Refinement of mouse protocols for the study of platelet thromboembolic responses in vivo

Mouse models of thromboembolism are frequently used to investigate platelet function in vivo and, according to European Union (EU) legislation, must be conducted in the context of replacement, refinement and reduction. We have previously developed a refined real-time mouse model of thromboembolism as an alternative to models of thromboembolic mortality which inflict considerable pain and suffering. Real-time monitoring involves infusion of radiolabelled platelets into the circulation of anaesthetized mice, and platelet aggregation is measured as increases in platelet-associated counts in the pulmonary vasculature following injection of platelet agonists. This gives a definitive data set on the tissue localization and extent of platelet activation. We developed an additional, more simplistic alternative to mortality models based on blood microsampling which entails the measurement of circulating platelet counts following agonist stimulation. Blood microsamples were collected from the tail vein of anaesthetized mice at three different time points leading to a reduction in animal numbers. Platelet counts significantly dropped 1 minute after stimulation with collagen or thrombin and were restored over 10 minutes. These results correlate with those obtained via real-time monitoring and were confirmed by immunohistochemistry. Pre-treatment of mice with aspirin significantly inhibited the decrease in platelet counts following collagen. These data suggest that blood microsampling may be implemented as a simplistic refined alternative to mortality models of thromboembolism when specialized monitoring equipment, or use of radioactive isotopes for real-time monitoring, which remains the ‘gold standard’, is not feasible. Microsampling refines and reduces animal procedures in compliance with current EU legislatio

Incorporating chemical signalling factors into cell-based models of growing epithelial tissues

In this paper we present a comprehensive computational framework within which the effects of chemical signalling factors on growing epithelial tissues can be studied. The method incorporates a vertex-based cell model, in conjunction with a solver for the governing chemical equations. The vertex model provides a natural mesh for the finite element method (FEM), with node movements determined by force laws. The arbitrary Lagrangian–Eulerian formulation is adopted to account for domain movement between iterations. The effects of cell proliferation and junctional rearrangements on the mesh are also examined. By implementing refinements of the mesh we show that the finite element (FE) approximation converges towards an accurate numerical solution. The potential utility of the system is demonstrated in the context of Decapentaplegic (Dpp), a morphogen which plays a crucial role in development of the Drosophila imaginal wing disc. Despite the presence of a Dpp gradient, growth is uniform across the wing disc. We make the growth rate of cells dependent on Dpp concentration and show that the number of proliferation events increases in regions of high concentration. This allows hypotheses regarding mechanisms of growth control to be rigorously tested. The method we describe may be adapted to a range of potential application areas, and to other cell-based models with designated node movements, to accurately probe the role of morphogens in epithelial tissues

Active Tension Network model suggests an exotic mechanical state realized in epithelial tissues.

Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behavior remains an open problem. Here we formulate and analyze the Active Tension Network (ATN) model, which assumes that the mechanical balance of cells within a tissue is dominated by cortical tension and introduces tension-dependent active remodeling of the cortex. We find that ATNs exhibit unusual mechanical properties. Specifically, an ATN behaves as a fluid at short times, but at long times supports external tension like a solid. Furthermore, an ATN has an extensively degenerate equilibrium mechanical state associated with a discrete conformal - "isogonal" - deformation of cells. The ATN model predicts a constraint on equilibrium cell geometries, which we demonstrate to approximately hold in certain epithelial tissues. We further show that isogonal modes are observed in the fruit y embryo, accounting for the striking variability of apical areas of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, the study of which helps to understand biological phenomena

Mechanical Stress Inference for Two Dimensional Cell Arrays

Many morphogenetic processes involve mechanical rearrangement of epithelial tissues that is driven by precisely regulated cytoskeletal forces and cell adhesion. The mechanical state of the cell and intercellular adhesion are not only the targets of regulation, but are themselves likely signals that coordinate developmental process. Yet, because it is difficult to directly measure mechanical stress {\it in vivo} on sub-cellular scale, little is understood about the role of mechanics of development. Here we present an alternative approach which takes advantage of the recent progress in live imaging of morphogenetic processes and uses computational analysis of high resolution images of epithelial tissues to infer relative magnitude of forces acting within and between cells. We model intracellular stress in terms of bulk pressure and interfacial tension, allowing these parameters to vary from cell to cell and from interface to interface. Assuming that epithelial cell layers are close to mechanical equilibrium, we use the observed geometry of the two dimensional cell array to infer interfacial tensions and intracellular pressures. Here we present the mathematical formulation of the proposed Mechanical Inverse method and apply it to the analysis of epithelial cell layers observed at the onset of ventral furrow formation in the {\it Drosophila} embryo and in the process of hair-cell determination in the avian cochlea. The analysis reveals mechanical anisotropy in the former process and mechanical heterogeneity, correlated with cell differentiation, in the latter process. The method opens a way for quantitative and detailed experimental tests of models of cell and tissue mechanics

Practice Considerations for Adapting In-Person Groups to Telerehabilitation

The Coronavirus-2019 (COVID-19) pandemic has shifted research and healthcare system priorities, stimulating literature on implementation and evaluation of telerehabilitation for a variety of patient populations. While there is substantial literature on individual telerehabilitation, evidence about group telerehabilitation remains limited despite its increasing use by rehabilitation providers. Therefore, the purpose of this manuscript is to describe our expert team’s consensus on practice considerations for adapting in-person group rehabilitation to group telerehabilitation to provide rapid guidance during a pandemic and create a foundation for sustainability of group telerehabilitation beyond the pandemic’s end. &nbsp

SÁNCHEZ, VIOLETA [Material gráfico]

Copia digital. Madrid : Ministerio de Educación, Cultura y Deporte. Subdirección General de Coordinación Bibliotecaria, 201

Inherited human group IVA cytosolic phospholipase A(2) deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

This research was supported by an Imperial College Junior Research Fellowship (to N.S.K.), Wellcome Trust program grant (0852551Z108/Z to J.A.M. and T.D.W.), British Heart Foundation Ph.D. studentship (FS/10/033/28271 to F.R.), British Heart Foundation project grant (PG/11/39/28890 to D.B.-B.), and by the Intramural Research Program of the U.S. National Institutes of Health, National Institute of Environmental Health Sciences (Z01 ES025034 to D.C.Z.)

RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding.American Cancer Society (125792-RSG-14-039-01-CSM

Comparative study of non-invasive force and stress inference methods in tissue

In the course of animal development, the shape of tissue emerges in part from mechanical and biochemical interactions between cells. Measuring stress in tissue is essential for studying morphogenesis and its physical constraints. Experimental measurements of stress reported thus far have been invasive, indirect, or local. One theoretical approach is force inference from cell shapes and connectivity, which is non-invasive, can provide a space-time map of stress and relies on prefactors. Here, to validate force- inference methods, we performed a comparative study of them. Three force-inference methods, which differ in their approach of treating indefiniteness in an inverse problem between cell shapes and forces, were tested by using two artificial and two experimental data sets. Our results using different datasets consistently indicate that our Bayesian force inference, by which cell-junction tensions and cell pressures are simultaneously estimated, performs best in terms of accuracy and robustness. Moreover, by measuring the stress anisotropy and relaxation, we cross-validated the force inference and the global annular ablation of tissue, each of which relies on different prefactors. A practical choice of force-inference methods in distinct systems of interest is discussed.Comment: 12 pages, 8 figures, EPJ E: Topical issue on "Physical constraints on morphogenesis and evolution

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin–Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization