An experimental system for real-time fluorescence recordings of cell membrane changes induced by electroporation.

The electroporation of cells is nowadays used for a large variety of purposes, from basic research to cancer therapy and food processing. Understanding molecular mechanisms of the main processes involved in electroporation is thus of significant interest. In the present work, we propose an experimental system to record in real time the evolution of any cell parameter which can be evaluated by fluorescence (before, during and after application of the electroporation pulses to cells in suspension). The system is based on the design of adequate electroporation electrodes, compatible with a standard spectrofluorometer cuvette housing. The electric field intensity generated when pulses are applied was carefully characterized for different geometries of the electrodes, to choose a construction ensuring the greatest homogeneity of the field in combination with the best possible illumination of the sample. As an example of the method's application, we present here generalized polarization kinetics for a varying number of electroporation pulses applied to a cell suspension; the general polarization parameter is strongly correlated to water presence in the hydrophobic membrane core. The system may be used for many other fluorescence measurements useful for the characterization of the electroporation process.

Noninvasive detection of changes in cells' cytosol conductivity by combining dielectrophoresis with optical tweezers.

Cellular electrical properties are modulated by various physical and/or chemical stresses and detection of these changes is a challenging issue. Optical tweezers (OT) and dielectrophoresis (DEP) are frequently integrated to devices dedicated to the investigation of cells properties. Here we provide a technique to detect changes in cytosol conductivity of cells by using a combination of DEP and OT. The method was exemplified for the case of cells electroporation and is based on balancing the DEP force by a controlled OT force. We observed a decrease of the DEP force in the case of electroporated cells which was correlated to a decrease of cytosol conductivity by means of Clausius-Mossotti factor modeling. For highly stressing electroporation pulses, the cytosol conductivity drops to values close to those of the cells suspending medium. These results are consistent with those reported in the literature proving the robustness of our proposed sensing method.

Control by Low Levels of Calcium of Mammalian Cell Membrane Electropermeabilization.

Electric pulses, when applied to a cell suspension, induce a reversible permeabilization of the plasma membrane. This permeabilized state is a long-lived process (minutes). The biophysical molecular mechanisms supporting the membrane reorganization associated to its permeabilization remain poorly understood. Modeling the transmembrane structures as toroidal lipidic pores cannot explain why they are long-lived and why their resealing is under the control of the ATP level. Our results describe the effect of the level of free Calcium ions. Permeabilization induces a Ca2+ burst as previously shown by imaging of cells loaded with Fluo-3. But this sharp increase is reversible even when Calcium is present at a millimolar concentration. Viability is preserved to a larger extent when submillimolar concentrations are used. The effect of calcium ions is occurring during the resealing step not during the creation of permeabilization as the same effect is observed if Ca2+ is added in the few seconds following the pulses. The resealing time is faster when Ca2+ is present in a dose-dependent manner. Mg2+ is observed to play a competitive role. These observations suggest that Ca2+ is acting not on the external leaflet of the plasma membrane but due to its increased concentration in the cytoplasm. Exocytosis will be enhanced by this Ca2+ burst (but hindered by Mg2+) and occurs in the electropermeabilized part of the cell surface. This description is supported by previous theoretical and experimental results. The associated fusion of vesicles will be the support of resealing.

Evaluation of the metastatic potential of malignant cells by image processing of digital holographic microscopy data.

The cell refractive index has been proposed as a putative cancer biomarker of great potential, being correlated with cell content and morphology, cell division rate and membrane permeability. We used digital holographic microscopy to compare the refractive index and dry mass density of two B16 murine melanoma sublines of different metastatic potential. Using statistical methods, the distribution of phase shifts within the reconstructed quantitative phase images was analyzed by the method of bimodality coefficients. The observed correlation of refractive index, dry mass density and bimodality profile with the metastatic potential of the cells was validated by real time impedance-based assay and clonogenic tests. We suggest that the refractive index and bimodality analysis of quantitative phase image histograms could be developed as optical biomarkers useful in label-free detection and quantitative evaluation of cell metastatic potential.

Changes in optical properties of electroporated cells as revealed by digital holographic microscopy.

Changes in optical and shape-related characteristics of B16F10 cells after electroporation were investigated using digital holographic microscopy (DHM). Bipolar rectangular pulses specific for electrochemotherapy were used. Electroporation was performed in an "off-axis" DHM set-up without using exogenous markers. Two types of cell parameters were monitored seconds and minutes after pulse train application: parameters addressing a specifically defined area of the cell (refractive index and cell height) and global cell parameters (projected area, optical phase shift profile and dry mass). The biphasic behavior of cellular parameters was explained by water and mannitol dynamics through the electropermeabilized cell membrane.

Hemorrhagic risk due to platelet dysfunction in myelodysplastic patients, correlations with anemia severity and iron overload.

Platelet function is influenced by changes in membrane fluidity that has an important role in the expression of platelet receptors and in modulating the activity of proteins like phospholipase C or proteinkinase C. In freshly prepared platelets, membrane fluidity modifies the aggregation/agglutination function. Reactive oxygen species (ROS) represent another important parameter involved in platelet receptor activation. There is a certain association of high levels of ROS and iron overload. Patients with hemochromatosis have low platelet aggregation induced by thrombin; little is known about the anemia and effects of iron overload on platelet activation in myelodysplastic syndromes (MDS) patients. Study of platelet membrane fluidity and ROS production changes in patients with MDS and possible correlations with altered platelet function as reflected in aggregation curves and platelet receptor expression. To find out possible correlations of fluidity of platelet membrane and ROS level with hematologic parameters and iron levels. The prospective study included 34 patients with myelodysplastic syndromes classified according to French-American-British cooperative group proposals and 29 healthy volunteers. Platelet membrane fluidity was quantified by fluorescence anisotropy measurements using the marker 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. ROS production was evaluated by fluorescence measurements using 2',7'-dichlorodihydrofluorescein diacetate. Platelet function was analyzed by optical aggregometry using the agonists adenosine diphosphate, collagen, ristocetin and epinephrine. The expression of platelet receptors CD41/CD61, CD42a/CD42b and CD62P/CD63 was evaluated by flow cytometry. Platelet membrane fluidity in patients with MDS was similar to that of healthy volunteers and did not vary according to the risk category. Patients with MDS had increased platelet ROS production compared with the control group without statistical correlation with membrane fluidity. We found a negative correlation of ROS levels with the severity of anemia (R =  -0.587, P = 0.017). Platelet response was reduced in patients with MDS compared with volunteers, for all reagents. The response was different according to the risk category only in case of ristocetin or collagen. Patients with anemia presented a decreased platelet aggregation induced by collagen or ristocetin (collagen: R = 0.395, P = 0.003; ristocetin: R = 0.420, P = 0.002). The membrane fluidity of platelets from MDS patients appeared unmodified, but the ROS production was increased in all risk categories of MDS. The levels of ROS were correlated with the degree of anemia, which, in turn, had a negative impact on the platelet aggregation function induced by collagen or ristocetin.

Iron oxide nanoparticles modulate the interaction of different antibiotics with cellular membranes.

The interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging and drug/gene delivery. These applications require a firm control over nanoparticle-cell interactions, which are mainly dictated by surface properties of the nanoparticles. The aim of this study was to investigate the interaction of Fe3O4 nanoparticles functionalized with several wide use antibiotics with opossum kidney (OK) cellular membranes in order to reveal changes in the membrane organization at different temperatures. We also investigated the in vivo biodistribution of the tested nanoparticles in a mouse model. Our results showed that, at low temperatures (31-35°C), plain Fe3O4 nanoparticles induced a drop of the membrane fluidity, while at physiological or higher temperatures (37-39°C) the membrane fluidity was increased. On the other hand, when nanoparticles functionalized with the tested antibiotics were used, we observed that the effect was opposite as compared to control Fe3O4 nanoparticles. Although most of antibiotics, used as plain solutions or linked on magnetite nanoparticles, proved heterogeneous effect on in vitro OK cells membrane fluidity, the aminoglycosides streptomycin and neomycin, used both as plain solutions and also combined with nanoparticles kept the same effect in all experimental conditions, increasing the membrane fluidity of OK cells plasma membrane. In vivo results showed that the antibiotic functionalized nanoparticles have a similar biodistribution pattern within the mouse body, being transported through the blood flow and entering the macrophages through endocytosis. Functionalized magnetite nanoparticles manifested a preferential biodistribution pattern, clustering within the lungs and spleen of treated mice. These results demonstrate that antibiotics manifest a different effect on plasma membrane fluidity depending on their type and temperature. Magnetite nanoparticles may interfere with antibiotic-cellular interactions by changing the plasma membrane fluidity. The fact that the antibiotic functionalized magnetite nanoparticles have a similar biodistribution pattern, are transported through the blood flow, and they increase the cellular uptake of the drug, suggest that they may be used for further studies aiming to develop personalized targeted delivery and controlled release nanoshuttles for treating localized and systemic infections.

Assessment of changes in membrane properties of platelets from patients with chronic myeloid leukaemia in different stages of the disease.

Patients with chronic myeloproliferative leukemia (CML) have frequent haemorrhage and/or thrombosis in their medical history. The mechanisms of these major and life-threatening complications remain unclear. Membrane organization influences many of the unique cellular functions and is strongly correlated, among other factors, to the membrane lipid composition; it may be evaluated by following up the membrane fluidity and aggregation properties of the platelet. In this study, we evaluated the platelet aggregation, the expression of platelet surface receptors, the membrane fluidity (as evaluated by fluorescence anisotropy) and its correlation to reactive oxygen species (ROS) production, in patients with chronic myeloid leukaemia (CML). It was found that the patients in accelerated and blastic phase of CML present an altered platelet aggregation response to all reagents except for ristocetin as compared with chronic phase group, which shows only epinephrine-altered response. We also found that BCR/ABL transcript leads to higher levels of ROS in accelerated and blastic CML phases. Patients without molecular remission have lower platelet membrane fluidity. We obtained a positive correlation between ROS level and membrane fluorescence anisotropy changes. The CD41 expression was decreased in CML patients and P selectin expression was found to be higher in these patients than in healthy volunteers. Platelets of CML patients have altered aggregation parameters in accelerated and blastic phases, in which BCR/ABL transcript level is increased. The increased level of ROS in CML patients without molecular remission is associated with a decrease in fluidity of platelet membrane and expression of CD41/CD61 receptors. These findings may contribute to understanding the mechanism of the altered platelet response reported in CML patients.

Changes of cell electrical parameters induced by electroporation. A dielectrophoresis study.

Dielectrophoresis was employed to distinguish the electroporated from non-electroporated cells. It was found that the electric field frequency at which cells change the direction of their movement (the crossover frequency f(CO)) is higher when cells are electroporated. The contribution to the cell dielectrophoretic behavior of four electric and geometrical cell parameters was analyzed using a single shell model. f(CO) measurements were performed in media with conductivities of 0.001-0.09S/m, on B16F10 cells which were electroporated in a Mannitol solution (0.001S/m), using rectangular or exponential pulses. The control cells' f(CO) was found in a domain of 2 to 105 kHz, while the electroporated cells' f(CO) was in a domain of 5 to 350 kHz, depending on the external media conductivities. At exterior conductivities above ~0.02S/m, f(CO) of electroporated cells became significantly higher compared to controls. Even though the possible contribution of membrane permittivity to explain the observed f(CO) shift toward higher values cannot be excluded, the computations highlight the fact that the variation of cytosol conductivity might be the major contributor to the dielectrophoretic behavior change. Our experimental observations can be described by considering a linear dependence of electroporated cells' cytosol conductivity against external conductivity.

Interaction of gentamicin polycation with model and cell membranes.

The interaction of positively-charged antibiotic gentamicin with cell membranes was studied to determine if any changes in membrane organization were induced by the drug. Opossum kidney epithelia (OK) cells were used as models of eukaryotic cells. Two methods were used: laurdan fluorescence spectroscopy and fluorescence anisotropy recordings on 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) labeled cell suspensions. Both methods showed an altered membrane hydration and fluidity of gentamicin treated cells. Liposomes prepared from dimyristoyl-phosphatidylcholine (DMPC) mixed with cardiolipin, which mimics the heterogeneous charge composition of the natural cell membrane, were used to determine the effect of gentamicin on artificial bilayers. The membrane lipid packing as revealed by generalized polarization (GP) and fluorescence anizotropy variation with increasing temperature was studied. It was found that the generalized polarization of liposomal membranes containing a negatively charged lipid (cardiolipin) is higher in the presence of gentamicin; in the membrane of living cell (OK), gentamicin induces, on the contrary, a decrease of general polarization. Considering the role of membrane organization in the function of transmembrane channels and receptors, our findings suggest hypotheses that may explain the permeation of gentamicin through the living cell membrane by using these channels.

900 MHz modulated electromagnetic fields accelerate the clathrin-mediated endocytosis pathway.

We report new data regarding the molecular mechanisms of GSM-induced increase of cell endocytosis rate. Even though endocytosis represents an important physical and biological event for cell physiology, studies on modulated electromagnetic fields (EMF) effects on this process are scarce. In a previous article, we showed that fluid phase endocytosis rate increases when cultured cells are exposed to 900 MHz EMF similar to mobile phones' modulated GSM signals (217 Hz repetition frequency, 576 micros pulse width) and to electric pulses similar to the GSM electrical component. Trying to distinguish the mechanisms sustaining this endocytosis stimulation, we exposed murine melanoma cells to Lucifer Yellow (LY) or to GSM-EMF/electric pulses in the presence of drugs inhibiting the clathrin- or the caveolin-dependent endocytosis. Experiments were performed at a specific absorption rate (SAR) of 3.2 W/kg in a wire patch cell under homogeneously distributed EMF field and controlled temperature (in the range of 28.5-29.5 degrees C). Thus, the observed increase in LY uptake was not a thermal effect. Chlorpromazine and ethanol, but not Filipin, inhibited this increase. Therefore, the clathrin-dependent endocytosis is stimulated by the GSM-EMF, suggesting that the cellular mechanism affected by the modulated EMF involves vesicles that detach from the cell membrane, mainly clathrin-coated vesicles.

Otoacoustic emissions analysers for monitoring aminoglycosides ototoxicity.

OBJECTIVES: Aminoglycosides (AG) are widely prescribed despite their notorious toxicity. These antibiotics cause irreversible hearing loss, starting with high frequencies and progressing toward conversational frequencies (0.5-2 kHz), by destroying the acoustic hair cells in the inner ear. The integrity of these cells could be analysed by recording faint sounds that they produce otoacoustic emissions (OAE). The aim of the present study was to monitor and to characterise the acoustic toxicity of the AG using an OAE analyser. METHOD: We performed a prospective study of 49 patients receiving gentamicin (G), during 2007-2008. We made serial OAE recordings with an ILO 92 analyser (1-8 kHz) on at least 3 occasions: at the start, during treatment and after the cessation of G therapy (1-6 months). The recordings were performed at the patient's bedside and did not require the active participation of the patient. The method is fast, non-invasive, accurate and does not request an ENT specialist. We included patients presenting OAE. Ototoxicity was defined using adapted ASHA (American Speech Hearing Association) criteria. RESULTS: We studied 49 patients (24/25 female/male), mean age of 37.24 (3-70 years old), who received G for 4-42 days: less than 10 days--24 patients and more than 10 days--25 patients. Hearing impairment was observed in 10 patients (20.4%) and appeared during the therapy or in the next 3 months, with a loss of one or 2 high frequencies that occurred unilaterally in 9 out of 10 cases. The acoustic damage correlates with the length of G treatment. We did not find a statistical correlation between the degree of impairment and the age, regimen or addition of another ototoxic drug, although the hearing loss was slightly higher in those with concomitant exposure. CONCLUSION: OAE monitoring of AG treatment is a very useful way for detecting and preventing acoustic toxicity, because it could warn about hearing loss before damage of the conversational frequencies. The accuracy is similar to the classical methods, but it is faster and easier to perform.

Microscopic observation of living cells during their exposure to modulated electromagnetic fields.

Studying cell behaviour under irradiation with radiofrequency electromagnetic fields (RF-EMF) is often impeded by the difficulty to monitor cell characteristics during irradiation. Here we report the design and the application of a complete device for continuous microscopic observation of cells exposed to modulated EMF similar to mobile phones signals. The system allows the follow up of cell progression into mitosis under controlled temperature and CO(2) environment. Protocols are proposed in which the same cells are the controls before and after the EMF exposure and we demonstrate the interest of the "before exposure" controls. The exposure system was validated by cell endocytosis measurements. While the endocytosis rate was increased, no alteration of mitosis progression and mitosis duration was observed in cells exposed to 900 MHz modulated EMF for 1 h, at 30 degrees C and at a Specific Absorption Rate of 2.2 W/kg.

Evaluation of trapping efficiency of optical tweezers by dielectrophoresis.

A relatively new method for measuring optically induced forces on microparticles and cells, different from the conventional Brownian motion and viscous drag force calibration methods widely used, is introduced. It makes use of the phenomenon of dielectrophoresis for the calibration of optical tweezers through the dielectrophoretic force calculations. A pair of microelectrodes is fabricated by photolithography on a microscope slide and it is connected to a high-frequency generator. The calibration of the optical tweezers setup is performed by the manipulation of polystyrene beads and yeast cells. Calibration diagrams of the transverse forces versus power are deduced for different cell radii and numerical apertures of the objective lenses. The optical system and the related technique provide a fast and easy method for optical tweezers calibration.

Correlation between different components of synovial fluid and pathogenesis of rheumatic diseases.

UNLABELLED: Biochemical analysis of synovial fluid is important in orientating the diagnosis of joint effusions. Its composition reflects the metabolic status of synovial tissue correlated with different rheumatic pathologies. The aim of this study was to compare the information provided by usual biochemical tests and proton magnetic resonance spectroscopy (MRS) analysis of synovial fluid, in order to detect potential characteristics of joint effusion correlated to the pathogenesis of arthritis. PATIENTS AND METHODS: This study included 99 synovial fluid samples obtained from patients with different etiology of arthritis--rheumatoid arthritis (RA), osteoarthritis (OA), gout, seronegative spondylarthropathies and septic arthritis, which underwent routine laboratory tests available in the Department of Rheumatology, Cantacuzino Clinical Hospital, Bucharest. Patients were admitted in the hospital between January 2003 and June 2005. Synovial samples were examined in parallel using MRS determination. Spectra have been recorded on a Bruker Avance DRX 400 MHz spectrometer. RESULTS: We obtained a good correlation between biochemical analysis and MRS determination. RA samples have offered the most important information regarding the complex composition of pathological synovial fluid. In addition to the markedly elevated lactate (p < 0.009) and diminished glucose concentration (p < 0.02) in RA samples vs. OA, MRS analysis provides evidence for reduced mean chain length of very-low-density-lipoprotein (VLDL)-associated triacylglycerols (p < 0.05), high levels of ketone bodies (p < 0.05), ceramide (p < 0.02) and citrulline (p < 0.04). OA samples have shown a characteristically increased level of N-glucosamine (p < 0.04) and creatine (p < 0.04). For the other pathologies, there are no characteristic markers detected, except for the extremely increased level of lactate and the decreased concentration of glucose in septic arthritis. CONCLUSIONS: This study gave us the possibility to provide a complex exploration of the biochemical environment of synovial fluid using the MRS method, that offers us the opportunity to understand more about the pathogenesis of rheumatic diseases, to provide information regarding the degree of inflammation, immune infiltration and apoptosis in RA, cartilage degradation and muscular secondary atrophy in OA. The most valuable information is focused on the possibility to have a simultaneous detection of more than 26 different metabolites in synovial fluid samples.

Effects of 1800 MHz GSM Radiation on Human Monocytes and Trombocytes Membrane Anisotropy.

The purpose of our experiments was to measure the effects of 1800 MHz microwave radiation on membrane anisotropy of a human blood monocitar and trombocitar cell population. Power level of the applied CW microwave radiation was low enough to consider it to be athermal. Experience demonstrates that the natural tendency to decrease of cell membrane anisotropy is favourised by microwave irradiation. This tendency maintains after the ceasing of microwave irradiation.

Evaluation of viability of retinal photoreceptor cells by using their endogenous electrical field.

The rod photoreceptor cells are electrical dipoles sustained by cell metabolic energy. Polarity of photoreceptor cells is directly connected to the so-called "dark current" which circulate along the living photoreceptors. Since only the living cells in a good functional state display electrical polarity, the orientation of photoreceptors in static electric field reflects their viability as long as it depends on the functionality of molecular mechanisms that maintain the dark current. Studying the rod cells' orientation in static electric field at different times after their isolation is thus an accurate way to evaluate the cell viability/degeneration. Retinal transplant experiments in animals and humans, which are presently in progress, require a quick and reliable viability test of cells/tissue to be transplanted. Checking the orientation pattern of rod photoreceptors in static electric field prior to transplantation is a candidate method for an accurate cell viability test.

The effects of low level microwaves on the fluidity of photoreceptor cell membrane.

Due to the extensive use of electromagnetic fields in everyday life, more information is required for the detection of mechanisms of interaction and the possible side effects of electromagnetic radiation on the structure and function of the organism. In this paper, we study the effects of low-power microwaves (2.45 GHz) on the membrane fluidity of rod photoreceptor cells. The retina is expected to be very sensitive to microwave irradiation due to the polar character of the photoreceptor cells [Biochim. Biophys. Acta 1273 (1995) 217] as well as to its high water content [Stud. Biophys. 81 (1981) 39].