Systematic Planning of Genome-Scale Experiments in Poorly Studied Species

Genome-scale datasets have been used extensively in model organisms to screen for specific candidates or to predict functions for uncharacterized genes. However, despite the availability of extensive knowledge in model organisms, the planning of genome-scale experiments in poorly studied species is still based on the intuition of experts or heuristic trials. We propose that computational and systematic approaches can be applied to drive the experiment planning process in poorly studied species based on available data and knowledge in closely related model organisms. In this paper, we suggest a computational strategy for recommending genome-scale experiments based on their capability to interrogate diverse biological processes to enable protein function assignment. To this end, we use the data-rich functional genomics compendium of the model organism to quantify the accuracy of each dataset in predicting each specific biological process and the overlap in such coverage between different datasets. Our approach uses an optimized combination of these quantifications to recommend an ordered list of experiments for accurately annotating most proteins in the poorly studied related organisms to most biological processes, as well as a set of experiments that target each specific biological process. The effectiveness of this experiment- planning system is demonstrated for two related yeast species: the model organism Saccharomyces cerevisiae and the comparatively poorly studied Saccharomyces bayanus. Our system recommended a set of S. bayanus experiments based on an S. cerevisiae microarray data compendium. In silico evaluations estimate that less than 10% of the experiments could achieve similar functional coverage to the whole microarray compendium. This estimation was confirmed by performing the recommended experiments in S. bayanus, therefore significantly reducing the labor devoted to characterize the poorly studied genome. This experiment-planning framework could readily be adapted to the design of other types of large-scale experiments as well as other groups of organisms.     

Genome-Wide Association Analyses Point to Candidate Genes for Electric Shock Avoidance in Drosophila melanogaster

Electric shock is a common stimulus for nociception-research and the most widely used reinforcement in aversive associative learning experiments. Yet, nothing is known about the mechanisms it recruits at the periphery. To help fill this gap, we undertook a genome-wide association analysis using 38 inbred Drosophila melanogaster strains, which avoided shock to varying extents. We identified 514 genes whose expression levels and/ or sequences co-varied with shock avoidance scores. We independently scrutinized 14 of these genes using mutants, validating the effect of 7 of them on shock avoidance. This emphasizes the value of our candidate gene list as a guide for follow-up research. In addition, by integrating our association results with external protein-protein interaction data we obtained a shock avoidance-associated network of 38 genes. Both this network and the original candidate list contained a substantial number of genes that affect mechanosensory bristles, which are hair-like organs distributed across the fly’s body. These results may point to a potential role for mechanosensory bristles in shock sensation. Thus, we not only provide a first list of candidate genes for shock avoidance, but also point to an interesting new hypothesis on nociceptive mechanisms.     

The most frequent short sequences in non-coding DNA

The purpose of this work is to determine the most frequent short sequences in non-coding DNA. They may play a role in maintaining the structure and function of eukaryotic chromosomes. We present a simple method for the detection and analysis of such sequences in several genomes, including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We also study two chromosomes of man and mouse with a length similar to the whole genomes of the other species. We provide a list of the most common sequences of 9–14 bases in each genome. As expected, they are present in human Alu sequences. Our programs may also give a graph and a list of their position in the genome. Detection of clusters is also possible. In most cases, these sequences contain few alternating regions. Their intrinsic structure and their influence on nucleosome formation are not known. In particular, we have found new features of short sequences in C. elegans, which are distributed in heterogeneous clusters. They appear as punctuation marks in the chromosomes. Such clusters are not found in either A. thaliana or D. melanogaster. We discuss the possibility that they play a role in centromere function and homolog recognition in meiosis.     

Evolutionary Distance of Amino Acid Sequence Orthologs across Macaque Subspecies: Identifying Candidate Genes for SIV Resistance in Chinese Rhesus Macaques

We use the Reciprocal Smallest Distance (RSD) algorithm to identify amino acid sequence orthologs in the Chinese and Indian rhesus macaque draft sequences and estimate the evolutionary distance between such orthologs. We then use GOanna to map gene function annotations and human gene identifiers to the rhesus macaque amino acid sequences. We conclude methodologically by cross-tabulating a list of amino acid orthologs with large divergence scores with a list of genes known to be involved in SIV or HIV pathogenesis. We find that many of the amino acid sequences with large evolutionary divergence scores, as calculated by the RSD algorithm, have been shown to be related to HIV pathogenesis in previous laboratory studies. Four of the strongest candidate genes for SIV_mac resistance in Chinese rhesus macaques identified in this study are CDK9, CXCL12, TRIM21, and TRIM32. Additionally, ANKRD30A, CTSZ, GORASP2, GTF2H1, IL13RA1, MUC16, NMDAR1, Notch1, NT5M, PDCD5, RAD50, and TM9SF2 were identified as possible candidates, among others. We failed to find many laboratory experiments contrasting the effects of Indian and Chinese orthologs at these sites on SIV_mac pathogenesis, but future comparative studies might hold fertile ground for research into the biological mechanisms underlying innate resistance to SIV_mac in Chinese rhesus macaques.     

Identifying components of protein complexes in C. elegans using co-immunoprecipitation and mass spectrometry

Mass spectrometry-based proteomics is rapidly becoming an essential tool for biologists. One of the most common applications is identifying the components of protein complexes isolated by co-immunoprecipitation. In this review, we discuss the co-immunoprecipitation, mass spectrometry and data analysis techniques that have been used successfully to define protein complexes in C. elegans research. In this discussion, two strategies emerged. One approach is to use stringent biochemical purification methods and attempt to identify a small number of complex components with a high degree of certainty based on MS data. A second approach is to use less stringent purification and identification parameters, and ultimately test a longer list of potential binding partners in biological validation assays. This should provide a useful guide for biologists planning proteomic experiments.     

FitSNPs: highly differentially expressed genes are more likely to have variants associated with disease

Background

Candidate single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWASs) were often selected for validation based on their functional annotation, which was inadequate and biased. We propose to use the more than 200,000 microarray studies in the Gene Expression Omnibus to systematically prioritize candidate SNPs from GWASs.

Results

We analyzed all human microarray studies from the Gene Expression Omnibus, and calculated the observed frequency of differential expression, which we called differential expression ratio, for every human gene. Analysis conducted in a comprehensive list of curated disease genes revealed a positive association between differential expression ratio values and the likelihood of harboring disease-associated variants. By considering highly differentially expressed genes, we were able to rediscover disease genes with 79% specificity and 37% sensitivity. We successfully distinguished true disease genes from false positives in multiple GWASs for multiple diseases. We then derived a list of functionally interpolating SNPs (fitSNPs) to analyze the top seven loci of Wellcome Trust Case Control Consortium type 1 diabetes mellitus GWASs, rediscovered all type 1 diabetes mellitus genes, and predicted a novel gene (KIAA1109) for an unexplained locus 4q27. We suggest that fitSNPs would work equally well for both Mendelian and complex diseases (being more effective for cancer) and proposed candidate genes to sequence for their association with 597 syndromes with unknown molecular basis.

Conclusions

Our study demonstrates that highly differentially expressed genes are more likely to harbor disease-associated DNA variants. FitSNPs can serve as an effective tool to systematically prioritize candidate SNPs from GWASs.     

Nymphal and adult performance of Apolygus lucorum (Hemiptera: Miridae) on a preferred host plant, mungbean Vigna radiata

Mungbean, Vigna radiata (L.) Wilczek, a preferred host plant of Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae), is used in China as a trap crop for this pest in cotton fields. Little is known, though, about the relationship between adult oviposition preference and offspring performance of A. lucorum on mungbean. In this study, we compared the oviposition preference of A. lucorum on mungbean versus cotton plants in both open-field plots and field cages. Field-plot experiments demonstrated that the population density of first instar nymphs of A. lucorum and the ratio of first instar nymphs to adults were significantly higher on mungbean than on cotton. Choice and no-choice cage experiments revealed that the number of eggs laid on mungbean was significantly higher than on cotton. We also compared adult longevity and fecundity, as well as offspring survival and development on these two host plants. We found that A. lucorum adult females survived longer and laid more eggs on mungbean than on cotton (P?<?0.05). Mungbean also promoted higher rates of development than did cotton (P?<?0.05), but did not affect nymphal survival (P?>?0.05). In summary, the preference of A. lucorum adults for mungbean is consistent with both adult and nymphal performance. This study furthers the understanding of the relationship between the polyphagous species A. lucorum and its host plants, which will help improve trap-cropping strategies for the control of A. lucorum that are based on the use of mungbean.     

On the use of chemically derived distance constraints in the prediction of protein structure with myoglobin as an example

Chemical techniques have been applied to proteins to determine the spatial proximity of certain sequentially distant amino acid residues. We present a specific example of how chemical information can aid in the prediction of the structure of myoglobin. The algorithm of Cohen et al. (1979a) for predicting the myoglobin fold produced a list of 20 possible structures from 10⁸ alternatives by introducing two filters. We added a third filter by exploiting non-crystallographically derived chemical data to constrain the distal-proximal histidine separation. Of the 20 possible structures, only two very similar structures satisfied this additional filter. The two remaining structures have root-mean-square deviations of 4.48 and 4.53 Å from the crystal structure. The relative effects of the various constraints used to reduce the number of candidates for the myoglobin fold from 10³² in a random structure to two are explained.     

sdef: an R package to synthesize lists of significant features in related experiments

Background  

In microarray studies researchers are often interested in the comparison of relevant quantities between two or more similar experiments, involving different treatments, tissues, or species. Typically each experiment reports measures of significance (e.g. p-values) or other measures that rank its features (e.g genes). Our objective is to find a list of features that are significant in all experiments, to be further investigated. In this paper we present an R package called sdef, that allows the user to quantify the evidence of communality between the experiments using previously proposed statistical methods based on the ranked lists of p-values. sdef implements two approaches that address this objective: the first is a permutation test of the maximal ratio of observed to expected common features under the hypothesis of independence between the experiments. The second approach, set in a Bayesian framework, is more flexible as it takes into account the uncertainty on the number of genes differentially expressed in each experiment.     

Monoclonal antibodies to antigen binding protein of annelids (Lumbricus terrestris)

• 1.1. Monoclonal antibodies (mAb) to antigen binding protein (ABP) of the earthworm Lumbricus terrestris have been prepared.
• 2.2. The specificity of mAb for a determinant located outside the antigen binding site was determined and verified in inhibition experiments.
• 3.3. The mAb were used for isolation of a 56 kDa ABP by an immunoprecipitation technique.
• 4.4. The binding of mAb to coelomocytes has demonstrated the existence of two cell populations, one with low and the other with high densities of ABP molecules on the cell membranes.

     

Effect of Twisted Fiber Anisotropy in Cardiac Tissue on Ablation with Pulsed Electric Fields

BackgroundAblation of cardiac tissue with pulsed electric fields is a promising alternative to current thermal ablation methods, and it critically depends on the electric field distribution in the heart.MethodsWe developed a model that incorporates the twisted anisotropy of cardiac tissue and computed the electric field distribution in the tissue. We also performed experiments in rabbit ventricles to validate our model. We find that the model agrees well with the experimentally determined ablation volume if we assume that all tissue that is exposed to a field greater than 3 kV/cm is ablated. In our numerical analysis, we considered how tissue thickness, degree of anisotropy, and electrode configuration affect the geometry of the ablated volume. We considered two electrode configurations: two parallel needles inserted into the myocardium (“penetrating needles” configuration) and one circular electrode each on epi- and endocardium, opposing each other (“epi-endo” configuration).ResultsFor thick tissues (10 mm) and moderate anisotropy ratio (a = 2), we find that the geometry of the ablated volume is almost unaffected by twisted anisotropy, i.e. it is approximately translationally symmetric from epi- to endocardium, for both electrode configurations. Higher anisotropy ratio (a = 10) leads to substantial variation in ablation width across the wall; these variations were more pronounced for the penetrating needle configuration than for the epi-endo configuration.For thinner tissues (4 mm, typical for human atria) and higher anisotropy ratio (a = 10), the epi-endo configuration yielded approximately translationally symmetric ablation volumes, while the penetrating electrodes configuration was much more sensitive to fiber twist.ConclusionsThese results suggest that the epi-endo configuration will be reliable for ablation of atrial fibrillation, independently of fiber orientation, while the penetrating electrode configuration may experience problems when the fiber orientation is not consistent across the atrial wall.     

Population Growth of Pratylenchus penetrans on Winter Cover Crops Grown in the Pacific Northwest

Population growth of Pratylenchus penetrans on 13 fall and winter cover crops was studied in the greenhouse and field. All crops except oat cv. Saia supported population growth of P. penetrans in greenhouse experiments, although the response of P. penetrans to oat cv. Saia varied considerably between experiments. The mean ratio of the final population density/initial population density (Pf/Pi) after 16 weeks for P. penetrans added to a greenhouse soil mix was 0.09, whereas Pf/Pi values after 10 weeks for two experiments with naturally infested soil were 0.95 and 2.3. Although P. penetrans increased on sudangrass cv. Trudan 8 and sudangrass × sorghum hybrid cv. SS 222, subsequent incorporation of sudangrass vegetation into soil reduced P. penetrans populations to preplant levels. Field experiments were inconclusive but suggested that oat cv. Saia or rye cv. Wheeler may be better choices for winter cover than weed-contaminated fallow or other crops on P. penetrans-infested sites in the Pacific Northwest.     

Restoration of pool margin communities in cutover peatlands

Two experiments were conducted for developing restoration techniques for pool margin communities in cutover peatlands. We first aimed to measure the regeneration potential of a typical edge pool liverwort, Cladopodiella fluitans (Nees) H. Buch. We introduced C. fluitans in floating baskets in a restored peatland. We tested three fragment sizes (patches of 2 cm², stretched patches and shredded fine fragments of 0.1-1 mm), two introduction densities (ratio between surface of collected areas and surface of restored areas of 1:5 and 1:10) as well as the effect of a straw mulch. After two years, the percentage covers of C. fluitans were five times larger in experimental units protected with straw than in those without protection. Yet, the fragment sizes and the densities tested had no effect on the regeneration of the liverwort. The second experiment aimed to test a moss layer transfer approach to restore plant diversity around pool margins. We tested four communities, dominated by (1) Sphagnum cuspidatum Hoffman, (2) Sphagnum fallax (H. Klinggraff) H. Klinggraff, (3) Sphagnum papillosum Linberg as well as (4) a mixed community composed of equal quantities of C. fluitans, S. cuspidatum and S. papillosum. We introduced plant material in two density ratios (1:5 and 1:10). Sphagnum mosses did colonize pool margins, and showed even more than 60% cover for some treatments after three growing seasons, but the recovery of the introduced vascular plants remained below 5% for most species. The establishment of pool vascular species thus seems to be more intricate than for bryophytes and specific introduction techniques might be needed.     

Phenotypic Plasticity in Response to the Social Environment: Effects of Density and Sex Ratio on Mating Behaviour Following Ecotype Divergence

The ability to express phenotypically plastic responses to environmental cues might be adaptive in changing environments. We studied phenotypic plasticity in mating behaviour as a response to population density and adult sex ratio in a freshwater isopod (Asellus aquaticus). A. aquaticus has recently diverged into two distinct ecotypes, inhabiting different lake habitats (reed Phragmites australis and stonewort Chara tomentosa, respectively). In field surveys, we found that these habitats differ markedly in isopod population densities and adult sex ratios. These spatially and temporally demographic differences are likely to affect mating behaviour. We performed behavioural experiments using animals from both the ancestral ecotype (“reed” isopods) and from the novel ecotype (“stonewort” isopods) population. We found that neither ecotype adjusted their behaviour in response to population density. However, the reed ecotype had a higher intrinsic mating propensity across densities. In contrast to the effects of density, we found ecotype differences in plasticity in response to sex ratio. The stonewort ecotype show pronounced phenotypic plasticity in mating propensity to adult sex ratio, whereas the reed ecotype showed a more canalised behaviour with respect to this demographic factor. We suggest that the lower overall mating propensity and the phenotypic plasticity in response to sex ratio have evolved in the novel stonewort ecotype following invasion of the novel habitat. Plasticity in mating behaviour may in turn have effects on the direction and intensity of sexual selection in the stonewort habitat, which may fuel further ecotype divergence.     

Rapid Establishment of a Regular Distribution of Adult Tropical Drosophila Parasitoids in a Multi-Patch Environment by Patch Defence Behaviour

Females of the larval parasitoid of Drosophila, Asobara citri, from sub-Saharan Africa, defend patches with hosts by fighting and chasing conspecific females upon encounter. Females of the closely related, palearctic species Asobara tabida do not defend patches and often search simultaneously in the same patch. The effect of patch defence by A. citri females on their distribution in a multi-patch environment was investigated, and their distributions were compared with those of A. tabida. For both species 20 females were released from two release-points in replicate experiments. Females of A. citri quickly reached a regular distribution across 16 patches, with a small variance/mean ratio per patch. Conversely, A. tabida females initially showed a clumped distribution, and after gradual dispersion, a more Poisson-like distribution across patches resulted (variance/mean ratio was closer to 1 and higher than for A. citri). The dispersion of A. tabida was most probably an effect of exploitation: these parasitoids increasingly made shorter visits to already exploited patches. We briefly discuss hypotheses on the adaptive significance of patch defence behaviour or its absence in the light of differences in the natural history of both parasitoid species, notably the spatial distribution of their hosts.     

Taxonomic revision of the Pyrgulopsis gilae (Caenogastropoda,Hydrobiidae) species complex,with descriptions of two new species from the Gila River basin,New Mexico

We describe two new species of springsnails (genus Pyrgulopsis) for populations from the middle Fork and upper East Fork of the Gila River Basin (New Mexico) that had been previously identified as P. gilae. We also restrict P. gilae to its originally circumscribed geographic range which consists of a short reach of the East Fork Gila River and a single spring along the Gila River (below the East Fork confluence). These three species form genetically distinct lineages that differ from each other by 3.9–6.3% for mtCOI and 3.7–8.7% for mtNDI (the latter data were newly obtained for this study), and are diagnosable by shell and penial characters. Collectively the three species form a strongly supported clade that is distinguished from other congeners by the unique presence of two glandular strips on the dorsal surface of the penial filament. These findings suggest that the conservation status of P. gilae, which was recently removed from the list of candidates for listing as endangered or threatened by the United States Fish and Wildlife Service, should be revisited and that the two new species may also merit protective measures given their narrow geographic ranges.