Abstract 17056: High-Throughput Physiological Assay for Force and Stiffness Quantification in IPS Derived Cardiomyocytes

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Ricardo Serrano ◽  
Wesley L McKeithan ◽  
Mark Mercola ◽  
Juan C del Álamo
Keyword(s):  
High Throughput ◽  
Traction Force ◽  
Optical Measurements ◽  
Response Curves ◽  
Force Microscopy ◽  
Novel Device ◽  
Cell Substrate ◽  
Omecamtiv Mecarbil ◽  
Set Up

Introduction: Cardiovascular disease persists as one of the leading causes of death in the U.S. Physiological assays with iPSC-cardiomyocytes have been quickly adopted for cardiac drug discovery, and the study of cardiac disease in vitro. Whereas most of these assays focus on the kinematics of voltage and calcium, assays that quantify contraction forces are less common. Moreover, there is no current high-throughput method to characterize the passive elastic properties of cardyomyocytes that contribute to dyastolic function. Methods and Results: We have developed a method to measure and characterize force of contraction and stiffness of beating cardiomyocytes, solely based on optical measurements. First, we created a device consisting of 96 well plates with soft deformable polyacrylamide gels, doped with fluorescent beads (A). IPSc cardiomyocytes are seeded on the gels and stained with Wheat Germ Agluttining (WGA). We then acquire videos of both membrane and beads (B). Our algorithm processes the videos with Particle Image Velocimetry to obtain instantaneous deformation fields on the cells (C) and the gel. Next, the traction stresses at the cell-substrate interface (D) are computed using Traction Force Microscopy, and the intracellular monolayer stress using Monolayer Stress Microscopy. Our algorithm computes temporal traces of strain on the cells (E), traction forces on the gel (F), and retrieve relevant parameters that characterize the signal (G). The elastic modulus of the cells is inferred by fitting the relationship between the measured intracellular stress and strain maps. We have validated our analysis by performing dose response curves on cardiomyocytes treated with isoproterenol, mavacamten, and omecamtiv mecarbil (H). Conclusions: To our knowledge, we present the first method that measures force and elasticity of cardiomyocytes in a high-throughput set-up, as well as a novel device that allows for the execution of such experiments.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Systematic Identification of Antiprion Drugs by High-Throughput Screening Based on Scanning for Intensely Fluorescent Targets

2005 ◽  
Vol 79 (12) ◽  
pp. 7785-7791 ◽  
Author(s):  
Uwe Bertsch ◽  
Konstanze F. Winklhofer ◽  
Thomas Hirschberger ◽  
Jan Bieschke ◽  
Petra Weber ◽  
...  
Keyword(s):  
High Throughput ◽  
Conformational Changes ◽  
High Throughput Screening ◽  
Prion Diseases ◽  
Inhibitory Effect ◽  
Helical Conformation ◽  
Response Curves ◽  
Vitro Assay ◽  
Systematic Identification

ABSTRACT Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrPSc), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrPC/PrPSc interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 μM. Among these, six compounds displayed an inhibitory effect on PrPSc propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N′-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrPC and PrPSc is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

scholarly journals Quantifying the mechanics of locomotion of the schistosome pathogen with respect to changes in its physical environment

2019 ◽  
Vol 16 (150) ◽  
pp. 20180675 ◽  
Author(s):  
Shun Zhang ◽  
Danielle Skinner ◽  
Prateek Joshi ◽  
Ernesto Criado-Hidalgo ◽  
Yi-Ting Yeh ◽  
...  
Keyword(s):  
Physical Environment ◽  
Traction Force ◽  
New Drugs ◽  
Force Microscopy ◽  
Forward Locomotion ◽  
Biomechanical Parameters ◽  
Hepatic Portal ◽  
Marine Worms ◽  
Wave Properties

Schistosomiasis is a chronic and morbid disease of poverty affecting approximately 200 million people worldwide. Mature schistosome flatworms wander in the host's hepatic portal and mesenteric venous system where they encounter a range of blood flow conditions and geometrical confinement. However, the mechanisms that support schistosome locomotion and underlie the pathogen's adaptation to its physical environment are largely unknown. By combining microfabrication and traction force microscopy, we developed various in vitro assays to quantify the mechanics of locomotion of adult male Schistosoma mansoni in different physiologically relevant conditions. We show that in unconfined settings, the parasite undergoes two-anchor marching mediated by the coordinated action of its oral and ventral suckers. This mode of locomotion is maintained when the worm faces an external flow, to which it responds by adjusting the strength of its suckers. In geometrically confined conditions, S. mansoni switches to a different crawling modality by generating retrograde peristaltic waves along its body, a mechanism shared with terrestrial and marine worms. However, while the surface of most worms has backward-pointing bristles that rectify peristaltic waves and facilitate forward locomotion, S. mansoni has isotropically oriented tubercles. This requires tight coordination between muscle contraction and substrate friction but gives S. mansoni the ability to reverse its direction of locomotion without turning its body, which is likely advantageous to manoeuvre in narrow-bore vessels. We show that the parasite can also coordinate the action of its suckers with its peristaltic body contractions to increase crawling speed. Throughout this study, we report on a number of biomechanical parameters to quantify the motility of adult schistosomes (e.g. sucker grabbing strength, the rate of detachment under flow, peristaltic wave properties and traction stresses). The new series of in vitro assays make it possible to quantify key phenotypical aspects of S. mansoni motility that could guide the discovery of new drugs to treat schistosomiasis.

scholarly journals Advanced in silico validation framework for three-dimensional Traction Force Microscopy and application to an in vitro model of sprouting angiogenesis

2020 ◽  
Author(s):  
J. Barrasa-Fano ◽  
A. Shapeti ◽  
J. de Jong ◽  
A. Ranga ◽  
J.A. Sanz-Herrera ◽  
...  
Keyword(s):  
In Silico ◽  
Inverse Method ◽  
In Vitro Model ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Traction Force ◽  
Force Microscopy ◽  
Validation Framework ◽  
Vitro Model

AbstractIn the last decade, cellular forces in three-dimensional hydrogels that mimic the extracellular matrix have been calculated by means of Traction Force Microscopy (TFM). However, characterizing the accuracy limits of a traction recovery method is critical to avoid obscuring physiological information due to traction recovery errors. So far, 3D TFM algorithms have only been validated using simplified cell geometries, bypassing image processing steps or arbitrarily simulating focal adhesions. Moreover, it is still uncertain which of the two common traction recovery methods, i.e., forward and inverse, is more robust against the inherent challenges of 3D TFM. In this work, we established an advanced in silico validation framework that is applicable to any 3D TFM experimental setup and that can be used to correctly couple the experimental and computational aspects of 3D TFM. Advancements relate to the simultaneous incorporation of complex cell geometries, simulation of microscopy images of varying bead densities and different focal adhesion sizes and distributions. By measuring the traction recovery error with respect to ground truth solutions, we found that while highest traction recovery errors occur for cases with sparse and small focal adhesions, our implementation of the inverse method improves two-fold the accuracy with respect to the forward method (average error of 23% vs. 50%). This advantage was further supported by recovering cellular tractions around angiogenic sprouts in an in vitro model of angiogenesis. The inverse method recovered more realistic traction patterns than the forward method, showing higher traction peaks and a clearer pulling pattern at the sprout protrusion tips.

scholarly journals Ultrafast and Predictive Mass Spectrometry–Based Autotaxin Assays for Label-Free Potency Screening

2017 ◽  
Vol 22 (4) ◽  
pp. 425-432 ◽  
Author(s):  
Tom Bretschneider ◽  
Andreas Harald Luippold ◽  
Helmut Romig ◽  
Daniel Bischoff ◽  
Klaus Klinder ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Whole Blood ◽  
Label Free ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Data Points ◽  
Set Up

Autotaxin (ATX) is a promising drug target for the treatment of several diseases, such as cancer and fibrosis. ATX hydrolyzes lysophosphatidyl choline (LPC) into bioactive lysophosphatidic acid (LPA). The potency of ATX inhibitors can be readily determined by using fluorescence-based LPC derivatives. While such assays are ultra-high throughput, they are prone to false positives compared to assays based on natural LPC. Here we report the development of ultrafast mass spectrometry–based ATX assays enabling the measurement of data points within 13 s, which is 10 times faster than classic liquid chromatography–mass spectrometry. To this end, we set up a novel in vitro and whole-blood assay. We demonstrate that the potencies determined with these assays are in good agreement with the in vivo efficacy and that the whole-blood assay has the best predictive power. This high-throughput label-free approach paired with the translatable data quality is highly attractive for appropriate guidance of medicinal chemists for constructing strong structure-activity relationships.

scholarly journals In vitro anti-motility and antispasmodic effects of Garcinia mangostana extracts in isolated chicken ileum preparation

2019 ◽  
Vol 10 (2) ◽  
pp. 1444-1447
Author(s):  
Michelle Ooi Yi Ching ◽  
Sasikala Chinnappan ◽  
Mogana Sundari Rajagopal
Keyword(s):  
Effective Concentration ◽  
Aqueous Extracts ◽  
Inhibitory Effects ◽  
Garcinia Mangostana ◽  
Response Curves ◽  
Half Maximal Effective Concentration ◽  
Set Up ◽  
Smooth Muscle Contractions ◽  
Significant Concentration

Garcinia mangostana pericarps have been traditionally used in Southeast Asia for a variety of medicinal conditions. The present study was carried out to determine the anti-motility and antispasmodic effects of methanolic and aqueous G. mangostana extracts (MEM and AEM) on isolated chicken ileum. Extracts were prepared from the pericarp of G. mangostana using maceration technique with methanol and distilled water. Isolated ileum preparations were set up for recording in Tyrode’s solution at 37°C. Dose-response curves were plotted using various doses of agonist as control such as acetylcholine (ACh) and histamine. Atropine, mepyramine and extracts were used as an antagonist. The results showed that methanolic and aqueous extracts possess significant concentration-dependent inhibitory effects (p<0.05) on agonist-induced contractions. The half maximal effective concentration (EC50) of extracts and standard antagonists were higher than the agonist alone. Both methanolic and aqueous extract of G. mangostana exerts anti-motility and antispasmodic effects on smooth muscle contractions. The study provides findings that support G. mangostana can be the potential treatment for diarrhoea and spasm.

scholarly journals Antibacterial potentiality of antiulcer and antispasmodic drugs with selected antibiotics against methicillin resistant Staphylococcus aureus: In vitro and in silico studies

2015 ◽  
Vol 10 (4) ◽  
pp. 875
Author(s):  
Krishnan Akilandeswari ◽  
Kandasamy Ruckmani
Keyword(s):  
Antibacterial Properties ◽  
Docking Studies ◽  
Penicillin Binding Protein ◽  
Force Microscopy ◽  
Atomic Force ◽  
Mebeverine Hydrochloride ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Set Up

<p class="Abstract">In the present study, the antispasmodic drug mebeverine hydrochloride and the antiulcer drug troxipide were tested for their possible antibacterial properties <em>in vitro</em>. The antimicrobial assays of the above drugs were determined with ampicillin, penicillin and ciprofloxacin against sensitive and resistant strains and their resistance were confirmed through Polymerase Chain Reaction by identifying the presence of the <em>mec</em>A gene. A computer-aided method was used for screening the effectiveness of the drug interactions. Mebeverine and  troxipide inhibited most of the sensitive and resistant strains tested<em> in vitro</em> from 32.5 to 125 µg/mL. The loss of structural alterations of the cell wall was analyzed by atomic force microscopy. In docking studies, troxipide and mebeverine were found to have substantial inhibition against penicillin binding protein 2a (IVQQ) and UDP-N-acetylglucosamine 1-carboxyvinyltransferase (2YVW) receptor proteins that seem to have interacted with most of the residues.</p><p class="Abstract"> </p><p class="Abstract"><strong>Video Clips</strong></p><p class="Abstract"><a href="https://www.youtube.com/v/CpfdUxdNvt0">Set up</a>                2 min 27 sec</p><p class="Abstract"><a href="https://www.youtube.com/v/IJlkGtIViUU">Broth and drug</a>     4 min 40 sec</p><p class="Abstract"><a href="https://www.youtube.com/v/_jZHZ8zOJlc">Inoculation</a>          2 min 29 sec</p><p class="Abstract"><a href="https://www.youtube.com/v/VYT5slycgdE">Incubation and result</a>     46 sec</p><p> </p><p> </p><p align="center"><strong><br /></strong></p>

scholarly journals Calcium oscillations in wounded fibroblast monolayers are spatially regulated through substrate mechanics

2017 ◽  
Author(s):  
Josephine Lembong ◽  
Benedikt Sabass ◽  
Howard A. Stone
Keyword(s):  
Physiological Role ◽  
Traction Force ◽  
Cell Types ◽  
Cytosolic Calcium ◽  
Skin Wound ◽  
Calcium Oscillations ◽  
Force Microscopy ◽  
Substrate Rigidity ◽  
Cell Substrate ◽  
Multicellular Organisms

ABSTRACTThe maintenance of tissue integrity is essential for the life of multicellular organisms. Healing of a skin wound is a paradigm for how various cell types localize and repair tissue perturbations in an orchestrated fashion. To investigate biophysical mechanisms associated with wound localization, we focus on a model system consisting of a fibroblast monolayer on an elastic substrate. We find that the creation of an edge in the monolayer causes cytosolic calcium oscillations throughout the monolayer. The oscillation frequency increases with cell density, which shows that wound-induced calcium oscillations occur collectively. Inhibition of myosin II reduces the number of oscillating cells, demonstrating a coupling between actomyosin activity and calcium response. The spatial distribution of oscillating cells depends on the stiffness of the substrate. For soft substrates with a Young’s modulus E ~ 360 Pa, oscillations occur on average within 0.2 mm distance from the wound edge. Increasing substrate stiffness leads to an average localization of oscillations away from the edge (up to ~0.6 mm). In addition, we use traction force microscopy to determine stresses between cells and substrate. We find that an increase of substrate rigidity leads to a higher traction magnitude. For E < ~2 kPa, the traction magnitude is strongly concentrated at the monolayer edge, while for E > ~8 kPa, traction magnitude is on average almost uniform beneath the monolayer. Thus, the spatial occurrence of calcium oscillations correlates with the cell-substrate traction. Overall, the experiments with fibroblasts demonstrate a collective, chemomechanical localization mechanism at the edge of a wound with a potential physiological role.

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