Dynamics of centipede locomotion revealed by large-scale traction force microscopy.

We present a novel approach to traction force microscopy (TFM) for studying the locomotion of 10 cm long walking centipedes on soft substrates. Leveraging the remarkable elasticity and ductility of kudzu starch gels, we use them as a deformable gel substrate, providing resilience against the centipedes' sharp leg tips. By optimizing fiducial marker size and density and fine-tuning imaging conditions, we enhance measurement accuracy. Our TFM investigation reveals traction forces along the centipede's longitudinal axis that effectively counterbalance inertial forces within the 0-10 mN range, providing the first report of non-vanishing inertia forces in TFM studies. Interestingly, we observe waves of forces propagating from the head to the tail of the centipede, corresponding to its locomotion speed. Furthermore, we discover a characteristic cycle of leg clusters engaging with the substrate: forward force (friction) upon leg tip contact, backward force (traction) as the leg pulls the substrate while stationary, and subsequent forward force as the leg tip detaches to reposition itself in the anterior direction. This work opens perspectives for TFM applications in ethology, tribology and robotics.

Microphase separation of living cells.

Self-organization of cells is central to a variety of biological systems and physical concepts of condensed matter have proven instrumental in deciphering some of their properties. Here we show that microphase separation, long studied in polymeric materials and other inert systems, has a natural counterpart in living cells. When placed below a millimetric film of liquid nutritive medium, a quasi two-dimensional, high-density population of Dictyostelium discoideum cells spontaneously assembles into compact domains. Their typical size of 100 µm is governed by a balance between competing interactions: an adhesion acting as a short-range attraction and promoting aggregation, and an effective long-range repulsion stemming from aerotaxis in near anoxic condition. Experimental data, a simple model and cell-based simulations all support this scenario. Our findings establish a generic mechanism for self-organization of living cells and highlight oxygen regulation as an emergent organizing principle for biological matter.

Aerotaxis and aerokinesis of Dictyostelium discoideum under hypoxic microenvironments.

Although spatiotemporal changes of oxygen in a microenvironment are known to affect the cellular dynamics of various eukaryotes, the details are not fully understood. Here, we describe the aerotaxis and aerokinesis of Dictyostelium discoideum (Dd), which has long been employed as a model organism for eukaryotic cells. We developed a microfluidic device capable of time-lapse observation of cultured cells while controlling oxygen concentrations in microchannels. Migratory behaviors of Dd were observed and quantitatively evaluated under an oxygen concentration gradient from 0% to 21% O2, as well as in various uniform oxygen conditions. In a hypoxic region within the oxygen concentration gradient, Dd migrated toward regions of higher oxygen concentration at increased velocity, which was independent of cell density. Observed under uniform oxygen concentrations of 1%, 2%, 3%, and 21%, the migration velocity of Dd increased significantly in hypoxic environments of 2% O2 or less. Thus, Dd shows aerotaxis, directed by the oxygen concentration gradient, and simultaneously shows aerokinesis, changing the migration velocity according to the oxygen concentration itself.

A new agarose-based microsystem to investigate cell response to prolonged confinement.

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and its porosity enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all cell lines tested. The soft cell confiner is thus a valuable tool to decipher the effects of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.

Charged particles interacting with a mixed supported lipid bilayer as a biomimetic pulmonary surfactant.

This study shows the interactions of charged particles with mixed supported lipid bilayers (SLB) as biomimetic pulmonary surfactants. We tested two types of charged particles: positively charged and negatively charged particles. Two parameters were measured: adsorption density of particles on the SLB and the diffusion coefficient of lipids by FRAPP techniques as a measure of interaction strength between particles and lipids. We found that positively charged particles do not adsorb on the bilayer, probably due to the electrostatic repulsion between positively charged parts of the lipid head and the positive groups on the particle surface, therefore no variation in diffusion coefficient of lipid molecules was observed. On the contrary, the negatively charged particles, driven by electrostatic interactions are adsorbed onto the supported bilayer. The adsorption of negatively charged particles increases with the zeta-potential of the particle. Consecutively, the diffusion coefficient of lipids is reduced probably due to binding onto the lipid heads which slows down their Brownian motion. The results are directly relevant for understanding the interactions of particulate matter with pulmonary structures which could lead to pulmonary surfactant inhibition or deficiency causing severe respiratory distress or pathologies.

Stability and tribological performances of fluid phospholipid bilayers: effect of buffer and ions.

We have investigated the mechanical and tribological properties of supported Dioleoyl phosphatidylcholine (DOPC) bilayers in different solutions: ultrapure water (pH 5.5), saline solution (150 mM NaCl, pH 5.8), Tris buffer (pH 7.2) and Tris saline buffer (150 mM NaCl, pH 7.2). Friction forces are measured using a homemade biotribometer. Lipid bilayer degradation is controlled in situ during friction tests using fluorescence microscopy. Mechanical resistance to indentation is measured by force spectroscopy with an atomic force microscope. This study confirms that mechanical stability under shear or normal load is essential to obtain low and constant friction coefficients. In ultrapure water, bilayers are not resistant and have poor lubricant properties. On the other hand, in Tris saline buffer, they fully resist to indentation and exhibit low (micro=0.035) and stable friction coefficient with no visible wear during the 50 min of the friction test. The unbuffered saline solution improves the mechanical resistance to indentation but not the lubrication. These results suggest that the adsorption of ions to the zwiterrionic bilayers has different effects on the mechanical and tribological properties of bilayers: higher resistance to normal indentation due to an increase in bilayer cohesion, higher lubrication due to an increase in bilayer-bilayer repulsion.

4D traction force microscopy reveals asymmetric cortical forces in migrating Dictyostelium cells.

We present a 4D (x; y; z; t) force map of Dictyostelium cells crawling on a soft gel substrate. Vertical forces are of the same order as the tangential ones. The cells pull the substratum upward along the cell, medium, or substratum contact line and push it downward under the cell except for the pseudopods. We demonstrate quantitatively that the variations in the asymmetry in cortical forces correlates with the variations of the direction and speed of cell displacement.

Changes in the magnitude and distribution of forces at different Dictyostelium developmental stages.

The distribution of forces exerted by migrating Dictyostelium amebae at different developmental stages was measured using traction force microscopy. By using very soft polyacrylamide substrates with a high fluorescent bead density, we could measure stresses as small as 30 Pa. Remarkable differences exist both in term of the magnitude and distribution of forces in the course of development. In the vegetative state, cells present cyclic changes in term of speed and shape between an elongated form and a more rounded one. The forces are larger in this first state, especially when they are symmetrically distributed at the front and rear edge of the cell. Elongated vegetative cells can also present a front-rear asymmetric force distribution with the largest forces in the crescent-shaped rear of the cell (uropod). Pre-aggregating cells, once polarized, only present this last kind of asymmetric distribution with the largest forces in the uropod. Except for speed, no cycle is observed. Neither the force distribution of pre-aggregating cells nor their overall magnitude are modified during chemotaxis, the later being similar to the one of vegetative cells (F(0) approximately 6 nN). On the contrary, both the force distribution and overall magnitude is modified for the fast moving aggregating cells. In particular, these highly elongated cells exert lower forces (F(0) approximately 3 nN). The location of the largest forces in the various stages of the development is consistent with the myosin II localization described in the literature for Dictyostelium (Yumura et al.,1984. J Cell Biol 99:894-899) and is confirmed by preliminary experiments using a GFP-myosin Dictyostelium strain.

Diffusion and deformations of single hydra cells in cellular aggregates.

Cell motion within cellular aggregates consists of both random and coherent components. We used confocal microscopy to study the center of mass displacements and deformations of single endodermal Hydra cells in two kinds of cellular aggregates, ectodermal and endodermal. We first carefully characterize the center of mass displacements using standard statistical analysis. In both aggregates, cells perform a persistent random walk, with the diffusion constant smaller in the more cohesive endodermal aggregate. We show that a simple parametric method is able to describe cell deformations and relate them to displacements. These deformations are random, with their amplitude and direction uncorrelated with the center of mass motion. Unlike for an isolated cell on a substrate, the random forces exerted by the surrounding cells predominate over the deformation of the cell itself, causing the displacements of a cell within an aggregate.

Evidence for partial agonist properties of daltroban (BM 13,505) at TP receptors in the anaesthetized open-chest rat.

We sought to determine whether the intrinsic pulmonary hypertensive activity of the purported thromboxane A2/prostanoid (TP) receptor antagonist, daltroban, was mediated by TP receptors, using the high efficacy TP receptor agonist, U-46619, and the silent TP receptor antagonist, SQ 29,548. In pentobarbitone-anesthetized, open-chest rats (n = 4-10 per group), non-cumulative injections of U-46619, dose-dependently increased mean pulmonary arterial pressure (MPAP) with an ED50 (geometric mean with 95% confidence limits in parentheses) of 1.4 (1.1-2.3) microg/kg i.v.. Daltroban increased MPAP in a bell-shaped manner, with an apparent ED50 [29 (21-35) microg/kg i.v.] being 21 fold less potent than that of U-46619. The maximal pulmonary hypertensive responses evoked by daltroban represented about half those induced by U-46619 (25.4+/-1.0 vs. 12.7+/-2 mmHg; P < 0.05 between groups). The TP receptor antagonist SQ 29,548 fully antagonized increases in MPAP evoked by equihypertensive doses of U-46619 (1.25 microg/kg) or daltroban (80 microg/kg). Further experiments were carried out to determine whether daltroban antagonized the pulmonary hypertensive responses evoked by the high efficacy agonist, U-46619, or by itself as receptor theory would predict for a partial agonist. Daltroban (10-2500 microg/kg) antagonized, although not fully, U-46619 (20 microg/kg)-evoked pulmonary hypertensive responses, since prominent intrinsic pulmonary hypertensive effects of daltroban were observed in the same range of doses. Furthermore, in contrast to U-46619 (1.25 microg/kg), daltroban (80 microg/kg) failed to evoke a second pulmonary hypertensive response following a previous injection, as would be expected for a partial agonist. Collectively, the results strongly suggest that daltroban behaves as a partial agonist at TP receptors in the pulmonary vascular bed of the rat in vivo.

Investigation of the mechanism by which ketanserin prolongs the duration of the cardiac action potential.

Action potential duration (APD) lengthening is believed to underlie the cardiac arrhythmogenicity of ketanserin, a serotonin (5-HT)2A/2C receptor antagonist. We wished to determine (a) whether this activity involves blockade of 5-HT2A/2C receptors and (b) the precise mechanism of ketanserin-induced APD prolongation. APs were recorded in guinea pig isolated papillary muscles by conventional "floating" microelectrodes, and potassium currents in guinea pig isolated myocytes were recorded in the whole-cell configuration. Ketanserin (1-10 microM) increased APD (EC50 value for enhancing APD at 90% repolarization (APD90) 3.1 +/- 2.7 microM, n = 24), without affecting resting potential, maximum upstroke velocity (Vmax) or AP amplitude (APA). Pirenperone (10 microM), a ketanserin congener, similarly increased APD90 from 204 +/- 3 to 241 +/- 7 ms (p < 0.001, n = 6). No increase in APD was observed, however, with ritanserin or ICI 170809, even at high concentrations (10 microM, n = 6, respectively), two 5-HT2A/2C receptor antagonists chemically distinct from ketanserin, thereby excluding the involvement of 5-HT2A/2C receptors in mediating APD lengthening. That APD prolongation was mediated specifically by the benzolyl-piperidine moiety of ketanserin and pirenperone was confirmed by 1-propyl-4(4-fluorobenzoyl)piperidine (PFBP), which evoked APD lengthening effects remarkably similar to those produced by ketanserin and pirenperone (EC50 3.73 +/- 2.6 microM, n = 12). In isolated cardiomyocytes, ketanserin (1-32 microM) selectively and concentration-dependently reduced the IKr component of the delayed outward current (IK) without affecting the inward rectifier current, IK1. Thus, ketanserin (32 microM) significantly reduced IK at a potential value of -20 mV from 813 +/- 65 to 569 +/- 55 pA (p < 0.001, n = 6), whereas at a potential value of -110 mV, IK1 was not significantly affected (730 +/- 103 vs. 603 +/- 143 pA, respectively; n=6). The results demonstrate that APD is prolonged by ketanserin and congeners but not be chemically different 5-HT2A/2C receptor antagonists. The benzoyl-piperidine moiety appears to mediate the APD-prolonging effects of ketanserin and pirenperone specifically. Furthermore, ketanserin-induced APD lengthening does not appear to involve 5-HT2A/2C receptors but is consecutive to direct blockade of myocardial potassium channels.

Comparison of the metabolism and pharmacokinetics of metbufen and itanoxone and their analogues in rats.

Metbufen, II, itanoxone, I, and two other derivatives of gamma-aryl-gamma-keto-substituted butyric acids labelled with 14C in their carbonyl group were synthesized for a metabolic investigation in rats. Profound changes in pharmacokinetic parameters, most specifically in the distribution, elimination, and metabolic pathways, were induced by substitution in the aromatic nucleus or changes in saturation of the aliphatic chain. The metabolites isolated from plasma and urine were identified by gas chromatography and mass spectrometry, by comparison with chemical controls, revealing the processes of metabolism of these structural analogues. This difference in metabolism further understanding of the diversity of biological effects inherent in these compounds.

[Liposarcoma of the spermatic cord]. / Liposarcome du cordon spermatique.

We review a new case of spermatic cord liposarcoma followed over a period of two years. After simple excision and 50 grays of radiotherapy had a local recurrence one year later; the patient a wide radical orchiectomy was then performed. A review of the literature illustrates the various clinicopathological data and their prognosis. The various treatments described are also analysed.

Secondary products of itanoxone.

Itanoxone synthesis by Friedel-Crafts reaction between itaconic anhydride and 2-chlorobiphenyl was studied. Five isomers corresponding to possible impurities were prepared and studied to perfect a reliable and practical method to detect these impurities in itanoxone.