Characterization of Curli A Production on Living Bacterial Surfaces by Scanning Probe Microscopy

AbstractCurli are adhesive surface fibers produced by many Enterobacteriaceae, such as Escherichia coli and Salmonella enterica. They are implicated in bacterial attachment and invasion to epithelial cells. In this study, atomic force microscopy was used to determine the effects of curli on topology and mechanical properties of live E. coli cells. Young's moduli of both curli-deficient and curli-overproducing mutants were significantly lower than that of their wild-type (WT) strain, while decay lengths of the former strains were higher than that of the latter strain. Surprisingly, topological images showed that, unlike the WT and curli-overproducing mutant, the curli-deficient mutant produced a large number of flagella-like fibers, which may explain why the strain had a lower Young's modulus than the WT. These results suggest that the mechanical properties of bacterial surfaces are greatly affected by the presence of filamentous structures such as curli and flagella

The implication of glycans on the ACE2: SARS-CoV-2 spike interaction

Since its emergence in 2019 the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) continues to a profoundly impact and threaten human health. For the development of novel prophylactic and therapeutic measures a detailed understanding of the virus-host interaction and features that modulate the interaction is of utmost importance. Attachment of the SARS-CoV-2 virus to human host cells predominantly relies on the specific interaction of the viral spike (S) surface glycoprotein with the receptor angiotensin-converting enzyme 2 (ACE-2). Glycans within or surrounding the binding interface have been demonstrated to play an important role in the ACE2:S interaction. The quality of this interaction is multifaceted and affected by several parameters, such as the speed, the number, the strength and duration of bond formation. As mutations within the coding sequences of the interaction partners may affect their binding capacity, they should be thoroughly studied. In this respect, viral evolution and the effect of mutations within the S-protein have received much attention, while human ACE2 polymorphisms naturally occurring throughout the population have so far been largely ignored. Of note, natural ACE2 polymorphisms and viral spike mutants that result in the loss of glycans within the binding interface should receive our particular attention. Please click Download on the upper right corner to see the full abstract

Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM

The surface topography of red blood cells (RBCs) was investigated under nearphysiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but noninvasive attachment of the cells. Using tappingmode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circularshaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane

AFM imaging of functionalized double-walled carbon nanotubes

We present a comparative study of several non-covalent approaches to disperse, debundle and noncovalently functionalize double-walled carbon nanotubes (DWNTs). We investigated the ability of bovine serum albumin (BSA), phospholipids grafted onto amine-terminated polyethylene glycol (PLPEG2000-NH2), as well as a combination thereof, to coat purified DWNTs. Topographical imaging with the atomic force microscope (AFM) was used to assess the coating of individual DWNTs and the degree of debundling and dispersion. Topographical images showed that functionalized DWNTs are better separated and less aggregated than pristine DWNTs and that the different coating methods differ in their abilities to successfully debundle and disperse DWNTs. Height profiles indicated an increase in the diameter of DWNTs depending on the functionalization method and revealed adsorption of single molecules onto the nanotubes. Biofunctionalization of the DWNT surface was achieved by coating DWNTs with biotinylated BSA, providing for biospecific binding of streptavidin in a simple incubation step. Finally, biotin-BSA-functionalized DWNTs were immobilized on an avidin layer via the specific avidin–biotin interaction

Hidden multiple bond effects in dynamic force spectroscopy

In dynamic force spectroscopy, a (bio-)molecular complex is subjected to a steadily increasing force until the chemical bond breaks. Repeating the same experiment many times results in a broad distribution of rupture forces, whose quantitative interpretation represents a formidable theoretical challenge. In this study we address the situation that more than a single molecular bond is involved in one experimental run, giving rise to multiple rupture events which are even more difficult to analyze and thus are usually eliminated as far as possible from the further evaluation of the experimental data. We develop and numerically solve a detailed model of a complete dynamic force spectroscopy experiment including a possible clustering of molecules on the substrate surface, the formation of bonds, their dissociation under load, and the post processing of the force extension curves. We show that the data, remaining after elimination of obvious multiple rupture events, may still contain a considerable number of "hidden" multiple bonds, which are experimentally indistinguishable from "true" single bonds, but which have considerable effects on the resulting rupture force statistics and its consistent theoretical interpretation.Comment: 31 pages, 7 figure

AFM imaging of functionalized carbon nanotubes on biological membranes

Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging

Molecular Recognition in Confined Space Elucidated with DNA Nanopores and Single-Molecule Force Microscopy

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions

Outline of Synthesis of Cognitive and Socio-cultural Foundations of Scientific Knowledge Evolution in Research Programs of Western Philosophy of Science

The article analyses the development of cognitive sociology of science, in the object field of which connection of cognitive and social structures of science is traced. The role of context in scientific knowledge formation is defined. It is stated that the basis for development of research program of cognitive sociology of science appeared to be reconsideration of the standard concept of science as a complex of gnoseological, epistemological and methodological interpretations of nature and morphology of the produced scientific knowledge, methods for its explanation and scientificity ideals. The difference between "strong" and «weak» varieties of scientific knowledge evolution, developed in western philosophy of science, is considered. "Social studies of science" are reviewed as a form of social constructivism and relativism, exhibiting their specific nature in macro-analytical and micro- analytical strategies of scientific knowledge evolution analysis. The thesis that multidimensionality of science cannot be adequately interpreted focusing only on conceptual history of science is proved

Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses

first_page settings Order Article Reprints Open AccessHypothesis Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses by Markus Kastner 1,†,‡, Andreas Karner 1,†,§ [ORCID] , Rong Zhu 1,† [ORCID] , Qiang Huang 2 [ORCID] , Andreas Geissner 3,4,‖, Anne Sadewasser 5,¶, Markus Lesch 6, Xenia Wörmann 6, Alexander Karlas 6,**, Peter H. Seeberger 3,4 [ORCID] , Thorsten Wolff 5 [ORCID] , Peter Hinterdorfer 1 [ORCID] , Andreas Herrmann 7 and Christian Sieben 8,9,* [ORCID] 1 Institute for Biophysics, Johannes Kepler University Linz, 4020 Linz, Austria 2 State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China 3 Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, 14476 Potsdam, Germany 4 Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany 5 Division of Influenza and other Respiratory Viruses, Robert Koch-Institute, 13353 Berlin, Germany 6 Molecular Biology Department, Max Planck Institute for Infection Biology, 10117 Berlin, Germany 7 Institut für Chemie und Biochemie, Freie Universität Berlin, Altensteinstraße 23a, 14195 Berlin, Germany 8 Nanoscale Infection Biology Group, Department of Cell Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany 9 Institute for Genetics, Technische Universität Braunschweig, 38106 Braunschweig, Germany * Author to whom correspondence should be addressed. † These authors contributed equally to this work. ‡ Current address: Materials Characterization Lab (MCL), Materials Research Institute (MRI), Pennsylvania State University, University Park, PA 16802, USA. § Current address: University of Applied Sciences Upper Austria, School of Medical Engineering and Applied Social Sciences, Garnisonstr. 21, 4020 Linz, Austria. ‖ Current address: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada. ¶ Current address: Secarna Pharmaceuticals GmbH & Co. KG, Am Klopferspitz 19, 82152 Planegg, Germany. ** Current address: Viral Vectors and Gene Therapeutics, ProBioGen AG, 13086 Berlin, Germany. Viruses 2023, 15(7), 1507; https://doi.org/10.3390/v15071507 Received: 9 May 2023 / Revised: 21 June 2023 / Accepted: 28 June 2023 / Published: 5 July 2023 (This article belongs to the Special Issue Physical Virology - Viruses at Multiple Levels of Complexity) Download Browse Figures Review Reports Versions Notes Abstract Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA’s receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus–cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA’s receptor binding preference does not necessarily reflect virus–cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells