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Abstract We present measurements of the bulk Young's moduli of early chick embryos at Hamburger-Hamilton stage 10. Using a micropipette probe with a force constant k ~0.025 N/m, we applied a known force in the plane of the embryo in the anterior-posterior direction and imaged the resulting tissue displacements. We used a two-dimensional finite-element simulation method to model the embryo as four concentric elliptical elastic regions with dimensions matching the embryo's morphology. By correlating the measured tissue displacements to the displacements calculated from the in-plane force and the model, we obtained the approximate short time linear-elastic Young's moduli: 2.4 ± 0.1 kPa for the midline structures (notocord, neural tube, and somites), 1.3 ± 0.1 kPa for the intermediate nearly acellular region between the somites and area pellucida, 2.1 ± 0.1 kPa for the area pellucida, and 11.9 ± 0.8 kPa for the area opaca.

Estradiol (E2) prevents cardiac hypertrophy, and these protective actions are mediated by estrogen receptor (ER)α and ERβ. The G protein-coupled estrogen receptor (GPER) mediates many estrogenic effects, and its activation in the heart has been observed in ischemia and reperfusion injury or hypertension models; however, the underlying mechanisms need to be fully elucidated. Herein, we investigated whether the protective effect of E2 against cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) is mediated by GPER and the signaling pathways involved. Isolated neonatal female rat cardiomyocytes were treated with ET-1 (100 nmol/l) for 48 h in the presence or absence of E2 (10 nmol/l) or GPER agonist G-1 (10 nmol/l) and GPER antagonist G-15 (10 nmol/l). ET-1 increased the surface area of cardiomyocytes, and this was associated with increased expression of atrial and brain natriuretic peptides. Additionally, ET-1 increased the phosphorylation of extracellular signal-related protein kinases-1/2 (ERK1/2). Notably, E2 or G-1 abolished the hypertrophic actions of ET-1, and that was reversed by G-15. Likewise, E2 reversed the ET-1-mediated increase of ERK1/2 phosphorylation as well as the decrease of phosphorylated Akt and its upstream activator 3-phosphoinositide-dependent protein kinase-1 (PDK1). These effects were inhibited by G-15, indicating that they are GPER dependent. Confirming the participation of GPER, siRNA silencing of GPER inhibited the antihypertrophic effect of E2. In conclusion, E2 plays a key role in antagonizing ET-1-induced hypertrophy in cultured neonatal cardiomyocytes through GPER signaling by a mechanism involving activation of the PDK1 pathway, which would prevent the increase of ERK1/2 activity and consequently the development of hypertrophy. [ABSTRACT FROM AUTHOR]

We use a quantitative phase imaging technique, defocusing microscopy (DM), to measure morphological, chemical, and mechanical parameters of individual red blood cells (RBCs) immersed in solutions with different osmolalities.We monitor the RBCs’ radius, volume, surface area, sphericity index, and hemoglobin content and concentration. The complete shape of cells is recovered and the effects of their adhesion to the glass substrate are observed. Finally, membrane fluctuation measurements give us information about the cells deformability. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated. Methods: Concentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group. Results: Anaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the nonanaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance. Conclusions: These findings revealed that vivax-infected patients with anaemia have increased levels of IgG autoantibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies. [ABSTRACT FROM AUTHOR]

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal. [ABSTRACT FROM AUTHOR]

Background: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. Methodology/Principal Finding: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. Conclusion/Significance: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex. Author Summary: Trypanosoma cruzi, is the etiological agent of a neglected tropical malady known as Chagas' disease, which affects about 8 million people in Latin America. 30–40% of affected individuals develop a symptomatic chronic infection, with cardiomyopathy being the most prevalent condition. T. cruzi utilizes an interesting strategy for entering cells: T. cruzi enhances intracellular calcium levels, which in turn trigger the exocytosis of lysosomal contents. Lysosomes then donate their membrane for the formation of the parasitophorous vacuole. Membrane rafts, cholesterol-enriched microdomains in the host cell plasma membrane, have also been implicated in T. cruzi invasion process. Since both plasma membrane and lysosomes collaborate in parasite invasion, we decided to study the importance of these membrane domains for lysosomal recruitment and fusion during T. cruzi invasion into host cells. Our results show that drug dependent depletion of plasma membrane cholesterol changes raft organization and induces excessive lysosome exocytosis in the earlier stages of treatment, leading to a depletion of lysosomes near the cell cortex, which in turn compromises T. cruzi invasion. Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events of pre-docked lysosomes, reducing lysosome availability at the cell cortex and consequently compromising T. cruzi infection. [ABSTRACT FROM AUTHOR]

We use the Glazier-Graner-Hogeweg model to simulate three-dimensional (3D), single-phenotype, avascular tumors growing in an homogeneous tissue matrix (TM) supplying a single limiting nutrient. We study the effects of two parameters on tumor morphology: a diffusion-limitation parameter defined as the ratio of the tumor-substrate consumption rate to the substrate-transport rate, and the tumor-TM surface tension. This initial model omits necrosis and oxidative/hypoxic metabolism effects, which can further influence tumor morphology, but our simplified model still shows significant parameter dependencies. The diffusion-limitation parameter determines whether the growing solid tumor develops a smooth (noninvasive) or fingered (invasive) interface, as in our earlier two-dimensional (2D) simulations. The sensitivity of 3D tumor morphology to tumor-TM surface tension increases with the size of the diffusion-limitation parameter, as in 2D. The 3D results are unexpectedly close to those in 2D. Our results therefore may justify using simpler 2D simulations of tumor growth, instead of more realistic but more computationally expensive 3D simulations. While geometrical artifacts mean that 2D sections of connected 3D tumors may be disconnected, the morphologies of 3D simulated tumors nevertheless correlate with the morphologies of their 2D sections, especially for low-surface-tension tumors, allowing the use of 2D sections to partially reconstruct medically-important 3D-tumor structures. [ABSTRACT FROM AUTHOR]

We present measurements of the bulk Young's moduli of early chick embryos at Hamburger-Hamilton stage 10. Using a micropipette probe with a force constant k ∼0.025 N/m, we applied a known force in the plane of the embryo in the anterior-posterior direction and imaged the resulting tissue displacements. We used a two-dimensional finite-element simulation method to model the embryo as four concentric elliptical elastic regions with dimensions matching the embryo's morphology. By correlating the measured tissue displacements to the displacements calculated from the inplane force and the model, we obtained the approximate short time linear-elastic Young's moduli: 2.4 ± 0.1 kPa for the midline structures (notocord, neural tube, and somites), 1.3 ± 0.1 kPa for the intermediate nearly acellular region between the somites and area pellucida, 2.1 ± 0.1 kPa for the area pellucida, and 11.9 ± 0.8 kPa for the area opaca. [ABSTRACT FROM AUTHOR]

Thin transparent objects (phase objects) can become visible in a bright-field light microscope, if the microscope is slightly defocused. Thick transparent objects, like red blood cells (RBC), are seen because some of their parts are always out of focus. By applying our recently developed defocusing microscopy technique to RBC, we are able to interpret RBC bright-field light microscopy images, an old standing problem. From the average image contrast we obtain RBC shape, size, and refractive index. From contrast fluctuations caused by the flicker phenomenon, we obtain RBC bending modulus and cytoplasm viscosity. [ABSTRACT FROM AUTHOR]

The mechanical properties of erythrocytes have been investigated by different techniques. However, there are few reports on how the viscoelasticity of these cells varies during malaria disease. Here, we quantitatively map the viscoelastic properties of Plasmodium falciparum -parasitized human erythrocytes. We apply new methodologies based on optical tweezers to measure the viscoelastic properties and defocusing microscopy to measure the erythrocyte height profile, the overall cell volume, and its form factor, a crucial parameter to convert the complex elastic constant into complex shear modulus. The storage and loss shear moduli are obtained for each stage of parasite maturation inside red blood cells, while the former increase, the latter decrease. Employing a soft glassy rheology model, we obtain the power-law exponent for the storage and loss shear moduli, characterizing the soft glassy features of red blood cells in each parasite maturation stage. Ring forms present a liquid-like behavior, with a slightly lower power-law exponent than healthy erythrocytes, whereas trophozoite and schizont stages exhibit increasingly solid-like behaviors. Finally, the surface elastic shear moduli, low-frequency surface viscosities, and shape recovery relaxation times all increase not only in a stage-dependent manner but also when compared to healthy red blood cells. Overall, the results call attention to the soft glassy characteristics of Plasmodium falciparum -parasitized erythrocyte membrane and may provide a basis for future studies to better understand malaria disease from a mechanobiological perspective. [ABSTRACT FROM AUTHOR]

The red blood cell membrane-cytoskeleton is a complex structure mainly responsible for giving the cell rigidity and shape. It also provides the erythrocyte with the ability to pass through narrow capillaries of the vertebrate blood circulatory system. Although the red blood cell viscoelastic properties have been extensively studied, reported experimental data differ by up to three orders of magnitude. This could be attributed to the natural cell variability, to the different techniques employed, and also to the models used for the cell response, which are highly dependent on cell geometry. Here, we use two methodologies based on optical tweezers to investigate the viscoelastic behavior of healthy human red blood cells, one applying small cell deformations (microrheology) and another imposing large deformations (tether extraction). We also establish a defocusing microscopy-based method to characterize the cell geometry and thus the erythrocyte form factor, an essential parameter that allows comparisons among the viscoelastic properties at different conditions. Moreover, for small deformations, a soft glassy rheology model is used to discuss the results, while for large deformations two surface shear moduli and one surface viscosity are determined, together with the surface tension and bending modulus of the erythrocyte membrane lipid component. We also show that F-actin is not detected in tethers, although the erythrocyte membrane has physical properties like those of other adherent cells, known to have tethers containing F-actin inside. Altogether, our results show good agreement with the reported literature and we argue that, to properly compare the viscoelastic properties of red blood cells in different situations, the task of cell geometry characterization must be accomplished. This may be especially important when the influence of agents, like the malaria parasite, induces changes in both the geometry and chemical constituents of the erythrocyte membrane. Together, the new methodologies and procedures used in this study would allow the erythrocyte community to better explore the mechanical behavior of red blood cells and may be useful to characterize erythrocyte viscoelasticity changes in several blood diseases. [ABSTRACT FROM AUTHOR]

Plasma membrane repair (PMR) is an important process for cell homeostasis, especially for cells under constant physical stress. Repair involves a sequence of Ca2+-dependent events, including lysosomal exocytosis and subsequent compensatory endocytosis. Cholesterol sequestration from plasma membrane causes actin cytoskeleton reorganization and polymerization, increasing cell stiffness, which leads to exocytosis and reduction of a peripheral pool of lysosomes involved in PMR. These changes in mechanical properties are similar to those observed in cells exposed to oxidized Low Density Lipoprotein (oxLDL), a key molecule during atherosclerosis development. Using a human umbilical vein endothelial cell line (EAhY926) we evaluated the influence of mechanical modulation induced by oxLDL in PMR and its effect in endothelial fragility. Similar to MβCD (a drug capable of sequestering cholesterol) treatment, oxLDL exposure led to actin reorganization and de novo polymerization, as well as an increase in cell rigidity and lysosomal exocytosis. Additionally, for both MβCD and oxLDL treated cells, there was an initial increase in endocytic events, likely triggered by the peak of exocytosis induced by both treatments. However, no further endocytic events were observed, suggesting that constitutive endocytosis is blocked upon treatment and that the reorganized cytoskeleton function as a mechanical barrier to membrane traffic. Finally, the increase in cell rigidity renders cells more prone to mechanical injury. Together, these data show that mechanical modulation induced by oxLDL exposure not only alters membrane traffic in cells, but also makes them more susceptible to mechanical injury, which may likely contribute to the initial steps of atherosclerosis development. Unlabelled Image • oxLDL induces actin rearrangements and de novo polymerization, leading to increased endothelial cell stiffness. • oxLDL disturbs vesicle trafficking events, such as constitutive endocytosis, between cell cytoplasm and plasma membrane. • Cell stiffness increases cell susceptibility to mechanical injury, which may contribute to atherosclerotic plaque formation [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to investigate the vascular effects of photobiomodulation using a light-emitting diode on the chorioallantoic embryonic membrane of chicken eggs grouped into different times of exposure and to detect the morphological changes induced by the light on the vascular network architecture using quantitative metrics., Methods: We used a phototherapy device with light-emitting diode (670 nm wavelength) as the source of photobiomodulation. We applied the red light at a distance of 2.5 cm to the surface of the chorioallantoic embryonic membrane of chicken eggs in 2, 4, or 8 sessions for 90 s and analyzed the vascular network architecture using AngioTool software (National Cancer Institute, USA). We treated the negative control group with 50 μl phosphate-buffered-saline (pH 7.4) and the positive control group (Beva) with 50 μl bevacizumab solution (Avastin, Produtos Roche Químicos e Farmacêuticos, S.A., Brazil)., Results: We found a decrease in total vessel length in the Beva group (24.96% ± 12.85%) and in all the groups that received 670 nm red light therapy (2× group, 34.66% ± 8.66%; 4× group, 42.42% ± 5.26%; 8× group, 38.48% ± 6.96%), compared with the negative control group. The fluence of 5.4 J/cm2 in 4 sessions (4×) showed more regular vessels. The number of junctions in the groups that received a higher incidence of 670 nm red light (4× and 8×) significantly decreased (p<0.0001)., Conclusion: Photo-biomodulation helps reduce vascularization in chorioallantoic embryonic membrane of chicken eggs and changes in the network architecture. Our results open the possibility of future clinical studies on using this therapy in patients with retinal diseases with neovascular components, especially age-related macular degeneration.

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Defocusing microscopy (DM) is a bright-field optical microscopy technique often used to obtain structural parameters of objects with low difference in refractive index in relation to the surrounding medium (phase objects). We show a use of this technique to measure the refractive index (n) profile of waveguides produced by femtosecond laser micromachining inside the bulk of a sodalime glass. The results are used to analyze the influence of production parameters on n. The methodology requires only a bright-field optical microscope and has proved to be easily applied. Results provide important insights on the waveguide microfabrication process, since translation speed, rather than intensity, has shown to be more important for achieving greater variations in refractive indices. Index of refraction differences between the waveguide and the substrate of the order of 10 -4 were measured for a series of straight waveguides fabricated with different parameters. Low sample scan speeds and pulse energies near 1.20 μJ used for fabrication showed the highest values of refractive index change for waveguides in sodalime glasses.

Defocusing microscopy (DM) is a recently developed technique that allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope. According to DM, the contrast of defocused images is proportional to cell surface curvature. Although, until now, this technique was used mainly to determine size and amount of membrane shape fluctuations, such as ruffles and small random membrane fluctuations, in macrophages, its applications on cell biology extend beyond that. We show how DM can be used to measure optical and mechanical properties of a living macrophage, such as cell refractive index n, membrane bending modulus K(c), and effective cell viscosity eta for membrane-actin meshwork relaxation. Experimental data collected from defocused images of bone marrow-derived macrophages were used to evaluate these parameters. The obtained values, averaged over several different macrophages, are n = (1.384 +/- 0.015), K(c) approximately 3.2 x 10(-19) J, and eta approximately 459 Pa.s. We also estimate the amplitude of the small fluctuations to be of the order of 3 nm, which is around the step size of a polymerizing actin filament.

Defocusing microscopy was used for real-time observation and quantification of membrane surface dynamics in murine bone marrow macrophages. Small random membrane fluctuations (SRMF), possibly metabolic driven, were detected uniformly over all membrane surface. Morphological and dynamical parameters of ruffles, such as shape, dimensions, and velocity of propagation, were analyzed. Optical tweezers were used to promote phagocytosis of single Leishmania amazonensis amastigotes by selected macrophages. Analysis of ruffling activity on the macrophages before and during phagocytosis of the parasites indicated that increased ruffling response near forming phagosomes, most likely induced by the parasite, accelerates phagocytosis. The effects of temperature decrease on the dynamics of membrane surface fluctuations and on the phagocytosis of parasites were used to determine the overall activation energies involved in these processes. The values obtained support the existence of strong correlation between membrane motility and phagocytic capacity.