Evaluation of the In Vitro Stability of Stimuli-Sensitive Fatty Acid-Based Microparticles for the Treatment of Lung Cancer.

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 (IC50 values for both lauric acid-based and myristic/palmitic-based microparticles containing paclitaxel were below 0.375 µg/mL), while reference microparticles were noncytotoxic.

New oral liposomal vitamin C formulation: properties and bioavailability.

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.

Changes in lipid membrane mechanics induced by di- and tri-phenyltins.

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.

High temporal resolution data reveal low bat and insect activity over managed meadows in central Europe.

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.

Environmental factors shaping ungulate abundances in Poland.

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.

Risk of African swine fever virus transmission among wild boar and domestic pigs in Poland.

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.

How do genetic relatedness and spatial proximity shape African swine fever infections in wild boar?

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.

Pan-European phylogeography of the European roe deer (Capreolus capreolus).

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.

Analyses of least cost paths for determining effects of habitat types on landscape permeability: wolves in Poland.

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.

Molecular biogeography of red deer Cervus elaphus from eastern Europe: insights from mitochondrial DNA sequences.

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.

Environmental factors shaping stable isotope signatures of modern red deer (Cervus elaphus) inhabiting various habitats.

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.

Settle Down! Ranging Behaviour Responses of Roe Deer to Different Capture and Release Methods.

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.

Fluorescence techniques for determination of the membrane potentials in high throughput screening.

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.

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid beta-spectrin on the properties of natural membranes and skeletal structures.

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.

Spatial epidemiology of African swine fever: Host, landscape and anthropogenic drivers of disease occurrence in wild boar.

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./km2. 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.

The level of habitat patchiness influences movement strategy of moose in Eastern Poland.

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.

Multispecies reservoir of Spirometra erinaceieuropaei (Cestoda: Diphyllobothridae) in carnivore communities in north-eastern Poland.

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 Bialowieza 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, Bialowieza 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.

Stimuli-sensitive fatty acid-based microparticles for the treatment of lung cancer.

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.

Fluorescence approach to evaluating conformational changes upon binding of beta-spectrin ankyrin-binding domain mutants with the lipid bilayer.

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.

Winter temperature correlates with mtDNA genetic structure of yellow-necked mouse population in NE Poland.

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.