Publications by authors named "Martin Benoit"

61 Publications

Ventricular lead malposition after TAVR causing ischaemic stroke.

Acta Cardiol 2021 Jan 20:1-3. Epub 2021 Jan 20.

Department of Cardiology, Cliniques Universitaires Saint-Luc, Brussels, Belgium.

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http://dx.doi.org/10.1080/00015385.2020.1858249DOI Listing
January 2021

Ensemble Learning of Convolutional Neural Network, Support Vector Machine, and Best Linear Unbiased Predictor for Brain Age Prediction: ARAMIS Contribution to the Predictive Analytics Competition 2019 Challenge.

Front Psychiatry 2020 15;11:593336. Epub 2020 Dec 15.

Paris Brain Institute, ICM, Paris, France.

We ranked third in the Predictive Analytics Competition (PAC) 2019 challenge by achieving a mean absolute error (MAE) of 3.33 years in predicting age from T1-weighted MRI brain images. Our approach combined seven algorithms that allow generating predictions when the number of features exceeds the number of observations, in particular, two versions of best linear unbiased predictor (BLUP), support vector machine (SVM), two shallow convolutional neural networks (CNNs), and the famous ResNet and Inception V1. Ensemble learning was derived from estimating weights via linear regression in a hold-out subset of the training sample. We further evaluated and identified factors that could influence prediction accuracy: choice of algorithm, ensemble learning, and features used as input/MRI image processing. Our prediction error was correlated with age, and absolute error was greater for older participants, suggesting to increase the training sample for this subgroup. Our results may be used to guide researchers to build age predictors on healthy individuals, which can be used in research and in the clinics as non-specific predictors of disease status.
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http://dx.doi.org/10.3389/fpsyt.2020.593336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770104PMC
December 2020

Development and characterization of a PLGA-HA composite material to fabricate 3D-printed scaffolds for bone tissue engineering.

Mater Sci Eng C Mater Biol Appl 2021 Jan 8;118:111334. Epub 2020 Aug 8.

Univ. Bordeaux, INSERM, BioTis U1026, CHU Bordeaux, Dentistry and Oral Health Department, 33076 Bordeaux, France. Electronic address:

Additive manufacturing is a rising field in bone tissue engineering. Additive fabrication offers reproducibility, high precision and rapid manufacture of custom patient-specific scaffolds. The development of appropriate composite materials for biomedical applications is critical to reach clinical application of these novel biomaterials. In this work, medical grade poly(lactic-co-glycolic) acid (PLGA) was mixed with hydroxyapatite nanoparticles (nHA) to fabricate 3D porous scaffolds by Fused Deposition Modeling. We have first confirmed that the composite material could be printed in a reproductive manner. Physical characterization demonstrated a low degradation of the material during manufacturing steps and an expected loading and homogeneous distribution of nHA. In vitro biodegradation of the scaffolds showed modifications of morphological and physicochemical properties over time. The composite scaffolds were biocompatible and high cell viability was observed in vitro, as well as a maintain of cell proliferation. As expected, the addition of nHA displayed a positive impact on osteodifferentiation in vitro. Furthermore, a limited inflammatory reaction was observed after subcutaneous implantation of the materials in the rat. Overall, this study suggests that this composite material is suitable for bone tissue engineering applications.
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http://dx.doi.org/10.1016/j.msec.2020.111334DOI Listing
January 2021

Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion.

Circulation 2021 Jan 10;143(3):254-266. Epub 2020 Nov 10.

Institute for Cardiovascular Prevention (IPEK), LMU Munich Hospital, Germany (A.S., A.O.-G., C.W., P. Lemnitzer, J.R.V., C.P., L.P.O., J.W., Y.D., O.S.).

Background: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy.

Results: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia.

Conclusions: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.046677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914394PMC
January 2021

Designed anchoring geometries determine lifetimes of biotin-streptavidin bonds under constant load and enable ultra-stable coupling.

Nanoscale 2020 Nov 20;12(41):21131-21137. Epub 2020 Oct 20.

Department of Physics and Center for NanoScience, LMU Munich, Germany.

The small molecule biotin and the homotetrameric protein streptavidin (SA) form a stable and robust complex that plays a pivotal role in many biotechnological and medical applications. In particular, the SA-biotin linkage is frequently used in single-molecule force spectroscopy (SMFS) experiments. Recent data suggest that SA-biotin bonds show strong directional dependence and a broad range of multi-exponential lifetimes under load. Here, we investigate engineered SA variants with different valencies and a unique tethering point under constant forces using a magnetic tweezers assay. We observed orders-of-magnitude differences in the lifetimes under force, which we attribute to the distinct force-loading geometries in the different SA variants. Lifetimes showed exponential dependencies on force, with extrapolated lifetimes at zero force that are similar for the different SA variants and agree with parameters determined from constant-speed dynamic SMFS experiments. We identified an especially long-lived tethering geometry that will facilitate ultra-stable SMFS experiments.
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http://dx.doi.org/10.1039/d0nr03665jDOI Listing
November 2020

Inadequate tissue mineralization promotes cancer cell attachment.

PLoS One 2020 28;15(8):e0237116. Epub 2020 Aug 28.

Center for Applied Tissue Engineering and Regenerative Medicine (CANTER), Munich University of Applied Sciences, Munich, Germany.

Bone metastases are a frequent complication in prostate cancer, and several studies have shown that vitamin D deficiency promotes bone metastases. However, while many studies focus on vitamin D's role in cell metabolism, the effect of chronically low vitamin D levels on bone tissue, i.e. insufficient mineralization of the tissue, has largely been ignored. To investigate, whether poor tissue mineralization promotes cancer cell attachment, we used a fluorescence based adhesion assay and single cell force spectroscopy to quantify the adhesion of two prostate cancer cell lines to well-mineralized and demineralized dentin, serving as biomimetic bone model system. Adhesion rates of bone metastases-derived PC3 cells increased significantly on demineralized dentin. Additionally, on mineralized dentin, PC3 cells adhered mainly via membrane anchored surface receptors, while on demineralized dentin, they adhered via cytoskeleton-anchored transmembrane receptors, pointing to an interaction via exposed collagen fibrils. The adhesion rate of lymph node derived LNCaP cells on the other hand is significantly lower than that of PC3 and not predominately mediated by cytoskeleton-linked receptors. This indicates that poor tissue mineralization facilitates the adhesion of invasive cancer cells by the exposure of collagen and emphasizes the disease modifying effect of sufficient vitamin D for cancer patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237116PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454967PMC
October 2020

Audiogenic seizure as a model of sudden death in epilepsy: A comparative study between four inbred mouse strains from early life to adulthood.

Epilepsia 2020 02 24;61(2):342-349. Epub 2020 Jan 24.

Univ Rennes, CHU Rennes, Inserm, LTSI (Laboratoire de Traitement du Signal et de l'Image), UMR-1099, F-35000, Rennes, France.

Objective: Mouse models of sudden unexpected death in epileptic patients (SUDEP) using audiogenic seizures (AGS) are valuable because death can occur following a sound-induced seizure in the absence of any pharmacologic or electric component. However, only a few strains of mice are AGS prone, and the vast majority of studies involve DBA/2 or DBA/1 inbred strains. With the goal of characterizing the variation of AGS susceptibility with age, and of offering a larger panel of mice available for AGS studies, we performed a comparative study of the variability in AGS responses.

Methods: The variation of AGS with age was determined in two classically used inbred strains of mice, DBA/2 and DBA/1, and two additional strains, BALB/c and 129/SvTer. As AGS-stimulated tonic seizures can be lethal or nonlethal, even in the same inbred strain, in a second experiment, we addressed whether there is an innate capacity to reproduce the same response after a tonic AGS, referred to as "determinism," in the DBA/2J, DBA/1J, and 129/SvTer mouse strains.

Results: Results show that the 129/SvTer mouse is a more versatile model of SUDEP due to its wider age range of susceptibility compared to the DBA/2J and DBA/1J mouse strains. In addition, we show that determinism is not consistently evident in DBA/2J and 129/SvTer strains after AGS. Hence, one cannot be certain that a lethal AGS will always be lethal in successive testing after resuscitation and vice versa in these two mouse strains.

Significance: These studies highlight the phenotypic variability of AGS in different mouse strains, show the value of an additional mouse strain, 129/SvTer, for studies using AGS, and thus provide valuable information for future studies of AGS and SUDEP.
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http://dx.doi.org/10.1111/epi.16432DOI Listing
February 2020

Quantification of Neural Conduction Block on the Rat Sciatic Nerve based on EMG Response.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:6450-6453

Neural conduction block performed by balanced-charge kilohertz frequency alternating currents (KHFAC) has been identified as a potential technique for therapy delivery in different clinical setups. The underlying mechanisms that contribute to this phenomenon have been studied through computational models and animal experiments. However, the optimal stimulation parameters to achieve axonal conduction block are difficult to define, since they depend on the species, the nerve being targeted, as well as the technical and experimental setup. This study proposes an experimental setup along with an original data processing approach for the quantification of the effectiveness of neural conduction block. Experiments were performed on the sciatic nerve of two Sprague-Dawley rats, by evaluating different groups of stimulation parameters with varying amplitudes and frequencies, ranging from 1 to 10 mA and from 2 to 10 kHz, respectively. Results suggest that the effectiveness of axonal conduction block strongly depends on the selection of the stimulation parameters. In this work, more effective blockages were achieved for frequencies around 4 kHz and within an approximate amplitude range of 2 to 8 mA.
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http://dx.doi.org/10.1109/EMBC.2019.8856943DOI Listing
July 2019

Publisher Correction: The effect of attention and working memory on the estimation of elapsed time.

Sci Rep 2019 Nov 18;9(1):17265. Epub 2019 Nov 18.

CEA, DRF/Joliot, NeuroSpin; INSERM, U992, Cognitive Neuroimaging Unit, Université Paris-Sud; Université Paris-Saclay, Gif/Yvette, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-019-53224-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861268PMC
November 2019

Multiplexed protein force spectroscopy reveals equilibrium protein folding dynamics and the low-force response of von Willebrand factor.

Proc Natl Acad Sci U S A 2019 09 28;116(38):18798-18807. Epub 2019 Aug 28.

Department of Physics, LMU Munich (Ludwig Maximilian University of Munich), 80799 Munich, Germany;

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We directly resolve the stabilization of the VWF A2 domain by Ca and discover transitions in the VWF C domain stem at low forces that likely constitute the first steps of VWF's mechano-activation. Probing the force-dependent lifetime of biotin-streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime.
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http://dx.doi.org/10.1073/pnas.1901794116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754583PMC
September 2019

Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging.

PLoS One 2019 15;14(1):e0210963. Epub 2019 Jan 15.

Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210963PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333368PMC
November 2019

PEDOT:PSS electrodes for acute experimental evaluation of vagus nerve stimulation on rodents.

Annu Int Conf IEEE Eng Med Biol Soc 2018 Jul;2018:4760-4763

The vagus nerve (VN) is involved in the autonomic regulation of many physiological systems (cardiovascular, respiratory, gastrointestinal, etc.) and its stimulation is already an approved therapy for refractory epilepsy and depression. Other pathologies are thought to be treatable through vagus nerve stimulation (VNS), such as heart failure, cardiac arrhythmia, inflammation or auto-immune diseases. However, the efficacy of the stimulation is not always optimal, partly due to the materials and the architecture of currently available electrodes. Standard electrodes, composed of metallic rings that stimulate the whole diameter of the nerve, are not adapted to experimentations involving spatial selectivity. Efficient and selective charge injection is usually difficult to achieve simultaneously, especially in experimental setups using rodents, due to the thin diameter of their VN. In this paper, we show that we can take advantage of the high charge injection property of conducting polymers to acutely stimulate the vagus nerve in rodents, using individual active electrodes with dimensions $725\,\,\mu \mathrm{m}\times \,450\,\,\mu\mathrm{m}$. A particular PEDOT:PSS architecture integrating 12 active electrodes is developed and applied to the VN of one rat. A closed-loop VNS system developed in our previous works is used to stimulate the VN while analyzing the heart rate response. Results show the feasibility of this kind of electrodes for acute VNS on rodents and open the path towards new experimentations focused on selective stimulation and recording.
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http://dx.doi.org/10.1109/EMBC.2018.8513235DOI Listing
July 2018

Long-term negative impact of an inappropriate first antiepileptic medication on the efficacy of a second antiepileptic medication in mice.

Epilepsia 2018 07 14;59(7):e109-e113. Epub 2018 Jun 14.

Univ Rennes, CHU Rennes, INSERM, LTSI - UMR 1099, Rennes, France.

Childhood absence epilepsy (CAE) is one of the most frequent epilepsies in infancy. The first-line recommended therapy for CAE is based on the prescription of the narrow-spectrum ethosuximide and the broad-spectrum valproic acid, which have similar efficacy in the first 12 months. Nevertheless, some antiepileptic drugs (AEDs) may worsen seizure duration and type in this syndrome. In line with this, we have encountered a case of identical twins with CAE and early exposure to different antiseizure drugs leading to divergent outcomes. From this, we hypothesized that the first AED to treat CAE may determine the long-term prognosis, especially in the developing brain, and that some situations leading to drug resistance may be explained by use of an inappropriate first AED. Therefore, we investigated this hypothesis by using a genetic mouse model of absence epilepsy (BS/Orl). Mice received a first appropriate or inappropriate AED followed by the same appropriate AED. Our data demonstrate that an inappropriate first AED has a negative impact on the long-term efficacy of a second appropriate AED. This work supports the necessity to effectively diagnose epileptic syndromes prior to medication use, particularly in children, in order to prevent the deleterious effects of an inappropriate initial AED.
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http://dx.doi.org/10.1111/epi.14454DOI Listing
July 2018

The effect of attention and working memory on the estimation of elapsed time.

Sci Rep 2018 04 27;8(1):6690. Epub 2018 Apr 27.

CEA, DRF/Joliot, NeuroSpin; INSERM, U992, Cognitive Neuroimaging Unit, Université Paris-Sud; Université Paris-Saclay, Gif/Yvette, France.

Psychological models of time perception involve attention and memory: while attention typically regulates the flow of events, memory maintains timed events or intervals. The precise, and possibly distinct, roles of attention and memory in time perception remain debated. In this behavioral study, we tested 48 participants in a prospective duration estimation task while they fully attended to time or performed a working memory (WM) task. We report that paying attention to time lengthened perceived duration in the range of seconds to minutes, whereas diverting attention away from time shortened perceived duration. The overestimation due to attending to time did not scale with durations. To the contrary, increasing WM load systematically decreased subjective duration and this effect scaled with durations. Herein, we discuss the dissociation between attention and WM in timing and scalar variability from the perspective of Bayesian models of time estimations.
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http://dx.doi.org/10.1038/s41598-018-25119-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923266PMC
April 2018

A Bayesian Perspective on Accumulation in the Magnitude System.

Sci Rep 2017 04 4;7(1):630. Epub 2017 Apr 4.

CEA, DRF/I2BM, NeuroSpin, INSERM, U992, Cognitive Neuroimaging Unit, Université Paris-Sud, Université Paris-Saclay, F-Gif/Yvette, France.

Several theoretical and empirical work posit the existence of a common magnitude system in the brain. Such a proposal implies that manipulating stimuli in one magnitude dimension (e.g. duration in time) should interfere with the subjective estimation of another magnitude dimension (e.g. size in space). Here, we asked whether a generalized Bayesian magnitude estimation system would sample sensory evidence using a common, amodal prior. Two psychophysical experiments separately tested participants on their perception of duration, surface, and numerosity when the non-target magnitude dimensions and the rate of sensory evidence accumulation were manipulated. First, we found that duration estimation was resilient to changes in surface and numerosity, whereas lengthening (shortening) the duration yielded under- (over-) estimations of surface and numerosity. Second, the perception of surface and numerosity were affected by changes in the rate of sensory evidence accumulation, whereas duration was not. Our results suggest that a generalized magnitude system based on Bayesian computations would minimally necessitate multiple priors.
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http://dx.doi.org/10.1038/s41598-017-00680-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428809PMC
April 2017

Structural and functional changes during epileptogenesis in the mouse model of medial temporal lobe epilepsy.

Annu Int Conf IEEE Eng Med Biol Soc 2016 Aug;2016:4005-4008

An important issue in epilepsy research is to understand the structural and functional modifications leading to chronic epilepsy, characterized by spontaneous recurrent seizures, after initial brain insult. To address this issue, we recorded and analyzed electroencephalography (EEG) and quantitative magnetic resonance imaging (MRI) data during epileptogenesis in the in vivo mouse model of Medial Temporal Lobe Epilepsy (MTLE, kainate). Besides, this model of epilepsy is a particular form of drug-resistant epilepsy. The results indicate that high-field (4.7T) MRI parameters (T2-weighted; T2-quantitative) allow to detect the gradual neuro-anatomical changes that occur during epileptogenesis while electrophysiological parameters (number and duration of Hippocampal Paroxysmal Discharges) allow to assess the dysfunctional changes through the quantification of epileptiform activity. We found a strong correlation between EEG-based markers (invasive recording) and MRI-based parameters (non-invasive) periodically computed over the `latent period' that spans over two weeks, on average. These results indicated that both structural and functional changes occur in the considered epilepsy model and are considered as biomarkers of the installation of epilepsy. Additionally, such structural and functional changes can also be observed in human temporal lobe epilepsy. Interestingly, MRI imaging parameters could be used to track early (day-7) structural changes (gliosis, cell loss) in the lesioned brain and to quantify the evolution of epileptogenesis after traumatic brain injury.
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http://dx.doi.org/10.1109/EMBC.2016.7591605DOI Listing
August 2016

Biophysical approaches promote advances in the understanding of von Willebrand factor processing and function.

Adv Biol Regul 2017 Jan 28;63:81-91. Epub 2016 Sep 28.

Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

The large multimeric plasma glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis by recruiting platelets to sites of vascular injury. VWF multimers respond to elevated hydrodynamic forces by elongation, thereby increasing their adhesiveness to platelets. Thus, the activation of VWF is force-induced, as is its inactivation. Due to these attributes, VWF is a highly interesting system from a biophysical point of view, and is well suited for investigation using biophysical approaches. Here, we give an overview on recent studies that predominantly employed biophysical methods to gain novel insights into multiple aspects of VWF: Electron microscopy was used to shed light on the domain structure of VWF and the mechanism of VWF secretion. High-resolution stochastic optical reconstruction microscopy, atomic force microscopy (AFM), microscale thermophoresis and fluorescence correlation spectroscopy allowed identification of protein disulfide isomerase isoform A1 as the VWF dimerizing enzyme and, together with molecular dynamics simulations, postulation of the dimerization mechanism. Advanced mass spectrometry led to detailed identification of the glycan structures carried by VWF. Microfluidics was used to illustrate the interplay of force and VWF function. Results from optical tweezers measurements explained mechanisms of the force-dependent functions of VWF's domains A1 and A2 and, together with thermodynamic approaches, increased our understanding of mutation-induced dysfunctions of platelet-binding. AFM-based force measurements and AFM imaging enabled exploration of intermonomer interactions and their dependence on pH and divalent cations. These advances would not have been possible by the use of biochemical methods alone and show the benefit of interdisciplinary research approaches.
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http://dx.doi.org/10.1016/j.jbior.2016.09.010DOI Listing
January 2017

pH-Dependent Interactions in Dimers Govern the Mechanics and Structure of von Willebrand Factor.

Biophys J 2016 Jul;111(2):312-322

Department of Physics and Center for Nanoscience, LMU Munich, Munich, Germany.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is activated for hemostasis by increased hydrodynamic forces at sites of vascular injury. Here, we present data from atomic force microscopy-based single-molecule force measurements, atomic force microscopy imaging, and small-angle x-ray scattering to show that the structure and mechanics of VWF are governed by multiple pH-dependent interactions with opposite trends within dimeric subunits. In particular, the recently discovered strong intermonomer interaction, which induces a firmly closed conformation of dimers and crucially involves the D4 domain, was observed with highest frequency at pH 7.4, but was essentially absent at pH values below 6.8. However, below pH 6.8, the ratio of compact dimers increased with decreasing pH, in line with a previous transmission electron microscopy study. These findings indicated that the compactness of dimers at pH values below 6.8 is promoted by other interactions that possess low mechanical resistance compared with the strong intermonomer interaction. By investigating deletion constructs, we found that compactness under acidic conditions is primarily mediated by the D4 domain, i.e., remarkably by the same domain that also mediates the strong intermonomer interaction. As our data suggest that VWF has the highest mechanical resistance at physiological pH, local deviations from physiological pH (e.g., at sites of vascular injury) may represent a means to enhance VWF's hemostatic activity where needed.
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http://dx.doi.org/10.1016/j.bpj.2016.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968480PMC
July 2016

Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction.

Proc Natl Acad Sci U S A 2016 Feb 19;113(5):1208-13. Epub 2016 Jan 19.

Department of Physics and Center for Nanoscience, Ludwig Maximilian University of Munich, 80799 Munich, Germany;

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF's hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above [Formula: see text]50 pN and provided [Formula: see text]80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF's effective length contributing to force sensing, they can be expected to tune VWF's sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF's function in hemostasis and thrombosis.
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http://dx.doi.org/10.1073/pnas.1516214113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747740PMC
February 2016

Decoding Cytoskeleton-Anchored and Non-Anchored Receptors from Single-Cell Adhesion Force Data.

Biophys J 2015 Oct;109(7):1330-3

Center for NanoScience, Ludwig-Maximilians-Universität München, Munich, Germany; Center for Applied Tissue Engineering and Regenerative Medicine, University of Applied Sciences, Munich, Germany. Electronic address:

Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking β1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that β1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions.
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http://dx.doi.org/10.1016/j.bpj.2015.07.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601042PMC
October 2015

A fast recoiling silk-like elastomer facilitates nanosecond nematocyst discharge.

BMC Biol 2015 Jan 16;13. Epub 2015 Jan 16.

Department of Molecular Evolution and Genomics, University of Heidelberg, Centre for Organismal Studies, Im Neuenheimer Feld 329, 69120, Heidelberg, Germany.

Background: The discharge of the Cnidarian stinging organelle, the nematocyst, is one of the fastest processes in biology and involves volume changes of the highly pressurised (150 bar) capsule of up to 50%. Hitherto, the molecular basis for the unusual biomechanical properties of nematocysts has been elusive, as their structure was mainly defined as a stress-resistant collagenous matrix.

Results: Here, we characterise Cnidoin, a novel elastic protein identified as a structural component of Hydra nematocysts. Cnidoin is expressed in nematocytes of all types and immunostainings revealed incorporation into capsule walls and tubules concomitant with minicollagens. Similar to spider silk proteins, to which it is related at sequence level, Cnidoin possesses high elasticity and fast coiling propensity as predicted by molecular dynamics simulations and quantified by force spectroscopy. Recombinant Cnidoin showed a high tendency for spontaneous aggregation to bundles of fibrillar structures.

Conclusions: Cnidoin represents the molecular factor involved in kinetic energy storage and release during the ultra-fast nematocyst discharge. Furthermore, it implies an early evolutionary origin of protein elastomers in basal metazoans.
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http://dx.doi.org/10.1186/s12915-014-0113-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321713PMC
January 2015

Seizure expression, behavior, and brain morphology differences in colonies of Genetic Absence Epilepsy Rats from Strasbourg.

Epilepsia 2014 Dec 6;55(12):1959-68. Epub 2014 Nov 6.

Department of Medicine, The University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria, Australia.

Objective: Originally derived from a Wistar rat strain, a proportion of which displayed spontaneous absence-type seizures, Genetic Absence Epilepsy Rats from Strasbourg (GAERS) represent the most widely utilized animal model of genetic generalized epilepsy. Here we compare the seizure, behavioral, and brain morphometric characteristics of four main GAERS colonies that are being actively studied internationally: two from Melbourne (MELB and STRAS-MELB), one from Grenoble (GREN), and one from Istanbul (ISTAN).

Methods: Electroencephalography (EEG) recordings, behavioral examinations, and structural magnetic resonance imaging (MRI) studies were conducted on GAERS and Non-Epileptic Control (NEC) rats to assess and compare the following: (1) characteristics of spike-and-wave discharges, (2) anxiety-like and depressive-like behaviors, and (3) MRI brain morphology of regions of interest.

Results: Seizure characteristics varied between the colonies, with MELB GAERS exhibiting the least severe epilepsy phenotype with respect to seizure frequency, and GREN GAERS exhibiting four times more seizures than MELB. MELB and STRAS-MELB colonies both displayed consistent anxiety and depressive-like behaviors relative to NEC. MELB and GREN GAERS showed similar changes in brain morphology, including increased whole brain volume and increased somatosensory cortical width. A previously identified mutation in the Cacna1h gene controlling the CaV 3.2 T-type calcium channel (R1584P) was present in all four GAERS colonies, but absent in all NEC rats.

Significance: This study demonstrates differences in epilepsy severity between GAERS colonies that were derived from the same original colony in Strasbourg. This multi-institute study highlights the potential impact of environmental conditions and/or genetic drift on the severity of epileptic and behavioral phenotypes in rodent models of epilepsy.
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http://dx.doi.org/10.1111/epi.12840DOI Listing
December 2014

Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes.

Nat Genet 2014 Dec 2;46(12):1327-32. Epub 2014 Nov 2.

Section of Complex Genetics, Department of Medical Genetics, University Medical Center Utrecht, Utrecht, the Netherlands.

Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes.
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http://dx.doi.org/10.1038/ng.3130DOI Listing
December 2014

Glutamatergic neuron-targeted loss of LGI1 epilepsy gene results in seizures.

Brain 2014 Nov 17;137(Pt 11):2984-96. Epub 2014 Sep 17.

1 INSERM, U 1127, F-75013, Paris, France 2 CNRS, UMR 7225, F-75013, Paris, France 3 Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, ICM, F-75013 Paris, France 4 Institut du Cerveau et de la Moelle épinière (ICM), F-75013, Paris, France

Leucin-rich, glioma inactivated 1 (LGI1) is a secreted protein linked to human seizures of both genetic and autoimmune aetiology. Mutations in the LGI1 gene are responsible for autosomal dominant temporal lobe epilepsy with auditory features, whereas LGI1 autoantibodies are involved in limbic encephalitis, an acquired epileptic disorder associated with cognitive impairment. We and others previously reported that Lgi1-deficient mice have early-onset spontaneous seizures leading to premature death at 2-3 weeks of age. Yet, where and when Lgi1 deficiency causes epilepsy remains unknown. To address these questions, we generated Lgi1 conditional knockout (cKO) mice using a set of universal Cre-driver mouse lines. Selective deletion of Lgi1 was achieved in glutamatergic pyramidal neurons during embryonic (Emx1-Lgi1cKO) or late postnatal (CaMKIIα-Lgi1cKO) developmental stages, or in gamma amino butyric acidergic (GABAergic) parvalbumin interneurons (PV-Lgi1cKO). Emx1-Lgi1cKO mice displayed early-onset and lethal seizures, whereas CaMKIIα-Lgi1cKO mice presented late-onset occasional seizures associated with variable reduced lifespan. In contrast, neither spontaneous seizures nor increased seizure susceptibility to convulsant were observed when Lgi1 was deleted in parvalbumin interneurons. Together, these data showed that LGI1 depletion restricted to pyramidal cells is sufficient to generate seizures, whereas seizure thresholds were unchanged after depletion in gamma amino butyric acidergic parvalbumin interneurons. We suggest that LGI1 secreted from excitatory neurons, but not parvalbumin inhibitory neurons, makes a major contribution to the pathogenesis of LGI1-related epilepsies. Our data further indicate that LGI1 is required from embryogenesis to adulthood to achieve proper circuit functioning.
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http://dx.doi.org/10.1093/brain/awu259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208469PMC
November 2014

Delineation of small mobile tumours with FDG-PET/CT in comparison to pathology in breast cancer patients.

Radiother Oncol 2014 Sep 9;112(3):407-12. Epub 2014 Sep 9.

Department of Nuclear Medicine, Henri Becquerel Cancer Centre, Rouen, France; Rouen University Hospital, France; QuantIF - LITIS [UPRES EA 4108], Rouen, France. Electronic address:

Purpose: Various segmentation methods for 18F-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography/computed tomography (PET/CT) images were correlated with pathological volume in breast cancer patients as a model of small mobile tumours.

Methods: Thirty women with T2-T3/M0 breast invasive ductal carcinoma (IDC) were included prospectively. A FDG-PET/CT was acquired 4 ± 3d before surgery in prone and supine positions, with/without respiratory gating. The segmentation methods were as follows: manual (Vm), relative (Vt%) and adaptive (Va) standard uptake value (SUV) threshold and semi-automatic on CT (Vct). Pathological volumes (Vpath) were measured for 26 lesions.

Results: The mean (±SD) Vpath was 4.1 ± 2.9 mL, and the lesion displacements were 3.9 ± 2.8 mm (median value: 3 mm). The delineated VOIs did not vary with the acquisition position nor with respiration, regardless of the segmentation method. The Vm, Va, Vct and Vt% methods, except Vt30%, were correlated with Vpath (0.5
Conclusions: When compared with pathology, small lesions (diameter <50mm) with limited respiratory displacement (i.e., breast or apical lung lesions) are best delineated on FDG-PET/CT using a 50% SUVmax threshold. The acquisition position and respiratory gating did not modify the delineated volumes.
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http://dx.doi.org/10.1016/j.radonc.2014.08.005DOI Listing
September 2014

Shape features of epileptic spikes are a marker of epileptogenesis in mice.

Epilepsia 2013 Dec 17;54(12):2219-27. Epub 2013 Oct 17.

U1099, INSERM, Rennes, France; Université de Rennes 1, LTSI, Rennes, France.

Purpose: To identify reliable biomarkers for quantitatively assessing the development of epilepsy in brain.

Methods: In a kainate mouse model of temporal lobe epilepsy, we performed long-term video-electroencephalography (EEG) monitoring (several weeks) of freely moving animals, from kainic acid injection to chronic epileptic stage. Using signal processing techniques, we automatically detected single epileptic spikes (ESs), and we quantified the evolution of shape features during the epileptogenesis process. Using a computational model of hippocampal activity (neuronal population level), we investigated excitatory-related and inhibitory-related parameters involved in morphologic changes of ESs.

Key Findings: The frequency of ESs increases during epileptogenesis. Regarding shape features, we found that both the initial spike component and the wave component of opposite polarity of ESs gradually increase during epileptogenesis. These very specific alterations of the shape of ESs were reproduced in a computational physiologically relevant neuronal population model. Using this model, we disclosed some key parameters (related to glutamatergic and γ-aminobutyric acid [GABA]ergic synaptic transmission) that explain the shape features of simulated ESs. Of interest, the model predicted that the decrease of GABAergic inhibition is responsible for the increase of the wave component of ESs. This prediction (at first sight counterintuitive) was verified in both in vivo and in vitro experiments. Finally, from aforementioned electrophysiologic features, we devised a novel and easily computable index (wave area/spike amplitude ratio) indicative of the progression of the disease (early vs. late stage).

Significance: Results suggest that dendritic inhibition in hippocampal circuits undertake dramatic changes over the latent period. These changes are responsible for observed modifications in the shape of ESs recorded in local field potential (LFP) signals. The proposed index may constitute a biomarker of epileptogenesis.
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http://dx.doi.org/10.1111/epi.12406DOI Listing
December 2013

Increase in lens capsule stiffness caused by vital dyes.

J Cataract Refract Surg 2013 Nov 4;39(11):1749-52. Epub 2013 Sep 4.

From the Department of Ophthalmology (Haritoglou, Schumann, Wolf, Kernt) and the Department of Applied Physics and Center for NanoScience (Mauell, Benoit), Ludwig-Maximilians-University, Munich, Germany; the Department of Ophthalmology (Henrich), University Hospital Basel, Basel, Switzerland. Electronic address:

Purpose: To assess potential changes in lens capsule mechanical properties after staining with brilliant blue, indocyanine green (ICG), and trypan blue.

Setting: Department of Ophthalmology and Applied Physics and Center for NanoScience, Ludwig-Maximilians-University, Munich, Germany.

Design: Experimental study.

Methods: Fifteen unstained lens capsules were dissected into 7 wedge-shaped parts. Three fragments were stained with brilliant blue 0.025%, ICG 0.05%, and trypan blue 0.06%, respectively, for 1 minute. Another 3 specimens were additionally illuminated using a standard light source. The seventh part served as an untreated control. All specimens were analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 μm × 10 μm were chosen, and stiffness was determined using AFM in a force spectroscopy mode. The force curves were performed with a data rate of 5000 Hz.

Results: Staining of the samples resulted in an increase in tissue stiffness (brilliant blue: P<.001; ICG: P<.01; trypan blue: P<.05). Additional illumination after staining further increased tissue stiffness, but not significantly. Mean increase in the relative elasticity values were 1.61 ± 0.15 (SD) for brilliant blue, 2.04 ± 0.21 for brilliant blue with illumination, 1.63 ± 0.22 for ICG, 2.01 ± 0.22 for ICG with illumination, 1.23 ± 0.11 for trypan blue, and 1.39 ± 0.11 for trypan blue with illumination. In relation to unstained tissue, the relative elasticity of the stained tissue increased 1.2-fold after illumination.

Conclusion: Staining significantly increased the mechanical properties of the human lens capsule.

Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.
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http://dx.doi.org/10.1016/j.jcrs.2013.02.057DOI Listing
November 2013

Exponential size distribution of von Willebrand factor.

Biophys J 2013 Sep;105(5):1208-16

Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany.

Von Willebrand Factor (VWF) is a multimeric protein crucial for hemostasis. Under shear flow, it acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Here, we quantify for the first time, to our knowledge, the size distribution of recombinant VWF and VWF-eGFP using a multilateral approach that involves quantitative gel analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy. We find an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization process during VWF biosynthesis. The distribution is solely described by the extent of polymerization, which was found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution is monitored using fluorescence correlation spectroscopy and compared to a computer simulation of a random cleavage process relating ADAMTS13 concentration to the degree of VWF breakdown. Quantitative assessment of VWF size distribution in terms of an exponential might prove to be useful both as a valuable biophysical characterization and as a possible disease indicator for clinical applications.
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http://dx.doi.org/10.1016/j.bpj.2013.07.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762346PMC
September 2013

Thalamocortical relationships and network synchronization in a new genetic model "in mirror" for absence epilepsy.

Brain Res 2013 Aug 3;1525:39-52. Epub 2013 Jun 3.

Epilepsie de l'Enfant et Plasticité Cérébrale, INSERM U 663, Paris, France.

Electroencephalographic generalized spike and wave discharges (SWD), the hallmark of human absence seizures, are generated in thalamocortical networks. However, the potential alterations in these networks in terms of the efficacy of the reciprocal synaptic activities between the cortex and the thalamus are not known in this pathology. Here, the efficacy of these reciprocal connections is assessed in vitro in thalamocortical slices obtained from BS/Orl mice, which is a new genetic model of absence epilepsy. These mice show spontaneous SWD, and their features can be compared to that of BR/Orl mice, which are free of SWD. In addition, since gap junctions may modulate the efficacy of these connections, their implications in pharmacologically-induced epileptiform discharges were studied in the same slices. The thalamus and neocortex were independently stimulated and the electrically-evoked responses in both structures were recorded from the same slice. The synaptic efficacy of thalamocortical and corticothalamic connections were assessed by measuring the dynamic range of synaptic field potential changes in response to increasing stimulation strengths. The connection efficacy was weaker in epileptic mice however, this decrease in efficacy was more pronounced in thalamocortical afferents, thus introducing an imbalance in the reciprocal connections between the cortex and thalamus. However, short-term facilitation of the thalamocortical responses were increased in epileptic mice compared to non-epileptic animals. These features may favor occurrence of rhythmical activities in thalamocortical networks. In addition, carbenoxolone (a gap junction blocker) decreased the cumulative duration of 4-aminopyridine-induced ictal-like activities, with a slower time course in epileptic mice. However, the 4-aminopyridine-induced GABA-dependent negative potentials, which appeared to trigger the ictal-like activities, remained. Our results show that the balance of the reciprocal connections between the thalamus and cortex is altered in favor of the corticothalamic connections in epileptic mice, and suggest that gap junctions mediate a stronger cortical synchronization in this strain.
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http://dx.doi.org/10.1016/j.brainres.2013.05.044DOI Listing
August 2013

Probing the interaction forces of prostate cancer cells with collagen I and bone marrow derived stem cells on the single cell level.

PLoS One 2013 5;8(3):e57706. Epub 2013 Mar 5.

Chair of Biophysics and New Materials, Ludwig-Maximilians-University, Munich, Germany.

Adhesion of metastasizing prostate carcinoma cells was quantified for two carcinoma model cell lines LNCaP (lymph node-specific) and PC3 (bone marrow-specific). By time-lapse microscopy and force spectroscopy we found PC3 cells to preferentially adhere to bone marrow-derived mesenchymal stem cells (SCP1 cell line). Using atomic force microscopy (AFM) based force spectroscopy, the mechanical pattern of the adhesion to SCP1 cells was characterized for both prostate cancer cell lines and compared to a substrate consisting of pure collagen type I. PC3 cells dissipated more energy (27.6 aJ) during the forced de-adhesion AFM experiments and showed significantly more adhesive and stronger bonds compared to LNCaP cells (20.1 aJ). The characteristic signatures of the detachment force traces revealed that, in contrast to the LNCaP cells, PC3 cells seem to utilize their filopodia in addition to establish adhesive bonds. Taken together, our study clearly demonstrates that PC3 cells have a superior adhesive affinity to bone marrow mesenchymal stem cells, compared to LNCaP. Semi-quantitative PCR on both prostate carcinoma cell lines revealed the expression of two Col-I binding integrin receptors, α1β1 and α2β1 in PC3 cells, suggesting their possible involvement in the specific interaction to the substrates. Further understanding of the exact mechanisms behind this phenomenon might lead to optimized therapeutic applications targeting the metastatic behavior of certain prostate cancer cells towards bone tissue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057706PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589411PMC
December 2013