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Platinum corrosion products from electrode contacts of human cochlear implants induce cell death in cell culture models
Kirsten Wissel ,
Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing
* E-mail: wissel.kirsten@mh-hannover.de
Affiliations Department of Otorhinolaryngology, Hannover Medical School, Hannover, Germany, Cluster of Excellence 'Hearing 4 all', NIFE, Hannover, Germany
Gudrun Brandes,
Roles Conceptualization, Data curation, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing
Affiliation Institute of Neuroanatomy and Cell Biology, Center of Anatomy and Cell Biology, Hannover Medical School, Hannover, Germany
Nils Pütz,
Roles Data curation, Formal analysis, Validation, Writing – review & editing
Affiliations Department of Otorhinolaryngology, Hannover Medical School, Hannover, Germany, Biotechnology Center, TU Dresden, Dresden, Germany
Gian Luigi Angrisani,
Roles Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing
Affiliation Institute of Materials Science, Leibniz University Hannover, Garbsen, Germany
Jan Thieleke,
Roles Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing
Affiliation Institute of Inorganic Chemistry, Leibniz University Hannover, Hannover, Germany
Thomas Lenarz,
Roles Conceptualization, Funding acquisition, Project administration, Writing – review & editing
Martin Durisin
Roles Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing
Affiliation Department of Otorhinolaryngology, Hannover Medical School, Hannover, Germany
Kirsten Wissel,
Kirsten Wissel Gudrun Brandes ... Martin Durisin
Despite the technological progress made with cochlear implants (CI), impedances and their diagnosis remain a focus of interest. Increases in impedance have been related to technical defects of the electrode as well as inflammatory and/or fibrosis along the electrode. Recent studies have demonstrated highly increased impedances as the result of corroded platinum (Pt) electrode contacts. This in vitro study examined the effects of Pt ions and compounds generated by corrosion of the electrode contacts of a human CI on cell metabolism. Since traces of solid Pt in surrounding cochlear tissues have been reported, the impact of commercially available Pt nanoparticles (Pt-NP, size 3 nm) on the cell culture model was also determined. For this purpose, the electrode contacts were electrically stimulated in a 0.5% aqueous NaCl solution for four weeks and the mass fraction of the platinum dissolute (Pt-Diss) was determined by mass spectrometry (ICP-MS). Metabolic activity of the murine fibroblasts (NIH 3T3) and the human neuroblastoma (SH-SY5Y) cells was determined using the WST-1 assay following exposure to Pt-Diss and Pt-NP. It was found that 5–50 μg/ml of the Pt-NP did not affect the viability of both cell types. In contrast, 100 μg/ml of the nanoparticles caused significant loss in metabolic activity. Furthermore, transmission electron microscopy (TEM) revealed mitochondrial swelling in both cell types indicating cytotoxicity. Additionally, TEM demonstrated internalized Pt-NP in NIH 3T3 cells in a concentration dependent manner, whereas endocytosis in SH-SY5Y cells was virtually absent. In comparison with the Pt-NP, the corrosion products (Pt-Diss) with concentrations between 1.64 μg/ml and 8.2 μg/ml induced cell death in both cell lines in a concentration dependent manner. TEM imaging revealed both mitochondrial disintegration and swelling of the endoplasmic reticulum, suggesting that Pt ions trigger cytotoxicity in both NIH 3T3 and SH-SY5Y cell lines by interacting with the respiratory chain.
Citation: Wissel K, Brandes G, Pütz N, Angrisani GL, Thieleke J, Lenarz T, et al. (2018) Platinum corrosion products from electrode contacts of human cochlear implants induce cell death in cell culture models. PLoS ONE 13(5): e0196649. https://doi.org/10.1371/journal.pone.0196649
Editor: Mária A. Deli, Hungarian Academy of Sciences, HUNGARY
Received: May 29, 2017; Accepted: April 17, 2018; Published: May 15, 2018
Copyright: © 2018 Wissel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This work was supported by a grant by the Deutsche Forschungsgemeinschaft, 240032230, recipients: Martin Durisin and Thomas Lenarz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Abbreviations: BSE, Backscattered electrons; CI, cochlear implant; FBS, fetal bovine serum; DIN, German Institute for Standardisation; ICP-MS, inductive coupled plasma-mass spectrometry; OD, optical density; Pt-NP, Pt nanoparticles; Pt-Diss, Pt dissolute; SEM, Standard error of mean; SEM, scanning electron microscopy; TEM, transmission electron microscopy
Cochlear implantation (CI) represents the only therapeutic intervention for patients with profound sensory neural hearing loss to date [1–2]. However, insertion of the CI into the scala tympani induces trauma in turn resulting in inflammation processes, fibrosis on the implant surface and new bone formation inside the scala tympani [3–8]. It was shown that those foreign-body reactions result in an increase of electrode impedance and consequently in a decrease of the electrical dynamic range for stimulation. Thus, greater electrical stimuli are required for sufficient neuronal stimulation leading to higher energy consumption [9–12]. In cases of persistent high-impedance values neural response telemetry and reprogramming of the implant in conjunction with administration of cortisone and antibiotics usually lead to normalization of the impedance values. Recently, several cases of recurrent increases in impedance in the same channels have been found which could not be explained by inflammation processes alone [13–14]. Investigation of explanted electrodes by scanning electron microscopy (SEM) revealed not only the presence of connective tissue with traces of blood, but also corrosion of the platinum surface of the electrode contacts [8, 14–15]. Furthermore, SEM micrographs of platinum microelectrodes demonstrated corrosion of Pt following stimulation in various biological buffer solutions [16].
Due to its electrochemical stability and biocompatibility Pt has been preferentially used as electrode material for neuroprostheses for some decades now. There are, however, electrochemical processes at the electrode-electrolyte interface involving corrosion of noble metals: Intrinsic charge injection is limited for every electrode and, therefore, the voltage that can be safely generated at the electrode surface is restricted. In the event that this voltage is exceeded, it is not possible to maintain purely capacitive charge transfer and, subsequently, irreversible faradaic processes occur. Those reactions comprise both cathodic and anodic water hydrolysis, the oxidation of chloride ions, release of oxygen gas and the oxidation of platinum [17–20].
Early studies on Pt dissolution suggested that Pt2+ ions and their complexes with chloride oxidation products as like as ClO- and ClO3- are the most likely dissolved species at the Pt/saline interface [17, 21]. Additionally, time of flight secondary ion mass spectrometry (TOF-SIMS) analysis of the tissue-platinum electrode interface following CI explantation identified not only oxidized PtO- and PtO2-, but also associates of Pt with sodium and calcium ions, Pt-protein-complexes and carbohydrates [8]. In previous studies, it was found that the presence of Pt-protein-complexes significantly reduced the extent of Pt dissolution. They were, by physical adsorption, able to form protective films which restrict the diffusion of reactants and products to and from the metal surface [22–24]. Furthermore, both platinum oxidation and oxide reduction were blocked following absorption of certain amino acids on the platinum surface. It has been suggested that several amino acids compete with chloride derived from phosphate-buffered saline [24]. However, amino acids with sulfur-hydrogen groups such as cysteine were known to interact strongly with platinum increasing the corrosion in a concentration-dependent manner [24].
Regarding the potential cytotoxicity of Pt ions and complexes, there is evidence of long-term toxic effects on bacteria, eukaryotic cells, and both mammalian and non-mammalian tissues [25–29]. In particular, cis-platinum compounds, e.g. cis-diamminedichloroplatinum(II) (cis-[Pt(NH3)2Cl2]), are used as potent anti-cancer drugs [26, 30–31].
In addition to Pt ions and its complexes traces of solid Pt particles were also found in the spiral ligament, connective tissue around the electrode, macrophages and even in the middle ear [8, 14, 32–34]. In vitro cytotoxic and genotoxic effects of Pt nanoparticles with average size below 10 nm have not been demonstrated at concentrations below 80–100 μg/ml in several cell lines thus far [35–38]. However, it has also been reported that–depending on the cell type and organism–Pt-NP induced oxidative stress, DNA damage, inflammation or other negative biological effects in a concentration-depending manner [39–44].
Therefore, it is of great clinical interest that we comprehend the process involved in formation of Pt corrosion products at the electrode-tissue interface, their distribution within the cochlea and their interaction with the neuronal cells. The present study examined the effects of Pt particles and compounds on an in vitro cell culture model. This involved electrically stimulating the electrode contacts of a human CI electrode for four weeks in an aqueous 0.5% NaCl solution. The Pt-containing dissolute (Pt-Diss) was administered to both the murine fibroblast cell line NIH 3T3 and the human neuroblastoma cell line SH-SY5Y in varying concentrations. Since previous studies reported traces of solid Pt within cochlear structures [8, 14, 32–34], commercially available Pt nanoparticles (Pt-NP, size 3 nm) were included in this study. The potential cytotoxicity of both Pt-Diss and Pt-NP in a concentration-dependent manner was examined using the WST-1 assay, and the ultrastructural changes were documented by transmission electron microscopy (TEM).
Electrical stimulation of the human CI electrode
The cochlear implant electrode used for this study was constructed in accordance with the design of the RE24 Contour Advance™ electrode for research purposes (Cochlear Ltd., Macquarie University, NSW, Australia). Electrical stimulation was performed in an electrolyte consisting of 0.5% NaCl (Braun, Melsungen, Germany) in deionized water for 600 h at room temperature; it was carried out. using a function generator (Agilent Technologies, Santa Clara, CA, USA) and amplified using a linear amplifier (Brüel & Kjær, Nærum, Denmark). The stimulation signal was a biphasic square wave with a duty cycle of 50% and a frequency of 5,000 Hertz resulting in alternating impulses of 100 μs with no interpulse delay. The voltage amplitude was 20.0 volts with a current of 0.3 amperes.
Determination of the platinum (Pt) content following electrical stimulation
The total amount of platinum (Pt) in the 0.5% NaCl solution was analysed by inductive coupled plasma-mass spectrometry (ICP-MS). The sample solution was diluted 1:10 with Millipore® high purity water and ultrapure nitric acid (p.a., Merck KGaA, Darmstadt, Germany), further purified by sub-boiling distillation to achieve a matrix matching of standards and samples. The standards were prepared by using ICP platinum standards (Carl Roth GmbH, Karlsruhe, Germany) and diluted ultrapure nitric acid (ω = 2.5%). Quantification was performed with external calibration by ICP-MS (X-Series 2, Thermo Fisher Scientific Inc., Waltham, USA). Evaluation and validation of the analysis was carried out in accordance with relevant DIN standards [45–46].
Dispersion of platinum nanoparticles (Pt-NP)
A 20 mg/ml stock solution of hydrophilic Pt-NP powder (average particle size: 3 nm), stabilized with polyvinylpyrrolidone (PlasmaChem, Berlin, Germany) was prepared by dispersion in sterile aqua bidest., followed by sonication in a water bath for 15 min.
Seeding and cultivation of NIH 3T3 and SH-SY5Y cells followed by supplementation of Pt nanoparticles (Pt-NP) and dissolutes (Pt-Diss)
Both the murine cell line NIH3T3 and the human neuroblastoma cell line SH-SY5Y (Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) were seeded into 96-well microtiter plates (Nunclon, Thermo Fisher Scientific, Kempen, Germany) at a density of 2 x 103 NIH 3T3 and 5 x 103 SH-SY5Y cells per well. Cultivation was performed in 100 μl high glucose DMEM containing 10% fetal bovine serum (FBS) (all components by Biochrom AG, Berlin, Germany) at 37°C and 5% CO2 for 24 h. The culture medium was removed and replaced by 100 μl of the Pt-containing solutions at their final concentrations (Pt-Diss, pH 5.0: 0.82–8.2 μg/ml in culture medium, pH 7.5–8.0, Pt-NP: 5–100 μg/ml in culture medium, pH 7.5–8.0). Cell cultivation was continued for another 48 h (NIH 3T3 cells) and 6 d (SH-SY5Y cells).
As described above, NaCl was used as the electrolyte for electrical stimulation and, therefore, changes in the NaCl concentration of the culture medium have to be considered due to supplementation of the cell culture assays with varying Pt-Diss concentrations. In order to exclude adverse effects of NaCl on cell metabolism, cultivation assays containing medium with final NaCl concentrations of 6.26, 6.12 and 5.93 mg/ml were included in this study. For this purpose NaCl (Merck GmbH, Darmstadt, Germany) was dissolved in bi-distilled water to a stock concentration of 5 mg/ml and sterile filtrated. Cultivation assays for both cell lines were set up as described above, and cultivated for 24 h prior to incubation in culture medium supplemented with the NaCl solution.
Since the completed DMEM culture medium also contains NaCl at a concentration of 6.4 mg/ml, dilution with 1:10, 1:5 and 1:3 NaCl stock corresponded to the final NaCl concentration of 6.26, 6.12 and 5.93 mg/ml respectively.
All cultivation assays were analysed using light microscopy (Zeiss Axio Observer Z1, Zeiss, Jena, Germany) and the images were captured digitally by a CCD colour camera (Hitachi HV-D30, Hitachi Kokusai Electric, Japan).
Determination of the effects of Pt-NP and Pt-Diss on the metabolic activity of NIH 3T3 and SH-SY5Y cells using the WST-1 assay
The metabolic activity of both cell types was determined by the conversion of the tetrazolium compound WST-1 to a formazan dye as the result of the reduction of WST-1 by mitochondrial dehydrogenases [47]. It was found that an increase in the optical density (OD) of the formazan dye directly correlates with the metabolic activity of the cells following exposure to either Pt-Diss or Pt-NP. For measurement of the formazan dye intensities, the cell culture medium was exchanged with those containing WST-1 solution at the final concentration of 1 mg/ml. Depending on cell type, the samples were incubated at 37°C for 45 min (NHI 3T3 cells) and 2 h (SH-SY5Y cells). The absorption of the formazan dye was measured at λ = 450 nm and at λ = 650 nm (the reference wave length) using the microplate reader (SynergyH1, Biotek, Bad Friedrichshall, Germany). Cells without any treatment were used as a reference, and wells containing culture medium alone served as a background control. To determine the relative metabolic activity of the culture assays supplemented with either Pt-Diss or Pt-NP, the resulting ODs were related to those obtained from the reference and calculated as a percentage [%]. In accordance with ISO 10993–5 [48], cell viability lower than 70% (in comparison with the reference) was deemed cytotoxic.
Each assay was performed at least in triplicate in n = 3 independent experiments.
Scanning electron microscopy (SEM)
The Pt electrode contacts of the human cochlear implant that underwent electrical stimulation were examined using SEM (Supra 55 VP, Carl Zeiss GmbH, Jena, Germany). Using the BSE detector, images were generated at a pressure of 6.64 x 10−6 mbar, a working distance of 11.5 mm and a tube voltage of 15 kV.
Transmission electron microscopy (TEM)
Both cell lines were seeded into a 6-well microtiter plate (Nunclon, Thermo Fisher Scientific, Kempen, Germany) at densities of 3.5 x 105 cells per well and cultivated in 3 ml supplemented high glucose DMEM for 24 h as described above. In order to investigate the effects of Pt–as either nanoparticles or ionic components–on the ultrastructure both cell lines were cultivated in 5 ml culture medium containing either 5–100 μg/ml of Pt-NP or 0.02–6 μg/ml of the Pt-Diss for another 48 h and 6 d, respectively. Additionally, untreated cells–both in the medium alone and in culture medium supplemented with the same NaCl concentration as present in the Pt-Diss solution–were included as controls. The cells were collected using a cell scraper and centrifuged at 1000 rpm for 4 min (Micro Star 17, VWR, Radnor, PA, USA) and fixed with 2.5% glutardialdehyde (Polysciences, Warrington, PA, USA) in 0.1 M sodium cacodylate (Th. Geyer, Hamburg, Germany). After post-fixation with 2% osmium tetroxide (Polysciences) in 0.1 M sodium cacodylate the cell pellets were embedded in epoxide resin (Serva, Heidelberg, Germany). Ultra-thin sections stained with 2% uranyl acetate (Serva) and lead citrate (Serva) were examined under the transmission electron microscope (Tecnai G2 200 kV TEM, Eindhoven, Netherlands). The digital images were processed using Adobe Photoshop CS6.
All data achieved from the in vitro cell culture assays were presented as mean ± standard error of mean (SEM). One-way nonparametric analysis of variance (ANOVA) and Newman-Keuls multiple comparison tests were used for statistical assessment.
Determination of the effects of additional NaCl in the cell cultivation assay
To exclude adverse effects of NaCl on cell growth and functionality cell culture assays containing medium with final NaCl concentrations of 6.26, 6.12 and 5.93 mg/ml were included in the investigation of potential cytotoxicity of Pt-Diss. In comparison to the reference with 6.4 mg/ml NaCl in the completed DMEM culture medium (OD 1.445 ± 0.01, standard cultivation assay), the metabolic activity of the NIH 3T3 cells did not significantly change in the different mixtures of 0.5% NaCl and DMEM medium (Fig 1A): The OD of the fibroblast cultivation assays with final NaCl concentrations of 6.26, 6.12 and 5.93 mg/ml varied between 1.175 ± 0.079 (NaCl 6.26 mg/ml) and 1.324 ± 0.092 (NaCl 5.93 mg/ml). Additionally, microscopic control revealed stable attachment and normal morphology of the NIH 3T3 cells independently of NaCl supplementation. As shown in Fig 1B, no significant impairment of cell metabolism could be found in cultivation assays with varying NaCl concentrations: Metabolic activity in relation to the reference ranged between 81.04% ± 3.31 (NaCl 6.26 mg/ml) and 87.5% ± 3.95 (NaCl 5.93 mg/ml).
Fig 1. Determination of the effects of additional NaCl input in cell cultivation.
Metabolic activity of both NIH 3T3 (A, B) and SH-SY5Y cells (C, D) grown in culture medium containing 6.4 (Pos.Ctrl., reference), 6.26, 6.12 and 5.93 mg/ml NaCl corresponding to 1:10, 1:5 and 1:3 dilution of the NaCl stock in culture medium was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 13–23; SH-SY5Y, n = 24–27). The resulting formazan dye intensities were also related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SEM. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, *p ≤ 0.05).
https://doi.org/10.1371/journal.pone.0196649.g001
As presented in Fig 1C, the neuroblastoma cell line SH-SY5Y demonstrated a significant decrease in metabolic activity at final NaCl concentrations of 5.93 mg/ml (OD 0.348 ± 0.010) and 6.12 mg/ml (OD 0.394 ± 0.019) in comparison with the reference (OD 0.467 ± 0.019). By contrast, 6.26 mg/ml NaCl in the medium resulted in a slight, but distinct, decline in metabolic activity (OD 0.410 ± 0.016) compared with the reference. Relative to the cell metabolism of the reference, however, cell viability in culture medium containing 6.26, 6.12 and 5.93 mg/ml was diminished to 88.50% (± 1.88), 86.17% (± 2.57) and 77.37% (± 2.76) respectively, presenting a significant difference in relative metabolic activity between 6.26 mg/ml and 5.93 mg/ml NaCl content. According to these findings, none of the cultivation assays with differing NaCl concentrations resulted in a decrease in cell viability below 70% relative to the reference indicating any cytotoxic effects. By contrast, microscopic imaging revealed stable adhesion and intact morphology of the SH-SY5Y cells in medium containing 6.26 mg/ml, 6.12 mg/ml and 5.93 mg/ml NaCl. Only the growth rate seemed to be retarded (Fig 2B–2D). We concluded that dilution of the culture medium with NaCl supplemention may limit the SH-SY5Y cell growth (as indicated by a decrease in overall metabolic activity), but did not impair cell differentiation.
Fig 2. Microscopic characterization of the morphology of SH-SY5Y cells following exposure to varying NaCl concentrations.
SH-SY5Y cells were cultivated either in normal cell culture medium containing 6.4 mg/ml NaCl as reference (A) or in culture medium containing 6.26 mg/ml (1:10) (B), 6.12 mg/ml (1:5) (C) and 5.93 mg/ml (1:3) NaCl (D). Microscopic images demonstrated reduced cell attachment and growth in a concentration dependent manner without any signs of cytotoxicity.
SEM view of eroded Pt electrode contacts of a human CI
Following electrical stimulation of the human CI the surface structure of the electrode contacts were examined by SEM. Fig 3 shows the electrode contacts before and after electrical stimulation for 600 h. While the Pt contact had a glossy surface without any irregularities prior to electrical stimulation (Fig 3A), strong corrosion was demonstrated as the result of induction of faradaic processes (Fig 3B).
Fig 3. SEM characterization of eroded Pt electrode contacts of a human CI.
Representative BSE-image of the CI-array used for electrical stimulation for 600 h at room temperature in an electrolyte consisting of 0.5% NaCl in deionized water. (A) represents an intact Pt-electrode contact, whereas corrosion of the Pt-electrode contact following electrical stimulation could be demonstrated (B).
Pt-Diss showed strong cytotoxic effects in both cell lines
After electrical stimulation of the Pt-electrode contacts the Pt-Diss was added to the NIH 3T3 and SH-SY5Y cell cultures in varying concentrations.
The microscopic view of NIH 3T3 fibroblasts showed–in comparison with the reference (cultivation assay without additional NaCl and Pt)–a strong reduction in cell growth (Fig 4A and 4B). Additionally, shrunk cell bodies probably undergoing cell death were observed at the Pt-Diss concentration of 8.2 μg/ml Pt (Fig 4B). Adhesion and growth were inversely correlated to Pt-Diss concentration (Fig 4). Beyond a concentration of 1.64 μg/ml Pt cell adhesion and morphology appeared normal (Fig 4D).
Fig 4. Microscopic characterization of the morphology of NIH 3T3 cells following exposure to Pt-Diss.
NIH 3T3 cells were cultivated either without any additional Pt-Diss as reference (A) or in culture medium containing 8.2 μg/ml (B), 4.1 μg/ml (C), 1.64 μg/ml (D) and 0.82 μg/ml (E) of the Pt components. Microscopic images demonstrated emerging cytotoxic effects of Pt-Diss in a concentration-dependent manner between 1.64 μg/ml and 8.2 μg/ml Pt-Diss concentration. Beyond Pt-Diss concentration of 1.64 μg/ml cell adhesion and morphology appeared normal, whereas a Pt-Diss concentration of 8.2 μg/ml strongly reduced NIH 3T3 cell growth and probably induced cell death. Size of bars: 200 μm.
Our observations were confirmed by TEM imaging that revealed induction of cytotoxic signals, especially in the mitochondria of fibroblasts at Pt-Diss concentrations of 6 μg/ml Pt (Fig 5B). The cells had pronounced phagocytic activity at Pt-Diss of 0.11 μg/ml Pt (Fig 5C). However, cells exposed to 0.02 μg/ml Pt could not be distinguished from those cultivated in medium alone (reference) as or in NaCl-diluted culture mixtures (Fig 5A and 5D).
Fig 5. Ultrastructural morphology of NIH 3T3 cells following exposure to Pt-Diss.
After cultivation either without Pt as reference (A) or in culture medium containing 6.0 μg/ml (B), 0.11 μg/ml (C) or 0.02 μg/ml Pt (D) the ultrastructure of 3T3 fibroblasts was compared. They demonstrated mitochondrial swelling (arrow in B) only at the highest tested Pt concentration. At 0.11 μg/ml Pt the cells showed greater phagocytic activity (arrowhead in C). Lower amount of Pt in the culture medium induced no morphological changes in comparison with the control. Size of bars: 2 μm.
The neuroblastoma cell line SH-SY5Y demonstrated (in comparison with the reference) a growth reduction over 6 d depending on the concentration of Pt-Diss in the medium (Fig 6). In a similar manner to observations of the NIH 3T3 cells after Pt-Diss exposure, few SH-SY5Y cells attached to the tissue culture plate following application of the Pt-Diss concentration of 8.2 μg/ml Pt. The somata partially exhibited spherical morphology indicating induction of apoptosis (Fig 6B). Neuroblastoma cells cultivated with 0.82 μg/ml Pt indicated normal cell adhesion and morphology–even spontaneous neurite sprouting could be observed (Fig 6A and 6E).
Fig 6. Microscopic characterization of the morphology of SH-SY5Y cells following exposure to Pt-Diss.
SH-SY5Y cells were cultivated either without any additional Pt-Diss as reference (A) or in culture medium containing 8.2 μg/ml (B), 4.1 μg/ml (C), 1.64 μg/ml (D) and 0.82 μg/ml (E) of the Pt components. Microscopic images demonstrated emerging cytotoxic effects of Pt in a concentration dependent-manner between 1.64 μg/ml and 8.2 μg/ml Pt-Diss concentration. Cell adhesion and growth appeared stable following exposure to Pt-Diss concentration of around 1.64 μg/ml. Even spontanous neurite sprouting could be observed, whereas the 8.2 μg/ml of the Pt-Diss concentration strongly induced detachment of the SH-SY5Y cells and subsequent cell death. Size of bars: 150 μm.
The ultrastructure of the neuroblastoma cells confirmed cytotoxic signals, especially in the mitochondria, but only at Pt-Diss concentrations of 6 μg/ml Pt (Fig 7B). As also described for the NIH 3T3 cells, the morphology of the SH-SY5Y cells following incubation in culture medium supplemented with 0.02 μg/ml or 0.11 μg/ml Pt was similar in to the cells of the reference and of the corresponding NaCl control. As shown in Fig 7A, 7C and 7D, cells exhibited euchromatic nuclei, abundant endoplasmic reticulum and electron-dense synaptic granules. Additionally, no phagocytic activity could be observed (Fig 7A, 7C and 7D).
Fig 7. Ultrastructure of the SH-SY5Y cell line cultivated in absence and presence of Pt-Diss.
After cultivation either without any Pt as reference (A) or in culture medium containing 6.0 μg/ml (B), 0.11 μg/ml (C) or 0.02 μg/ml Pt (D) SH-SY5Y were characterized by large euchromatic nucleus, abundant endoplasmic reticulum and a few synaptic granules (arrowheads). At the highest tested Pt concentration these cells were adversely affected, as proven by mitochondrial swelling (arrow in B). A smaller amount of Pt in the culture medium induced no morphological changes in comparison with the control. Size of bars: 2 μm.
Cell viability following 48 h and 6 d cultivation was determined using the WST-1 assay. The optical densities of the samples resulting from the indirect reduction of the tetrazolium salt to the formazan dye were related to the reference. In accordance with the microscopic characterization, we found that metabolic activity decreased in a concentration-dependent manner (Fig 8). Highly significant downregulation of metabolic activity was found in both cell types (NIH 3T3: 9.44% ± 0.93; SH-SY5Y: 7.10% ± 0.51) following administration of 8.2 μg/ml Pt-Diss (Fig 8). However, at the Pt-Diss concentration of 4.1 μg/ml metabolism of the neuroblastoma cells seemed to be more affected (25.70% ± 1.32) in comparison with the fibroblasts (57.86% ± 5.39). The administration of Pt-Diss at a concentration of 1.64 μg/ml Pt resulted in significantly improved cell viability to 76.70% ± 1.32 and 73.32% ± 2.89 in in NIH 3T3 and SH-SY5Y cells, respectively. Finally, the tenfold lower Pt-Diss concentration of 0.82 μg/ml did not impair the metabolic activity in either of the cell lines (NIH 3T3 101.60% ± 8.55; SH-SY5Y 89.77% ± 4.52).
Fig 8. Determination of the effects of Pt-Diss with varying Pt-Diss concentrations in cell cultivation.
Metabolic activity of both NIH 3T3 (A) and SH-SY5Y cells (B) grown in culture medium supplied with 0.82 μg/ml– 8.2 μg/ml Pt-Diss concentration was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–16; SH-SY5Y, n = 10). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SEM. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).
Pt-NP of up to 50 μg/ml did not cause cell damage in NIH 3T3 and SH-SY5Y cells
Since it was assumed that the electrolyte may contain not only ionic Pt, but also Pt particles following electrical stimulation, Pt-NP in concentrations from 5 μg/ml up to 100 μg/ml were added to the cell culture assays 24 h following cell seed. Fig 9 shows a representative microscopic view of the cell morphology of NIH 3T3 cells following Pt-NP exposure. Normal cell adhesion and growth could be observed up to 50 μg/ml Pt-NP. At 100 μg/ml Pt-NP the cell number appeared slightly reduced.
Fig 9. Microscopic characterization of the morphology of NIH 3T3 cells following exposure to Pt-NP.
NIH 3T3 cells were cultivated either without any additional Pt particles as reference (A) or in culture medium containing 25 μg/ml (B), 50 μg/ml (C) and 100 μg/ml (D) of the Pt-NP. The images demonstrated highly uniform cell adhesion without any morphological impairment throughout the cell cultures assays with varying Pt-NP concentrations. Size of bars: 200 μm.
After incubation with 5–50 μg/ml Pt-NP NIH 3T3 cells revealed no cytotoxic effect (Fig 10). However, these cells endocytozed the electron-dense nanoparticles in a concentration-dependent manner (Fig 10B and 10D). In particular, the Pt-NP accumulated in the multi-vesicular bodies (Fig 10D). At 100 μg/ml Pt-NP early signs of necrosis, i.e. mitochondrial swelling, were recognized in the cells.
Fig 10. Ultrastructure of NIH 3T3 cells following exposure to Pt-NP.
NIH 3T3 cells were cultivated either without any additional Pt particles as reference (A, insert in C) or in culture medium containing 50 μg/ml (B, insert in D) of the Pt-NP. No cytotoxic effect of Pt-NP could be found. Fibroblasts were highly active in endocytosis (rectangles in A and B). In the multi-vesicular bodies (arrowheads in C and D) accumulation of adsorbed material such as Pt-NP (arrow in D) was detected. Size of bars: 1 μm.
The viability of the SH-SY5Y cells was not adversely affected following exposure to Pt-NP at concentrations up to 100 μg/ml (Fig 11). As observed by TEM the neuroblastoma cells showed normal cellular ultrastructure up to 50 μg/ml Pt-NP in the culture medium (Fig 12B). Interestingly, the SH-SY5Y cells exhibited virtually no endocytosis (Fig 12A and 12B). Therefore, no multi-vesicular bodies could be seen in the control cells (Fig 12A) or in cells exposed to Pt-NP at all concentrations used in this study. As shown in Fig 12B, the biosynthesis of synaptic granules remained unchanged in cell cultivation assays supplemented with 50 μg/ml Pt-NP (Fig 12B). However, Pt-NP at a concentration of 100 μg/ml induced mitochondrial disintegration as well as swelling of the endoplasmic reticulum indicating cytotoxic effects of Pt.
Fig 11. Microscopic characterization of the morphology of SH-SY5Y cells following exposure to Pt-NP.
SH-SY5Y cells were cultivated either without any additional Pt particles as reference (A) or in culture medium containing 25 μg/ml (B), 50 μg/ml (C) and 100 μg/ml (D) of the Pt-NP. Morphology and adhesion behavior of the SH-SY5Y cells did not change throughout the cultivation assays at varying Pt-NP concentrations. Size of bars: 200 μm.
Fig 12. Ultrastructural morphology of SH-SY5Y cells following exposure to Pt-NP.
SH-SY5Y cells were cultivated either without any additional Pt particles as reference (A) or in culture medium containing 50 μg/ml (B) of the Pt-NP. The biosynthesis of synaptic granules as seen in the control cells around the Golgi apparatus (arrowhead in A) is typical for those cells; no multi-vesicular bodies of the endocytosis were detected. Neuroblastoma cells in contact with Pt-NP were active only in the secretory pathway. No Pt-NP were evident inside the cell. Size of bars: 200 μm.
As shown in Fig 13, the metabolic assay with WST-1 revealed no significant influence of the Pt-NP concentrations 5 μg/ml, 10 μg/ml, 25 μg/ml and 50 μg/ml in NIH 3T3 fibroblasts (96.44% ± 4.69, 92.94% ± 4.26, 85.40% ± 3.89 and 89.54% ± 4.66) as well as in neuroblastoma cells (107.3% ± 6.12, 92.61% ± 5.19, 93.16% ± 5.80 and 86.77% ± 5.07), even though a slight downward trend in metabolic activity was observed. By contrast, the administration of 100 μg/ml Pt-NP to the cultivation assays resulted in a significant decrease in cellular activity of both NIH 3T3 (65.42% ± 5.91) and SH-SY5Y cells (68.35% ± 5.07). In accordance with ISO 10993–5 [48], this Pt-NP concentration was deemed cytotoxic (Fig 13).
Fig 13. Determination of the effects of Pt-NP with varying concentrations in cell cultivation.
Metabolic activity of both NIH 3T3 (A) and SH-SY5Y cells (B) grown in culture medium supplied with 5 μg/ml– 100 μg/ml Pt-NP was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–14; SH-SY5Y, n = 11–15). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SEM. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).
For the first time, this study presents data on potential neuronal cell damage by Pt derived from electrical stimulation of a human CI electrode in a concentration-dependent manner.
At first, to determine whether the lower NaCl content had any adverse influence of on NIH 3T3 and SH-SY5Y cell metabolism, both cell lines were exposed to 5.93–6.26 mg/ml NaCl. WST assay data and microscopical control of the NIH 3T3 cells allowed to rule out negative biological effects due to the reduced NaCl concentration and medium osmolarity. Despite a greater sensitivity of the SH-SY5Y cells to the lower NaCl content in the cell culture medium, cytotoxic effects could be also excluded as determined by the WST-1 assays.
The Pt-Diss solution used in this study may contain a mixture of both Pt particles of various sizes and shapes, and Pt ions or complexes as also reported in numerous studies [8, 15, 32–34]. Since ICP-MS only enables analysis of the Pt-mass content in total, it is not possible to estimate the contribution of individual components to the cytotoxicity. Nevertheless, our findings demonstrated strong reduction of mitochondrial activity and, thus, induction of oxidative stress following supplementation of the culture medium with Pt-Diss in a concentration-dependent manner. The small contribution of the lower NaCl content in the SH-SY5Y cell culture has to be considered as described above. Stress induction resulting in cell death by incubation of Pt(I-IV) compounds has been described in numerous studies. Only the lethal dosage may differ depending on tissue type, its resistance in cellular uptake of the Pt ions and effective nuclear repair mechanisms of the cells [25–29]. Exposure of 0.5 mM PtCl4 to A549 and HaCaT cells and 10 μM cisplatin to HT29 cells induced severe DNA damage and subsequent cell death as the consequence of drastic increase of intracellular reactive oxygen species and lipid peroxidation levels [36, 44]. As previously reviewed, Pt complexes form stable covalent bonds with nuclear DNA., Guanine, in particular, is a susceptible target at the 7-nitrogen atom to be able to form inter- and intrastrand crosslinks [30–31].
The present study demonstrated mitochondrial swelling in both NIH 3T3 and SH-SY5Y cells, which corresponds to the reduction in metabolic activity in the presence of ≥1.64 μg/ml Pt-Diss. Indeed, previous studies found that Pt2+ ions accumulate in mitochondria. It follows that cytotoxicity and genotoxicity may be mediated by Pt2+ ions [37, 49]. This assumption was supported by Pelka et al. [40] who observed DNA strand breaks in human colon carcinoma (HT29) cells. Although no significant reactive oxygen formation occurred, the authors concluded that Pt ions could be the cause.
To consider the potential biological effects of solid Pt particles contained in the electrolyte, the biocompatibility of Pt-NP 3 nm in size on both cell lines was determined. Additionally, the cellular uptake of Pt-NP and their effects on the ultrastructure were investigated by means of TEM. Furthermore, the particle size chosen allowed their localization inside the cells to be detected by TEM. We found no significant influence of increasing Pt-NP concentrations between 5 μg/ml and 50 μg/ml on either ultrastructure or the metabolism of NIH 3T3 and SH-SY5Y cells. Only at 100 μg/ml Pt-NP concentration cell metabolism and mitochondrial activity were reduced. Our results were in line with previous in vitro studies exposing murine L929 fibroblasts, several human cell lines such as RAW264, A549, HaCaT, IMR90 and U251 to Pt-NP with a primary size less than 10 nm: no or only moderate cell death induction were observed [35–38]. Significant toxicity was only found in IMR90, U251 L929 and RAW264 cells following exposure to 80–100 μg/ml Pt-NP [35, 38–39]. In vivo administration of 3–10 nm Pt-NP to zebrafish embryos showed dosage dependent mortality rate, hatching delay, phenotypic defects and metal accumulation at concentrations above 50 μg/ml [50]. However, another in vivo study reported inhibitory effects on cardiac rate of zebrafish embryos induced by Pt-NP at lower concentrations around 25 μg/ml [42].
Even though particles below 10 nm in size may enable nuclear penetration [36–37, 44], TEM imaging revealed cellular uptake of most of the Pt-NP into the lysosomes, but not into the nuclei and mitochondria of the examined cell lines. It is suggested that either endocytosis or phagocytosis play key roles in cellular uptake of Pt-NP into mammalian cells [36–38, 51–52]. Another study demonstrated both diffusion and endocytosis of Pt-NP into the cytosol of IMR90 and U251 cells depending on the size of the nanoparticles. Electron micrographs revealed agglomerates of Pt-NP in the endosomal-lysosomal compartment, whereas single nanoparticles with sizes limited to less than 10 nm were able to diffuse into the cytosol [35]. Our TEM imaging also demonstrated dose-dependent internalisation of Pt-NP into the multi-vesicular bodies of the NIH 3T3 cells, but no diffusion into the cytosol or nucleus. Interestingly, no Pt-NP were internalized into the SH-SY5Y cells. The Pt-NP used in this study were administered to the cell culture assays as colloidal solution stabilized with polyvinylpyrrolidone (PVP) for effective dispersion. This is well known not only as a nontoxic and bioinert compound [53], but also as a means of protecting metallic nanoparticles from growing and agglomerating [54–55]. Therefore, as expected, Pt-NP dispersion did not form large aggregates accumulating on the surface of both NIH 3T3 and SH-SY5Y cells, as revealed by TEM analysis. However, protein adsorption, especially of medium components such as serum proteins and salts, may significantly enlarge the size of the Pt-NP forming 'secondary' nanoparticles, and influence cellular uptake [36]. Admittedly, it has been suggested that those adsorbed proteins are digested in phagolysosomes leaving the nanoparticles 'naked' [51]. Nevertheless, the size of the secondary Pt-NP following dispersion in FBS-medium affected neither endocytosis-mediated uptake nor biological activities as compared with the tenfold smaller primary particles [36]. Hence, we assume that dispersion of the commercial Pt-NP colloids in supplemented cell culture medium did not falsify our findings.
Cytotoxicity of Pt was not only found after incubation with ≥1.64 μg/ml Pt-Diss, but also at 100 μg/ml Pt-NP. However, no internalization of the Pt-NP was found in the SH-SY5Y cells prompting the question of whether Pt2+ ions could trigger the decrease in mitochondrial activity in SH-SY5Y cells as well. The surface of not only Pt electrodes but also of Pt-NP could corrode, releasing the cytotoxic Pt2+ ions into the medium [37, 40, 49].
Based on our results, we assumed that the contribution of the metallic Pt particles in the electrolyte to cell damage may be negligible. Instead, the content of Pt ions and complexes seems to be the main factor inducing oxidative stress and subsequent cell death. By our data disturbations of the cell metabolism in both NIH 3T3 and SH-SY5Y cells appeared in vitro at Pt-Diss concentrations between 1.64 μg/ml and 4.1 μg/ml. Previous in vivo studies demonstrated low levels of dissolved Pt ranging from 5 ng/ml to 1 μg/ml depending on the electrical-stimulation parameters [17, 24]. In comparison with our data, those findings indicate that corrosion of the electrode contacts may not result in tissue damage. Additionally, diffusion of the corrosion products within the inner ear via the lymphatic fluids may dilute the concentration of toxic Pt compounds, so that the potential of Pt ions or particles to harm living cells would be negligible. Furthermore, new formation of metallic Pt particles may hypothetically function as scavenger of reactive oxygen species to a certain extent [56–59], protecting the tissue from toxic effects of ionic Pt. However–as shown in the present study and reported by several authors, and depending on the cell type and organism–Pt-NP induced oxidative stress, inflammation or other negative biological effects in a concentration-depending manner [39–43]. It is assumed that endocytosis-mediated internalization of the Pt particles results in etching of the surface of the metal particles and their oxidation to ionic Pt due to exposure to low pH and hydrolytic enzymes in the endosomes. Subsequent reduction of the Pt2+ ions by H2O2 generated in the mitochondria may allow the new formed Pt particles to start a new cycle [40, 50]. Consequently, Pt particles may indirectly contribute to cytotoxicity and genotoxicity [37, 49]. In particular, long-term electrical stimulation over several years may cause Pt particles to accumulate in the cell, thus enhancing their cytotoxicity. Furthermore, the complex interactions between the corrosion products and the tissues of the auditory nerve at the Pt/lymphatic fluid interface require more attention. It also has to be considered that, in particular, children need to benefit from the CI throughout their lifetime, so that limitation of Pt corrosion should be of great clinical interest.
Recently, Piret et al., 2015 [60] reported that 3D-nanostructured boron doped diamond (BDD) may be the electrode material of choice for long-term neural implants. The electrode contacts consisted of vertically aligned carbon nanotubes inter-layer template encapsulated within two BDD nanolayers [60, 61]. It was shown that this novel electrode material not only enabled neural cell attachment, survival and neurite outgrowth, but also demonstrated low impedances and high safe charge injection capacity [60].
This study is an in vitro evaluation of the murine fibroblast NIH 3T3 and human neuroblastoma SH-SY5Y cell culture model exposed to commercially manufactured Pt-NP and corrosion products following electrical stimulation of a human CI electrode (Pt-Diss). We found negligible oxidative stress in both NIH 3T3 and SH-SY5Y cells up to 50 μg/ml Pt-NP, which was in line with several other studies. Interestingly, no diffusion of the very small Pt-NP into the cells was found. In contrast to Pt-NP, corrosion products (Pt-Diss) with platinum mass fractions between 1.64 μg/ml and 8.2 μg/ml induced cell death in both murine and human cell lines. Taken together, our data indicate that corrosion of Pt electrode contacts may not lead to toxic concentrations in the surrounding inner ear tissues, since low levels of dissolved Pt ranging from 5 ng/ml to 1 μg/ml have been previously demonstrated. However, long-term cytotoxicity over a period of several years during Pt electrode corrosion requires investigation, as do the interactions of the corrosion products with the tissues of the auditory nerve at the Pt/lymphatic fluid interface.
We are grateful to Anja Krabbenhöft and Elke Mallon for technical assistance with SEM and TEM preparation, respectively.
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text:
Q: Constant in enum not seen in define I have such a code in C
enum {
     MYVAR = 1
};

#ifdef MYVAR
#define VAR 1
#else
#define VAR 2
#endif

printf("VAR = %d", VAR);

in this case it will prints "VAR = 2".
Is there any way to get preprocessor see the definition in enum?

A: No, this is not possible. #ifdef and #if are part of preprocessor, which completes its run before the portion of the compiler that "understands" enums.
#ifdef works only with preprocessor constants (i.e. things defined with #define, or passed to the compiler on the command line, say, with a -DMYVAR=123 option).
#if works with integer and character constants, and preprocessor constants. All identifiers which are not preprocessor constants are considered undefined, and interpreted as if they were zeros when evaluating #if conditions. This includes enum constants.

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Understanding and Evaluating Research and Scholarly Publishing in the Social Sciences and Humanities (SSH)
Gunnar Sivertsen
expected and must be respected in more typical cultural and societal sectors and domains, such as democratic development, policy design, public administration, international affairs, integration and understanding of different languages and cultures, education at all levels, cultural life, media and information, and in investigating and informing about history, the "memory of society". In these sectors and domains, one will find that societal impact usually is in place as organized interaction and responsibilities between the SSH and other institutions of civilization
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What Motives Users to Participate in Danmu on Live Streaming Platforms? The Impact of Technical Environment and Effectance
Kun Wang, Zhao Pan, Yaobin Lu and Sumeet Gupta
International 55 3 273 286 10.1080/09523987.2018.1512447 Liu, L., Suh, A., & Wagner, C. (2016). Watching online videos interactively: The impact of media capabilities in Chinese Danmaku video sites. Chinese Journal of Communication , 9(3), 283–303. 10.1080/17544750.2016.1202853 Liu L. Suh A. Wagner C. 2016 Watching online videos interactively: The impact of media capabilities in Chinese Danmaku video sites Chinese Journal of Communication 9 3 283 303 10.1080/17544750.2016.1202853 Liu, L., Suh, A., & Wagner, C. (2017). Who is with You? Integrating a Play Experience into
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Farhan Khan, Xiangyun Si and Kashif Ullah Khan
• Convenient to use • Communicate with top and lower staff quickly • Quick connection and prompt • Communication and association • Reduce the frequency of face-to-face reports • Efficiency and quick response • Interact with my colleague and share express information ideas • Meeting and social groups those are created on social media • A useful way of sharing information • Better connection • Media groups for coordination process • Convenient to integrate • A platform for communicating quickly
When Econometrics Meets Machine Learning
Eric Zheng, Yong Tan, Paulo Goes, Ramnath Chellappa, D.J. Wu, Michael Shaw, Olivia Sheng and Alok Gupta
invite Yong Tan to help answer the question first. 2.2 Yong Tan 2.2.1 Idea 1: Integration of machine learning and econometrics will be of great value Actually, I have been talking about this topic many times from the methodological point of view. However, this time I would like to start with a disclaimer that methodology itself is just a tool. We want to advance the tool in order to solve more interesting business problems. The objective of using econometrics is to find out the causal relationship of data, which is our main purpose. On the other hand, from
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g st -store: Querying Large Spatiotemporal RDF Graphs
Dong Wang, Lei Zou and Dongyan Zhao
day )} In this paper, we extend the semantics of the SPARQL language by integrating the spatial and temporal feature assertions (the extended SPARQL query is called the ST query, short for spatial-temporal SPARQL query). The spatial and the temporal constraints assert the location of an entity/event and the event's valid time, for instance, distance ( place (? y ), place (49, 10)) < 300 and time (? t 1) < date (1900.01.01) in Example 1 . In order to answer S-T queries in a uniform manner, we propose a tree-style index structure (called the ST
Identifier Services: Modeling and Implementing Distributed Data Management in Cyberinfrastructure
Maria Esteva, Ramona L. Walls, Andrew B. Magill, Weijia Xu, Ruizhu Huang, James Carson and Jawon Song
different aspects of a project (e.g., specimen, analysis, sequences). The COPO interface makes helpful suggestions regarding what information you might want to submit and normalizes metadata, which can be uploaded in bulk, to controlled vocabularies, ontologies, and community standards assisting with data integration. In addition, publications can be submitted through COPO to long-term storage repositories. IDS could be a complement to this project by tracking the evolution of the different components of a dataset. At the end of a research project's lifecycle, data
Improving Publication Pipeline with Automated Biological Entity Detection and Validation Service
Weijia Xu, Amit Gupta, Pankaj Jaiswal, Crispin Taylor, Patti Lockhart and Jennifer Regala
through an interactive web application for curation and evaluation by authors and other domain experts. Through the web interface, a user can make additional annotations and corrections to the current results. The updates are stored and managed via the relational database for future improvements to the entity detection process. The service includes several components: automated informational vocabulary extraction based on existing domain ontologies, experts' validation and curation, and integration of results using the formal publication process. The contribution of the
The Contingency Effect of Relational Competency on the Relationship between Information Technology Competency and Firm Performance
Shaobo Wei and Kwok-Kee Wei
). Firms generally leverage two distinct strategic mechanisms: resource-picking and capability-building ( Makadok 2001 ). Specifically, resource-picking mechanism creates economic rents when the firm applies superior information and knowledge to gain the value from the resources, while capability-building mechanism highlights the firm's ability to integrate, build, and reconfigure internal and external resources and competencies to achieve competitive advantage in a rapidly changing business environment ( Eisenhardt et al. 2000 ; Teece et al. 1997 ). It is also
Smart and Connected Health: What Can We Learn from Funded Projects?
Minghong Chen, Jingye Qu, Yuan Xu and Jiangping Chen
1 Introduction Increasing health awareness and widespread adoption of wearable devices, wireless communications and big data in the context of smart cities have led to the needs for smart and connected health (SCH). SCH refers to any digital healthcare solutions or systems that can operate remotely with integration of innovative computational and engineering approaches to support the transformation of health and medicine services ( Clancy, 2006 ; National Science Foundation). It is a young but promising field of study at the intersection of medical
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text:
Legal update on applying the legislation regulating employment of company CEOs
Legal update No 535
Goltsblat BLP advises that the Plenum of the Russian Supreme Court has adopted Resolution No. 21 dated 2 June 2015 clarifying in detail the most disputable aspects of applying the legislation regulating employment of company CEOs and summarising court judgements on application of the relevant Chapter of the Russian Labour Code (the "Resolution").
The concept of CEO has been clarified
Pursuant to the Russian Labour Code, a company CEO means "a person who manages a company, including by performing the functions of its sole executive body". There used to be quite a number of disputes as to whether the special rules regulating CEO employment can be applicable to employees who actually manage certain areas of company activities, such as a head of the branch, representative office or separate structural subdivisions and so on. For instance, may an employer dismiss the Head of Branch under the rules applicable to CEO dismissal, i.e., due to a decision of the authorised corporate body with payment of at least three average earnings?
In its Resolution, the Supreme Court clearly explains that the above-mentioned special provisions applicable to CEOs do not apply in regulating relations with employees managing certain areas of company activities or structural subdivisions, including branches, representative offices and other separate structural subdivisions, since they are not charged with the duties of the company's sole executive body. Consequently, an editor-in-chief or a branch director may not be dismissed under the rules applicable to CEOs.
Specifics of dismissing a CEO by a decision of competent corporate body and compensation payment
Summing up recent court practice, the Plenum of the Supreme Court advises that a competent company corporate body may cancel an employment contract with a CEO by taking a relevant decision without specifying any reasons.
At the same time, the Supreme Court points out that cancellation of an employment contract on the above grounds does not constitute a legal liability measure, so it is possible only if compensation of at least three average earnings is paid. Yet this position of the Supreme Court does not imply that compensation has to be paid under any circumstances.
In particular, the position that cancellation of a CEO's employment by decision of a competent corporate body is possible only if compensation is paid originally appeared in Rulings of the Constitutional Court. Yet both the Constitutional and the Supreme Courts consider dismissal of a CEO by decision of a competent corporate body solely in the context of the employer's right to dismiss a CEO "without specifying any reason". In this case, dismissing a CEO without either explaining the reasons or paying compensation evidently testifies to abuse by the employer of its rights.
At the same time, both the Constitutional Court and the Supreme Court consider it possible to dismiss a CEO by decision of a competent corporate body without paying any compensation if the CEO has culpably failed to perform his/her duties. Before the termination, however, the employer should follow the formal disciplinary process and must carefully prepare the dismissal documents and proof to support its positions in case the matter goes to court.
It should be specifically noted, however, that the Supreme Court expressly states that failure to pay compensation does not suffice as grounds for reinstatement of the CEO in the previous position anyway.
Severance amount may, in certain cases, be established by a court
In its Resolution, the Supreme Court significantly extends the powers of courts in relation to severance pay, compensation and other payments due to a CEO as a result of employment contract termination.
In particular, employment contracts with CEOs may sometimes establish significant compensation to be paid to a CEO in the event of dismissal. Sometimes, such compensation is due to the CEO in the event of dismissal on any termination ground whatsoever (even voluntary resignation or dismissal owing to culpable actions). Previously, if the employer did not pay out the agreed severance, courts often recovered the full amount agreed between the parties.
Under the new rules, the court is empowered to reject claims seeking payments from the employer due to employment contract termination or significantly reduce them, should it be established that the relevant employment contract terms are in breach of:
legislative requirements;
the general legal principle of inadmissibility of abuse of rights;
legitimate interests of a company;
legitimate interests of other employees;
legitimate interests of other persons (for example, a company's shareholder(s)).
The court is, however, obliged to specify the reason and justify the rejection of a claim or reduction of the amount.
In certain cases, the court may, on the basis of its own view and decision, determine the severance amount payable to the CEO. In particular, the Russian Labour Code states that, if an employment contract is terminated in the absence of culpable actions (omissions) by a CEO, the latter is paid the compensation specified in the employment contract but no less than three average monthly earnings.
Previously, therefore, if no amount of severance compensation was agreed in the employment contract, the court would confine itself to recovering the minimum statutory amount (three average monthly earnings) in favour of the CEO.
Now the Plenum of the Supreme Court states that if, for some reason, an employment contract is silent about the specific amount of severance or there is a dispute over the agreed amount, the court is allowed to decide the sum itself, without considering the statutory minimum. This can be done on the basis of the following criteria: the period for which the dismissed person was employed as CEO, the time left to expiry of the employment contract, the amounts the dismissed person might have received if they continued to hold their position, etc. (CEOs of state organisations and companies with more than 50% state or municipal participation are the only exceptions to this rule).
Nadezhda Ilyushina
Head of Employment Practice (Russia), Advocate
Yuri Ivanov
Counsel, Advocate


text:
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This will be the OOC thread linked to the IC thread here. Try to keep any out-of-game chatter here - this is also where we will be discussing point costs, mechanics, and playtest notes.
This game will be a PbP playtest of my own Mutants and Masterminds port of Sand and Steam's Iron Edda Fate-based War of Metal and Bone by Tracy Barnett. (details here) Despite not being reached as a stretch goal in the Iron Edda Kickstarter, Tracy has expressed interest in pushing for some of the stretch goals in the future anyway - so I'm continuing development and playtesting.
Recruitment for the game is tentatively closed. I have four players (who at the time of this posting may or may not already have ATT logins) who I know and trust to help me focus on playtesting. If you have an excellent case for being a player, I'll review it. Something must be understood by both current and prospective players: though I am going to start with the 3e RAW from the Deluxe Player's Guide (which I assume has errata published between the original and deluxe), emergent trends from playing the game might cause me to introduce house rules suddenly and often.
(some of this will be review, since we've gone over this in emails - I just want it in the forums for completeness) Our first order of business, before the players even generate characters, is something called Prologue Generation. This is a direct (Mutants and Masterminds flavored) descendant of Tracy's Holdfast Creation, which is itself credited to Travis Scott. Each player rolls 2d20 (feel free to use an online roller or real dice - I trust you) and finds the corresponding entry on this table. Post your result here, consult with the other players, then post a short fiction in the IC post describing the impact of your roll on the game world.
The Holdfast itself, as a shared character resource, will be a Headquarters, built with 10EP. To expand the Holdfast beyond this, characters can spend personal PPs. The name, location, and nature of the Holdfast (mobile/stationary, populated/just the group, high-tech/low-tech) may be defined during Prologue Generation, or after.
Player Characters will be PL8, 120PP builds. Every PC either has a rune marking them as one of the Nine Clans, or they have become Bonebonded. Bonebonded is a 10PP power that acts like the Metamorph power - the big departure from base rules here is that the alternate form is a PL12 build. In other words, a Bonebonded player will create two 110PP builds at different PLs.
Last edited by angille on Sun Mar 16, 2014 5:56 pm, edited 2 times in total.
Hey, lurkers! So if you're wondering where the posts are, it's because my players are still using email instead of the forum to communicate and plan. They'll be here soon enough.
But guess what!One of my players has backed down, so I've got a vacancy. You fine forum folks know what to do. I will take two new players - PM me first.
Sorry for lagging. Things have been crazy lately. I'm planning on playing a Bonebonded but haven't had a chance to actually sit down and dig through M&M's chargen, which I haven't looked at in a few years. This WILL be fine tomorrow though, even if I have to lock myself in a closet with a laptop.
Raj was is an engineer in Delhi. When the dwarves took over the city, Raj helped them integrate human and Dwarven tech as he was fascinated with it. Life was good for him until he found out the dwarves were making humans into cyborgs and using them as the foot soldiers for the next invasion.
Knowing dwarven tech (worked with them for a while), Raj stole a small destroyer and led a slave escape. Of course of the around 500 people he lead, only about 20 people survived. The destroyer was nearly destroyed; my character's been working on repairing the destroyer ever since. The survivors of the escape know who he was (a Dwarven collaborator) and blame him for the massacre.
Raj has be laying low for a while now. He claims that he figured out Dwarven technology from the broken destroyer he "found". He doesn't talk about his past.
My idea for the roll I got was I'm giving the alliance my knowledge of the dwarven city for the invasion (of course this is from my time as a "slave"). While the pubic horribly wrong is my information is a few years out of date, the real problem is one of the survivors IDing me.
Things are crazy? You don't say! Trying to schedule for a F2F game is why we're all here, right?
I've put some pregens on Drive, including my conception of a Bonebonded. kanly also has a build, which I'm sure will be shared once some of the Prologue rolls have hit this thread.
Just wanted to let everyone know that my Prologue Generation rolls were 12 and 3, so it looks like plague is coming to town. What do the rest of you know about life in our holdfast?
I got a 17 and a 13 for my prologue rolls, so it looks like dwarves and humans are managing to peacefully co-exist on some sort of tech-magic Second Life. I.... don't know exactly what to do with that, but it sounds interesting.
Maybe that has something to do with the Second Life thing? They've taken over ours, but we've got someone who knows a bit about Dwarf tech, so maybe with his insight we're getting back in to things anyway and some people from our holdfast and some on the other side are sick of the war thing and wanting to do some escapism. Might be dangerous though if people are caught. On the other hand, might end up causing a little bit of harmony, or help from someone's buddy who doesn't want to see them get killed. But for which side? Hmm.
Kurt and Ian are in first post moderation limbo, so I'll throw their rolls down here too, and aggregate the Prologue Generation so far.
Humans and Dwarves have been living second, peaceful, lives on virtual pockets in Yggdrasil and Jormungandr. How does the leadership of each faction deal with this? What about the general populace?
Rumors of plague are spreading across the holdfast. Everyone who so much as coughs is looked at sideways. How is the Jarl handling the situation? Why?
Your Holdfast has banded together with several others, and with multiple Bonebonded warriors, are planning an attack on one of the Nine Cities. What's going to make this successful? How could it go horribly wrong?
Dwarven technology has taken over Earth's satellite belt. What does this mean for Yggdrasil? How are they using it in the War? What stake does your Holdfast have in this?
Oh, btw, I'm leaving the "Recruiting" in the title - I'll take a fifth player (along with the Prologue roll) right up until we start playing.
Well, my thought is that there are two sets of things going on here. First, the warriors brought the plague or at least rumors of it. Something that the jotunn that were used for bone bonding carried but were immune to? Second, the peaceful interactions have been going on, on the new tech out in the satellite belt. Attempting to establish a third independent system? Thoughts, ideas, alternates, like/don't like.
Might also be good to discuss the nine cities, and which one we are going after. The counterpoint, what our holdfast is like, is also important.
Seeing if maybe my entire character was just too long to post (it hasn't posted yet and I don't know why).
That... was... weird. Had some back-dated posts there! Well, looks like everyone's here now. I'm gonna stand back a bit and let you folks craft the situation.
Once you've got the general stuff down, go ahead and start posting Prologue replies against the IC thread. It doesn't have to be necessarily "in character" for your PC. The vision I have for it is to get a feel similar to prologues in comic books? Kinda like the first chapter in each of the Song of Ice and Fire books - sets something up for the main characters, but doesn't have to involve them directly.
I don't know if I'm explaining myself perfectly. Lemme know if you have questions. Otherwise, brainstorm!
I got posted What do all you all think of the back story? Do you like the holdfast?


We have recently moved out fish into a bigger tank (you may have seen on my Instagram or Twitter). I really like their new home, I think they do too!
We were contacted by Swell UK to see if we would be interested in trying out one of the fish tanks they sell and of course because we wanted to upgrade our current tank we said yes, why not!
We opted for a Fluval Edge Aquarium (priced at £99.99 from Swell UK), mainly because of how it looks and also because its holds a decent amount of water (23 liters) without taking up too much space.
I absolutely love how flawless this tank looks. The way you can fill the water right up to the top, how all 6 sides of the tank are glass and how it sits so smartly, looking as if it's floating.
Another great thing about the tank is that it has two light modes with, one of which is a beautiful moonlight glow, night mode where everything gets covered in a lovely light blue glow. (It's very calming and relaxing to sit and watch at night).
If your children keep asking for a pet I would definitely recommend starting with fish. Our children just love watching our and helping clean and look after them.
This tank is lovely! My fish could do with a new home too!


text:
Winter Weather Update
Friday 1 February 2019 – 9am - Update on affected schools and council services
London is currently experiencing adverse weather conditions. Barnet Council is working to ensure that the borough is well prepared for the the weather
Last updated: 1 February, 2019
Victoria Lodge update
Planning consent for six flats to be built on the site of the Victoria Lodge was refused by Barnet Council's Planning Committee in September 2018. The Planning Inspectorate subsequently granted permission following an appeal by the applicant.
Last updated: 26 November, 2018
Urgency Committee report
Barnet Council has today (Thursday, 22 November) published a report, which proposes that councillors agree a £4.12m payment from Capita to settle historical commercial issues, including delivery of IT improvements in Adult Social Care (MOSAIC) and delays in delivering housing on council land.
International Fraud Awareness Week
Council counter-fraud officers will once again be taking part in a week of action during International Fraud Awareness Week (12-18 November).
Servicemen and women to be remembered
This weekend Barnet will pause to pay tribute to those who have lost their lives in conflict exactly 100 years after the guns fell silent in the First World War.
Last updated: 6 November, 2018
Views sought on Council Tax Support scheme
Barnet Council has launched a public consultation in relation to its Council Tax Support scheme. The consultation began on Thursday, 18 October and will run until Thursday, 29 November.
RAF Museum awarded Freedom of the Borough
Barnet Council has awarded the Freedom of the Borough to the RAF Museum to celebrate its diligent preservation of the proud history of our nation's 100-year-old Royal Air Force.
Last updated: 26 October, 2018
Direct payments fraudster prosecuted
A woman who continued to receive and spend her father's social care payments despite the fact he had passed away has been brought before the courts and prosecuted.
Last updated: 3 October, 2018
Jail for builder who ignored enforcement action
A builder who blighted his street by storing debris and construction materials outside his home, has been jailed for failing to clear the land.
Resident fined more than £50,000 for illegal extensions
A Magistrate has found a Barnet resident guilty of breaching three separate planning enforcement notices.
(-) News Story
(-) Planning, conservation and building control
(-) Your council
(-) Council tax and benefits


The City proposes to construct 4.8 miles of Class 1 multi-use trial along the abandoned railway corridor west of Freeport Blvd. from south of Meadowview Rd/Pocket Rd. to the Sacramento River Parkway north of Sutterville Rd. The proposed Del Rio Trail Project consists of a Class I multi-use trail (12 to 16 feet of pavement with unpaved shoulders ranging from 2 to 3 feet) and when feasible, an adjacent 5 to 6 foot wide unpaved walking trail. The Del Rio Trail would include at-grade crossings and intersection modifications at each location where the trail intersects a vehicular roadway.


text:
Diabetes Mellitus and Pregnancy
Daniel H Mintz,
Jay S Skyler and
Ronald A Chez
Department of Medicine, Division of Endocrinology and Metabolism, and Diabetes Research Institute, University of Miami School of Medicine Miami, Florida Department of Obstetrics and Gynecology, Howard University School of Medicine Washington, D. C
Reprint requests: Daniel H. Mintz, Department of Medicine, Division of Endocrinology and Metabolism, Miami, Florida 33152.
Diabetes Care 1978 Jan; 1(1): 49-63. https://doi.org/10.2337/diacare.1.1.49
Proper therapy in pregnancy complicated by diabetes mellitus affords the physician an opportunity that is unparalleled in immediate impact and over-all therapeutic dimension for his or her patient. The immediate survival of the mother can be virtually assured and that of the unborn child brought close to that of the child of the nondiabetic gravida. Further, the benefit of this care extends considerably beyond these traditional clinical targets. Such relatively remote events as intellectual and psychologic performance in offspring from these pregnancies may be jeopardized by inadequate attention to details of medical management during pregnancy. Lastly, an opportunity is presented to both physician and patient to beneficially influence future diabetes therapy. The intense motivation of the mother, together with her positive experiences, can be channeled to promote an enduring attitudinal and behavioral approach to future
Copyright © 1978 by the American Diabetes Association
January 1978, 1(1)
You are going to email the following Diabetes Mellitus and Pregnancy
Daniel H Mintz, Jay S Skyler, Ronald A Chez
Diabetes Care Jan 1978, 1 (1) 49-63; DOI: 10.2337/diacare.1.1.49
Gene-Environment and Gene-Treatment Interactions in Type 2 Diabetes
Relationships Between Gastric Emptying, Postprandial Glycemia, and Incretin Hormones
Retinal Vascular Caliber as a Biomarker for Diabetes Microvascular Complications
Show more Review Articles


text:
The Mobile Workforce
In an Era of Change
Countries Impose Travel Restrictions to Slow Spread of Omicron Variant
By María E. Ferré & John Corgan on November 29, 2021
Posted in Canada, Chile, COVID-19
In response to the identification of the omicron variant of the SARS-CoV-2 virus, several countries around the world have implemented new travel restrictions aimed at slowing the spread of the disease. In the Americas region alone, several countries—including Brazil, Canada, Chile, and the United States—have announced new restrictions on the entry of foreign nationals. Other countries, such as Mexico, have insisted that such measures are of little use.
A Range of Travel Restrictions in Response to Omicron Variant
International travel restrictions adopted in response to the identification of the omicron variant widely vary from one country to the next. In the Americas region, some of the notable restrictions include:
Brazil: On November 26, 2021, President Jair Bolsonaro's chief of staff announced that Brazil would close its borders to entry from six African countries, including South Africa, Botswana, Eswatini, Lesotho, Namibia, and Zimbabwe. Entry restrictions are set to begin on November 29, 2021. According to the new restrictions, no flights from these six countries may land in Brazil. Foreign nationals physically present in or passing through these countries within the past 14 days will also be barred from entering Brazil. In addition, Brazilian citizens and permanent residents who are physically present in or passing through these six countries in the fourteen days before embarking for Brazil must quarantine for fourteen days in the city of their final destination.
Canada: On November 26, 2021, the Canadian government announced a new set of measures designed to address the emergence of the omicron variant. Under the new measures, foreign nationals who have been in the Southern Africa region – including South Africa, Eswatini, Lesotho, Botswana, Zimbabwe, Mozambique, and Namibia – within the last 14 days before arriving in Canada will not be allowed to enter Canada. Canadian citizens, permanent residents, and people with status under the Indian Act, who have been in these countries in the previous 14 days will be subject to enhanced testing, screening, and quarantine measures, regardless of their vaccination status or previous recovery from COVID-19. The measures will be in effect through at least January 31, 2022.
Chile: Effective December 1, 2021, foreign nationals who have been physically present in seven Southern African countries – namely, South Africa, Zimbabwe, Namibia, Botswana, Lesotho, Eswatini, and Mozambique – will be prohibited from entering Chile. Chilean citizens and resident foreign nationals who have been physically present in these seven countries during the previous fourteen days may still enter Chile, subject to additional testing and quarantine requirements. Specifically, such individuals must submit to PCR testing at the port of entry, and must complete a seven-day quarantine, regardless of vaccination status and even if the PCR test comes back negative.
Mexico: To date, Mexico has not adopted any new restrictions on international travel in response to the omicron variant. On November 28, 2021, the country's deputy health secretary stated that travel bans are of little use and that the responses of other countries have been disproportionate in consideration of what is presently known about the virulence of the omicron variant and its ability to evade the immune system.
United States: On November 26, 2021, the Biden Administration issued a Presidential Proclamation banning the entry into the United States, as immigrants or nonimmigrants, of noncitizens who were physically present within Botswana, Eswatini, Lesotho, Malawi, Mozambique, Namibia, South Africa, and Zimbabwe during the past 14-day period. The ban came only 19 days after the U.S. government ended its COVID-19 travel bans worldwide and replaced them with a vaccine mandate for travel to the United States. The Proclamation became effective at 12:01 a.m. Eastern standard time on November 29, 2021. Among the stated exceptions to the travel ban are U.S. citizens, lawful permanent residents, spouses of U.S. citizens and lawful permanent residents, and any noncitizen whose entry would be in the national interest, as determined by the Secretary of State, the Secretary of Homeland Security, or their designee.
These policies are sure to evolve as more is learned about the omicron variant in the coming days and weeks. Please continue to follow updates on our blog, The Mobile Workforce.
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