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Jacak J.,Johannes Kepler University | Schnidar H.,University of Salzburg | Schnidar H.,Biologics | Muresan L.,Johannes Kepler University | And 6 more authors.
Journal of Biotechnology | Year: 2013

We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138neg) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138neg cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material. © 2013 Elsevier B.V.


Zhang J.,CAS Changchun Institute of Applied Chemistry | Zhang J.,University of Chinese Academy of Sciences | Chtcheglova L.A.,Center for Advanced Bioanalysis Gmb | Zhu R.,Johannes Kepler University | And 4 more authors.
Analytical Chemistry | Year: 2014

Human gonadotropin-releasing hormone receptor (GnRH-R; or type I GnRH-R), which is expressed in tumor cells, has gained more and more attention as a specific target for cancer therapy. Given the clinical utility, the improved characterization of both the subcellular distribution and surface organization of GnRH-R is an important step in the development of more effective and possibly new therapeutic strategies. In the present study, the nano-organization of human GnRH-R was analyzed on fixed human bladder cancer cells (T24) by atomic force microscopy (AFM). The recognition images reveal that GnRH-Rs have a tendency to assemble in nanodomains (or clusters) that are irregularly distributed on the T24 cell surface. The locations of the GnRH-Rs were identified on the topographical images with nanometer accuracy. The obtained results enrich our understanding of the local distribution of GnRH-Rs on the bladder cancer cell membrane and demonstrate the ability of biological AFM to provide more complete and exact information at the single molecule level. © 2014 American Chemical Society.


Chtcheglova L.A.,Center for Advanced Bioanalysis Gmb | Hinterdorfer P.,Johannes Kepler University
Methods in Molecular Biology | Year: 2013

The real-time visualization of specific binding sites on biological samples with high spatial resolution, in order of several nanometers, is an important undertaking in many fields of biology. During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nanomaps on complex heterogeneous biosurfaces, such as cells and membranes. In this chapter, we present the TREC technique and explain how to unravel the nano-landscape of cells of the immune system, such as macrophages. We describe the procedures for all steps of the experiment including tip functionalization with Fc fragments via flexible PEG-linker, sample preparation, and localization of Fcγ receptors on macrophages. © 2013 Springer Science+Business Media, LLC.


Chtcheglova L.A.,Center for Advanced Bioanalysis GmbH | Hinterdorfer P.,Center for Advanced Bioanalysis GmbH | Hinterdorfer P.,Johannes Kepler University
Methods in Molecular Biology | Year: 2012

One of the challenging tasks in molecular cell biology is to identify and localize speci fic binding sites on biological samples with high spatial accuracy (in order of several nm). During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful AFM-based technique for quick and easy high-resolution receptor mapping. In this chapter, we provide a flavor of TREC application on vascular endothelial cells by describing the detailed procedures for all stages of the experiment from tip and sample preparations through the operating principles and visualization ©Springer Science+Business Media, LLC 2012.


Ahmad S.F.,University of Rostock | Chtcheglova L.A.,Johannes Kepler University | Chtcheglova L.A.,Center for Advanced Bioanalysis Gmb | Mayer B.,Johannes Kepler University | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2011

Determining the distribution of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometer) is an important challenge in many fields of biological science. Combination of high-resolution atomic force microscope (AFM) topography imaging with single-molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Simultaneous topography and recognition imaging was used to unravel the nanolandscape of cells of the immune system such as macrophages. The most studied phagocytic receptors include the Fc receptors that bind to the Fc portion of immunoglobulins. Here, nanomapping of FcγRs (Fc receptors for immunoglobulin G (IgG)) was performed on fixed J774.A1 mouse macrophage cell surfaces with magnetically coated AFM tips functionalized with Fc fragments of mouse IgG via long and flexible poly(ethylene glycol) linkers. Because of possible AFM tip engulfment on living macrophages, appropriate cell fixation procedure leaving the binding activity of FcγRs practically intact was elaborated. The recognition maps revealed prominent spots (microdomains) more or less homogeneously distributed on the macrophage surface with the sizes from 4 to 300 nm. Typical recognition image contained about ∼4% of large clusters (>200 nm), which were surrounded by a massive number (∼50%) of small-size (4-30 nm) and the rest by middle-size (50, 150 nm) domains. These spots were detected from the decrease of oscillation amplitude during specific binding between Fc-coated tip and FcγRs on macrophage surfaces. In addition, the effect of osmotic swelling on the topographical landscape of macrophage surfaces and on the reorganization of FcγRs was investigated. © 2010 Springer-Verlag.


Zhang J.,CAS Changchun Institute of Applied Chemistry | Zhang J.,University of Chinese Academy of Sciences | Liu H.,CAS Changchun Institute of Applied Chemistry | Liu H.,University of Chinese Academy of Sciences | And 5 more authors.
Analyst | Year: 2013

The understanding of ligand binding interactions is an important component of understanding the fundamental mechanism of receptor function. In this study, the binding abilities of EGF and TGF-α to EGFR on human bladder cancer (T24) cells were investigated by single molecular force spectroscopy (SMFS) based on atomic force microscopy (AFM). By approaching the specifically functionalized AFM tips to the T24 cell surface and subsequent retraction, specific unbinding events of the EGF/EGFR complexes and TGF-α/EGFR complexes were investigated. Further, the unbinding forces and the kinetic off rate constants that govern the bond stabilities were calculated through varying the external mechanic forces applied. Meanwhile, the distances from the energy minimum to the transition states along the separation paths of the EGF/EGFR complexes and TGF-α/EGFR complexes were deduced. This study at single-molecule level may enrich our understanding of the ligand binding properties of EGFR and provide some new information to the development of improved EGFR inhibitors. In addition, the results present new insight into the study of the energy landscape of the dissociation of ligand-EGFR system. © 2013 The Royal Society of Chemistry.


PubMed | Heidelberg Institute for Theoretical Studies, University of Hamburg, Goethe University Frankfurt, Free University of Berlin and 3 more.
Type: | Journal: Data in brief | Year: 2016

We here give information for a deeper understanding of single molecule force spectroscopy (SMFS) data through the example of the blood protein von Willebrand factor (VWF). It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD) loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK) was used for this demonstration. Further, Brownian dynamics (BD) simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, Mutual A domain interactions in the force sensing protein von Willebrand Factor (Posch et al., 2016) [1].


PubMed | Heidelberg Institute for Theoretical Studies, University of Hamburg, Goethe University Frankfurt, Free University of Berlin and 3 more.
Type: | Journal: Journal of structural biology | Year: 2016

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.


Chtcheglova L.A.,Center for Advanced Bioanalysis GmbH
Methods in molecular biology (Clifton, N.J.) | Year: 2013

One of the challenging tasks in molecular cell biology is to identify and localize specific binding sites on biological samples with high spatial accuracy (in order of several nm). During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful AFM-based technique for quick and easy high-resolution receptor mapping. In this chapter, we provide a flavor of TREC application on vascular endothelial cells by describing the detailed procedures for all stages of the experiment from tip and sample preparations through the operating principles and visualization.


PubMed | Center for Advanced Bioanalysis GmbH
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2012

One of the challenging tasks in molecular cell biology is to identify and localize specific binding sites on biological samples with high spatial accuracy (in order of several nm). During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful AFM-based technique for quick and easy high-resolution receptor mapping. In this chapter, we provide a flavor of TREC application on vascular endothelial cells by describing the detailed procedures for all stages of the experiment from tip and sample preparations through the operating principles and visualization.

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