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Reactive oxygen species (ROS) are signaling molecules essential for plant responses to abiotic and biotic stimuli as well as for multiple developmental processes. They are produced as byproducts of aerobic metabolism and are affected by adverse environmental conditions. The ROS content is controlled on the side of their production but also by scavenging machinery. Antioxidant enzymes represent a major ROS-scavenging force and are crucial for stress tolerance in plants. Enzymatic antioxidant defense occurs as a series of redox reactions for ROS elimination. Therefore, the deregulation of the antioxidant machinery may lead to the overaccumulation of ROS in plants, with negative consequences both in terms of plant development and resistance to environmental challenges. The transcriptional activation of antioxidant enzymes accompanies the long-term exposure of plants to unfavorable environmental conditions. Fast ROS production requires the immediate mobilization of the antioxidant defense system, which may occur via retrograde signaling, redox-based modifications, and the phosphorylation of ROS detoxifying enzymes. This review aimed to summarize the current knowledge on signaling processes regulating the enzymatic antioxidant capacity of plants.

Drivers of the speciation in bacteria, including geographical isolation and horizontal gene transfer, are still poorly understood. Here, we characterized a new lineage within an anciently diverged thermophilic cyanobacteria. We sequenced the whole genome of a strain Thermostichus vulcanus isolated from the Rupite spring (Bulgaria), which is closely related to Theromstichus strains JA-2-3Aa and JA-2-3B′a(2-13). We performed phylogenetic inference, horizontal gene transfer estimation, and CRISPR/Cas system characterization. We found that the Rupite strain is a distinct species from strains JA-2-3Aa, and JA-2-3B′a(2-13). Furthermore, the horizontal gene transfer seemed to be more frequent among the geographically distant species than between the two species within the same hot spring. The CRISPR/Cas system had variable complexity among the species of Thermostichus in terms of both the number of spacers and genes. The Rupite strain had the highest, and JA-2-3Aa the lowest number of spacers among the analyzed strains, and the CRISPR spacers were only rarely shared among the strains. We conclude that the CRISPR/Cas system size varied among the lineages as well as the gene flow.

Annexin 1 (ANN1) is the most abundant member of the evolutionary conserved multigene protein superfamily of annexins in plants. Generally, annexins participate in diverse cellular processes, such as cell growth, differentiation, vesicle trafficking, and stress responses. The expression of annexins is developmentally regulated, and it is sensitive to the external environment. ANN1 is expressed in almost all Arabidopsis tissues, while the most abundant is in the root, root hairs, and in the hypocotyl epidermal cells. Annexins were also occasionally proposed to associate with cytoskeleton and vesicles, but they were never developmentally localized at the subcellular level in diverse plant tissues and organs. Using advanced light-sheet fluorescence microscopy (LSFM), we followed the developmental and subcellular localization of GFP-tagged ANN1 in post-embryonic Arabidopsis organs. By contrast to conventional microscopy, LSFM allowed long-term imaging of ANN1-GFP in Arabidopsis plants at near-environmental conditions without affecting plant viability. We studied developmental regulation of ANN1-GFP expression and localization in growing Arabidopsis roots: strong accumulation was found in the root cap and epidermal cells (preferentially in elongating trichoblasts), but it was depleted in dividing cells localized in deeper layers of the root meristem. During root hair development, ANN1-GFP accumulated at the tips of emerging and growing root hairs, which was accompanied by decreased abundance in the trichoblasts. In aerial plant parts, ANN1-GFP was localized mainly in the cortical cytoplasm of trichomes and epidermal cells of hypocotyls, cotyledons, true leaves, and their petioles. At the subcellular level, ANN1-GFP was enriched at the plasma membrane (PM) and vesicles of non-dividing cells and in mitotic and cytokinetic microtubular arrays of dividing cells. Additionally, an independent immunolocalization method confirmed ANN1-GFP association with mitotic and cytokinetic microtubules (PPBs and phragmoplasts) in dividing cells of the lateral root cap. Lattice LSFM revealed subcellular accumulation of ANN1-GFP around the nuclear envelope of elongating trichoblasts. Massive relocation and accumulation of ANN1-GFP at the PM and in Hechtian strands and reticulum in plasmolyzed cells suggest a possible osmoprotective role of ANN1-GFP during plasmolysis/deplasmolysis cycle. This study shows complex developmental and subcellular localization patterns of ANN1 in living Arabidopsis plants.

For several decades, researchers are working to develop improved major crops with better adaptability and tolerance to environmental stresses. Forage legumes have been widely spread in the world due to their great ecological and economic values. Abiotic and biotic stresses are main factors limiting legume production, however, alfalfa (Medicago sativa L.) shows relatively high level of tolerance to drought and salt stress. Efforts focused on alfalfa improvements have led to the release of cultivars with new traits of agronomic importance such as high yield, better stress tolerance or forage quality. Alfalfa has very high nutritional value due to its efficient symbiotic association with nitrogen-fixing bacteria, while deep root system can help to prevent soil water loss in dry lands. The use of modern biotechnology tools is challenging in alfalfa since full genome, unlike to its close relative barrel medic (Medicago truncatula Gaertn.), was not released yet. Identification, isolation, and improvement of genes involved in abiotic or biotic stress response significantly contributed to the progress of our understanding how crop plants cope with these environmental challenges. In this review, we provide an overview of the progress that has been made in high-throughput sequencing, characterization of genes for abiotic or biotic stress tolerance, gene editing, as well as proteomic and metabolomics techniques bearing biotechnological potential for alfalfa improvement.

Anaerobic digestion (AD) plants undoubtedly represent an integral and irreplaceable element in rural energy transition and sustainable waste management. In our study, we focus on an advanced understanding of the dynamics behind the changing perceptions of AD plants in host communities in Poland, Slovakia, and the Czech Republic. The surveyed AD plant case studies were selected to represent best-practice examples of AD plant operations in their countries. By means of a comparative survey in three host communities, we seek to learn more about the shift in perceptions of AD plants between their planning and operational phases. We find that, although our cases are considered best-practice examples of AD plant operations, their overall support in their communities dramatically dropped in the operational phase consequent to real-life experience with living in the neighbourhood of an AD plant. Additionally, our findings indicate that respondents who reported the most severe deterioration of AD plant perceptions were, surprisingly, those who had participated in the planning process more than other respondents. The most frequently mentioned type of participation in the planning phase was reported to be visits to the local administration office to inspect an AD plant’s planning documentation. We argue that deteriorating perceptions of best-practice examples of AD plant operations negatively impact the further development and acceptance of the biogas sector in Eastern Europe. The element of acceptance by the locals of AD plant operations urgently requires reflection in the definition of best practices to inspire and upgrade both existing and planned biogas energy projects.

The aim of the study was to evaluate total antioxidant activity (TAA) and total content of carotenoids (TCC), anthocyanins (TAC), phenolics (TPC) and phenolic acids (PAs) in grain of selected pigmented wheat genotypes and traditional control cultivar cultivated under organic and conventional cropping systems in two-year trials. All of the evaluated parameters were significantly affected both by genotype and evaluated environmental factors. While in TPC, PAs and TCC the effect of years prevailed, TAC was affected mainly by genotype. The effect of genotype and year in TAA was comparable. TPC ranged from 581.71 mg/kg (control cv. Annie) to 723.60 mg/kg (cultivar with purple pericarp PS Karkulka), total PAs content from 711.77 mg/kg (cv. PS Karkulka) to 849.47 mg/kg (cv. Skorpion with blue aleurone). TCC varied from 1.56 mg/kg (cv. PS Karkulka) to 5.32 mg/kg (cv. Citrus with yellow endosperm). The highest TAC (63.23 mg/kg) was found in cv. Skorpion, the lowest (12.70 mg/kg) in cv. AF Jumiko with purple pericarp. Anthocyanins were not detected in cvs. Annie and Citrus. TAA varied from 162.68 mg/kg in cv. Annie to 226.71 mg/kg in breeding line KM 53-14 with blue aleurone. Higher TAA and antioxidants contents and lower grain yields were observed in organic cropping system and in drier year 2016.

Facilities for generating renewable energy form important elements in the rural landscape of the Czech Republic. The distribution of these facilities is highly uneven due to various natural and socio-economic factors. In our paper, we are focusing our attention on one of the important facilities for the generation of renewable energy in the Czech Republic; anaerobic digestion (AD) plants. In 2016, more than five hundred AD plants could be found in the country with a total installed capacity of 360 MW. By means of analyses of data on location of individual AD plants, quality of soils, size of agricultural farms (in which majority of AD plants is incorporated) we found that agricultural AD plants in the Czech Republic tend to concentrate in areas with larger agricultural farms and also in areas with average and slightly below average conditions for agricultural activities. Core areas where agricultural AD plants are the most densely located were also identified. In these areas, large farms with AD plants tend to crowd out smaller farms. Pressure to grow maize for ADs significantly competes here with growing of other agricultural crops for food. This finding is in strong contradiction with the official national agricultural policy where support for small farms is systematically emphasized. Core areas with AD plants make a belt that mostly covers sub-mountainous central parts of the Czech Republic where traditional agricultural plants are now significantly being displaced by maize to feed AD plants.

Common carp (Cyprinus carpio) aquaculture is one of the most important and rapidly growing productions around the world. However, for consumers, carp is often not acceptable due to its distinctive colour and odour. In this study, we investigated the effects of bleeding of common carp on fillet quality. The obtained results show that carp bleeding by cutting the gill arches is an effective way of reducing the total haem content, which here decreased from (9.6±1.6) in unbled carp to (2.34±0.8) μmol/kg of haemoglobin in bled carp. Furthermore, fillets from bled carp showed reduced formation of primary and secondary lipid oxidation products and growth of microorganisms during 12 days of refrigerated aerobic storage. On the last day of storage, the amount of lipid hydroperoxides decreased from (88.9±4.2) in unbled to (62.1±2.9) μmol/kg of cumene hydroperoxide in bled carp, TBARS decreased from (4.2±0.5) in unbled to (2.6±0.4) μmol/kg of malondialdehyde in bled carp, mesophilic and psychrotrophic bacteria count decreased from (6.4±0.1) and (6.2±0.3) log CFU/g in unbled to (4.0±0.2) and (4.2±0.2) log CFU/g in bled carp, respectively. These raw bled fillets showed increased lightness L*, and reduced redness a* and yellowness b* compared to unbled fillets. Sensory analysis showed improved colour, odour and overall acceptability of bled raw fillets. Overall, bleeding improves the quality of carp fillets. Thus, inclusion of bleeding into processing of carp fillets has the potential to improve their acceptance by consumers and prolong their shelf-life.

Fortification structures, both military and civilian, are designed to resist a blast explosion to some extent. Their technical condition after a blast load must be assessed in a fast and reliable way to enable the users’ decision about the future use of the structure. Preferably, for the assessment of the protective structure, the non-destructive testing method should be used. To assess the suitability of ultrasound testing method for fortification structures built from steel fiber reinforced concrete, an investigation in a laboratory and in situ was conducted, together with numerical simulation and statistical evaluation. The numerical simulation of the blast load of a steel fiber reinforced concrete slab was conducted using multiphysics simulation software with the aim to verify basic parameters of the field experiment. During the field tests, several slabs were loaded by plastic explosive and changes in the structure of the slabs, before and after the blast load, were examined using the ultrasound pass-through method. After the field tests, the slabs were subjected to a destructive laboratory test to determine their residual strength. Subsequently, the data sets obtained from the measurements were tested using functional data analysis. The results from the ultrasound pulse method show that specimens after a dynamic blast load can in some cases increase the strength of their cement matrix.

Our paper deals with a micro-study of one residential building in the city center of Brno (Czech Republic) where we strived to identify and better understand the main factors behind the successful implementation of environmentally friendly solutions during the regeneration process. We followed the unique, complicated, and often conflictual story of the regeneration (conducted during the years 2010–2020) of the residential building, which was originally built in the 1930s. In total, 18 solutions were discussed—all four solutions on the state level of centralization were realized, only two of six solutions on the building level of centralization were materialized, and six of eight decentralized solutions were realized during the regeneration process. In the field of energy savings requiring high investments, a significant dominance of centralized solutions (on the state level) was identified. Centralized solutions on the building level such as heat pumps or solar panels were not realized. In the area of waste management and care for community greenery (that did not require large investments), we see as the most beneficial the promotion of decentralized solutions in the form of community-funded communal composting or the planting of new greenery. The formation of various regeneration options, which is discussed in detail, appeared as an integral instrument for dealing with conflicts among residents during the planning phase.

Green marketing is possibly the newest type of marketing. Recently, it has also been the most discussed one by organizations, companies and even states. Green marketing tries to produce, promote and recycle products that are friendly to the environment. Green marketing is a global concern and it is going to have a better future. However, this type of marketing meets a few problems such as much effort to replace conventional products and a lack of confidence. Many companies produce and promote such products as much as possible. In spite of this, the public is still sceptical. The paper presents the theoretical important knowledge on green marketing, its definitions, customer behaviour formulas, and its potential.

This study elucidated the stage-specific roles of FGF2 signaling during neural development using in-vitro human embryonic stem cell-based developmental modeling. We found that the dysregulation of FGF2 signaling prior to the onset of neural induction resulted in the malformation of neural rosettes (a neural tube-like structure), despite cells having undergone neural induction. The aberrant neural rosette formation may be attributed to the misplacement of ZO-1, which is a polarized tight junction protein and shown co-localized with FGF2/FGFR1 in the apical region of neural rosettes, subsequently led to abnormal neurogenesis. Moreover, the FGF2 signaling inhibition at the stage of neural rosettes caused a reduction in cell proliferation, an increase in numbers of cells with cell-cycle exit, and premature neurogenesis. These effects may be mediated by NUMB, to which expression was observed enriched in the apical region of neural rosettes after FGF2 signaling inhibition coinciding with the disappearance of PAX6+/Ki67+ neural stem cells and the emergence of MAP2+ neurons. Moreover, our results suggested that the hESC-based developmental system reserved a similar neural stem cell niche in vivo.

Currently, due to the elapsed time and the nature of the Chernobyl accident, the only artificial radionuclide present in the soil is 137Cs, with a physical half-life conversion of 30.17 years. The 137Cs is quickly integrated into a biological cycle, similar to potassium. Generally, radionuclides are characterized by their mobility in soil. Contamination of materials and food by radionuclides represent a serious problem and has a negative impact on human health. The threat of international terrorism and the inability to forestall the impact of natural disasters on nuclear energetic (Fukushima accident), are also reasons for continuous monitoring of food safety. According screening measurement performed in European countries, high radioactivity levels were reported in the wild boars muscles from Sumava (Czech Republic). Seasonal fluctuation of 137Cs activity in the wild boar meat samples was observed in the forests on the southern Rhineland. Monitoring of 137Cs activity in the wild boar meat samples in the hunting grounds in Slovakia was initiated based on the reports on exceeding limits of the content of radiocaesium in the meat of wild boar from the surrounding countries. The aim of this study was to determine the 137Cs post Chernobyl contamination of wild boars population in different hunting districts of Slovakia during 2013 - 2014. A total of 60 thigh muscle samples from wild boars of different age categories (4 months - 2 years) were evaluated. 137Cs activity was measured by gamma spectrometry (Canberra). Despite the fact Slovakia is closer to Chernobyl as Czech Republic and Germany, the 137Cs activity measured was very low and far below the permitted limit. The highest radiocaesium activity level measured in muscle was 37.2 Bq.kg-1 ±4.7%. Wild boar originated from Zlate Moravce district. The measurement results show, that 137Cs contamination levels of game in Slovakia are low. Radiocaesium activity in examined samples was very low and therefore consumption of wild boar meat does not represent a health risk problem.

We report on the fabrication of metasurface phase plates consisting of gold nanoantenna arrays that generate Laguerre-Gaussian modes from a circularly polarized Gaussian input beam. The corresponding helical phase profiles with radial discontinuities are encoded in the metasurfaces by the orientation of the nanoantennas. A common-path interferometer is used to determine the orbital angular momentum of the generated beams. Additionally, we employ digital holography to record the detailed phase profiles of the Laguerre-Gaussian modes. This method allows a simple and direct quantitative comparison of the measured phase profiles of the generated Laguerre-Gaussian beams with the theoretically expected phase profiles. Experiments with different laser sources demonstrate the broadband operation of the metasurfaces.

The rapid spread of invasive plants makes their management increasingly difficult. Remote sensing offers a means of fast and efficient monitoring, but still the optimal methodologies remain to be defined. The seasonal dynamics and spectral characteristics of the target invasive species are important factors, since, at certain time of the vegetation season (e.g., at flowering or senescing), plants are often more distinct (or more visible beneath the canopy). Our aim was to establish fast, repeatable and a cost-efficient, computer-assisted method applicable over larger areas, to reduce the costs of extensive field campaigns. To achieve this goal, we examined how the timing of monitoring affects the detection of noxious plant invaders in Central Europe, using two model herbaceous species with markedly different phenological, structural, and spectral characteristics. They are giant hogweed (Heracleum mantegazzianum), a species with very distinct flowering phase, and the less distinct knotweeds (Fallopia japonica, F. sachalinensis, and their hybrid F. × bohemica). The variety of data generated, such as imagery from purposely-designed, unmanned aircraft vehicle (UAV), and VHR satellite, and aerial color orthophotos enabled us to assess the effects of spectral, spatial, and temporal resolution (i.e., the target species' phenological state) for successful recognition. The demands for both spatial and spectral resolution depended largely on the target plant species. In the case that a species was sampled at the most distinct phenological phase, high accuracy was achieved even with lower spectral resolution of our low-cost UAV. This demonstrates that proper timing can to some extent compensate for the lower spectral resolution. The results of our study could serve as a basis for identifying priorities for management, targeted at localities with the greatest risk of invasive species' spread and, once eradicated, to monitor over time any return. The best mapping strategy should reflect morphological and structural features of the target plant and choose appropriate spatial, spectral, and temporal resolution. The UAV enables flexible data acquisition for required time periods at low cost and is, therefore, well-suited for targeted monitoring; while satellite imagery provides the best solution for larger areas. Nonetheless, users must be aware of their limits.

The coal has been mined in the Karviná area for more than 150 years. During the course of time mining areas were continuously extended at the expense of the settled areas. At the beginnings the mining was limited, but later, namely under conditions of centrally planned economy in the period between 1950s and 1980s, it was heavily intensified. Then, as a result of economic restructuring of the Czech Republic mining was reduced in the region to be re-developed in the last decade again. The expansion of coal mining has been continuously affecting the socio-economic structure of local population (huge working immigration, industrialisation, construction of mass housing for miners – miners dormitories, later housing estates etc., displacement of settlements), surrounding landscape (subsidence of terrain, undermining, hydrological changes and formation of artificial lakes, occurrence of plenty of post-mining brownfields after the reduction of mining etc.), but also contemporary outward, image and socio-spatial structure of cities (dominance housing in housing estates, effects of communists spatial urban planning etc.). Currently, negotiations about expansion of coal mining are in progress in this region, namely the city parts of Karviná (Staré Město) and Orlová (Výhoda) should be affected. In the introductory parts of this paper social, economical and environmental aspects of coal mining on the development of regions and connected problems are discussed and both cities are shortly presented. The questionnaire survey focused on perceptions of after-mining renewal and potential expansion of mining in the area of cities of Karviná and Orlová was conducted (n=1000). As the most important predictor that influences perception of mining and renewal, employment in mining companies has been identified. The higher education respondents achieved, the higher level of opposition against mining was manifested. The correlation was also found between the level of income and the agreement with expansion of mining. It can be concluded that employees of mining company, less educated population and people with higher income agreed with economic development of the city that is based on mining.

This article deals with the issue of brownfield redevelopment for the needs of solar energy projects from different perspectives. Attention is first paid to a brief characterisation of the data and information available both for brownfields and for solar energy projects in the Czech Republic. Then there is description from the most important results of GIS analysis, which (based on a comparison of aerial images from different years) identified the previous uses at the 127 solar power plants with an output of over 1 MWp in the South Moravian Region. The results of the quantitative analyses are accompanied by knowledge from a qualitative survey conducted on selected actors participating in this kind of brownfield regeneration for solar energy development. The conclusion contains proposals which could contribute to the more intensive future use of brownfields for the needs of solar energy in the Czech Republic (a similar process to what has already successfully been realised in Germany). The research results can be inspiring not only for the Czech Republic but also for other EU-member counrties where brownfields have not been re-used for the needs of solar energy projects or where brownfields have been used in this way only marginally.

The possible use of leaching residue from leaching deep-sea nodules in SO2/H2SO4/H2O medium as a low-cost adsorbent of heavy metals (Pb(II), Cd(II), Cu(II), Ni(II), Co(II), As(V)) was studied. The leaching residue was found to be an effective adsorbent for all of the tested elements; however, it was inactive in the solution containing As(V). The chemical activation of adsorbent in 10 vol. % HCl resulted in the greatest improvement of adsorption properties, while the activation in 10 vol. % HNO3 and heat treatment at 250 °C did not significantly affect the sorption characteristics of treated adsorbents compared with the original leaching residue. After HCl activation, the maximal adsorption capacities for lead (12.0 mg/g at pH 5.0 after 1 h), nickel (3.1 mg/g at pH 5.5 after 4 h) and cobalt (2.0 mg/g at pH 5.0 after 2 h) were achieved. Additional mechanical treatment connected with HCl activation provided the highest adsorption capacities for cadmium (11.5 mg/g at pH 4.0) and copper (5.7 mg/g at pH 4.5). Coprecipitation of Fe/Al-based particles on the surface of the leaching residue increased As(V) removal of the adsorbent. Surface coating based on AlIII was extremely effective, causing the increase of the adsorption capacity from 0 with the original leaching residue, to 28.1 mg/g (pH 7.0, 24 min). Kinetics studies showed the rapid progress of adsorption for Pb(II), Cd(II), and As(V) in tens of minutes, while the adsorption of Cu(II), Ni(II) and Co(II) approached a steady state after 2 h.

In this study, processes based on roasting-leaching-crystallization steps and condensation-precipitation steps for Li2CO3 separation from spent Li/MnO2 batteries and lithium-containing wastewaters were developed and verified on a laboratory scale. Spent Li/MnO2 batteries were roasted under reduced pressure at 650°C, which split the castings and deactivated the batteries by reduction of LiMnO2 and MnO2 with residual lithium metal and graphite to form MnO and Li2CO3. The resultant lithium carbonate was selectively solubilised in water with manganese remaining in the leach residue. Li2CO3 of 99.5 % purity was obtained after evaporation of 95 % water. Processing of lithium-containing alkaline wastewaters from the production of liquid rubber comprises condensation up to lithium concentration of 12-13 g/l Li and a two-step precipitation of lithium carbonate using CO2 as a precipitation agent. Sparingly soluble Li2CO3 was produced in the second step at 95°C, whilst most impurities remain in the solution. Obtained lithium carbonate products contained on average more than 99.5 % Li2CO3. The lithium precipitation efficiency was about 90 %.

We present three cases of pneumopericardium following blunt chest trauma injury. All three patients were victims of road traffic accidents. All had multiple associated injuries and pneumopericardium was found as the additional finding. Pneumopericardium was treated conservatively with thoracic drains placement and patients observation. Transesophageal echocardiography was used as a method of choice for exclusion of cardiac air tamponade. All three patients survived.

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH4 cycling in rivers.

It has long been assumed that cyanobacteria have, as with other free-living microorganisms, a ubiquitous occurrence. Neither the geographical dispersal barriers nor allopatric speciation has been taken into account. We endeavoured to examine the spatial and temporal patterns of global distribution within populations of the cyanobacterium Microcoleus vaginatus, originated from three continents, and to evaluate the role of dispersal barriers in the evolution of free-living cyanobacteria. Complex phylogeographical approach was applied to assess the dispersal and evolutionary patterns in the cyanobacterium Microcoleus vaginatus (Oscillatoriales). We compared the 16S rRNA and 16S-23S ITS sequences of strains which had originated from three continents (North America, Europe, and Asia). The spatial distribution was investigated using a phylogenetic tree, network, as well as principal coordinate analysis (PCoA). A temporal characterization was inferred using molecular clocks, calibrated from fossil DNA. Data analysis revealed broad genetic diversity within M. vaginatus. Based on the phylogenetic tree, network, and PCoA analysis, the strains isolated in Europe were spatially separated from those which originated from Asia and North America. A chronogram showed a temporal limitation of dispersal barriers on the continental scale. Dispersal barriers and allopatric speciation had an important role in the evolution of M. vaginatus. However, these dispersal barriers did not have a permanent character; therefore, the genetic flow among populations on a continental scale was only temporarily present. Furthermore, M. vaginatus is a recently evolved species, which has been going through substantial evolutionary changes.

The efficient and reversible control of transgene expression is a powerful tool for the correct manipulation of embryonic stem cells in both cell therapy and transgenesis. The aim of this work was to investigate the possibilities of recently developed reverse tetracycline-controlled transactivator rtTA2$^{\rm S}$-S2. We show that the rtTA2$^{\rm S}$-S2 is useful for transient inducible expression of genes in embryonic stem cells. However, we found that it was not possible to establish mouse embryonic stem cell lines stably expressing this transactivator. Using the viral IRES sequence which couples the expression of rtTA2$^{\rm S}$-S2 and neomycin phosphotransferase, we found that embryonic stem cells expressing rtTA2$^{\rm S}$-S2 are not capable of growing in the presence of G418. Our results indicate that this transactivator is toxic to ES cells and raise the need for the development of other strategies for stable and inducible expression of genes in ES cells. [ABSTRACT FROM AUTHOR]