| 1. |
- Bano, Fouzia, et al.
(författare)
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A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force
- 2016
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG·protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG·protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices.
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| 2. |
- Bano, Fouzia, et al.
(författare)
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Interaction of hyaluronan with cationic nanoparticles
- 2015
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 31:30, s. 8411-8420
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Tidskriftsartikel (refereegranskat)abstract
- The polysaccharide hyaluronan (HA) is a main component of peri- and extracellular matrix, and an attractive molecule for materials design in tissue engineering and nanomedicine. Here, we study the morphology of complexes that form upon interaction of nanometer-sized amine-coated gold particles with this anionic, linear, and regular biopolymer in solution and grafted to a surface. We find that cationic nanoparticles (NPs) have profound effects on HA morphology on the molecular and supramolecular scale. Quartz crystal microbalance (QCM-D) shows that depending on their relative abundance, cationic NPs promote either strong compaction or swelling of films of surface-grafted HA polymers (HA brushes). Transmission electron and atomic force microscopy reveal that the NPs do also give rise to complexes of distinct morphologies–compact nanoscopic spheres and extended microscopic fibers–upon interaction with HA polymers in solution. In particular, stable and hydrated spherical complexes of single HA polymers with NPs can be prepared when balancing the ionizable groups on HA and NPs. The observed self-assembly phenomena could be useful for the design of drug delivery vehicles and a better understanding of the reorganization of HA-rich synthetic or biological matrices.
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| 3. |
- Bano, Fouzia, et al.
(författare)
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Single-molecule unbinding forces between the polysaccharide hyaluronan and its binding proteins
- 2018
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Ingår i: Biophysical Journal. - : Biophysical society. - 0006-3495 .- 1542-0086. ; 114:12, s. 2910-2922
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Tidskriftsartikel (refereegranskat)abstract
- The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.
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| 4. |
- Bano, Fouzia, et al.
(författare)
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Toward multiprotein nanoarrays using nanografting and DNA directed immobilization of proteins
- 2009
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 9:7, s. 2614-2618
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Tidskriftsartikel (refereegranskat)abstract
- Atomic force microscopy nanografting was utilized to prepare DNA nanopatches of different sizes (200 × 200 to 1000 × 1000 nm2) onto which DNA−protein conjugates can be anchored through DNA-directed immobilization. Height measurements were used to assess the binding of the proteins as well as their subsequent interaction with other components, such as antibodies. The results indicate that nanografted patch arrays are well suited for application in biosensing and could enable the fabrication of multifeature protein nanoarrays.
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| 5. |
- Bano, Fouzia, et al.
(författare)
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Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy
- 2015
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Ingår i: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 7:46, s. 19528-19533
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Tidskriftsartikel (refereegranskat)abstract
- Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold – a substrate commonly used in nanotechnology – and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA–gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide–Au bond strength. Force data show that, at physiological ionic strength, adenine–Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine–Au and guanine–Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.
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| 6. |
- Bar, Laure, et al.
(författare)
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Impact of antigen density on recognition by monoclonal antibodies
- 2020
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Ingår i: Analytical Biochemistry. - : American Chemical Society (ACS). - 0003-2697 .- 1096-0309 .- 0003-2700 .- 1520-6882. ; 92:7, s. 5396-5403
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Tidskriftsartikel (refereegranskat)abstract
- Understanding antigen–antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.
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| 7. |
- Bosco, Alessandro, et al.
(författare)
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Hybridization in nanostructured DNA monolayers probed by AFM : theory versus experiment
- 2012
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Ingår i: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 4:5, s. 1734-1741
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Tidskriftsartikel (refereegranskat)abstract
- Nanografted monolayers (NAMs) of DNA show novel physico-chemical properties that make them ideally suited for advanced biosensing applications. In comparison with alternative solid-phase techniques for diagnostic DNA detection, NAMs have the advantage of combining a small size with a high homogeneity of the DNA surface coverage. These two properties favour the extreme miniaturization and ultrasensitivity in high-throughput biosensing devices. The systematic use of NAMs for quantitative DNA (and protein) detection has so far suffered from the lack of a control on key fabrication parameters, such as the ss- or ds-DNA surface coverage. Here we report on a combined experimental–computational study that allows us to estimate the surface density of the grafted DNA by analyzing the sample mechanical response, that is the DNA patch height vs. applied tip load curves. It is shown that the same analysis scheme can be used to detect the occurrence of hybridization with complementary strands in solution and estimate its efficiency. Thanks to these quantitative relationships it is possible to use a single AFM-based setup to: (i) fabricate a DNA NAM, (ii) control the DNA surface coverage, and (iii) characterize its level of hybridization helping the design of NAMs with pre-determined fabrication parameters.
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| 8. |
- Castronovo, Matteo, et al.
(författare)
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Mechanical stabilization effect of water on a membrane-like system
- 2007
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 129:9, s. 2636-2641
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Tidskriftsartikel (refereegranskat)abstract
- The penetration resistance of a prototypical model-membrane system (HS−(CH2)11−OH self-assembled monolayer (SAM) on Au(111)) to the tip of an atomic force microscope (AFM) is investigated in the presence of different solvents. The compressibility (i.e., height vs tip load) of the HS−(CH2)11−OH SAM is studied differentially, with respect to a reference structure. The reference consists of hydrophobic alkylthiol molecules (HS−(CH2)17−CH3) embedded as nanosized patches into the hydrophilic SAM by nanografting, an AFM-assisted nanolithography technique. We find that the penetration resistance of the hydrophilic SAM depends on the nature of the solvent and is much higher in the presence of water than in 2-butanol. In contrast, no solvent-dependent effect is observed in the case of hydrophobic SAMs. We argue that the mechanical resistance of the hydroxyl-terminated SAM is a consequence of the structural order of the solvent−SAM interface, as suggested by our molecular dynamics simulations. The simulations show that in the presence of 2-butanol the polar head groups of the HS−(CH2)11−OH SAM, which bind only weakly to the solvent molecules, try to bind to each other, disrupting the local order at the interface. On the contrary, in the presence of water the polar head groups bind preferentially to the solvent that, in turn, mediates the release of the surface strain, leading to a more ordered interface. We suggest that the mechanical stabilization effect induced by water may be responsible for the stability of even more complex, real membrane systems.
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| 9. |
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| 10. |
- Conca, Dario Valter, et al.
(författare)
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The role of membrane complexity in the early entry stages of SARS-CoV-2 variants
- 2023
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Ingår i: European Biophysics Journal. - 1432-1017 .- 0175-7571. ; 52:SUPPL 1, s. S176-S176
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The highest density of mutations in SARS-CoV-2 variants is located on the spike glycoprotein (S), which is responsible for receptor ACE2 engagement. This suggests that SARS-CoV-2 is evolving to optimize viral entry. Several molecular studies report differences in the affinity between isolated S and ACE2 among variants. However, overall ACE2 affinity poorly correlates with the increased infectivity of recent variants. We address this discrepancy by considering the virus interaction with the whole plasma membrane and study the role of avidity and membrane complexity in modulating virus-host binding. To this end, we employ an in-vitro model system combining single-particle tracking and native supported lipid bilayers (nSLBs) made from lung epithelial cells. As virion mimics, we developed S-decorated liposomes that allow for direct comparison between variants and BSL-1 handling. Sliposome interaction with nSLBs showed a significant increase in avidity for Omicron compared to Delta and Wuhan strains. Further, using single-molecule force spectroscopy, we reveal a higher affinity for Omicron and Delta S to sensor immobilise heparan sulfate (HS). Our results indicate a shift in the variants’ attachment strategy towards more efficient use of coreceptors and the role of HS as an initial docking site that facilitates virus accumulation at the membrane and ACE2 engagement.
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