Real-time PCR genotyping and frequency of the myostatin F94L mutation in beef cattle breeds

This research developed two real-time PCR assays, employing high-resolution melt and allele-specific analysis to accurately genotype the F94L mutation in cattle. This mutation (g.433C > A) in the growth differentiation factor 8 or myostatin gene has recently been shown to be functionally associated with increased muscle mass and carcass yield in cattle. The F94L mutation is not, like other myostatin mutations, associated with reduced fertility and dystocia. It is therefore a candidate for introgression into other breeds to improve retail beef yield and the development of a simple and accurate test to genotype this specific mutation is warranted. Variations in the efficiency of enzyme cleavage compromised the accuracy of genotyping by published methods, potentially resulting in an overestimation of the frequency of the mutant allele. The frequency of the F94L mutation was determined by real-time PCR in 1140 animals from 15 breeds of cattle in Australia. The mutation was present in Simmental (0.8%), Piedmontese (2%), Droughtmaster (4%) and Limousin (94.2%) but not found in Salers, Angus, Poll Hereford, Hereford, Gelbvieh, Charolais, Jersey, Brahman, Holstein, Shorthorn or Maine Anjou. The low prevalence of F94L in all beef breeds except Limousin indicates the significant potential for this mutation to improve retail yield in Australian beef cattle.     

Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells

We report phosphorylated and ubiquitinated aggregates of TAR DNA binding protein of 43 kDa (TDP-43) in SH-SY5Y cells similar to those in brains of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). Two candidate sequences for the nuclear localization signal were examined. Deletion of residues 78-84 resulted in cytoplasmic localization of TDP-43, whereas the mutant lacking residues 187-192 localized in nuclei, forming unique dot-like structures. Proteasome inhibition caused these to assemble into phosphorylated and ubiquitinated TDP-43 aggregates. The deletion mutants lacked the exon skipping activity of cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Our results suggest that intracellular localization of TDP-43 and proteasomal function may be involved in inclusion formation and neurodegeneration in TDP-43 proteinopathies.     

Engineered Cystine-Knot Peptides that Bind αvβ3 Integrin with Antibody-Like Affinities

The α_vβ₃ integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to α_vβ₃ integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized α_vβ₃ integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing α_vβ₃ integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to α_vβ₃ integrins and had only minimal or no binding to α_vβ₅, α₅β₁, and α_iibβ₃ integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for α_vβ₃ and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against α_vβ₃ integrins resulted in peptides that retained high affinities for α_vβ₃ and had increased specificities for α_vβ₃ over α_iibβ₃ integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.     

Crystal structure of peptidoglycan recognition protein SA in Apis mellifera (Hymenoptera: Apidae)

Peptidoglycan recognition protein SA (PGRP‐SA) is a key pattern recognition receptor in the insect innate immune system. PGRP‐SA can bind to bacterial PGN and activate the Toll pathway, which triggers the expression and release of antimicrobial peptides to prevent bacterial infection. Here, we report the first structure of Apis mellifera PGRP‐SA from Hymenoptera at 1.86 Å resolution. The overall architecture of Am‐PGRP‐SA was similar to the Drosophila PGRP‐SA; however, the residues involved in PGN binding groove were not conserved, and the binding pocket was narrower. This structure gives insight into PGN binding characteristics in honeybees.     

Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild‐type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight‐binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30‐fold higher affinity of Ubp8^C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.     

Harder,better, faster,stronger: Weapon size is more sexually dimorphic than weapon biomechanical components in two freshwater anomuran species

Sexual selection influences the evolution of morphological traits that increase the likelihood of monopolizing scarce resources. When such traits are used during contests, they are termed weapons. Given that resources are typically linked to monopolizing mating partners, theory expects only males to bear weapons. In some species, however, females also bear weapons, although typically smaller than male weapons. Understanding why females bear smaller weapons can thus help us understand the selective pressures behind weapon evolution. However, most of our knowledge comes from studies on weapon size, while the biomechanics of weapons, such as the size of the muscles, efficiency, and shape are seldom studied. Our goal was to test if the theoretical expectations for weapon size sexual dimorphism also occur for weapon biomechanics using two aeglid crab species. Males of both species had larger claws which were also stronger than female claws. Male claws were also more efficient than females' claws (although we used only one species in this analysis). For weapon shape, though, only one species differed in the mean claw shape. Regarding scaling differences, in both species, male claws had higher size scaling than females, while only one species had a higher shape scaling. However, male weapons did not have higher scaling regarding strength and efficiency than females. Thus, males apparently allocate more resources in weapons than females, but once allocated, muscle and efficiency follow a similar developmental pathway in both sexes. Taken together, our results show that sexual dimorphism in weapons involves more than differences in size. Shape differences are especially intriguing because we cannot fully understand its causes. Yet, we highlight that such subtle differences can only be detected by measuring and analysing weapon shape and biomechanical components. Only then we might better understand how weapons are forged.     

Evolution under pH stress and high population densities leads to increased density-dependent fitness in the protist Tetrahymena thermophila

Abiotic stress is a major force of selection that organisms are constantly facing. While the evolutionary effects of various stressors have been broadly studied, it is only more recently that the relevance of interactions between evolution and underlying ecological conditions, that is, eco-evolutionary feedbacks, have been highlighted. Here, we experimentally investigated how populations adapt to pH-stress under high population densities. Using the protist species Tetrahymena thermophila, we studied how four different genotypes evolved in response to stressfully low pH conditions and high population densities. We found that genotypes underwent evolutionary changes, some shifting up and others shifting down their intrinsic rates of increase (r₀). Overall, evolution at low pH led to the convergence of r₀ and intraspecific competitive ability (α) across the four genotypes. Given the strong correlation between r₀ and α, we argue that this convergence was a consequence of selection for increased density-dependent fitness at low pH under the experienced high density conditions. Increased density-dependent fitness was either attained through increase in r₀, or decrease of α, depending on the genetic background. In conclusion, we show that demography can influence the direction of evolution under abiotic stress.     

A chalcid wasp acts chiefly as a hyperparasitoid by mostly using small uncommon hosts

Although ovipositing insects may predominantly use resources that lead to high offspring quality, exceptions to this rule have considerably aided understanding of oviposition decisions. We report the frequency of host species use by a solitary facultative hyperparasitoid, Brachymeria subrugosa Blanchard (Hymenoptera: Chalcididae). In our samples, the wasp attacks the large pupae of the moth Gonioterma indecora Zeller (Lepidoptera: Elachistidae), as well as the considerably smaller, and rarer, pupae of two of its other parasitoids. Consistent with conditional sex allocation models, the wasp produced mainly female offspring on the largest (moth) host, an unbiased sex ratio on the middle‐sized (parasitoid) host, and only males on the smallest (parasitoid) host. Adult offspring size was correlated with the size of the host attacked. These features strongly suggest that the two smaller, primary parasitoid, hosts produce lower‐quality offspring. Despite being more common, the proportion of hosts from which parasitoids emerged was lowest (14%) on the largest host species, and highest on the rarer middle‐sized (34%) and smallest (30%) hosts. This suggests that costs or constraints on attacking high‐quality primary hosts may be a selective force favouring the evolution of hyperparasitism.     

The strength of assortative mating for flowering date and its basis in individual variation in flowering schedule

Although it has been widely asserted that plants mate assortatively by flowering time, there is virtually no published information on the strength or causes of phenological assortment in natural populations. When strong, assortative mating can accelerate the evolution of plant reproductive phenology through its inflationary effect on genetic variance. We estimated potential assortative mating for flowering date in 31 old‐field species in Ontario, Canada. For each species, we constructed a matrix of pairwise mating probabilities from the individual flowering schedules, that is the number of flower deployed on successive dates. The matrix was used to estimate the phenotypic correlation between mates, ρ, for flowering date. We also developed a measure of flowering synchrony within species, S, based upon the eigenstructure of the mating matrix. The mean correlation between pollen recipients and potential donors for flowering date was  = 0.31 (range: 0.05–0.63). A strong potential for assortative mating was found among species with high variance in flowering date, flowering schedules of short duration and skew towards early flower deployment. Flowering synchrony, S, was negatively correlated with potential assortment (r = ?0.49), but we go on to show that although low synchrony is a necessary condition for phenological assortative mating, it may not be sufficient to induce assortment for a given phenological trait. The potential correlation between mates showed no seasonal trend; thus, as climate change imposes selection on phenology through longer growing seasons, spring‐flowering species are no more likely to experience an accelerated evolutionary response than summer species.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.