| 1. |
- Bally, Marta, 1981, et al.
(author)
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Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context
- 2021
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In: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 413, s. 7157-7178
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Journal article (peer-reviewed)abstract
- The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.
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| 2. |
- Berg, F., et al.
(author)
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AFM-Based Quantification of Conformational Changes in DNA Caused by Reactive Oxygen Species
- 2015
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 119:1, s. 25-32
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Journal article (peer-reviewed)abstract
- Radical induced modification of DNA plays an important role in many pathological pathways like cancer development, aging, etc. In this work, we quantify radical-induced DNA damage that causes transitions from double to single stranded DNA using atomic force microscopy (AFM). The plasmid pBR322 is attacked by free hydroxyl radicals that are produced by Fenton's reaction; the strength of the radical attack is controlled via the ratio of hydroxyl radical molecules to DNA base pairs. The extent of DNA modification is assessed by AFM tapping mode (TM) imaging of the plasmids (after adsorption onto PAH-functionalized mica) in air. As single stranded DNA chains (height similar to 2 angstrom) are much smaller than intact DNA strands (similar to 5 angstrom), their fraction can be quantified based on the height distribution, which allows a simplified data analysis in comparison to similar AFM-based approaches. It is found that the amount of damaged DNA strands increases with increasing strength of radical attack, and decreases if ROS scavengers like sodium acetate are added. Competition curves are calculated for the interaction of hydroxyl radicals with DNA and sodium acetate, which finally allows calculation of relative rate constants for the respective reactions.
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| 3. |
- Berg, Johan, et al.
(author)
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Single Proteoliposomes with E.coli Quinol Oxidase : Proton Pumping without Transmembrane Leaks
- 2017
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In: Israel Journal of Chemistry. - : Wiley. - 0021-2148. ; 57:5, s. 437-445
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Journal article (peer-reviewed)abstract
- Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochromebo(3) from E.coli at single-molecule level. Liposomes with reconstituted cytochromebo(3) were loaded with a pH-sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single-molecule approach allowed us to determine the orientation of cytochromebo(3) in the membrane; in approximate to 70% of the protein-containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a pH (in the presence of the K+ ionophore valinomycin), which was stable over at least approximate to 800s. No rapid changes in pH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E.coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li etal. J. Am. Chem. Soc. (2015) 137, 16055-16063).
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| 4. |
- Block, Stephan, 1978, et al.
(author)
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Antenna-Enhanced Fluorescence Correlation Spectroscopy Resolves Calcium-Mediated Lipid-Lipid Interactions
- 2018
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In: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 12:4, s. 3272-3279
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Journal article (peer-reviewed)abstract
- Fluorescence correlation spectroscopy (FCS) has provided a wealth of information on the composition, structure, and dynamics of cell membranes. However, it has proved challenging to reach the spatial resolution required to resolve biophysical interactions at the nanometer scale relevant to many crucial membrane processes. In this work, we form artificial cell membranes on dimeric, nanoplasmonic antennas, which shrink the FCS probe volume down to the ∼20 nm length scale. By analyzing the autocorrelation functions associated with the fluorescence bursts from individual fluorescently tagged lipids moving through the antenna "hotspots", we show that the confinement of the optical readout volume below the diffraction limit allows the temporal resolution of FCS to be increased by up to 3 orders of magnitude. Employing this high spatial and temporal resolution to probe diffusion dynamics of individual dye-conjugated lipids, we further show that lipid molecules diffuse either as single entities or as pairs in the presence of calcium ions. Removal of calcium ions by addition of the chelator EDTA almost completely removes the complex contribution, in agreement with previous theoretical predications on the role of calcium ions in mediating transient interactions between zwitterionic lipids. We envision that antenna-enhanced FCS with single-molecule burst analysis will enable resolving a broad range of challenging membrane biophysics questions, such as stimuli-induced lipid clustering and membrane protein dynamics.
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| 5. |
- Block, Stephan, 1978, et al.
(author)
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Characterization of bonds formed between platelet factor 4 and negatively charged drugs using single molecule force spectroscopy
- 2014
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In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 10:16, s. 2775-2784
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Journal article (peer-reviewed)abstract
- Immunogenicity (i.e., the ability to initiate immune reactions) is one of the major challenges for the development of new drugs, as it may turn the developed drug therapeutically ineffective or cause severe immune-related effects. Using single molecule force spectroscopy, we study rupture forces between the positively charged, endogenous protein platelet factor 4 (PF4; also known as CXC chemokine ligand 4, CXCL4) and the antithrombotic drug heparin and other negatively charged glycosaminoglycans (GAGs), which are known to form immunogenic PF4/GAG-complexes (e. g., heparin and dextran sulfate) as well as non-immunogenic complexes (e. g., chondroitin sulfate A). Our measurements suggest that the average number of sulfate groups per monosaccharide unit (i.e., the degree of sulfation DS) does not affect the unbinding characteristics of single PF4/GAG-bonds (reaction coordinate x(0) = 2.2 +/- 0.2 angstrom, energy barrier Delta G approximate to -1 k(B)T). However, the average number of GAG bonds formed to a single PF4 molecule increases with increasing DS as indicated by a rising frequency of unbinding events, suggesting a multivalent binding scheme between PF4 and GAGs. Our studies show that at least three GAG bonds have to be formed to each PF4 molecule to induce epitope formation on the PF4/GAG-complex to which PF4/GAG-complex specific antibodies bind. Hence, GAG-based drugs that form less than three bonds per PF4 molecule are unlikely to constitute PF4/drug-complexes that are of immunologic relevance.
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| 6. |
- Block, Stephan, 1978
(author)
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Imaging and characterization of magnetic micro- and nanostructures using force microscopy
- 2015
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In: Surface Science Tools for Nanomaterials Characterization. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 9783662445518 ; , s. 489-529
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Book chapter (other academic/artistic)abstract
- This chapter introduces into the principles of different force microscopic approaches that sense a magnetic probe-sample force to study magnetism of micro- and (sub)nanometer-sized objects. Although all of them are capable to characterize magnetic properties on small length scales, their applicability depends strongly on the object (e.g., nm-thin magnetic films, magnetic nanoparticles, electronic and nuclear spins) to be investigated. A comparison of their application range will be given, which allows identifying the method most suitable for the intended measurement. Finally, the discussion of each approach is complemented by an overview about current exemplary applications.
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| 7. |
- Block, Stephan, 1978, et al.
(author)
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Quantification of Multivalent Interactions by Tracking Single Biological Nanoparticle Mobility on a Lipid Membrane
- 2016
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In: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 16:7, s. 4382-4390
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Journal article (peer-reviewed)abstract
- Macromolecular association commonly occurs via dynamic engagement of multiple weak bonds referred to as multivalent interactions. The distribution of the number of bonds, combined with their strong influence on the residence time, makes it very demanding to quantify this type of interaction. To address this challenge in the context of virology, we mimicked the virion association to a cell membrane by attaching lipid vesicles (100 nm diameter) to a supported lipid bilayer via multiple, identical cholesterol based DNA linker molecules, each mimicking an individual virion receptor link. Using total internal reflection microscopy to track single attached vesicles combined with a novel filtering approach, we show that histograms of the vesicle diffusion coefficient D exhibit a spectrum of distinct peaks, which are associated with vesicles differing in the number, n, of linking DNA tethers. These peaks are only observed if vesicles with transient changes in n are excluded from the analysis. D is found to be proportional to 1/n, in excellent agreement with the free draining model, allowing to quantify transient changes of n on the single vesicle level and to extract transition rates between individual linking states. Necessary imaging conditions to extend the analysis to multivalent interactions in general are also reported.
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| 8. |
- Block, Stephan, 1978, et al.
(author)
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Two-dimensional flow nanometry of biological nanoparticles for accurate determination of their size and emission intensity
- 2016
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 7, s. art no 12956 -
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Journal article (peer-reviewed)abstract
- Biological nanoparticles (BNPs) are of high interest due to their key role in various biological processes and use as biomarkers. BNP size and composition are decisive for their functions, but simultaneous determination of both properties with high accuracy remains challenging. Optical microscopy allows precise determination of fluorescence/scattering intensity, but not the size of individual BNPs. The latter is better determined by tracking their random motion in bulk, but the limited illumination volume for tracking this motion impedes reliable intensity determination. Here, we show that by attaching BNPs to a supported lipid bilayer, subjecting them to hydrodynamic flows and tracking their motion via surface-sensitive optical imaging enable determination of their diffusion coefficients and flow-induced drifts, from which accurate quantification of both BNP size and emission intensity can be made. For vesicles, the accuracy of this approach is demonstrated by resolving the expected radius-squared dependence of their fluorescence intensity for radii down to 15 nm.
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| 9. |
- Friedrich, R., et al.
(author)
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A nano flow cytometer for single lipid vesicle analysis
- 2017
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In: Lab on a Chip - Miniaturisation for Chemistry and Biology. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 17:5, s. 830-841
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Journal article (peer-reviewed)abstract
- We present a nanofluidic device for fluorescence-based detection and characterization of small lipid vesicles on a single particle basis. The device works like a nano flow cytometer where individual vesicles are visualized by fluorescence microscopy while passing through parallel nanochannels in a pressure-driven flow. An experiment requires less than 20 mu l sample volume to quantify both the vesicle content and the fluorescence signals emitted by individual vesicles. We show that the device can be used to accurately count the number of fluorescent synthetic lipid vesicles down to a vesicle concentration of 170 fM. We also show that the size-distribution of the vesicles can be resolved from their fluorescence intensity distribution after calibration. We demonstrate the applicability of the assay in two different examples. In the first, we use the nanofluidic device to determine the particle concentration in a sample containing cell-derived extracellular vesicles labelled with a lipophilic dye. In the second, we demonstrate that dual-color detection can be used to probe peptide binding to synthetic lipid vesicles; we identify a positive membrane-curvature sensing behavior of an arginine enriched version of the Antennapedia homeodomain peptide penetratin. Altogether, these results illustrate the potential of this nanofluidic-based methodology for characterization and quantification of small biological vesicles and their interactors without ensemble averaging. The device is therefore likely to find use as a quantitative analytical tool in a variety of fields ranging from diagnostics to fundamental biology research. Moreover, our results have potential to facilitate further development of automated lab-on-a-chip devices for vesicle analysis.
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| 10. |
- Hernandez, Victor Agmo, et al.
(author)
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The adhesion and spreading of thrombocyte vesicles on electrode surfaces
- 2008
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In: Bioelectrochemistry. - : Elsevier BV. - 1567-5394 .- 1878-562X. ; 74, s. 210-216
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Journal article (peer-reviewed)abstract
- The interaction of thrombocyte vesicles with the surface of metal electrodes, i.e., mercury, gold and gold electrodes modified with self assembled monolayers (SAM), was studied with the help of chronoamperometry, atomic force microscopy, and quartz crystal microbalance measurements. The experimental results show that the interaction of the thrombocyte vesicles with the surface of the electrodes depends on the hydrophobicity of the latter: whereas on very hydrophobic surfaces (mercury and gold functionalized with SAM) the thrombocyte vesicles disintegrate and form a monolayer of lipids. on the less hydrophobic gold surface a bilayer is formed. The chronoamperometric measurements indicate the possibility of future applications to probe membrane properties of thrombocytes. (C) 2008 Elsevier B.V. All rights reserved.
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