Effect of daptomycin and vancomycin on Staphylococcus epidermidis biofilms: An in vitro assessment using fluorescence in situ hybridization

Colonization of in-dwelling catheters by microbial biofilms is a major concern in patient health eventually leading to catheter-related blood stream infections. Biofilms are less susceptible to standard antibiotic therapies that are effective against planktonic bacteria. Standard procedure for the detection of microorganisms on the catheter tip is culture. However, viable but non-culturable cells (VBNCs) may be missed. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) as an indicator to visualize and quantify the effect of the antibiotics daptomycin and vancomycin on biofilms in situ. We established an in vitro catheter biofilm model of Staphylococcus epidermidis biofilms on polyurethane catheters. Biofilm activity was measured by FISH and correlated to colony forming units (CFU) data. Digital image analysis was used for quantification of total biofilm mass and the area of the FISH positive biofilm cells. FISH showed a pronounced effect of both antibiotics on the biofilms, with daptomycin having a significantly stronger effect in terms of both reduction of biofilm mass and number of FISH-positive cells. This supports the anti-biofilm capacity of daptomycin. Interestingly, neither antibiotic was able to eradicate all of the FISH-positive cells. In summary, FISH succeeded in visualization, quantification, and localization of antibiotic activity on biofilms. This technique adds a new tool to the arsenal of test systems for anti-biofilm compounds. FISH is a valuable complementary technique to CFU since it can be highly standardized and provides information on biofilm architecture and quantity and localization of survivor cells

Diffusion of single active-dipolar cubes in applied fields

"Active matter" refers to a class of out-of-equilibrium systems whose ability to transform environmental energy to kinetic energy is sought after in multiple fields of science and at very different length scales. At microscopic scales, an important challenge lies in overpowering the particles reorientation due to thermal fluctuations, especially in nano-sized systems, to create non-random, directed motion, needed for a wide range of possible applications. In this article, we employ molecular dynamics simulations to show that the diffusion of a self-propelling dipolar nanocube can be enhanced in a pre-defined direction with the help of a moderately strong applied magnetic field, overruling the effect of the thermal fluctuations. Furthermore, we show that the direction of diffusion is given by the orientation of the net internal magnetisation of the cube. This can be used to determine experimentally the latter in synthetically crafted active cobalt ferrite nanocubes.Comment: 10 pages, 7 Figures, 1 Tabl

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor

Adipose tissue arachidonic acid content is associated with the expression of 5-lipoxygenase in atherosclerotic plaques

BACKGROUND: The content of arachidonic acid in adipose tissue is positively associated with the risk of myocardial infarction, whereas the content of eicosapentaenoic acid in adipose tissue has been reported to be negatively associated with the risk of myocardial infarction. Both arachidonic acid and eicosapentaenoic acid are substrates for the synthesis of pro-inflammatory leukotrienes and leukotrienes derived from eicosapentaenoic acid are generally much less potent. In this study we hypothesized that a high content of arachidonic acid in adipose tissue would reflect a high formation of arachidonic acid derived leukotrienes and a high expression of 5-lipoxygenase in atherosclerotic plaques. Likewise, we hypothesized that a high content of eicosapentaenoic acid in adipose tissue would reflect a low formation of arachidonic acid derived leukotrienes and a low expression of 5-lipoxygenase in plaques. METHODS: In a cross sectional study we included 45 consecutive subjects undergoing femoral thrombendarterectomy. The expression of 5-lipoxygenase in plaques was assessed by a semi-automated image analysis computer programme after immunohistochemical staining with mono-clonal 5-lipoxygenase antibodies. Leukotriene B(4) and cysteinyl leukotriene formation from stimulated femoral artery plaques was quantified using ELISA methods. The fatty acid content of adipose tissue biopsies from the thigh was analyzed using gas chromatography. Associations between variables were assessed by Pearson correlations and were further explored in a multivariable linear regression model adjusting for potential confounders. RESULTS: A high content of arachidonic acid in adipose tissue was associated with a higher expression of 5-lipoxygenase in plaques (r = 0.32, p = 0.03), but no significant associations with leukotriene B(4) (r = 0.22, p = 0.14) and cysteinyl leukotriene (r = −0.11, p = 0.46) formation was seen. No significant associations were found between the content of eicosapentaenoic acid in adipose tissue and 5-lipoxygenase expression or leukotriene formation in plaque. CONCLUSIONS: Adipose tissue arachidonic acid contents correlated positively with the expression of 5-lipoxygenase in plaques. This association might represent a causal link between adipose tissue arachidonic acid and the risk of myocardial infarction but confirmatory studies are needed

Contrasting macrophage activation by fine and ultrafine titanium dioxide particles is associated with different uptake mechanisms

Inhalation of (nano)particles may lead to pulmonary inflammation. However, the precise mechanisms of particle uptake and generation of inflammatory mediators by alveolar macrophages (AM) are still poorly understood. The aim of this study was to investigate the interactions between particles and AM and their associated pro-inflammatory effects in relation to particle size and physico-chemical properties

High environmental ozone levels lead to enhanced allergenicity of Birch pollen

BACKGROUND: Evidence is compelling for a positive correlation between climate change, urbanisation and prevalence of allergic sensitisation and diseases. The reason for this association is not clear to date. Some data point to a pro-allergenic effect of anthropogenic factors on susceptible individuals. OBJECTIVES: To evaluate the impact of urbanisation and climate change on pollen allergenicity. METHODS: Catkins were sampled from birch trees from different sites across the greater area of Munich, pollen were isolated and an urbanisation index, NO(2) and ozone exposure were determined. To estimate pollen allergenicity, allergen content and pollen-associated lipid mediators were measured in aqueous pollen extracts. Immune stimulatory and modulatory capacity of pollen was assessed by neutrophil migration assays and the potential of pollen to inhibit dendritic cell interleukin-12 response. In vivo allergenicity was assessed by skin prick tests. RESULTS: The study revealed ozone as a prominent environmental factor influencing the allergenicity of birch pollen. Enhanced allergenicity, as assessed in skin prick tests, was mirrored by enhanced allergen content. Beyond that, ozone induced changes in lipid composition and chemotactic and immune modulatory potential of the pollen. Higher ozone-exposed pollen was characterised by less immune modulatory but higher immune stimulatory potential. CONCLUSION: It is likely that future climate change along with increasing urbanisation will lead to rising ozone concentrations in the next decades. Our study indicates that ozone is a crucial factor leading to clinically relevant enhanced allergenicity of birch pollen. Thus, with increasing temperatures and increasing ozone levels, also symptoms of pollen allergic patients may increase further

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were(13)C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring(13)C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp281(6.48)of the highly conserved SWLP motif and Trp327(7.55)adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp116(23.50)was identified