Eugenia Kovacs

Microfabrication of polystyrene microbead arrays by laser induced forward transfer

In this study we describe a simple method to fabricate microarrays of polystyrene microbeads (PS-μbeads) on Thermanox coverslip surfaces using laser induced forward transfer (LIFT). A triazene polymer layer which acts as a dynamic release layer and propels the closely packed microspheres on the receiving substrate was used for this approach. The deposited features were characterized by optical microscopy, scanning electron microscopy, atomic force microscopy, and Raman spectroscopy. Ultrasonication was used to test the adherence of the transferred beads. In addition, the laser ejection of the PS-μbead pixels was investigated by time resolved shadowgraphy. It was found that stable PS-μbeads micropatterns without any specific immobilization process could be realized by LIFT. These results highlight the increasing role of LIFT in the development of biomaterials, drug delivery, and tissue engineering.

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].

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.

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.

Membrane Damage of Human Red Blood Cells Induced by Low-Power Microwave Irradiation

The effect of low-level, 2.45 GHz microwaves on human erythrocyte membrane was studied by measuring the induced hemolysis of the exposed erythrocytes at different power densities (0.025-10.000 mW/cm2). A significant increase of the hemoglobin loss by the microwave-exposed erythrocytes compared to controls was observed. Red blood cell count was essentially the same in irradiated and control samples while the mean cellular hemoglobin concentration decreased in the exposed samples. These observations indicate that the hemoglobin loss from the microwave-irradiated cells is due to the membrane permeabilization of the exposed erythrocytes rather than to their lysis.

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.

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.

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.

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.

Relationship between urinary metabolites and type 2 diabetes mellitus by proton nuclear magnetic resonance spectroscopy method (1H-NMR)

Control Group: 334 individuals (161 males, 173 females), averaged age of 38.3 years, ranging between 23-67 years old, without metabolic diseases such as diabetes, hypertension, urinary infection, clinical evidence of renal disease; without alcohol consumption for 24 h before sampling, in condition of no drugs administration. Biochemical determinations in Control Group: Urine Uro, Bil, Ket, pH, SG, Leu, Blood, Nitrite, Ascorbic Acid, Glu, Pro. Type 2 DM Group: 388 patients (173 males, 215 females), averaged age 55, ranging between 34-75 years old. The patients had a history of type 2 DM less than 5 years and were hospitalized in Craiova County Emergency Clinical Hospital, Department of Diabetes, Nutrition and Metabolic Diseases. Biochemical determinations in type 2 DM patients: Blood: urea (33.9±8.69 mg/dl), creatinine (0.84±0.14 mg/dl), fasting glycemia (179±54 mg/dl) and Glomerular Filtration Rate (eGFR)=101.56±9.55 ml/min/1.73m2. Urine Uro, Bil, Ket, pH, SG, Leu, Blood, Nitrite, Ascorbic Acid, Glu, Pro, Creatinine (154.14±32.05 mg/dl). Nuclear Magnetic Resonance Spectroscopy Method: NMR spectra recorded with 10 % D2O and 5 mM sodium 3-(trimethylsilyl)-[2,2,3,3-d4 ]-1-propionate (TSP) at 400 MHz Brucker Avance DRX spectrometer, in 5 mm NMR tubes, with 32 scans, and water presaturation. Statistical analysis: The data were calculated using Graph Pad Prism 5.0 and were given as mean±SD; P<0.05 was taken as significant. The results are evaluated in mmol/mol of Creatinine.