Wolf population genetics in Europe: a systematic review,meta‐analysis and suggestions for conservation and management

The grey wolf (Canis lupus) is an iconic large carnivore that has increasingly been recognized as an apex predator with intrinsic value and a keystone species. However, wolves have also long represented a primary source of human–carnivore conflict, which has led to long‐term persecution of wolves, resulting in a significant decrease in their numbers, genetic diversity and gene flow between populations. For more effective protection and management of wolf populations in Europe, robust scientific evidence is crucial. This review serves as an analytical summary of the main findings from wolf population genetic studies in Europe, covering major studies from the ‘pre‐genomic era’ and the first insights of the ‘genomics era’. We analyse, summarize and discuss findings derived from analyses of three compartments of the mammalian genome with different inheritance modes: maternal (mitochondrial DNA), paternal (Y chromosome) and biparental [autosomal microsatellites and single nucleotide polymorphisms (SNPs)]. To describe large‐scale trends and patterns of genetic variation in European wolf populations, we conducted a meta‐analysis based on the results of previous microsatellite studies and also included new data, covering all 19 European countries for which wolf genetic information is available: Norway, Sweden, Finland, Estonia, Latvia, Lithuania, Poland, Czech Republic, Slovakia, Germany, Belarus, Russia, Italy, Croatia, Bulgaria, Bosnia and Herzegovina, Greece, Spain and Portugal. We compared different indices of genetic diversity in wolf populations and found a significant spatial trend in heterozygosity across Europe from south‐west (lowest genetic diversity) to north‐east (highest). The range of spatial autocorrelation calculated on the basis of three characteristics of genetic diversity was 650?850 km, suggesting that the genetic diversity of a given wolf population can be influenced by populations up to 850 km away. As an important outcome of this synthesis, we discuss the most pressing issues threatening wolf populations in Europe, highlight important gaps in current knowledge, suggest solutions to overcome these limitations, and provide recommendations for science‐based wolf conservation and management at regional and Europe‐wide scales.     

Global ecological impacts of invasive species in aquatic ecosystems

The introduction of invasive species, which often differ functionally from the components of the recipient community, generates ecological impacts that propagate along the food web. This review aims to determine how consistent the impacts of aquatic invasions are across taxa and habitats. To that end, we present a global meta‐analysis from 151 publications (733 cases), covering a wide range of invaders (primary producers, filter collectors, omnivores and predators), resident aquatic community components (macrophytes, phytoplankton, zooplankton, benthic invertebrates and fish) and habitats (rivers, lakes and estuaries). Our synthesis suggests a strong negative influence of invasive species on the abundance of aquatic communities, particularly macrophytes, zooplankton and fish. In contrast, there was no general evidence for a decrease in species diversity in invaded habitats, suggesting a time lag between rapid abundance changes and local extinctions. Invaded habitats showed increased water turbidity, nitrogen and organic matter concentration, which are related to the capacity of invaders to transform habitats and increase eutrophication. The expansion of invasive macrophytes caused the largest decrease in fish abundance, the filtering activity of filter collectors depleted planktonic communities, omnivores (including both facultative and obligate herbivores) were responsible for the greatest decline in macrophyte abundance, and benthic invertebrates were most negatively affected by the introduction of new predators. These impacts were relatively consistent across habitats and experimental approaches. Based on our results, we propose a framework of positive and negative links between invasive species at four trophic positions and the five different components of recipient communities. This framework incorporates both direct biotic interactions (predation, competition, grazing) and indirect changes to the water physicochemical conditions mediated by invaders (habitat alteration). Considering the strong trophic links that characterize aquatic ecosystems, this framework is relevant to anticipate the far‐reaching consequences of biological invasions on the structure and functionality of aquatic ecosystems.     

A framework for exhaustively mapping functional missense variants

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin‐like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.     

Pendrin overexpression affects cell volume recovery, intracellular pH and chloride concentration after hypotonicity-induced cell swelling

The pendrin (SLC26A4 or PDS) gene is responsible, when mutated, for the Pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid dysfunctions. Pendrin protein is an anion exchanger and we focused on a still unexplored function that it might play in view of its importance in the inner ear: Cl(-) fluxes regulation during cellular volume control. We challenged HEK-293 Phoenix cells over-expressing wild type pendrin (PDS HEK cells) together with the EYFP (Enhanced Yellow Fluorescent Protein) or over-expressing the EYFP alone (control HEK cells) with hypo-osmolar solutions. Taking advantage of the confocal optical sectioning we measured the cell volume. In addition, we determined the intracellular pH and chloride concentration with fluorescent probes (EYFP and seminaphthorhodafluor-5F, SNARF-5F). Consequently, we could estimate simultaneously Cl(-) fluxes, cellular volume and intracellular pH variations. Cl(-) movements markedly differed between PDS and control HEK cells upon hypotonic shock and are accompanied by an attenuation of the swelling induced pH drop in PDS HEK cells. The contemporary measurements of the three variables not yet reported in living cells, allowed to assess a possible influence of pendrin upregulation in volume homeostasis and evidenced its participation to Cl(-) fluxes.     

Multiscale modeling of the cardiovascular system: application to the study of pulmonary and coronary perfusions in the univentricular circulation

The objective of this study is to compare the coronary and pulmonary blood flow dynamics resulting from two configurations of systemic-to-pulmonary artery shunts currently utilized during the Norwood procedure: the central (CS) and modified Blalock Taussig (MBTS) shunts. A lumped parameter model of the neonatal cardiovascular circulation and detailed 3-D models of the shunt based on the finite volume method were constructed. Shunt sizes of 3, 3.5 and 4 mm were considered. A multiscale approach was adopted to prescribe appropriate and realistic boundary conditions for the 3-D models of the Norwood circulation. Results showed that the average shunt flow rate is higher for the CS option than for the MBTS and that pulmonary flow increases with shunt size for both options. Cardiac output is higher for the CS option for all shunt sizes. Flow distribution between the left and the right pulmonary arteries is not completely balanced, although for the CS option the discrepancy is low (50-51% of the pulmonary flow to the right lung) while for the MBTS it is more pronounced with larger shunt sizes (51-54% to the left lung). The CS option favors perfusion to the right lung while the MBTS favors the left. In the CS option, a smaller percentage of aortic flow is distributed to the coronary circulation, while that percentage rises for the MBTS. These findings may have important implications for coronary blood flow and ventricular function.     

JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs

We have examined wound healing during regeneration of Drosophila wing imaginal discs fragments by confocal microscopy and assessed the role of components of the JNK pathway in this process. After cutting, columnar and peripodial epithelia cells at the wound edge start to close the wound through formation and contraction of an actin cable. This is followed by a zipping process through filopodial protrusions from both epithelia knitting the wound edges from proximal to distal areas of the disc. Activation of the JNK pathway is involved in such process. puckered (puc) expression is induced in several rows of cells at the edge of the wound, whereas absence of JNK pathway activity brought about by hemipterous, basket, and Dfos mutants impair wound healing. These defects are accompanied by lowered or loss of expression of puc. In support of a role of puc in wound healing, hep mutant phenotypes are rescued by reducing puc function, whereas overexpression of puc inhibits wound healing. Altogether, these results demonstrate a role for the JNK pathway in imaginal disc wound healing, similar to that reported for other healing processes such as embryonic dorsal closure, thoracic closure, and adult epithelial wound healing in Drosophila. Differences with such processes are also highlighted.     

Circadian rhythms in mouse blood coagulation

The circadian clock, influencing many biological processes, has been demonstrated to modulate levels of specific coagulation factors, but its impact on the coagulation efficiency is unknown. In a mouse model, the authors evaluated the temporal variations in the initial rate of activated factor X (FXa) and thrombin generation. Upon coagulation activation through the FVIIa-TF pathway (extrinsic activation), both parameters showed rhythmic variations with a significant peak at ZT 12, the light-to-dark transition. In mice subjected to a 6-h delayed light-dark cycle, the peak was shifted as expected. These cyclic oscillations were also observed in constant darkness, thus demonstrating, for the first time, the existence of strong circadian rhythms of the initial rate of either FXa or thrombin generation activity levels. These circadian variations overlapped with those that have been recently described in factor VII (FVII) activity. The peak of FXa generation activity was simulated by the addition of purified human FVII, thus indicating that circadian variations in FVII activity are important determinants of the circadian rhythm of the procoagulant cascade efficiency. These findings help to elucidate the complex control on the coagulation process and might contribute in explaining the temporal variations in the frequency of cardiovascular events observed in humans.     

Role of polyols in thermal inactivation of shark ornithine transcarbamoylase

The ability of activity modulators of ornithine transcarbamoylase (OCT) from the liver of the thresher shark Alopias vulpinus to stabilize the enzyme against thermal denaturation was investigated in the tri-buffer at pH 7.8, at temperatures ranging from 60 to 70 (o)C, in the presence of polyhydroxylic molecules such as glycerol and sugars. The study indicated that in the presence of 0.5 M sugars and 1.6 M glycerol in the preincubation medium the OCT activity increases. When trehalose is introduced directly in the reaction mixture in a range of concentration of 0.25-0.5 M, the activity is lower than that with maltose, glycerol and buffer alone. Kinetic data for carbamoyl phosphate and ornithine with and without maltose and glycerol are similar, whereas trehalose increases the kinetic values. Arrhenius plots show an increase of activation energy due to trehalose, whereas values obtained with maltose and glycerol are similar to the control.     

Production of fuculose-1-phosphate aldolase using operator-repressor titration for plasmid maintenance in high cell density Escherichia coli fermentations

We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.