Properties and Potential Antiproliferative Activity of Thrombin-Binding Aptamer (TBA) Derivatives with One or Two Additional G-Tetrads

In this paper, we study the biological properties of two TBA analogs containing one and two extra G-tetrads, namely TBAG3 and TBAG4, respectively, and two further derivatives in which one of the small loops at the bottom (TBAG41S) or the large loop at the top (TBAG4GS) of the TBAG4 structure has been completely modified by replacing all loop residues with abasic site mimics. The therapeutical development of the TBA was hindered by its low thermodynamic and nuclease stability, while its potential as an anticancer/antiproliferative molecule is also affected by the anticoagulant activity, being a side effect in this case. In order to obtain suitable TBA analogs and to explore the involvement of specific aptamer regions in biological activity, the antiproliferative capability against DU 145 and MDAMB 231 cancer cell lines (MTT), the anticoagulant properties (PT), the biological degradability (nuclease stability assay) and nucleolin (NCL) binding ability (SPR) of the above described TBA derivatives have been tested. Interestingly, none of the TBA analogs exhibits an anticoagulant activity, while all of them show antiproliferative properties to the same extent. Furthermore, TBAG4 displays extraordinary nuclease stability and promising antiproliferative properties against breast cancer cells binding NCL efficiently. These results expand the range of G4-structures targeting NCL and the possibility of developing novel anticancer and antiviral drugs.     

Role of the purinergic P2Z receptor in spontaneous cell death in J774 macrophage cultures

J774 mouse macrophages express an ionotropic receptor gated by extracellular ATP. Activation of this receptor, currently named purinergic P2Z, causes transmembrane ion fluxes, plasma membrane depolarization, cell swelling and eventual cell death. The physiological role of this receptor is as yet unknown. In the present report we show that macrophage cell clones that hypo-express the P2Z receptor showed a very low degree of spontaneous cell death in culture, while hyper-expressing clones were exceedingly susceptible to cell death. To further support a role for ATP receptors in spontaneous cell death, addition to the macrophage cell cultures of oxidized ATP, a selective inhibitor of ionotropic purinergic receptors, or the ATP-hydrolysing enzyme apyrase, also reduced spontaneous death.     

Mouse microglial cells express a plasma membrane pore gated by extracellular ATP

We have investigated responses to extracellular ATP (ATPe) in the microglial cell lines N9 and N13 and in freshly isolated mouse microglial cells. Upon stimulation with this nucleotide, N9 and N13 cells underwent an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i), a sustained depolarization of the plasma membrane, and an uptake of extracellular markers such as ethidium bromide and lucifer yellow; increases in plasma membrane permeability were paralleled by striking morphologic changes. ATPe, as well as other nucleotides, activated a spiking Ca2+ release from intracellular stores; however, only ATPe was also able to cause a massive transmembrane Ca2+ influx. The ATP analogue 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) triggered a sustained Ca2+ influx accompanied by little release from stores. The ATP derivative oxidized ATP (oATP) strongly inhibited Ca2+ influx, minimally affecting Ca2+ release. From ATPe-sensitive microglial cell lines, we selected several ATPe-resistant clones that showed complete lack of ATPe-mediated plasma membrane permeability changes, although they retained the Ca2+ mobilization response from intracellular stores. ATPe-dependent plasma membrane permeability changes were also greatly reduced in growth-arrested microglial cells. Finally, ATPe triggered IL-1 beta release from wild-type but not ATPe-resistant microglial cells. These results show that microglial cells express at least two purinergic receptor subtypes, metabotropic (P2Y) and ionotropic (P2Z), and that the latter is modulated during cell cycle and coupled to IL-1 beta release.     

SiO2 nanoparticles biocompatibility and their potential for gene delivery and silencing

Despite the extensive use of silica nanoparticles (SiO(2)NPs) in many fields, the results about their potential toxicity are still controversial. In this work, we have performed a systematic in vitro study to assess the biological impact of SiO(2)NPs, by investigating 3 different sizes (25, 60 and 115 nm) and 2 surface charges (positive and negative) of the nanoparticles in 5 cell lines (3 in adherence and 2 in suspension). We analyzed the cellular uptake and distribution of the NPs along with their possible effects on cell viability, membrane integrity and generation of reactive oxygen species (ROS). Experimental results show that all the investigated SiO(2)NPs do not induce detectable cytotoxic effects (up to 2.5 nM concentration) in all cell lines, and that cellular uptake is mediated by an endocytic process strongly dependent on the particle size and independent of its original surface charge, due to protein corona effects. Once having assessed the biocompatibility of SiO(2)NPs, we have evaluated their potential in gene delivery, showing their ability to silence specific protein expression. The results of this work indicate that monodisperse and stable SiO(2)NPs are not toxic, revealing their promising potential in various biomedical applications.     

Measurement of fetal cells in the maternal circulation

There is evidence that the quantitation of fetal-maternal hemorrhage may be useful in reducing some of the RhoGAM failures in preventing Rh sensitization. The Betke-Kleihauer technique is a well-established method that has been used to estimate the amount of transplacental hemorrhage. Recently, a kit (Fetaldex) has become available for use in the detection and quantitation of fetal-maternal hemorrhage. Using control blood smears, we compared the results of the Fetaldex kit with those results obtained by the Betke-Kleihauer technique. The data indicate that the Fetaldex method is as accurate as the Betke-Kleihauer technique in detecting and measuring fetal red cells in the maternal circulation.     

Cross-linked polystyrene-dithiocarbamate polymers: Use in heavy metal removal

Cross-linked polystyrene polymers were converted to dithiocarbamate polymers. These polymers were used to remove heavy metal cations from water. Dithiocarbamate polymers can be understood to be inherently more stable toward hydrolysis than xanthate polymers.     

Subwavelength size determination by spatially modulated illumination virtual microscopy

A new approach for determining the sizes of individual, small fluorescent objects with diameters considerably below the optical resolution limit is described in which spatially modulated illumination (SMI) microscopy and 360-647-nm excitation wavelengths are used. The results of SMI virtual microscopy computer simulations indicate that, in this wavelength range, reliable measurements of sizes as small as approximately 20 nm are feasible if the low numbers of fluorescence photons that are usually detected from such small objects are taken into account. This method is based on the well-known fact that the modulation of the diffraction image in a SMI microscope is disturbed by the size of the object. Using appropriately calculated calibration functions, one can use this disturbance of the modulation to determine the size of the original object.     

Rapid and Controllable Digital Microfluidic Heating by Surface Acoustic Waves

Fast and controllable surface acoustic wave (SAW) driven digital microfluidic temperature changes are demonstrated. Within typical operating conditions, the direct acoustic heating effect is shown to lead to a maximum temperature increase of about 10 °C in microliter water droplets. The importance of decoupling droplets from other on‐chip heating sources is demonstrated. Acoustic‐heating‐driven temperature changes reach a highly stable steady‐state value in ≈3 s, which is an order of magnitude faster than previously published. This rise time can even be reduced to ≈150 ms by suitably tailoring the applied SAW‐power excitation profile. Moreover, this fast heating mechanism can lead to significantly higher temperature changes (over 40 °C) with higher viscosity fluids and can be of much interest for on‐chip control of biological and/or chemical reactions.