| 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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| 11. |
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| 12. |
- Giubertoni, Giulia, et al.
(författare)
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Strong reduction of the chain rigidity of hyaluronan by selective binding of Ca2+ ions
- 2021
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Ingår i: Macromolecules. - : American Chemical Society (ACS). - 0024-9297 .- 1520-5835. ; 54:3, s. 1137-1146
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Tidskriftsartikel (refereegranskat)abstract
- The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intramolecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of ∼10–15 mol %. This saturation indicates that the binding of Ca2+ strongly reduces the probability of subsequent binding of Ca2+ at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.
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| 13. |
- Herrero, M. Antonia, et al.
(författare)
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Synthesis and characterization of a carbon nanotube−dendron series for efficient sirna delivery
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131:28, s. 9843-9848
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Tidskriftsartikel (refereegranskat)abstract
- A new series of dendron-functionalized multiwalled carbon nanotube (MWNT) derivatives, characterized by the presence of numerous positively charged tetraalkyl ammonium salts at the periphery of the dendron, has been synthesized. The positive charges on the MWNT surface, coupled with the unique ability of carbon nanotubes (CNTs) to penetrate cell membranes, make the new derivatives potentially ideal vectors for siRNA delivery. Using a fluorescently labeled, noncoding siRNA sequence, we demonstrate that cytoplasmic delivery of the nucleic acid is remarkably increased throughout the different dendron generations. The work reported here highlights the fact that dendron-functionalized CNTs can be rationally designed as efficient carriers of siRNA that can eventually lead to gene silencing.
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| 14. |
- Heudt, Laetitia, et al.
(författare)
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Raman spectroscopy and laser desorption mass spectrometry for minimal destructive forensic analysis of black and color inkjet printed documents
- 2012
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Ingår i: Forensic Science International. - : Elsevier. - 0379-0738 .- 1872-6283. ; 219:1-3, s. 64-75
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Tidskriftsartikel (refereegranskat)abstract
- Inkjet ink analysis is the best way to discriminate between printed documents, or even though more difficult, to connect an inkjet printed document with a brand or model of printers. Raman spectroscopy and laser desorption mass spectrometry (LDMS) have been demonstrated as powerful tools for dyes and pigments analysis, which are ink components. The aim of this work is to evaluate the aforementioned techniques for inkjet inks analysis in terms of discriminating power, information quality, and nondestructive capability. So, we investigated 10 different inkjet ink cartridges (primary colors and black), 7 from the HP manufacturer and one each from Epson, Canon and Lexmark. This paper demonstrates the capabilities of three methods: Raman spectroscopy, LDMS and MALDI-MS. Raman spectroscopy, as it is preferable to try the nondestructive approach first, is successfully adapted to the analysis of color printed documents in most cases. For analysis of color inkjet inks by LDMS, we show that a MALDI matrix (9-aminoacridine, 9AA) is needed to desorb and to ionize dyes from most inkjet inks (except Epson inks). Therefore, a method was developed to apply the 9AA MALDI matrix directly onto the piece of paper while avoiding analyte spreading. The obtained mass spectra are very discriminating and lead to information about ink additives and paper compositions. Discrimination of black inkjet printed documents is more difficult because of the common use of carbon black as the principal pigment. We show for the first time the possibility to discriminate between two black-printed documents coming from different, as well as from the same, manufacturers. Mass spectra recorded from black inks in positive ion mode LDMS detect polyethylene glycol polymers which have characteristic mass distributions and end groups. Moreover, software has been developed for rapid and objective comparison of the low mass range of these positive mode LDMS spectra which have characteristic unknown peaks.
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| 15. |
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| 16. |
- Mirmomtaz, Elham, et al.
(författare)
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Quantitative study of the effect of coverage on the hybridization efficiency of surface-bound DNA nanostructures
- 2008
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 8:12, s. 4134-4139
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate that, contrary to current understanding, the density of probe molecules is not responsible for the lack of hybridization in high density single-stranded DNA (ss-DNA) self-assembled monolayers (SAMs). To this end, we use nanografting to fabricate well packed ss-DNA nanopatches within a “carpet matrix” SAM of inert thiols on gold surfaces. The DNA surface density is varied by changing the “writing” parameters, for example, tip speed, and number of scan lines. Since ss-DNA is 50 times more flexible than ds-DNA, hybridization leads to a transition to a “standing up” phase. Therefore, accurate height and compressibility measurements of the nanopatches before and after hybridization allow reliable, sensitive, and label-free detection of hybridization. Side-by-side comparison of self-assembled and nanografted DNA-monolayers shows that the latter, while denser than the former, display higher hybridization efficiencies.
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| 17. |
- Nadeem, Aftab, et al.
(författare)
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A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:47
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Tidskriftsartikel (refereegranskat)abstract
- Vibrio cholerae, responsible for outbreaks of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe how the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto-unrecognized genomic island. The proteins MakA/B/E can form a tripartite toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the cytolytic activity of the Mak proteins was obtained by X-ray crystallography. Flagellum-facilitated secretion ensuring spatially coordinated delivery of Mak proteins revealed a role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of diagnostics and therapeutic strategies against pathogenic Vibrionaceae.
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| 18. |
- Prittinen, Juha, 1989-, et al.
(författare)
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Microstructured collagen films for 3D corneal stroma modelling
- 2022
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Ingår i: Connective Tissue Research. - : Taylor & Francis Group. - 0300-8207 .- 1607-8438. ; 63:5, s. 443-452
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Tidskriftsartikel (refereegranskat)abstract
- Purpose/aim: Corneal injury is a major cause of impaired vision around the globe. The fine structure of the corneal stroma plays a pivotal role in the phenotype and behavior of the embedded cells during homeostasis and healing after trauma or infection. In order to study healing processes in the cornea, it is important to create culture systems that functionally mimic the natural environment.Materials and methods: Collagen solution was vitrified on top of a grated film to achieve thin collagen films with parallel microgrooves. Keratocytes (corneal stromal cells) were cultured on the films either as a single layer or as stacked layers of films and cells. SEM and F-actin staining were used to analyze the pattern transference onto the collagen and the cell orientation on the films. Cell viability was analyzed with MTS and live/dead staining. Keratocytes, fibroblasts, and myofibroblasts were cultured to study the pattern’s effect on phenotype.Results: A microstructured collagen film-based culture system that guides keratocytes (stromal cells) to their native, layerwise perpendicular orientation in 3D and that can support fibroblasts and myofibroblasts was created. The films are thin and transparent enough to observe cells at least three layers deep. The cells maintain viability in 2D and 3D cultures and the films can support fibroblast and myofibroblast phenotypes.Conclusions: The films provide an easily reproducible stroma model that maintains high cell viability and improves the preservation of the keratocyte phenotype in keratocytes that are differentiated to fibroblasts.
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| 19. |
- Szuba, Agata, et al.
(författare)
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Membrane binding controls ordered self-assembly of animal septins
- 2021
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.
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| 20. |
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