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  • Pharmacological modulation of spontaneous Ca2+ oscillations and effects of altered extracellular ionic milieu in Fluo-4 loaded differentiating chondrocytes of 2-day-old HDC. Representative confocal line-scan images and time courses of Fluo-4 fluorescence intensities are shown; horizontal and vertical calibrations are the same for all traces in panels (A)–(D). Horizontal lines under traces show the duration of treatments with pharmacons or altered extracellular ionic milieu. Acquisition of line-scan images started immediately after changing the bath solution on the cultures. Prior to that, normal functions were detected on each culture. (A) Spontaneous Ca2+ oscillations in normal ([Ca2+]e=1.8mM) Tyrode's solution. (B) After the non-selective cation channel-mediated Ca2+ entry blocker LaCl3 (500μM) and the store-operated Ca2+ entry and Ca2+ release-activated Ca2+ (CRAC) channel blocker YM-58483 (1μM) were applied in normal ([Ca2+]e=1.8mM) Tyrode's, Ca2+ oscillations ceased, although irregular fluctuations in basal cytosolic Ca2+ concentration remained detectable. (C) When the SERCA-blocker CPA (10μM) was co-administered with 500μM LaCl3 and 1μM YM-58483 in 1.8mM [Ca2+]e, Ca2+ oscillations were totally eliminated. (D) 3min after changing the bath solution to Ca2+-free Tyrode's, periodic oscillations could not be detected. Line-scan diagrams on panels (A)–(D) are representative data out of 4 independent experiments. ... Pooled data of Ca2+ oscillations gathered from series of X–Y images acquired from random visual fields of Fluo-4 loaded HDC on culturing days 1 and 2 with Zeiss LIVE 5 Laser Scanning Confocal Microscope. (A) Ratio of oscillating cells and frequency of repetitive Ca2+ transients on days 1 and 2 of culturing. Numbers above bars indicate the number of oscillating cells compared to all cells recorded. (B) Amplitude and full time at half maximum (FTHM) of Ca2+ oscillations in differentiating cells of HDC on culturing days 1 and 2. For both panels (A) and (B), while calculating the parameters of Ca2+ oscillations, only oscillating cells with round, chondroblast-like morphology were considered. Measurements were carried out on cultures from 4 independent experiments. Data represent mean±standard error of the mean (SEM). Numbers in parentheses above bars indicate the number of cells measured. Asterisks (*) mark significant differences (*Poscillating cells in 1- and 2-day-old HDC. ... Spontaneous Ca2+ oscillations in cells of HDC on day 2 of culturing. Prior to measurements, cells were loaded with Fluo-4-AM for 30min Ca2+ oscillations were observed without agonist stimulation in Tyrode's solution containing 1.8mM Ca2+ at room temperature. (A) Series of X–Y images were recorded from random visual fields of chondrifying cultures with Zeiss LIVE 5 Laser Scanning Confocal Microscope. These four representative frames were acquired at 6.5, 18.3, 29.6 and 54.4s during measurements. Arrows indicate differentiating chondrocytes with repetitive intracellular Ca2+ oscillations. (B) Time course of fluorescence intensities of the cells marked with arrows in panel (A). Fluo-4 fluorescence intensity values normalised to baseline fluorescence (F/F0) are plotted vs. time. Wide ranges of frequency and amplitude of oscillating cells were observed. ... Pooled data of Ca2+ oscillations obtained from series of X–Y images acquired from Fluo-4 loaded HDC in response to various treatments. Measurements were carried out with Zeiss LIVE 5 Laser Scanning Confocal Microscope. A total number of 500 images were recorded at each time point for each visual field; frame acquisition rate was 10s−1. (A) Percentage of oscillating cells before treatment (control), and 1, 3 or 5min after the application of bath solution containing 10μM nifedipine, or 500μM LaCl3 and 1μM YM-58483. Values were normalised to the untreated cells measured at 0min (control), and then at 1, 3 and 5min. Numbers in parentheses above bars show the number of cells measured. (B) Amplitude of Ca2+ oscillations, normalised to values of untreated control cells. Numbers in parentheses above bars represent the number of oscillating cells measured. (C) Frequency of Ca2+ oscillations normalised to the control. Numbers in parentheses above bars show the number of cells measured. For panels (A)–(C), oscillating cells with round morphology in the same random visual field were recorded at all four time points. Differentiating cartilage colonies were only used for a single measurement series and then were discarded. Graphs represent pooled data of 3 independent experiments, measuring random visual fields of 5 colonies for each treatment. Asterisks (*) mark significant differences (*P<0.05) between parameters of treated vs. control cells at respective time points. ... Refined model showing known components of Ca2+ homeostasis and signalling pathways that modulate Ca2+ oscillations in developing chondrocytes. Ca2+ can enter the cell via voltage-operated Ca2+ channels (VOCCs), P2X4 purinergic receptors, NMDA receptors or TRP channels; changes in resting membrane potential are mediated by voltage-gated K+ channels. ATP is secreted to the extracellular space via putative connexin 43 hemichannels. Activation of G-protein coupled P2Y purinergic receptors cause Ca2+-release from the endoplasmic reticulum (ER) via inositol-1,4,5-trisphosphate receptors (IP3R). Depletion of the Ca2+ stores cause aggregation of stromal interaction molecules (STIM1/STIM2), which are the Ca2+ sensors of the ER and trigger the opening of the store-operated Orai1 channels. Reuptake of Ca2+ to the ER is mediated by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and the Na+–Ca2+ exchanger (NCX). See text for details.
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  • Oscillation at outer edge with damping effect for a DEA with λ0=3.5 under harmonic electronic load. (a) ΩR0ρ/μ=0.00145. (b) ΩR0ρ/μ=0.00725. (c) ΩR0ρ/μ=0.0725Hz. (d) ΩR0ρ/μ=0.725. ... Oscillation at outer edge without damping effect for a DEA with λ0=3.5 under harmonic electronic load. (a) ΩR0ρ/μ=0.00145. (b) ΩR0ρ/μ=0.00725. (c) ΩR0ρ/μ=0.0725Hz. (d) ΩR0ρ/μ=0.725. ... Theoretical oscillation amplitudes at various dimensionless excitation frequencies and the dynamic range for a DEA. The inserted figure is a magnification of the measured data and theoretical prediction with actual dimensionless damping coefficient derived from the transient response.
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  • Frequency response and optimal output power. (a) Peak-to-peak voltage and (b) output current of the Hy-TENG versus various vibration frequencies. Straight lines represents the half of maximum value. Both the output voltage and the current show resonance at 5Hz, and the FWHM of the output voltage and current are 22Hz and 250Hz, respectively. (c) Dependence of the output voltage and current at various load resistances. (d) Instantaneous output power density and average power of the Hy-TENG versus various load resistances.
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  • (a) Results of PSMA detection in diluted bovine serum, with D2B functionalized pillars devices. Full blue circles and blue line are the same already shown in Fig. 3a, empty purple triangles represent data obtained detecting PSMA in diluted bovine serum (1:20 in PBS). Two concentrations, 10nM and 100nM, and a control sample (only bovine serum) were tested. (b) Dark squares show frequency shifts induced by PSMA at 100nM in PBS, containing BSA 0.2%w/v, as a function of the antigen incubation time. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) ... (a) Results of PSMA detection in PBS, containing BSA 0.2%w/v, with D2B functionalized pillars devices. 7 concentrations, ranging from 300pM to 100nM, were tested, full blue circles. On the left axis, frequency shifts induced at each PSMA concentration are displayed. On the right axis, corresponding values of PSMA density are shown. Green line indicates the initial frequency shift occurring after D2B adsorption, while the orange one indicates the frequency shift induced by BSA passivation. Each value is the mean shift and the error bar is the standard deviation of at least 30 independent pillars detected in parallel. Experimental data are fitted with a second order Langmuir curve (blue line) which provides a KD=18nM. Red empty circles represent data acquired using three different devices to demonstrate the reproducibility of the detection system. (b) Boxplot representation of data obtained detecting PSMA at 10nM in PBS containing BSA 0.2%w/v, with D2B functionalized pillars using three different devices. Statistical analysis, one way ANOVA test provides p=0.1132, means and variances are not significantly different (significance p<0.05). (c) Box plot representation of data obtained detecting PSMA in PBS at 100nM with D2B functionalized pillars using three different devices. Statistical analysis, one way ANOVA test provides p=0.1489, means and variances are not significantly different (significance p<0.05). (d) Box plot representation of data obtained detecting 7 different concentrations of PSMA in PBS containing BSA 0.2%w/v, with D2B functionalized pillars devices; concentrations ranging from 300pM to 100nM. Data distributions are compared by t-test (significance p<0.05). Data at 300pM, assumed as baseline, are compared with all the other concentrations. Data at 3nM and 10nM are also compared. Significance is represented for each couple as (*) significant p≤0.05, (**) very significant p≤0.01, (***) extremely significant p<0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) ... (a) Optical image of a “T” shaped pillar array actuated by a piezo at the resonance frequency of one of them, in the middle a pillar is oscillating. (b) A schematic representation of pillar detection: when pillars oscillate, the light reflection path is slightly deviated and the light intensity recorded by the CCD slightly decreased. (c) 53 traces corresponding to 53 different pillars as a function of actuation frequency. (d) Individual actuation mode. (e) Multiple actuation mode is obtained driving separate frequencies in parallel through the same piezo actuator and detecting simultaneously more pillars with separate resonance frequencies. ... Real time video of the dynamic response of pillars during a frequency scan. In the upper part, a real time image of the pillar array is shown. In the lower part, the signals corresponding to the image intensity of the top of pillars are acquired. At the resonance frequency, in the array image, the pillar oscillation is visible, while in the lower part, the corresponding signal of the image intensity decreases and the resonance peak appears. ... The following is the Supplementary material related to this article Movie 1.Movie 1Real time video of the dynamic response of pillars during a frequency scan. In the upper part, a real time image of the pillar array is shown. In the lower part, the signals corresponding to the image intensity of the top of pillars are acquired. At the resonance frequency, in the array image, the pillar oscillation is visible, while in the lower part, the corresponding signal of the image intensity decreases and the resonance peak appears.
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  • Frequency as a function of Reynolds number. ... h-dependence of the frequency, Re=350, Stage II. ... Oscillating flow... h-dependence of the frequency, Re=350, Stage I.
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  • Relative power evolution during a period of oscillation of 1.06s.
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  • Ca2+ oscillations... Effect of ACh on endothelial cell [Ca2+]i in isolated sheets of cells. (A) Confocal brightfield (top) and fluorescence (bottom) images of an isolated endothelial cell sheet loaded with fluo-4. (B andC) The time course of endothelial cell fluorescence changes in response to 1μM ACh added at the time indicated (by ▴). (B) The average whole-cellular fluorescence intensity over time. The coloured lines correspond to the individual cells marked in (A). Note that the oscillations in fluorescence are partially synchronized. (C) The average multi-cellular relative fluorescence (F/F0) over time. The region of interest for measuring fluorescence intensity was increased to include all the cells depicted in (A). (D) Time course of increases in membrane potential (Em) in response to 3μM ACh added at the time indicated (by ▾). The average resting membrane potential was −16.7±2.4mV (n=3 cells), which was increased to −28.8±3.9mV (n=3 cells) by current injection (to cell 1: black trace; and cell 3: red trace). ACh hyperpolarized these cells to a peak of −67.1±2.1mV (n=3 cells). The response to ACh was reproducible (cell 2: green and blue traces correspond to first and second additions), and in two of the three cells, the membrane potential oscillated (frequency: 0.14±0.02Hz, n=3 responses). (E) The cell marked with the arrow in (A) was used to illustrate the time course of sub-cellular fluorescence changes in response to 1μM ACh (added at ▴). Top panel: Changes in F/F0 in the regions corresponding to the coloured boxes shown over the cell below. Note that the increases in F/F0 were synchronous. Bottom panels: The difference in fluorescence intensity (F−F0) across the length of the cell (white line over image) over time (single oscillation indicated by the bar in the top panel). The dotted line in each panel corresponds to the x-coordinate in the image above (white line), and t=0s. There was no evidence for a Ca2+ wave. Note that the longitudinal axis of the artery was horizontally orientated, as in Fig. 1A. A movie of this experiment is available online (Movie 2, Supplementary material). ... Effect of ACh on endothelial cell [Ca2+]i in pressurized arteries. (A) Confocal fluorescence image of arterial endothelial cells loaded with fluo-4. (B and C) The time course of endothelial cell fluorescence changes in response to 0.3μM ACh added at the time indicated (by ▴). (B) The average whole-cellular fluorescence intensity over time. The coloured lines correspond to the individual cells marked in (A), note that the oscillations in fluorescence were asynchronous. (C) The average multi-cellular relative fluorescence (F/F0) over time. The region of interest for measuring fluorescence intensity was increased to include all the cells in the field of view (>50 cells). Note that the pattern of oscillations was markedly dampened compared to the individual cells, reflecting the asynchronous signals. (D) Summarized data showing the time course of increases in F/F0 in response to 0.3μM ACh (n=10) or 3μM ACh (added at t=0s, n=3). (E) The cell marked with an arrow in (A) was enlarged (image below) and used to illustrate the time course of sub-cellular fluorescence changes in response to 0.3μM ACh (added at ▴). Top panel: changes in F/F0 in the regions corresponding to the coloured boxes shown over the cell below. Note that the increases in F/F0 started moving from right to left, were not uniform, and changed direction. Bottom five panels: The difference in fluorescence intensity (F−F0) across the length of the cell (white line in image) over time (a single oscillation indicated by the bar in the top panel). The dotted line in each panel corresponds to the x-coordinate in the image above (white line), and t=0s. The wave travelled from right to left at a speed of approximately 30μm/s, and appeared to quench at the position of the nucleus. A movie of this experiment is available online (Movie 1, Supplementary material). ... Effect of treatments on the oscillations in endothelial cell [Ca2+]i in pressurized arteries. The effect of the various treatments on the oscillations in endothelial cell [Ca2+]i in response to ACh in pressurized arteries. The values were obtained from the experiments presented in Figs. 1, 3 and 4. The time course following the addition of 0.3μM ACh to the bath was divided into two phases, initial: t=0–30s; and sustained: t=30–100s. The percentage of cells oscillating (A) was calculated from the 16 cells analyzed per artery, and the frequency of oscillations (B) calculated using cells that oscillated in (A).
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  • Changes in intracellular Ca2+/Sr2+ levels of quiescent monolayer NRK cells, upon stimulation with PGF2α inside the ring, at different positions outside, inside and under the ring. Cells inside the ring were stimulation with PGF2α (n=7 experiments). Distribution of the interval time between calcium oscillations is depicted at right panels. (A) Response of 62 cells measured 5mm outside the ring at position A (see inset) after addition inside the ring of 0.8μM PGF2α. (B) Response of 47 cells measured under the ring (position B in inset) in the presence of PGF2α inside the ring. (C) Response of 67 cells measured at the inner edge of the ring (position C in inset) in the presence of PGF2α inside the ring. Individual traces are shown in gray, the mean response of the imaged cells is represented by the black line. Cells outside the ring were perfused with 5mM Sr2+, while inside the ring 1mM Ca2+ was present (see Materials and methods). The lower gray bar indicates presence of PGF2α inside the ring. Inset shows different positions of the imaged regions in relation to the ring location on the monolayer. Time (5min) is indicated by the lower black bar. ... The inset shows a schematic two-dimensional network of 400×400 cells. At the centre of the network cells, within a ring with an inner radius of 1mm (equivalent to 100 cells) have an IP3 concentration randomly distributed within a range between 5 and 15μM. (A) IP3 gradient due to diffusion and degradation of IP3 as described by Eq. (A.4) in Appendix A. For cells under the ring (0.5mm thick) the IP3 concentration decreases according to Eq. (A.4) from 10μM (inner side of the ring) to 0.1μM (outside the ring). Cells outside the ring have an IP3-concentration of 0.1μM and do not exhibit spontaneous calcium oscillations. (B) Membrane potential gradient in the network with (dashed line) and without (solid line) taking into account IP3 diffusion. The solid line shows the average membrane potential if there would be no diffusion of IP3 from the inside of the ring to cells under the ring. The dashed line shows the average membrane potential as a result of simulations for an IP3 distribution in the network according to Eq. (A.4) in Appendix A.
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  • Longitudinal flame oscillations for a 10vol.% H2 experiment, re-interpreted with the theoretical considerations given in previous sections. The white arrow indicates the moment when strong external noise affects the flame. ... Stability graphs for a 10vol.% H2–air mixture at normal conditions and an excitation frequency of 110Hz corresponding to the auto excitation of the flame (left) and 1000Hz corresponding to the major frequency of the horn (right). ... Longitudinal flame oscillations for the 10% H2 experiment. Time and distance along horizontal and vertical directions respectively. The white arrow indicates the moment when the strong external noise was turned on and subsequently started affecting the flame propagation. ... Stability graphs for H2–air mixtures at normal conditions. From left to right and from top to bottom: (a) 10, (b) 10.5, (c) 11, (d) 12, (e) 13 and (f) 14vol.% H2. Excitation frequencies are 110, 122, 121, 130, 145 and 170Hz respectively. ... Positions and average velocity of the flame for 12% vol. H2 experiment. For clarity, the origin of coordinates for time and space was selected as the instant in which oscillations appear for the first time.
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  • Oscillating Water Column... Water elevation into the converter (wave gauge 4) for free oscillation test. Natural frequency found are 0.91±0.03s-1 for laboratory test (plain line) and 0.91±0.04s-1 for simulation (dashed line).
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