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

2019 ◽  
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

AbstractSchistosomiasis 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 S. 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. But 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 confers S. mansoni the ability to reverse its direction of locomotion without turning its body, which is likely advantageous to maneuver 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, 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 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.

Biophysical tools to assess the interaction of PF4 with polyanions

2016 ◽  
Vol 116 (11) ◽  
pp. 783-791 ◽  
Author(s):  
Mihaela Delcea ◽  
Andreas Greinacher
Keyword(s):  
Threshold Energy ◽  
New Drugs ◽  
Titration Calorimetry ◽  
Platelet Factor ◽  
Force Microscopy ◽  
X Ray Crystallography ◽  
Thermal Stability Of Complexes ◽  
Endogenous Protein ◽  
Stability Of Complexes

SummaryThe antigen in heparin-induced thrombocytopenia (HIT) is expressed on platelet factor 4 (PF4) when PF4 complexes with polyanions. In recent years, biophysical tools (e. g. circular dichroism spectroscopy, atomic force microscopy, isothermal titration calorimetry, x-ray crystallography, electron microscopy) have gained an important role to complement immunological and functional assays for better understanding the interaction of heparin with PF4. This allowed identification of those features that make PF4 immunogenic (e. g. a certain conformational change induced by the polyanion, a threshold energy of the complexes, the existence of multimeric complexes, a certain number of bonds formed by PF4 with the polyanion) and to characterize the morphology and thermal stability of complexes formed by the protein with polyanions. These findings and methods can now be applied to test new drugs for their potential to induce the HIT-like adverse drug effect by preclinical in vitro testing. The methods and techniques applied to characterize the antigen in HIT may also be helpful to better understand the mechanisms underlying other antibody-mediated disorders in thrombosis and hemostasis (e. g. acquired hemophilia, thrombotic thrombocytopenic purpura). Furthermore, understanding the mechanisms making the endogenous protein PF4 immunogenic may help to understand the mechanisms underlying other autoimmune disorders.

scholarly journals How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes

2021 ◽  
Vol 8 (12) ◽  
pp. 213
Author(s):  
Dominik Müller ◽  
Sören Donath ◽  
Emanuel Georg Brückner ◽  
Santoshi Biswanath Devadas ◽  
Fiene Daniel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Troponin I ◽  
Troponin T ◽  
Laser System ◽  
Traction Force ◽  
Force Generation ◽  
Proper Function ◽  
Force Microscopy ◽  
Vitro Model

The proper function of cardiomyocytes (CMs) is highly related to the Z-disc, which has a pivotal role in orchestrating the sarcomeric cytoskeletal function. To better understand Z-disc related cardiomyopathies, novel models of Z-disc damage have to be developed. Human pluripotent stem cell (hPSC)-derived CMs can serve as an in vitro model to better understand the sarcomeric cytoskeleton. A femtosecond laser system can be applied for localized and defined damage application within cells as single Z-discs can be removed. We have investigated the changes in force generation via traction force microscopy, and in gene expression after Z-disc manipulation in hPSC-derived CMs. We observed a significant weakening of force generation after removal of a Z-disc. However, no significant changes of the number of contractions after manipulation were detected. The stress related gene NF-kB was significantly upregulated. Additionally, α-actinin (ACTN2) and filamin-C (FLNc) were upregulated, pointing to remodeling of the Z-disc and the sarcomeric cytoskeleton. Ultimately, cardiac troponin I (TNNI3) and cardiac muscle troponin T (TNNT2) were significantly downregulated. Our results allow a better understanding of transcriptional coupling of Z-disc damage and the relation of damage to force generation and can therefore finally pave the way to novel therapies of sarcomeric disorders.

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.

scholarly journals TFMLAB: a MATLAB toolbox for 4D traction force microscopy

2020 ◽  
Author(s):  
Jorge Barrasa Fano ◽  
Apeksha Shapeti ◽  
Alvaro Jorge-Penas ◽  
Mojtaba Barzegari ◽  
Jose A Sanz Herrera ◽  
...  
Keyword(s):  
User Interfaces ◽  
Traction Force ◽  
Cell Segmentation ◽  
Traction Force Microscopy ◽  
Force Microscopy ◽  
In Vitro Systems ◽  
Force Recovery ◽  
Segmentation Image ◽  
Cellular Forces

We present TFMLAB, a MATLAB software package for 4D (x;y;z;t) Traction Force Microscopy (TFM). While various TFM computational workflows are available in the literature, open-source programs that are easy to use by researchers with limited technical experience and that can analyze 4D in vitro systems do not exist. TFMLAB integrates all the computational steps to compute active cellular forces from confocal microscopy images, including image processing, cell segmentation, image alignment, matrix displacement measurement and force recovery. Moreover, TFMLAB eases usability by means of interactive graphical user interfaces. This work describes the package's functionalities and analyses its performance on a real TFM case.

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.

Natural Anti-inflammatory compounds as Drug candidates in Alzheimer’s disease

2020 ◽  
Vol 27 ◽  
Author(s):  
Reyaz Hassan Mir ◽  
Abdul Jalil Shah ◽  
Roohi Mohi-ud-din ◽  
Faheem Hyder Potoo ◽  
Mohd. Akbar Dar ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Natural Products ◽  
Protective Action ◽  
New Drugs ◽  
Huperzine A ◽  
Brain Disorder ◽  
Anti Inflammatory

: Alzheimer's disease (AD) is a chronic neurodegenerative brain disorder characterized by memory impairment, dementia, oxidative stress in elderly people. Currently, only a few drugs are available in the market with various adverse effects. So to develop new drugs with protective action against the disease, research is turning to the identification of plant products as a remedy. Natural compounds with anti-inflammatory activity could be good candidates for developing effective therapeutic strategies. Phytochemicals including Curcumin, Resveratrol, Quercetin, Huperzine-A, Rosmarinic acid, genistein, obovatol, and Oxyresvertarol were reported molecules for the treatment of AD. Several alkaloids such as galantamine, oridonin, glaucocalyxin B, tetrandrine, berberine, anatabine have been shown anti-inflammatory effects in AD models in vitro as well as in-vivo. In conclusion, natural products from plants represent interesting candidates for the treatment of AD. This review highlights the potential of specific compounds from natural products along with their synthetic derivatives to counteract AD in the CNS.

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