L-Arginine-Dependent Nitric Oxide Production in the Blood of Patients with Type 2 Diabetes: A Pilot, Five-Year Prospective Study.

Backgound: Type 2 diabetes mellitus (T2DM) is a major cardiovascular risk factor. Nitric oxide (NO) is one of the many molecules that regulate vascular tone, and red blood cells (RBCs) are known to play an important role in adjusting cardiac function through NO export from RBCs. Our study prospectively investigated the L-arginine (L-arg)-nitric oxide (NO) metabolic pathway in the erythrocytes and plasma of subjects with T2DM. Methods: RBCs and plasma were collected from patients with T2DM (n = 10), at first clinical onset (baseline) and after five years of disease evolution (follow-up). L-arg content was assayed by competitive enzyme-linked immunoassay. Arginase activity and nitrate/nitrite levels were measured using spectrophotometry. Results: When compared to baseline, L-arg content decreased in RBCs and remained similar in the plasma; NO production decreased in RBCs and the plasma; and arginase activity was lower in RBCs and increased in plasma. Conclusions: The L-arg/NO metabolic pathway decreases in the RBCs of patients with T2DM five years after the first clinical onset. The persistent decrease in RBCs' arginase activity fails to compensate for the sustained decrease in RBCs' NO production in the diabetic environment. This pilot study indicates that the NO-RBC pool is depleted during the progression of the disease in the same cohort of T2DM patients.

Early differentiation of mesenchymal stem cells is reflected in their dielectrophoretic behavior.

The therapeutic use of mesenchymal stem cells (MSCs) becomes more and more important due to their potential for cell replacement procedures as well as due to their immunomodulatory properties. However, protocols for MSCs differentiation can be lengthy and may result in incomplete or asynchronous differentiation. To ensure homogeneous populations for therapeutic purposes, it is crucial to develop protocols for separation of the different cell types after differentiation. In this article we show that, when MSCs start to differentiate towards adipogenic or osteogenic progenies, their dielectrophoretic behavior changes. The values of cell electric parameters which can be obtained by dielectrophoretic measurements (membrane permittivity, conductivity, and cytoplasm conductivity) change before the morphological features of differentiation become microscopically visible. We further demonstrate, by simulation, that these electric modifications make possible to separate cells in their early stages of differentiation by using the dielectrophoretic separation technique. A label free method which allows obtaining cultures of homogenously differentiated cells is thus offered.

OpenDEP: An Open-Source Platform for Dielectrophoresis Spectra Acquisition and Analysis.

Dielectrophoretic (DEP) cell separation, which utilizes electric fields to selectively manipulate and separate cells based on their electrical properties, has emerged as a cutting-edge label-free technique. DEP separation techniques rely on differences in the electrical and morphological properties of cells, which can be obtained by a thorough analysis of DEP spectra. This article presents a novel platform, named OpenDEP, for acquiring and processing DEP spectra of suspended cells. The platform consists of lab-on-a-chip and open-source software that enables the determination of DEP spectra and electric parameters. The performance of OpenDEP was validated by comparing the results obtained using this platform with the results obtained using a commercially available device, 3DEP from DEPtech. The lab-on-a-chip design features two indium tin oxide-coated slides with a specific geometry, forming a chamber where cells are exposed to an inhomogeneous alternating electric field with different frequencies, and microscopic images of cell distributions are acquired. A custom-built software written in the Python programing language was developed to convert the acquired images into DEP spectra, allowing for the estimation of membrane and cytoplasm conductivities and permittivities. The platform was validated using two cell lines, DC3F and NIH 3T3. The OpenDEP platform offers several advantages, including easy manufacturing, statistically robust computations due to large cell population analysis, and a closed environment for sterile work. Furthermore, continuous observation using any microscope allows for integration with other techniques.

Changes in the packing of bilayer lipids triggered by electroporation: real-time measurements on cells in suspension.

Electropermeabilization of the cell membrane is a technique used to facilitate penetration of impermeant molecules into cells. Although there are studies regarding the mechanism of processes occurring after electropermeabilization, the relationship between electropermeabilization and associated phenomena (e.g. generation of reactive oxygen species, endocytosis, lipid peroxidation, etc.) is yet to be elucidated. This work aimed to get information on the changes in the packing of the bilayer lipids and their peroxidation induced by application of electroporation pulses. We used a specially designed system of electrodes which allowed performing electropermeabilization of cells in suspension simultaneously with time-dependent measurements of fluorescence and temperature. The kinetics of membrane packing and production of reactive oxygen species were studied using various conductivity buffers (0.01, 0.04 and 0.14 S/m) and different number of 1 kV/cm bipolar pulses (1-50). Two categories of effects were observed: a thermal effect, consisting in an increased bilayer disorder (a deeper penetration of water into the hydrophobic core), and a nonthermal effect, leading to a higher degree of lipids packing, the latter being attributed to a peroxidation process. An analysis of the permeabilization conditions in which one of these two processes predominates was performed.

Influence of surfactant-tailored Mn-doped ZnO nanoparticles on ROS production and DNA damage induced in murine fibroblast cells.

The present study concerns the in vitro oxidative stress responses of non-malignant murine cells exposed to surfactant-tailored ZnO nanoparticles (NPs) with distinct morphologies and different levels of manganese doping. Two series of Mn-doped ZnO NPs were obtained by coprecipitation synthesis method, in the presence of either polyvinylpyrrolidone (PVP) or sodium hexametaphosphate (SHMTP). The samples were investigated by powder X-ray Diffraction, Transmission Electron Microscopy, Fourier-Transform Infrared and Electron Paramagnetic Resonance spectroscopic methods, and N2 adsorption-desorption analysis. The observed surfactant-dependent effects concerned: i) particle size and morphology; ii) Mn-doping level; iii) specific surface area and porosity. The relationship between the surfactant dependent characteristics of the Mn-doped ZnO NPs and their in vitro toxicity was assessed by studying the cell viability, intracellular reactive oxygen species (ROS) generation, and DNA fragmentation in NIH3T3 fibroblast cells. The results indicated a positive correlation between the specific surface area and the magnitude of the induced toxicological effects and suggested that Mn-doping exerted a protective effect on cells by diminishing the pro-oxidative action associated with the increase in the specific BET area. The obtained results support the possibility to modulate the in vitro toxicity of ZnO nanomaterials by surfactant-controlled Mn-doping.

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.