Using motion planning to study RNA folding kinetics.

We propose a novel, motion planning based approach to approximately map the energy landscape of an RNA molecule. A key feature of our method is that it provides a sparse map that captures the main features of the energy landscape which can be analyzed to compute folding kinetics. Our method is based on probabilistic roadmap motion planners that we have previously successfully applied to protein folding. In this paper, we provide evidence that this approach is also well suited to RNA. We compute population kinetics and transition rates on our roadmaps using the master equation for a few moderately sized RNA and show that our results compare favorably with results of other existing methods.     

Simulating protein motions with rigidity analysis.

Protein motions, ranging from molecular flexibility to large-scale conformational change, play an essential role in many biochemical processes. Despite the explosion in our knowledge of structural and functional data, our understanding of protein movement is still very limited. In previous work, we developed and validated a motion planning based method for mapping protein folding pathways from unstructured conformations to the native state. In this paper, we propose a novel method based on rigidity theory to sample conformation space more effectively, and we describe extensions of our framework to automate the process and to map transitions between specified conformations. Our results show that these additions both improve the accuracy of our maps and enable us to study a broader range of motions for larger proteins. For example, we show that rigidity-based sampling results in maps that capture subtle folding differences between protein G and its mutants, NuG1 and NuG2, and we illustrate how our technique can be used to study large-scale conformational changes in calmodulin, a 148 residue signaling protein known to undergo conformational changes when binding to Ca(2+). Finally, we announce our web-based protein folding server which includes a publicly available archive of protein motions: (http://parasol.tamu.edu/foldingserver/).     

Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb–A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K_D ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP–sdAb gave a yield of approximately 100 mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb–A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1 mg/ml for 1 h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1 h at 90 °C. The PfuMBP–sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb–A5 both as a capture reagent and as a detection reagent.     

A predator's perspective of nest predation: predation by red squirrels is learned,not incidental

Nest predation has been used to explain aspects of avian ecology ranging from nest site selection to population declines. Many arguments rely on specific assumptions regarding how predators find nests, yet these predatory mechanisms remain largely untested. Here we combine artificial nest experiments with behavioural observations of individual red squirrels Tamiasciurus hudsonicus to differentiate between two common hypotheses: predation is incidental versus learned. Specifically, we tested: 1) whether nest survival could be explained solely by a squirrel's activity patterns or habitat use, as predicted if predation was incidental; or 2) if predation increased as a squirrel gained experience preying on a nest, as predicted if predation was learned. We also monitored squirrel activity after predation to test for evidence of two search mechanisms: area‐restricted searching and use of microhabitat search images. Contrary to incidental predation and in support of learning, squirrels did not find nests faster in areas with high use (e.g. forest edges). Instead, survival of artificial nests was strongly related to a squirrel's prior experience preying on artificial nests. Experience reduced nest survival times by over half and increased predation rates by 150–200%. Squirrels returned to and doubled their activity at the site of a previously preyed on nest. However, neither area‐restricted searching nor microhabitat search images can explain how squirrels located artificial nests more readily with experience. Instead, squirrels likely used cues associated with the nests or eggs themselves. Learning implies that squirrels could be increasingly effective predators as the density or profitability of nests increases. Our results add support to the view that nest predation is complex and broadly influenced (e.g. by predator experience, motivation), and is unlikely to be predicted consistently by simple relationships with predator activity, abundance or habitat.     

Limitations of cross‐ and multigenerational plasticity for marine invertebrates faced with global climate change

Although cross generation (CGP) and multigenerational (MGP) plasticity have been identified as mechanisms of acclimation to global change, the weight of evidence indicates that parental conditioning over generations is not a panacea to rescue stress sensitivity in offspring. For many species, there were no benefits of parental conditioning. Even when improved performance was observed, this waned over time within a generation or across generations and fitness declined. CGP and MGP studies identified resilient species with stress tolerant genotypes in wild populations and selected family lines. Several bivalves possess favourable stress tolerance and phenotypically plastic traits potentially associated with genetic adaptation to life in habitats where they routinely experience temperature and/or acidification stress. These traits will be important to help ‘climate proof’ shellfish ventures. Species that are naturally stress tolerant and those that naturally experience a broad range of environmental conditions are good candidates to provide insights into the physiological and molecular mechanisms involved in CGP and MGP. It is challenging to conduct ecologically relevant global change experiments over the long times commensurate with the pace of changing climate. As a result, many studies present stressors in a shock‐type exposure at rates much faster than projected scenarios. With more gradual stressor introduction over longer experimental durations and in context with conditions species are currently acclimatized and/or adapted to, the outcomes for sensitive species might differ. We highlight the importance to understand primordial germ cell development and the timing of gametogenesis with respect to stressor exposure. Although multigenerational exposure to global change stressors currently appears limited as a universal tool to rescue species in the face of changing climate, natural proxies of future conditions (upwelling zones, CO₂ vents, naturally warm habitats) show that phenotypic adjustment and/or beneficial genetic selection is possible for some species, indicating complex plasticity–adaptation interactions.     

A survey of Penstemon's genome size

Penstemon is the largest genus in North America with more than 270 reported species. However, little is known about its genome size. This information may be useful in developing hybrids for landscape use and for gaining insight into its current taxonomy. Using flow cytometry, we estimated the genome size of approximately 40% of the genus (115 accessions from 105 different species). Genome sizes for both reported and probable diploids range from P. dissectus 2C = 0.94 pg (1C = 462 Mbp) to P. pachyphyllus var. mucronatus 2C = 1.88 pg (1C = 919 Mbp), and the polyploids range from P. attenuatus var. attenuatus 2C = 2.35 pg (1C = 1148 Mbp) to P. digitalis 2C = 6.45 pg (1C = 3152 Mbp). Chromosome counts were done for ten previously published and four previously unreported Penstemon species (P. dissectus, P. navajoa, P. caespitosus var. desertipicti, and P. ramaleyi). These counts were compiled with all previously published chromosome data and compared with the flow cytometry results. Ploidy within this study ranged from diploid to dodecaploid. These data were compared and contrasted with the current taxonomy of Penstemon and previously published internal transcribed spacer and chloroplast DNA phylogenetic work. Based on genome size and previous studies, reassigning P. montanus to the subgenus Penstemon and P. personatus to the subgenus Dasanthera, would better reflect the phylogeny of the genus. Furthermore, our data concur with previous studies suggesting that the subgenus Habroanthus be included in the subgenus Penstemon. The DNA content of subgenus Penstemon exhibits high plasticity and spans a sixfold increase from the smallest to the largest genome (P. linarioides subsp. sileri and P. digitalis, respectively). Our study found flow cytometry to be useful in species identification and verification.     

Spatial variability in airborne bacterial communities across land-use types and their relationship to the bacterial communities of potential source environments

Although bacteria are ubiquitous in the near-surface atmosphere and they can have important effects on human health, airborne bacteria have received relatively little attention and their spatial dynamics remain poorly understood. Owing to differences in meteorological conditions and the potential sources of airborne bacteria, we would expect the atmosphere over different land-use types to harbor distinct bacterial communities. To test this hypothesis, we sampled the near-surface atmosphere above three distinct land-use types (agricultural fields, suburban areas and forests) across northern Colorado, USA, sampling five sites per land-use type. Microbial abundances were stable across land-use types, with ∼10⁵–10⁶ bacterial cells per m³ of air, but the concentrations of biological ice nuclei, determined using a droplet freezing assay, were on average two and eight times higher in samples from agricultural areas than in the other two land-use types. Likewise, the composition of the airborne bacterial communities, assessed via bar-coded pyrosequencing, was significantly related to land-use type and these differences were likely driven by shifts in the sources of bacteria to the atmosphere across the land-uses, not local meteorological conditions. A meta-analysis of previously published data shows that atmospheric bacterial communities differ from those in potential source environments (leaf surfaces and soils), and we demonstrate that we may be able to use this information to determine the relative inputs of bacteria from these source environments to the atmosphere. This work furthers our understanding of bacterial diversity in the atmosphere, the terrestrial controls on this diversity and potential approaches for source tracking of airborne bacteria.