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Blood is a two-component system with two levels of hierarchy: the macrosystem of blood formed elements and the dispersed system of blood nanoparticles. Biological nanoparticles are the key participants in communication between the irradiated and non-irradiated cells and inducers of the non-targeted effects of ionizing radiation. The work aimed at studying by atomic force microscopy the structural, mechanical, and electrical properties of exosomes and lipoproteins (LDL/VLDL) isolated from rat blood after its exposure to X-rays in vitro. MATERIALS AND METHODS: The whole blood of Wistar rats fed with a high-fat diet was irradiated with X-rays (1 and 100â¯Gy) in vitro. The structural and mechanical properties (the elastic modulus and nonspecific adhesion force) of exosome and lipoprotein isolates from the blood by ultracentrifugation method were studied using Bruker Bioscope Resolve atomic force microscope in PF QNM mode, their electric properties (the zeta-potential) was measured by electrophoretic mobility. RESULTS: Lipoproteins isolated from non-irradiated blood were softer (Me(LQ; UQ): 7.8(4.9;12.1) MPa) compared to blood nanoparticles of its exosome fraction (34.8(22.6;44.9) MPa) containing both exosomes and non-membrane nanoparticles. X-ray blood irradiation with a dose of 1â¯Gy significantly weakened the elastic properties of lipoproteins. Exposure of the blood to 100â¯Gy X-rays made lipoproteins stiffer and their nonspecific adhesive properties stronger. The radiation effects on the mechanical parameters of exosomes and non-membrane nanoparticles in exosome fractions differed. The significant radiation-induced change in electric properties of the studied nanoparticles was detected only for lipoproteins in the blood irradiated with 1â¯Gy X-rays. The low-dose radiation-induced changes in zeta-potential and increase in lipoprotein size with the appearance of a soft thick surface layer indicate the formation of the modified lipoproteins covered with a corona from macromolecules of irradiated blood. CONCLUSION: Our data obtained using the nanomechanical mapping mode of AFM are the first evidence of the significant radiation-induced changes in the structural and mechanical properties of the dispersed system of blood nanoparticles after the X-ray irradiation of the blood.
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Exosomas , Lipoproteínas , Microscopía de Fuerza Atómica , Ratas Wistar , Animales , Microscopía de Fuerza Atómica/métodos , Rayos X , Exosomas/efectos de la radiación , Exosomas/ultraestructura , Exosomas/química , Ratas , Lipoproteínas/sangre , Lipoproteínas/efectos de la radiación , MasculinoRESUMEN
Cells of two molecular genetic types of breast cancer-hormone-dependent breast cancer (ZR-75 cell line) and triple-negative breast cancer (BT-20 cell line)-were studied using atomic force microscopy and an optical nanomotion detection method. Using the Peak Force QNM and Force Volume AFM modes, we revealed the unique patterns of the dependence of Young's modulus on the indentation depth for two cancer cell lines that correlate with the features of the spatial organization of the actin cytoskeleton. Within a 200-300 nm layer just under the cell membrane, BT-20 cells are stiffer than ZR-75 cells, whereas in deeper cell regions, Young's modulus of ZR-75 cells exceeds that of BT-20 cells. Two cancer cell lines also displayed a difference in cell nanomotion dynamics upon exposure to cytochalasin D, a potent actin polymerization inhibitor. The drug strongly modified the nanomotion pattern of BT-20 cells, whereas it had almost no effect on the ZR-75 cells. We are confident that nanomotion monitoring and measurement of the stiffness of cancer cells at various indentation depths deserve further studies to obtain effective predictive parameters for use in clinical practice.
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Citoesqueleto de Actina , Neoplasias de la Mama Triple Negativas , Humanos , Microscopía de Fuerza Atómica/métodos , Citoesqueleto de Actina/metabolismo , Módulo de Elasticidad , Línea Celular , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Vascular aging is a major contributing factor to cardiovascular disease. The aged blood vessels, characterized by vascular wall thickening and stiffening, are instigated by endothelial cell dysfunction induced by oxidative stress and inflammation. von Willebrand Factor (vWF) is a glycoprotein known for its role in coagulation, and plasma levels of vWF are increased with age. Elevated vWF promotes thrombosis, atherosclerotic plaque formation, inflammation and proliferation of vascular smooth muscle cells. Cadmium (Cd) is an environmental pollutant associated with increased morbidity and mortality of cardiovascular disease. At low concentrations, Cd activates pro-survival signaling in endothelial cells, however enhances intima-media thickness and atherogenesis. A non-cytotoxic dose of Cd also increases endothelial vWF expression and secretion in vivo and in vitro. In this review, we summarize the molecular mechanisms underlying vWF-promoted vascular aging-associated pathologies and Cd-induced vWF expression. In addition, we propose that exposure to low-dose Cd is a risk factor for vascular aging, through elevation of plasma vWF.
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Introduction: Patients undergoing cancer treatment by radiation therapy commonly develop Candida albicans infections (candidiasis). Such infections are generally treated by antifungals that unfortunately also induce numerous secondary effects in the patient. Additional to the effect on the immune system, ionizing radiation influences the vital activity of C. albicans cells themselves; however, the reaction of C. albicans to ionizing radiation acting simultaneously with antifungals is much less well documented. In this study, we explored the effects of ionizing radiation and an antifungal drug and their combined effect on C. albicans. Methods: The study essentially relied on a novel technique, referred to as optical nanomotion detection (ONMD) that monitors the viability and metabolic activity of the yeast cells in a label and attachment-free manner. Results and discussion: Our findings demonstrate that after exposure to X-ray radiation alone or in combination with fluconazole, low-frequency nanoscale oscillations of whole cells are suppressed and the nanomotion rate depends on the phase of the cell cycle, absorbed dose, fluconazole concentration, and post-irradiation period. In a further development, the ONMD method can help in rapidly determining the sensitivity of C. albicans to antifungals and the individual concentration of antifungals in cancer patients undergoing radiation therapy.
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CD109 antigen on the endothelial cell surface plays an important role in vascular pathology. The aim of the work was to investigate the effect of the immobilization of CD109 antigen with specific antibodies on nanomechanical properties of human umbilical endothelial cells (HUVECs) using atomic force microscopy in quantitative nanomechanical property mapping mode (PeakForce QNM). Anti-CD109 antibodies induced significant stiffening of the cell surface Me(LQ; UQ): in 1.45(1.07;2.29) times with respect to control cells for fixed cells and in 4.9(3.6;5.9) times with respect to control cells for living cells, and changes in the spatial distribution of cell surface mechanical properties. The changes in the HUVEC's mechanical properties were accompanied by the activation of the TGF-/Smad2/3 signaling pathway and reorganization of the vimentin and actin cytoskeletal elements. Our findings show that blocking CD109 antigen using anti-CD109 antibodies leads in HUVECs to the processes similar to that occur after cell TGF-ß-signaling activation. Therefore, we suggest that CD109 antigen may be involved in regulating the mechanical behavior of endothelial cells.
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Células Endoteliales , Transducción de Señal , Humanos , Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Microscopía de Fuerza Atómica/métodos , Transducción de Señal/fisiología , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Fungal infections can pose great threat to sight. Immediate treatment is usually required; antifungal agents are widely accepted and are effective in most cases. The present experimental study aims to investigate the probable effects of intravitreal injection of antifungal agents on the structure and mechanical properties of the surface of peripheral blood erythrocytes. METHODS: Nine albino New Zealand white rabbits, aged five months old, were chosen for the experiment. Solutions of micafungin, voriconazole, or balanced salt solution (BSS) were injected into the midvitreous. Animals were divided into two experimental groups and one control group. Blood sampling from an intravenous (IV) line was performed after 10 days from the last IV injection. An atomic force microscope (AFM) was used to study the structural and mechanical properties of cell surfaces. RESULTS: The analysis results showed that the parameters of the cytoskeleton's spatial organization changed insignificantly with the antifungal drug treatment. CONCLUSIONS: Our findings suggest that locally administered antifungal drugs can cause significant changes to the structure and frictional properties of the erythrocyte surface. These effects occur in the long-term period after administration of the drugs and represent a potential possibility for violation of blood supply to tissues, and the further development of negative side effects.
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Antifúngicos , Micosis , Animales , Antifúngicos/uso terapéutico , Eritrocitos , Micafungina/uso terapéutico , Micosis/tratamiento farmacológico , Conejos , Voriconazol/farmacologíaRESUMEN
BACKGROUND: To date, EVs characterization techniques are extremely diverse. The contribution of AFM, in particular, is often confined to size distribution. While AFM provides a unique possibility to carry out measurements in situ, nanomechanical characterization of EVs is still missing. METHODS: Blood plasma EVs were isolated by ultracentrifugation, analyzed by flow cytometry and NTA. Followed by cryo-EM, we applied PeakForce AFM to assess morphological and nanomechanical properties of EVs in liquid. RESULTS: Nanoparticles were subdivided by their size estimated for their suspended state into sub-sets of small S1-EVs (< 30 nm), S2-EVs (30-50 nm), and sub-set of large ones L-EVs (50-170 nm). Non-membranous S1-EVs were distinguished by higher Young's modulus (10.33(7.36;15.25) MPa) and were less deformed by AFM tip (3.6(2.8;4.4) nm) compared to membrane exosomes S2-EVs (6.25(4.52;8.24) MPa and 4.8(4.3;5.9) nm). L-EVs were identified as large membrane exosomes, heterogeneous by their nanomechanical properties (22.43(8.26;53.11) MPa and 3.57(2.07;7.89) nm). Nanomechanical mapping revealed a few non-deformed L-EVs, of which Young's modulus rose up to 300 MPa. Taken together with cryo-EM, these results lead us to the suggestion that two or more vesicles could be contained inside a large one being a multilayer vesicle. CONCLUSIONS: We identified particles similar in morphology and showed differences in nanomechanical properties that could be attributed to the features of their inner structure. GENERAL SIGNIFICANCE: Our results further elucidate the identification of EVs and concomitant nanoparticles based on their nanomechanical properties.
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Exosomas , Nanopartículas , Módulo de Elasticidad , Microscopía de Fuerza Atómica , PlasmaRESUMEN
Endothelial cells, due to heterogeneity in the cell structure, can potentially form an inhomogeneous on structural and mechanical properties of the inner layer of the capillaries. Using quantitative nanomechanical mapping mode of atomic force microscopy, the parameters of the structural, elastic, and adhesive properties of the cell surface for living and glutaraldehyde-fixed human umbilical vein endothelial cells were studied. A significant difference in the studied parameters for three cell surface zones (peripheral, perinuclear, and nuclear zones) was established. The perinuclear zone appeared to be the softest zone of the endothelial cell surface. The heterogeneity of the endothelial cell mechanical properties at the nanoscale level can be an important mechanism in regulating the endothelium functions in blood vessels.
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Membrana Celular/fisiología , Núcleo Celular/fisiología , Citoesqueleto/fisiología , Células Endoteliales de la Vena Umbilical Humana/fisiología , Mecanotransducción Celular , Adhesividad , Células Cultivadas , Módulo de Elasticidad , Humanos , Microscopía de Fuerza Atómica , Nanotecnología , Fenotipo , Análisis de la Célula Individual , Estrés MecánicoRESUMEN
Hereditary spherocytosis (HS), an erythrocyte membranopathy, is a heterogeneous disease, even at the level of the erythrocyte population. The paper aims at studying the mechanical properties (the Young's modulus, median and RMS roughness of friction force maps; fractal dimension, lacunarity and spatial distribution parameters of lateral force maps) of the cell surface layer of the erythrocytes of two different morphologies (discocytes and spherocytes) in HS using atomic force microscopy. The results of spatial-spectral and fractal analysis showed that the mechanical property maps of the HS spherocyte surface were more structurally homogeneous compared to the maps of HS discocytes. HS spherocytes also had a reduced RMS roughness and lacunarity of the mechanical property maps. The Young's modulus and averaged friction forces over the microscale HS spherocyte surface regions were approximately 20% higher than that of HS discocytes. The revealed significant difference at the nano- and microscales in the structural and mechanical properties of main (discoidal and spheroidal) morphological types of HS erythrocytes can potentially cause blood flow disturbance in the vascular system in HS.
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Eritrocitos/patología , Fenómenos Mecánicos , Microtecnología , Nanotecnología , Esferocitosis Hereditaria/sangre , Fenómenos Biomecánicos , Niño , Módulo de Elasticidad , Femenino , Fricción , Humanos , MasculinoRESUMEN
Fractal dimension (DF) is one of the important parameters in the description of object's properties in different fields including biology and medicine. The present paper is focused on the application of the fractal dimension (the box counting dimension) in the analysis of the properties of cell surface on the base of its images obtained by atomic force microscopy (AFM). Fractal dimension of digital 3D AFM images depends on interpoint distances determined by the scanning step in the XY-plane and Z-scale factor t. We have studied the dependence of DF of AFM images on the Z-scale factor (DF=φ(t)) with purpose to reveal the features of the dependence and its usefulness in the analysis of the maps of surface properties. Using the model digital surfaces such as the plane, sinusoidal surfaces and "hilly" surface, we revealed that the sizes and spatial frequency of surface structural elements determined the basic features of the dependence (the parameters of peaks on the curve DF=φ(t)) and the element of chance in the localization of the structural elements on the surface had no significant influence on the dependence. Our findings demonstrate that the dependence of the fractal dimension on the Z-scale factor characterizes the structure of the AFM images more comprehensively than the roughness Ra and fractal dimension DF evaluated at a certain t. The dependence DF=φ(t) can be considered as a novel characteristic of AFM images. On analyzing the AFM images (lateral force maps) of glutaraldehyde-fixed adhered human fibroblasts and A549 human lung epithelial cells we found the significant difference in the dependences DF=φ(t) for different cell types that could be related to the difference of structural and mechanical surface properties of the studied cells.
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Membrana Celular/ultraestructura , Eritrocitos/ultraestructura , Fibroblastos/ultraestructura , Microscopía de Fuerza Atómica/métodos , Células A549 , Línea Celular Tumoral , Fractales , Humanos , Neoplasias Pulmonares/ultraestructura , Piel/citología , Propiedades de SuperficieRESUMEN
Atomic force microscopy (AFM) in lateral force mode was applied to assess the microscale thermo-mechanical (frictional) properties of the air-dried cell surface in the wide temperature range (288-363K/15-90°C). AFM-investigated cell surface layer can be represented as a biocomposite composed of several layers including the glycocalyx, the membrane and the intercellular layer containing membrane (cortical) cytoskeleton. The cells with two different cytoskeleton structures, erythrocytes and thymocytes, were studied. Above a certain temperature (T(g)), the significant change in friction force with temperature was revealed for the both cell types whereas there was no similar change in their topography parameters. The experimentally determined value T(g) for erythrocyte samples was lower than that for thymocyte ones. Treating living cells with the cross-linking agent, glutaraldehyde, led to the weakening of the temperature dependence of air-dried cell surface frictional properties in the studied temperature range. Addition of oxidizing agent, peroxynitrite, to living cell suspensions changed the temperature dependence of air-dried cell surface frictional properties depending on cell type and peroxynitrite concentration. The obtained data indicate that the study of thermo-mechanical properties of air-dried cells with AFM in lateral force mode provides expanded information on the structural characteristics of the living cell surface layer, and sets the stage for the development of AFM-based method (with using a lateral force mode) for the cell pathology diagnostics.
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Técnicas Citológicas/métodos , Microscopía de Fuerza Atómica/métodos , Propiedades de Superficie , Animales , Fenómenos Químicos , Eritrocitos/química , Eritrocitos/fisiología , Fricción , Humanos , Fenómenos Mecánicos , Ratas , Suspensiones , Temperatura , Timocitos/química , Timocitos/fisiologíaRESUMEN
Mechanical properties are fundamental properties of the cells and tissues of living organisms. The mechanical properties of a single cell as a biocomposite are determined by the interdependent combination of cellular components mechanical properties. Quantitative estimate of the cell mechanical properties depends on a cell state, method of measurement, and used theoretical model. Predominant tendency for the majority of cells with ageing is an increase of cell stiffness and a decrease of cell ability to undergo reversible large deformations. The mechanical signal transduction in old cells becomes less effective than that in young cells, and with ageing, the cells lose the ability of the rapid functional rearrangements of cellular skeleton. The article reviews the theoretical and experimental facts touching the age-related changes of the mechanical properties of cellular components and cells in the certain systems of an organism (the blood, the vascular system, the musculoskeletal system, the lens, and the epithelium). In fact, the cell mechanical parameters (including elastic modulii) can be useful as specific markers of cell ageing.
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Envejecimiento/fisiología , Fenómenos Fisiológicos Celulares , Fenómenos Mecánicos , Animales , Células Sanguíneas/fisiología , Forma de la Célula/fisiología , Humanos , Fenómenos Fisiológicos Musculoesqueléticos , Estrés MecánicoRESUMEN
The changes in passive ion permeability of the red blood cell membrane after peroxynitrite action (3 microM-3 mM) have been studied by biophysical (using radioisotopes of rubidium, sodium and sulphur (sulphate)) and electrophysiological methods. The enhancement of passive membrane permeability to cations (potassium and sodium ions) and the inhibition of anion flux through the anion exchanger in peroxynitrite-treated red blood cells were revealed. In patch-clamp experiments the whole-cell conductance after peroxynitrite (80 microM) treatment of red blood cells increased 3-3.5-fold with a shift in the reversal potential from -7.0+/-1.5 mV to -4.3+/-0.9 mV (n=7, p=0.005). The addition of cobalt and nickel ions to red blood cell suspensions before peroxynitrite treatment had no effect on the peroxynitrite-induced cation flux but zinc ions in the same condition decreased cation flux about 2-fold. Using atomic force microscopy methods we revealed an increase in red blood cell membrane stiffness and the membrane skeleton complexity after peroxynitrite action. We conclude that the peroxynitrite-induced water and ion imbalance and reorganization in membrane structure lead to crenation of red blood cells.
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Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Ácido Peroxinitroso/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cloruros/metabolismo , Eritrocitos/metabolismo , Eritrocitos/ultraestructura , Humanos , Potasio/metabolismo , Elementos de Transición/metabolismoRESUMEN
Atomic force microscopy (AFM) was used to study surface layers of fixed intact erythrocytes. Advantages of simultaneous analysis of surface topography and lateral force maps in the investigation of cytoskeleton structure were shown. Fractal analysis was applied to the lateral force maps of erythrocyte surfaces to evaluate the complexity of the cytoskeleton. Peroxynitrite was used as an oxidant to induce changes in the cytoskeleton structure of intact erythrocytes. Peroxynitrite action on whole blood leads to local abnormalities in the erythrocyte cytoskeleton structure, as well as cytoskeleton reorganization in protruded regions of crenated erythrocytes.
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Membrana Eritrocítica/ultraestructura , Microscopía de Fuerza Atómica/métodos , Ácido Peroxinitroso/farmacología , Membrana Eritrocítica/efectos de los fármacos , HumanosRESUMEN
Atomic force microscopy (AFM) has recently provided the great progress in the study of micro- and nanostructures including living cells and cell organelles. Modern AFM techniques allow solving a number of problems of cell biomechanics due to simultaneous evaluation of the local mechanical properties and the topography of the living cells at a high spatial resolution and force sensitivity. Particularly, force spectroscopy is used for mapping mechanical properties of a single cell that provides information on cellular structures including cytoskeleton structure. This entry is aimed to review the recent AFM applications for the study of dynamics and mechanical properties of intact cells associated with different cell events such as locomotion, differentiation and aging, physiological activation and electromotility, as well as cell pathology. Local mechanical characteristics of different cell types including muscle cells, endothelial and epithelial cells, neurons and glial cells, fibroblasts and osteoblasts, blood cells and sensory cells are analyzed in this paper.