Computational model of chromosome aberration yield induced by high- and low-LET radiation exposures

We present a computational model for calculating the yield of radiation-induced chromosomal aberrations in human cells based on a stochastic Monte Carlo approach and calibrated using the relative frequencies and distributions of chromosomal aberrations reported in the literature. A previously developed DNA-fragmentation model for high- and low-LET radiation called the NASARadiationTrackImage model was enhanced to simulate a stochastic process of the formation of chromosomal aberrations from DNA fragments. The current version of the model gives predictions of the yields and sizes of translocations, dicentrics, rings, and more complex-type aberrations formed in the G(0)/G(1) cell cycle phase during the first cell division after irradiation. As the model can predict smaller-sized deletions and rings (<3 Mbp) that are below the resolution limits of current cytogenetic analysis techniques, we present predictions of hypothesized small deletions that may be produced as a byproduct of properly repaired DNA double-strand breaks (DSB) by nonhomologous end-joining. Additionally, the model was used to scale chromosomal exchanges in two or three chromosomes that were obtained from whole-chromosome FISH painting analysis techniques to whole-genome equivalent values.     

Interactions between nematodes and their microbial enemies in coastal sand dunes

European foredunes are almost exclusively colonised by Ammophila arenaria, and both the natural succession and the die-out of this plant have been linked to populations of plant-parasitic nematodes (PPN). The overarching aim of this study was to investigate top-down control processes of PPN in these natural ecosystems through comparative analyses of the diversity and dynamics of PPN and their microbial enemies. Our specific aims were, first, to identify and quantify PPN microbial enemies in European sand dunes; second, to assess their life history traits, their spatial and temporal variation in these ecosystems, and third, to evaluate their control potential of PPN populations. This was done by seasonal sampling of a range of sites and making observations on both the nematode and the microbial enemy communities in rhizosphere sand. Nine different nematode microbial enemies belonging to different functional groups were detected in European sand dunes. Their high diversity in these low productivity ecosystems could both result from or lead to the lack of dominance of a particular nematode genus. The distribution of microbial enemies was spatially and temporally variable, both among and within sampling sites. Obligate parasites, either with low host-specificity or having the ability to form an environmentally resistant propagule, are favoured in these ecosystems and are more frequent and abundant than facultative parasites. Three microbial enemies correlated, either positively or negatively, with PPN population size: Catenaria spp., Hirsutella rhossiliensis and Pasteuria penetrans. Microbial-enemy supported links in the food-web may be involved in the control of PPN populations through indirect effects. The endospore-forming P. penetrans was the most successful top-down control agent, and was implicated in the direct control of Meloidogyne spp. and indirect facilitation of Pratylenchus spp. Overall, our findings suggest strong and diverse top-down control effects on the nematode community in these natural ecosystems.     

Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed at the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to aid molecular identifications of unknowns via the FUSARIUM-ID and Fusarium MLST online databases and analysis packages. Analyses of a 190-taxon, two-locus dataset, which included 159 isolates from insects, indicated that: (i) insect-associated fusaria were nested within 10 species complexes spanning the phylogenetic breadth of Fusarium, (ii) novel, putatively unnamed insecticolous species were nested within 8/10 species complexes and (iii) Latin binomials could be applied with confidence to only 18/58 phylogenetically distinct fusaria associated with pest insects. Phylogenetic analyses of an 82-taxon, three-locus dataset nearly fully resolved evolutionary relationships among the 10 clades containing insecticolous fusaria. Multilocus typing of isolates within four species complexes identified surprisingly high genetic diversity in that 63/65 of the fusaria typed represented newly discovered haplotypes. The DNA sequence data, together with corrected ABI sequence chromatograms and alignments, have been uploaded to the following websites dedicated to identifying fusaria: FUSARIUM-ID (http://isolate.fusariumdb.org) at Pennsylvania State University's Department of Plant Pathology and Fusarium MLST (http://www.cbs.knaw.nl/fusarium) at the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center.     

Comparative demography of an epiphytic lichen: support for general life history patterns and solutions to common problems in demographic parameter estimation

Lichens are major components in many terrestrial ecosystems, yet their population ecology is at best only poorly understood. Few studies have fully quantified the life history or demographic patterns of any lichen, with particularly little attention to epiphytic species. We conducted a 6-year demographic study of Vulpicida pinastri, an epiphytic foliose lichen, in south-central Alaska. After testing multiple size-structured functions to describe patterns in each V. pinastri demographic rate, we used the resulting estimates to construct a stochastic demographic model for the species. This model development led us to propose solutions to two general problems in construction of demographic models for many taxa: how to simply but accurately characterize highly skewed growth rates, and how to estimate recruitment rates that are exceptionally difficult to directly observe. Our results show that V. pinastri has rapid and variable growth and, for small individuals, low and variable survival, but that these traits are coupled with considerable longevity (e.g., >50?years mean future life span for a 4-cm(2) thallus) and little deviation of the stochastic population growth rate from the deterministic expectation. Comparisons of the demographic patterns we found with those of other lichen studies suggest that their relatively simple architecture may allow clearer generalities about growth patterns for lichens than for other taxa, and that the expected pattern of faster growth rates for epiphytic species is substantiated.     

Multi-scale assessment of greater sage-grouse fence collision as a function of site and broad scale factors

Previous research in Europe and North America suggested grouse are susceptible to collision with infrastructure, and anecdotal observation suggested greater sage-grouse (Centrocercus urophasianus) fence collision in breeding habitats may be prevalent. However, no previous research systematically studied greater sage-grouse fence collision in any portion of their range. We used data from probability-based sampling of fences in greater sage-grouse breeding habitats of southern Idaho, USA, to model factors associated with collision at microsite and broad spatial scales. Site-scale modeling suggested collision may be influenced by technical attributes of fences, with collisions common at fence segments absent wooden fence posts and with segment widths >4 m. Broad-scale modeling suggested relative probability of collision was influenced by region, a terrain ruggedness index (TRI), and fence density per square km. Conditional on those factors, collision counts were also influenced by distance to nearest active sage-grouse lek. Our models provide a conceptual framework for prioritizing sage-grouse breeding habitats for collision mitigation such as fence marking or moving, and suggest mitigation in breeding habitats should start in areas with moderate-high fence densities (>1 km/km²) within 2 km of active leks. However, TRI attenuated other covariate effects, and mean TRI/km² >10 m nearly eliminated sage-grouse collision. Thus, our data suggested mitigation should focus on sites with flat to gently rolling terrain. Moreover, site-scale modeling suggested constructing fences with larger and more conspicuous wooden fence posts and segment widths <4 m may reduce collision. © 2012 The Wildlife Society.     

Rapid, long-distance dispersal by pumice rafting

Pumice is an extremely effective rafting agent that can dramatically increase the dispersal range of a variety of marine organisms and connect isolated shallow marine and coastal ecosystems. Here we report on a significant recent pumice rafting and long-distance dispersal event that occurred across the southwest Pacific following the 2006 explosive eruption of Home Reef Volcano in Tonga. We have constrained the trajectory, and rate, biomass and biodiversity of transfer, discovering more than 80 species and a substantial biomass underwent a >5000 km journey in 7-8 months. Differing microenvironmental conditions on the pumice, caused by relative stability of clasts at the sea surface, promoted diversity in biotic recruitment. Our findings emphasise pumice rafting as an important process facilitating the distribution of marine life, which have implications for colonisation processes and success, the management of sensitive marine environments, and invasive pest species.     

Multiple biosynthetic trafficking routes for apically secreted proteins in MDCK cells

Many newly synthesized membrane proteins traverse endocytic intermediates en route to the surface in polarized epithelial cells; however, the biosynthetic itinerary of secreted proteins has not been elucidated. We monitored the trafficking route of two secreted proteins with different apical sorting signals: the N-glycan-dependent cargo glycosylated growth hormone (gGH) and Ensol, a soluble version of endolyn whose apical sorting is independent of N-glycans. Both proteins were observed to colocalize in part with apical recycling endosome (ARE) markers. Cargo that lacks an apical targeting signal and is secreted in a nonpolarized manner did not localize to the ARE. Expression of a dominant-negative mutant of myosin Vb, which disrupts ARE export of glycan-dependent membrane proteins, selectively inhibited apical release of gGH but not Ensol. Fluorescence recovery after photobleaching (FRAP) measurements revealed that gGH in the ARE was less mobile than Ensol, consistent with tethering to a sorting receptor. However, knockdown of galectin-3 or galectin-4, lectins implicated in apical sorting, had no effect on the rate or polarity of gGH secretion. Together, our results suggest that apically secreted cargoes selectively access the ARE and are exported via differentially regulated pathways.     

Human tumor cell proliferation evaluated using manganese-enhanced MRI

Background

Tumor cell proliferation can depend on calcium entry across the cell membrane. As a first step toward the development of a non-invasive test of the extent of tumor cell proliferation in vivo, we tested the hypothesis that tumor cell uptake of a calcium surrogate, Mn²⁺ [measured with manganese-enhanced MRI (MEMRI)], is linked to proliferation rate in vitro.

Methodology/Principal Findings

Proliferation rates were determined in vitro in three different human tumor cell lines: C918 and OCM-1 human uveal melanomas and PC-3 prostate carcinoma. Cells growing at different average proliferation rates were exposed to 1 mM MnCl₂ for one hour and then thoroughly washed. MEMRI R₁ values (longitudinal relaxation rates), which have a positive linear relationship with Mn²⁺ concentration, were then determined from cell pellets. Cell cycle distributions were determined using propidium iodide staining and flow cytometry. All three lines showed Mn²⁺-induced increases in R₁ compared to cells not exposed to Mn²⁺. C918 and PC-3 cells each showed a significant, positive correlation between MEMRI R₁ values and proliferation rate (p≤0.005), while OCM-1 cells showed no significant correlation. Preliminary, general modeling of these positive relationships suggested that pellet R₁ for the PC-3 cells, but not for the C918 cells, could be adequately described by simply accounting for changes in the distribution of the cell cycle-dependent subpopulations in the pellet.

Conclusions/Significance

These data clearly demonstrate the tumor-cell dependent nature of the relationship between proliferation and calcium influx, and underscore the usefulness of MEMRI as a non-invasive method for investigating this link. MEMRI is applicable to study tumors in vivo, and the present results raise the possibility of evaluating proliferation parameters of some tumor types in vivo using MEMRI.     

Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity

Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.