Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification

The blood–brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma‐targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high‐throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d‐ amino acids, this approach screens only protease‐resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state‐of‐the‐art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix‐and‐split technique to generate a library based on the following: Ac‐d‐ Arg‐XXXXX‐NH₂, where X were: d‐ Ala (a), d‐ Arg (r), d‐ Ile (i), d‐ Glu (e), d‐ Ser (s), d‐ Trp (w) or d‐ Pro (p). The assays used comprised the in vitro cell‐based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two‐step mass spectrometry approach combining LTQ‐Orbitrap and Q‐trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease‐resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Protective role of ubiquinone in vitamin E and selenium-deficient plasma membranes.

We have studied the effects of dietary depletion of vitamin E and selenium on endogenous ubiquinone-dependent antioxidant system. Deficiency induced an increase in both coenzyme Q9 and Q10 in liver tissue, reaching a maximum between 4 and 7 weeks of deficient diet consumption. Cytochrome b5 reductase polypeptide was also enriched in membranes after 5 weeks of deficient diet consumption. Substantial DT-diaphorase activity was found in deficient, but not in control plasma membranes. Deficient membranes were very sensitive to lipid peroxidation, although a great protection was observed after incubation with NAD(P)H. Our results show that liver cells can boost endogenous ubiquinone-dependent protective mechanisms in response to deficiency in vitamin E and selenium.     

Recent Male-Mediated Gene Flow over a Linguistic Barrier in Iberia, Suggested by Analysis of a Y-Chromosomal DNA Polymorphism

We have examined the worldwide distribution of a Y-chromosomal base-substitution polymorphism, the T/C transition at SRY-2627, where the T allele defines haplogroup 22; sequencing of primate homologues shows that the ancestral state cannot be determined unambiguously but is probably the C allele. Of 1,191 human Y chromosomes analyzed, 33 belong to haplogroup 22. Twenty-nine come from Iberia, and the highest frequencies are in Basques (11%; n=117) and Catalans (22%; n=32). Microsatellite and minisatellite (MSY1) diversity analysis shows that non-Iberian haplogroup-22 chromosomes are not significantly different from Iberian ones. The simplest interpretation of these data is that haplogroup 22 arose in Iberia and that non-Iberian cases reflect Iberian emigrants. Several different methods were used to date the origin of the polymorphism: microsatellite data gave ages of 1,650, 2,700, 3,100, or 3,450 years, and MSY1 gave ages of 1,000, 2,300, or 2,650 years, although 95% confidence intervals on all of these figures are wide. The age of the split between Basque and Catalan haplogroup-22 chromosomes was calculated as only 20% of the age of the lineage as a whole. This study thus provides evidence for direct or indirect gene flow over the substantial linguistic barrier between the Indo-European and non-Indo-European-speaking populations of the Catalans and the Basques, during the past few thousand years.     

Birds and people: from conflict to coexistence

Negative interactions between humans and animals are becoming increasingly frequent, as wild habitats shrink and human presence and activities expand throughout the world. Conflicts between people over conservation are one of the outcomes of this increased interaction, with severe consequences for both wildlife and people. Globally, conflicts can arise across diverse ecosystems, species and circumstances. Even if most attention in wildlife-related conflicts has been on mammals, birds are also often at the centre of such conflicts, but conflict research is still not explicitly present in ornithological literature. Examples of such conflicts include those related to birds and agriculture, forestry, hunting, fishing and public health interests. Conflicts are often more complex than initial assessments might suggest, involving ecological, economic, cultural, social and political elements. Reflecting the complexity of these issues and their increasing relevance to bird conservation, a British Ornithologists' Union conference was organized in November 2021 that aimed to highlight examples of conflicts that exist between people over birds and their conservation. Building on this conference, we provide here a review of key themes relating to the understanding of conflicts, including the importance of conflict perceptions, the collaboration between multiple disciplines and the different types of knowledge needed to better understand conflicts. We then consider the management of bird conservation conflicts, including the key issues of dealing with uncertainty, the role of technical solutions and the importance of collaboration and building trust, illustrating each theme with real-world examples. Finally, we outline potential future conflicts around bird conservation and how best to address them proactively.     

Regional and temporal variation in diet and provisioning rates suggest weather limits prey availability for an endangered raptor

Understanding variation in food requirements of wild animals is of central importance in population ecology and conservation, as it helps to identify where and when food may be limiting. Studies on diet variation or prey provisioning rates may give useful insights when direct information on prey availability is lacking. We assess spatial and temporal variation in the diet of an endangered predator, the Black Harrier Circus maurus. This raptor is endemic to southern Africa and specializes on small mammals but also feeds on birds and reptiles as alternative prey. Using data on 1679 prey identified in 953 pellets collected in inland and coastal regions from 2006 to 2015, we show that diet composition changed little throughout the breeding season in the coastal region, whereas there was a marked seasonal decline in the occurrence of small mammal prey in the inland region, with a concomitant increase in alternative prey. The proportion of small mammals in the diet declined with increasing maximum temperature, the latter being highest at the inland region late in the breeding season. Using camera recordings at nests in 2014, we further analysed daily patterns of prey provisioning to nestlings. A marked reduction in small mammal provisioning rates occurred during the middle of the day in the hotter inland region but not in the cooler coastal region. Reduced availability of the primary prey, small mammals, in hotter conditions, through a reduction in activity or overall abundance, could explain these patterns. Finally, we show a positive relationship between winter rainfall and interannual differences in the proportion of small mammals in the diet of Black Harriers breeding in the coastal region, suggesting relationships between diet and prey abundance that are mediated through rainfall. We discuss the need to consider spatial variation in food availability in conservation strategies.     

Intraspecific and interspecific competition induces density‐dependent habitat niche shifts in an endangered steppe bird

Interspecific competition is a dominant force in animal communities that induces niche shifts in ecological and evolutionary time. If competition occurs, niche expansion can be expected when the competitor disappears because resources previously inaccessible due to competitive constraints can then be exploited (i.e., ecological release). Here, we aimed to determine the potential effects of interspecific competition between the little bustard (Tetrax tetrax) and the great bustard (Otis tarda) using a multidimensional niche approach with habitat distribution data. We explored whether the degree of niche overlap between the species was a density‐dependent function of interspecific competition. We then looked for evidences of ecological release by comparing measures of niche breadth and position of the little bustard between allopatric and sympatric situations. Furthermore, we evaluated whether niche shifts could depend not only on the presence of great bustard but also on the density of little and great bustards. The habitat niches of these bustard species partially overlapped when co‐occurring, but we found no relationship between degree of overlap and great bustard density. In the presence of the competitor, little bustard's niche was displaced toward increased use of the species' primary habitat. Little bustard's niche breadth decreased proportionally with great bustard density in sympatric sites, in consistence with theory. Overall, our results suggest that density‐dependent variation in little bustard's niche is the outcome of interspecific competition with the great bustard. The use of computational tools like kernel density estimators to obtain multidimensional niches should bring novel insights on how species' ecological niches behave under the effects of interspecific competition in ecological communities.     

Regional differences in land use affect population performance of the threatened insectivorous plant Drosophyllum lusitanicum (Droseraceae)

Abstract. We analyse populations of the insectivorous plant Drosophyllum lusitanicum (L.) Link (Droseraceae) and their habitats throughout the species' distribution range (Portugal, Spain and Morocco), and examine their relations. We have surveyed 32 populations and we analyse their demographic structures, accompanying plant communities (by twinspan analyses), and relationships between population features and several edaphic and nonedaphic environmental variables (by multiple regression analyses). Plant communities, their floristic richness and degree of endemism change across the geographical range of Drosophyllum . Populations vary strongly in size, density and age structure. Their recruitment seems largely determined by competition with the surrounding vegetation, while only weak relationships were detected between population performance and other environmental factors. The demographic structure of populations exhibits a marked geographical differentiation, with populations becoming older and sparser towards the north-western part of the range. The largest and presumably most stable populations are mostly found in southern Spanish heathlands, which are nowadays scarcely affected by man. Moroccan populations suffer from browsing by cattle and experience an accelerated generation turnover, while most Portuguese populations occur in afforested areas and experience strongly reduced recruitment. The historical distribution range of Drosophyllum within its peculiar habitat has probably been relatively stable, but recent regional differences in human land use have resulted in present-day differences in population performance, as well as in types and degrees of threat across the three countries involved. Conservation strategies should take into account this range-wide variation and combine approaches on different spatial scales.