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Throughout their life, plant root are submitted to mechanical stresses due to pressure exerted by the soil. So far, few studies addressed root cell deformation and calcium signaling elicited by soil compression. In this study, we designed a microchip inspired by pneumatic microvalve concept in order to deliver a lateral pressure to the root of a plant expressing the RGECO1-mTurquoise calcium reporter. Lateral pressure applied on the root induced a moderate elastic deformation of root cortical cells and elicited a multicomponent calcium signal at the onset of the pressure pulse, followed by a second one at the release of the pressure. This indicates that straining rather than stressing of tissues is relevant to trigger the calcium signal. The calcium elevation was restricted to the tissue under pressure and did not propagate. Additionally, the calcium signals exhibited a remarkable attenuation upon repetitive stimulations.Significance statementIn natural conditions, roots are subjected to diurnal hydraulic pressure variations producing a periodic root diameter fluctuation moreover during its progression, the lateral confinement of the root increases, generating additional lateral forces. Thus it is crucial for the root to be able to perceive and transduce these stimulations in order to progress in the heterogeneous soil network. Our microfluidic set-up, mimicking lateral soil pressure, revealed new features of the early root cellular responses. A cytosolic calcium elevation is observed upon increase but also upon release of the pressure, corresponding to variations in the shape of the cells of the cortex. Although the intensity of the calcium response increases with the pressure applied, successive pressure stimuli lead to attenuation of the calcium signal. Strain sensing and habituation to repetitive stimulation represent the fundamental properties of the “sensing system” allowing the root to adapt to the stresses generated by the soil.

Stroke is a leading cause of disability and death worldwide. Ivermectin is a broad-spectrum anti-parasitic agent with potential anti-bacterial, anti-viral, and anti-cancer effects. However, the effects of ivermectin on the brain are poorly described. This study examined the effects of ivermectin on cerebral ischemia-reperfusion (IR) in rats. A rat model of transient global IR was induced by bilateral carotid artery occlusion for 20 min. Rats received ivermectin (2 mg/kg/day, ip) one hour after inducing cerebral IR for three consecutive days at 24-h intervals. Next, we examined the effects of ivermectin on brain infarction, histopathology, malondialdehyde levels, myeloperoxidase activity, spatial learning and memory, and phospho-AMPK protein levels. The results showed that ivermectin reduced brain infarct size (P

Stroke is a serious life-threatening medical condition and is one of the principal reasons for death and disabilities worldwide. The aim of the present study was to determine the neuroprotective effects of hydroxychloroquine (HCQ) and the timing of its administration in cerebral ischemia/reperfusion (I/R) in rats. A global I/R model was used, and HCQ was administered in either pre- or post-treatment doses of 25 and 50 mg/kg. Effects of HCQ on infarct size, histological changes, oxidative stress, and learning and memory were evaluated. Phospho-AMPK and SQSTM1/p62 protein levels were also measured to elucidate the possible mechanisms involved. HCQ in both pre- (at doses of 25 and 50 mg/kg) or post-treatment (at a dose of 50 mg/kg) protocols reduces brain infarct size and histopathological changes and improves learning and memory after cerebral I/R. Pre-treatment with HCQ reduced AMPK activity with no significant effect on SQSTM1/p62 increment. Post-treatment with HCQ increased AMPK activity and SQSTM1/p62 protein levels. Our results show the neuroprotective effects of HCQ on cerebral I/R through the reduction in infarct size, histopathological changes, and improvement in memory and learning functions. Moreover, AMPK and autophagy may play a role in this protective effect.

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The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Using proteomics, we identified PPP2R1A among proteins differentially associated with the WAVE complex subunit ABI1 when RAC1 was activated and downstream generation of branched actin was blocked. PPP2R1A was found to associate at the lamellipodial edge with a novel form of WAVE complex, the WAVE Shell Complex (WSC), that contains NHSL1 instead of the Arp2/3 activating subunit WAVE as in the canonical WAVE Regulatory Complex (WRC). PPP2R1A was required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement was abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impaired WSC binding and migration regulation, suggesting that this novel function of PPP2R1A is critical for its tumor suppressor activity.

Phagocytic cells form the first line of defense in an organism, engulfing microbial pathogens. Phagocytosis involves cell mechanical changes that are not yet well understood. Understanding these mechanical modifications promises to shed light on the immune processes that trigger pathological complications. Previous studies showed that phagocytes undergo a sequence of spreading events around their target followed by an increase in cell tension. Seemingly in contradiction, other studies observed an increase in cell tension concomitant with membrane expansion. Even though phagocytes are viscoelastic, few studies have quantified viscous changes during phagocytosis. It is also unclear whether cell lines behave mechanically similarly to primary neutrophils. We addressed the question of simultaneous versus sequential spreading and mechanical changes during phagocytosis by using immunoglobulin-G-coated 8- and 20-μm-diameter beads as targets. We used a micropipette-based single-cell rheometer to monitor viscoelastic properties during phagocytosis by both neutrophil-like PLB cells and primary human neutrophils. We show that the faster expansion of PLB cells on larger beads is a geometrical effect reflecting a constant advancing speed of the phagocytic cup. Cells become stiffer on 20- than on 8-μm beads, and the relative timing of spreading and stiffening of PLB cells depends on target size: on larger beads, stiffening starts before maximal spreading area is reached but ends after reaching maximal area. On smaller beads, the stiffness begins to increase after cells have engulfed the bead. Similar to PLB cells, primary cells become stiffer on larger beads but start spreading and stiffen faster, and the stiffening begins before the end of spreading on both bead sizes. Our results show that mechanical changes in phagocytes are not a direct consequence of cell spreading and that models of phagocytosis should be amended to account for causes of cell stiffening other than membrane expansion.

Pericytes are mural cells of the microvasculature, recognized by their thin processes and protruding cell body. Pericytes wrap around endothelial cells and play a central role in regulating various endothelial functions, including angiogenesis and inflammation. They also serve as a vascular support and regulate blood flow by contraction. Prior reviews have examined pericyte biological functions and biochemical signaling pathways. In this Review, we focus on the role of mechanics and mechanobiology in regulating pericyte function. After an overview of the morphology and structure of pericytes, we describe their interactions with both the basement membrane and endothelial cells. We then turn our attention to biophysical considerations, and describe contractile forces generated by pericytes, mechanical forces exerted on pericytes, and pericyte responses to these forces. Finally, we discuss 2D and 3D engineered in vitro models for studying pericyte mechano-responsiveness and underscore the need for more evolved models that provide improved understanding of pericyte function and dysfunction.

In many cell types, shape and function are intertwined. In vivo, vascular endothelial cells (ECs) are typically elongated and aligned in the direction of blood flow; however, near branches and bifurcations where atherosclerosis develops, ECs are often cuboidal and have no preferred orientation. Thus, understanding the factors that regulate EC shape and alignment is important. In vitro , EC morphology and orientation are exquisitely sensitive to the composition and topography of the substrate on which the cells are cultured; however, the underlying mechanisms remain poorly understood. Different strategies of substrate patterning for regulating EC shape and orientation have been reported including adhesive motifs on planar surfaces and micro- or nano-scale gratings that provide substrate topography. Here, we explore how ECs perceive planar bio-adhesive versus microgrooved topographic surfaces having identical feature dimensions. We show that while the two types of patterned surfaces are equally effective in guiding and directing EC orientation, the cells are considerably more elongated on the planar patterned surfaces than on the microgrooved surfaces. We also demonstrate that the key factor that regulates cellular morphology is focal adhesion clustering which subsequently drives cytoskeletal organization. The present results promise to inform design strategies of novel surfaces for the improved performance of implantable cardiovascular devices.

Organs-on-chip is one of the most promising tools for producing more realistic in vitro models of human organs and tissues. One limitation of current organ-on-chip systems is the difficulty of deve...

In the microvasculature, blood flow-derived forces are key regulators of vascular structure and function. Consequently, the development of hydrogel-based microvessel-on-chip systems that strive to mimic the in vivo cellular organization and mechanical environment has received great attention in recent years. However, despite intensive efforts, current microvessel- on-chip systems suffer from several limitations, most notably failure to produce physiologically relevant wall strain levels. In this study, a novel microvessel-on-chip based on the templating technique and using luminal flow actuation to generate physiologically relevant levels of wall shear stress and circumferential stretch is presented. Normal forces induced by the luminal pressure compress the surrounding soft collagen hydrogel, dilate the channel, and create large circumferential strain. The fluid pressure gradient in the system drives flow forward and generates realistic pulsatile wall shear stresses. Rigorous characterization of the system reveals the crucial role played by the poroelastic behavior of the hydrogel in determining the magnitudes of the wall shear stress and strain. The experimental measurements are combined with an analytical model of flow in both the lumen and the porous hydrogel to provide an exceptionally versatile user manual for an application-based choice of parameters in microvessels-on-chip. This unique strategy of flow actuation adds a dimension to the capabilities of microvessel-on-chip systems and provides a more general framework for improving hydrogel-based in vitro engineered platforms.Abstract Figure

To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. Using an innovative micropipette rheometer, we show in three different cell types that when stimulated by microbeads mimicking target cells, leukocytes become up to ten times stiffer and more viscous. These mechanical changes initiate within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. The current results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new way to investigate cell mechanical processes. Our findings also suggest that dynamic immuno-mechanical measurements provide an identifier of leukocyte type and an indicator of the cell’s state of activation.

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T-lymphocyte passive deformation when squeezing through narrow capillaries is limited by the excess membrane contained in microvilli and membrane folds. During active processes such as transendothelial migration, larger deformations are made possible by an increase in membrane area, possibly through recruitment of internal membrane reservoirs., T-lymphocytes in the human body routinely undergo large deformations, both passively, when going through narrow capillaries, and actively, when transmigrating across endothelial cells or squeezing through tissue. We investigate physical factors that enable and limit such deformations and explore how passive and active deformations may differ. Employing micropipette aspiration to mimic squeezing through narrow capillaries, we find that T-lymphocytes maintain a constant volume while they increase their apparent membrane surface area upon aspiration. Human resting T-lymphocytes, T-lymphoblasts, and the leukemic Jurkat T-cells all exhibit membrane rupture above a critical membrane area expansion that is independent of either micropipette size or aspiration pressure. The unfolded membrane matches the excess membrane contained in microvilli and membrane folds, as determined using scanning electron microscopy. In contrast, during transendothelial migration, a form of active deformation, we find that the membrane surface exceeds by a factor of two the amount of membrane stored in microvilli and folds. These results suggest that internal membrane reservoirs need to be recruited, possibly through exocytosis, for large active deformations to occur.

this file contains figures supporting the manuscript; .raw files contains manuscript dataset

We describe the micropipette force probe, a novel technique that uses a micropipette as a flexible cantilever that aspirates a coated microbead and brings it into contact with a cell. We apply the technique to quantify mechanical and morphological events occurring during T-cell activation., In response to engagement of surface molecules, cells generate active forces that regulate many cellular processes. Developing tools that permit gathering mechanical and morphological information on these forces is of the utmost importance. Here we describe a new technique, the micropipette force probe, that uses a micropipette as a flexible cantilever that can aspirate at its tip a bead that is coated with molecules of interest and is brought in contact with the cell. This technique simultaneously allows tracking the resulting changes in cell morphology and mechanics as well as measuring the forces generated by the cell. To illustrate the power of this technique, we applied it to the study of human primary T lymphocytes (T-cells). It allowed the fine monitoring of pushing and pulling forces generated by T-cells in response to various activating antibodies and bending stiffness of the micropipette. We further dissected the sequence of mechanical and morphological events occurring during T-cell activation to model force generation and to reveal heterogeneity in the cell population studied. We also report the first measurement of the changes in Young’s modulus of T-cells during their activation, showing that T-cells stiffen within the first minutes of the activation process.

International audience; RUNNING HEAD Micropipette Force Probe to quantify single-cell force generation

International audience; We investigate the mechanical conditions leading to the rupture of the plasma membrane of an endothelial cell subjected to a local, compressive force. Membrane rupture is induced by tilted microindentation, a technique used to perform mechanical measurements on adherent cells. In this technique, the applied force can be deduced from the measured horizontal displacement of a microindenter’s tip, as imaged with an inverted microscope and without the need for optical sensors to measure the microindenter’s deflection. We show that plasma membrane rupture of endothelial cells occurs at a well-defined value of the applied compressive stress. As a point of reference, we use numerical simulations to estimate the magnitude of the compressive stresses exerted on endothelial cells during the deployment of a stent.

Flexible superparamagnetic filaments (‘fleximags’) are very slender elastic filaments, which can be driven by distributed magnetic torques to mimic closely the behaviour of biological flagella. Previously, fleximags have been used as a basis for artificial micro-swimmers capable of transporting small cargos Dreyfus et al. (Nature, vol. 437, 2005, p. 862). Here, we demonstrate how these filaments can be anchored to a wall to make carpets of artificial micro-magnetic cilia with tunable densities. We analyse the dynamics of an artificial cilium under both planar and three-dimensional beating patterns. We show that the dynamics are controlled by a single characteristic length scale varying with the inverse square root of the driving frequency, providing a mechanism to break the fore and aft symmetry and to generate net fluxes and forces. However, we show that an effective geometrical reciprocity in the filament dynamics creates intrinsic limitations upon the ability of the artificial flagellum to pump fluid when driven in two dimensions.

Purpose: The ability of remotely tagging tissues in a controlled and three-dimensional manner during preoperative imaging could greatly help surgeons to identify targets for resection. The authors’ objective is to selectively and noninvasively deposit markers under image guidance for such internal tattooing. Methods: This study describes the production of new ultrasound-inducible droplets carrying large payloads of fluorescent markers and thein vivo proof of concept of their remote and controlled deposition via focused ultrasound. The droplets are monodispersed multiple emulsions produced in a microfluidic system, consisting of aqueous fluorescein in perfluorocarbon in water. Their conversion (either by vaporization or cavitation) is performed remotely using a clinical ultrasonic imaging probe. Results: When submitted to 5 MHz imaging pulses, the droplets vaporizein vitro at 1.4 MPa peak-negative pressure and eject their content. After several seconds, a brightly fluorescent spot (0.5 mm diameter) is observed at the focus of the transducer. Experiments in the chorioallantoique membrane of chicken eggs and chicken embryo demonstrate that the spot is stable and is easily seen by naked eye. Conclusions: These ultrasound-inducible multiple emulsions could be used to deliver large amounts of contrast agents, chemotherapy, and genetic materialsin vivo using a conventional ultrasound scanner.

We have developed a simple and relatively inexpensive system to visualize adherent cells in profile while measuring their mechanical properties using microindentation. The setup allows simultaneous control of cell microenvironment by introducing a micropipette for the delivery of soluble factors or other cell types. We validate this technique against atomic force microscopy measurements and, as a proof of concept, measure the viscoelastic properties of vascular endothelial cells in terms of an apparent stiffness and a dimensionless parameter that describes stress relaxation. Furthermore, we use this technique to monitor the time evolution of these mechanical properties as the cells’ actin is depolymerized using cytochalasin-D.

We have developed a technique to directly quantify cell-substrate adhesion force using micropipette aspiration. The micropipette is positioned perpendicular to the surface of an adherent cell and a constant-rate aspiration pressure is applied. Since the micropipette diameter and the aspiration pressure are our control parameters, we have direct knowledge of the aspiration force, whereas the cell behavior is monitored either in brightfield or interference reflection microscopy. This setup thus allows us to explore a range of geometric parameters, such as projected cell area, adhesion area, or pipette size, as well as dynamical parameters such as the loading rate. We find that cell detachment is a well-defined event occurring at a critical aspiration pressure, and that the detachment force scales with the cell adhesion area (for a given micropipette diameter and loading rate), which defines a critical stress. Taking into account the cell adhesion area, intrinsic parameters of the adhesion bonds, and the loading rate, a minimal model provides an expression for the critical stress that helps rationalize our experimental results.

Previous records of chromosome number for species belonging to the genus Salvia L. (Lamiaceae) are summarized and the frequency of different chromosome base numbers, the number of taxa having a particular base number, and the occurrence of aneuploidy and polyploidy in the genus are discussed. In addition, chromosome numbers and meiotic behaviour in 12 species of Salvia from Iran, S. syriaca (2n = 2x = 20), S. leriifolia (2n = 2x = 20), S. macrosiphon (2n = 2x = 20), S. reuteriana (2n = 2x = 20), S. persepolitana (2n = 2x = 20), S. palaestina (2n = 2x = 20), S. ceratophylla (2n = 2x = 22), S. atropatana (2n = 2x = 22), S. chloroleuca (2n = 2x = 22), S. hypoleuca (2n = 2x = 22), S. aethiopis (2n = 2x = 22) and S. indica (2n = 2x = 22), are presented here. Comparison of the data in the literature revealed that the variation in base numbers supports the reported polyphyletic status of the genus. The relationships between geographical distribution, chromosome numbers and ploidy levels indicate that both aneuploidy and polyploidy have played an important role in the speciation processes in Salvia.