An extensive individual particle analysis of solid airborne particles collected in a moderately urbanized area.

Detailed individual particle characterization of PM10, in terms of particle size, morphology, and elemental composition, was done using scanning electron microscopy combined with energy-dispersive X-ray spectroscopy. The samples were collected in four localities in the Czech Republic (Central Europe), three of which are medium-sized cities, and one is a natural locality in the mountains. More than 1600 particles obtained from each locality were evaluated. During the sampling period (1.9.-8.9.2019), the atmospheric conditions were similar in the localities, which enabled the identification of PM10 characteristics common to all the sampling sites. Some differences in the particles' morphology and composition, arising from site-specific conditions, were observed too. The most abundant elements in the PM10 were C, O, Si, Fe, Al, Ca, Na, K, Mg, and S, but some toxic elements (Cr, Cu, and Ni) were also detected. The main component of the PM10 is carbon, whose multimodal distribution indicates that the particles contain different carbonaceous chemical compounds. The distribution of carbon in the natural locality was different compared to the other sites, suggesting a specific character of the sources of carbonaceous compounds in this region. Last but not least, a relationship between Al, Si, and O concentrations was found, which implies the presence of aluminosilicates and silicon dioxide (possibly sand) of crustal origin in the particles.

In-solution structure and oligomerization of human histone deacetylase 6 - an integrative approach.

Human histone deacetylase 6 (HDAC6) is a structurally unique, multidomain protein implicated in a variety of physiological processes including cytoskeletal remodelling and the maintenance of cellular homeostasis. Our current understanding of the HDAC6 structure is limited to isolated domains, and a holistic picture of the full-length protein structure, including possible domain interactions, is missing. Here, we used an integrative structural biology approach to build a solution model of HDAC6 by combining experimental data from several orthogonal biophysical techniques complemented by molecular modelling. We show that HDAC6 is best described as a mosaic of folded and intrinsically disordered domains that in-solution adopts an ensemble of conformations without any stable interactions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration-dependent manner, and its oligomerization is mediated via the positively charged N-terminal microtubule-binding domain. Our findings provide the first insights into the structure of full-length human HDAC6 and can be used as a basis for further research into structure function and physiological studies of this unique deacetylase.

Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed ß-propeller fold.

Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria - BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed ß-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for ß-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

Synthesis of ß-d-galactopyranoside-Presenting Glycoclusters, Investigation of Their Interactions with Pseudomonas aeruginosa Lectin A (PA-IL) and Evaluation of Their Anti-Adhesion Potential.

Pseudomonas aeruginosa is an opportunistic human pathogen associated with cystic fibrosis. This bacterium produces, among other virulence factors, a soluble d-galactose-specific lectin PA-IL (LecA). PA-IL plays an important role in the adhesion to the host cells and is also cytotoxic. Therefore, this protein is an interesting therapeutic target, suitable for inhibition by carbohydrate-based compounds. In the current study, ß-d-galactopyranoside-containing tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol equipped with propargyl groups were chosen as multivalent scaffolds and the galactoclusters were built from the above-mentioned cores by coupling ethylene or tetraethylene glycol-bridges and peracetylated propargyl ß-d-galactosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between galactoside derivatives and PA-IL was investigated by several biophysical methods, including hemagglutination inhibition assay, isothermal titration calorimetry, analytical ultracentrifugation, and surface plasmon resonance. Their ability to inhibit the adhesion of P. aeruginosa to bronchial cells was determined by ex vivo assay. The newly synthesized multivalent galactoclusters proved to be significantly better ligands than simple d-galactose for lectin PA-IL and as a result, two representatives of the dendrimers were able to decrease adhesion of P. aeruginosa to bronchial cells to approximately 32% and 42%, respectively. The results may provide an opportunity to develop anti-adhesion therapy for the treatment of P. aeruginosa infection.

Selecting appropriate variables for detecting grassland to cropland changes using high resolution satellite data.

Grassland is one of the most represented, while at the same time, ecologically endangered, land cover categories in the European Union. In view of the global climate change, detecting its change is growing in importance from both an environmental and a socio-economic point of view. A well-recognised tool for Land Use and Land Cover (LULC) Change Detection (CD), including grassland changes, is Remote Sensing (RS). An important aspect affecting the accuracy of change detection is finding the optimal indicators of LULC changes (i.e., variables). Inappropriately selected variables can produce inaccurate results burdened with a number of uncertainties. The aim of our study is to find the most suitable variables for the detection of grassland to cropland change, based on a pair of high resolution images acquired by the Landsat 8 satellite and from the vector database Land Parcel Identification System (LPIS). In total, 59 variables were used to create models using Generalised Linear Models (GLM), the quality of which was verified through multi-temporal object-based change detection. Satisfactory accuracy for the detection of grassland to cropland change was achieved using all of the statistically identified models. However, a three-variable model can be recommended for practical use, namely by combining the Normalised Difference Vegetation Index (NDVI), Wetness and Fifth components of Tasselled Cap. Increasing number of variables did not significantly improve the accuracy of detection, but rather complicated the interpretation of the results and was less accurate than detection based on the original Landsat 8 images. The results obtained using these three variables are applicable in landscape management, agriculture, subsidy policy, or in updating existing LULC databases. Further research implementing these variables in combination with spatial data obtained by other RS techniques is needed.

Structure and properties of AB21, a novel Agaricus bisporus protein with structural relation to bacterial pore-forming toxins.

We report the characterization of the dimeric protein AB21 from Agaricus bisporus, one of the most commonly and widely consumed mushrooms in the world. The protein shares no significant sequence similarity with any protein of known function, and it is the first characterized member of its protein family. The coding sequence of the ab21 gene was determined and the protein was expressed in E. coli in a recombinant form. We demonstrated a high thermal and pH stability of AB21 and proved the weak affinity of the protein to divalent ions of some transition metals (nickel, zinc, cadmium, and cobalt). The reported crystallographic structure exhibits an interesting rod-like helical bundle fold with structural similarity to bacterial toxins of the ClyA superfamily. By immunostaining, we demonstrated an abundance of AB21 in the fruiting bodies of A. bisporus.

Structural insights into Aspergillus fumigatus lectin specificity: AFL binding sites are functionally non-equivalent.

The Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications.

X-ray vs. NMR structure of N-terminal domain of δ-subunit of RNA polymerase.

The crystal structure of the N-terminal domain of the RNA polymerase δ subunit (Nδ) from Bacillus subtilis solved at a resolution of 2.0Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important ß sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nδ, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni(2+) ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.

A soluble fucose-specific lectin from Aspergillus fumigatus conidia--structure, specificity and possible role in fungal pathogenicity.

Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, Le(Y) being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,L-selenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus' conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection.

Stacking interactions between carbohydrate and protein quantified by combination of theoretical and experimental methods.

Carbohydrate-receptor interactions are an integral part of biological events. They play an important role in many cellular processes, such as cell-cell adhesion, cell differentiation and in-cell signaling. Carbohydrates can interact with a receptor by using several types of intermolecular interactions. One of the most important is the interaction of a carbohydrate's apolar part with aromatic amino acid residues, known as dispersion interaction or CH/π interaction. In the study presented here, we attempted for the first time to quantify how the CH/π interaction contributes to a more general carbohydrate-protein interaction. We used a combined experimental approach, creating single and double point mutants with high level computational methods, and applied both to Ralstonia solanacearum (RSL) lectin complexes with α-L-Me-fucoside. Experimentally measured binding affinities were compared with computed carbohydrate-aromatic amino acid residue interaction energies. Experimental binding affinities for the RSL wild type, phenylalanine and alanine mutants were -8.5, -7.1 and -4.1 kcal x mol(-1), respectively. These affinities agree with the computed dispersion interaction energy between carbohydrate and aromatic amino acid residues for RSL wild type and phenylalanine, with values -8.8, -7.9 kcal x mol(-1), excluding the alanine mutant where the interaction energy was -0.9 kcal x mol(-1). Molecular dynamics simulations show that discrepancy can be caused by creation of a new hydrogen bond between the α-L-Me-fucoside and RSL. Observed results suggest that in this and similar cases the carbohydrate-receptor interaction can be driven mainly by a dispersion interaction.