Surface-templated fibril growth of peptide fragments from the shaft domain of the adenovirus fibre protein

Here we present a study of five analogues of a fragment from the shaft domain of the adenovirus fibre protein that readily form fibrils under a range of conditions. Using atomic force microscopy the fibrillisation of these peptides at the liquid/solid interface utilizing ordered crystalline substrates has been investigated. Our results demonstrate that the assembly pathway at the liquid/solid interface enables only the formation of truncated fibrillar structures, which align along the substrate's underlying atomic lattice during growth. Furthermore, that the concentration and volume of solution applied can be used to directly control the density of fibrillar coverage at the surface.     

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above-ground community structure. We collected 3800 root fragments from four randomly positioned, 1-m-deep soil profiles (two vertical transects per plot), located in an old-field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below-ground was correlated with their frequency above-ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above-ground quadrats. Multivariate analyses indicated that diversity was highly structured below-ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above-ground. Furthermore, analyses of species co-occurrence indicates strong species segregation overall but random co-occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below-ground, and also demonstrate the utility of barcoding for the identification of plant roots.     

Efficient mammalian germline transgenesis by cis-enhanced <Emphasis Type="Italic">Sleeping Beauty</Emphasis> transposition

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.     

No reversion to single mating in a socially parasitic ant

Social Hymenoptera are ideal biological models for the study of the selective forces affecting the evolution of multiple mating (polyandry), because sister species can evolve different lifestyles and mating strategies. Single mating is predicted in workerless social parasites, because the key benefit of multiple mating in social insects, that is, the increase in genetic diversity among worker offspring, does not hold for workerless species. We compared the queen mating frequency between the ant Plagiolepis pygmaea and its derived social parasite P. xene. Previous studies showed that queens of the host P. pygmaea are obligately polyandrous. Here, pedigree analyses of mother–offspring combinations indicate that queens of the parasite P. xene did not revert to single mating; more than 50% of queens mated multiply, with 2–4 males. This result shows that reversal from multiple to single mating may be not selected in polyandrous social insect workerless parasites. We propose that such reversion does not occur when multiple mating is virtually cost free.     

Rapid membrane protein topology prediction

State-of-the-art methods for topology of α-helical membrane proteins are based on the use of time-consuming multiple sequence alignments obtained from PSI-BLAST or other sources. Here, we examine if it is possible to use the consensus of topology prediction methods that are based on single sequences to obtain a similar accuracy as the more accurate multiple sequence-based methods. Here, we show that TOPCONS-single performs better than any of the other topology prediction methods tested here, but ~6% worse than the best method that is utilizing multiple sequence alignments. AVAILABILITY AND IMPLEMENTATION: TOPCONS-single is available as a web server from http://single.topcons.net/ and is also included for local installation from the web site. In addition, consensus-based topology predictions for the entire international protein index (IPI) is available from the web server and will be updated at regular intervals.     

Membrane depolarization and NADPH oxidase activation in aortic endothelium during ischemia reflect altered mechanotransduction

Vascular endothelial growth factor (VEGF) is considered to be important in promotion of capillary growth in skeletal muscles exposed to increased activity. We studied its interactions with nitric oxide (NO) by examining the expression of endothelial NO synthase (NOS), VEGF, and VEGF receptor-2 (VEGFR-2) proteins in relation to capillary growth in rat extensor digitorum longus muscles electrically stimulated for 2, 4, or 7 days with and without NOS inhibition by N(omega)-nitro-L-arginine (L-NNA, 3 mg/day). Stimulation increased all proteins from 2 days onward, concomitantly with capillary proliferation (labeling for proliferating cell nuclear antigen). Capillary-to-fiber ratio was elevated by 25% after 7 days. Concurrent oral administration of L-NNA did not affect the increase in endothelial NOS but depressed its activity, as shown by increased blood pressure and decreased arteriolar diameters in 2-day-stimulated muscles. NOS inhibition eliminated the increased expression of VEGFR-2 and VEGF proteins in muscles stimulated for 2 and 4 days but not for 7 days. However, it depressed capillary proliferation and the increase in C/F at all time points. We conclude that, in stimulated muscles, NO, generated by activation of neuronal NOS by muscle activity or endothelial NOS by increased blood flow and capillary shear stress, may increase capillary proliferation in the early stages of stimulation through upregulation of VEGFR-2 and VEGF. With longer stimulation, capillary growth appears to require NO, and high levels of VEGF and VEGFR-2 may be contributing to maintenance of the increased capillary bed.     

AFM studies on the role of the protein RdgC in bacterial DNA recombination

Genetic studies of rdgC in different bacterial systems suggest that it may play a role in replication and recombination. However, the exact function of the corresponding protein, RdgC, is unknown. In this study, we have imaged complexes of RdgC with both linear and supercoiled circular plasmid DNA using atomic force microscopy. We confirm that RdgC does not target any specific sequences in double-stranded DNA, as has been suggested from biochemical data. However, we detect an increased affinity of the protein to DNA ends, and an ability to promote bending of DNA. Similar binding preferences have been reported for enzymes involved in recombination. Protein complexes with supercoiled plasmid DNA further enabled us to study the effect of RdgC on DNA superstructure. At high concentrations of protein we observed promotion of DNA condensation. Recombination is largely enhanced by close contacts of distant regions along the DNA strands, as can occur, for instance, through condensation. Our data thus support a possible function of RdgC as a midwife of recombination.     

Biophysical and transfection studies of an amine-modified poly(vinyl alcohol) for gene delivery

Novel, multifunctional polymers remain an attractive objective for drug delivery, especially for hydrophilic macromolecular drugs candidates such as peptides, proteins, RNA, and DNA. To facilitate intracellular delivery of DNA, new amine-modified poly(vinyl alcohol)s (PVAs) were synthesized by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution. The resulting polymers were characterized using NMR, thermogravimetric analysis (TGA), and gelpermation chromatography (GPC). Atomic force microscopy (AFM), dynamic light scattering photon correlation spectroscopy (PCS), and zeta-potential were used to investigate polyplexes of DNA with PVA copolymers. These studies suggest an influence of the polycation structure on the morphology of condensed DNA in polyplexes. Significant differences were observed by changing both the degrees of amine substitution and the structure of the PVA backbone, demonstrating that both electrostatic and hydrophobic interactions affect DNA condensation. DNA condensation measured by an ethidium bromide intercalation assay showed a higher degree of condensation with pDNA with increasing degrees of amine substitution and more hydrophobic functional groups. These findings are in line with transfection experiments, in which a good uptake of these polymer DNA complexes was noted, unfortunately, with little endosomal escape. Co-administration of chloroquine resulted in increased endosomal escape and higher transfection efficiencies, due to disruption of the endosomal membrane. In this study, the structural requirements for DNA complexation and condensation were characterized to provide a basis for rational design of nonviral gene delivery systems.     

Inbreeding and kinship in the ant Plagiolepis pygmaea

In ants the presence of multiple reproductive queens (polygyny) decreases the relatedness among workers and the brood they rear, and subsequently dilutes their inclusive fitness benefits from helping. However, adoption of colony daughters, low male dispersal in conjunction with intranidal (within nest) mating and colony reproduction by budding may preserve local genetic differences, and slow down the erosion of relatedness. Reduced dispersal and intranidal mating may, however, also lead to detrimental effects owing to competition and inbreeding. We studied mating and dispersal patterns, and colony kinship in three populations of the polygynous ant Plagiolepis pygmaea using microsatellite markers. We found that the populations were genetically differentiated, but also a considerable degree of genetic structuring within populations. The genetic viscosity within populations can be attributed to few genetically homogeneous colony networks, which presumably have arisen through colony reproduction by budding. Hence, selection may act at different levels, the individuals, the colonies and colony networks. All populations were also significantly inbred (F=0.265) suggesting high frequencies of intranidal mating and low male dispersal. Consequently the mean regression relatedness among workers was significantly higher (r = 0.529-0.546) than would be expected under the typically reported number (5-35) of queens in nests of the species. Furthermore, new queens were mainly recruited from their natal or a neighbouring related colony. Finally, the effective number of queens coincided with that found upon excavation, suggesting low reproductive skew.