Identification of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in patients with erythema migrans

Anaplasma phagocytophilum has been first isolated from the blood of two Czech patients simultaneously with a cultivation of Borrelia burgdorferi sensu lato from their erythema migrans lesions. Cultivation of different Borrelia spp. from 12 erythema migrans biopsies, from 2 blood, one liquor and one placenta sample in BSK-H medium was successful. Adapted conventional methods targeting 16S rRNA and OspA genes for real-time polymerase chain reaction (PCR) and partial sequencing of these genes together with microscopical examinations of the blood smears provided a direct detection of the B. afzelii, B. burgdorferi, B. garinii, B. valaisiana and B. bissettii in the skin, B. garinii in the blood, placenta and liquor in 24 (36.3 %) patients, and A. phagocytophilum in 10 (15 %) patients with erythema migrans. Positive indirect IgM immunofluorescence against Anaplasma sp. was obtained in 7 cases, specific IgG antibodies were detected in 12 patients. Three women suffering from erythema migrans in the first trimester had positive PCR for Anaplasma and/or for Borrelia in the blood and two of them, later, in the placenta. Interpretation of laboratory data can bring important contribution to establishing the role of Anaplasma sp. in erythema migrans and forming the principle of precaution with laboratory diagnosis during pregnancy which always should be reflected in the resistance of Anaplasma sp. toward penicillins.     

Growth strategy of the pathogenic yeast <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> submerged culture under different cultivation formats

Growth patterns of Cryptococcus neoformans submerged culture in different culture volumes, intensity of agitation and types of sealing were evaluated to better understand the physiological role of hypoxia response in this yeast. When low intensity agitation was set at high culture volumes and air exchange between the cultivation vessel and external environment was not abolished completely, the cells proliferated slowly but steadily. On the other hand, when the intensity of agitation was high but the vessel was withheld from fresh air supply, the cells first proliferated rapidly, then arrested completely and finally died. Therefore, the central strategy of C. neoformans here seems to lie in its proliferation-rate adjustment to the available oxygen levels and not in its capacity to survive under anoxia. The data support the opinion that the cultures grown under limited aeration (even though not completely withheld from fresh air supply) are much closer to the real cryptococcal life in human tissues than conventional well-aerated exponential cultures.     

Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AM946602–AM946620 and FM872474–FM872476.     

Biocatalytic application of nitrilases from Fusarium solani O1 and Aspergillus niger K10

The nitrilases from Fusarium solani O1 and Aspergillus niger K10 showed a broad substrate specificity for carbocyclic and nonaromatic heterocyclic amino nitriles, the preferred substrates being five-membered γ-amino nitrile (±)-1a, six-membered γ-amino nitriles (±)-3a, (±)-5a and (±)-6a, pyrrolidine-3-carbonitriles (±)-9a and (±)-10a as well as piperidine-4-carbonitriles 14a and 15a. Both enzymes showed a strong diastereopreference for cis- vs. trans-γ-amino nitriles. The electronic and steric effects of N-protecting groups affected the reactivity of the nitriles. Amides as by-products of the nitrilase-catalyzed reaction were produced from heterocyclic amino nitriles (±)-9a, (±)-10a, 14a and 15a by the A. niger enzyme but only from nitrile (±)-9a by the F. solani enzyme.     

An evaluation of field and noninvasive genetic methods for estimating Eurasian otter population size

Successful conservation and management of rare and elusive species requires reliable estimates of population size, but acquisition of such data is often challenging. We compare the two most frequently used methods of assessing abundance of Eurasian otter (Lutra lutra) populations, noninvasive genetic sampling (NGS) based on genotyping of faeces and field surveys using snow tracking. In a 100-km² oligotrophic otter habitat with linear water bodies, both methods yielded very similar estimates (10–12 individuals). However, in a 100-km² fishpond area, consisting of a complex network of rivers, fishponds, channels and marshes, genotyping of faeces revealed the presence of a higher number of individuals (46–50 genotypes) than the snow survey (38 individuals). NGS data analysed by capture-mark-recapture (CMR)-based software CAPWIRE provided even higher estimates, being twice the number assessed through snow tracking (76–81 individuals, CI_95% = 49–96 and 55–89). Our results suggest that the performance of both NGS and snow tracking is comparable in simple linear habitats, but in complex habitats with very high otter density a combination of genetic and field methods, or CMR analysis using genetic data, is recommended. We emphasise that to obtain reliable estimates using NGS it is necessary to follow strict protocols for detection and elimination of genotyping errors. Based on a literature review and our experience, we suggest improvements that may increase the success rate and efficiency of NGS for otters.     

High Arctic vegetation after 70 years: a repeated analysis from Svalbard

We asked whether vegetation mapping repeated after 70 years revealed vegetation changes in the high Arctic. The study site is located at 78°38′N, 16°45′E, near Brucebyen at the Adolfbukta Bay (head of Billjefjorden) in central Spitsbergen (Svalbard), and encompasses an area of 2,042 × 521 m. The mapping carried out in 2008 did not reveal any changes in vegetation, since a previous study in 1936–1937, that could be attributed to climate change. We argue that our finding can be interpreted as evidence of a slow ecological response of constituent plants in such a harsh environment. Moreover, geographic isolation may limit establishment and expansion of new species. Some successional changes were only due to erosion–accumulation processes connected especially to stream activity.     

β-N-Acetylhexosaminidase: What's in a name…?

β-N-Acetylhexosaminidases (EC 3.2.1.52, belonging to CAZy GH families 3, 20 and 84) have recently gained a lot of attention, not only due to their implication in human physiology and disease, but also due to their great potential in the enzymatic synthesis of carbohydrates and glycomimetics. GH family 20 β-N-acetylhexosaminidases, and GH family 3 and 84 β-N-acetylglucosaminidases from all kinds of organisms have been intensively studied from the point of view of their physiological roles, reaction mechanisms, structure and inhibition. Thanks to their outstanding substrate promiscuity, extracellular β-N-acetylhexosaminidases from filamentous fungi are able to cleave and transfer substrates bearing various functionalities, ranging from carboxylates, sulfates, acylations to azides, and even 4-deoxy glycosides. Thus, they have proved to be versatile biosynthetic tools for the preparation of both natural and modified hexosaminides under mild conditions with good yields.     

Applications of the Cartesian coordinate tensor transfer technique in the simulations of vibrational circular dichroism spectra of oligonucleotides

The application of the Cartesian coordinate tensor transfer (CCT) technique for simulations of the IR absorption and vibrational circular dichroism (VCD) spectra of relatively large nucleic acid fragments is demonstrated on several case studies. The approach is based on direct ab initio calculations of atomic tensors, determining molecular properties, for relatively small fragments, and subsequent transfer of these tensors to the larger systems in Cartesian coordinates. This procedure enables precise computations of vibrational spectra for large biomolecular systems, currently with up to several thousands of atoms. The versatile ability of the CCT methods is emphasized on the examples of VCD and IR absorption spectra calculations for B- and Z-forms of DNA, single-, double-, and triple-stranded RNA helices and DNA structures with different base content and sequences. The development and recent improvements of the methodology are followed, including utilization of the constrained normal mode optimization (NMO) strategy and combined quantum mechanics and molecular dynamics simulations. Advantages, drawbacks, and recommendations for future improvements of the CCT method as applied to nucleic acid spectra calculations are discussed.     

Understory vegetation in reclaimed and unreclaimed post-mining forest stands

In the Sokolov coal mining district of the Czech Republic, spoil heaps are reclaimed by forest plantations, which are planted directly into the rough substrate (alkaline tertiary clay). We compared the understory that spontaneously developed in seven types of forest stands: one type was unreclaimed stands (spontaneously overgrown by Betula pendula and Salix caprea) and six were plantations, each dominated by one tree genus (Alnus, Larix, Picea, Pinus, Quercus, and Tilia). The age of the stands ranged from 22 to 33 years. The cover of understory plant species in each stand was estimated, and 16 other environmental and community variables were quantified. The number of plant species was highest in Quercus, Larix, and unreclaimed stands, and was negatively correlated with forest canopy cover and with the cover of the understory dominant, the grass Calamagrostis epigejos. Understory composition differed considerably among the types of forest stands and was significantly explained by the measured environmental variables. Forward selection in redundancy analysis indicated that the most important variable driving understory composition was thickness of the fermentation layer, which is clearly connected with soil development. Environmental variables, including fermentation layer, were also affected by the type of forest stand. Therefore, all of the explained variability in understory composition could be attributed to the type of forest stand, which apparently affected the understory by its impact on soil formation. However, the most favorable soil conditions were not favorable for understory development, as they supported mainly C. epigejos, which suppressed other species. Our study also showed that even in the absence of reclamation measures, mining sites can be successfully restored due to spontaneous succession.     

Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes

Epithelial–mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these growth factors was also detected in tumor samples.