Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus

Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A(2) catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.     

Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C

Xenotransplantation of pig organs is complicated by the existence of polytropic replication-competent porcine endogenous retroviruses (PERV) capable of infecting human cells. The potential for recombination between ecotropic PERV-C and human-tropic PERV-A and PERV-B adds another level of infectious risk. Proviral PERV-C were characterized in MAX-T cells derived from d/d haplotype miniature swine. Three proviruses were cloned from a genomic library. Clone PERV-C(1312) generated infectious particles after transfection into porcine ST-IOWA cells. Electron microscopy revealed the same morphologies of virions in MAX-T cells and in ST-IOWA cells infected with cell-free PERV-C(1312) particles, indicating that MAX-T cells harbor one functional PERV-C provirus.     

Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1

DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway.     

A quantitative approach to catabolite repression in Escherichia coli

A dynamic mathematical model was developed to describe the uptake of various carbohydrates (glucose, lactose, glycerol, sucrose, and galactose) in Escherichia coli. For validation a number of isogenic strains with defined mutations were used. By considering metabolic reactions as well as signal transduction processes influencing the relevant pathways, we were able to describe quantitatively the phenomenon of catabolite repression in E. coli. We verified model predictions by measuring time courses of several extra- and intracellular components such as glycolytic intermediates, EII-ACrr phosphorylation level, both LacZ and PtsG concentrations, and total cAMP concentrations under various growth conditions. The entire data base consists of 18 experiments performed with nine different strains. The model describes the expression of 17 key enzymes, 38 enzymatic reactions, and the dynamic behavior of more than 50 metabolites. The different phenomena affecting the phosphorylation level of EIIACrr, the key regulation molecule for inducer exclusion and catabolite repression in enteric bacteria, can now be explained quantitatively.     

A Tractable Experimental Model for Study of Human and Animal Scabies

Background

Scabies is a parasitic skin infestation caused by the burrowing mite Sarcoptes scabiei. It is common worldwide and spreads rapidly under crowded conditions, such as those found in socially disadvantaged communities of Indigenous populations and in developing countries. Pruritic scabies lesions facilitate opportunistic bacterial infections, particularly Group A streptococci. Streptococcal infections cause significant sequelae and the increased community streptococcal burden has led to extreme levels of acute rheumatic fever and rheumatic heart disease in Australia''s Indigenous communities. In addition, emerging resistance to currently available therapeutics emphasizes the need to identify potential targets for novel chemotherapeutic and/or immunological intervention. Scabies research has been severely limited by the availability of parasites, and scabies remains a truly neglected infectious disease. We report development of a tractable model for scabies in the pig, Sus domestica.

Methodology/Principal Findings

Over five years and involving ten independent cohorts, we have developed a protocol for continuous passage of Sarcoptes scabiei var. suis. To increase intensity and duration of infestation without generating animal welfare issues we have optimised an immunosuppression regimen utilising daily oral treatment with 0.2mg/kg dexamethasone. Only mild, controlled side effects are observed, and mange infection can be maintained indefinitely providing large mite numbers (>6000 mites/g skin) for molecular-based research on scabies. In pilot experiments we explore whether any adaptation of the mite population is reflected in genetic changes. Phylogenetic analysis was performed comparing sets of genetic data obtained from pig mites collected from naturally infected pigs with data from pig mites collected from the most recent cohort.

Conclusions/Significance

A reliable pig/scabies animal model will facilitate in vivo studies on host immune responses to scabies including the relations to the associated bacterial pathogenesis and more detailed studies of molecular evolution and host adaption. It is a most needed tool for the further investigation of this important and widespread parasitic disease.     

Different mechanisms for heterogeneity in leprosy susceptibility can explain disease clustering within households

The epidemiology of leprosy is characterized by heterogeneity in susceptibility and clustering of disease within households. We aim to assess the extent to which different mechanisms for heterogeneity in leprosy susceptibility can explain household clustering as observed in a large study among contacts of leprosy patients.We used a microsimulation model, parameterizing it with data from over 20,000 contacts of leprosy patients in Bangladesh. We simulated six mechanisms producing heterogeneity in susceptibility: (1) susceptibility was allocated at random to persons (i.e. no additional mechanism), (2) a household factor, (3, 4) a genetic factor (dominant or recessive), or (5, 6) half a household factor and half genetic. We further assumed that a fraction of 5%, 10%, and 20% of the population was susceptible, leading to a total of 18 scenarios to be fitted to the data. We obtained an acceptable fit for each of the six mechanisms, thereby excluding none of the possible underlying mechanisms for heterogeneity of susceptibility to leprosy. However, the distribution of leprosy among contacts did differ between mechanisms, and predicted trends in the declining leprosy case detection were dependent on the assumed mechanism, with genetic-based susceptibility showing the slowest decline. Clustering of leprosy within households is partially caused by an increased transmission within households independent of the leprosy susceptibility mechanism. Even a large and detailed data set on contacts of leprosy patients could not unequivocally reveal the mechanism most likely responsible for heterogeneity in leprosy susceptibility.     

Dynamic proteomic overview of glioblastoma cells (A172) exposed to perillyl alcohol

Perillyl alcohol (POH) is a naturally occurring terpene and a promising chemotherapeutic agent for glioblastoma multiform; yet, little is known about its molecular effects. Here we present results of a semi-quantitative proteomic analysis of A172 cells exposed to POH for different time-periods (1′, 10′, 30′, 60′, 4 h, and 24 h). The analysis identified more than 4000 proteins; which were clustered using PatternLab for proteomics and then linked to Ras signaling, tissue homeostasis, induction of apoptosis, metallopeptidase activity, and ubiquitin-protein ligase activity. Our results make available one of the most complete protein repositories for the A172. Moreover, we detected the phosphorylation of GSK3β (Glycogen synthase kinase) and the inhibition of ERK's (extracellular signal regulated kinase) phosphorylation after 10′, which suggests a new mechanism of POH's activation for apoptosis.     

Solving the aerodynamics of fungal flight: how air viscosity slows spore motion

Viscous drag causes the rapid deceleration of fungal spores after high-speed launches and limits discharge distance. Stokes' law posits a linear relationship between drag force and velocity. It provides an excellent fit to experimental measurements of the terminal velocity of free-falling spores and other instances of low Reynolds number motion (Re<1). More complex, non-linear drag models have been devised for movements characterized by higher Re, but their effectiveness for modeling the launch of fast-moving fungal spores has not been tested. In this paper, we use data on spore discharge processes obtained from ultra-high-speed video recordings to evaluate the effects of air viscosity predicted by Stokes' law and a commonly used non-linear drag model. We find that discharge distances predicted from launch speeds by Stokes' model provide a much better match to measured distances than estimates from the more complex drag model. Stokes' model works better over a wide range projectile sizes, launch speeds, and discharge distances, from microscopic mushroom ballistospores discharged at <1 m s(-1) over a distance of <0.1mm (Re<1.0), to macroscopic sporangia of Pilobolus that are launched at >10 m s(-1) and travel as far as 2.5m (Re>100).     

Environmental effects on temperature stress resistance in the tropical butterfly Bicyclus anynana

Background

The ability to withstand thermal stress is considered to be of crucial importance for individual fitness and species'' survival. Thus, organisms need to employ effective mechanisms to ensure survival under stressful thermal conditions, among which phenotypic plasticity is considered a particularly quick and effective one.

Methodology/Principal Findings

In a series of experiments we here investigate phenotypic adjustment in temperature stress resistance following environmental manipulations in the butterfly Bicyclus anynana. Cooler compared to warmer acclimation temperatures generally increased cold but decreased heat stress resistance and vice versa. In contrast, short-time hardening responses revealed more complex patterns, with, e.g., cold stress resistance being highest at intermediate hardening temperatures. Adult food stress had a negative effect on heat but not on cold stress resistance. Additionally, larval feeding treatment showed interactive effects with adult feeding for heat but not for cold stress resistance, indicating that nitrogenous larval resources may set an upper limit to performance under heat stress. In contrast to expectations, cold resistance slightly increased during the first eight days of adult life. Light cycle had marginal effects on temperature stress resistance only, with cold resistance tending to be higher during daytime and thus active periods.

Conclusions/Significance

Our results highlight that temperature-induced plasticity provides an effective tool to quickly and strongly modulate temperature stress resistance, and that such responses are readily reversible. However, resistance traits are not only affected by ambient temperature, but also by, e.g., food availability and age, making their measurement challenging. The latter effects are largely underexplored and deserve more future attention. Owing to their magnitude, plastic responses in thermal tolerance should be incorporated into models trying to forecast effects of global change on extant biodiversity.