Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly

Cell-extracellular matrix (cell-ECM) interactions mediated by integrin receptors are essential for providing positional and environmental information necessary for many cell functions, such as proliferation, differentiation and survival. In vitro studies on cell adhesion to randomly adsorbed molecules on substrates have been limited to sub-micrometer patches, thus preventing the detailed study of structural arrangement of integrins and their ligands. In this article, we illustrate the role of the distance between integrin ligands, namely the RGD (arginine-glycine-aspartate) sequence present in ECM proteins, in the control of cell adhesion. By using substrates, which carry cyclic RGD peptides arranged in highly defined nanopatterns, we investigated the dynamics of cell spreading and the molecular composition of adhesion sites in relation to a fixed spacing between the peptides on the surface. Our novel approach for in vitro studies on cell adhesion indicates that not only the composition, but also the spatial organization of the extracellular environment is important in regulating cell-ECM interactions.     

Two-week longitudinal survey of bone architecture alteration in the hindlimb-unloaded rat model of bone loss: sex differences

The goal of this study was to determine, through a longitudinal follow-up, whether sex influences bone adaptation during simulated weightlessness. Twelve-week-old male and female Wistar rats were hindlimb unweighted for 2 wk, and the time course of bone alteration was monitored in vivo by means of densitometry and unbiased three-dimensional quantitative microcomputed tomography at 7 and 14 days. Compared with male rats, female rats had twice more cancellous bone volume at the proximal tibia at baseline, and this bone volume continued to increase, whereas in males it stabilized. Conversely, cortical area was greater in males than in females, and in both sexes cortical bone was still expanding. Hindlimb unloading resulted in larger reductions in males than in females in both cortical and cancellous compartments. In females, trabecular thickness and number decreased mildly, whereas in males trabecular number was dramatically reduced. In both sexes, the trabecular network became less connected and more rod-like shaped. Bone cellular activities evaluated by histomorphometry showed decreased bone formation rate in both sexes and increased resorption activity only in males. In conclusion, in female rats unloaded-related cancellous alterations reversed the growing process, whereas in males, which show lower growth process, it induced an accentuation of age-related cancellous bone changes for most of the parameters.     

Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve-muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(-/-);fyn(-/-) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-beta-cyclodextrin disassembled AChR clusters and decreased AChR-rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(-/-);fyn(-/-) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.     

Seven day oral supplementation with Cardax TM (disodium disuccinate astaxanthin) provides significant cardioprotection and reduces oxidative stress in rats

In the current study, the improved oral bioavailability of a synthetic astaxanthin derivative (Cardax^TM; disodium disuccinate astaxanthin) was utilized to evaluate its potential effects as a cardioprotective agent after 7-day subchronic oral administration as a feed supplement to Sprague-Dawley rats. Animals received one of two concentrations of Cardax^TM in feed (0.1 and 0.4%; ∼125 and 500 mg/kg/day, respectively) or control feed without drug for 7 days prior to the infarct study carried out on day 8. Thirty minutes of occlusion of the left anterior descending (LAD) coronary artery was followed by 2 h of reperfusion prior to sacrifice, a regimen which resulted in a mean infarct size (IS) as a percentage (%) of the area at risk (AAR; IS/AAR,%) of 61 ± 1.8%. The AAR was quantified by Patent blue dye injection, and IS was determined by triphenyltetrazolium chloride (TTC) staining. Cardax^TM at 0.1 and 0.4% in feed for 7 days resulted in a significant mean reduction in IS/AAR,% to 45 ± 2.0% (26% salvage) and 39 ± 1.5% (36% salvage), respectively. Myocardial levels of free astaxanthin achieved after 7-day supplementation at each of the two concentrations (400 ± 65 nM and 1634 ± 90 nM, respectively) demonstrated excellent solid-tissue target organ loading after oral supplementation. Parallel trends in reduction of plasma levels of multiple lipid peroxidation products with disodium disuccinate astaxanthin supplementation were observed, consistent with the documented in vitro antioxidant mechanism of action. These results extend the potential utility of this compound for cardioprotection to the elective human cardiovascular patient population, for which 7-day oral pre-treatment (as with statins) provides significant reductions in induced periprocedural infarct size.     

Life-history characters and phylogeny are correlated with extinction risk in the Australian angiosperms

Aim To determine whether life‐history characters that affect population persistence (e.g. habit and life span) and those that influence reproductive success (e.g. sexual system and fruit type) are non‐randomly correlated with extinction risk (i.e. threat category) in the Australian flora (c. 19,000 species, of which c. 14% is threatened). To identify patterns that present useful conservation directions. To understand patterns of extinction risk in the Australian flora at a broad scale. Location Continental Australia. Methods A country‐wide exploration of four life‐history characters in the Australian flora (n = 18,822 species) was undertaken using reference texts, expert opinion, herbarium records and field work. For each character and threat‐category combination, a G‐test (using a log‐linear model) was performed to test the null hypothesis that the two factors were independent in their effects on count. A generalized linear model (GLM) with a logit link and binomial error distribution was constructed with the proportion of taxa in each extinction risk category as the response variable and the habit, sex and fruit‐type characters as explanatory terms. In a separate approach, we investigated patterns across the threat categories of non‐endangered extant, endangered, and extinct using a multinomial model. We examined whether or not species‐poor genera were more likely to contain threatened or extinct species than species‐rich genera. A GLM with a binomial error distribution and logit link function was constructed to obtain a weighted regression on the proportion of species listed as extinct or endangered within a genus versus the log of the size of the genus. We also used a supertree analysis and character tracing to investigate the role of phylogeny on extinction risk. Results We found that the Australian flora is primarily composed of bisexual shrubs with dry‐dehiscent fruits. Dioecious breeding systems (separate female and male flowers on separate plants) in many floras are the predominant unisexual system, but in Australia there are unexpectedly high levels of monoecy (separate female and male flowers on the same plant). Within the extinct data set of 31 species we detected a significant departure from that expected for habit but not for life span, sexual system or fruit type. There are significantly fewer trees on the extinct list than expected. This may reflect the greater resilience of trees than of other growth habits to extinction processes as well as the observation time‐frame. Within the endangered data set of 450 species we found significant differences in the representation of the observed characters from that expected within sex systems and fruit types. We show that, depending on the life form, unisexual breeding systems can be significantly and positively associated with endangered species compared with non‐threatened species. For example, there are more monoecious species than expected by chance among the tree species listed as endangered but fewer among the herbaceous life forms. Threat category was found to be non‐randomly clustered in some clades. Main conclusions Life‐history characters in certain combinations are predictive of extinction risk. Phylogeny is also an important component of extinction risk. We suggest that specific life‐history characters could be used for conservation planning and as an early warning sign for detecting vulnerability in lists of species.     

Information-driven protein-DNA docking using HADDOCK: it is a matter of flexibility

Intrinsic flexibility of DNA has hampered the development of efficient protein−DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731–1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor−DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein−DNA complexes from unbound constituents using non-structural experimental data to drive the docking.     

Overyielding and species diversity: what should we expect?

Recent empirical studies have found evidence of increased biomass production ('overyielding') in species mixtures relative to monoculture, but the interpretation of these results remains controversial, in part, because of the lack of a theoretical expectation. Here, we examined the expected frequency and stability of overyielding species mixtures using Lotka-Volterra models of species dynamics in two- and four-species systems in conjunction with community, population, and specific rate of biomass production (SRP) definitions of overyielding. Overyielding plant mixtures represented > 55% of potential species assemblages under community definitions and approximately 100% of species were either overyielding or underyielding under the population definition. Our species simulations approached their equilibria in 1-2 yr, supporting the relevancy of an equilibrial analysis. The range of parameter space that we explored produced realistic values of plot biomass, supporting their biological relevance. We show that overyielding is expected to be common under community definitions and population definitions. Overyielding, under community or population definitions, does not imply an actual increase in the specific rate of biomass production. In addition, assemblages of overyielding and underyielding species under all three definitions can be stable over time with underyielding species persisting in the presence of overyielding species.     

Does ecological filtering across a productivity gradient explain variation in species pool‐richness relationships?

Concerns about the impact of species diversity loss has heightened the importance of determining what limits local diversity and the relative roles of the available species pool and local interactions. Recent theory suggests that local diversity may exhibit linear or asymptotic responses to regional variation in species pools depending resource supply rates. We tested this idea by augmenting species pools in three sites along a natural productivity gradient (a surrogate for resource supply) in a mid-successional old-field. Augmentation of the species pool, enhanced local diversity more at medium than low productivity sites in this field and seed addition had no effect at high productivity, suggesting that diversity was saturated in high resource areas of this field. These results suggest that resource supply rates may mediate species pool-richness relationships. Species additions into cleared plots demonstrated that the observed differences in species recruitment along this gradient were largely driven by abiotic filtering at low and competitive filtering at high productivity. Furthermore, we observed that augmentation of the species pool shifted the productivity–diversity relationship from a negative linear to unimodal suggesting that species pools may influence reported productivity–diversity patterns.