Sigma-1 receptor as a potential pharmacological target for the treatment of neuropathology

Sigma receptors are usually classified as a separate class of intracellular receptors. Among them the sigma-1 receptor has been the most studied regarding its pharmacological applications. This receptor with average or high affinity binds a wide range of chemical compounds of very different structural classes and a variety of therapeutic and pharmacological properties. The sigma-1 receptor is a trans-membrane protein placed in the endoplasmic reticulum (ER), which regulates the function of inositol-3-phosphate receptor, stabilizing the calcium signaling between ER and mitochondria. There are studies that the sigma-1 receptor is involved in the formation of many neurological and psychiatric conditions. It is assumed that the sigma-1 receptor acts as a sensor of normal calcium operation. The studies over the recent years have shown the role of the violation in calcium signaling in the pathogenesis of Alzheimer's and Huntington's diseases. In particular, changes in calcium homeostasis of the endoplasmic reticulum lead to the break of synaptic connections in the neurons. Thus, the sigma-1 receptor holds promise in application as a potential therapeutic target for the treatment of neuropathological diseases

Modification of Polydiallyldimethylammonium Chloride with Sodium Polystyrenesulfonate Dramatically Changes the Resistance of Polymer-Based Coatings towards Wash-Off from Both Hydrophilic and Hydrophobic Surfaces

Polymer coatings based on polycations represent a perspective class of protective antimicrobial coatings. Polydiallyldimethylammonium chloride (PDADMAC) and its water-soluble complexes with sodium polystyrenesulfonate (PSS) were studied by means of dynamic light-scattering, laser microelectrophoresis and turbidimetry. It was shown that addition of six mol.% of polyanion to polycation results in formation of interpolyelectrolyte complex (IPEC) that was stable towards phase separation in water-salt media with a concentration of salts (NaCl, CaCl2, Na2SO4, MgSO4) up to 0.5 M. Most of the polyelectrolyte coatings are made by layer-by-layer deposition. The utilization of water-soluble IPEC for the direct deposition on the surface was studied. The coatings from the PDADMAC and the PSS/PDADMAC complex were formed on the surfaces of hydrophilic glass and hydrophobic polyvinylchloride. It was found that formation IPEC allows one to increase the stability of the coating towards wash-off with water in comparison to individual PDADMAC coating on both types of substrates. The visualization of the coatings was performed by atomic force microscopy and scanning electron microscopy

Hydrophilization of Hydrophobic Mesoporous High-Density Polyethylene Membranes via Ozonation

This work addresses hydrophilization of hydrophobic mesoporous membranes based on high-density polyethylene (HDPE) via ozonation. Mesoporous HDPE membranes were prepared by intercrystallite environmental crazing. Porosity was 50%, and pore dimensions were below 10 nm. Contact angle of mesoporous membranes increases from 96° (pristine HDPE) to 120° due to the formation of nano/microscale surface relief and enhanced surface roughness. The membranes are impermeable to water (water entry threshold is 250 bar). The prepared membranes were exposed to ozonation and showed a high ozone uptake. After ozonation, the membranes were studied by different physicochemical methods, including DSC, AFM, FTIR spectroscopy, etc. Due to ozonation, wettability of the membranes was improved: their contact angle decreased from 120° down to 60°, and they became permeable to water. AFM micrographs revealed a marked smoothening of the surface relief, and the FTIR spectra indicated the development of new functionalities due to ozonolysis. Both factors contribute to hydrophilization and water permeability of the ozonated HDPE membranes. Hence, ozonation was proved to be a facile and efficient instrument for surface modification of hydrophobic mesoporous HDPE membranes and can also provide their efficient sterilization for biomedical purposes and water treatment

Ultrasonic Film Rehydration Synthesis of Mixed Polylactide Micelles for Enzyme-Resistant Drug Delivery Nanovehicles

A facile technique for the preparation of mixed polylactide micelles from amorphous poly-D,L-lactide-block-polyethyleneglycol and crystalline amino-terminated poly-L-lactide is described. In comparison to the classical routine solvent substitution method, the ultrasonication assisted formation of polymer micelles allows shortening of the preparation time from several days to 15–20 min. The structure and morphology of mixed micelles were analyzed with the assistance of electron microscopy, dynamic and static light scattering and differential scanning calorimetery. The resulting polymer micelles have a hydrodynamic radius of about 150 nm and a narrow size distribution. The average molecular weight of micelles was found to be 2.1 × 107 and the aggregation number was calculated to be 6000. The obtained biocompatible particles were shown to possess low cytotoxicity, high colloid stability and high stability towards enzymatic hydrolysis. The possible application of mixed polylactide micelles as drug delivery vehicles was studied for the antitumor hydrophobic drug paclitaxel. The lethal concentration (LC50) of paclitaxel encapsulated in polylactide micelles was found to be 42 ± 4 µg/mL—a value equal to the LC50 of paclitaxel in the commercial drug Paclitaxel-Teva

Addition of H₂O to the medium causes filopodial bleb-like protrusions.

<p>(A) Confocal time-lapse montage of a HEK-293 cell stably overexpressing GFP-AQP9. During acquisition, 20 µl of H₂O was added to the medium (2 ml) with a pipette directed towards the cell, yielding a rapid but transient reduction in local osmolarity. The images are pseudo-colored in fire scale to visualize variations in fluorescence intensity. White arrows are pointing towards a representative bleb-like protrusions formed during image acquisition. Scale bar 10 µm. (B, upper panel) An enlarged image of a single filopodium during acquisition, before and after the addition of H₂O. The white arrow shows the direction and length of measurement presented in the lower panel. The images are linearly adjusted and pseudo-colored in fire scale to visualize variations in fluorescent intensity. (B, lower panel) Intensity profile plots measured along the filopodia as shown by the white arrow. The red arrows are pointing towards peaks in fluorescent intensity before and after the addition of H₂O. (C) Quantification of the percentage of filopodia that developed filopodial bleb-like protrusions subsequent to the addition of 20 µl of H₂O after pre-treatment with AQP9-inhibitors. HEK-293 cells overexpressing GFP-AQP9 were pretreated with 1, 5 and 10 µM Hg²⁺ or with 25 µM of HTS13286. Control cells represents untreated HEK-293 cell overexpressing GFP-AQP9. Data is presented as mean (±SEM, n = 4–7 experiements/group). (D, left panel) Phase contrast images of primary human macrophages. The cell in the lower panel is treated with 25 µM of the novel AQP9 inhibitor HTS13286. (D, right panel) Cropped and inverted time lapse montage of the cells displayed in the left panel. During image acquisition 20 µl of H₂O was added to the medium (2 ml). Magenta arrows are pointing towards filopodial bleb-like protrusions. Scale bar 10 µm.</p

Model for AQP9-induced membrane protrusion.

<p>(A) A migrating cell with lamellipodia, filopodia, and blebs where an increased influx of water corresponds to a darker blue tone. (B1) Local accumulation of AQP9 by vesicle transport and/or lateral membrane diffusion enables a localized increased influx of water across the cell membrane. The influx is driven by an osmotic gradient, likely created by the transmembrane ion distribution (not shown). (B2) The rapid influx of water creates a localized hydrostatic pressure between the membrane and the cytoskeleton pushing the membrane outwards, thus initiating a membrane protrusion. (B3) The influx of water increases the hydrostatic pressure locally. In parallel, actin polymerization is promoted by the exposure of previously membrane-anchored barbed ends and the rapid diffusion of actin monomers in the now diluted, less viscous cytoplasm leading to an elongating filopodium. (B4) Then the rapid water-induced elongation reaches a critical distance from the actin, resulting in termination of the filopodial elongation likely due to equilibration of the water along the filopodium and loss of counter-pressure obtained from the actin cytoskeleton. (B5) The rate of the actin polymerization catches up with the water-induced protrusion and thereby stabilizes the structure. Based on the rate of water flux and equilibration, the filopodium can either protrude once more, or remain at its present length.</p

Size and location of H₂O-induced bleb like protrusions.

<p>(A) Time lapse montage of the formation of filopodial bleb-like protrusions. H₂O was delivered with a micropipette in close proximity to the cell. The pressure (4000hPa) was applied to the micropipette for 1, 2, 4 or 8 s to the same cell. The white arrow is pointing toward a filopodial bleb-like protrusion. Magenta arrow is pointing toward a bleb-like protrusion originating from the cell body. (A, lower panel) Following an 8 s localized water release in close vicinity of a cell that was pre-treated with 25 µM HTS13286 no bleb-like formations were observed. Scale bar 5 µm. (B) Quantification of the filopodial diameter at the site of the bleb-like protrusion. Time 0 equals the image before localized H₂O release. The lower graph shows the mean diameter for 8 s water release to cells untreated (green line), or treated with 25 µM of HTS13286 (black line).The data is displayed as mean±SEM, n = 8–18 filopodia/water release period. (C) Quantification of mean percentage of bleb-like protrusions originating from filopodia or the cell body after 1, 2, 4 or 8 s injection of micropipette-delivered H₂O (n = 3 experiments).</p

Disruption of actin dynamics inhibits the formation of new filopodia.

<p>(A) Representative confocal images of HEK-293 cells stably overexpressing GFP-AQP9 before and 15 min after treatment with 1 µM Cyt D or 500 nM Jasplakinolide. The red arrows point towards distended filopodia. Scalebar 10 µm. (B) Quantification of peripheral filopodia before, and 10–15 min after treatment with 1 µM Cyt D. The data is presented as mean (±SEM, n = 5–43 cells/group). (C, left panel) Quantification of the relative filopodial tip area of GFP-AQP9 expressing cells before (Ctrl), and 15 min after the addition of 1 µM Cytochalsin D. The filopodial tips are defined by the fluorescent area occupied in a 2×2 µm ROI of the filopodial tips. The data is presented as mean (±SEM) of fold change compared to untreated cells (Ctrl; n = 13–22 filopodia/group).(C, right panel) Representative examples of a filopodia before and after treatment with Cyt D. The red box illustrates the area of measurement for the data presented in the left panel. (D) A confocal time-lapse montage of GFP-AQP9 fluorescence, pseudo-colored in fire scale, in HEK-293 cells 10 min after treatment with 10 µM Cytochalsin D. The images illustrate a bleb-like protrusion that recoils back towards the cell body after treatment with actin dynamics inhibitors. The linear intensity is adjusted to visualize differences in fluorescence intensity. Scalebar 1 µm.</p

Characterization of AQP9-induced filopodia.

<p>(A) Representative confocal images of HEK-293 cells transfected with tagRFP-AQP9 or empty vector together with GFP-Mem to label the membrane. Intensities have been adjusted linearly to visualize the relative expression and localization of both fluorophores. Scalebar 10 µm. (B) Quantification of peripheral filopodia in HEK-293 cells transfected with GFP-AQP9 or GFP-Mem. The data is presented as mean number of filopodia/µm perimeter(±SEM; n = 34–43 cells/group). (C) Representative confocal images of HEK-293 cells transfected with tagRFP-AQP9 and GFP-Mem. Images are pseudo-colored in fire scale to visualize the differences between the two vectors in the filopodia. The intensities have been adjusted linearly to visualize the relative distribution of both fluorophores. The lower panel represents enlargement of the green box. Scalebar 10 µm. (D) Ratiometric measurements of mean fluorescence intensity (MFI) in the filopodial membrane divided by MFI in the cell body membrane in HEK-293 cells transfected with both tagRFP-AQP9 and GFP-Mem. Measurement areas are illustrated in the schematic image. The data is presented as mean (± SEM, n = 51 filopodia/group). (E) Montage of a representative confocal time-lapse of a HEK-293 cell overexpressing GFP-AQP9 pseudo-colored in fire scale to visualize AQP9 localization in growing filopodia. The linear intensity has been adjusted to visualize differences in fluorescent intensity. Scalebar 2 µm. (F, left panel) An enlarged image from (E) showing the points of measurements for the profile plots presented in the right panel. (F, right panel) Intensity profile plots of filopodia during growth to visualize AQP9 accumulation in filopodial tips.</p