Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR

Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb–HEXIM1–7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb–HEXIM1–7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5′ UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR

Increased antigen specific T cell numbers in the absence of altered migration or division rates as a result of mucosal cholera toxin administration

Cholera toxin (CT) is a mucosal adjuvant capable of inducing strong immune responses to co-administered antigens following oral or intranasal immunization of mice. To date, the direct effect of CT on antigen-specific CD4(+) T cell migration and proliferation profiles in vivo is not well characterized. In this study, the effect of CT on the migration pattern and proliferative responses of adoptively transferred, CD4(+) TCR transgenic T cells in orally or intranasally vaccinated mice, was analyzed by flow cytometry. GFP-expressing or CFSE-labeled OT-II lymphocytes were adoptively transferred to naïve C57BL/6 mice, and mice were subsequently vaccinated with OVA with or without CT via the oral or intranasal route. CT did not alter the migration pattern of antigen-specific T cells, regardless of the route of immunization, but increased the number of transgenic CD4(+) T cells in draining lymphoid tissue. This increase in the number of transgenic CD4(+) T cells was not due to cells undergoing more rounds of cellular division in vivo, suggesting that CT may exert an indirect adjuvant effect on CD4(+) T cells. The findings reported here suggest that CT functions as a mucosal adjuvant by increasing the number of antigen specific CD4(+) T cells independent of their migration pattern or kinetics of cellular division.Grant support was received from the National Health and Medical Research Council of Australia (NHMRC). OLW is a recipient of an R.D. Wright Career Development Award

Diffusion measurements to understand dynamics and structuring in solutions involving a homologous series of ionic liquids

The self-diffusion coefficients of each of the components in mixtures containing pyridine and each of the homologous series 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imides in acetonitrile were determined using NMR diffusometry (i. e., Pulsed Gradient Spin Echo). The nature of solvation was found to change significantly with the proportion of salt in the mixtures. Increased diffusion coefficients (when corrected for viscosity) for the molecular components were observed with increasing proportion of ionic liquid and with increasing alkyl chain length on the cation. Comparison of the molecular solvents suggests increased interactions in solution of the pyridine with other components of the mixture, consistent with the proposed interactions shown previously to drive changes in reaction kinetics. Discontinuities were seen in the diffusion data for each species in solution across different ionic liquids between the hexyl and octyl derivatives, suggesting a change in the structuring in solution as the alkyl chain on the cation changes and demonstrating the importance of such when considering homologous series

A Complete Catalog of Swift GRB Spectra and Durations: Demise of a Physical Origin for Pre-Swift High-Energy Correlations

We calculate durations and spectral paramaters for 218 Swift bursts detected by the BAT instrument between and including GRBs 041220 and 070509, including 77 events with measured redshifts. Incorporating prior knowledge into the spectral fits, we are able to measure the characteristic $\nu F_{\nu}$ spectral peak energy $E_{\rm pk,obs}$ and the isotropic equivalent energy $E_{\rm iso}$ (1--$10^4$ keV) for all events. This complete and rather extensive catalog, analyzed with a unified methodology, allows us to address the persistence and origin of high-energy correlations suggested in pre-Swift observations. We find that the $E_{\rm pk,obs}$-$E_{\rm iso}$ correlation is present in the Swift sample; however, the best-fit powerlaw relation is inconsistent with the best-fit pre-Swift relation at >5 sigma significance. Moreover, it has a factor >~ 2 larger intrinsic scatter, after accounting for large errors on $E_{\rm pk,obs}$. A large fraction of the Swift events are hard and subluminous relative to (and inconsistent with) the pre-Swift relation, in agreement with indications from BATSE GRBs without redshift. Moreover, we determine an experimental threshold for the BAT detector and show how the $E_{\rm pk,obs}$--$E_{\rm iso}$ correlation arises artificially due to partial correlation with the threshold. We show that pre-Swift correlations found by Amati et al.(2002), Yonetoku et al. (2004), Firmani et al.(2006) (and independently by others) are likely unrelated to the physical properties of GRBs and are likely useless for tests of cosmology. Also, an explanation of these correlations in terms of a detector threshold provides a natural and quantitative explanation for why short-duration GRBs and events at low redshift tend to be outliers to the correlations.Comment: 25 pages, 9 figures, 2 tables, Accepted to Ap

A New Constraint on the Escape Fraction in Distant Galaxies Using Gamma-ray Burst Afterglow Spectroscopy

We describe a new method to measure the escape fraction fesc of ionizing radiation from distant star-forming galaxies using the afterglow spectra of long-duration gamma-ray bursts (GRBs). Optical spectra of GRB afterglows allow us to evaluate the optical depth of the host ISM, according to the neutral hydrogen column density N(HI) observed along the sightlines toward the star-forming regions where the GRBs are found. Different from previous effort in searching for faint, transmitted Lyman continuum photons, our method is not subject to background subtraction uncertainties and does not require prior knowledge of either the spectral shape of the host galaxy population or the IGM Lya forest absorption along these GRB sightlines. Because most GRBs occur in sub-L_* galaxies, our study also offers the first constraint on fesc for distant low-mass galaxies that dominate the cosmic luminosity density. We have compiled a sample of 27 GRBs at redshift z>2 for which the underlying N(HI) in the host ISM are known. These GRBs together offer a statistical sampling of the integrated optical depth to ionizing photons along random sightlines from star-forming regions in the host galaxies, and allow us to estimate the mean escape fraction averaged over different viewing angles. We find =0.02\pm 0.02 and place a 95% c.l. upper limit <= 0.075 for these hosts. We discuss possible biases of our approach and implications of the result. Finally, we propose to extend this technique for measuring at z~0.2 using spectra of core-collapse supernovae.Comment: Five journal pages, including one figure; ApJL in pres

Chemical Cartography of the Sagittarius Stream with Gaia

The stellar stream connected to the Sagittarius (Sgr) dwarf galaxy is the most massive tidal stream that has been mapped in the Galaxy, and is the dominant contributor to the outer stellar halo of the Milky Way. We present metallicity maps of the Sgr stream, using 34,240 red giant branch stars with inferred metallicities from Gaia BP/RP spectra. This sample is larger than previous samples of Sgr stream members with chemical abundances by an order of magnitude. We measure metallicity gradients with respect to Sgr stream coordinates $(\Lambda, B)$, and highlight the gradient in metallicity with respect to stream latitude coordinate $B$, which has not been observed before. We find $\nabla \mathrm{[M/H]} = -2.48 \pm 0.08 \times 10^{-2}$ dex/deg above the stream track ($B>B_0$ where $B_0=1.5$ deg is the latitude of the Sgr remnant) and $\nabla \mathrm{[M/H]} =- 2.02 \pm 0.08 \times 10^{-2}$ dex/deg below the stream track ($B<B_0$). By painting metallicity gradients onto a tailored N-body simulation of the Sgr stream, we find that the observed metallicities in the stream are consistent with an initial radial metallicity gradient in the Sgr dwarf galaxy of $\sim -0.1$ to $-0.2$ dex/kpc, well within the range of observed metallicity gradients in Local Group dwarf galaxies. Our results provide novel observational constraints for the internal structure of the dwarf galaxy progenitor of the Sgr stream. Leveraging new large datasets in conjunction with tailored simulations, we can connect the present day properties of disrupted dwarfs in the Milky Way to their initial conditions.Comment: 20 pages, 12 figures. Submitted to ApJ; comments welcome

PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8+ T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8+ T cells and increased on memory CD8+ T cells according to antigen specificity. Memory CD8+ T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8+ T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8+ T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8+ T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8+ T cell numbers, but possibly not all functions in vivo

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome

CD4+ T Cell Depletion during all Stages of HIV Disease Occurs Predominantly in the Gastrointestinal Tract

The mechanisms underlying CD4+ T cell depletion in human immunodeficiency virus (HIV) infection are not well understood. Comparative studies of lymphoid tissues, where the vast majority of T cells reside, and peripheral blood can potentially illuminate the pathogenesis of HIV-associated disease. Here, we studied the effect of HIV infection on the activation and depletion of defined subsets of CD4+ and CD8+ T cells in the blood, gastrointestinal (GI) tract, and lymph node (LN). We also measured HIV-specific T cell frequencies in LNs and blood, and LN collagen deposition to define architectural changes associated with chronic inflammation. The major findings to emerge are the following: the GI tract has the most substantial CD4+ T cell depletion at all stages of HIV disease; this depletion occurs preferentially within CCR5+ CD4+ T cells; HIV-associated immune activation results in abnormal accumulation of effector-type T cells within LNs; HIV-specific T cells in LNs do not account for all effector T cells; and T cell activation in LNs is associated with abnormal collagen deposition. Taken together, these findings define the nature and extent of CD4+ T cell depletion in lymphoid tissue and point to mechanisms of profound depletion of specific T cell subsets related to elimination of CCR5+ CD4+ T cell targets and disruption of T cell homeostasis that accompanies chronic immune activation