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According to the Trivers-Willard hypothesis, females of polygynous mammal species can adaptively modify offspring sex ratio, which would provide them evolutionary benefits in terms of a higher number of grand-offspring. In red deer ( Cervus elaphus), a species where the reproductive success of males is significantly more variable than that of females, mothers that are in good condition are expected to have a male-biased offspring sex ratio but only in a population below carrying capacity. We aimed at testing the Trivers-Willard hypothesis on a low-density population of red deer in large woodlands of north-eastern Poland. The overall foetal sex ratio did not differ from parity. The mother's probability of having male offspring increased with her body mass independently of age and population density. Such condition-based adjustment of the offspring sex ratio in female red deer from a low-density population conforms to the Trivers-Willard hypothesis. However, full support of Trivers-Willard predictions would require further investigations comparing survival and reproductive output of male and female offspring. [ABSTRACT FROM AUTHOR]

Purpose: Habitat selection in animals is a hierarchal process that operates across multiple temporal and spatial scales, adapting to changes in environmental conditions, human disturbances, and predation risks. Despite its significance, previous research often oversimplifies temporal dynamics by categorizing them into broad seasonal and diel patterns, overlooking the continuous nature of temporal variability and habitat specificity., Methods: We investigated the temporal patterns in habitat selection of moose (Alces alces) in highly heterogenous landscapes at the southwestern edge of their European range using step-selection functions. Utilizing over 700,000 GPS locations from 34 adult moose, we aimed to assess seasonal and diel patterns in their selectivity for both natural and human-related habitats., Results: Our findings revealed significant overall temporal variation in moose habitat selection at both seasonal and diel scales. Moose selectivity toward different habitats showed low repeatability over time, with 35% of cases displaying negative correlation between selectivity in different time windows. Diel changes were more pronounced, showing 5.6-fold difference in cumulative selectivity, compared to 1.4-fold difference in seasonal dynamics. Notably, moose exhibited lower selectivity during nighttime hours throughout the year compared to daytime hours. The study also highlighted distinct habitat selection patterns across different habitat types: natural habitats (deciduous forests, coniferous forests, wetlands) exhibited pronounced seasonal variation, while anthropogenic habitats (grasslands, arable land, roads and settlements) showed more diel variability. Moose generally avoided human-related habitats during daytime hours, but their preferences during nighttime varied depending on the habitat type and time of year., Conclusion: This research advances our understanding of the complex temporal patterns in habitat selection by large herbivores and underscores the importance of considering temporal dynamics in habitat selection modelling., (© 2024. The Author(s).)

Increasing agriculture and pesticide use have led to declines in insect populations and biodiversity worldwide. In addition to insect diversity, it is also important to consider insect abundance, due to the importance of insects as food for species at higher trophic levels such as bats. We monitored spatiotemporal variation in abundance of nocturnal flying insects over meadows, a common open landscape structure in central Europe, and correlated it with bat feeding activity. Our most important result was that insect abundance was almost always extremely low. This was true regardless of management intensity of the different meadows monitored. We also found no correlation of insect abundance or the presence of insect swarms with bat feeding activity. This suggests that insect abundance over meadows was too low and insect swarms too rare for bats to risk expending energy to search for them. Meadows appeared to be poor habitat for nocturnal flying insects, and of low value as a foraging habitat for bats. Our study highlights the importance of long-term monitoring of insect abundance, especially at high temporal scales to identify and protect foraging habitats. This will become increasingly important given the rapid decline of insects., (© 2024. The Author(s).)

Introduction: African swine fever (ASF) is a notifiable disease of swine that impacts global pork trade and food security. In several countries across the globe, the disease persists in wild boar (WB) populations sympatric to domestic pig (DP) operations, with continued detections in both sectors. While there is evidence of spillover and spillback between the sectors, the frequency of occurrence and relative importance of different risk factors for transmission at the wildlife-livestock interface remain unclear., Methods: To address this gap, we leveraged ASF surveillance data from WB and DP across Eastern Poland from 2014-2019 in an analysis that quantified the relative importance of different risk factors for explaining variation in each of the ASF surveillance data from WB and DP., Results: ASF prevalence exhibited different seasonal trends across the sectors: apparent prevalence was much higher in summer (84% of detections) in DP, but more consistent throughout the year in WB (highest in winter with 45%, lowest in summer at 15%). Only 21.8% of DP-positive surveillance data included surveillance in WB nearby (within 5 km of the grid cell within the last 4 weeks), while 41.9% of WB-positive surveillance samples included any DP surveillance samples nearby. Thus, the surveillance design afforded twice as much opportunity to find DP-positive samples in the recent vicinity of WB-positive samples compared to the opposite, yet the rate of positive WB samples in the recent vicinity of a positive DP sample was 48 times as likely than the rate of positive DP samples in the recent vicinity of a positive WB sample. Our machine learning analyses found that positive samples in WB were predicted by WB-related risk factors, but not to DP-related risk factors. In contrast, WB risk factors were important for predicting detections in DP on a few spatial and temporal scales of data aggregation., Discussion: Our results highlight that spillover from WB to DP might be more frequent than the reverse, but that the structure of current surveillance systems challenge quantification of spillover frequency and risk factors. Our results emphasize the importance of, and provide guidance for, improving cross-sector surveillance designs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pepin, Borowik, Frant, Plis and Podgórski.)

The importance of social and spatial structuring of wildlife populations for disease spread, though widely recognized, is still poorly understood in many host-pathogen systems. In particular, system-specific kin relationships among hosts can create contact heterogeneities and differential disease transmission rates. Here, we investigate how distance-dependent infection risk is influenced by genetic relatedness in a novel host-pathogen system: wild boar (Sus scrofa) and African swine fever (ASF). We hypothesized that infection risk would correlate positively with proximity and relatedness to ASF-infected individuals but expected those relationships to weaken with the distance between individuals due to decay in contact rates and genetic similarity. We genotyped 323 wild boar samples (243 ASF-negative and 80 ASF-positive) collected in north-eastern Poland in 2014-2016 and modelled the effects of geographic distance, genetic relatedness and ASF virus transmission mode (direct or carcass-based) on the probability of ASF infection. Infection risk was positively associated with spatial proximity and genetic relatedness to infected individuals with generally stronger effect of distance. In the high-contact zone (0-2 km), infection risk was shaped by the presence of infected individuals rather than by relatedness to them. In the medium-contact zone (2-5 km), infection risk decreased but was still associated with relatedness and paired infections were more frequent among relatives. At farther distances, infection risk further declined with relatedness and proximity to positive individuals, and was 60% lower among un-related individuals in the no-contact zone (33% in10-20 km) compared among relatives in the high-contact zone (93% in 0-2 km). Transmission mode influenced the relationship between proximity or relatedness and infection risk. Our results indicate that the presence of nearby infected individuals is most important for shaping ASF infection rates through carcass-based transmission, while relatedness plays an important role in shaping transmission rates between live animals., (© 2021 Wiley-VCH GmbH.)

To provide the most comprehensive picture of species phylogeny and phylogeography of European roe deer ( Capreolus capreolus ), we analyzed mtDNA control region (610 bp) of 1469 samples of roe deer from Central and Eastern Europe and included into the analyses additional 1541 mtDNA sequences from GenBank from other regions of the continent. We detected two mtDNA lineages of the species: European and Siberian (an introgression of C .  pygargus mtDNA into C .  capreolus ). The Siberian lineage was most frequent in the eastern part of the continent and declined toward Central Europe. The European lineage contained three clades (Central, Eastern, and Western) composed of several haplogroups, many of which were separated in space. The Western clade appeared to have a discontinuous range from Portugal to Russia. Most of the haplogroups in the Central and the Eastern clades were under expansion during the Weichselian glacial period before the Last Glacial Maximum (LGM), while the expansion time of the Western clade overlapped with the Eemian interglacial. The high genetic diversity of extant roe deer is the result of their survival during the LGM probably in a large, contiguous range spanning from the Iberian Peninsula to the Caucasus Mts and in two northern refugia., Competing Interests: The authors declare that they have no conflict of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The fitting of tracking devices to wild animals requires capture and handling which causes stress and can potentially cause injury, behavioural modifications that can affect animal welfare and the output of research. We evaluated post capture and release ranging behaviour responses of roe deer ( Capreolus capreolus ) for five different capture methods. We analysed the distance from the centre of gravity and between successive locations, using data from 14 different study sites within the EURODEER collaborative project. Independently of the capture method, we observed a shorter distance between successive locations and contextual shift away from the home range centre of gravity after the capture and release event. However, individuals converged towards the average behaviour within a relatively short space of time (between 10 days and one month). If researchers investigate questions based on the distance between successive locations of the home range, we recommend (1) initial investigation to establish when the animals start to behave normally again or (2) not using the first two to three weeks of data for their analysis. We also encourage researchers to continually adapt methods to minimize stress and prioritize animal welfare wherever possible, according to the Refinement of the Three R's.

Stable isotope analyses of bone collagen are often used in palaeoecological studies to reveal environmental conditions in the habitats of different herbivore species. However, such studies require valuable reference data, obtained from analyses of modern individuals, in habitats of well-known conditions. In this article, we present the stable carbon and nitrogen isotope composition of bone collagen from modern red deer (N = 242 individuals) dwelling in various habitats (N = 15 study sites) in Europe. We investigated which of the selected climatic and environmental factors affected the δ13C and δ15N values in bone collagen of the studied specimens. Among all analyzed factors, the percent of forest cover influenced the carbon isotopic composition most significantly, and decreasing forest cover caused an increase in δ13C values. The δ15N was positively related to the proportion of open area and (only in the coastal areas) negatively related to the distance to the seashore. Using rigorous statistical methods and a large number of samples, we confirmed that δ13C and δ15N values can be used as a proxy of past habitats of red deer., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Both imagery rescripting and imaginal exposure have been proven to be effective in the treatment of chronic nightmares when compared to a waitlist condition. Little is known about their comparative efficacy and their efficacy compared to an active control., Objective: The aims of this study were to compare the two treatments to one another and to positive imagery as an active control, and to explore covariates of the treatment effect., Methods: In this single-blinded randomized controlled trial, 96 patients with nightmare disorder (idiopathic nightmares) from an outpatient clinic were randomly assigned to a single individual treatment session of rescripting, exposure, or positive imagery and 4 weeks of practice at home. The primary outcome was nightmare distress, and the secondary outcomes were nightmare frequency, nightmare effects, self-efficacy, and general psychopathology., Results: Nightmare distress was reduced in all groups (imagery rescripting: Cohen's d = -1.04, imaginal exposure: d = -0.68, positive imagery: d = -0.57), as were nightmare frequency, nightmare effects, and psychopathology. Self-efficacy was enhanced. No differential treatment effects were found on any primary or secondary measure. Treatment gains were not associated with demographic or disorder characteristics, baseline values, treatment credibility, or the number of practice sessions., Conclusions: Even short nightmare treatments are effective regardless of personal characteristics, and different interventions produce similar results. Future research should aim to clarify the mechanisms of action. Health care should make more use of these powerful and easy-to-administer nightmare treatments., (© 2021 S. Karger AG, Basel.)

Background: Spirometra erinaceieuropaei is a diphylobothriid tapeworm with a complex life-cycle including definitive, intermediate and paratenic (transport) hosts. Multiple routes of parasite transmission often make it impossible to determine what type of host a specific infected animal is considered to be. Spargana larvae cause sparganosis, a severe food- and water-borne disease mainly found in Asia. In Poland, Spirometra sp. was reported in large carnivores in Białowieża Primeval Forest for the first time in the 1940s and was recently confirmed as S. erinaceieuropaei in several mammals and snakes using molecular methods., Methods: In total, 583 carcasses of 9 carnivore species were necropsied between 2013 and 2019 in north-eastern (NE) Poland. The larvae of S. erinaceieuropaei (spargana) were isolated from subcutaneous tissue, counted, and preserved for genetic analyses. We calculated the prevalence and intensity of infection. To assess spatial variation in S. erinaceieuropaei infection probability in NE Poland, we applied a generalized additive model (GAM) with binomial error distribution. To confirm the species affiliation of isolated larvae, we amplified a partial fragment of the 18S rRNA gene (240 bp in length)., Results: Spirometra larvae were found in the subcutaneous tissue of 172 animals of 7 species and confirmed genetically as S. erinaceieuropaei. The overall prevalence in all studied hosts was 29.5% with a mean infection intensity of 14.1 ± 33.8 larvae per individual. Native European badgers and invasive raccoon dogs were characterized by the highest prevalence. An analysis of parasite spread showed a spatially diversified probability of infection with the highest values occurring in the biodiversity hot spot, Białowieża Primeval Forest., Conclusions: Our study revealed that various mammal species (both native and non-native) can serve as S. erinaceieuropaei reservoirs. The frequency and level of infection may differ between selected hosts and likely depend on host diversity and habitat structure in a given area. Further studies are needed to assess the distribution of the parasite throughout Europe and the environmental and biological factors influencing infection severity in wild mammals.

The fatty acid-based microparticles containing iron oxide nanoparticles and paclitaxel (PAX) are a viable proposition for the treatment of lung cancer. The microparticles inhaled as a dry powder can be guided to selected locations using an external magnetic field, and when accumulated there, the active compound release can be triggered by local hyperthermia. However, this general strategy requires that the active compound is released from microparticles and can reach the targeted cells before microparticles are removed. Isothermal titration calorimetry was used to demonstrate that the components of microparticles were released and transferred to albumins and lipid bilayers. The morphology of the measured particulates was studied with scanning electron microscopy and dynamic light scattering. To determine the cytotoxicity of microparticles, cell culture studies were done. It has been shown that the transfer efficiency depends predominantly on the fatty acid composition of microparticles, which, together with the active ingredient, accumulate predominantly in membrane structures after being released from microparticles and before entering the cytoplasm. The release process is sufficient; hence, paclitaxel-loaded microparticles effectively suppressed the proliferation of A549 human lung epithelial cells of malignant origin (IC 50 values for both lauric acid-based and myristic/palmitic-based microparticles containing paclitaxel were below 0.375 μg/mL), while reference microparticles were noncytotoxic.

Vitamin C is the exogenous compound necessary for a variety of metabolic processes; therefore, the efficient delivery is critical for the maintenance of body homeostasis. Vitamin C pharmacokinetics and low quantities in processed foodstuff, necessitates its continuous supplementation. In the paper, we present the new liposomal formulation of vitamin C free of harmful organic solvents. The formulation was quantitatively characterized with respect to its chemically composition and nano-structuring. The vitamin C accessibility to cells from the formulation was evaluated using evidence derived from experiments performed on cell cultures. Finally, the enhanced bioavailability of vitamin C from the formulation was demonstrated in the medical experiment.

Despite recent advancements in medicine, lung cancer still lacks an effective therapy. In the present study we have decided to combine superparamagnetic iron oxide nanoparticles (SPION) with solid lipid microparticles to develop novel, stimuli-sensitive drug carriers that increase the bioavailability of the anticancer drug (paclitaxel - PAX) through guided accumulation directly at the tumour site and controlled drug delivery. SPION and PAX-loaded microparticles (MPs) were fabricated from lauric acid (LAU) and a mixture of myristic and palmitic acids (MYR/PAL) using hot oil-in-water emulsification method. MP size, surface properties, melting temperature and magnetic mobility were evaluated along with their in vitro efficacy against malignant lung epithelial cells (A549). MPs were spherical in shape with the average particle size between 2 and 3.5 μm and responded to external magnetic field up to the distance of 15 mm. MPs were effectively internalised by the cells. Unloaded or NP-loaded MPs were cytocompatible with A549 cells, while NP + PAX-loaded MPs significantly decreased cell viability and effectively suppressed colony formation. The developed stimuli-sensitive, inhalable MPs have shown promising results as PAX carriers for controlled pulmonary delivery for the treatment of lung cancer., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Host abundance and landscape structure often interact to shape spatial patterns of many wildlife diseases. Emergence, spread, and persistence of African swine fever (ASF) among wild boar in eastern Europe has raised questions on the factors underlying ASF dynamics in this novel host-pathogen system. This work identifies drivers of ASF occurrence in natural wild boar population. We evaluated factors shaping the probability of ASF-postitive wild boar during the first three years (2014-2016) of the ASF epidemic in Poland. We expected to observe positive effects of wild boar density, proportion of forested area, human activity, and proximity to previous infections on ASF case probability. We tested these predictions using the infection status of 830 wild boar samples and generalized mixed-effects models. The probability of ASF case increased from 3 to 20% as population density rose from 0.4 to 2 ind./km 2 . The positive effect of population density on ASF case probability was stronger at locations near previous ASF incidents. ASF was more likely to occur in forested areas, with the probability of detecting an ASF positive sample rising from 2 to 11% as forest cover around the sample increased from 0.5 to 100%. This pattern was consistent at both low and high wild boar densities. Indicators of human activity were poor predictors of ASF occurrence. Disease control efforts, such as culling and carcass search, should be focused on high-density populations where chances of detecting and eliminating ASF-positive wild boar are higher. The intensity of control measures should decrease with distance from the infected area to match the observed spatial pattern of ASF case probability. Woodlands represent areas of the highest risk of ASF case occurrence. Distribution and connectivity of suitable habitats over the landscape can be used to prioritize disease-management actions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Spatio-temporal variation in resource availability leads to a variety of animal movement strategies. In the case of ungulates, temporally unpredictable landscapes are associated with nomadism, while high predictability in the resource distribution favours migratory or sedentary behaviours depending on the spatial and temporal scale of landscape dynamics. As most of the surveys on moose (Alces alces) movement behaviours in Europe have been conducted on Scandinavian populations, little is known about the movement strategies of moose at the southern edge of the species' range. We expected that decreasing habitat patchiness in central Europe would be associated with the prevalence of migratory behaviours. To verify this hypothesis, we analysed 32 moose fitted with GPS collars from two study areas in eastern Poland which differed in a level of habitat patchiness. We classified moose movements using the net squared displacement method. As presumed, lower patchiness in the Biebrza study site was associated with the predominance of individuals migrating short-distance, while in more patchy landscape of Polesie, resident moose dominated. At the individual level, the propensity of moose to migrate decreased with increasing abundance of forest habitats in their summer ranges. In addition, the parameters (migration distance, timing and duration) for migratory individuals varied substantially between individuals and years. Yet, in spring individual moose expressed a consistent migration timing across years. There was little synchronization of migration timing between individuals from the same population both in spring and autumn, which may have been related to mild weather conditions. We observed that moose postponed their migrations and started movement toward summer ranges at a similar time window in years when spring was delayed due to harsh weather. Hence, in light of global warming, we presume further changes in animal movements will arise., Competing Interests: The authors have declared that no competing interests exist.

We analysed a fragment (247 bp) of cytochrome b of mitochondrial DNA sequenced using 353 samples of yellow-necked mice Apodemus flavicollis trapped in seven forests and along three woodlot transects in north-eastern Poland. Our aims were to identify the phylogeographic pattern and mtDNA structure of the population and to evaluate the role of environmental conditions in shaping the spatial pattern of mtDNA diversity. We found out that three European haplogroups occurred sympatrically in north-eastern Poland. Inferences based on mtDNA haplotype distribution and frequency defined five subpopulations. The mtDNA-based structure of mice significantly correlated with winter temperature: frequency of Haplogroup 1 was positively, and that of Haplogroup 3 negatively correlated to mean temperature of January in the year of trapping. Synthesis of the published pan-European data on the species phylogeography also showed that the possibly 'thermophilous' Haplogroup 1 has the westernmost occurrence, whereas the more 'cold-resistant' Haplogroup 3 occurs much further to north-east than the other haplogroups. The observed patter may be a byproduct of the tight coevolution with nuclear genes, as we have earlier found that - in mice population in NE Poland - the spatial pattern of nuclear DNA was best explained by January temperature. Alternatively, the observed association of mitochondrial genetic variation with temperature is possible to be adaptive as cytochrome b is involved in the process of ATP production via oxidative phosphorylation., Competing Interests: The authors have declared that no competing interests exist.

Background: Deviation in blood collection procedures is a central source of preanalytical variation affecting overall analytical and diagnostic precision. The procedure of venous blood collection for ionized calcium is hypothesized to affect analytical results. Here, we evaluate the effect of blood collection with and without a discard tube, and storage duration on results of P-Ionized Calcium (pH adjusted = 7.4)., Methods: We collected 100 paired venous blood tubes from randomly selected outpatients using a winged blood collection. No discard tube was drawn before the first tube. The samples were divided in five subsamples, stored at 4°-6 °C at 24 (n = 20), 48 (n = 20), 72 (n = 20), 96 (n = 20) and 120 h (n = 20) after venipuncture, and analyzed for P-Ionized Calcium (pH adjusted = 7.4) on Konelab 60i (Thermo Scientific, Finland). Differences between first and second tubes were evaluated for all samples (n = 100) and for subsamples divided by storage duration, using Bland-Altman plot and Wilcoxon's rank-sum test., Results: P-Ionized Calcium (pH adjusted = 7.4) results ranged from 1.13 to 1.37 mmol/L. We observed no statistical significant differences between the first and the second tube when comparing all samples. Dividing samples by storage duration, a statistically significant difference was found (p = .0068) after 120 h, but the difference of individual samples was not clinically relevant., Conclusions: Our study has shown no significant difference between P-Ionized Calcium (pH adjusted = 7.4) values for the first and second tubes. Hence, the use of a discard tube is not required. A statistically significant difference was found on samples stored 120 h but was not considered clinically relevant., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Large mammals are often a source of conflict, and consequently there has been increasing interest in close encounters with them. Knowledge of wildlife responses to human disturbance is crucial for the management of increasing and expanding populations of large animals. We investigated flight initiation distance (FID) and aggressive responses of the European bison-the largest terrestrial mammal of Europe-to human disturbance in the Białowieża Forest (NE Poland). When encountered by humans, bison usually flee. Aggression was observed in only 0.4% of approach attempts. Mean FID was 77±46 m and was influenced by habitat, sex, and supplementary feeding intensity. Females showed greater timidity than males, FID was lower in forest than in open habitats, and supplementary feeding caused a drop in FID. In 84.5% of all documented aggression cases, bison attacks were provoked by humans approaching too close to the bison or by deliberate scaring them. Males were more aggressive than females, and attacked mainly during the rut, while females attacked during the winter and calving. Bison attacked in built-up areas significantly more often than expected. The mean critical distance of attacks was 21±2 m. Most attacks took the form of a short chase preceded by warning signs. Goring was observed in 22.7% of all aggression cases and no fatalities were recorded. Our study shows that bison are not dangerous animals and only manifest aggression in response to prolonged disturbance at close ranges. The education of people and recommendations for minimum approach distances should ensure a low rate of disturbance and safety when encountering large mammals., Competing Interests: The authors have declared that no competing interests exist.

The goal of this study, conducted in seven large woodlands and three areas with small woodlots in northeastern Poland in 2004-2008, was to infer genetic structure in yellow-necked mouse Apodemus flavicollis population and to evaluate the roles of environmental and population ecology variables in shaping the spatial pattern of genetic variation using 768 samples genotyped at 13 microsatellite loci. Genetic variation was very high in all studied regions. The primal genetic subdivision was observed between the northern and the southern parts of the study area, which harbored two major clusters and the intermediate area of highly admixed individuals. The probability of assignment of individual mice to the northern cluster increased significantly with lower temperatures of January and July and declined in regions with higher proportion of deciduous and mixed forests. Despite the detected structure, genetic differentiation among regions was very low. Fine-scale structure was shaped by the population density, whereas higher level structure was mainly shaped by geographic distance. Genetic similarity indices were highly influenced by mouse abundance (which positively correlated with the share of deciduous forests in the studied regions) and exhibited the greatest change between 0 and 1 km in the forests, 0 and 5 km in small woodlots. Isolation by distance pattern, calculated among regions, was highly significant but such relationship between genetic and geographic distance was much weaker, and held the linearity at very fine scale (~1.5 km), when analyses were conducted at individual level.

We investigated how do environmental and climatic factors, but also management, affect the carbon (δ13C) and nitrogen (δ15N) stable isotope composition in bone collagen of the two largest contemporary herbivores: European bison (Bison bonasus) and moose (Alces alces) across Europe. We also analysed how different scenarios of population recovery- reintroduction in bison and natural recovery in moose influenced feeding habitats and diet of these two species and compared isotopic signatures of modern populations of bison and moose (living in human-altered landscapes) with those occurring in early Holocene. We found that δ13C of modern bison and moose decreased with increasing forest cover. Decreasing forest cover, increasing mean annual temperature and feeding on farm crops caused an increase in δ15N in bison, while no factor significantly affected δ15N in moose. We showed significant differences in δ13C and δ15N among modern bison populations, in contrast to moose populations. Variation in both isotopes in bison resulted from inter-population differences, while in moose it was mainly an effect of intra-population variation. Almost all modern bison populations differed in δ13C and δ15N from early Holocene bison. Such differences were not observed in moose. It indicates refugee status of European bison. Our results yielded evidence that habitat structure, management and a different history of population recovery have a strong influence on foraging behaviour of large herbivores reflected in stable isotope signatures. Influence of forest structure on carbon isotope signatures of studied herbivores supports the "canopy effect" hypothesis.

If protected areas are to remain relevant in our dynamic world they must be adapted to changes in species ranges. In the EU one of the most notable such changes is the recent recovery of large carnivores, which are protected by Natura 2000 at the national and population levels. However, the Natura 2000 network was designed prior to their recent recovery, which raises the question whether the network is sufficient to protect the contemporary ranges of large carnivores. To investigate this question we evaluated Natura 2000 coverage of the three wolf Canis lupus populations in Poland. Wolf tracking data showed that wolves have recolonised almost all suitable habitat in Poland (as determined by a recent habitat suitability model), so we calculated the overlap between the Natura 2000 network and all wolf habitat in Poland. On the basis of published Natura 2000 criteria, we used 20% as the minimum required coverage. At the national level, wolves are sufficiently protected (22% coverage), but at the population level, the Baltic and Carpathian populations are far better protected (28 and 47%, respectively) than the endangered Central European Lowland population (12%). As Natura 2000 insufficiently protects the most endangered wolf population in Poland, we recommend expansion of Natura 2000 to protect at least an additional 8% of wolf habitat in western Poland, and discuss which specific forests are most in need of additional coverage. Implementation of these actions will have positive conservation implications and help Poland to fulfil its Habitats Directive obligations. As it is likely that similar gaps in Natura 2000 are arising in other EU member states experiencing large carnivore recoveries, particularly in Central Europe, we make the case for a flexible approach to Natura 2000 and suggest that such coverage evaluations may be beneficial elsewhere.

Organotin compounds, being biologically active, affect a variety of cellular functions due to their ability to accumulate in and penetrate biological membranes. These compounds influence the distribution of electrostatic charges, alter organization, disrupt molecular dynamics and change mechanical properties of biological membranes. It was found that the membrane/water partition coefficient equals 4, a value significantly higher than octanol/water partition coefficient. In addition, the effect of di- and tri-phenyltin chlorides on the mechanics of model lipid membranes was measured for the first time. It has been determined that phenyltins affect the global model lipid bilayer properties by reducing the membrane expansion modulus, when measured using micromanipulation technique, and elevating the bending rigidity coefficient of the lipid bilayer, as determined with the flickering noise spectroscopy. In addition, the elevated water permeability shows that phenyltins also cause the local defects formation in the lipid bilayer, i.e. lipid pores. These data shows that phenyltins may interfere indirectly with variety cellular processes by altering non-specifically the entire cellular membrane system. Accordingly, when phenyltins are added to macrophages in culture, they inflict massive alterations of cell morphology and interfere with membrane-associated processes, as visualized using fluorescence labelling of selected subcellular compartments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The full course of new parasite introductions in wild animals is difficult to accurately trace. We documented and analysed the invasive blood-sucking nematode Ashworthius sidemi (Trichostrongylidae) introduction and spread in European bison ( Bison bonasus ) from the initial phase of its progression. In the Polish part of the Białowieża Primeval Forest (BPF) the parasite was first found in 2000. From 2002 to 2015, 165 culled bison were investigated. The prevalence and intensity of A. sidemi Schulz, 1933 infection increased over the following years, reaching 100% of investigated bison four years after introduction and a maximal median intensity of 8200 nematodes per animal in the winter of 2008/2009. Afterwards, a significant decline of median infection intensity was observed to the minimum value of 410 nematodes per animal. Between 2011 and 2014 prevalence varied from 89 to 100%. Among the factors analysed, the number of years since introduction, herd size, age and sex proved to significantly influence infection intensity. A higher infection intensity was recorded in sub-adults compared to juveniles and adults. Males had significantly lower infection intensity than females, but this was the case for adults only. The highest infection intensities were recorded in the biggest bison herds, where the winter supplementary feeding of bison is intense. Moreover, the longer the parasite was present in the host population, the more important herd size became as a factor. Our study indicates that it is not solely biological factors that determine the spread of a newly detected parasite in wildlife, but that management practices can also have a strong influence. This is especially important in endangered species under intensive human care as the management practices may pose a threat to the species.

The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Carniolan honey bees (Apis mellifera carnica) are considered as an indigenous subspecies in Hungary adapted to most of the ecological and climatic conditions in this area. However, during the last decades Hungarian beekeepers have recognized morphological signs of the Italian honey bee (Apis mellifera ligustica). As the natural distribution of the honey bee subspecies can be affected by the importation of honey bee queens or by natural gene flow, we aimed at determining the genetic structure and characteristics of the local honey bee population using molecular markers. All together, 48 Hungarian and 84 foreign (Italian, Polish, Spanish, Liberian) pupae and/or workers were used for mitochondrial DNA analysis. Additionally, 53 sequences corresponding to 10 subspecies and the Buckfast hybrid were downloaded from GenBank. For the nuclear analysis, 236 Hungarian and 106 foreign honey bees were genotyped using nine microsatellites. Heterozygosity values, population-specific alleles, FST values, principal coordinate analysis, assignment tests, structure analysis, and dendrograms were calculated. Haplotype and nucleotide diversity values showed moderate values. We found that one haplotype (H9) was dominant in Hungary. The presence of the black honey bee (Apis mellifera mellifera) was negligible, but a few individuals resembling other subspecies were identified. We proved that the Hungarian honey bee population is nearly homogeneous but also demonstrated introgression from the foreign subspecies. Both mitochondrial DNA and microsatellite analyses corroborated the observations of the beekeepers. Molecular analyses suggested that Carniolan honey bee in Hungary is slightly affected by Italian and black honey bee introgression. Genetic differences were detected between Polish and Hungarian Carniolan honey bee populations, suggesting the existence of at least two different gene pools within A. m. carnica.

Ecological and environmental heterogeneity can produce genetic differentiation in highly mobile species. Accordingly, local adaptation may be expected across comparatively short distances in the presence of marked environmental gradients. Within the European continent, wolves (Canis lupus) exhibit distinct north-south population differentiation. We investigated more than 67-K single nucleotide polymorphism (SNP) loci for signatures of local adaptation in 59 unrelated wolves from four previously identified population clusters (northcentral Europe n = 32, Carpathian Mountains n = 7, Dinaric-Balkan n = 9, Ukrainian Steppe n = 11). Our analyses combined identification of outlier loci with findings from genome-wide association study of individual genomic profiles and 12 environmental variables. We identified 353 candidate SNP loci. We examined the SNP position and neighboring megabase (1 Mb, one million bases) regions in the dog (C. lupus familiaris) genome for genes potentially under selection, including homologue genes in other vertebrates. These regions included functional genes for, for example, temperature regulation that may indicate local adaptation and genes controlling for functions universally important for wolves, including olfaction, hearing, vision, and cognitive functions. We also observed strong outliers not associated with any of the investigated variables, which could suggest selective pressures associated with other unmeasured environmental variables and/or demographic factors. These patterns are further supported by the examination of spatial distributions of the SNPs associated with universally important traits, which typically show marked differences in allele frequencies among population clusters. Accordingly, parallel selection for features important to all wolves may eclipse local environmental selection and implies long-term separation among population clusters.

We investigated contemporary and historical influences on the pattern of genetic diversity of European roe deer (Capreolus capreolus). The study was conducted in northeastern Poland, a zone where vast areas of primeval forests are conserved and where the European roe deer was never driven to extinction. A total of 319 unique samples collected in three sampling areas were genotyped at 16 microsatellites and one fragment (610 bp) of mitochondrial DNA (mtDNA) control region. Genetic diversity was high, and a low degree of genetic differentiation among sampling areas was observed with both microsatellites and mtDNA. No evidence of genetic differentiation between roe deer inhabiting open fields and forested areas was found, indicating that the ability of the species to exploit these contrasting environments might be the result of its phenotypic plasticity. Half of the studied individuals carried an mtDNA haplotype that did not belong to C. capreolus, but to a related species that does not occur naturally in the area, the Siberian roe deer (C. pygargus). No differentiation between individuals with Siberian and European mtDNA haplotypes was detected at microsatellite loci. Introgression of mtDNA of Siberian roe deer into the genome of European roe deer has recently been detected in eastern Europe. Such introgression might be caused by human-mediated translocations of Siberian roe deer within the range of European roe deer or by natural hybridization between these species in the past.

The wild boar (Sus scrofa) is one of the most widely distributed mammals in Europe. Its demography was affected by various events in the past and today populations are increasing throughout Europe. We examined genetic diversity, structure and population dynamics of wild boar in Central and Eastern Europe. MtDNA control region (664 bp) was sequenced in 254 wild boar from six countries (Poland, Hungary, Belarus, Ukraine, Moldova and the European part of Russia). We detected 16 haplotypes, all known from previous studies in Europe; 14 of them belonged to European 1 (E1) clade, including 13 haplotypes from E1-C and one from E1-A lineages. Two haplotypes belonged respectively to the East Asian and the Near Eastern clade. Both haplotypes were found in Russia and most probably originated from the documented translocations of wild boar. The studied populations showed moderate haplotype (0.714±0.023) and low nucleotide diversity (0.003±0.002). SAMOVA grouped the genetic structuring of Central and Eastern European wild boar into three subpopulations, comprising of: (1) north-eastern Belarus and the European part of Russia, (2) Poland, Ukraine, Moldova and most of Belarus, and (3) Hungary. The multimodal mismatch distribution, Fu's Fs index, Bayesian skyline plot and the high occurrence of shared haplotypes among populations did not suggest strong demographic fluctuations in wild boar numbers in the Holocene and pre-Holocene times. This study showed relatively weak genetic diversity and structure in Central and Eastern European wild boar populations and underlined gaps in our knowledge on the role of southern refugia and demographic processes shaping genetic diversity of wild boar in this part of Europe.

Suppressing unwanted thoughts can lead to an increased occurrence of the suppressed thought in dreams. This is explainable by the ironic control theory, which theorizes why the suppression of thoughts might make them more persistent. The present study examined the influence of thought suppression on dream rebound, dream distress, general psychiatric symptomatology, depression, sleep quality and perceived stress. Thirty healthy participants (good sleepers) were investigated over a period of 1 week. Half were instructed to suppress an unwanted thought 5 min prior to sleep, whereas the other half were allowed to think of anything at all. Dream content was assessed through a dream diary. Independent raters assessed whether or not the dreams were related to the suppressed target thought. The results demonstrated increased target-related dreams and a tendency to have more distressing dreams in the suppression condition. Moreover, the data imply that thought suppression may lead to significantly increased general psychiatric symptomatology. No significant effects were found for the other secondary outcomes., (© 2013 European Sleep Research Society.)

This study presents a meta-analysis of the effectiveness of psychological treatments for chronic nightmares using imaginal confrontation with nightmare contents (ICNC) or imagery rescripting and rehearsal (IRR). Pre-post effect sizes (Hedges' g) were calculated for the outcome measures of nightmare frequency, nights per week with nightmares, sleep quality, depression, anxiety, and PTSD severity. Fixed-effects and random-effects models were applied. High effect sizes were found for nightmare frequency (g=1.04), nights per week with nightmares (g=0.99), and PTSD severity (g=0.92). Most of the effect sizes for the secondary outcomes were moderate. One objective was to clarify whether ICNC or IRR is more important for nightmare reduction. The results indicate that a higher duration of time for ICNC is associated with greater improvements: The minutes of applied ICNC moderate the effect sizes for nightmare frequency at follow-up 2 and for nights per week with nightmares at post and follow-up 1. The percentage of applied ICNC moderates the effect sizes for nightmare frequency and nights per week with nightmares at follow-up 1. Thus, dismantling studies are necessary to draw conclusions regarding whether ICNC or IRR is the most effective in the psychological treatment of chronic nightmares., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Population densities of large herbivores are determined by the diverse effects of density-dependent and independent environmental factors. In this study, we used the official 1998-2003 inventory data on ungulate numbers from 462 forest districts and 23 national parks across Poland to determine the roles of various environmental factors in shaping country-wide spatial patterns of ungulate abundances. Spatially explicit generalized additive mixed models showed that different sets of environmental variables explained 39 to 50 % of the variation in red deer Cervus elaphus , wild boar Sus scrofa , and roe deer Capreolus capreolus abundances. For all of the studied species, low forest cover and the mean January temperature were the most important factors limiting their numbers. Woodland cover above 40-50 % held the highest densities for these species. Wild boar and roe deer were more numerous in deciduous or mixed woodlands within a matrix of arable land. Furthermore, we found significant positive effects of marshes and water bodies on wild boar abundances. A juxtaposition of obtained results with ongoing environmental changes (global warming, increase in forest cover) may indicate future growth in ungulate distributions and numbers.

Purpose: The aim of this study is to search for more effective derivatives of the superoxide dismutase mimetic tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). Although tempol is neuroprotective in a rat partial optic nerve crush (PONC) model, relatively high doses are required to exert this effect., Methods: Tempol acyl esters with different-length fatty acids (tempol-C4, tempol-C8, tempol-C12 and tempol-C16) were synthesized and the following properties were evaluated: water-octanol partition coefficient, liposome-liposome energy transfer, and electron paramagnetic resonance (EPR). Brown Norway rats underwent PONC and received tempol or acyl esters intraperitoneally once daily for 7 consecutive days. We then compared the effects of tempol and its four esters on retinal ganglion cell (RGC) damage using a retrograde labelling method., Results: The water-octanol partition coefficient increased with increasing length of attached acyl chain. However, the energy of the liposome-liposome transfer seemed to be optimal for tempol-C8 and tempol-C12. The EPR signal was very similar for all tested compounds, suggesting similar efficiency of superoxide scavenging. Partial optic nerve crush in vehicle-treated animals reduced RGC numbers by approx. 59% when compared with sham-operated eyes. Tempol did not affect RGC loss at a dose of 1 mg/kg. In contrast, at molar doses equivalent to 1 mg/kg of tempol, tempol-C8 showed a significant neuroprotective effect, whereas tempol-C4, tempol-C12 and tempol-C16 did not act neuroprotectively., Conclusion: Manipulating the hydrophobicity of tempol seems to be a promising tool for developing more potent neuroprotectants in the PONC degeneration model. However, the resulting compounds need further pharmacological evaluation., (© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.)

European red deer are known to show a conspicuous phylogeographic pattern with three distinct mtDNA lineages (western, eastern and North-African/Sardinian). The western lineage, believed to be indicative of a southwestern glacial refuge in Iberia and southern France, nowadays covers large areas of the continent including the British Isles, Scandinavia and parts of central Europe, while the eastern lineage is primarily found in southeast-central Europe, the Carpathians and the Balkans. However, large parts of central Europe and the whole northeast of the continent were not covered by previous analyses. To close this gap, we produced mtDNA control region sequences from more than 500 red deer from Denmark, Germany, Poland, Lithuania, Belarus, Ukraine and western Russia and combined our data with sequences available from earlier studies to an overall sample size of almost 1,100. Our results show that the western lineage extends far into the European east and is prominent in all eastern countries except for the Polish Carpathians, Ukraine and Russia where only eastern haplotypes occurred. While the latter may actually reflect the natural northward expansion of the eastern lineage after the last ice age, the present distribution of the western lineage in eastern Europe may in large parts be artificial and a result of translocations and reintroduction of red deer into areas where the species became extinct in historical times.

Determining ecological corridors is crucial for conservation efforts in fragmented habitats. Commonly employed least cost path (LCP) analysis relies on the underlying cost matrix. By using Ecological Niche Factor Analysis, we minimized the problems connected with subjective cost assessment or the use of presence/absence data. We used data on the wolf presence/absence in Poland to identify LCPs connecting patches of suitable wolf habitat, factors that influence patch occupancy, and compare LCPs between different genetic subpopulations. We found that a lower proportion of cities and roads surrounds the most densely populated patches. Least cost paths between areas where little dispersal takes place (i.e., leading to unpopulated patches or between different genetic subpopulations) ran through a higher proportion of roads and human settlements. They also crossed larger maximal distances over deforested areas. We propose that, apart from supplying the basis for direct conservation efforts, LCPs can be used to determine what factors might facilitate or hinder dispersal by comparing different subsets of LCPs. The methods employed can be widely applicable to gain more in-depth information on potential dispersal barriers for large carnivores.

The characterization of small molecules requires identification and evaluation of several predictive parameters, when selecting compounds for pharmacological applications and/or determining their toxicity. A number of them are correlated with the compound interaction with biological membranes and/or capacity to cross them. The knowledge of the extent of adsorption, partition coefficient and permeability along with the compound ability to alter membrane properties are critical for such studies. Lipid bilayers are frequently used as the adequate experimental models of a biological membrane despite their simple structure and a limited number of components. A significant number of the biologically relevant lipid bilayer properties are related to its electrostatics. Three electrostatic potentials were defined for the lipid bilayer; the intrinsic or induced surface electrostatic potential, the dipole potential and the membrane potential. Each of them was measured with dedicated methodologies. The complex measurement protocols and technically demanding instrumentation made the development of efficient HTS approaches for complete characterization of membrane electrostatics practically impossible. However, the rapid development of fluorescence techniques accompanied by rapid growth in diversity and number of dedicated fluorescent probes enabled characterization of lipid bilayer electrostatics in a moderately simple manner. Technically advanced, compact and automated workstations, capable of measuring practically all fluorescence parameters, are now available. Therefore, the proper selection of fluorescent probes with measuring procedures can be designed to evaluate drug candidates in context of their ability to alter membrane electrostatics. In the paper we present a critical review of available fluorescence methods, useful for the membrane electrostatics evaluation and discuss the feasibility of their adaptation to HTS procedures. The significance of the presented methodology is even greater considering the rapid growth of advanced drug formulations, where electrostatics is an important parameter for production processes and pharmacokinetics of the product. Finally, the potential of the membrane electrostatics to emerge as a viable pharmacological target is indicated and fluorescence techniques capable to evaluate this potential are presented.

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of beta-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482-94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na(+)K(+)ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the beta-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.

The major component of the cell membrane skeleton, spectrin, is anchored in the cell membrane via interactions with membrane proteins. It has been previously shown that both erythroid and non-erythroid spectrin interact directly with membrane phospholipids (mainly aminophospholipids). One of the binding sites responsible for these interactions is located in the ankyrin-binding domain. In the present study, in order to better understand the character of binding, a more detailed investigation of the interactions between the beta-spectrin fragments corresponding to the truncated mutants of the ankyrin-binding domain (Frag1 and Frag3) and liposomes of different compositions were carried out. The obtained results suggest that the binding of both spectrin fragments with liposomes induces conformational changes within the protein. Analysis of the changes in intrinsic tryptophan fluorescence spectra upon binding with liposomes, together with quenching studies (from the water and membrane hydrocarbon environment), allows for qualitative description of changes in proteins conformation. Our results suggest that the largest conformational changes occur for Frag1 bound to PC : PE (2 : 3) liposomes what is consistent with previous studies on monolayers. They are also in good agreement with those obtained previously for native erythroid and nonerythroid spectrin molecules.

Complex molecular ensembles are frequently considered an element of new pharmacological formulations. This is especially evident in the therapies based on genetic information. In order to obtain an effective drug, it is necessary to associate a nucleic acid molecule with the components to ensure the desired aggregate structure and properties. To evaluate the progress of the supromolecular aggregate formation a range of methodologies and techniques are needed to test the quality and uniformity of the formulations. In this paper we propose a procedure which measures the association of a small molecule with nucleic acid using propidium iodide and oligonucleotides as an example. To measure propidium iodide binding constant the oligonucleotide was covalently labeled with fluorescein and then the changes in fluorescence resonance energy transfer (FRET) were determined and handled according to the acceptor-donor titration methodology. The calculated binding constants were in a good agreement with the values published previously. The developed method was then used to evaluate the extent of an oligonucleotide association with the lipid aggregates. It was found that two populations of oligonucleotides are present in all lipid samples studied. The fraction of oligonucleotides associated with liposomes rises with the increase of a cationic lipid content, reaching the constant value when the fraction of cationic lipid exceeded 20 mol%. Energy transfer data combined with these obtained in quenching experiments show that the orientation of the oligonucleotide associated with a lipid bilayer depends on the amount of surface charge.