration to a model containing conventional risk factors was associated with a C-index increase of 0·012 (0·010-0·014) and a net reclassification improvement of 0·027 (0·019-0·036) for the combination of coronary heart disease and stroke and a C-index increase of 0·019 (0·016-0·022) and a net reclassification improvement of 0·028 (0·019-0·038) for the combination of coronary heart disease, stroke, and heart failure. INTERPRETATION: In people without baseline cardiovascular disease, NT-proBNP concentration assessment strongly predicted first-onset heart failure and augmented coronary heart disease and stroke prediction, suggesting that NT-proBNP concentration assessment could be used to integrate heart failure into cardiovascular disease primary prevention

The glitch-induced identity changes of PSR J1119-6127

We demonstrate that the high-magnetic field pulsar J1119-6127 exhibits three different types of behaviour in the radio band. Trailing the "normal" profile peak there is an "intermittent" peak and these components are flanked by two additional components showing very erratic "RRAT-like" emission. Both the intermittent and RRAT-like events are extremely rare and are preceded by a large amplitude glitch in the spin-down parameters. The post-glitch spin-down rate is smaller than the pre-glitch rate. This type of relaxation is very unusual for the pulsar population as a whole, but is observed in the glitch recovery of a RRAT. The abnormal emission behaviour in PSR J1119-6127 was observed up to three months after the epoch of the large glitch, suggestive of changes in the magnetospheric conditions during the fast part of the recovery process. We argue that both the anomalous recoveries and the emission changes could be related to reconfigurations of the magnetic field. Apart from the glitches, the spin-down of PSR J1119-6127 is relatively stable, allowing us to refine the measurement of the braking index (n=2.684\pm0.002) using more than 12 years of timing data. The properties of this pulsar are discussed in light of the growing evidence that RRATs do not form a distinct class of pulsar, but rather are a combination of different extreme emission types seen in other neutron stars. Different sub-classes of the RRATs can potentially be separated by calculating the lower limit on the modulation index of their emission. We speculate that if the abnormal behaviour in PSR J1119-6127 is indeed glitch induced then there might exist a population of neutron stars which only become visible in the radio band for a short duration in the immediate aftermath of glitch activity. These neutron stars will be visible in the radio band as sources that only emit some clustered pulses every so many years.Comment: 20 pages, 10 figures, Accepted for publication in MNRA

A High-Velocity Narrow Absorption Line Outflow in the Quasar J212329.46-005052.9

We report on a variable high-velocity narrow absorption line outflow in the redshift 2.3 quasar J2123-0050. Five distinct outflow systems are detected with velocity shifts from -9710 to -14,050 km/s and CIV 1548,1551 line widths of FWHM = 62-164 km/s. These data require five distinct outflow structures with similar kinematics, physical conditions and characteristic sizes of order 0.01-0.02 pc. The most likely location is ~5 pc from the quasar. The coordinated line variations in <0.63 yr (rest) are best explained by global changes in the outflow ionization caused by changes in the quasar's ionizing flux. The absence of strong X-ray absorption shows that radiative shielding is not needed to maintain the moderate ionizations and therefore, apparently, it is not needed to facilitate the radiative acceleration to high speeds. The kinetic energy yield of this flow is at least two orders of magnitude too low to be important for feedback to the host galaxy's evolution.Comment: 20 pages. In press with MNRA

Genomic identification of a novel co-trimoxazole resistance genotype and its prevalence amongst Streptococcus pneumoniae in Malawi.

OBJECTIVES: This study aimed to define the molecular basis of co-trimoxazole resistance in Malawian pneumococci under the dual selective pressure of widespread co-trimoxazole and sulfadoxine/pyrimethamine use. METHODS: We measured the trimethoprim and sulfamethoxazole MICs and analysed folA and folP nucleotide and translated amino acid sequences for 143 pneumococci isolated from carriage and invasive disease in Malawi (2002-08). RESULTS: Pneumococci were highly resistant to both trimethoprim and sulfamethoxazole (96%, 137/143). Sulfamethoxazole-resistant isolates showed a 3 or 6 bp insertion in the sulphonamide-binding site of folP. The trimethoprim-resistant isolates fell into three genotypic groups based on dihydrofolate reductase (encoded by folA) mutations: Ile-100-Leu (10%), the Ile-100-Leu substitution together with a residue 92 substitution (56%) and those with a novel uncharacterized resistance genotype (34%). The nucleotide sequence divergence and dN/dS of folA and folP remained stable from 2004 onwards. CONCLUSIONS: S. pneumoniae exhibit almost universal co-trimoxazole resistance in vitro and in silico that we believe is driven by extensive co-trimoxazole and sulfadoxine/pyrimethamine use. More than one-third of pneumococci employ a novel mechanism of co-trimoxazole resistance. Resistance has now reached a point of stabilizing evolution. The use of co-trimoxazole to prevent pneumococcal infection in HIV/AIDS patients in sub-Saharan Africa should be re-evaluated