Molecular analysis of scats revealed diet and prey choice of grey wolves and Eurasian lynx in the contact zone between the Dinaric Mountains and the Alps.

A comprehensive understanding of the dietary habits of carnivores is essential to get ecological insights into their role in the ecosystem, potential competition with other carnivorous species, and their effect on prey populations. Genetic analysis of non-invasive samples, such as scats, can supplement behavioural or microscopic diet investigations. The objective of this study was to employ DNA metabarcoding to accurately determine the prey species in grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) scat samples collected in the Julian Alps and the Dinaric Mountains, Slovenia. The primary prey of wolves were red deer (Cervus elaphus) (detected in 96% scat samples), European roe deer (Capreolus capreolus) (68%), and wild boar (Sus scrofa) (45%). A smaller portion of their diet consisted of mesocarnivores, small mammals, and domestic animals. In contrast, the lynx diet mostly consisted of European roe deer (82%) and red deer (64%). However, small mammals and domestic animals were also present in lynx diet, albeit to a lesser extent. Our findings indicate that the dietary habits of wolves and lynx are influenced by geographical location. Snapshot dietary analyses using metabarcoding are valuable for comprehending the behaviour and ecology of predators, and for devising conservation measures aimed at sustainable management of both their natural habitats and prey populations. However, to gain a more detailed understanding of wolf and lynx dietary habits and ecological impact, it would be essential to conduct long-term genetic monitoring of their diet.

Wolf genetic diversity compared across Europe using the yardstick method.

Integrating data across studies with traditional microsatellite genetic markers requires careful calibration and represents an obstacle for investigation of wide-ranging species where populations require transboundary management. We used the "yardstick" method to compare results published across Europe since 2002 and new wolf (Canis lupus) genetic profiles from the Carpathian Mountains in Central Europe and the Dinaric Mountains in Southeastern Europe, with the latter as our reference population. We compared each population with Dinaric wolves, considering only shared markers (range 4-17). For each population, we calculated standard genetic diversity indices plus calibrated heterozygosity (Hec) and allelic richness (Ac). Hec and Ac in Dinaric (0.704 and 9.394) and Carpathian wolves (0.695 and 7.023) were comparable to those observed in other large and mid-sized European populations, but smaller than those of northeastern Europe. Major discrepancies in marker choices among some studies made comparisons more difficult. However, the yardstick method, including the new measures of Hec and Ac, provided a direct comparison of genetic diversity values among wolf populations and an intuitive interpretation of the results. The yardstick method thus permitted the integration of diverse sources of publicly available microsatellite data for spatiotemporal genetic monitoring of evolutionary potential.

Inventory and DNA-barcode library of ground-dwelling predatory arthropods from Krokar virgin forest, Slovenia.

Background: At a time of immense human pressure on nature and the resulting global environmental changes, the inventory of biota - especially of undisturbed natural areas - is of unprecedented value as it provides a baseline for future research. Krokar, an example of such an undisturbed area, is the largest virgin forest remnant in Slovenia. It is located in the Dinaric Alps, which are believed to harbour the most diverse fauna of soil invertebrates in Europe. Nevertheless, the soil fauna of the Krokar virgin forest has not been thoroughly studied. Moreover, modern taxonomic approaches often rely on genetic information (e.g. DNA-barcodes), while extensive reference libraries from the Dinaric area are lacking. Our work, therefore, focused on addressing this lack of faunistic and genetic data from the Dinaric area. New information: A total of 2336 specimens belonging to 100 taxa (45 spiders, 30 centipedes, 25 ground-dwelling beetles) were collected and deposited to GBIF. DNA-barcodes of 124 specimens belonging to 73 species were successfully obtained and deposited in GenBank and BOLD databases.

The current knowledge on centipedes (Chilopoda) in Slovenia: faunistic and ecological records from a national database.

In spite of Slovenia's very high biodiversity, it has only a few animal groups that have been significantly investigated and are well known in this area. Slovenian researchers have studied only about half of the species known to be living in the country (Mrsic 1997), but among well investigated species are centipedes. All available data about centipedes in Slovenia collected from 1921 to 2014 have been consolidated and constitute a general electronic database called "CHILOBIO", which was created to provide an easy overview of the Slovenian centipede fauna and to allow entry and interpretation of new data collected in future research. The level of investigation has been studied with this database, in conjunction with a geographic information system (GIS). In the study period, 109 species were identified from 350 localities in 109 of the 236 UTM 10 × 10 km quadrants which cover the study area. The south-central part of the country has been the subject of the best investigations, whereas there is an absence of data from the south-eastern, eastern and north-eastern regions The highest number of species (52) has been recorded near the Iska valley (Central Slovenia, quadrant VL68). In 48% of the UTM quadrants investigated fewer than 10 species were recorded and just 5 species were found in one locality. Seventeen species were reported only in the Dinaric region, 4 in the Prealpine-subpannonian region and 7 in the Primorska-submediterranean region.

Thermal analysis of N-carbamoyl benzotriazole derivatives.

Thermal properties of N-carbamoyl benzotriazole derivatives and N,N',N''-tribenzyloxyisocyanuric acid were investigated using thermogravimetric analysis and differential scanning calorimetry. The results revealed a difference between structural analogs of N-carbamoyl benzotriazole derivatives. They seem to be in agreement with the previously proposed formation of N,N',N''-tribenzyloxyisocyanuric acid from 1-(N-benzyloxycarbamoyl) benzotriazole, via an intermediary N-benzyloxyisocyanate acid, during heating. Substantially different thermal properties were observed for structural analogues, 1-(N-methoxycarbamoyl) benzotriazole and 1-(N-ethoxycarbamoyl) benzotriazole. In contrast to N-benzyloxyisocyanate, no corresponding reactions were observed for their decomposition products, i.e., methoxyisocyanate and ethoxyisocyanate.

Recovery of large carnivores in Europe's modern human-dominated landscapes.

The conservation of large carnivores is a formidable challenge for biodiversity conservation. Using a data set on the past and current status of brown bears (Ursus arctos), Eurasian lynx (Lynx lynx), gray wolves (Canis lupus), and wolverines (Gulo gulo) in European countries, we show that roughly one-third of mainland Europe hosts at least one large carnivore species, with stable or increasing abundance in most cases in 21st-century records. The reasons for this overall conservation success include protective legislation, supportive public opinion, and a variety of practices making coexistence between large carnivores and people possible. The European situation reveals that large carnivores and people can share the same landscape.

[DIABETIC NEPHROPATHY AS A CAUSE OF CHRONIC KIDNEY DISEASE]. / DIJABETICKA NEFROPATIJA KAO UZROK KRONICNE BUBREZNE BOLESTI.

Diabetic nephropathy is the leading cause of end-stage chronic kidney disease in most developed countries. Hyperglycemia, hypertension and genetic predisposition are the main risk factors for the development of diabetic nephropathy. Elevated serum lipids, smoking habits, and the amount and origin of dietary protein also seem to play a role as risk factors. Clinical picture includes a progressive increase in albuminuria, decline in glomerular filtration, hypertension, and a high risk of cardiovascular morbidity and mortality. Screening for albuminuria should be performed yearly, starting 5 years after diagnosis in type 1 diabetes or earlier in the presence of adolescence or poor metabolic control. In patients with type 2 diabetes, screening should be performed at diagnosis and yearly thereafter. Patients with albuminuria should undergo evaluation regarding the presence of associated comorbidities, especially retinopathy and macrovascular disease. Achieving the best metabolic control (HbA1c < 7%), treating hypertension (target blood pressure < 140/85 mm Hg), using drugs with blockade effect on the renin-angiotensin-aldosterone system, treating dyslipidemia and anemia are effective strategies for preventing the development of albuminuria, delaying the progression to more advanced stages of nephropathy and reducing cardiovascular mortality in patients with type 1 and type 2 diabetes.

[Flexion and version of the uterus on pelvic ultrasound examination]. / Fleksija I verzija maternice na ultrazvucnon pregledu zdjelice kroz trbusnu stijenku.

In the longitudinal (sagittal) plane, the angle between the axis of the uterine body and the cervix defines the flexion, whereas the angle between the axis of the cervix and the axis of the vagina defines the version of the uterus. In that regard, there are four uterine positions in the pelvis: anteflexion, retroflexion, anteversion and retroversion. The anteflexion with anteversion of the uterus is considered the natural position of the uterus in the pelvis. The transabdominal ultrasound examination of the female pelvis is most frequently performed if, for any reason, it is not possible to make a more appropriate transvaginal ultrasound examination. Suprapubic region is scanned with a high-frequency convex transducer in the longitudinal and transverse plane. The prerequisites for appropriate ultrasound examination are the filled urinary bladder, optimal quality of the ultrasound image, consistency in the sonographic technique and excellent knowledge of echomorphology of the pelvic organs. The commonest of the less common variants of the uterine position is retroflexion with retroversion. Although sometimes related with serious problems during childbirth as well as miscarriage, it usually does not cause any major problems. Yet, data on the uterine position may help the clinician in planning of various procedures.

Acid-base and electrochemical properties of manganese meso(ortho- and meta-N-ethylpyridyl)porphyrins: voltammetric and chronocoulometric study of protolytic and redox equilibria.

Growing interest in redox-active compounds as therapeutics for oxidative stress-related diseases led to the design of metalloporphyrins as some of the most potent functional SOD-mimics. Herein we report a detailed electrochemical study of the protolytic and redox equilibria of manganese ortho and meta substituted N-ethylpyridyl porphyrins (MnPs), MnTE-2-PyP(5+) and MnTE-3-PyP(5+), in aqueous solutions. The electrochemical parameters of redox processes for all experimentally available species have been determined, as well as their diffusion coefficients and estimated sizes of aqueous cavities. The results indicate that possible changes of the intracellular acidity cannot affect the antioxidant activity of MnPs in vivo, since no change in the E(Mn(III)P/Mn(II)P) values was observed below pH 10. Furthermore, the results confirm that both of these MnPs can be efficient redox scavengers of peroxynitrite (ONOO(-)), another major damaging species in vivo. This can occur by either single-electron reduction or two-electron reduction of ONOO(-), involving either the Mn(IV)P/Mn(III)P redox couple or Mn(IV)P/Mn(II)P redox couple. In addition to kred(ONOO(-)) reported previously, the thermodynamic parameters calculated herein imply a strong and identical driving force for the reaction of both ortho and meta isomeric MnPs with ONOO(-). An enlargement of both Mn(III)P complexes upon an increase of the solution pH was also observed and attributed to the reduction of positive charge on the central ion caused by deprotonation of the axial water molecules. This expansion of aqueous cavities suggests the formation of a solvent cage and the increased lipophilicity of Mn(III)P complexes caused by increased electron density on the Mn ion.

Synthesis, antibacterial and cytotoxic activity evaluation of hydroxyurea derivatives.

5 Synthesis and biological evaluation of a series (N = 16) of cyclic and acyclic hydroxyurea derivatives, including benzotriazole-, isocyanuric acid- and biuret-containing compounds, are disclosed. 1-N-(benzyloxycarbamoyl)benzotriazole was used as a benzyloxyisocyanate donor, a useful intermediate in the preparation of substituted hydroxyurea. Antibacterial activities of synthesized hydroxyurea derivatives were tested on three E. coli strains, i.e., a strain susceptible to antibiotics, a strain resistant to macrolide antibiotics and a strain resistant to aminoglycoside antibiotics. Six compounds (three acyclic and three cyclic hydroxyureas) showed growth inhibition of the tested E. coli strains, with different specificity toward each strain. Results of the cytotoxic activity evaluation revealed that twelve out of sixteen test compounds were cytotoxic to human acute monocytic leukemia THP-1 and/or human acute T cell leukemia Jurkat cell line. 1-(N-hydroxycarbamoyl) benzotriazole () increased the metabolic activity of both cell lines. Two compounds, 1-(N-hydroxycarbamoyl) benzotriazole (5) and N,N',N''-trihydroxybiuret (15), were identified as potential NO donors.

Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics, MnTE-2-PyP5+ and MnTnHex-2-PyP5+.

The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP(5+) (AEOL10113, FBC-007) and MnTnHex-2-PyP(5+) have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O2(•-) and ONOO(-) and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood-brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP(5+) is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP(5+), which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10mg/kg MnTE-2-PyP(5+) and 0.5 or 2mg/kg MnTnHex-2-PyP(5+). Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (Cmax, C24h, tmax, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUCoral/AUCiv) is 23% for MnTE-2-PyP(5+) and 21% for MnTnHex-2-PyP(5+). Despite the fivefold lower dose administered, the AUC values for liver, heart, and spleen are higher for MnTnHex-2-PyP(5+) than for MnTE-2-PyP(5+) (and comparable for other organs), clearly demonstrating the better tissue penetration and tissue retention of the more lipophilic MnTnHex-2-PyP(5+).

Endocrine, lifestyle, and genetic factors in the development of metabolic syndrome.

Metabolic syndrome (MetS) is a chronic, multi-component disease characterised by central obesity, hyperglycaemia, dyslipidaemia, and hypertension. Since MetS leads to type 2 diabetes, cardiovascular disease, development of certain cancers, and eventually to premature death, it is not surprising that it draws the attention of scientists around the world. The aetiopathology of MetS is complex and still not fully understood. This review focuses on the role of endocrine factors such as cortisol and insulin in the development of MetS. It also takes a look at some of the contributing lifestyle and genetic factors as well as at the current knowledge about its treatment.

Monitoring the effective population size of a brown bear (Ursus arctos) population using new single-sample approaches.

The effective population size (N(e) ) could be the ideal parameter for monitoring populations of conservation concern as it conveniently summarizes both the evolutionary potential of the population and its sensitivity to genetic stochasticity. However, tracing its change through time is difficult in natural populations. We applied four new methods for estimating N(e) from a single sample of genotypes to trace temporal change in N(e) for bears in the Northern Dinaric Mountains. We genotyped 510 bears using 20 microsatellite loci and determined their age. The samples were organized into cohorts with regard to the year when the animals were born and yearly samples with age categories for every year when they were alive. We used the Estimator by Parentage Assignment (EPA) to directly estimate both N(e) and generation interval for each yearly sample. For cohorts, we estimated the effective number of breeders (N(b) ) using linkage disequilibrium, sibship assignment and approximate Bayesian computation methods and extrapolated these estimates to N(e) using the generation interval. The N(e) estimate by EPA is 276 (183-350 95% CI), meeting the inbreeding-avoidance criterion of N(e) > 50 but short of the long-term minimum viable population goal of N(e) > 500. The results obtained by the other methods are highly consistent with this result, and all indicate a rapid increase in N(e) probably in the late 1990s and early 2000s. The new single-sample approaches to the estimation of N(e) provide efficient means for including N(e) in monitoring frameworks and will be of great importance for future management and conservation.

Genotyping success of historical Eurasian lynx (Lynx lynx L.) samples.

Historical samples, like tanned hides and trophy skulls, can be extremely important for genetic studies of endangered or elusive species. Selection of a sampling protocol that is likely to provide sufficient amount and quality of DNA with a minimum damage to the original specimen is often critical for a success of the study. We investigated microsatellite genotyping success of DNA isolated from three different types of Eurasian lynx historical samples. We analysed a total of 20 microsatellite loci in 106 historical samples from the endangered Dinaric lynx population, established from re-introduction of three pairs of lynx in 1973 from Slovakian Carpathians. Of the three tested sample types, turbinal bone and septum from the nasal cavity of the trophy skulls had the lowest percentage of samples successfully genotyped for all 20 microsatellite loci. Footpad samples, collected using a cork drill, exhibited better results in polymerase chain reaction amplification and genotyping than samples of footpad epidermis cut with a scalpel. We report simple and efficient sampling protocols, which could be widely applied for future studies utilizing historical samples.

Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant, ascorbate.

Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O(2)(·-), reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP(5+), MnTnHex-2-PyP(5+), and a meta isomer MnTnHex-3-PyP(5+), which differ greatly with regard to their metal-centered reduction potential, E(1/2) (Mn(III)P/Mn(II)P) and lipophilicity, were explored. Employing Mn(III)P/Mn(II)P redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP(5+) was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP(5+) is most prone to oxidative degradation with H(2) , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.

Bioavailability of metalloporphyrin-based SOD mimics is greatly influenced by a single charge residing on a Mn site.

In the cell Mn porphyrins (MnPs) likely couple with cellular reductants which results in a drop of total charge from 5+ to 4+ and dramatically increases their lipophilicity by up to three orders of magnitude depending upon the length of alkylpyridyl chains and type of isomer. The effects result from the interplay of solvation, lipophilicit and stericity. Impact of ascorbate on accumulation of MnPs was measured in E. coli and in Balb/C mouse tumours and muscle; for the latter measurements, the LC/ESI-MS/MS method was developed. Accumulation was significantly enhanced when MnPs were co-administered with ascorbate in both prokaryotic and eukaryotic systems. Further, MnTnHex-2-PyP(5+) accumulates 5-fold more in the tumour than in a muscle. Such data increase our understanding of MnPs cellular and sub-cellular accumulation and remarkable in vivo effects. The work is in progress to understand how coupling of MnPs with ascorbate affects their mechanism of action, in particular with respect to cancer therapy.

Acid-base and electrochemical properties of manganese meso(ortho- and meta-N-ethylpyridyl)porphyrins: potentiometric, spectrophotometric and spectroelectrochemical study of protolytic and redox equilibria.

The difference in electrostatics and reduction potentials between manganese ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP) and manganese meta-tetrakis(N-ethylpyridinium-3-yl)porphyrin (MnTE-3-PyP) is a challenging topic, particularly because of the high likelihood for their clinical development. Hence, a detailed study of the protolytic and electrochemical speciation of Mn(II-IV)TE-2-PyP and Mn(II-IV)TE-3-PyP in a broad pH range has been performed using the combined spectrophotometric and potentiometric methods. The results reveal that in aqueous solutions within the pH range ∼2-13 the following species exist: (H(2)O)Mn(II)TE-m-PyP(4+), (HO)Mn(II)TE-m-PyP(3+), (H(2)O)(2)Mn(III)TE-m-PyP(5+), (HO)(H(2)O)Mn(III)TE-m-PyP(4+), (O)(H(2)O)Mn(III)TE-m-PyP(3+), (O)(H(2)O)Mn(IV)TE-m-PyP(4+) and (O)(HO)Mn(IV)TE-m-PyP(3+) (m = 2, 3). All the protolytic equilibrium constants that include the accessible species as well as the thermodynamic parameters for each particular protolytic equilibrium have been determined. The corresponding formal reduction potentials related to the reduction of the above species and the thermodynamic parameters describing the accessible reduction couples were calculated as well.

Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification.

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.

High lipophilicity of meta Mn(III) N-alkylpyridylporphyrin-based superoxide dismutase mimics compensates for their lower antioxidant potency and makes them as effective as ortho analogues in protecting superoxide dismutase-deficient Escherichia coli.

Lipophilicity/bioavailibility of Mn(III) N-alkylpyridylporphyrin-based superoxide dismutase (SOD) mimics has a major impact on their in vivo ability to suppress oxidative stress. Meta isomers are less potent SOD mimics than ortho analogues but are 10-fold more lipophilic and more planar. Enhanced lipophilicity contributes to their higher accumulation in cytosol of SOD-deficient Escherichia coli, compensating for their lower potency; consequently, both isomers exert similar-to-identical protection of SOD-deficient E. coli. Thus meta isomers may be prospective therapeutics as are ortho porphyrins.

Determination of residual manganese in Mn porphyrin-based superoxide dismutase (SOD) and peroxynitrite reductase mimics.

The awareness of the beneficial effects of Mn porphyrin-based superoxide dismutase (SOD) mimics and peroxynitrite scavengers on decreasing oxidative stress injuries has increased the use of these compounds as mechanistic probes and potential therapeutics. Simple Mn2+ salts, however, have SOD-like activity in their own right both in vitro and in vivo. Thus, quantification/removal of residual Mn2+ species in Mn-based therapeutics is critical to an unambiguous interpretation of biological data. Herein we report a simple, sensitive, and specific method to determine residual Mn2+ in Mn porphyrin preparations that combines a hydrometallurgical approach for separation/speciation of metal compounds with a spectrophotometric strategy for Mn determination. The method requires only common chemicals and a spectrophotometer and is based on the extraction of residual Mn2+ by bis(2-ethylhexyl)hydrogenphosphate (D2EHPA) into kerosene, re-extraction into acid, and neutralization followed by UV-vis determination of the Mn2+ levels via a Cd2+-catalyzed metallation of the H2TCPP4- porphyrin indicator. The overall procedure is simple, sensitive, specific, and amenable to adaptation. This quantification method has been routinely used by us for a large variety of water-soluble porphyrins.