Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation,proteolytic activity,and cell surface localization

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.     

Integrative taxonomy provides evidence for the species status of the Ibero‐Maghrebian grass snake Natrix astreptophora

The grass snake (Natrix natrix) is Europe's most widely distributed and, in many regions, most common snake species, with many morphologically defined subspecies. Yet, the taxonomy of grass snakes is relatively little studied and recent work has shown major conflicts between morphologically defined subspecies and phylogeographical differentiation. Using external morphology, osteological characters, and information from 13 microsatellite loci and two mitochondrial markers, we examine differentiation of the subspecies N. n. astreptophora from the North African Maghreb region, the Iberian Peninsula and neighbouring France. According to previous studies, N. n. astreptophora corresponds to a deeply divergent mitochondrial clade and constitutes the sister taxon of all remaining grass snakes. In the French Pyrenees region, there is a contact zone of N. n. astreptophora with another subspecies, N. n. helvetica. Our analyses of microsatellites and mitochondrial DNA reveal that the distribution ranges of the two taxa abut there, but both hybridize only exceptionally. Even though many morphological characters are highly variable and homoplastic in grass snakes, N. n. astreptophora differs consistently from all other grass snakes by its reddish iris coloration and in having significantly fewer ventral scales and another skull morphology. Considering further the virtual absence of gene flow between N. n. astreptophora and N. n. helvetica, and acknowledging the morphological distinctiveness of N. n. astreptophora and its sister group relationship to all remaining subspecies of grass snakes, we conclude that Natrix astreptophora (Seoane, 1884) should be recognized as a distinct species. Further research is needed to explore whether N. astreptophora is polytypic because a single sample of N. astreptophora from Tunisia turned out to be genetically highly distinct from its European conspecifics.     

The Association Between Personality Traits,Morphological Traits and Alternative Mating Behaviour in Male Endler's Guppies,Poecilia wingei

Alternative mating behaviour, personality traits and morphological characters are predicted to be correlated. Bolder, larger and more colourful males are expected to preferentially court females, while shy, small and drab‐coloured individuals are predicted to sneak copulations. We used males of Endler's guppy, Poecilia wingei, to test this association over a long temporal period (hence including ontogenetic changes) and under two social environments (male‐biased and female‐biased). We found that personality traits (exploration, boldness, activity) of P. wingei males were highly repeatable across long time spans, but they were not correlated (formed no behavioural syndrome). Male age and social environment had no effect on any personality trait, despite their effects on alternative mating behaviour. Young males with higher activity levels were more likely to attempt sneaking. In older fish, there was an association between orange coloration, courtship and boldness, but this was not observed in young males. Our results suggest that alternative mating behaviour is more flexible than personality traits and is independent of them. Non‐colour‐based morphological traits (gonopodium length, body length, caudal straps length, dorsal fin length) were not correlated with any particular mating behaviour.     

Designing and Implementing an Assay for the Detection of Rare and Divergent NRPS and PKS Clones in European,Antarctic and Cuban Soils

The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovered.     

Nest Predation Deviates from Nest Predator Abundance in an Ecologically Trapped Bird

In human-modified environments, ecological traps may result from a preference for low-quality habitat where survival or reproductive success is lower than in high-quality habitat. It has often been shown that low reproductive success for birds in preferred habitat types was due to higher nest predator abundance. However, between-habitat differences in nest predation may only weakly correlate with differences in nest predator abundance. An ecological trap is at work in a farmland bird (Lanius collurio) that recently expanded its breeding habitat into open areas in plantation forests. This passerine bird shows a strong preference for forest habitat, but it has a higher nest success in farmland. We tested whether higher abundance of nest predators in the preferred habitat or, alternatively, a decoupling of nest predator abundance and nest predation explained this observed pattern of maladaptive habitat selection. More than 90% of brood failures were attributed to nest predation. Nest predator abundance was more than 50% higher in farmland, but nest predation was 17% higher in forest. Differences between nest predation on actual shrike nests and on artificial nests suggested that parent shrikes may facilitate nest disclosure for predators in forest more than they do in farmland. The level of caution by parent shrikes when visiting their nest during a simulated nest predator intrusion was the same in the two habitats, but nest concealment was considerably lower in forest, which contributes to explaining the higher nest predation in this habitat. We conclude that a decoupling of nest predator abundance and nest predation may create ecological traps in human-modified environments.     

Quantifying energetic and fitness consequences of seasonal heterothermy in an Arctic ungulate

Animals have adapted behavioral and physiological strategies to conserve energy during periods of adverse conditions. Heterothermy is one such adaptation used by endotherms. While heterothermy—fluctuations in body temperature and metabolic rate—has been shown in large vertebrates, little is known of the costs and benefits of this strategy, both in terms of energy and in terms of fitness. Hence, our objective was to model the energetics of seasonal heterothermy in the largest Arctic ungulate, the muskox (Ovibos moschatus), using an individual‐based energy budget model of metabolic physiology. We found that the empirically based drop in body temperature (winter max ~−0.8°C) overwinter in adult females resulted in substantial fitness benefits in terms of reduced daily energy expenditure and body mass loss. Body mass and energy reserves were 8.98% and 14.46% greater in modeled heterotherms compared to normotherms by end of winter. Based on environmental simulations, we show that seasonal heterothermy can, to some extent, buffer the negative consequences of poor prewinter body condition or reduced winter food accessibility, leading to greater winter survival (+20%–30%) and spring energy reserves (+10%–30%), and thus increased probability of future reproductive success. These results indicate substantial adaptive short‐term benefits of seasonal heterothermy at the individual level, with potential implications for long‐term population dynamics in highly seasonal environments.     

Retinal Glia Promote Dorsal Root Ganglion Axon Regeneration

Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.     

The Claudin Megatrachea Protein Complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.     

Distinct Contributions of the Dorsolateral Prefrontal and Orbitofrontal Cortex during Emotion Regulation

The lateral prefrontal and orbitofrontal cortices have both been implicated in emotion regulation, but their distinct roles in regulation of negative emotion remain poorly understood. To address this issue we enrolled 58 participants in an fMRI study in which participants were instructed to reappraise both negative and neutral stimuli. This design allowed us to separately study activations reflecting cognitive processes associated with reappraisal in general and activations specifically related to reappraisal of negative emotion. Our results confirmed that both the dorsolateral prefrontal cortex (DLPFC) and the lateral orbitofrontal cortex (OFC) contribute to emotion regulation through reappraisal. However, activity in the DLPFC was related to reappraisal independently of whether negative or neutral stimuli were reappraised, whereas the lateral OFC was uniquely related to reappraisal of negative stimuli. We suggest that relative to the lateral OFC, the DLPFC serves a more general role in emotion regulation, perhaps by reflecting the cognitive demand that is inherent to the regulation task.     

A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry

We present the demonstration of a rapid “middle-up” liquid chromatography mass spectrometry (LC–MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody–drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison—a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug–antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.