Mining the structural genomics pipeline: identification of protein properties that affect high-throughput experimental analysis

Structural genomics projects represent major undertakings that will change our understanding of proteins. They generate unique datasets that, for the first time, present a standardized view of proteins in terms of their physical and chemical properties. By analyzing these datasets here, we are able to discover correlations between a protein's characteristics and its progress through each stage of the structural genomics pipeline, from cloning, expression, purification, and ultimately to structural determination. First, we use tree-based analyses (decision trees and random forest algorithms) to discover the most significant protein features that influence a protein's amenability to high-throughput experimentation. Based on this, we identify potential bottlenecks in various stages of the structural genomics process through specialized "pipeline schematics". We find that the properties of a protein that are most significant are: (i.) whether it is conserved across many organisms; (ii). the percentage composition of charged residues; (iii). the occurrence of hydrophobic patches; (iv). the number of binding partners it has; and (v). its length. Conversely, a number of other properties that might have been thought to be important, such as nuclear localization signals, are not significant. Thus, using our tree-based analyses, we are able to identify combinations of features that best differentiate the small group of proteins for which a structure has been determined from all the currently selected targets. This information may prove useful in optimizing high-throughput experimentation. Further information is available from http://mining.nesg.org/.     

Quantitative pH assessment of small-volume samples using a universal pH indicator

We developed a hue-based pH determination method to analyze digital images of samples in a 384-well plate after the addition of a universal pH indicator. The standard error of calibration for 69 pH standards was 0.078 pH units, and no sample gave an error greater than 0.23 units. We then used in-solution isoelectric focusing to determine the isoelectric point of Wnt3A protein in conditioned medium and after purification and applied the described method to assess the pH of these small-volume samples. End users may access our standard to assay the pH of their own samples with no additional calibration.     

SENSITIVITY OF THE DISTRIBUTION OF MUTATIONAL FITNESS EFFECTS TO ENVIRONMENT,GENETIC BACKGROUND,AND ADAPTEDNESS: A CASE STUDY WITH DROSOPHILA

Heterogeneity in the fitness effects of individual mutations has been found across different environmental and genetic contexts. Going beyond effects on individual mutations, how is the distribution of selective effects, f(s), altered by changes in genetic and environmental context? In this study, we examined changes in the major features of f(s) by estimating viability selection on 36 individual mutations in Drosophila melanogaster across two different environments in two different genetic backgrounds that were either adapted or nonadapted to the two test environments. Both environment and genetic background affected selection on individual mutations. However, the overall distribution f(s) appeared robust to changes in genetic background but both the mean, E(s), and the variance, V(s) were dependent on the environment. Between these two properties, V(s) was more sensitive to environmental change. Contrary to predictions of fitness landscape theory, the match between genetic background and assay environment (i.e., adaptedness) had little effect on f(s).     

Correction to: Skeletal records of bleaching reveal different thermal thresholds of Pacific coral reef assemblages

Coral Reefs - Figure 2 in the original article has been updated with this figure 2 due to discrepancies related to incorrect mapping with one of the islands.     

Hemoglobin C modulates the surface topography of Plasmodium falciparum-infected erythrocytes

There is a well-established clinical association between hemoglobin genotype and innate protection against Plasmodium falciparum malaria. In contrast to normal hemoglobin A, mutant hemoglobin C is associated with substantial reductions in the risk of severe malaria in both heterozygous AC and homozygous CC individuals. Irrespective of hemoglobin genotype, parasites may induce knob-like projections on the erythrocyte surface. The knobs play a major role in the pathogenesis of severe malaria by serving as points of adherence for P. falciparum-infected erythrocytes to microvascular endothelia. To evaluate the influence of hemoglobin genotype on knob formation, we used a combination of atomic force and light microscopy for concomitant topographic and wide-field fluorescence imaging. Parasitized AA, AC, and CC erythrocytes showed a population of knobs with a mean width of approximately 70 nm. Parasitized AC and CC erythrocytes showed a second population of large knobs with a mean width of approximately 120 nm. Furthermore, spatial knob distribution analyses demonstrated that knobs on AC and CC erythrocytes were more aggregated than on AA erythrocytes. These data support a model in which large knobs and their aggregates are promoted by hemoglobin C, reducing the adherence of parasitized erythrocytes in the microvasculature and ameliorating the severity of a malaria infection.     

Visualization methods for statistical analysis of microarray clusters

Background  

The most common method of identifying groups of functionally related genes in microarray data is to apply a clustering algorithm. However, it is impossible to determine which clustering algorithm is most appropriate to apply, and it is difficult to verify the results of any algorithm due to the lack of a gold-standard. Appropriate data visualization tools can aid this analysis process, but existing visualization methods do not specifically address this issue.     

Seed-spitting Primates and the Conservation and Dispersion of Large-seeded Trees

Primate frugivory may reduce density-dependent predation on seeds and seedlings via effective seed dispersal. Accordingly, the tendency of cercopithecines to spit and scatter seeds > 4 mm wide could represent a prominent means of dispersal. However, the importance of seed-spitting may vary according to the life history adaptations of plants. Indeed, the actions of cercopithecines may be incongruent with the reproductive biology of plants that rely on large frugivores to swallow and defecate their seeds. This possibility raises conservation concerns because large frugivores are often susceptible to extirpation or extinction from hunting and habitat fragmentation. It is therefore important to determine if cercopithecines have a compensatory effect; that is, whether or not seed-spitting effectively conveys large seeds to recruitment sites. To test this concept, we used geospatial techniques to measure and analyze the dispersion of tree species dispersed by elephants, chimpanzees, and cercopithecines to different spatial extents. We studied adult trees of Balanites wilsoniana, Chrysophyllum gorungosanum, and Uvariopsis congensis in a 2.2-ha plot in Kibale National Park, Uganda. Despite the tendency of cercopithecines to spit the seeds of Uvariopsis congensis, adult trees were highly clumped, with a modal nearest-neighbor distance of < 5 m and a crown overlap of 1.5 m. Virtually identical results for Balanites wilsoniana and Chrysophyllum gorungosanum, the seeds of which are not spat, suggest that seed-spitting may be a poor mechanism of dispersal for some large-seeded plants.     

Evaluation of primary production in Lake Erie by multiple proxies

Direct measurements of rates of primary production in Lake Erie are few and uncertainties surround rate measurements based on radiocarbon and the light-dark bottle incubation methods. For these reasons, we conducted a series of simultaneous primary productivity measurements in Lake Erie in July and August of 2003, based on incubation with [14C]-NaHCO3, the light-dark bottle method, and incubation with (18)O enriched water. Significant differences in the rates of primary production obtained by incubations with [(18)O]-H2O (0.19-34.60 mmol-O2 m(-3) h(-1)), [14C]-NaHCO3 (0.03-90.50 mmol-C m(-3) h(-1)), and light-dark bottles (0.06-60.78 mmol-O2 m(-3) h(-1)) were evident in six out of nine comparisons. Within the epilimnion, [(18)O]-H2O rates of primary production were significantly different from rates based on [14C]-NaHCO3 and light-dark bottles in all four comparisons and lower rates were obtained in three out of four comparisons. Eutrophic conditions in Sandusky Bay, Lake Erie were evident from the high primary production rates of 20.50-34.60 mmol-O2 m(-3) h(-1) ([(18)O]-H2O), 34.39-90.50 mmol-C m(-3) h(-1) ([14C]-NaHCO3), and 46.66-60.78 mmol-O2 m(-3) h(-1) (light-dark bottle). The photosynthetic quotient (PQ), or ratio of O2 production to CO2 consumption during photosynthesis, averaged 0.64+/-0.33 and 1.93+/-1.93, respectively, based on a comparison of [(18)O]-H2O to [14C]-NaHCO3 rates or light-dark bottle to [14C]-NaHCO3 production rates, respectively, demonstrating that photosynthesis in Lake Erie communities primarily follows expected stochiometric trends. The average of the ratio of production rates based on incubation with [(18)O]-H2O relative to those obtained by the light-dark incubation method was 0.66+/-0.33, indicating a tendency for the [(18)O]-H2O method to provide slightly lower estimates of production in Lake Erie. Lower estimates of primary production based on [(18)O]-H2O incubation relative to the other two approaches is most likely a consequence of consumption of labeled O2 within the cell or dilution of label by the release of O2 from supersaturated cells. This latter effect may be particularly characteristic of eutrophic environments.