Prediction and Experimental Characterization of nsSNPs Altering Human PDZ-Binding Motifs

Single nucleotide polymorphisms (SNPs) are a major contributor to genetic and phenotypic variation within populations. Non-synonymous SNPs (nsSNPs) modify the sequence of proteins and can affect their folding or binding properties. Experimental analysis of all nsSNPs is currently unfeasible and therefore computational predictions of the molecular effect of nsSNPs are helpful to guide experimental investigations. While some nsSNPs can be accurately characterized, for instance if they fall into strongly conserved or well annotated regions, the molecular consequences of many others are more challenging to predict. In particular, nsSNPs affecting less structured, and often less conserved regions, are difficult to characterize. Binding sites that mediate protein-protein or other protein interactions are an important class of functional sites on proteins and can be used to help interpret nsSNPs. Binding sites targeted by the PDZ modular peptide recognition domain have recently been characterized. Here we use this data to show that it is possible to computationally identify nsSNPs in PDZ binding motifs that modify or prevent binding to the proteins containing the motifs. We confirm these predictions by experimentally validating a selected subset with ELISA. Our work also highlights the importance of better characterizing linear motifs in proteins as many of these can be affected by genetic variations.     

Vibrio Type III Effector VPA1380 Is Related to the Cysteine Protease Domain of Large Bacterial Toxins

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2), but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator.     

Extreme Genetic Fragility of the HIV-1 Capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies.     

Methane indicator values for peatlands: a comparison of species and functional groups

Previous studies have shown a correspondence between the abundance of particular plant species and methane flux. Here, we apply multivariate analyses, and weighted averaging, to assess the suitability of vegetation composition as a predictor of methane flux. We developed a functional classification of the vegetation, in terms of a number of plant traits expected to influence methane production and transport, and compared this with a purely taxonomic classification at species level and higher. We applied weighted averaging and indirect and direct ordination approaches to six sites in the United Kingdom, and found good relationships between methane flux and vegetation composition (classified both taxonomically and functionally). Plant species and functional groups also showed meaningful responses to management and experimental treatments. In addition to the United Kingdom, we applied the functional group classification across different geographical regions (Canada and the Netherlands) to assess the generality of the method. Again, the relationship appeared good at the site level, suggesting some general applicability of the functional classification. The method seems to have the potential for incorporation into large‐scale (national) greenhouse gas accounting programmes (in relation to peatland condition/management) using vegetation mapping schemes. The results presented here strongly suggest that robust predictive models can be derived using plant species data (for use in national‐scale studies). For trans‐national‐scale studies, where the taxonomic assemblage of vegetation differs widely between study sites, a functional classification of plant species data provides an appropriate basis for predictive models of methane flux.     

Linking the Trophic Fingerprint of Groundfishes to Ecosystem Structure and Function in the California Current

Mean trophic level (MTL) is one of the most widely used indicators of marine ecosystem health. It usually represents the relative abundance of fished species across a spectrum of TLs. The reality, ubiquity, and causes of a general decline in the MTL of fisheries catch through time, and whether fisheries catch tracks ecosystem level changes, have engendered much attention. However, the consequences of such patterns for broader ecosystem structure and function remain virtually unexplored. Along the Pacific U.S. Coast, previous work has documented fluctuations and a slow increase in ecosystem MTL from 1977 to 2004. Here, we document a decline in the ecosystem MTL of groundfishes in the same ecosystem from 2003 to 2011, the proximate cause of which was a decrease in the biomass of higher TL groundfishes. Using a food web model, we illustrate how these shifts in ecosystem structure may have resulted in short term, positive responses by many lower TL species in the broader ecosystem. In the longer term, the model predicts that initial patterns of prey release may be tempered in part by lagged responses of other higher TL species, such as salmon and seabirds. Although ecosystem functions related to specific groups like piscivores (excluding high-TL groundfishes) changed, aggregate ecosystem functions altered little following the initial reorganization of biomass, probably due to functional redundancy within the predator guild. Efforts to manage and conserve marine ecosystems will benefit from a fuller consideration of the information content contained within, and implied by, fisheries-independent TL indicators.     

Patch and soil characteristics of pastures with water‐spreading banks and woody encroachment in semi‐arid Australia

Water‐spreading banks are used in semi‐arid areas such as the Cobar pediplain in western New South Wales, Australia to encourage pasture growth, often after removal of woody encroachment. We studied the arrangement of bare inter‐patches and vegetated patches, and associated surface soil variables, in three pastures following installation of water‐spreading banks (2, 15, 38 years ago) and in an area of woody encroachment near Cobar. The aims of the study were as follows: (i) to determine the number and percent area of inter‐patches and vegetated patches, and associated surface soil variables at the three pasture sites and at the woody encroachment site and (ii) by inference, explore effects of establishing water‐spreading banks and pasture following removal of woody encroachment on these factors, to understand the role of water‐spreading banks as a management tool. The percent area of inter‐patches in pasture with 38‐year‐old water‐spreading banks was much lower, and the percent area of medium‐vegetated patches (but not of well‐vegetated patches) was substantially higher, than in the woody encroachment. Differences in soil carbon and nitrogen between the sites were related to their percent areas of inter‐patches and vegetated patches. The results suggest that the mosaic of bare inter‐patches and vegetated patches changes over time after clearing of woody encroachment and establishment of pasture with water‐spreading banks, from many large inter‐patches to a few small inter‐patches, and from small to large medium‐vegetated patches. Water‐spreading banks are a useful management tool in these landscapes because of their benefits for landscape function, that is, bare areas become less connected, the percent area of moderately vegetated patches increases, and soil carbon builds up with time following their installation.     

Tradescantia fluminensis,an exotic weed affecting native forest regeneration in New Zealand: Ecological surveys,safety tests and releases of four biocontrol agents from Brazil

In the native range of Tradescantia fluminensis in SE Brazil surveys revealed a natural enemy biota attacking the plant that was rich in potential biocontrol agents for New Zealand (NZ), including nine fungi and 10 herbivorous insect species. Similar surveys in NZ, where T. fluminensis is an invasive exotic weed, revealed no specialist insect herbivores or pathogens. The Brazilian insect herbivores included leafmining, stemboring and gall-forming feeding guilds that were absent in NZ. Mean foliar damage levels per site on T. fluminensis were 7.8× higher for folivores in Brazil cf. NZ, and 21.2× higher for pathogens. The presence of rust pustules, or ‘brown lesions’, on leaves in Brazil was negatively associated with damage by folivores, perhaps indicating an antagonistic interaction. In contrast, damage by the white smut fungus, Kordyana sp., was not negatively associated with folivore damage. Mean dry biomass of T. fluminensis was significantly lower in Brazil (164 g m^?2) cf. NZ (455 g m^?2). In NZ, 85% of sites had biomass measures >200 g m^?2 (the previously determined threshold above which native forest regeneration fails). In Brazil, only 27% of sites had biomass measures >200 g m^?2. Among the insect herbivores, three chrysomelid beetles, Neolema ogloblini, Neolema abbreviata and Lema basicostata were prioritised as potential biocontrol agents. Their larvae cause potentially complementary damage to leaves, shoot-tips and mature stems, respectively. Several pathogens, including a rust, were rejected before we selected the Kordyana species. Host range testing of all four agents showed sufficient host-specificity for consideration for release in NZ. Neolema ogloblini and L. basicostata were field-released in NZ in 2011 and 2012, with the field-release of N. abbreviata due late 2012. An application to release Kordyana sp. in NZ has been made.     

The Holes of Moles: Osteological Correlates of the Trigeminal Nerve in Talpidae

Talpidae consists of small insectivorous mammals exhibiting a range of environmental preferences. As all its members rely on a highly developed somatosensation system, they are an ideal study-group for investigating osteological correlates of the trigeminal nerve. We quantitatively studied cranial anatomy in 22 species of desman, shrew-mole, and true mole using microscopy and micro-CT imaging to investigate whether the infraorbital foramina within Talpidae is enlarged in semiaquatic forms and more broadly associated with habitat preference. We also investigated whether associated foramina were covariant. In order to account for a phylogenetic basis for any correlations, we reconstructed the best taxonomically sampled phylogeny for talpids in the literature to date, based on cytochrome b. Relationships among genera and species are broadly congruent with previous analyses; however, we report new placements for Neurotrichus gibbsii and Mogera tokudae. Although no correlation was found between habitat and the caliber of the V₃ associated mandibular canal and foramen ovale, our results indicate that semiaquatic forms show larger infraorbital foramina in comparison to terrestrial species, and that the caliber of the sphenorbital fissure can also serve as a proxy for habitat preference. This work, therefore, supports the use of certain osteological features to infer habitat preferences in fossil species and indicates this can be achieved even when studying ecologically diverse, closely related species within the same family.