Intracellular trafficking and subcellular distribution of a large array of HPMA copolymers

The basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges or contained short hydrophobic peptides. The copolymers were fractionated to create parallel "ladders" consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition. The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import.     

Increasing incidence of Geomyces destructans fungus in bats from the Czech Republic and Slovakia

Background

White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown.

Methodology/Principal Findings

We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval.

Conclusions/Significance

G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.     

Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors

This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a β-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental β1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental β1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.     

Genetic mapping of DArT markers in the Festuca�CLolium complex and their use in freezing tolerance association analysis

Species belonging to the Festuca-Lolium complex are important forage and turf species and as such, have been studied intensively. However, their out-crossing nature and limited availability of molecular markers make genetic studies difficult. Here, we report on saturation of F. pratensis and L. multiflorum genetic maps using Diversity Array Technology (DArT) markers and the DArTFest array.The 530 and 149 DArT markers were placed on genetic maps of L. multiflorum and F. pratensis, respectively, with overlap of 20 markers, which mapped in both species. The markers were sequenced and comparative sequence analysis was performed between L. multiflorum, rice and Brachypodium. The utility of the DArTFest array was then tested on a Festulolium population FuRs0357 in an integrated analysis using the DArT marker map positions to study associations between markers and freezing tolerance. Ninety six markers were significantly associated with freezing tolerance and five of these markers were genetically mapped to chromosomes 2, 4 and 7. Three genomic loci associated with freezing tolerance in the FuRs0357 population co-localized with chromosome segments and QTLs previously identified to be associated with freezing tolerance. The present work clearly confirms the potential of the DArTFest array in genetic studies of the Festuca-Lolium complex. The annotated DArTFest array resources could accelerate further studies and improvement of desired traits in Festuca-Lolium species.     

Forage quality of leaf-fodder from the main broad-leaved woody species and its possible consequences for the Holocene development of forest vegetation in Central Europe

Leaf-hay was the principal winter feed of livestock from the Neolithic until the first archaeological records of scythes dated to the Iron Age (700–0 b.c.). Despite the use of meadow hay, leaf-fodder remained an important winter supplement until the present. Archaeological evidence lists Quercus, Tilia, Ulmus, Acer, Fraxinus and Corylus as woody species harvested for leaf-fodder, while Fagus, Populus or Carpinus were rarely used. The aim of our study was to test whether the use of listed woody species followed the pattern of their forage quality (syn. nutritive value). In late May 2012, we collected leaf biomass at four localities in the Czech Republic and determined concentrations of N, P, K, Ca, Mg, neutral- and acid-detergent fibre and lignin. Species with leaves of low forage quality were Carpinus betulus, Fagus sylvatica and Quercus robur, species with leaves of intermediate quality were Corylus avellana and Populus tremula and species with leaves of high quality were Ulmus glabra, Fraxinus excelsior, Tilia cordata and Acer platanoides. Selective browsing and harvesting of high quality species Acer, Fraxinus, Tilia and Ulmus thus probably supported their decline in the Bronze and Iron ages and supported the expansion of Carpinus and Fagus. Our results indicate that our ancestors’ practice of exploiting woody species as leaf-hay for winter fodder followed their nutritive value.     

Exclusion of livestock grazing and wood collection in dryland savannah: an effect on long‐term vegetation succession

Sahelian savannah faces increasing pressure from human activities, leading to its degradation. The aim of this study was to investigate the possibility of restoration of dryland savannah vegetation by the elimination of disturbance factors on the ecosystem. Is degraded dryland savannah vegetation able to be restored by means of natural succession? What is the timescale for its recovery? The study took place in the Bandia Reserve, 65 km south‐east of Dakar (Senegal), a unique site with two successional stages due to the elimination of uncontrolled exploitation. The vegetation structure of 15 years (15YRS) and 5 years (5YRS) after fencing was compared with vegetation exposed to continuous livestock grazing and wood collection outside the fenced area. Calculated by redundancy analysis, a significant effect of selected areas on the cover of all species was revealed and successional stage explained more than 45% of data variability. Perennial forbs, annual forbs and perennial grasses achieved the highest cover in 5YRS, woody species in 15YRS and annual grasses in the area outside of the fenced reserve. The dominant woody species Acacia seyal, A. ataxacantha, A. nilotica subsp. adstringens and Balanites aegyptiaca reconstituted the dense formation of Acacia bushland by means of natural succession in the 15YRS area.     

The negative effect of magnetic nanoparticles with ascorbic acid on peritoneal macrophages

Neurochemical Research - Superparamagnetic iron oxide nanoparticles (SPIOn) are widely used as a contrast agent for cell labeling. Macrophages are the first line of defense of organisms in contact...