| 1. |
- Agnarsson, Björn, 1977, et al.
(author)
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Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells
- 2015
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In: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 9:12, s. 11849-11862
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Journal article (peer-reviewed)abstract
- Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 rim gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond.
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| 2. |
- Agnarsson, Björn, 1977, et al.
(author)
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Waveguide structure
- 2018
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Patent (other academic/artistic)abstract
- A waveguide structure for evanescent wave microscopy and/or spectroscopy, comprising an optically transparent core layer, a lower dielectric cladding layer and an upper dielectric cladding layer arranged on opposite sides of the core layer. The core layer has a refractive index higher than the refractive indices of the cladding layers. The upper cladding layer is made of an organic material. A sample well is arranged on an upper surface of the core layer formed by a cavity in the upper cladding layer, the sample well being adapted to contain a sample medium with one or more sample objects. The core layer is made of a first dielectric inorganic material, and the upper cladding layer has a refractive index which closely matches the refractive index of the sample medium. A method for manufacturing such waveguide structure, and a measurement system comprising the waveguide structure are also disclosed.
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| 3. |
- Altgärde, Noomi, 1983, et al.
(author)
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Mucin-like region of herpes simplex virus type 1 attachment protein gC modulates the virus-glycosaminoglycan interaction.
- 2015
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:35, s. 21473-21485
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Journal article (peer-reviewed)abstract
- Glycoprotein C (gC) mediates the attachment of herpes simplex virus type 1 (HSV-1) to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying an HSV-1 mutant lacking the mucin- like domain in gC and the corresponding purified mutant protein (gCΔmuc), in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared to native HSV-1, i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells, and reduced release of viral particles from the surface of infected cells. Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared to native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.
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| 4. |
- Bally, Marta
(author)
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Regulating the dynamic interactions between herpes simplex viruses and cell -surface glycosaminoglycans
- 2019
-
In: European Biophysics Journal. - : Springer. - 0175-7571 .- 1432-1017. ; 48, s. S41-S41
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Journal article (other academic/artistic)abstract
- Virus entry is a complex dynamic multistep process requiring a series of fine-tuned events mediating virus diffusion through the glycocalyx, its attachment to the cell membrane and lateral diffusion to the point of entry. A number of enveloped viruses, including herpes simplex viruses (HSV) attach to susceptible host cells via interaction between their glycoproteins and cell-surface glycosaminoglycans (GAGs). In our work, we study the molecular and physical mechanisms modulating HSV binding, diffusion and release from cell-surface glycosaminoglycans. Using single virus tracking in combination with either in vitro minimal models of the cell surface or live cell microscopy, we gain insights into the modulatory function of protein glycosylation (the presence of mucin-like regions on viral glycoproteins) and interrogate the role of GAG sulfation in the process. We show that mucin-like regions found on the glycoproteins of HSV-1 and HSV-2 play an important role in modulating the interaction, an observation further supported by cell experiments. We further show that the diffusion of virions on the surface depends on the type of GAGs and their degree of sulfation. Taken together, our research contributes to a better understanding of the mechanisms underlying the interaction between a virus and the surface of its host. Such insights will without doubt facilitate the design of more efficient antiviral drugs or vaccines.
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| 5. |
- Bernasconi, Valentina, 1989, et al.
(author)
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Mucosal Vaccine Development Based on Liposome Technology
- 2016
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In: Journal of Immunology Research. - : Hindawi Limited. - 2314-8861 .- 2314-7156.
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Journal article (peer-reviewed)abstract
- Immune protection against infectious diseases is most effective if located at the portal of entry of the pathogen. Hence, there is an increasing demand for vaccine formulations that can induce strong protective immunity following oral, respiratory, or genital tract administration. At present, only few mucosal vaccines are found on the market, but recent technological advancements and a better understanding of the principles that govern priming of mucosal immune responses have contributed to a more optimistic view on the future of mucosal vaccines. Compared to live attenuated vaccines, subcomponent vaccines, most often protein-based, are considered safer, more stable, and less complicated to manufacture, but they require the addition of nontoxic and clinically safe adjuvants to be effective. In addition, another limiting factor is the large antigen dose that usually is required for mucosal vaccines. Therefore, the combination ofmucosal adjuvantswith the recent progress in nanoparticle technology provides an attractive solution to these problems. In particular, the liposome technology is ideal for combining protein antigen and adjuvant into an effective mucosal vaccine. Here, we describe and discuss recent progress in nanoparticle formulations using various types of liposomes that convey strong promise for the successful development of the next generation of mucosal vaccines.
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| 6. |
- Delguste, Martin, et al.
(author)
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Regulatory Mechanisms of the Mucin-Like Region on Herpes Simplex Virus during Cellular Attachment
- 2019
-
In: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 14:3, s. 534-542
-
Journal article (peer-reviewed)abstract
- Mucin-like regions, characterized by a local high density of O-linked glycosylation, are found on the viral envelope glycoproteins of many viruses. Herpes simplex virus type 1 (HSV-1), for example, exhibits a mucin-like region on its glycoprotein gC, a viral protein involved in initial recruitment of the virus to the cell surface via interaction with sulfated glycosaminoglycans. So far, this mucin-like region has been proposed to play a key role in modulating the interactions with cellular glycosaminoglycans, and in particular to promote release of HSV-1 virions from infected cells. However, the molecular mechanisms and the role as a pathogenicity factor remains unclear. Using single virus particle tracking, we show that the mobility of chondroitin sulfate-bound HSV-1 virions is decreased in absence of the mucin-like region. This decrease in mobility correlates with an increase in HSV-1-chondroitin sulfate binding forces as observed using atomic force microscopy-based force spectroscopy. Our data suggest that the mucin-like region modulates virus-glycosaminoglycan interactions by regulating the affinity, type, and number of glycoproteins involved in the virus-glycosaminoglycan interaction. This study therefore presents new evidence for a role of the mucin-like region in balancing the interaction of HSV-1 with glycosaminoglycans and provides further insights into the molecular mechanisms used by the virus to ensure both successful cell entry and release from the infected cell.
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| 7. |
- Emilsson, Gustav, et al.
(author)
-
Nanoplasmonic Sensor Detects Preferential Binding of IRSp53 to Negative Membrane Curvature
- 2019
-
In: Frontiers in Chemistry. - : Frontiers Media SA. - 2296-2646. ; 7:FEB
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Journal article (peer-reviewed)abstract
- Biosensors based on plasmonic nanostructures are widely used in various applications and benefit from numerous operational advantages. One type of application where nanostructured sensors provide unique value in comparison with, for instance, conventional surface plasmon resonance, is investigations of the influence of nanoscale geometry on biomolecular binding events. In this study, we show that plasmonic "nanowells" conformally coated with a continuous lipid bilayer can be used to detect the preferential binding of the insulin receptor tyrosine kinase substrate protein (IRSp53) I-BAR domain to regions of negative surface curvature, i.e., the interior of the nanowells. Two different sensor architectures with and without an additional niobium oxide layer are compared for this purpose. In both cases, curvature preferential binding of IRSp53 (at around 0.025 nm(-1) and higher) can be detected qualitatively. The high refractive index niobium oxide influences the near field distribution and makes the signature for bilayer formation less clear, but the contrast for accumulation at regions of negative curvature is slightly higher. This work shows the first example of analyzing preferential binding of an average-sized and biologically important protein to negative membrane curvature in a label-free manner and in real-time, illustrating a unique application for nanoplasmonic sensors.
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| 8. |
- Friedrich, R., et al.
(author)
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A nano flow cytometer for single lipid vesicle analysis
- 2017
-
In: Lab on a Chip - Miniaturisation for Chemistry and Biology. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 17:5, s. 830-841
-
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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| 9. |
- Junesch, Juliane, 1987, et al.
(author)
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Location-specific nanoplasmonic sensing of biomolecular binding to lipid membranes with negative curvature
- 2015
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In: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 7:37, s. 15080-15085
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Journal article (peer-reviewed)abstract
- The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic "nanowells" provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids.
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| 10. |
- Lubart, Quentin, 1989, et al.
(author)
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High throughput size-determination and multiplexed fluorescence analysis of single biological particles in a nanofluidic device
- 2019
-
In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. ; , s. 1420-1421
-
Conference paper (peer-reviewed)abstract
- Biological nanoparticles, such as exosomes and viruses, are responsible for a multitude of important functions, but methods to characterize them on the single particle level are rare. We here present a nanofluidic platform for multi-parametric characterization of biological nanoparticles with high throughput. The device consists of feeding microchannels and an array of ~100 nanochannels where the nanoparticles can be characterized. We determine the size by analyzing the Brownian motion of the particles and quantify their content based on fluorescence imaging of up to three different colors. We successfully benchmark our method against existing techniques, such as Nanoparticle Tracking Analysis (NTA).
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| 11. |
- Mazur, Federico, et al.
(author)
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Liposomes and lipid bilayers in biosensors
- 2017
-
In: Advances in Colloid and Interface Science. - : Elsevier BV. - 0001-8686. ; 249, s. 88-99
-
Research review (peer-reviewed)abstract
- Biosensors for the rapid, specific, and sensitive detection of analytes play a vital role in healthcare, drug discovery, food safety, and environmental monitoring. Although a number of sensing concepts and devices have been developed, many longstanding challenges to obtain inexpensive, easy-to-use, and reliable sensor platforms remain largely unmet. Nanomaterials offer exciting possibilities for enhancing the assay sensitivity and for lowering the detection limits down to single-molecule resolution. In this review, we present an overview of liposomes and lipid bilayers in biosensing applications. Lipid assemblies in the form of spherical liposomes or two-dimensional planar membranes have been widely used in the design of biosensing assays; in particular, we highlight a number of recent promising developments of biosensors based on liposomes in suspension, liposome arrays, and lipid bilayers arrays. Assay sensitivity and specificity are discussed, advantages and drawbacks are reviewed, and possible further developments are outlined.
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| 12. |
- Nasir, Waqas, et al.
(author)
-
Histo-Blood Group Antigen Presentation Is Critical for Binding of Norovirus VLP to Glycosphingolipids in Model Membranes
- 2017
-
In: Acs Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 12:5, s. 1288-1296
-
Journal article (peer-reviewed)abstract
- Virus entry depends on biomolecular recognition at the surface of cell membranes. In the case of glycolipid receptors, these events are expected to be influenced by how the glycan epitope close to the membrane is presented to the virus. This presentation of membrane associated glycans is more restricted than that of glycans in solution, particularly because of orientational constraints imposed on the glycolipid through its lateral interactions with other membrane lipids and proteins. We have developed and employed a total internal reflection fluorescence microscopy-based binding assay and. a scheme for molecular dynamics (MD) membrane simulations to investigate the consequences of various glycan presentation effects. The system studied was histo-blood group antigen (HBGA) epitopes of membrane-bound glycosphingolipids (GSLs) derived from small intestinal epithelium of humans (type 1 chain) and dogs (type 2 chain) interacting with GII.4 norovirus-like particles. Our experimental results showed strong binding to all lipid-linked type 1 chain HBGAs but no or only weak binding to the corresponding type 2 chain HBGAs. This is in contrast to results derived from STD experiments with free HBGAs in solution where binding was observed for Lewis x. The MD data suggest that the strong binding to type 1 chain glycolipids was due to the well-exposed (1,2)-linked alpha-L-Fucp and (1,4)- linked alpha-L-Fucp residues, while the weaker binding or lack of binding to type 2 chain HBGAs was due to the very restricted accessibility of the (1,3) -linked alpha-L-Fucp residue when the glycolipid is embedded in a phospholipid membrane. Our results not only contribute to a general understanding of protein carbohydrate interactions on model membrane surfaces, particularly in the context of virus binding, but also suggest a possible role of human intestinal GSLs as potential receptors for norovirus uptake.
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| 13. |
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| 14. |
- Nasir, Waqas, et al.
(author)
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Interaction of Virus-Like Particles with Vesicles Containing Glycolipids: Kinetics of Detachment
- 2015
-
In: The Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 119:35, s. 11466-11472
-
Journal article (peer-reviewed)abstract
- Many viruses interact with their host cells via glycosphingolipids (GSLs) and/or glycoproteins present on the outer cell membrane. This highly specific interaction includes virion attachment and detachment. The residence time determined by the detachment is particularly interesting, since it is directly related to internalization and infection as well as to virion egress and spreading. In an attempt to deepen the understanding of virion detachment kinetics, we have used total internal reflection fluorescence (TIRF) microscopy to probe the interaction between individual fluorescently labeled GSL-containing lipid vesicles and surface-bound virus-like particles (VLPs) of a norovirus genotype II.4 strain. The distribution of the VLP-vesicle residence time was investigated for seven naturally occurring GSLs, all of which are candidates for the not yet identified receptor(s) mediating norovirus entry into host cells. As expected for interactions involving multiple GSL binding sites at a viral capsid, the detachment kinetics displayed features typical for a broad activation-energy distribution for all GSLs. Detailed inspection of these distributions revealed significant differences among the different GSLs. The results are discussed in terms of strength of the interaction, vesicle size, as well as spatial distribution and clustering of GSLs in the vesicle membrane. (Figure Presented).
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| 15. |
- Norling, Karin, 1988, et al.
(author)
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Gel Phase 1,2-Distearoyl-sn-glycero-3-phosphocholine-Based Liposomes Are Superior to Fluid Phase Liposomes at Augmenting Both Antigen Presentation on Major Histocompatibility Complex Class II and Costimulatory Molecule Display by Dendritic Cells in Vitro
- 2019
-
In: ACS Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 5:11, s. 1867-1878
-
Journal article (peer-reviewed)abstract
- Lipid-based nanoparticles have in recent years attracted increasing attention as pharmaceutical carriers. In particular, reports of them having inherent adjuvant properties combined with their ability to protect antigen from degradation make them suitable as vaccine vectors. However, the physicochemical profile of an ideal nanoparticle for vaccine delivery is still poorly defined. Here, we used an in vitro dendritic cell assay to assess the immunogenicity of a variety of liposome formulations as vaccine carriers and adjuvants. Using flow cytometry, we investigated liposome-assisted antigen presentation as well as the expression of relevant costimulatory molecules on the cell surface. Cytokine secretion was further evaluated with an enzyme-linked immunosorbent assay (ELISA). We show that liposomes can successfully enhance antigen presentation and maturation of dendritic cells, as compared to vaccine fusion protein (CTA1-3E alpha-DD) administered alone. In particular, the lipid phase state of the membrane was found to greatly influence the vaccine antigen processing by dendritic cells. As compared to their fluid phase counterparts, gel phase liposomes were more efficient at improving antigen presentation. They were also superior at upregulating the costimulatory molecules CD80 and CD86 as well as increasing the release of the cytokines IL-6 and IL-1 beta. Taken together, we demonstrate that gel phase liposomes, while nonimmunogenic on their own, significantly enhance the antigen-presenting ability of dendritic cells and appear to be a promising way forward to improve vaccine immunogenicity.
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| 16. |
- Peerboom, Nadia, 1990, et al.
(author)
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Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans
- 2017
-
In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 113:6, s. 1223-1234
-
Journal article (peer-reviewed)abstract
- Many viruses, including herpes simplex (HSV), are recruited to their host cells via interaction between their envelope glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial attachment is of a multivalent nature, i.e., it requires the establishment of multiple bonds between amino acids of viral glycoproteins and sulfated saccharides on the GAG chain. To gain understanding of how this binding process is modulated, we performed binding kinetics and mobility studies using end-grafted GAG chains that mimic the end attachment of these chains to proteoglycans. Total internal reflection fluorescence microscopy was used to probe binding and release, as well as the diffusion of single HSV-1 particles. To verify the hypothesis that the degree of sulfation, but also the arrangement of sulfate groups along the GAG chain, plays a key role in HSV binding, we tested two native GAGs (chondroitin sulfate and heparan sulfate) and compared our results to chemically sulfated hyaluronan. HSV-1 recognized all sulfated GAGs, but not the nonsulfated hyaluronan, indicating that binding is specific to the presence of sulfate groups. Furthermore we observed that a notable fraction of GAG-bound virions exhibit lateral mobility, although the multivalent binding to the immobilized GAG brushes ensures firm virus attachment to the interface. Diffusion was faster on the two native GAGs, one of which, chondroitin sulfate, was also characterized by the highest association rate per GAG chain. This highlights the complexity of multivalent virus-GAG interactions and suggests that the spatial arrangement of sulfates along native GAG chains may play a role in modulating the characteristics of the HSV-GAG interaction. Altogether, these results, obtained with a minimal and well-controlled model of the cell membrane, provide, to our knowledge, new insights into the dynamics of the HSV-GAG interaction.
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| 17. |
- Peerboom, Nadia, 1990, et al.
(author)
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Cell Membrane Derived Platform To Study Virus Binding Kinetics and Diffusion with Single Particle Sensitivity
- 2018
-
In: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 4:6, s. 944-953
-
Journal article (peer-reviewed)abstract
- Discovery and development of new antiviral therapies essentially rely on two key factors: an in-depth understanding of the mechanisms involved in viral infection and the development of fast and versatile drug screening platforms. To meet those demands, we present a biosensing platform to probe virus-cell membrane interactions on a single particle level. Our method is based on the formation of supported lipid bilayers from cell membrane material. Using total internal reflection fluorescence microscopy, we report the contribution of viral and cellular components to the interaction kinetics of herpes simplex virus type 1 with the cell membrane. Deletion of glycoprotein C (gC), the main viral attachment glycoprotein, or deletion of heparan sulfate, an attachment factor on the cell membrane, leads to an overall decrease in association of virions to the membrane and faster dissociation from the membrane. In addition to this, we perform binding inhibition studies using the antiviral compound heparin to estimate its IC50 value. Finally, single particle tracking is used to characterize the diffusive behavior of the virus particles on the supported lipid bilayers. Altogether, our results promote this platform as a complement to existing bioanalytical assays, being at the interface between simplified artificial membrane models and live cell experiments.
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| 18. |
- Royes, Jorge, et al.
(author)
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Bacteria-Based Production of Thiol-Clickable, Genetically Encoded Lipid Nanovesicles
- 2019
-
In: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 58:22, s. 7395-7399
-
Journal article (peer-reviewed)abstract
- Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high-yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria-based proteoliposomes are an attractive eco-friendly alternative that can outperform currently used liposomes.
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| 19. |
- Rupert, Deborah, 1986, et al.
(author)
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Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles
- 2016
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:20, s. 9980-9988
-
Journal article (peer-reviewed)abstract
- Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.
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| 20. |
- Rupert, Deborah, 1986, et al.
(author)
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Effective Refractive Index and Lipid Content of Extracellular Vesicles Revealed Using Optical Waveguide Scattering and Fluorescence Microscopy
- 2018
-
In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 34:29, s. 8522-8531
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are generating a growing interest because of the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different subpopulations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Because of the large heterogeneity of EV samples, such information remains rather elusive, and there is accordingly a need for new and complementary characterization schemes that can help expand the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were preseparated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high-density (HD) population was on an average a factor of three lower than that of the low-density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single-particle level. The labeled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index (n approximate to 1.35) compared with the LD EVs (n approximate to 1.38), indicating that both the lipid and protein contents were indeed lower in the HD EVs. Although separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs, that is, the two gradient bands are more likely because of the differences in membrane permeability than to differences in biomolecular content of the EVs.
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| 21. |
- Rupert, Deborah, 1986, et al.
(author)
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Methods for the physical characterization and quantification of extracellular vesicles in biological samples
- 2017
-
In: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 1872-8006 .- 0304-4165. ; Epub ahead of print:1, s. 3164-3179
-
Research review (peer-reviewed)abstract
- BACKGROUND:Our body fluids contain a multitude of cell-derived vesicles, secreted by most cell types, commonly referred to as extracellular vesicles. They have attracted considerable attention for their function as intercellular communication vehicles in a broad range of physiological processes and pathological conditions. Extracellular vesicles and especially the smallest type, exosomes, have also generated a lot of excitement in view of their potential as disease biomarkers or as carriers for drug delivery. In this context, state-of-the-art techniques capable of comprehensively characterizing vesicles in biological fluids are urgently needed.SCOPE OF REVIEW:This review presents the arsenal of techniques available for quantification and characterization of physical properties of extracellular vesicles, summarizes their working principles, discusses their advantages and limitations and further illustrates their implementation in vesicle research.MAJOR CONCLUSIONS:The small size and physicochemical heterogeneity of extracellular vesicles make their physical characterization and quantification an extremely challenging task. Currently, structure, size, buoyant density, optical properties and zeta potential have most commonly been studied. The concentration of vesicles in suspension can be expressed in terms of biomolecular or particle content depending on the method at hand. In addition, common quantification methods may either provide a direct quantitative measurement of vesicle concentration or solely allow for relative comparison between samples.GENERAL SIGNIFICANCE:The combination of complementary methods capable of detecting, characterizing and quantifying extracellular vesicles at a single particle level promises to provide new exciting insights into their modes of action and to reveal the existence of vesicle subpopulations fulfilling key biological tasks.
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| 22. |
- Saletti, D., et al.
(author)
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The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model
- 2017
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 7
-
Journal article (peer-reviewed)abstract
- Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles. In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation, we have combined structural and in vitro reconstitution experiments with model membranes. We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers. Using negatively charged giant unilamellar vesicles (GUVs), we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses. Membrane tubule formation requires the C-terminal domain of M1-C, corroborating its essential role for M1-C polymerization. Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress.
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