Leaky scanning translation generates a second A49 protein that contributes to vaccinia virus virulence.

Vaccinia virus (VACV) strain Western Reserve gene A49L encodes a small intracellular protein with a Bcl-2 fold that is expressed early during infection and has multiple functions. A49 co-precipitates with the E3 ubiquitin ligase β-TrCP and thereby prevents ubiquitylation and proteasomal degradation of IκBα, and consequently blocks activation of NF-κB. In a similar way, A49 stabilizes β-catenin, leading to activation of the wnt signalling pathway. However, a VACV strain expressing a mutant A49 that neither co-precipitates with β-TrCP nor inhibits NF-κB activation, is more virulent than a virus lacking A49, indicating that A49 has another function that also contributes to virulence. Here we demonstrate that gene A49L encodes a second, smaller polypeptide that is expressed via leaky scanning translation from methionine 20 and is unable to block NF-κB activation. Viruses engineered to express either only the large protein or only the small A49 protein both have lower virulence than wild-type virus and greater virulence than an A49L deletion mutant. This demonstrates that the small protein contributes to virulence by an unknown mechanism that is independent of NF-κB inhibition. Despite having a large genome with about 200 genes, this study illustrates how VACV makes efficient use of its coding potential and from gene A49L expresses a protein with multiple functions and multiple proteins with different functions

NF-κB activation is a turn on for vaccinia virus phosphoprotein A49 to turn off NF-κB activation.

Vaccinia virus protein A49 inhibits NF-κB activation by molecular mimicry and has a motif near the N terminus that is conserved in IκBα, β-catenin, HIV Vpu, and some other proteins. This motif contains two serines, and for IκBα and β-catenin, phosphorylation of these serines enables recognition by the E3 ubiquitin ligase β-TrCP. Binding of IκBα and β-catenin by β-TrCP causes their ubiquitylation and thereafter proteasome-mediated degradation. In contrast, HIV Vpu and VACV A49 are not degraded. This paper shows that A49 is phosphorylated at serine 7 but not serine 12 and that this is necessary and sufficient for binding β-TrCP and antagonism of NF-κB. Phosphorylation of A49 S7 occurs when NF-κB signaling is activated by addition of IL-1β or overexpression of TRAF6 or IKKβ, the kinase needed for IκBα phosphorylation. Thus, A49 shows beautiful biological regulation, for it becomes an NF-κB antagonist upon activation of NF-κB signaling. The virulence of viruses expressing mutant A49 proteins or lacking A49 (vΔA49) was tested. vΔA49 was attenuated compared with WT, but viruses expressing A49 that cannot bind β-TrCP or bind β-TrCP constitutively had intermediate virulence. So A49 promotes virulence by inhibiting NF-κB activation and by another mechanism independent of S7 phosphorylation and NF-κB antagonism. Last, a virus lacking A49 was more immunogenic than the WT virus.Wellcome Trus

Multiple Bcl-2 family immunomodulators from vaccinia virus regulate MAPK/AP-1 activation.

Vaccinia virus (VACV) is a poxvirus and encodes many proteins that modify the host cell metabolism or inhibit the host response to infection. For instance, it is known that VACV infection can activate the mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathway and inhibit activation of the pro-inflammatory transcription factor NF-κB. Since NF-κB and MAPK/AP-1 share common upstream activators we investigated whether six different VACV Bcl-2-like NF-κB inhibitors can also influence MAPK/AP-1 activation. Data presented show that proteins A52, B14 and K7 each contribute to AP-1 activation during VACV infection, and when expressed individually outwith infection. B14 induced the greatest stimulation of AP-1 and further investigation showed B14 activated mainly the MAPKs ERK (extracellular signal-regulated kinase) and JNK (Jun N-terminal kinase), and their substrate c-Jun (a component of AP-1). These data indicate that the same viral protein can have different effects on distinct signalling pathways, in blocking NF-κB activation whilst leading to MAPK/AP-1 activation.This work was funded by grants from the Medical Research Council, the Wellcome Trust, the Minas Gerais State’s Foundation for Research Support (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq - Brazil).This is the author accepted manuscript. The final version is available from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00052

Microbial Communities in Pre-Columbian Coprolites

The study of coprolites from earlier cultures represents a great opportunity to study an “unaltered” composition of the intestinal microbiota. To test this, pre-Columbian coprolites from two cultures, the Huecoid and Saladoid, were evaluated for the presence of DNA, proteins and lipids by cytochemical staining, human and/or dog-specific Bacteroides spp. by PCR, as well as bacteria, fungi and archaea using Terminal Restriction Fragment analyses. DNA, proteins and lipids, and human-specific Bacteroides DNA were detected in all coprolites. Multidimensional scaling analyses resulted in spatial arrangements of microbial profiles by culture, further supported by cluster analysis and ANOSIM. Differences between the microbial communities were positively correlated with culture, and SIMPER analysis indicated 68.8% dissimilarity between the Huecoid and Saladoid. Proteobacteria, Bacteroidetes and methanogens were found in all coprolite samples. Propionebacteria, Shewanella and lactic acid bacteria dominated in the Huecoid samples, while Acidobacteria, and peptococci were dominant in Saladoid samples. Yeasts, including Candida albicans and Crypotococcus spp. were found in all samples. Basidiomycetes were the most notable fungi in Huecoid samples while Ascomycetes predominated in Saladoid samples, suggesting differences in dietary habits. Our study provides an approach for the study of the microbial communities of coprolite samples from various cultures

Community-engagement to support cardiovascular disease prevention in disparities populations: three case studies

Cardiovascular diseases remain the leading cause of death in the United States, and are characterized by socioeconomic, geographic, ethnic, and gender disparities in risk, morbidity and mortality. In response, public health efforts have moved beyond approaches focusing on individual-level behavior change toward culturally appropriate community-focused efforts. In specific, engagement of community partners is now recognized as essential to facilitate changes at multiple levels to improve cardiovascular disease outcomes. This paper shares lessons learned to deepen appreciation for the unique challenges community-engagement in health disparities research entails, including variations in practice, time commitment, and complexity. This paper presents three case studies documenting community-engagement in the planning, implementation and evaluation processes. All projects collaborated with community partners in contexts with disproportionately high rates of cardiovascular disease but with distinct programmatic foci: the East Los Angeles, California project focused on improving access to fresh fruit and vegetables through corner store makeovers; the Boston, Massachusetts project reached out to and engaged Puerto Rican community members in a lifestyle intervention study; and the Lenoir County, North Carolina project engaged local restaurant owners and a range of community agencies in healthy lifestyle promotion activities. These cases provide examples of the unique solutions and approaches to issues common in doing community-engagement work

CHC22 and CHC17 clathrins have distinct biochemical properties and display differential regulation and function

Clathrins are cytoplasmic proteins that play essential roles in endocytosis and other membrane traffic pathways. Upon recruitment to intracellular membranes, the canonical clathrin triskelion assembles into a polyhedral protein coat that facilitates vesicle formation and captures cargo molecules for transport. The triskelion is formed by trimerization of three clathrin heavy-chain subunits. Most vertebrates have two isoforms of clathrin heavy chains, CHC17 and CHC22, generating two clathrins with distinct cellular functions. CHC17 forms vesicles at the plasma membrane for receptor-mediated endocytosis and at the trans-Golgi network for organelle biogenesis. CHC22 plays a key role in intracellular targeting of the insulin-regulated glucose transporter 4 (GLUT4), accumulates at the site of GLUT4 sequestration during insulin resistance, and has also been implicated in neuronal development. Here, we demonstrate that CHC22 and CHC17 share morphological features, in that CHC22 forms a triskelion and latticed vesicle coats. However, cellular CHC22-coated vesicles were distinct from those formed by CHC17. The CHC22 coat was more stable to pH change and was not removed by the enzyme complex that disassembles the CHC17 coat. Moreover, the two clathrins were differentially recruited to membranes by adaptors, and CHC22 did not support vesicle formation or transferrin endocytosis at the plasma membrane in the presence or absence of CHC17. Our findings provide biochemical evidence for separate regulation and distinct functional niches for CHC17 and CHC22 in human cells. Furthermore, the greater stability of the CHC22 coat relative to the CHC17 coat may be relevant to its excessive accumulation with GLUT4 during insulin resistance. [Abstract copyright: Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

Planetary Construction Zones in Occultation: Discovery of an Extrasolar Ring System Transiting a Young Sun-like Star and Future Prospects for Detecting Eclipses by Circumsecondary and Circumplanetary Disks

The large relative sizes of circumstellar and circumplanetary disks imply that they might be seen in eclipse in stellar light curves. We estimate that a survey of ~10^4 young (~10 Myr old) post-accretion pre-MS stars monitored for ~10 years should yield at least a few deep eclipses from circumplanetary disks and disks surrounding low mass companion stars. We present photometric and spectroscopic data for a pre-MS K5 star (1SWASP J140747.93-394542.6), a newly discovered ~0.9 Msun member of the ~16 Myr-old Upper Cen-Lup subgroup of Sco-Cen at a kinematic distance of 128 pc. SuperWASP and ASAS light curves for this star show a remarkably long, deep, and complex eclipse event centered on 29 April 2007. At least 5 multi-day dimming events of >0.5 mag are identified, with a >3.3 mag deep eclipse bracketed by two pairs of ~1 mag eclipses symmetrically occurring +-12 days and +-26 days before and after. Hence, significant dimming of the star was taking place on and off over at least a ~54 day period in 2007, and a strong >1 mag dimming event occurred over a ~12 day span. We place a firm lower limit on the period of 850 days (i.e. the orbital radius of the eclipser must be >1.7 AU and orbital velocity must be <22 km/s). The shape of the light curve is similar to the lop-sided eclipses of the Be star EE Cep. We suspect that this new star is being eclipsed by a low-mass object orbited by a dense inner disk, girded by at least 3 dusty rings of lower optical depth. Between these rings are at least two annuli of near-zero optical depth (i.e. gaps), possibly cleared out by planets or moons, depending on the nature of the secondary. For possible periods in the range 2.33-200 yr, the estimated total ring mass is ~8-0.4 Mmoon (if the rings have optical opacity similar to Saturn's rings), and the edge of the outermost detected ring has orbital radius ~0.4-0.09 AU.Comment: Astronomical Journal, in press, 13 figure

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

The X-Ray Outburst of the Galactic Center Magnetar over Six Years of Chandra Observations

The magnetar SGR J1745−2900, discovered at a distance of parsecs from the Milky Way central black hole, Sagittarius A*, represents the closest pulsar to a supermassive black hole ever detected. Furthermore, its intriguing radio emission has been used to study the environment of the black hole, as well as to derive a precise position and proper motion for this object. The discovery of SGR J1745−2900 has led to interesting debates about the number, age, and nature of pulsars expected in the Galactic center region. In this work, we present extensive X-ray monitoring of the outburst of SGR J1745−2900 using the Chandra X-ray Observatory, the only instrument with the spatial resolution to distinguish the magnetar from the supermassive black hole (2"4 angular distance). It was monitored from its outburst onset in 2013 April until 2019 August, collecting more than 50 Chandra observations for a total of more than 2.3 Ms of data. Soon after the outburst onset, the magnetar emission settled onto a purely thermal emission state that cooled from a temperature of about 0.9–0.6 keV over 6 yr. The pulsar timing properties showed at least two changes in the period derivative, increasing by a factor of about 4 during the outburst decay. We find that the long-term properties of this outburst challenge current models for the magnetar outbursts.N.R., D.V., and A.B. are supported by the H2020 ERC Consolidator Grant “MAGNESIA” under grant agreement No. 817661 (PI: Rea). N.R., F.C.Z., D.V., A.B., and D.F.T. also acknowledge support from grants SGR2017-1383 and PGC2018-095512-BI00. F.C.Z. is supported by a Juan de la Cierva fellowship. A.P. acknowledges financial support from grants ASI/INAF I/037/12/0, ASI/INAF 2017-14-H.0 (PI: Belloni) and from INAF grant “Sostegno alla ricerca scientifica main streams dell’INAF,” Presidential Decree 43/2018 (PI: Belloni). D.H. acknowledges support from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant, the Fonds de recherche du Québec–Nature et Technologies (FRQNT) Nouveaux Chercheurs program, and the Canadian Institute for Advanced Research (CIFAR). G.L.I., S.M., and R.T. have been partially supported by PRIN-MIUR 2017. J.A.P. acknowledges support by the Generalitat Valenciana (PROMETEO/2019/071) and by Agencia Estatal de Investigación (PGC2018-095984-B-I00). G.P. is supported by the H2020 ERC Consolidator Grant “Hot Milk” under grant agreement No. 865637. L.S. acknowledges financial contributions from ASI-INAF agreements 2017-14-H.O and I/037/12/0 and from “iPeska” research grant (PI: Andrea Possenti) funded under the INAF call PRIN-SKA/CTA (resolution 70/2016). We acknowledge support from the PHAROS COST Action (CA16214)

Measurement of χ c1 and χ c2 production with s√ = 7 TeV pp collisions at ATLAS

The prompt and non-prompt production cross-sections for the χ c1 and χ c2 charmonium states are measured in pp collisions at s√ = 7 TeV with the ATLAS detector at the LHC using 4.5 fb−1 of integrated luminosity. The χ c states are reconstructed through the radiative decay χ c → J/ψγ (with J/ψ → μ + μ −) where photons are reconstructed from γ → e + e − conversions. The production rate of the χ c2 state relative to the χ c1 state is measured for prompt and non-prompt χ c as a function of J/ψ transverse momentum. The prompt χ c cross-sections are combined with existing measurements of prompt J/ψ production to derive the fraction of prompt J/ψ produced in feed-down from χ c decays. The fractions of χ c1 and χ c2 produced in b-hadron decays are also measured