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Aim of the study was to identify the mechanism of specific endothelial toxicity related to calcium phosphate bions (CPB). Material and methods. CPB and magnesium phosphate bions (MPB) were artificially synthesised through supersaturation of culture medium with respective salts and then added to human endothelial cells (EA.hy 926) and murine endothelial cells (2H-11) to study: 1) spatiotemporal aspects of bion internalisation by means of transmission electron microscopy and confocal microscopy; 2) whether blocking of H+-ATPase by lysosomal inhibitor bafilomycin A1 affects endothelial toxicity of bions; 3) expression of caspase-3 and its substrate poly(ADP-ribose) polymerase (PARP-1). Results. CPB were internalized by endothelial cells as early as 1 h upon their addition and were localized in lysosomes; after 4 h, we detected release of calcium ions (Ca2+) from lysosomes to cytosol accompanied by multifold increase in cleaved caspase 3 and its substrate PARP-1. Bafilomycin A1 rescued endothelial cells from death induced by slightly soluble CPB regardless of exposure time and dose; however, freely soluble MPB did not evince endothelial toxicity regardless of bafilomycin A1 addition. Conclusion. Upon internalization by endothelial cells, CPB cause their death due to dissolution in lysosomes and subsequent release of calcium ions into the cytosol, ultimately leading to cleavage of executioner caspases. MPB lack endothelial toxicity because their dissolution does not lead to release of calcium ions. Therefore, specific endothelial toxicity of CPB is defined by lysosome-dependent cell death.

Aim of the study was to investigate whether endothelial cells internalise magnesium phosphate and calcium phosphate bions. Material and methods. Magnesium phosphate and calcium phosphate bions synthesised through supersaturation of the culture medium with the respective salts were further added to the confluent cultures of primary human coronary artery endothelial cells for 4 h. Internalisation was evaluated by backscattered scanning electron microscopy (BSEM) and confocal microscopy. For BSEM, cells were fixed in 10% neutral phosphate buffered formalin, postfixed/stained by 2 % osmium tetroxide, dehydrated in a graded ethanol series, stained by 2% uranyl acetate, impregnated into acetone:epoxy resin (1:1) and into the fresh epoxy resin with its subsequent polymerisation, grinding, and polishing. Samples were then counterstained by lead citrate, sputter coated with carbon, and visualised utilising BSEM. Confocal microscopy was conducted after exposure of endothelial cells to FITC-labeled magnesium phosphate bions or calcein-labeled calcium phosphate bions with the following staining by a pH sensor LysoTracker Red and nuclear stain Hoechst 33342. Results. All types of bions were internalised by endothelial cells within 4 h. BSEM after long-term postfixation/ staining by osmium tetroxide and uranyl acetate, embedding into epoxy resin and counterstaining by lead citrate visualised multiple black electron-dense mineral inclusions which were not detected both within the control cells and in the intercellular space. Confocal microscopy also detected internalised MPB and CPB in cytosol and lysosomes. Conclusion. BSEM visualised MPB and CPB internalised by endothelial cells after 4 h exposure while confocal microscopy detected their colocalisation with lysosomes. MPB is an appropriate comparison group to study the specificity of CPB-related pathogenic effects as both of these bion types have close physico-chemical properties and are similarly internalised.

Aim. To study the expression of scavenger-receptor genes SCARF1 , MSR1 , CD36 , LDLR , VLDLR and cell adhesion genes VCAM1 , ICAM1 , PECAM1 , SELE , SELP , CDH5 potentially responsible for endothelial toxicity of mineral-organic nanoparticles (bions) in cultures of endothelial cells exposed to magnesium phosphate (MPB) and calcium phosphate bions (CPB). Materials and methods. In this study we used the culture of human immortalized venous endothelial cells (EA.hy 926 line), as well as commercial cultures of primary human coronary (HCAEC) and internal thoracic artery endothelial cells (HITAEC). Cells were cultured in the presence of synthesized MPB and CPB and pure phosphate-saline buffer (control). Immediately after cultivation, total RNA was isolated from samples of the cell lines. Based on the isolated RNA, a single-stranded cDNA was synthesized using a reverse transcription reaction. Evaluation of gene expression in cell cultures was carried out using Real-Time quantitative PCR with a SYBR Green fluorescent dye. Statistical analysis of the results was performed in GraphPad Prism 6 software. Results. Exposure of EA.hy 926 cells by MPB did not lead to significant changes in the expression of the studied genes except SELP and SELE compared with non-exposed control. Two-fold change in gene expression was shown for the SCARF1 and CD36 genes in culture exposed to spherical CPB (SCPB), as well as for the ICAM1 gene in culture exposed to needle-like CPB (NCPB). CPB exposure to HCAEC and HITAECcultures resulted in a pronounced decrease of expression of the CD36 and SELP genes and an increase of expression of the ICAM1 gene both in HCAEC and HITAEC.Expression of the VCAM1 and SELE genes was 2.5-fold higher in HCAEC exposed to NCPB and 2-fold increase of expression of the VLDLR gene HITAEC exposed to both types of CPB and the PECAM1 exposed to SCPB. MPB had practically no effect on the expression of the studied genes in the EA.hy 926 and HCAEC cultures, but at the same time they increased the expression of the PECAM1 gene and reduced the expression of the SELP and CDH5 genes in HITAEC. Conclusion.The chemical composition and morphology of bions, as well as the physiological characteristics of vessels can affect to gene expression signature in cultures of endothelial cells.

Aim. To evaluate specificity of endothelial toxicity of calcium-phosphate bions (CPB) in vivo.Material and methods. Toxicity of calcium-phosphate bions and magnesiumphosphate bions (MPB) in relation to intima of abdominal aorta of the Wistar rats, was assessed by single intravenous injection after balloon angioplastics with further explanting of aortas in five weeks. Bioptates were analyzed: 1) with classical histological methods (hematoxilin-eosine, alizarin red, van Gison, Russell-Movat) with light microscopy; 2) immune fluorescence coloring of cryoslices (combinational coloring for marker of mature endothelial cells CD31 and marker of progenitory CD34, for CD31 and marker of vascular smooth muscle cells α-smoothmuscle actin (α-SMA), for vimentin and α-SMA, for extracellular matrix marker collagen type IV and α-SMA, after all colorings there was additional nuclear 4’,6-diamidine-2-phenylindol color) with further confocal microscopy. In all animals the blood was collected with serum extraction for systemic inflammation molecules analysis, as chemoattractant protein (МСР-1/CCL2) and ceruloplasmin via the immune enzyme analysis.Results. With the difference from CPB, MPB did not lead to intimal hypertrophy in abdominal aorta in rats. Shaping of neointima in aorta is related with CPB-induced endothelium damage that induces a phenotype shift in mesenchymal cells (smooth muscle cells and fibroblasts) from contractile (for smooth muscle) and non-active (for fibroblasts) towards synthetizing.Conclusion. Intravenous load of MPB did not lead to intimal hypertrophy that witness on specificity of endothelial toxicity of CPB, with phenotypical shift of the mesenchymal cells in neointima.

Aim. To identify death subroutine of endothelial cells upon the exposure to calcium phosphate bions (CPB) and to define whether CPB induce specific molecular response. Materials and Methods. Immortalized murine lymphatic endothelial cells (2H-11, 85-90 % confluence) were exposed to either magnesium phosphate bions (MPB), spherical calcium phosphate bions (SCPB), or needle-shaped calcium phosphate bions (NCPB) for 24 hours with the subsequent protein extraction using radioimmunoprecipitation assay (RIPA) buffer followed by Western blotting to: 1) apoptosis effector proteins (cleaved caspase-3 (cCasp-3), cleaved poly (ADP-ribose) polymerase (cParp-1)); 2) intrinsic apoptosis proteins ((X-linked inhibitor of apoptosis protein (Xiap), survivin, plasminogen activator inhibitor 1 (Pai-1), HtrA2/Omi, cytochromec, p53 upregulated modulator of apoptosis (Puma)); 3) proteins relate to central signaling pathways (phosphorylated extracellular signal-regulated kinase (pErk), phosphorylated mitogen-activated proteinkinase (pMapk), phosphorylated focal adhesion kinase (pFak), phosphorylated nuclear factor kappa-light-chain-enhancer of activated Bcells(pNF-kB), Itch, and Gli). Results were assessed by chemiluminescence detection and using the standard ImageJ algorithm for analysis of chemiluminescent gels. Results. We found increased levels of cleaved effector caspase 3 and its cleaved substrate, cPARP-1, in endothelial cells exposed to SCPB or NCPB as compared to those exposed to MPB or control phosphate buffered saline. Further, we documented decreased level of Xiap, a key inhibitor of intrinsic apoptosis, in endothelial cells exposed to SCPB or NCPB. In contrast, survivin, an other major inhibitor of intrinsic apoptosis, and anti-apoptotic protein Pai-1 were abated upon the SCPB or NCPB exposure, possibly due to the activation of negative feedback loop. In addition, we did not detect any significant changes in master regulators of cell signaling pathways after exposure to CPB. Conclusion. CPB induce intrinsic apoptosis in endothelial cells but do not cause any specific molecular signaling response.

Study aim. To investigate whether the shape of calcium phosphate bions (CPB) affects their endothelial toxicity via evaluating the cytokine secretion profile of endothelial cells upon the exposure to either spherical or spindle-shaped CPB.Material and methods. For the experiments, we used an immortalized human vein endothelial cell line EA.hy 926. Cells were seeded into 6-well plates (3*105 cells) with the further: 1) addition of 100 |jL either spherical CPB, spindle-shaped CPB, or 1x phosphate buffered saline (PBS) upon 1 h following culture for 24 h (non-confluent cell culture); 2) culture for 44 h and subsequent addition of 100 jL either spherical CPB, spindle-shaped CPB, or PBS following culture for 4 h (confluent cell culture). Upon the collection of cell culture supernatant (n=11 wells per group), the levels of proatherosclerotic cytokines (interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12, IL-23, tumor necrosis factor (TNF)-a, interferon (IFN)-y, and soluble vascular cell adhesion molecule (sVCAM)-1) were measured utilizing an enzyme-linked immunosorbent assay.Results. In a non-confluent cell culture, exposure to spindleshaped CPB increased the secretion of several proatherosclerotic cytokines (IL-1 ß, IL-10, IL-12, IL-23, IFN-y) compared to either spherical CPB-treated or control cells. In a confluent cell culture, exposure to either of CPB types decreased the release of IL-1 ß, IL- 10, and IFN-y; however, their concentration was still higher upon the exposure to spindle-shaped CPB in comparison with exposure to spherical CPB. Discriminant analysis and principal component analysis demonstrated that the cytokine secretion profile of spindle-shaped CPB-treated endothelial cells significantly differed from those of either spherical CPB-treated or control cells.Conclusion. Spindle-shaped CPB induce the secretion of proatherosclerotic cytokines by endothelial cells compared to spherical CPB; this suggests higher endothelial toxicity of spindleshaped CPB.

Wilhelm Reich is the focus of this second in a series of papers on a group of independent figures from the 1930s into the 1950s--also including Jung, the later Heidegger, Toynbee, Bergson, Teilhard de Chardin, and Simone Weil--who in the context of those years of crisis articulated overlapping visions of a future "New Age" spirituality that might in some more distant future serve to balance and even transform a globalizing materialism and disenchantment with traditional religion. The later Reich developed a highly original version of a "vitalistic" transpersonal psychology, as his "religion for the children of the future," which needs to be differentiated from its more doubtful supportive research in his orgone physics and biology. The spiritual nature of his larger intuitions of a transformative life energy is also reflected in the parallels between Reich's personal development and the classical purgation/illumination phases of unitive mysticism, and the "spiritual emergency" of his final "dark night" crisis. A later paper will concentrate on largely unrealized implications of Reich's work for still evolving directions in consciousness studies, neoshamanism, the historical Jesus, emergent systems approaches in science, and a future planetary identity. [ABSTRACT FROM AUTHOR]

Calcium phosphate bions (CPBs) are formed under blood supersaturation with calcium and phosphate owing to the mineral chaperone fetuin-A and representing mineralo-organic particles consisting of bioapatite and multiple serum proteins. While protecting the arteries from a rapid medial calcification, CPBs cause endothelial injury and aggravate intimal hyperplasia in balloon-injured rat aortas. Here, we asked whether CPBs induce intimal hyperplasia in intact rat arteries in the absence of cardiovascular risk factors. Normolipidemic Wistar rats were subjected to regular (once/thrice per week over 5 weeks) tail vein injections of either spherical (CPB-S) or needle-shaped CPBs (CPB-N), magnesium phosphate bions (MPBs), or physiological saline (n = 5 per group). Neointima was revealed in 3/10 and 4/10 rats which received CPB-S or CPB-N, respectively, regardless of the injection regimen or blood flow pattern in the aortic segments. In contrast, none of the rats treated with MPBs or physiological saline had intimal hyperplasia. The animals also did not display signs of liver or spleen injury as well as extraskeletal calcium deposits. Serum alanine/aspartate transaminases, interleukin-1β, MCP-1/CCL2, C-reactive protein, and ceruloplasmin levels did not differ among the groups. Hence, CPBs may provoke intimal hyperplasia via direct endothelial injury regardless of their shape or type of blood flow.

Abstract: This paper presents the design, fabrication, and functional testing of a fully implantable, flexible, Parylene-enable neurostimulator that features single-channel wireless stimulation capability . This system comprises a fold-and-bond microelectromechanical systems (MEMS) coil for wireless power and data transmission, a BION 1-3 CMOS stimulator chip, discrete capacitors, as well as a carrier substrate with two platinum stimulating electrodes and interconnections for system assembly. The physical geometries of the devices are designed for use in retinal implantation with the specifications determined from the implantation results in canine eyes. The coil and carrier substrate are separately fabricated using a Parylene–metal skin technology. The unique properties of Parylene C allow these MEMS devices to be flexible and biocompatible, facilitating surgical procedure. The system assembly is achieved by interconnecting individual components together on the carrier substrate with a biocompatible silver epoxy. A 10μm layer of Parylene C is selectively deposited on the integrated system to protect it from corrosive eye environment. The system functionality is verified using a telemetry link setup, and single-phase pulses with amplitudes ranging from 7 to 8.5V are detected. [Copyright &y& Elsevier]

Calcium phosphate bions (CPBs) are formed under blood supersaturation with calcium and phosphate owing to the mineral chaperone fetuin-A and representing mineralo-organic particles consisting of bioapatite and multiple serum proteins. While protecting the arteries from a rapid medial calcification, CPBs cause endothelial injury and aggravate intimal hyperplasia in balloon-injured rat aortas. Here, we asked whether CPBs induce intimal hyperplasia in intact rat arteries in the absence of cardiovascular risk factors. Normolipidemic Wistar rats were subjected to regular (once/thrice per week over 5 weeks) tail vein injections of either spherical (CPB-S) or needle-shaped CPBs (CPB-N), magnesium phosphate bions (MPBs), or physiological saline (n = 5 per group). Neointima was revealed in 3/10 and 4/10 rats which received CPB-S or CPB-N, respectively, regardless of the injection regimen or blood flow pattern in the aortic segments. In contrast, none of the rats treated with MPBs or physiological saline had intimal hyperplasia. The animals also did not display signs of liver or spleen injury as well as extraskeletal calcium deposits. Serum alanine/aspartate transaminases, interleukin-1β, MCP-1/CCL2, C-reactive protein, and ceruloplasmin levels did not differ among the groups. Hence, CPBs may provoke intimal hyperplasia via direct endothelial injury regardless of their shape or type of blood flow. [ABSTRACT FROM AUTHOR]

We observed earlier that mineralo-organic nanoparticles form in human body fluids when the concentrations of calcium, carbonate and phosphate exceed saturation. The particles have been shown to represent mineral precursors in developing bones and teeth as well as in ectopic calcification and kidney stones. Recent studies suggest that the mineral particles may also be involved in other physiological processes, including immune tolerance against the gut microbiota and food antigens. We review here the involvement of mineralo-organic nanoparticles in physiological and pathological processes and discuss recent findings that reveal novel and unexpected roles for these particles in the human body. [ABSTRACT FROM AUTHOR]