Critical Stages in Viral Replication - Entry, Gene Regulation and Egress

Despite the amazing biological diversity exhibited by viruses, their very existence relies upon their ability to overcome a set of common barriers. The nature of these barriers reflects the nature of viruses themselves. During their extracellular phase, viruses are metabolically inert obligate parasites. Upon encountering a host cell, productive infection necessitates that the virus successfully enter the cell, regulate the expression of its genes, and after assembling new progeny particles, egress such that the cycle of infection can continue. These three basic processes are not only attractive candidates for therapeutic intervention, but also reveal much about virus biology in the most basic sense. That is why these processes are the focus of the studies described herein. We have identified vimentin, an intermediate filament protein expressed primarily in cells of mesenchymal origin, as a cellular factor required for the efficient onset of human cytomegalovirus (CMV) infection in fibroblasts. We observed that an endotheliotropic (EC-tropic) strain of CMV relies more heavily on vimentin than the fibroblast-adapted strain, possibly reflecting different modes of entry utilized by these two strains. We have also performed the first functional study of the 55R E1A protein encoded by human adenovirus (HAdV). This protein was expressed at late times post-infection and was able to both transactivate expression of viral genes, and promote productive replication of HAdV in the absence of all other E1A isoforms. Finally, our focus returns to CMV where we describe a novel protein that we have dubbed \u27 nuclear rim-associated cytomegaloviral protein\u27 (RASCAL). RASCAL is expressed with early-late kinetics and localizes to the nuclear rim, in deep intranuclear invaginations, and in unusual lamin B-positive vesicular structures at late times post-infection. RASCAL could be immunoprecipitated with pUL50, a member of the CMV nuclear egress complex (NEC) and pUL50 was sufficient to recruit RASCAL to the nuclear rim. These studies have illuminated novel processes through which two important human viruses enter cells, regulate viral gene expression and ultimately egress. Considered together, they have also expanded our understanding of three central aspects of virus biology upon which further studies can build

The Role of Projection in the Control of Bird Flocks

Swarming is a conspicuous behavioural trait observed in bird flocks, fish shoals, insect swarms and mammal herds. It is thought to improve collective awareness and offer protection from predators. Many current models involve the hypothesis that information coordinating motion is exchanged between neighbors. We argue that such local interactions alone are insufficient to explain the organization of large flocks of birds and that the mechanism for the exchange of long-ranged information necessary to control their density remains unknown. We show that large flocks self-organize to the maximum density at which a typical individual is still just able to see out of the flock in many directions. Such flocks are marginally opaque - an external observer can also just still see a substantial fraction of sky through the flock. Although seemingly intuitive we show that this need not be the case; flocks could easily be highly diffuse or entirely opaque. The emergence of marginal opacity strongly constrains how individuals interact with each other within large swarms. It also provides a mechanism for global interactions: An individual can respond to the projection of the flock that it sees. This provides for faster information transfer and hence rapid flock dynamics, another advantage over local models. From a behavioural perspective it optimizes the information available to each bird while maintaining the protection of a dense, coherent flock.Comment: PNAS early edition published online at http://www.pnas.org/cgi/doi/10.1073/pnas.140220211

Nitric oxide as a mediator of inflammation?—You had better believe it

Nitric oxide has enigmatic qualities in inflammation. In order to appreciate the precise contributions of nitric oxide to a pathophysiological process, one must account for enzyme source, coproduction of oxidants and antioxidant defences, time, rate of nitric oxide production, cellular source, peroxynitrite formation and effects on DNA (mutagenesis/apoptosis). We contend that there is ample evidence to consider nitric oxide as a molecular aggressor in inflammation, particularly chronic inflammation. Therapeutic benefit can be achieved by inhibition of inducible nitric oxide synthase and not the donation of additional nitric oxide. Furthermore, there is growing appreciation that nitric oxide and products derived thereof, are critical components linking the increased incidence of cancer in states of chronic inflammation

Age-specific mortality during the 1918 influenza pandemic: unravelling the mystery of high young adult mortality.

The worldwide spread of a novel influenza A (H1N1) virus in 2009 showed that influenza remains a significant health threat, even for individuals in the prime of life. This paper focuses on the unusually high young adult mortality observed during the Spanish flu pandemic of 1918. Using historical records from Canada and the U.S., we report a peak of mortality at the exact age of 28 during the pandemic and argue that this increased mortality resulted from an early life exposure to influenza during the previous Russian flu pandemic of 1889-90. We posit that in specific instances, development of immunological memory to an influenza virus strain in early life may lead to a dysregulated immune response to antigenically novel strains encountered in later life, thereby increasing the risk of death. Exposure during critical periods of development could also create holes in the T cell repertoire and impair fetal maturation in general, thereby increasing mortality from infectious diseases later in life. Knowledge of the age-pattern of susceptibility to mortality from influenza could improve crisis management during future influenza pandemics

The characterization of widespread fatigue damage in fuselage structure

The characteristics of widespread fatigue damage (WSFD) in fuselage riveted structure were established by detailed nondestructive and destructive examinations of fatigue damage contained in a full size fuselage test article. The objectives of this were to establish an experimental data base for validating emerging WSFD analytical prediction methodology and to identify first order effects that contribute to fatigue crack initiation and growth. Detailed examinations were performed on a test panel containing four bays of a riveted lap splice joint. The panel was removed from a full scale fuselage test article after receiving 60,000 full pressurization cycles. The results of in situ examinations document the progression of fuselage skin fatigue crack growth through crack linkup. Detailed tear down examinations and fractography of the lap splice joint region revealed fatigue crack initiation sites, crack morphology, and crack linkup geometry. From this large data base, distributions of crack size and locations are presented and discussions of operative damage mechanisms are offered

A universal density matrix functional from molecular orbital-based machine learning: Transferability across organic molecules

We address the degree to which machine learning (ML) can be used to accurately and transferably predict post-Hartree-Fock correlation energies. Refined strategies for feature design and selection are presented, and the molecular-orbital-based machine learning (MOB-ML) method is applied to several test systems. Strikingly, for the second-order Møller-Plessett perturbation theory, coupled cluster with singles and doubles (CCSD), and CCSD with perturbative triples levels of theory, it is shown that the thermally accessible (350 K) potential energy surface for a single water molecule can be described to within 1 mhartree using a model that is trained from only a single reference calculation at a randomized geometry. To explore the breadth of chemical diversity that can be described, MOB-ML is also applied to a new dataset of thermalized (350 K) geometries of 7211 organic models with up to seven heavy atoms. In comparison with the previously reported Δ-ML method, MOB-ML is shown to reach chemical accuracy with threefold fewer training geometries. Finally, a transferability test in which models trained for seven-heavy-atom systems are used to predict energies for thirteen-heavy-atom systems reveals that MOB-ML reaches chemical accuracy with 36-fold fewer training calculations than Δ-ML (140 vs 5000 training calculations)

Oral dosing for antenatal corticosteroids in the Rhesus macaque.

Antenatal corticosteroids (ACS) are standard of care for women at risk of preterm delivery, although choice of drug, dose or route have not been systematically evaluated. Further, ACS are infrequently used in low resource environments where most of the mortality from prematurity occurs. We report proof of principle experiments to test betamethasone-phosphate (Beta-P) or dexamethasone-phosphate (Dex-P) given orally in comparison to the clinical treatment with the intramuscular combination drug beta-phosphate plus beta-acetate in a Rhesus Macaque model. First, we performed pharmacokinetic studies in non-pregnant monkeys to compare blood levels of the steroids using oral dosing with Beta-P, Dex-P and an effective maternal intramuscular dose of the beta-acetate component of the clinical treatment. We then evaluated maternal and fetal blood steroid levels with limited fetal sampling under ultrasound guidance in pregnant macaques. We found that oral Beta is more slowly cleared from plasma than oral Dex. The blood levels of both drugs were lower in maternal plasma of pregnant than in non-pregnant macaques. Using the pharmacokinetic data, we treated groups of 6-8 pregnant monkeys with oral Beta-P, oral Dex-P, or the maternal intramuscular clinical treatment and saline controls and measured pressure-volume curves to assess corticosteroid effects on lung maturation at 5d. Oral Beta-P improved the pressure-volume curves similarly to the clinical treatment. Oral Dex-P gave more variable and nonsignificant responses. We then compared gene expression in the fetal lung, liver and hippocampus between oral Beta-P and the clinical treatment by RNA-sequencing. The transcriptomes were largely similar with small gene expression differences in the lung and liver, and no differences in the hippocampus between the groups. As proof of principle, ACS therapy can be effective using inexpensive and widely available oral drugs. Clinical dosing strategies must carefully consider the pharmacokinetics of oral Beta-P or Dex-P to minimize fetal exposure while achieving the desired treatment responses