Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices

Endothelial cell invasion is a key step in angiogenic blood vessel formation. Sphingosine-1-phosphate (S1P) has been previously reported to play a role in endothelial cell proliferation, survival, migration, and angiogenesis. Here, we examine the ability of S1P to regulate human endothelial cell invasion into three-dimensional collagen or fibrin matrices. We show that S1P potently stimulated human endothelial cell invasion, lumen formation, and branching morphogenesis in collagen, and fibrin matrices, (5- and 15-fold increases in invasion were observed, respectively). The S1P-induced invasion response was pertussis-toxin sensitive and completely dependent on integrins. Addition of integrin blocking reagents revealed that the alpha2beta1 integrin regulated invasion in collagen matrices, while a combination of alphavbeta3 and alpha5beta1 integrins regulated invasion in fibrin. Additionally, the S1P-induced invasion response was dependent on matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinase-3 (TIMP-3) was the only physiologic inhibitor of metalloproteinases that completely inhibited the potent stimulation of invasion induced by S1P. In contrast, TIMP-1 had no blocking effect on invasion or morphogenesis, while TIMP-2 and TIMP-4 partially reduced invasion but completely blocked lumen formation events. Collectively, these data reveal a marked ability of S1P to induce metalloproteinase- and integrin-dependent human endothelial cell invasion and morphogenesis in both collagen and fibrin three-dimensional matrices, the two most physiologically relevant matrices for angiogenesis.     

Population structure and reticulate evolution of Saccharomyces eubayanus and its lager‐brewing hybrids

Reticulate evolution can be a major driver of diversification into new niches, especially in disturbed habitats and at the edges of ranges. Industrial fermentation strains of yeast provide a window into these processes, but progress has been hampered by a limited understanding of the natural diversity and distribution of Saccharomyces species and populations. For example, lager beer is brewed with Saccharomyces pastorianus, an alloploid hybrid of S. cerevisiae and S. eubayanus, a species only recently discovered in Patagonia, Argentina. Here, we report that genetically diverse strains of S. eubayanus are readily isolated from Patagonia, demonstrating that the species is well established there. Analyses of multilocus sequence data strongly suggest that there are two diverse and highly differentiated Patagonian populations. The low nucleotide diversity found in the S. eubayanus moiety of hybrid European brewing strains suggests that their alleles were drawn from a small subpopulation that is closely related to one of the Patagonian populations. For the first time, we also report the rare isolation of S. eubayanus outside Patagonia, in Wisconsin, USA. In contrast to the clear population differentiation in Patagonia, the North American strains represent a recent and possibly transient admixture of the two Patagonian populations. These complex and varied reticulation events are not adequately captured by conventional phylogenetic methods and required analyses of Bayesian concordance factors and phylogenetic networks to accurately summarize and interpret. These findings show how genetically diverse eukaryotic microbes can produce rare but economically important hybrids with low genetic diversity when they migrate from their natural ecological context.     

3D models of human ERα and ERβ complexed with 5-androsten-3β,17β-diol

Recently, binding of 5-androsten-3β,17β-diol (Δ(5)-androstenediol) to human estrogen receptor-beta (ERβ) was found to repress microglia-mediated inflammation, which is associated with various neurodegenerative diseases, such as multiple sclerosis. In contrast, binding of estradiol to ERβ resulted in little or no repression of microglia-mediated inflammation. Binding of Δ(5)-androstenediol to ERβ, as well as to ERα, is unexpected because unlike estradiol, Δ(5)-androstenediol has a saturated A ring and a C19 methyl group. To begin to elucidate the interaction of Δ(5)-androstenediol with both ERs, we constructed 3D models of Δ(5)-androstenediol with human ERα and ERβ for comparison with the crystal structures of estradiol in ERα and ERβ. Conformational flexibility in human ERα and ERβ accommodates the C19 methyl on Δ(5)-androstenediol. This conformational flexibility may be relevant for binding of other Δ(5)-steroids with C19 methyl substituents, such as 25-hydroxycholesterol and 27-hydroxycholesterol, to ERs.     

Bloody‐minded parasites and sex: the effects of fluctuating virulence

Asexual lineages can grow at a faster rate than sexual lineages. Why then is sexual reproduction so widespread? Much empirical evidence supports the Red Queen hypothesis. Under this hypothesis, coevolving parasites favour sexual reproduction by adapting to infect common asexual clones and driving them down in frequency. One limitation, however, seems to challenge the generality of the Red Queen: in theoretical models, parasites must be very virulent to maintain sex. Moreover, experiments show virulence to be unstable, readily shifting in response to environmental conditions. Does variation in virulence further limit the ability of coevolving parasites to maintain sex? To address this question, we simulated temporal variation in virulence and evaluated the outcome of competition between sexual and asexual females. We found that variation in virulence did not limit the ability of coevolving parasites to maintain sex. In fact, relatively high variation in virulence promoted parasite‐mediated maintenance of sex. With sufficient variation, sexual females persisted even when mean virulence fell well below the threshold virulence required to maintain sex under constant conditions. We conclude that natural variation in virulence does not limit the relevance of the Red Queen hypothesis for natural populations; on the contrary, it could expand the range of conditions over which coevolving parasites can maintain sex.     

Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase

Background

Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections.

Methodology/Principal Findings

Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC₅₀s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC₅₀ = 12 µM and EC₉₀ = 20 µM).

Significance

We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.     

Embryonic stem cells induce pluripotency in somatic cell fusion through biphasic reprogramming

It is a long-held paradigm that cell fusion reprograms gene expression but the extent of reprogramming and whether it is affected by the cell types employed remain unknown. We recently showed that the silencing of somatic genes is attributable to either trans-acting cellular environment or cis-acting chromatin context. Here, we examine how trans- versus cis-silenced genes in a somatic cell type behave in fusions to another somatic cell type or to embryonic stem cells (ESCs). We demonstrate that while reprogramming of trans-silenced somatic genes occurs in both cases, reprogramming of cis-silenced somatic genes occurs only in somatic-ESC fusions. Importantly, ESCs reprogram the somatic genome in two distinct phases: trans-reprogramming occurs rapidly, independent of DNA replication, whereas cis-reprogramming occurs with slow kinetics requiring DNA replication. We also show that pluripotency genes Oct4 and Nanog are cis-silenced in somatic cells. We conclude that cis-reprogramming capacity is a fundamental feature distinguishing ESCs from somatic cells.     

Evolution to environmental contamination ablates the circadian clock of an aquatic sentinel species

Environmental contamination is a common cause of rapid evolution. Recent work has shown that Daphnia pulex, an important freshwater species, can rapidly evolve increased tolerance to a common contaminant, sodium chloride (NaCl) road salt. While such rapid evolution can benefit organisms, allowing them to adapt to new environmental conditions, it can also be associated with unforeseen tradeoffs. Given that exposure to environmental contaminants can cause circadian disruption, we investigated whether the circadian clock was affected by evolving a tolerance to high levels of road salt. By tracking the oscillations of a putative clock gene, period, we demonstrated that D. pulex express per mRNA with approximately 20‐hr oscillations under control conditions. This putative circadian rhythm was ablated in response to high levels of salinity; populations adapted to high NaCl concentrations exhibited an ablation of period oscillation. Moreover, we showed that while gene expression is increased in several other genes, including clock, actin, and Na⁺/K⁺‐ATPase, upon the adaptation to high levels of salinity, per expression is unique among the genes we tracked in that it is the only gene repressed in response to salt adaptation. These results suggest that rapid evolution of salt tolerance occurs with the tradeoff of suppressed circadian function. The resultant circadian disruption may have profound consequences to individuals, populations, and aquatic food webs by affecting species interactions. In addition, our research suggests that circadian clocks may also be disrupted by the adaptation to other environmental contaminants.     

Friendship Network Characteristics Are Associated with Physical Activity and Sedentary Behavior in Early Adolescence

Introduction

There is limited understanding of the association between peer social networks and physical activity (PA), sedentary and screen-related behaviors. This study reports on associations between personal network characteristics and these important health behaviors for early adolescents.

Methods

Participants were 310 students, aged 11–13 years, from fifteen randomly selected Victorian primary schools (43% response rate). PA and sedentary behaviors were collected via accelerometer and self-report questionnaire, and anthropometric measures via trained researchers. Participants nominated up to fifteen friends, and described the frequency of interaction and perceived activity intensity of these friends. Personal network predictors were examined using regression modelling for PA and sedentary/screen behavior.

Results

Perceived activity levels of friends, and friendships with very frequent interaction were associated with outside-of-school PA and/or sedentary/screen time. Differences according to sex were also observed in the association between network characteristics and PA and sedentary time. A higher number of friends and greater proportion of same sex friends were associated with boys engaging in more moderate-to-vigorous PA outside of school hours. PA intensity during school-day breaks was positively associated with having a greater proportion of friends who played sports for girls, and a greater proportion of male friends for boys.

Conclusion

Friendship network characteristics are associated with PA and sedentary/screen time in late childhood/early adolescence, and these associations differ by sex. The positive influence of very active peers may be a promising avenue to strengthen traditional interventions for the promotion of PA and reduction in screen time.