Predicting survival in out-of-hospital cardiac arrest patients undergoing targeted temperature management: The Polish Hypothermia Registry Risk Score.

BACKGROUND: Prompt reperfusion and post-resuscitation care, including targeted temperature management (TTM), improve survival in out-of-hospital cardiac arrest (OHCA) patients. To predict inhospital mortality in OHCA patients treated with TTM, the Polish Hypothermia Registry Risk Score (PHR-RS) was developed. The use of dedicated risk stratification tools may support treatment decisions. METHODS: Three hundred seventy-six OHCA patients who underwent TTM between 2012 and 2016 were retrospectively analysed and whose data were collected in the Polish Hypothermia Registry. A multivariate logistic regression model identified a set of predictors of in-hospital mortality that were used to develop a dedicated risk prediction model, which was tested for accuracy. RESULTS: The mean age of the studied population was 59.2 ± 12.9 years. 80% of patients were male, 73.8% had shockable rhythms, and mean time from cardiac arrest (CA) to cardiopulmonary resuscitation (CPR) was 7.2 ± 8.6 min. The inputs for PHR-RS were patient age and score according to the Mild Therapeutic Hypothermia (MTH) Scale. Criteria for the MTH score consisted of time from CA to CPR above 10 min, time from CA to the return of spontaneous circulation above 20 min, in-hospital CA, unwitnessed CA, and non-shockable rhythm, each counted as 1 point. The predictive value of PHR-RS was expressed as an area under the curve of 0.74. CONCLUSIONS: PHR-RS is one of the simplest and easiest models to use and enables a reliable prediction of in-hospital mortality in OHCA patients treated with TTM.

Sex-biased polyparasitism in moose (Alces alces) based on molecular analysis of faecal samples.

Simultaneous infection with multiple parasite species in an individual host is often observed in wild populations. The understanding of parasite species distribution across populations of wild animals is of basic and applied importance, because parasites can have pronounced effects on the dynamics of host population. Here, we quantified prevalence and endoparasite species richness in moose and explored sex-biased polyparasitism using diagnostic PCR method coupled with DNA sequencing of moose faecal samples from the Biebrza River valley, North-Eastern Poland. This is the largest moose population in Central Europe that has not been harvested for almost 20 years. We also evaluated the appropriate quantity of faeces for detecting DNA of parasite species. Faecal samples were screened for molecular markers of 10 different species of endoparasites. Endoparasite prevalence was high in the studied population. Almost all of the samples (98%) tested positive for at least one parasite species, and we found polyparasitism in the majority of the tested individuals. The number of different parasite species found in a single individual ranged from 0 to 9. The parasite species richness was significantly higher in male than in female individuals. The most prevalent were liver fluke Parafasciolopsis fasciolaemorpha and gastrointestinal nematodes Ostertargia sp. Of the ten endoparasite species detected, only the prevalence of the tapeworm Moniezia benedeni was significantly higher in males than in females. Additionally, we identified co-occurrence associations of parasite species, which tended to be random, but we noted some evidence of both positive and negative associations. Our findings promote applications of molecular methods for parasite species identification from non-invasively collected faecal samples in management and scientific study of moose population, which should include investigation of parasite status, and in health monitoring programs for other wild cervids.

Development of microsatellite loci and optimization of a multiplex assay for Latibulus argiolus (Hymenoptera: Ichneumonidae), the specialized parasitoid of paper wasps.

Microsatellite loci are commonly used markers in population genetic studies. In this study, we present 40 novel and polymorphic microsatellite loci elaborated for the ichneumonid parasitoid Latibulus argiolus (Rossi, 1790). Reaction condition optimisation procedures allowed 14 of these loci to be co-amplified in two PCRs and loaded in two multiplex panels onto a genetic analyser. The assay was tested on 197 individuals of L. argiolus originating from ten natural populations obtained from the host nests of paper wasps. The validated loci were polymorphic with high allele numbers ranging from eight to 27 (average 17.6 alleles per locus). Both observed and expected heterozygosity values were high, ranging between 0.75 and 0.92 for HO (mean 0.83) and from 0.70 to 0.90 for HE (mean 0.85). The optimized assay showed low genotyping error rate and negligible null allele frequency. The designed multiplex panels could be successfully applied in relatedness analyses and genetic variability studies of L. argiolus populations, which would be particularly interesting considering the coevolutionary context of this species with its social host.

Population Genetic Structure and Demography of the Critically Endangered Chequered Blue Butterfly (Scolitantides orion) in a Highly Isolated Part of Its Distribution Range.

Scolitantides orion is a butterfly species threatened in many European countries. In Poland, it survived in a single highly isolated area (Vistula River valley), which is an example of the dramatic decline in the population number. We studied the two largest remaining populations inhabiting opposite banks of the river. Mark-release-recapture studies showed that both populations were small, and they fluctuated in numbers, but adult individuals were twice as numerous on the western site. Genetic analyses were carried out using a mitochondrial (COI, ND5) and nuclear markers (Wgl, EF-1α, and microsatellite loci). We found out that genetic variation was low at both sites but higher in the smaller eastern population. This pattern is likely to be better explained by past distribution, when the butterfly, as a continental species used to be much more widespread in the east. However, the genetic differentiation between populations was low. This could suggest that the existing gene flow is facilitated by dominant regional wind direction, which may also contribute to a better genetic condition of the western population. Finally, a comparison of the obtained COI sequences with others available enabled us to reveal the phylogeographic pattern of the S. orion from different localities within its range.

The impact of early therapeutic intervention on the central pattern generator in premature newborns - a preliminary study and literature review.

OBJECTIVE: The aim: To study the effect of therapeutic intervention on the improvement of the rhythmicity of non-nutritive and nutritive sucking in premature newborns and on the suck central pattern generator. PATIENTS AND METHODS: Material and methods: Stimulation of the breast zone was performed in two premature newborns by means of the Vojta method. Intraoral pressure was measured during non-nutritive and nutritive sucking before and after this therapeutic intervention. The maximum negative pressures generated during individual sucks and the intervals between sucks were analysed. RESULTS: Results: The stimulation of the breast zone using the Vojta method seems to have no impact on the duration of individual sucking episodes. However, a significant improvement in the rhythmicity and regularity of sucking was observed in both newborns: the coefficient of quartile deviation for the intervals decreased from 15% and 11% to 13% and 6%, respectively, and for the maximum negative pressures it decreased from 24% and 27% to 9% and 19%, respectively. Additionally, the median value of the maximum negative pressure decreased in both newborns: from -39 and -37 mmHg to -45 and -60 mmHg, respectively. CONCLUSION: Conclusions: The stimulation of the breast zone using the Vojta method seems to have a direct impact on the central pattern generator, which improves the rhythmicity as well as the regularity of both non-nutritive and nutritive sucking.

A novel polymorphism in the fatty acid desaturase 2 gene (Fads2): A possible role in the basal metabolic rate.

Fatty acyl composition of cell membrane lipids, particularly the abundance of highly unsaturated docosahexaenoic fatty acid (22:6n-3, DHA), is likely to be an important predictor of basal metabolic rate (BMR). Our study was performed using two lines of laboratory mice divergently selected for either high or low BMR. We describe a novel single nucleotide polymorphism in the Fads2 gene encoding Δ6-desaturase, a key enzyme in the metabolic pathways of polyunsaturated fatty acids (PUFAs). The allele frequencies of Fads2 were significantly different in both lines of mice. The analysis of genetic distances revealed that the genetic differentiation between the two studied lines developed significantly faster at the Fads2 locus than it did at neutral loci. Such a pattern suggests that the Fads2 polymorphism is related to the variation in BMR, i.e. the direct target of selection. The Fads2 polymorphism significantly affected abundance of several PUFAs; however, the differences in PUFA composition between lines were compatible with the difference in frequency of Fads2 alleles only for DHA. We hypothesize that the polymorphism in the Fads2 gene affects the BMR through modification of DHA abundance in cell membranes. This may be the first example of a significant link between a polymorphism in a gene responsible for fatty acyl composition and variation in BMR.

Genetic differentiation and asymmetric gene flow among Carpathian brown bear (Ursus arctos) populations-Implications for conservation of transboundary populations.

The abundance and distribution of large carnivores in Europe have been historically reduced. Their recovery requires multilevel coordination, especially regarding transboundary populations. Here, we apply nuclear and mitochondrial genetic markers to test for admixture level and its impact on population genetic structure of contemporary brown bears (Ursus arctos) from the Eastern, Southern, and Western Carpathians. Carpathian Mountains (Europe). Nearly 400 noninvasive brown bear DNA samples from the Western (Poland) and Eastern Carpathians (Bieszczady Mountains in Poland, Slovakia, Ukraine) were collected. Together with DNA isolates from Slovakia and Romania, they were analyzed using the set of eight microsatellite loci and two mtDNA regions (control region and cytochrome b). A set of 113 individuals with complete genotypes was used to investigate genetic differentiation across national boundaries, genetic structuring within and between populations, and movement between populations. Transboundary brown bear subpopulations (Slovakia and Poland) did not show significant internal genetic structure, and thus were treated as cohesive units. All brown bears from the Western Carpathians carried mitochondrial haplotypes from the Eastern lineage, while the Western lineage prevailed in the brown bears from the Bieszczady Mountains. Despite similar levels of microsatellite variability, we documented significant differentiation among the studied populations for nuclear markers and mtDNA. We also detected male-biased and asymmetrical movement into the Bieszczady Mountains population from the Western Carpathians. Our findings suggest initial colonization of the Western Carpathians by brown bears possessing mtDNA from the Eastern lineage. Genetic structuring among populations at microsatellite loci could be a result of human-mediated alterations. Detected asymmetric gene flow suggests ongoing expansion from more abundant populations into the Bieszczady Mountains and thus supports a metapopulation model. The knowledge concerning this complex pattern can be implemented in a joint Carpathian brown bear management plan that should allow population mixing by dispersing males.

Purifying Selection, Density Blocking and Unnoticed Mitochondrial DNA Diversity in the Red Deer, Cervus elaphus.

The trajectories of postglacial range expansions, the occurrence of lineage patches and the formation and maintenance of secondary contact between lineages may mostly reflect neutral demographic processes, including density blocking, that may leave long-lasting genetic signatures. However, a few studies have recently shown that climate may also play a role. We used red deer, a large, mobile herbivore that is assumed to be sensitive to climate change, to test hypotheses of possible selection on the mitochondrial DNA cytochrome b gene (mtDNA cytb) and competitive and/or density-blocking (using mtDNA control region). We searched for a possible link between the phylogeographic structure and abiotic climatic variables. Finally, we tested for isolation by distance and isolation by environment and assessed the impact of human-mediated translocations on the genetic structure of red deer. Our analysis of 30 red deer populations in Poland using the mtDNA control region (N = 357) and cytochrome b (N = 50) markers not only confirmed the presence of the Western and South-Eastern lineages of the species but also indicated the presence of a previously unnoticed, rare relic haplotype that grouped together C. e. italicus from Italy (the Mesola deer). No significant signs of positive selection were detected for the mtDNA cytb gene in the studied red deer. However, a significant signal for purifying selection was found in our study that may explain the narrowness of the contact zone because gene flow between the Western and South-Eastern lineages should drive relatively strong mito-nuclear incompatibilities. MtDNA control region differentiation among red deer populations in Poland correlated with different abiotic climatic variables. Strikingly, the southernmost ice sheet limits during the Elsterian was the most important factor, and it explained the largest amount of variation. However, neither isolation by distance (IBD) nor isolation by environment (IBE) were recorded, and a very limited impact of human translocations was evident. The above-mentioned results suggest that in contemporary red deer populations in Poland, the phylogeographic pattern is well preserved, and long-term processes (density and/or competitive blocking) still play a major role.

Determination of coating thickness of minitablets and pellets by dynamic image analysis.

The study was focused on the feasibility of measuring film thickness on minitablets and pellets by means of dynamic image analysis using Camsizer XT. Minitablets with diameters of 2.0mm or 2.5mm and pellets in the size range of 0.7-0.8mm with the polymer coating thickness of 80-170 µm were prepared and analyzed. In minitablets the film thickness was measured on the walls and edges and the difference was significant. The results were compared with the results obtained with the use of three microscopic methods (scanning and stereoscopic microscopy). Good correlation between dynamic image analysis and microscopic techniques was shown in the case of the film thickness in pellets and 2.0mm minitablets, but the measurement of the coating layer of larger particles (minitablets 2.5mm) was not sufficiently precise due to the non-isometric shape of these objects and the limited measuring range of the apparatus. Short analysis time, no need for sample preparation and a large number of objects under examination are main advantages of the proposed film thickness measurement by use of a dynamic image analysis.

Fast and efficient DNA-based method for winter diet analysis from stools of three cervids: moose, red deer, and roe deer.

Effects of cervid browsing on timber production, especially during winter, lead to economic losses in forest management. The aim of this study was to present an efficient DNA-based method which allows qualitative assessment of the winter diet from stools of moose (Alces alces), roe deer (Capreolus capreolus), and red deer (Cervus elaphus). The preliminary results of the diet composition of the three cervids from Poland were also presented with a special emphasis on moose. The electropherograms of the chloroplast intron trnL (UAA) P6 loop amplification products using g (fluorescence-labeled) and h primers revealed differences in the length of PCR products among various plant species eaten by these herbivores. In addition, the usage of species-specific primers allowed unambiguous identification of different gymnosperms and angiosperms. The preliminary moose diet analysis, based on winter fecal samples from the entire range of moose occurrence in Poland, revealed the presence of 15 to 24 tree, shrub, and herbaceous species. This fast, cost-efficient, and simple method proved also to be reliable for the diet analysis of red deer and roe deer. It may be a valuable tool in forest and conservation management, as well as a way of enhancing ecological studies focusing on the impact of herbivores on the ecosystems and their possible food niche overlap.

Complex patterns of population genetic structure of moose, Alces alces, after recent spatial expansion in Poland revealed by sex-linked markers.

In recent years, human activity directly and indirectly influenced the demography of moose in Poland. The species was close to extinction, and only a few isolated populations survived after the Second World War; then, unprecedented demographic and spatial expansions had occurred, possibly generating a very complex pattern of population genetic structure at the present-day margins of the species range in Poland. Over 370 moose from seven populations were collected from Poland, and partial sequences of the mitochondrial control region (mtDNA-cr; 607 bp) were obtained. In addition, the entire mtDNA cytochrome b gene (1,140 bp) and Y-chromosome markers (1,982 bp in total) were studied in a chosen set of individuals. Twelve mtDNA haplotypes that all belonged to the European moose phylogroup were recorded. They could be divided into two distinct clades: Central Europe and the Ural Mountains. The first clade consists of three distinct groups/branches: Biebrza, Polesie, and Fennoscandia. The Biebrza group has experienced spatial and demographic expansion in the recent past. Average genetic differentiation among moose populations in Poland at mtDNA-cr was great and significant (ΦST = 0.407, p < 0.001). Using mtDNA-cr data, four separate groups of population were recognized using spatial analysis of molecular variance and principal coordinate analysis, including a relict population in Biebrza National Park, a reintroduced Kampinos National Park population, as well as populations that were descendants of moose that colonized Poland from the east (Lithuania, Belarus, and Ukraine) and the north (former East Prussia). Among all the sequenced Y-chromosome markers, polymorphisms were found in the DBY14 marker in three populations only; four haplotypes were recorded in total. No significant differentiation was detected for this Y-linked marker among moose populations in Poland. Our mtDNA study revealed that a variety of different factors-bottleneck, the presence of relict, autochthonous populations, translocations, limited female dispersal, and the colonization from the east and north-are responsible for the observed complex pattern of population genetic structure after demographic and spatial expansion of moose in Poland.

Activity of ozonated water and ozone against Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

BACKGROUND: The known bactericidal properties of ozone have not been checked in relation to its action on bacterial biofilms. This is especially true of ozonated fluids. The aim of this study was to investigate the bactericidal activity of ozonated water and that of a mixture of ozone and oxygen against biofilms. MATERIAL/METHODS: Eighteen clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa exhibiting various levels of antibiotic sensitivity were investigated. Bacteria were cultured in biofilm form on polystyrene titration plates for periods of 2 to 72 hours. The biofilms formed in this way were exposed to in statu nascendi ozonated water produced in a prototype device that had been tested in clinical conditions, or to a mixture of oxygen and ozone generated in the same device. Live cells in the biofilm were stained with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide solution. The degree of reduction of viable bacteria following ozone exposure was determined. RESULTS: Ozonated water was found to be an effective bactericidal agent against biofilms after as little as 30 seconds of exposure, while the bactericidal activity of the ozone-oxygen solution was much lower. Prolongation of the duration of biofilm exposure to the gaseous disinfectant to 40 minutes led to a reduction in the viable cell count, which nevertheless remained high. CONCLUSIONS: Unlike the ozone-oxygen mixture, ozonated water effectively destroys bacterial biofilms in vitro.

Antimicrobial activity of ozonated water.

BACKGROUND: The purpose of this study was to analyze basic bactericidal and fungicidal activity of ozonated water according to EN 1040 "Chemical disinfectants and antiseptics--Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics" and EN 1275 "Chemical disinfectants and antiseptics--Quantitative suspension test for the evaluation of basic fungicidal or basic yeasticidal activity of chemical disinfectants and antiseptics" with additional clinical multidrug-resistant bacterial strains and evaluate whether the ozonated water acts as a rapid and efficient antimicrobial agent and as such could be applied during intraoperative ozone treatment for tissue protection against infection with pathogenic bacteria. MATERIAL/METHODS: A prototype device for intraoperative ozone therapy was used. Besides standard bacterial and fungal strains, 60 clinical bacterial isolates were analyzed. RESULTS: The ozone concentration in ozonated water was sufficient to kill almost all cells of the bacterial and yeast strains tested after 30 seconds. Effective action against Aspergillus brasiliensis spores required a longer time than those required in the case of bacterial cells or vegetative cells of yeast. CONCLUSIONS: The prototype device used in our study produced high ozone concentrations in freshly prepared ozonated water. This liquid complied with the requirements of the EN Standards: basic bactericidal and basic yeasticidal activities.