A labile Ca storage pool exists on bone surfaces storing excess or supplying Ca when blood Ca is lowered. Aim was to examine minute-to-minute regulation of p-Ca2+ in the very early phase of acute uremia, as induced by total bilateral nephrectomy and to study the effect of absence of kidneys on the rapid recovery of p-Ca2+ from a brief induction of acute hypocalcemia.Understanding the regulation of mineral homeostasis and function of the skeleton as buffer for Calcium and Phosphate has regained new interest with introduction of the syndrome “Chronic Kidney Disease-Mineral and Bone Disorder”(CKD-MBD). The very rapid minute-to-minute regulation of plasma-Ca2+ was examined in sham-operated rats, acute nephrectomized rats(NX), acute thyroparathyrectomized(TPTX) rats and NX-TPTX rats.The rapid regulation of p-Ca+2 falls rapidly and significantly very early after acute NX, from 1.23 ± 0.02 to 1.06 ± 0.04 mM (p < 0.001). Further hypocalcemia was induced by a 30 min iv infusion of EGTA. Control groups had saline. After discontinuing EGTA a rapid increase in p-Ca2+ took place, but with a lower level in NX rats (p < 0.05). NX-TPTX model excluded potential effect of accumulation of Calcitonin and C-terminal PTH, both having potential hypocalcemic actions. Acute TPTX resulted in hypercalcemia, 1.44 ± 0.02 mM and less in NX-TPTX rats,1.41 ± 0.02 mM (p < 0.05). Recovery of p-Ca2+ from hypocalcemia resulted in lower levels in NX-TPTX than in TPTX rats, 1.20 ± 0.02 vs.1.30 ± 0.02 (p < 0.05) demonstrating that absence of kidneys significantly affected the rapid regulation of p-Ca2+ independent of PTH, C-PTH and CT.The results clearly showed that p-Ca2+ on a minute-to-minute basis is influenced by presence of kidneys. Hypocalcemia developed rapidly in acute uremia. Levels of p-Ca2+, obtained during recovery from hypocalcemia resulted in lower levels in acutely nephrectomized rats. This indicates that kidneys are of significant importance for the ‘set-point’ of p-Ca2+ on bone surface, independently of PTH and calcitonin. Our results point toward existence of an as yet unknown factor/mechanism, which mediates the axis between kidney and bone, and which is involved in the very rapid regulation of p-Ca2+.P-Ca Furthermore, extracellular Ca2+ participates in its own regulation through systemic and local Ca2+ sensing receptor (CaR) mediated actions in tissues related to the mineral ion homeostasis [Calcium (Ca) has important extracellular as well as intracellular functions. Its extracellular functions include its role in blood clotting, maintenance of plasma membrane integrity and intercellular adhesion. Extracellular Ca provides a source of calcium ions  [The free plasma Cariations . The det2+ after a brief induction of hypocalcemia [2+ concentration elicits a prompt increase in the rate of PTH secretion. The renal responses to the increased plasma PTH level are phosphaturia, enhanced distal tubular reabsorption of Ca2+ and increased generation of the active metabolite of vitamin D, 1,25(OH)2D , from 25(OH)D by stimulation of the 1α-hydroxylase in the proximal convoluted tubules. The increased level of calcitriol stimulates the intestinal absorption of phosphate (P) and Ca. PTH and calcitriol promote net release of P and Ca from bone. The increased flux of Ca2+ into the extracellular fluid, coupled with renal retention of Ca2+ restore circulating Ca2+ levels toward normal, and thereby inhibits PTH secretion and close the negative feedback loop. In this model of Ca2+ homeostasis the emphasis is on the key role of PTH and kidney. This classical model of plasma Ca2+ regulation does indeed apply to the long-term regulation and stability of plasma Ca2+, but does not apply to the very rapid minute-to-minute regulation of plasma Ca2+, as previously clearly shown from our lab. [The traditional understanding of the Ca homeostasis provides the following mechanism for the subsequent normalization of plasma Cacalcemia -8. Even our lab. -12.2+ is held constant has been expanded by Kurokawa [2+ flux between extracellular fluid volume (ECF) and bone, and the set point for renal tubular Ca2+ reabsorption in relation to the set point for the Ca2+ regulated PTH secretion from the parathyroids [2+ level in ECF by adjusting the set points in the different organs to the same level of Ca2+. As such the set points of the Ca2+ regulating tissues correspond to the resulting plasma Ca2+ level, which the organism is maintaining. This elegant concept is strongly supported by results from studies on the regulation of renal tubular reabsorption of Ca2+ [2+ channel, TRPV5 [The conceptual framework on how plasma CaKurokawa , who desthyroids . The int of Ca2+ ,14,15. T of Ca2+ -18 togetl, TRPV5 ,20.2+ homeostasis has been proposed, as fibroblast growth factor 23 (FGF23) was shown to promote renal Ca2+ reabsorption through the TRPV5 channel in the distal tubule, and hereby contributes to conserve Ca. In short time in vitro experiments FGF23 increased the TRPV5 protein abundance and the channel activity already after 45 minutes [Recently, a new factor in the rapid regulation of Ca minutes .2+ between bone and the ECF interphase is less clear. Kurokawa assumed that PTH and calcitriol, in concert, adjusted the set point for Ca2+ by regulating the rate of the coupled bone resorption and formation [2+.How and where the calciotropic hormones are setting the set point for Caormation . However2+ at the quiescent surface of bone which is in dynamic equilibrium with ECF has previously clearly been established by experiments using incorporation of radiolabelled Ca in bone. The magnitude of the rapid calcium exchange was estimated to be many fold higher than the daily flux from remodeling based bone turnover. This labile pool functions as a short-term buffer, taking up or releasing calcium to correct for changes in ECF [The existence of a labile pool of Cas in ECF . Knowleds in ECF ,12,22-25s in ECF .2+ [2+ was examined after a brief induction of hypocalcemia with an EGTA infusion in parathyroidectomized (PTX), thyroidectomized (TX), thyroparathyroidectomized (TPTX) and calcitriol depleted rats and rats treated with different doses of PTH, CT and calcitriol. The overall conclusion was that none of these calciotropic hormones were prerequisite for the rapid correction of plasma Ca2+ [2+ homeostasis, with the calciotropic hormones defining the plasma Ca2+ levels, by adjusting the set point for plasma Ca2+ in a dose-related manner.Results from our lab have previously clearly shown that neither PTH, calcitriol or CT were essential for the rapid minute-to-minute regulation of plasma Ca2+ -12. In tsma Ca2+ -12. Our 2+. This indicated that the effect of removing endogenous CT initially dominated over the effect of removing endogenous PTH, and resulted in an increase of calcium efflux from bone to the extracellular fluid. The role of PTH in the rapid, minute-to-minute plasma calcium regulation was characterized by stable plasma Ca2+ for 2 h after acute PTX [2+ levels, and as such that calcium homeostasis was hormone-controlled, even on a minute-to-minute basis [Furthermore, we have previously shown that the rapid calcemic recovery, which occurs after a brief induction of hypocalcemia does not result from suppression of CT levels. Thus, the calcemic recovery curves were similar in TPTX rats, normal and PTX rats and in rats given an exogenous salmon CT infusion. It was also clearly shown that TPTX and selective TX resulted in similar increments of plasma Cacute PTX . It was te basis . Thus, ote basis .The understanding of the regulation of the mineral homeostasis and the function of the skeleton as a buffer for Ca and P has regained new interest with the introduction of the concept of the syndrome “Chronic Kidney Disease - Mineral and Bone Disorder” (CKD-MBD) by Moe et al. . This coHypocalcemia is an important complication in uremia. Traditionally, hypocalcemia in uremia is explained by the associated decreased renal biosynthesis of calcitriol, P retention, decreased intestinal Ca absorption, and skeletal resistance to PTH, factors that all take time to respond. However, a very rapid skeletal response to the induction of acute kidney injury has recently been demonstrated , where a2+ in the very early phase of acute uremia, as induced by total bilateral nephrectomy and to study the effect of total absence of kidneys on the rapid recovery of plasma Ca2+ from a brief induction of acute hypocalcemia.Thus, the present investigation was conducted in order to examine the performance of plasma Ca2+ to nephrectomy and recovery from acute hypocalcemia, the data are presented combined. The rats were not fasting before the experiments.The experimental studies were performed in accordance with the National Institute for Health Guide for Care and Use of Laboratory Animals and approved by the Animal Experiments Inspectorate, the Ministry of Food, Agriculture and Fisheries, Denmark (Reference: 2012-DY-2934-00023). Adult male Wistar and DA rats weighing 250 g  were housed in a temperature-controlled environment with a 12-hour light/dark circle. The rats were given ad libitum access to a standard diet  and water. Rats were anesthetized with pentobarbital . Additional doses were administered when required in order to maintain a steady level of anesthesia. Since DA and Wistar rats had identical response of CaAcute TPTX was performed under stereomicroscope using a microsurgical procedure with special care to avoid trauma to the recurrent laryngeal nerve. The model has previously been described from our lab . SuccessNX was performed from the back by a one-step surgical removal of both kidneys , as prev2+ was measured by a Calcium Selective Electrode . Plasma phosphate was analyzed by Vitros 250 .Hypocalcemia was induced by an intravenous infusion of EGTA (ethylene-bis(oxyethylenenitrilo)tetraacetic acid; Sigma, USA), 40 mM, at a rate of 3.0 ml/h for 30 minutes (from time 60 to 90 minutes) through a catheter inserted into the femoral vein. Plasma Ca2+ was examined in sham-operated rats, NX rats, TPTX rats and NX-TPTX rats, as presented in Table 2+ and phosphate concentrations were obtained from a catheter in the femoral artery at times 0, 60, 90, 100, 120, 140 min. After each blood sample, the blood volume was replaced by infusion of 0.4 ml NaCl. The hypocalcemic model is described in more details from our lab [Rats were allocated to eight experimental models, and the rapid regulation of plasma CaP < 0.05 is considered statistically significant.The data are presented as mean ± SEM and compared with Mann–Whitney test or t-test,. 2+ concentrations is illustrated in Figure 2+ was significantly diminished in NX rats compared to sham rats (p < 0.01). Then a further reduction of plasma Ca2+ took place. Plasma Ca2+ diminished in NX rats from a basal value of 1.23 ± 0.02 to 1.06 ± 0.04 mmol/L at 140 min, p < 0.001, while sham rats, given saline only, had a slight decrease in plasma Ca2+ from 1.21 ± 0.01 to 1.17 ± 0.02 mmol/L (ns). TPTX resulted as expected in hypercalcemia with a significant increase in plasma Ca2+ from 1.29 ± 0.01 to 1.44 ± 0.02 mmol/L (p < 0.001). The increase in plasma Ca2+ was significantly less in NX-TPTX rats, 1.29 ± 0.02 mmol/L, than in TPTX rats, (p < 0.05)  developed within 30 minutes of EGTA infusion. Then subsequently a rapid and significant increase of plasma Ca2+ took place within 10 minutes after stopping the EGTA infusion and with further recovery towards basal levels.Further the minute-to-minute regulation of plasma Ca2+ regulation, sham operated rats were compared to total bilateral NX rats, as shown in Figure p < 0.01) and the concentration of plasma Ca2+ remained significantly lower in the NX group during the recovery from hypocalcemia, (p <0.01) at time 100, 120 and 140 minutes. Thus in the presence of acute alterations in plasma Ca2+ concentration, NX rats had the ability to restore calcium levels. The equilibrium is however now set by the new, lower set-point, indicating an important role of the kidneys in the very rapid regulation of the set-point for Ca2+ on bone surfaces.In order to examine any effect of the presence of kidneys on the rapid minute-to-minute plasma Ca2+ regulation. The nadir of hypocalcemia in the EGTA infused NX-TPTX rats was significantly lower than that of the EGTA infused TPTX rats, 0.81 ± 0.03 vs. 1.00 ± 0.02 mmol/L, (p < 0.01). Plasma Ca2+ concentrations were significantly lower in the NX-TPTX rats, as compared to the TPTX rats during the recovery from EGTA induced hypocalcemia, (p < 0.01) at time 100, 120 and 140 minutes. Thus, also in this experimental situation the NX group had a downward shift of the Ca recovery curve from acute hypocalcemia, once again providing further support for the existence of a rapid effect of NX on the set-point for Ca2+, which is independent of PTH and CT, although it still is respecting the degree of hypercalcemia, which developed post TPTX.A possible direct or indirect effect of intact PTH, C-terminal PTH or CT was further examined by comparing the responses in TPTX and NX-TPTX rats, as shown in Figure 2+ values and differed from 1.74 ± 0.04 to 2.40 ± 0.6 mmol/L between the 8 groups of rats at time 0 min, i.e. before any experimental procedure was performed. After that plasma P remained stable within the groups for the next 120 minutes, except in the NX-EGTA group, where a significant increase of plasma P from 1.43 ± 0.14 to 2.08 ± 0.13 mmol/L (p < 0.01) took place, as indicated in Figure The basal normal plasma P concentrations were more dispersed than basal Ca2+ falls rapidly very early after induction of acute uremia, and that plasma Ca2+ levels already 60 minutes after total nephrectomy became significantly decreased and further decreased for another 80 minutes. The mechanism for this rapid decline of plasma Ca2+ is not known. Hypocalcemia, as observed in patients with acute renal failure, was previously explained as being secondary to decreased synthesis of calcitriol and development of hyperphosphatemia [The results of present study clearly show that plasma Caphatemia ,35. The phatemia , even thOur initial hypothesis was that this rapid development of hypocalcemia in a model of total nephrectomy was caused by accumulation of C-terminal PTH fragments and/or of CT.2+ homeostasis [2+ [2+ in acute uremia, as previously hypothesized from our lab, could however not be confirmed by the results of the present investigation, as no influence of PTH, C-terminal PTH and CT was found on plasma Ca2+ concentrations when comparing the models of TPTX (without PTH and CT) and NX-TPTX rats . Thus, our present results interestingly points toward the existence of a kidney-bone axis, which is involved in the rapid regulation of plasma Ca2+, and that this very rapid regulation is not mediated by any of the classical calciotropic hormones, PTH and 1,25(OH)2D or CT.Mammalian intact PTH is a polypeptide consisting of 84 amino acids -39. The eostasis . Circulaeostasis ,42. The asis [2+ ,44. The asis [2+  as shownasis [2+ ,47 and casis [2+ . Similarasis [2+ . An impa2+ levels were not followed in the study. In models of acute uremia they found an increase in FGF23 levels, which was observed already at one hour after acute kidney injury (AKI) [Christov et al. recently added another interesting aspect to the possible existence of a rapid skeletal response to induction of acute uremia, , even thry (AKI) . This rary (AKI) . As suchry (AKI)  and the 2+ level was observed in NX-TPTX rats compared to TPTX rats, indicating that the Ca2+ lowering effect of NX was independent of a PTH signal. Similarly, in the study of Christov et al. [In the present study a decreased plasma Cav et al.  elevatio2+ concentrations throughout the study, once again indicating that presence of kidneys influences the plasma Ca2+ concentration on a minute-to-minute basis.TPTX rats developed significant hypercalcemia, which is in line with our previous results . This ob2+-EGTA complex has previously been carefully examined by our group and it was clearly shown that the rapid recovery of plasma Ca2+ was not due to dissociation of calcium from the complex, not due to induction of a decreased plasma P and was not a product of alterations in plasma pH [EGTA is a specific calcium chelator that induces hypocalcemia by binding calcium in a stable physiological inert complex. The stability of the Calasma pH . Thus EG2+ regulation. Surprisingly, despite development of hypocalcemia NX rats had stable plasma P for 60 minutes post NX, clearly demonstrating that acute hypocalcemia was not balanced by acute recipocal hyperphosphatemia. Furthermore, plasma P remained stable independent of EGTA induced changes in plasma Ca2+ and independent of the presence of PTH. This dissociation between plasma Ca2+ and plasma P levels is in accordance with results on diurnal variations of the two ions [Another question is whether plasma P levels are affected by the acute NX and thereby could have an effect on plasma Catwo ions .2+ values. At present this is not explained. Plasma P remained stable within the groups, except in the NX-EGTA group, where a significant increase of plasma P (p < 0.01) took place. Potentially this could be explained by a stimulatory effect of the EGTA induced hypocalcemia on the PTH secretion [Surprisingly, the basal normal plasma P concentrations were considerably more dispersed than the basal plasma Caecretion  in a sit2+. The demonstration, that the induction of and recovery from hypocalcemia is significantly lower in NX rats, independently of PTH, C-terminal PTH and CT in the circulation, might indirectly indicate that presence of kidney is of significant importance for the setting the set-point for Ca2+ on bone surfaces. We, therefore, suggest that there might exist an as yet unknown factor or mechanism, which is involved in the very rapid signaling between kidney and bone, and which is affecting the minute-to-minute regulation of plasma Ca2+. We suggest that this factor acts rapidly as a hormone or a mechanism with a hormone-like effect on bone, and thereby sets the ‘set-point’ for plasma Ca2+ regulation on bone.The results of the current investigation clearly demonstrated that the kidneys affect the rapid changes in plasma Ca2+ concentrations on a minute-to-minute basis are influenced by the presence of kidneys. This indicates that the kidneys are of significant importance for the ‘set-point’ of plasma Ca2+ on bone independent of PTH and calcitonin. Our results point toward the existence of an as yet unknown factor/mechanism, which mediates signals between kidney and bone, and which is involved in the very rapid regulation of plasma Ca2+.In conclusion, our results clearly demonstrate that plasma Ca


We show with confocal microscopy that 800 nm laser pulse application onto a neuron decorated with a few AuNPs triggers a transient increase in free Ca2+, measured optically with GCaMP6s. We show that action potentials, measured electrophysiologically, can be induced with this approach. We demonstrate activation of local Ca2+ transients and Ca2+ signaling via CaMKII in dendritic domains, by illuminating a single or few functionalized AuNPs specifically targeting genetically-modified neurons. This NP-Assisted Localized Optical Stimulation (NALOS) provides a new complement to light-dependent methods for controlling neuronal activity and cell signaling.Light-assisted manipulation of cells to control membrane activity or intracellular signaling has become a major avenue in life sciences. However, the ability to perform subcellular light stimulation to investigate localized signaling has been limited. Here, we introduce an all optical method for the stimulation and the monitoring of localized Ca While non-specific binding of bare AuNPs onto the neurons was possible through passive sedimentation, we show that AuNP functionalization with monoclonal antibodies can be used to specifically target their localization on neuronal membranes expressing the targeted antigen. We demonstrate with combined Ca2+ imaging and whole-cell patch-clamping that photo-stimulation of neurons decorated with bare or functionalized AuNPs can drive local or widespread Ca2+ responses and APs, depending on the intensity and localization of the illumination. Finally, we demonstrate that NALOS can be used to investigate local Ca2+ signaling in dendritic compartments, by monitoring the spatial dynamics of Ca2+/calmodulin-dependent protein kinase II (CaMKII).Here, we present a technique called Nanoparticle-Assisted Localized Optical Stimulation (NALOS) for localized, diffraction-unlimited stimulation of subcellular regions on cultured hippocampal neurons by the mean of plasmonic excitation of single AuNPs. To combine optical stimulation and optical read-out of neuronal activity, we used the off-resonance plasmonic excitation of AuNPs with near-IR light (NIR) at 800 nm as a source for localized neural stimulation, and optical imaging of Ca2+ imaging with laser scanning confocal microscopy (488 nm) on neurons transfected with the Ca2+ sensitive protein GCaMP6s2 at the focus of the objective) with fs pulsed 90 MHz laser at 800 nm  (2) to sub-regions on the soma or proximal dendrites of the neurons where a diffraction-limited single AuNP reflection spot was localized. We also observed transient widespread Ca2+ elevation, although the delay and amplitude varied with the location of the light stimulus  . At thisstimulus . Altogetggregate . We repe2+ elevation by applying irradiation over a very small region of interest (ROI) around one or few AuNPs. We observed an increase in GCaMP6s fluorescence directly after scanning over an ROI (5 μm2) with the 800 nm fs laser at an intensity of 0.3 MW/cm2  tag, using our recently optimized protocol2+ response, but also to determine visually the specificity of the binding of fAuNPs and targeted cells.We chose to use an antibody against a tag (HA), rather than an endogenous receptor, to ensure specificity in the binding, which can be tested by comparing binding on neurons transfected or not with an HA-tagged receptor gene. Neurons were transfected with an HA-GluA1 subunit of the AMPA receptor, which we have used previously with HA-tagged quantum dots to track receptor mobility2+ responses, confined around a single fAuNP, since no Ca2+ response was detected in regions 5 μm away from the same fAuNP on a dendrite. The amplitude and length of the Ca2+ transients were very similar for each of the 5 stimulation pulses .Following incubation and wash of the fAuNPs, we observed that their reflectance at 800 nm co-localized very specifically with the transfected fluorescent neurons, further confirmed by immunostaining the neurons with anti-HA antibodies . Furthern pulses . The res2), a single pulse indeed could induce an AP, which was correlated with a widespread but small Ca2+ transient (2) induced a stronger widespread Ca2+ transient and was characterized by a burst of APs  but miniature post-synaptic currents remained. To induce a local response, we applied a laser scan of 0.4 – 0.5 MW/cm2 (800 nm) in a small ROI around a AuNP on a proximal dendrite  in a small ROI triggered a rapid and localized Ca2+ transient accompanied by a slower and delayed translocation of GFP-CaMKII in the same region, while surrounding regions did not exhibit a Ca2+ transient, nor GFP-CaMKII translocation (n = 6) (2+ and CaMKII response (2) stimulated a widespread Ca2+ response, which triggered a long lasting translocation of CaMKII to spines throughout the dendritic arborisation  . Spontanrisation . These r2) was lower than what would be necessary for two-photon fluorescence excitation and therefore no cross-talk between the off-resonance plasmonic excitation of the AuNPs and the activity read-out with Ca2+ imaging was observed. The use of NIR light for the excitation of the AuNPs is advantageous for combining the use of fluorescent proteins for monitoring molecular signaling. This provides an all-optical stimulation and molecular imaging approach at the subcellular level.In this study, we show that NALOS allows for localized stimulation of neuronal cells with high spatial and temporal precision. This was achieved by generating off-resonance excitation of single 100 nm AuNPs with a pulsed fs laser at 800 nm on small ROIs on cultured hippocampal neurons. In the absence of AuNPs, the fs laser did not induce any measurable effect on the cells. The size of the responding area appeared to be dependent on the created near-field enhancement and the resulting plasmon generation. With this method, we were able to monitor neuronal response after stimulation with two independent fluorescence detection channels, without observing any cross-talk between fluorescence excitation and AuNPs excitation. This would not have been possible with the use of a visible cw laser to illuminate AuNPs as reported previously42+ indicator could be combined.The functionalization of the AuNPs further increased the specificity of the method and its reproducibility by limiting substantially AuNP aggregation. It can also provide a means to target the AuNPs to specific cell types or even subcellular compartments that would express a specific transmembrane protein. We used an HA-tag antibody as proof of concept and to demonstrate specificity of the binding on transfected cells, but a specific antibody against an extracellular epitope of a native transmembrane protein could be used, avoiding the need for gene transfer. In such case, an organic rather than a genetically-encoded Caet al.Since the size of the stimulated region depends of the presence of AuNPs, it is not diffraction-limited; thus future work combining NALOS with laser scanning super-resolution imaging techniques could allow diffraction-unlimited optical stimulation and recording of neuronal signaling in nanodomains. Furthermore, it is foreseen that the method could be extended to produce highly localized phenomena, such as heat, pressure wave or nanocavitation depending of the irradiation parameters and AuNPs characteristics as described in Boulais 2+ signaling in any small cellular compartments, without the need for caged neurotransmitters, such as caged glutamate, which needs to be used at very high concentration (mM), leading to high costs and potential off-target effects2+ response can be tuned by the laser intensity. However, beyond the threshold intensity, the Ca2+ response can become widespread. The ability to locally induce Ca2+ transient should provide a useful approach to study Ca2+ signaling in various subcellular compartments of neurons or other cell types. For example, NALOS could serve to investigate the mechanisms of Ca2+-induced Ca2+ release from internal stores, Ca2+-dependent growth and structural remodeling, Ca2+-regulated local translation, Ca2+-dependent kinase activation, and several more. We tested the effect of inducing local Ca2+ transients on CaMKII signaling in neuronal dendrites, since this enzyme was shown to exhibit a dynamic translocation to dendritic domains where Ca2+ activity was spontaneously high2+ signals in cells. By contrast, the production of a widespread Ca2+ signal with stronger local stimulation reproduced an effect observed by Rose et al.NALOS provides a method to study CaBezanilla and colleagues showed that diode laser-induced heating of a patch of cell membrane activates a measurable capacitive current2+ signaling, other local activity-dependent processes might also be available for investigation with this method.NALOS could serve as a complement to several light-dependent methods to control neuronal activity and cell signaling. It may be used in non-genetically altered cells, taking advantage of functionalization, or combined with genetically-encoded targets to select the cell type to be studied. The probably most useful feature is the capacity to target small subcellular domains for photo-stimulation at reasonably low light regime in NIR range. In addition to Cad-lysine-coated glass coverslips (12 or 18 mm) at a density of 4225 or 860 cells/mm2 respectively. Growth media consisted of Neurobasal and B27 (50:1), supplemented with penicillin/streptomycin  and 0.5 mM L-GlutaMAX (Invitrogen). Fetal bovine serum  was added at time of plating. After 5 days, half of the media was changed without serum and with Ara-C  to limit proliferation of non-neuronal cells. Twice a week thereon, half of the growth medium was replaced with serum- and Ara-C–free medium. The neurons were transfected at 11–14 days in vitro (DIV) with Lipofectamine 2000 (Invitrogen) as described previously2+ imaging, plasmids encoding GCaMP6sThe preparation of cultured dissociated hippocampal neurons was described previously272+ imaging. GCaMP6s and GFP-CaMKII were excited with a continuous wave 488 nm Argon laser and detected with a 500–530 nm bandpass filter. RGCaMP1.07 was excited with a continuous wave 543 nm Helium-Neon laser and detected with a 565–615 nm bandpass filter. Off-resonance plasmonic excitation of the AuNPs was achieved with a fs pulsed Ti:Sapphire laser at 800 nm with a diffraction-limited PSF . For the experiments combining NALOS and current clamp recording, a similar LSM confocal microscope was used with a 80 MHz pulsed Chameleon Ti:Sapphire laser at 800 nm. The power of the laser after the objective was tuned between 0.5 and 2 mW, which corresponds to a focal spot intensity of 0.27–1.02 MW/cm2 when using a 0.8 NA water immersion objective. When using 18 mm glass coverslip combined with a closed perfusion chamber, a 1.0 NA oil immersion objective was used and the laser intensity was adjusted to obtain the same focal intensity. For NALOS, a single scan over a ROI was performed with a pixel dwell time varying between 1.3 and 6.4 μs. The excitation of GCaMP6s with a laser at 488 nm had no measurable effect on the neurons with AuNPs at the imaging intensity of 0.2–0.9 kW/cm2.NALOS and confocal imaging were performed on a confocal laser scanning microscope . Neurons were imaged in HEPES-aCSF (pH 7.4) using a perfusion system with temperature adjusted to 29–30 °C, either an open chamber  or closed custom-made superfusion imaging chamber. GCaMP6s or RGCaMP1.07 were used as probes for CaImaging of the AuNPs was performed in reflectance mode by taking advantage of the small transmittance of the excitation filter at 800 nm. The reflected light was detected using a long pass filter (LP 560). With this method, the position and the movement of the AuNPs could be monitored during the entire experiment. The resulting power of the 800 nm fs laser at the back aperture of the objective was less than 100 nW and therefore insufficient to generate AuNP-induced stimulation. However, when imaging RGCaMP1.07 and GFP-CaMKII, a band pass filter (BP565–615) blocking 800 nm light was used for the red detection channel and therefore the reflection of the AuNPs could not be detected during the fluorescence measurements. In this case, mapping of the AuNPs positions was performed prior to the measurement.N-hydroxysuccinimide  in aqueous Na2CO3 10 mM pH 8.5 . The PEG-antibodies solution was mixed by vortexing and kept at 4 °C during 3h. Aliquots of the solution were kept at −20 °C until use. fAuNPs were prepared following our previously reported procedure2CO3 10 mM pH 8.5 (212 μL) and the solution of PEG-antibodies (10 μL) at 4 °C during 1h. Then, an aqueous solution of 50 μM PEG-SH  was added to the suspension to block the remaining free sites on the Au surface at 4 °C during 1h. The suspension was purified by centrifugation at 5000 rpm during 2 min. The supernatant was removed and replaced with Neurobasal and B27 (50:1) or phenol red-free Dulbecco’s Modified Eagle’s Medium . Aliquots of the unpurified or purified mixtures in DMEM (320 μL) were treated with water or 10% NaCl (35.5 μL) at 4 °C during 30 min and analyzed by UV-visible-NIR spectroscopy in a 96-well plate. The absorption spectra  were acquired with an Epoch microplate spectrophotometer (BioTek Instruments) controlled with the Gen5 Data Analysis software version 1.11.5. A blank spectrum (solvent without NPs) was subtracted from each sample spectrum.Rat monoclonal anti-HA antibodies  were tethered to AuNPs using heterobifunctional PEG linkers by using the strong covalent binding between sulfur and gold22319 AuNPs/mL, ε = 1.1 × 1011 M−1cm−1, potential zeta −46 mV) was mixed with 50 μL of neurobasal cell culture medium and added dropwise to the cells. For specific binding, fAuNPs solution was centrifuged at 2000 rpm during 2 min and the fAuNPs were resuspended in neurobasal culture medium. 100 μL of this solution was then added to the cells for 1 to 2 h incubation. Longer incubation times increased non-specific binding. The AuNPs and fAuNPs solutions were kept at 4 °C and experiments with fAuNPs were performed within 2 weeks after fAuNPs synthesis.Prior to imaging, incubation for 2h of the AuNPs onto the neurons inside the incubator allowed for sufficient sedimentation3, 20 CsCl, 10 diNa-phosphocreatine, 10 HEPES, 2.5 MgCl2, 0.6 EGTA, 4 ATP-Tris, 0.4 GTP-Tris, pH 7.28; 245 mOsm/L for voltage clamp and 111 KMeSO3, 10 diNa-phosphocreatine, 10 HEPES, 2.5 MgCl2, 2 ATP-Tris, 0.4 GTP-Tris, pH 7.25; 240 mOsm/L for current clamp. Data acquisition (filtered at 1.8–2 kHz and digitized at 10 kHz) was performed using a Multiclamp 700B amplifier and the Clampex 10.6 software (Molecular Devices). Data were analyzed using Clampfit 10.2 (Molecular Devices) and Igor Pro (WaveMetrics).Whole-cell voltage-clamp and current-clamp recordings were obtained from visually identified pyramidal cells. For recordings, the glass pipettes of 3.5–5 MΩ were filled with a solution containing the following (in mM): 96 CsMeSO2+ fluctuations was done as described previouslyAnalysis of the CaHow to cite this article: Lavoie-Cardinal, F. et al. Gold nanoparticle-assisted all optical localized stimulation and monitoring of Ca2+ signaling in neurons. Sci. Rep.6, 20619; doi: 10.1038/srep20619 (2016).


The present paper reports on the preliminary experimental results concerning a new concept of ultracold neutron production with a single crystal converter of ortho-deuterium lying in the ground rotational state at the low temperature of about 10 K, which should make it possible to utilize a guided cold neutron beam instead of irradiating the converter material in the inside of high radiation fields. The successful observation of the clear Bragg scattering pattern from the single crystal converter and the reasonable results from the first experimental trial of the ultracold neutron production with the single crystal are shown. Ultracold neutrons (UCN) with the velocity of about 6 m/s can be stored in a closed vessel called a neutron bottle and therefore are utilized in various kinds of fundamental physics experiments on neutrons . Experima- and the c-axes with the lattice parameters of about 0.35 nm and 0.58 nm, respectively, and thus the intersections between the phonon curves[Ortho-deuterium molecules have much attractive properties as the UCN converter material  with a von curves and the The present study reports on the results of our preliminary experiments of the orthodeuterium single crystal preparation as the UCN converter and its Bragg scattering observed, and further the first trial of UCN production with the single crystal of orthodeuterium.ϕ × 50 mml copper cell at the top of a two-stage helium refrigerator CryoMiniSince para-deuterium molecules are excited with the energy corresponding to about 90 K even at the lowest rotational state, the effective UCN lifetime in SD2 is determined by the para-content. In the present studies, the high purity ortho-deuterium gas was prepared with the magnetic catalyser OXISORBW × 6 cmH × 3 cmD crystallising cell connected above our catalyser cell in the refrigerator.The procedure to prepare a several cm cube single crystal of ortho-deuterium was specially developed for the present experiments of the UCN converter at at Prof. Momose’s laboratory in Kyoto University. The key factors for the optimized procedure are the direct and gradual growth of the single crystal from deuterium gas under well controlled optimum gas flow rate and also at the optimum temperature of the crystallising cell. After a number of trials at the Momose laboratory, the obvious perfect quality of the single crystal cube with the optical inspections was at last attained, and the optimim conditions for the crystallising procedure were defined. Then, in our neutron experiments, these conditions are exactly followed for our UCN converter preparation with the 2 cm6Li tiles and B4C plastic collimaters, a narrow slit chopper, a 2 m long evacuated extension guide tube, and related shieldings assembled in the upstream of our cryomachine and also around our gas line shown in The inspection and the identification of the crystal orientation prepared in our UCN converter cell was carried out with the time-of-flight method to observe the Bragg scattering of the chopper-pulsed cold neutron beam at the exit of C2 guide tube, JRR-3M reactor, JAERI. We specially prepared our experimental setup with 3 counter, where the solid circles are the results from the converter cell filled with about 2 cm thick deuterium crystal, while the open circles are those from the empty cell. They indicate clearly the contribution from the very pure lattice structure composed of the c-direction of the hexagonal deuterium crystal, just as complete agreement with our expectation to the crystal orientation.The typical time-of-flight spectrum measured is shown in Principally, the experiments of UCN production and measurement should be planned with the incident neutron beam as intense as possible since the UCN production rate per incident cold neutron is very small even for the supposed best UCN converter material. However, unfortunately in the present time we must collimate our incident beam very severely to minimize the leakage radiations in order to maintain very low background radiation level in our neutron guide hall to aviod possible influence to neighboring experiments. Actually, our chopper duty factor was about 1/100, and the beam cross section and the divergence reduced to about 1/50 with a series of slits. Our experimental arrangement of the UCN production and measurement after such beam tailoring is shown in 8 n/cm2s and the reduction factors mentioned above, and further with the velocity factors on the detectable UCN.Both kinds of the discriminations gave very similar results on the UCN countrate. One of such results obtained on the converter at 11 K for the time-of-flight discrimination is shown in Our present experimental situation of the source guide intensity would provide much higher UCN countrate sufficient to show obvious performance of the ortho-deuterium single crystal converter with preparing an improved shielding condition around the extension guide and the converter cell, and recovering the above mentioned reduction factors. Then, measurements on the crystal orientation and incident neutron energy dependences will be much interesting and important tasks for assuring the proposed concept of the ortho-deuterium single crystal UCN converter. Further progress to the continuous beam measurements will give the UCN countrate of about 300/s, enough high to perform a definite demonstration to store the produced UCN.Another possible utilization of the present concept with the ortho-deuterium single crystal will be the coupling of preferred crystal orientation with the space-dependent cold neutron premoderator spectrum, according to the strong dependence of the most effective incident neutron energy for UCN production on the relative angle between the crystal axes and the inflow direction of cold neutrons.


Oryctolagus cuniculus) as a reservoir for Coxiella burnetii in the Iberian region. High individual and population seroprevalences observed in wild and farmed rabbits, evidence of systemic infections, and vaginal shedding support the reservoir role of the European rabbit for C. burnetii.We studied the role of European rabbits ( The second scenario involved rabbits that had not been in contact with ruminants since 2002 (site C). Serum samples were collected from European wild rabbits in 13 locations in Spain, Portugal, and the Chafarinas Islands during 2003–2013 . Wild raIn addition to serum samples, spleen, uterus, and mammary gland samples and vaginal and uterus swab specimens were collected from rabbits surveyed at location LO. Each rabbit from this location was weighed and sexed. Serum samples were also collected from farmed rabbits on 4 farms in Spain . SamplesSerum samples were analyzed by using the LSIVet Ruminant Q Fever Serum/Milk ELISA Kit  and horseradish peroxidase–conjugated protein G  as secondary antibody . Swabs were incubated at 56°C for 30 min in 200 μL of AL buffer containing 20 μL of proteinase K. Swabs specimens were then vortexed for 15 s and removed. The remaining solution was incubated at 56°C for 30 min. The manufacturer’s blood extraction protocol was then used. DNA aliquots were frozen at −20°C. Negative controls  were included during DNA extraction.DNA samples were analyzed by using a conventional PCR , for the presence/absence of C. burnetii DNA in spleen (a proxy of systemic infection), and for the presence/absence of C. burnetii DNA in the reproductive tract . Location LO was included as a random factor, and sex, weight and ruminant presence/absence were also included as predictor variables . Prevalence-associated, Clopper-Pearson exact 95% CIs were estimated.>1 seropositive rabbit. The best model for C. burnetii exposure retained sampling year and season, and the risk for seropositivity was higher in summer (Serum samples from 572 rabbits (464 wild and 108 farmed)  were anan summer .C. burnetii DNA in vaginal swab specimens. Spleen PCR–positive rabbits were observed only at sites A and B ; it was slightly lower at site C than at sites A and B , panel A A and B , panel BC. burnetii DNA in spleen retained sampling year, season, presence of ruminants, and sex  of 64 female rabbits at sites A, B, and C (C. burnetii DNA in the reproductive tract (C. burnetii DNA.The best model for the presence of  and sex . Results and sex , panel BB, and C , panel Fve tract . None ofC. burnetii. These 3 results are high seroprevalence of this bacteria; systemic infections; and bacterial shedding in vaginal secretions, which, in other host species, constitutes the main source for environmental contamination and transmission between species (This study provides 3 results that suggest that European rabbits might be reservoirs of C. burnetii prevalence in livestock (C. burnetii. In addition, the European rabbit is a gregarious species with a high reproductive rate. This rate favors transmission of C. burnetii from infected to susceptible animals, which is enhanced by replacement of C. burnetii–negative rabbits and can contribute further to spread of this bacterium in the environment.Host density is a major factor in C. burnetii observed during the summer might be related to increased indirect interaction with C. burnetii shed by coexisting ruminants, whose main shedding season is late spring–early summer (C. burnetii.The higher risk of exposure to C. burnetii between rabbits, humans, livestock, and other wild species may be enhanced in regions with high-density rabbit populations and in regions in which the European rabbit is a major game or farm species. Hunters, game keepers, rabbit farmers, veterinarians, wildlife researchers, livestock producers and livestock might be exposed to C. burnetii from rabbits (,C. burnetii for infection of livestock and humans in Europe.Indirect transmission of


Current treatments mostly focus on the tight glycemic control and few of these treatments have been designed to directly recover the vascular dysfunction in diabetes. As a classical natural medicine, berberine has been explored as a possible therapy for DM. In addition, it is reported that berberine has an extra-protective effect in diabetic vascular dysfunction. However, little is known whether the berberine treatment could ameliorate the smooth muscle contractility independent of a functional endothelium under hyperglycemia. Furthermore, it remains unknown whether berberine affects the arterial contractility by regulating the intracellular Ca2+ handling of isolated cerebral VSMCs was investigated by recording the whole-cell L-type Ca2+ channel (CaL) currents, assessing the protein expressions of CaL channel, and measuring the intracellular Ca2+ in response to caffeine. Our results showed that chronic administration of 100 mg/kg/day berberine not only reduced glucose levels, but also inhibited the augmented contractile function of cerebral artery to KCl and 5-hydroxytryptamine (5-HT) in diabetic rats. Furthermore, chronic administration of 100 mg/kg/day berberine significantly inhibited the CaL channel current densities, reduced the α1C-subunit expressions of CaL channel, decreased the resting intracellular Ca2+ ([Ca2+]i) level, and suppressed the Ca2+ releases from RyRs in cerebral VSMCs isolated from diabetic rats. Correspondingly, acute application of 10 μM berberine could directly inhibit the hyperglycemia-induced CaL currents and suppress the hyperglycemia-induced Ca2+ releases from RyRs in cerebral VSMCs isolated from normal control rats.Sprague–Dawley rats were used to establish the diabetic model with a high-fat diet plus injections of streptozotocin (STZ). Berberine  were intragastrically administered to control and diabetic rats for 8 weeks since the injection of STZ. The intracellular Ca2+ handling of smooth muscle cells.Our study indicated that berberine alleviated the cerebral arterial contractility in the rat model of streptozotocin-induced diabetes via regulating the intracellular Ca W. WL chanCON Fig. a, which Experiment II and Experiment III, chronic treatment with 50 mg/kg/day berberine for 8 weeks had no obvious effects on CaL channel function in CON and diabetic rats, respectively. However, 200 mg/kg/day berberine for 8 weeks significantly inhibited CaL channel function in both CON and diabetic rats  for 8 weeks significantly reduced the augmented α1C-subunit expressions of CaL channel in cerebral arteries of diabetic rats. However, there was also a significant difference in the α1C-subunit expressions of CaL channel in cerebral arteries between Diabetic + berberine and CON rats, which indicated that berberine treatment did not restore the α1C-subunit expressions of CaL channel in cerebral arteries of diabetic rat to the normal control level. When the control rats were treated with berberine (100 mg/kg/day), there was no significant difference in the α1C-subunit expressions of CaL channel in cerebral arteries between CON + berberine and CON rats. These results suggested that berberine treatment significantly reduced the augmented α1C-subunit expressions of CaL channel in cerebral arteries isolated from diabetic rats.The apparent masses of 240 kD band were shown in the membrane, which correspond to the predicted sizes of αarteries . As the 2+]i which represents the Ca2+ release from the SR. Representative dot graph of Fluo-3/AM fluorescence recorded showed the typical transient increases of intracellular Ca2+ fluorescence intensity evoked by 10 mM caffeine  for 8 weeks not only significantly decreased the resting [Ca2+]i level, but also markedly suppressed the maximal increases of [Ca2+]i in cerebral VSMCs isolated from diabetic rats. However, there were also significant differences in the resting [Ca2+]i level and the maximal increases of [Ca2+]i in cerebral VSMCs between diabetic + berberine and CON rats, which indicated that berberine treatment did not restore the resting [Ca2+]i level and the Ca2+ releases from RyRs in cerebral VSMCs of diabetic rats to the normal control level. When the control rats were treated with berberine (100 mg/kg/day), there was no significant difference in the resting [Ca2+]i level and the Ca2+ releases from RyRs in cerebral VSMCs between CON + berberine and CON rats. These results indicated that berberine treatment not only markedly decreased the increased resting [Ca2+]i level, but also suppressed the augmented Ca2+ releases from RyRs in cerebral VSMCs isolated from diabetic rats.As previously described , a high ine Fig. A. Summarine Fig. B. We invin VSMCs , 5. The Experiment II and Experiment III, chronic treatment with 50 mg/kg/day berberine for 8 weeks had no obvious effects on maximal increases of Ca2+ fluorescence intensity in CON and diabetic rats, respectively. However, 200 mg/kg/day berberine for 8 weeks significantly inhibited maximal increases of Ca2+ fluorescence intensity in both CON and diabetic rats  signifieviously ) markedl2+ releases from RyRs diabetic rats is mainly due to a direct effect of berberine or only a secondary consequence of the lowing blood glucose in response to berberine treatment, we investigated the acute effects of berberine on the Ca2+ releases from RyRs in cerebral VSMCs isolated from normal control rats under high glucose condition. After the load of Fluo-3/AM, the isolated VSMCs were incubated with 20 mM d-glucose and the combination of 10 μM berberine and 20 mM d-glucose for 10 min at 37 °C, respectively. As shown in Fig. d-glucose significantly increased the resting [Ca2+]i level . This Ca2+ event not only contribute to the overall rise in the concentration of intracellular Ca2+, but also in turn activate nearby Ca2+-activated K+ (KCa) channels in plasma membrane, leading to membrane hyperpolarization, inhibition of CaL channels, and thereby favoring vasodilation by reducing the Ca2+ influx. In the present work, the rat model of STZ-induced diabetic rats showed a higher blood glucose level  i) , 5, up-r channel , and altthe RyRs , 24. It c SD rat . Howeverc SD rat . In addic SD rat .Fig. 8AcBerberine  is an isoquinoline alkaloid, which is extracted from berberine-containing herbs, such as Goldenseal (Hydrastis canadensis), Oregon Grape (Berberis aquifolium), and Coptis chinensis (Chinese goldthread) . It has L channel current densities (Fig. 1C-subunit expressions of CaL channel (Fig. 2+]i level, but also suppressed the augmented Ca2+ releases from RyRs (Fig. L currents (Fig. 2+ releases from RyRs (Fig. 2+ handling of VSMCs in diabetic rats or when exposed to hyperglycemia condition.In the present work, chronic administration of 100 mg/kg/day berberine not only significantly reduced glucose levels in diabetic rats (Table ies Fig. , but alsnel Fig.  in cerebyRs Fig.  in cerebnts Fig.  and suppyRs Fig.  in cerebL peak current density (Fig. 2+ fluorescence intensity (Fig. L peak current density (Fig. 2+ fluorescence intensity (Fig. L channel and [Ca2+]i.From our results, chronic administration of 50 mg/kg/day berberine had no obvious effects on contractile responsiveness Fig. , CaL peaity Fig. , and maxity Fig.  in CON aity Fig. , and maxity Fig.  in both 2+ handling with their related proteins in VSMCs. In addition, the safety and efficacy of berberine have been generally accepted as a kind of traditional Chinese medicine. It is believed that berberine performs a good clinical practice and our study provided new evidence that application of berberine is a novel potential application in the treatment of diabetic vascular dysfunction.It has been reported that several therapeutic strategies are administered to ameliorate the deteriorated vascular function in diabetes, such as increasing the endothelium-dependent NO production, reducing the oxidative stress damage, inhibiting the inflammatory responses , 8. Our L channel significantly increased in the rat model of STZ-induced diabetic rats (Figs. L channel currents significantly reduced in STZ-induced diabetic rats [2+ releases from RyRs significantly increased in the diabetic rats (Fig. 2+ release channels in the SR. Activation of IP3Rs could also evoke the localized Ca2+ transients and contribute to the global elevation of intracellular Ca2+. Thus, it is necessary to study the role of IP3Rs in the berberine treatment for the diabetic vascular dysfunction. Third, we observed that berberine inhibited the function of CaL channel in the present work. However, the present work did not study the underlying mechanism. It is believed that there are three subclasses of calcium channel blockers which are wildly used to block CaL channel by binding to the poreforming α1C subunit, such as dihydropyridine, phenylalkylamine, and benzothiazepine. Whether berberine inhibited the function of CaL channel by directly binding to poreforming α1C subunit or other indirect pathway needs the further investigation.First, there are some disparities between our work and other previous reports. The present work showed that the function of Cats Figs. , 5 or whts Figs. , which atic rats . In addiats Fig.  or when ats Fig. , which aL channels, but also suppressed the function of Ca2+ releases from the RyRs of cerebral VSMCs in diabetic rats or when exposed to hyperglycemia condition. Taken together, our study indicated that berberine alleviated the cerebral arterial contractility in the rat model of streptozotocin-induced diabetes via regulating the intracellular Ca2+ handling of smooth muscle cells.The present work showed that berberine treatment could alleviate the cerebral arterial contractility independent of a functional endothelium in the rat model of streptozotocin-induced diabetes. In addition, the berberine treatment not only directly inhibited the function of Ca


The objective of this study was to investigate the role of intracellular calcium overload in the in vitro apoptosis of C6 glioma cells mediated by low level ultrasound and hematoporphyrin monomethyl ether (HMME) therapy. The frequency of ultrasound was optimized by the cell viability assay using 3--2,5-diphenyltetrazolium bromide (MTT). The apoptotic rate, reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP) were determined by flow cytometry. Morphological changes were observed by the transmission electron microscope. Concentrations of intracellular Ca2+, [Ca2+]i were detected by a confocal microscopic laser scanning, and the release of cytochrome-c (cyt-c) was measured by western blotting. Results: The SDT-mediated apoptotic effect involved an overload of [Ca2+]i derived from the intra- and extracellular sources during the early progression of apoptotosis. The process was associated with an increased ROS production, a decreased MMP, and a release of cyt-c. In conclusion,the combined use of low level ultrasound and HMME improved the apoptotic rate of C6 glioma cells mediated by ultrasound alone. The [Ca2+]i overload involving activation of mitochondrial signaling played a pivotal role in the SDT-induced apoptosis. Various auxiliary therapies, including radiation, chemo, and photodynamic are used for the treatment of glioma ; however, the median survival rate remains far below the desired outcome . The ultApoptosis is controlled by many factors, such as reactive oxygen species (ROS), calcium overload, and mitochondrial damage –12. CalcAccumulating evidence has demonstrated that the SDT-induced apoptosis of C6 glioma cells is mediated through an excessive production of ROS incurred by the interaction of the ultrasonic cavitation and the sensitizers , 13. TheAccordingly, we hypothesized that treatment of the C6 glioma cells with low level ultrasound in combination with HMME might promote apoptosis by increasing the concentration of [Ca2+]i. It is also possible that increased levels of [Ca2+]i may trigger the production of ROS, decrease mitochondrial membrane potential (MMP), and release of cytochrome c (cyt-c). We determined the apoptotic rate and the concentration of [Ca2+]i in SDT- treated glioma cells. The L-type Ca2+ channel antagonist nimodipine was used to find out as to whether the source of [Ca2+]i overload was extracellular.The C6 glioma cell line was purchased from the Beijing Institute of Biology, Chinese Academy of Science ; 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), Rhodamine 123 and fluo-3/acetoxymethylester were purchased from Sigma . FITC-Annexin-V/PI and HBSS were purchased from Beyotime . Nimodipine was purchased from Bayer (Germany). HMME was purchased from Kangmei Chemical Co. Ltd. . Polyclonal antibodies against cyt-c were from purchased from Santa Cruz .The C6 glioma cells were cultured in RPMI 1640 containing 10 % fetal bovine serum (FBS). The cells were maintained at 37 °C in a humidified atmosphere containing 5 % CO2. Once grown to 70–80 % confluence, the cells were trypsinized, counted, and seeded in 6-well plates at a density of 1 × 106/ml cells per well for the subsequent experiments.2) was applied for a duration of 60 s. The ultrasound frequencies varied from 0 to 1.0 MHz. After this treatment, the cells were trypsinized and transferred to 96-well plates and incubated with 0.5 mg/ml of MTT  at 37 °C for 4 h. Then the medium was removed and 200 μl of dimethylsulfoxide  were added. The absorbance was read at 490 nm with a universal microplate spectrophotometer (BIO-RAD Model 550). The cell viability at 0 MHz was considered as control, and the others were expressed as percentage of control. Cell viability was statistically analyzed to select the optimal frequency for subsequent ultrasound treatments.To optimize the ultrasound frequency, the cell viability assay using 3--2,5-diphenyltetrazolium bromide  was carried out as described previously . Cells wThe ultrasound and SDT treatment of the C6 glioma cells were conducted as previously described i was observed to be further elevated with SDT treatment of the cells in HBSS containing calcium (408 ± 11.6 nM). Compared to the controls, it was significant (P < 0.05). Also, addition of nimodipine to HBSS was not found to augment the SDT-mediated concentration of [Ca2+]i.The concentration of intracellular free calcium was measured in 1800 s by confocal laser scanning microscopy in a single cell using fluo-3/acetoxymethylester, the fluorescent probe sensitive for Ca2+. The concentration of [Ca2+]i was significantly increased in the cells treated with ultrasound in PBS or HBSS, with or without nimodipine. In the cells treated with SDT, the concentration of [Ca2+]i was further enhanced and it was statistically significant (P < 0.05) in apoptotic rate compared to that in PBS  or treated with HMME, ultrasound, and SDT in calcium-free PBS or in HBSS containing calcium. The SDT and ultrasound treatments in HBSS showed a significant elevation  by ultrasound and SDT treatment  in MMP  up-regulated in the cells treated with ultrasound or SDT in both PBS and HBSS .Since MMP reduction in apoptotic cells leads to the release of cyt-c from the mitochondria, we measured the levels of this protein by Western blotting in the SDT-treated cells. The release of cyt-c was found to be significantly (BSS Fig. . The rel2 are considered as low i flux from both extra and intracellular sources. Furthermore, an impairment of MMP accompanied with increased cyt-c release suggests the contribution of mitochondrial signaling in the apoptotic effect of SDT.


Penicillium chrysogenum antifungal protein PAF is toxic against potentially pathogenic Ascomycetes. We used the highly sensitive aequorin-expressing model Aspergillus niger to identify a defined change in cytoplasmic free Ca2+ dynamics in response to PAF. This Ca2+ signature depended on an intact positively charged lysine-rich PAF motif. By combining Ca2+ measurements in A. niger mutants with deregulated cAMP/protein kinase A (PKA) signaling, we proved the interconnection of Ca2+ perturbation and cAMP/PKA signaling in the mechanistic function of PAF. A deep understanding of the mode of action of PAF is an invaluable prerequisite for its future application as new antifungal drug.The  Our knowledge about their mode of action, however, badly lacks behind, although scientists put major efforts into their characterization. This phenomenon may be based on variable degrees in similarity concerning primary sequence, solution structure, conformational dynamics, mechanistic function and antifungal spectrum 2+, both second messengers, allow the integration of information originating from multiple upstream inputs and enable quick transmission of signals through the cell. The interaction of cAMP mediated signaling and cytoplasmic Ca2+ has been intensively studied in animal, plant and yeast cells (reviewed in The cyclic nucleotide cAMP and cellular CaC) and two regulatory (PKAR) subunits forming a heterodimer. Upon activation, four molecules of cAMP bind to the PKAR subunits resulting in the release of PKAC, which phosphorylates down-stream targets. The PKA activity regulates most diverse cellular processes, e.g. morphology, hyphal growth, conidiation, virulence, pathogenicity and dimorphic switching PKA consists of two catalytic . The unique signature of the [Ca2+]c change determines the specificity in the signaling response that regulates a wide range of processes like hyphal tip growth, branching, differentiation, cell cycle, stress response and virulence Ca2+ homeostasis in response to the antifungal compound PAF as independent events in two different model organisms 2+ homeostasis in response to PAF by using one sensitive model organism.So far, we were able to observe the involvement of the cAMP/PKA signaling cascade and the perturbation of the CaAspergillus niger mutant test system previously characterized and described by A. niger strains lack the catalytic subunit (strain ΔpkaC) or have a multiple copy integration of the catalytic and the regulatory subunit (mcpkaCR). The mutant mcpkaCR contains an equal copy number (10 copies) of the genes pkaC and pkaR, which are simultaneously over-expressed. The activity of PKA was reported to be 6 times higher in this mutant than in the wt control . PKA activity is, however, still under the control of PKAR and cAMP pkaC has no detectable PKA activity 2+ sensitive photo-protein aequorin for the determination of the [Ca2+]c signature in response to external stimuli To achieve our objective we took advantage of the elegant 2+]c in response to PAF treatment are interconnected and regulate PAF toxicity. Our study further underline that this mechanistic function of the antifungal protein PAF is common in sensitive fungi.Our investigations proved for the first time our hypothesis that cAMP/PKA signaling and the sustained elevation of c and the determination of the [Ca2+]c signatures were performed as described in 33.1A. niger strains with different PKA activities to increasing concentrations of PAF and determined their growth on solid medium. To this end we used the aequorin-expressing mutants ΔpkaC and mcpkaCR and included also the strains ΔpkaCR and mcpkaC without aequorin expression to investigate the role of the regulatory subunit of PKA in PAF toxicity. Under control conditions (no PAF) the PKA over-expressing mutants, mcpkaC and mcpkaCR, showed similar growth and development as the wt, whereas the PKA deletion strains ΔpkaC and ΔpkaCR exhibited smaller colony diameters compared to the wt and the mcpka mutants , both multi-copy mutants were similarly susceptible to the antifungal protein as the wt, showing reduced colony diameters and delayed conidiation . Furthermore, neither the additional deletion nor the increased copy number of the regulatory subunits in the mutants, ΔpkaCR and mcpkaCR, respectively, resulted in any additional visible effects on the susceptibility of A. niger to PAF compared to the single mutants ΔpkaC and mcpkaC. This indicates that PKAC plays a dominant role over PKAR in the PAF-specific response. The asexual development of the ΔpkaC and ΔpkaCR strains, however, was negatively affected by PAF and seemed not to be under the direct control of PKA activity under the test conditions applied. On the other hand, we have to note here that deregulated expression of PKA may trigger so far undefined rescue mechanisms that result in enhanced conidiation at low PAF concentrations (50 μg/ml) as observed with the mutants mcpkaC and mcpkaCR. However, at high PAF concentrations (200 μg/ml) these mechanisms may not be efficient enough to overcome the toxic effect in both mutants.Our data therefore indicate that the lack of the catalytic subunit of PKA rescued A. niger strains ΔpkaC and mcpkaCR did not influence the sensitivity toward PAF (A. niger wt and the untransformed wt strain (data not shown).For completeness we note here that the expression of recombinant aequorin in the ward PAF , Table 23.22+ response to PAF, we used the aequorin-expressing A. niger wt strain. When exposing 12 h old A. niger wt germlings to PAF (0–400 μg/ml) a significant, PAF-concentration dependent and sustained elevation of the intracellular Ca2+ resting level could be observed (2+]c resting level of untreated samples was 0.04 μM (S.D. ⩽ 10%). When applying 400 μg/ml PAF, the [Ca2+]c reached 0.23 μM (S.D. ± 0.02) within the first five min before it decreased within the next five min to remain elevated at approx. 0.15 μM (S.D. ⩽ 10%) for the duration of measurement (30 min). Instead, the intracellular Ca2+ resting level of the untreated control remained at 0.04 μM (S.D. ⩽ 10%). Notably, we used up to 400 μg/ml PAF to monitor the Ca2+ response because fungal germlings are less sensitive to PAF than conidia 2+ response is PAF specific, we exposed the wt strain to the protein variant PAFK35A/K38A. This recombinant PAF variant carries the exchange of two lysine residues (K) at the positions 35 and 38 of the mature protein for two alanines, respectively, and was expressed in P. pastoris. For internal control, a recombinant PAF wt protein (mPAF) was produced in P. pastoris as well. In liquid growth inhibition assays mPAF exhibited a similar activity against A. niger as PAF, whereas the PAFK35A/K38A variant was significantly less active . In this mutant, PAF failed to trigger a specific Ca2+ response and the [Ca2+]c resting level of the treated sample remained at the level of the untreated control  for the time of measurement with no significant relative rise in Ca2+ (0.7% change to control)   . In contctively) . Neverth2+ channel activity is regulated by PKA-dependent phosphorylation pkaC mutant revealed that Ca2+ signaling was impaired and the [Ca2+]c kinetics in response to mechanical perturbation was significantly reduced 2+ resting level even without PAF challenge and PAF was unable to elicit a specific Ca2+ response. However, increased activation of Ca2+ channel activity in the mcpkaCR mutant did not further augment the Ca2+ response to PAF. We therefore hypothesize that PAF might itself directly or indirectly interfere with Ca2+ channel activity. However, deregulation of the PKA signaling might have other/additional effects on the Ca2+ homeostasis and explain the differences in strain-specific susceptibility for PAF: (i) The amount and composition of specific Ca2+ channels/pumps/transporters of the PKA mutants might be different to the wt strain, ultimately affecting the dynamics of the Ca2+ response. (ii) Jernejc and Benčina A. niger: PKA mutants exhibited differences in the lipid composition. The mutant lacking PKA activity had an increased content of total lipids and a 30% reduction in phospholipids, whereas the mutant with increased PKA activity showed basically a similar lipid content as the wt strain. This was suggested to affect the permeability and fluidity of the plasma membrane and consequently to have impact on the distribution and activity of Ca2+ channels/pumps/transporters 2+ channels/pumps/transporters. Some fungi contain two or more catalytic PKA subunits with overlapping or distinct functions A. nidulans PkaBC, has a congruent as well as opposite function to PKAC in growth, conidiation and germination depending on the nutrient availability A. niger genome indeed contains the annotated gene An07g05060 coding for a putative orthologue of the A. nidulans PkaB (AN4717), the Aspergillus fumigatus PkaC2 (Afu5g08570) and the Aspergillus oryzae PkaB (AO090120000207). The function of PkaB in Ca2+ signaling, however, has not been characterized so far and awaits detailed investigation in the future.It had been reported previously, that the Ca42+ and cAMP/PKA signaling in A. niger in response to the antifungal protein PAF. Our data indicate that the fast and sustained increase of the [Ca2+]c resting level is directly linked with PAF toxicity and is specific to a functional protein: the protein variant PAFK35A/K38A is less active and does not elicit a PAF-specific Ca2+ signature. Furthermore, activation of the cAMP/PKA signaling cascade is required for PAF activity and is closely connected to Ca2+ signaling. The A. niger mutant defective in cAMP/PKA signaling and Ca2+ response was more resistant toward PAF than the wt strain.This study provides for the first time new insights into the interrelation between Ca2+ signaling in mediating the toxicity of antifungal proteins has been reported in former studies that investigated the mode of action of PAF and the plant defensins MsDef1 and MtDef4 in N. crassaAspergillus giganteus antifungal protein AFPNN5353 in A. niger2+ homeostasis by antifungal proteins like PAF is a conserved mechanism common to sensitive fungi. Considering the fact that PAF is harmless for mammalian cells, in vitro 2+ homeostasis seems to be specific to fungal cells. The antifungal protein PAF, therefore, represents a promising molecule to develop new antifungal strategies to prevent and combat fungal infections.The prominent role of Ca


Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature . However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM  and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.Spatio-temporal dynamics of intracellular calcium, i dynamics throughout the cell cross-section at a much higher resolution than previously possible. We estimated the size of structural maladaptation that would cause disease-related alterations in [Ca2+]i dynamics. Using our methods for data integration, we also tested whether reducing the density of RyRs in human cardiomyocytes (~40% relative to rat) would have a significant effect on [Ca2+]i. We found that Ca2+ release patterns between the two species are similar, suggesting Ca2+ dynamics are robust to variations in cell ultrastructure.Calcium (Ca This Ca2+ triggers the opening of a set of ion channels located on the SR called the ryanodine receptors (RyRs) causing a large flux of Ca2+ to enter the dyadic cleft from the SR. Finally, the resulting increase in intracellular Ca2+ concentration ([Ca2+]i) activates the surrounding contractile machinery, thus effecting a contractile response.The cardiac myocyte possesses a highly organized assembly of membrane networks, contractile proteins, ion channels and buffering systems. The heartbeat is the result of tightly regulated electrical, chemical, and mechanical processes that repeatedly occur at the subcellular scale across the millions of cells that make up the heart—a phenomenon called excitation-contraction coupling (ECC) –3. ECC b2+ release and diffusion in the cardiac cytosol. Structural imaging using confocal, super-resolution, and electron microscopy [2+ dynamics that current [Ca2+]i imaging technologies cannot resolve i transient have been examined and used as a measure of t-tubule degradation  in many previous studies on structure-function relationships in cellular cardiology and heart failure [2+]i transient could result in reduced contractile power. Furthermore, cellular processes such as the calcineurin-NFAT pathway to cardiac hypertrophy, apoptosis, stress-response, and energy metabolism are [Ca2+]i dependent [2+]i transient—using biophysical equations to account for Ca2+ buffering and Ca2+ indicator-dye kinetics—and assessed two aspects of the relationship between cell structure and [Ca2+]i dynamics: (i) the effect of the realistic spatial organization of myofibrils, mitochondria, and RyR clusters on the spatial heterogeneity of [Ca2+]i; (ii) and the sensitivity of these effects to variations in RyR cluster organization that have been measured in previous experimental studies i, that we predicted computationally, would be visible in simulated line-scan images.We further demonstrate the power of this structurally realistic model in systematically assessing the degree of heterogeneity that can be introduced in the cell-wide Ca2+]i can partly be caused by the non-uniform distribution and clustering of mitochondria in relation to the myofibrils and the sarcolemma; (ii) spatial heterogeneity in [Ca2+]i can also be caused by localized aggregation of RyR clusters observed in the structural data; (iii) variations in RyR cluster organization could give rise to observable differences in line scan data; and (iv) differences in the spread and density of RyR clusters between different mammalian species (as reported in the literature) have only a marginal effect on [Ca2+]i at the peak of the transient, thus highlighting the robustness of the ECC system to structural variations in RyR cluster distributions. Furthermore, our mathematical model showed that in order to observe a reduction in regional fluorescence in line scan images , RyR clusters within a 1.29 μm diameter neighborhood (corresponding to 4 to 6 RyR clusters) must remain un-activated in an action-potential evoked transient.Our model showed that: (i) spatial heterogeneity in [Ca2+]i simulations on the hybrid-scale spatial model that were analyzed to derive the above stated findings. The basic study design and the biophysical model are outlined in the materials and methods. The method for generating the hybrid model and the biophysical equations for the simulations are detailed in 2+]i simulation codes available at our publically accessible repository: https://github.com/vraj004/cardiac_ecc. We have also made the RyR cluster simulation algorithm and the experimental data that were used to develop the algorithm and simulations available at: https://github.com/vraj004/RyR-simulator. We encourage cardiac computational scientists to make use of our resources in their research of cardiac biophysics at the subcellular scale. We conclude the study with a discussion of the limitations of the current analysis and future directions.The following sections present the results of the [Ca2+ release kinetics that have helped develop several quantitative models of Ca2+ biophysics [2+]i; current spatially extended models of cardiac cell structure use simplified representations of the organization of myofibrils and mitochondria [2+]i dynamics in a geometric model that was neither limited by the spatial resolution of confocal microscopy (with regards to the contractile machinery and mitochondria), nor by the lack of contrast and small field of view that is inherent in electron microscopy . We developed a novel algorithm that generates realistic confocal-scale RyR cluster distributions around nanometer-resolution 3D templates of myofibril and mitochondrial organization acquired using 3D electron tomography. This approach enabled us to create a hybrid-scale spatial model of the distribution of myofibrils, mitochondria, and RyR clusters  between each of the cells we studied see . These m±0.16 μm We tested the hypothesis that an algorithm will generate RyR cluster distributions that are similar in characteristics to experimentally measured spatial distributions observed in a given cell, provided that the simulated nearest-neighborhood distribution and the number of RyR clusters matched the experimentally measured values in the cell  Figs.Given, from (1), that the nearest-neighborhood distance and number of clusters are consistent across cells, we also showed that these two parameters were sufficient to recapitulate distributions of RyR clusters in all of the cell geometries we had imaged using the confocal microscope; the specific distribution of myofibrils within each cell does not affect the accuracy of the RyR cluster simulation.Three key observations could be made from the analysis of the spatial distribution of RyR clusters and the development of the algorithm that enable simulations of realistic distributions of RyR clusters on any given organization of myofibrils and mitochondria:2. With an average density of 1.3 couplons/μm2 of cell-cross-section  and Ca2+ bound Fluo-4 (F4Ca) were also simulated as coupled partial differential equations to simulate Ca2+-dye binding kinetics and diffusion. Troponin C was distributed homogeneously through the myofibrillar space and acted as a stationary buffer of the diffusing Ca2+. We restricted the simulations to incorporate minimal RyR cluster gating kinetics  and minimal Ca2+ buffering components to explore the sensitivity of [Ca2+]i dynamics to structural organization alone. The simulations were conducted for the first 30 milliseconds (ms) of the ECC cycle, representing the rising phase of the Ca2+ transient. The remaining, re-uptake phase of the ECC cycle was not considered in this study because our aim was to explore the role of the spatially realistic organization of the included components of the cell; modeling the re-uptake phase would require, among other things, a realistic distribution of sarco-endoplasmic reticulum calcium pumps (SERCA). The initiation of release from each RyR cluster was stochastically varied to simulate the experimentally observed stochasticity in the coupling latency between RyR clusters and L-type Ca2+ channels [2+]i and other buffer components are outlined in Details of the computational model and the biophysical equations are described in Materials and Methods and channels ,37. The 2+]i and [F4Ca]i at the z-disc of the half-sarcomere model. 2+]i transient rises to approximately 1 μM and the relative F4Ca transient (F/F0) rises approximately 5-fold. These values are within the range of many experimental measurements of Ca2+ transients in healthy rat ventricular myocytes found in the literature i has range of values between 0.3 and 12.7 μM; the F4Ca F/F0 signal ranges from 3 to 9.5. Careful examination of the 30 ms snapshots of the [Ca2+]i and [F4Ca]i signals shows several features that give rise to “hot-spots”. Some hot-spots occur due to the close proximity of several RyR clusters (red arrows), while others occur due to Ca2+ release into regions bounded by clusters of mitochondria (black arrows).2+]i (middle) and [F4Ca]i (right) taken along the line-position on the left figure. The peak [Ca2+]i of ~10 μM is close to a release site and is of a magnitude similar to that reported in the literature previously i and [F4Ca]i at 30 ms was also a function of the stochasticity in RyR cluster triggering. With the given RyR cluster distribution and mesh in i (and [Ca2+]i not shown) is not dependent on the release times of the RyR clusters. Instead, the spatial pattern is characteristic of the organization of the cell components in the mesh.We examined whether the spatial pattern of [Ca2+]i transient. 0 signal at 30 ms (A) with the default simulation settings; (B-C) with mitochondrial regions replaced with cytosolic properties; and (D) with RyR clusters being forced to have a nearest-neighbor at the mean nearest-neighborhood distance that was measured experimentally, but with zero variance as opposed to being distributed with nearest-neighborhood distances of the experimentally measured mean and variance. Note that (C) shows (B) without the mitochondria rendered and clearly shows the increase in homogeneity in the F/F0 signal. The red points depict the distribution of RyR clusters that are similar in characteristics to experimental data. (D) shows the effect of imposing a regular distribution of RyR clusters—by way of positioning nearest-neighbors of every cluster at a fixed distance. The encircled regions show that the F/ F0 signal is less intense where RyR clusters are less closely positioned. These results thus confirm that structural heterogeneities in the cell can introduce spatial heterogeneities in [Ca2+]i.We finally illustrate the impact that heterogeneous distributions of mitochondria and RyR clusters have on the heterogeneity of the [Ca2+]i release and diffusion in the absence of Fluo-4. 2+]i increases and the heterogeneous distribution of [Ca2+]i is not greatly affected. However, clusters of RyRs that are in close proximity of each other amplify the hot-spots more because of the lack of buffering by Fluo-4.Similar to previous studies , we exam2+]i. 2+]i transient, but affect where “hot-spots” in [Ca2+]i arise. As such, the RyR cluster algorithm provides valid RyR cluster distributions. Furthermore, the result shows that the distribution of RyR clusters (in addition to organization of mitochondria) also affect the spatial pattern of [Ca2+]i. These results have not been captured in previous modeling studies i due to the RyR cluster distribution. 2+]i, but had no effect on the range of [Ca2+]i in the cytosol. As such, [Ca2+]i is robust to variations in RyR cluster organization observed within our experimental data of RyR cluster distributions in adult rat ventricular myocytes.We have demonstrated how our method for generating realistic distributions of RyR clusters enables us to examine heterogeneities in [Ca2+]i.A previous comparison of RyR cluster distributions in rat and human cardiomyocytes ,23 foundWe simulated a human-equivalent distribution of RyR clusters (87 clusters with a mean nearest-neighborhood distance of 0.79 μm) within the electron-tomogram template distribution of myofibrils and mitochondria from the Wistar rat myocyte; the comparative studies between rat and human myocytes showed that the volume fraction of mitochondria and myofibrils were not different between the two species [2+ release and diffusion was simulated on this human-type myocyte to explore [Ca2+]i heterogeneity. Ca2+ release current from each of the 87 clusters was increased to 2.8 pA to compensate for the drop in the number of clusters in the model and maintain a whole-cell average [Ca2+]i of 1 μM that is also measured in human myocytes [2+] release is also consistent with recent experimental findings that the cardiac Ca2+ store and release systems maintain long-term balance in Ca2+ through alterations to SR Ca2+ content and the strength and duration of Ca2+ release from RyR clusters i (top panel) and Fluo-4-bound Ca2+, [F4Ca]i (bottom panel) in the rat (left column) and human (right column) models. The range of [F4Ca]i and [Ca2+]i are the same in both species and although the locations of local hot-spots may be in different regions in the two models, the spatial profiles are similar. This analysis illustrates the robustness of [Ca2+]i dynamics to a significant change in RyR cluster distribution properties.2+]i at a much higher resolution than was previously possible and has given several important insights on the role that structural organization can play in affecting [Ca2+]i in health and disease. We discuss some of the key advances and findings from this study in the following sections.We have presented a novel hybrid-scale spatial model of the organization of clusters of ryanodine receptors around cardiac cell myofibrils and mitochondria. The model is spatially realistic, owing to a novel computational fusion of 3D confocal microscopy data of RyR clusters with a 3D electron tomogram of myofibrils and mitochondria acquired from tissue samples of Wistar adult rat ventricular myocytes. This model was used to examine the spatio-temporal dynamics of i heterogeneity to the biophysical detail in RyR gating. As detailed in 2+ dependent RyR gating; sensitivity of RyR gating to Ca2+ in the junctional sarcoplasmic reticulum (JSR); and stochastic behavior of individual ion-channel gating i transient is homogeneous under control or normal physiological conditions. We have for the first time shown with a structurally realistic model, how the “normal” structural organization of RyR clusters alone can introduce spatial heterogeneities in the rising Ca2+ transient.These results, together with Figs 2+]i dynamics. A previous experimental analysis i between a simulated rat and a simulated human cardiomyocyte—defined by the different RyR cluster distributions measured in literature [2+ diffusion mechanism and the organization of myofibrils and mitochondria provide significant robustness to [Ca2+]i dynamics. In particular, a 40% drop in density of RyR clusters has virtually no effect on the spatial profile of [Ca2+]i. This comparison of the cluster distribution of human versus rat RyR is an important example of how parameterization of structure enables systematic analysis of the sensitivity of cardiac function to specific aspects of cell structure. Such analysis is not possible when generating a model from a single dataset.Our comparison see  of the sterature ,23–sugge2+, thus adding more robustness to the ECC system.Our simulations assumed that all RyR clusters were activated by the action potential. The impact of the difference in t-tubule organization between human and rat cardiomyocytes has not been thoroughly investigated in this study. However, the observation that t-tubules in the human myocyte are more coarsely arranged than in the rat myocyte is consistent with the more dispersed, and less dense RyR cluster parameters that we used to simulate human myocytes in this study . Previou2+]i. The ability to model RyR cluster distributions enabled us to explore an aspect of cardiac cell structure as a model parameter within experimental bounds. The degree of influence that this structural parameter may have on cell biophysics was systematically investigated with simplified and biophysically detailed models of [Ca2+]i dynamics and buffering.Our current analysis focused on the possible role that realistic distributions of RyR clusters, mitochondria and myofibrils could play in the spatio-temporal dynamics of [Ca2+]i transients are consistent with experimental records of Ca2+ in cardiomyocytes.Inherently, electron tomograms are low contrast, which can hamper the identification of boundaries between myofibrils. Being aware of the fact that myofibrils can bundle together and also twist through the cell volume , we segm2+ release and diffusion around realistic t-tubule geometries i dynamics in a structurally realistic three-dimensional topology.The binary image stacks of myofibril and mitochondria cover the entire cross-section of a cell, but only span 240 nm as explained above. Using the fact that RyR clusters predominantly localize to the z-disc and the observation that myofibrils follow straight paths over two or three sarcomeres , we comp2+ diffusion barriers and did not act as Ca2+ buffers; this assumption was based on recent experimental studies i dynamics simulations were conducted on the computational mesh using coupled reaction-diffusion equations for free [Ca2+]i, free Fluo-4-AM, [F4]i (a Ca2+- sensitive fluorescent indicator) and Fluo-4-bound-Ca2+ ([F4Ca]i). The role of myofibrillar Troponin C as an immobile buffer of Ca2+ was also incorporated. Mesh nodes that fell within a 200 nm diameter spherical neighborhood of simulated RyR cluster coordinates   and how the points of the pattern interact . The simplest model is the Poisson point process, where point locations are independent of one another.In most instances, point processes can be characterized by their λ(x) ≡ λ) is said to exhibit “Complete Spatial Randomness” (CSR). In this model, intensity (number of points per unit volume) does not vary spatially and there is no interaction between the points of the pattern. This is often the initial model fit when analyzing point patterns.The constant intensity Poisson process , is the probability that the shortest distance, d, from an arbitrary point u to a point of the point pattern X is less than or equal to r:  It is typically physically interpreted as a measure of empty space in the point pattern.The G function, G(r), is the probability that the shortest distance, d, from an arbitrary point u of the point pattern X to another point of X is less than or equal to r and is the cumulative nearest-neighborhood distance measure: G(r) = P{d ≤ r: u ∊ XThe λ is the average number of points per unit volume, B is a sphere centered on u with radius r, and E is the notation for the expected (or mean) number of points.The K-function  is derivThe pair correlation function (PCF) for a stationary point process is defined as as: A number of summary statistics exist for investigating departures from CSR. The four measures we use in this paper are:d. These bounds provide “envelopes” that can be used to determine whether there is evidence against the hypothesis that an observed point pattern could have been generated by the proposed point process model Using many instances of spatial point patterns that are simulated from a proposed point process model (termed Monte-Carlo simulations), one can then determine the maximum and minimum possible values of the above spatial measures out of all the simulated patterns for each radius neighborhood, rAs mentioned above, we performed an initial hypothesis test that the RyR clusters within the rectangular windows in reconstruction algorithm in spatial statistics theory, the method uses energy minimization techniques to match a target set of statistical measures of an observed point process i. Top plot shows rise in F/F0 signal in the bulk cytosol; the four panels below show the F/F0 signal at t = 30 ms for the four different patterns of RyR cluster triggering. Colour coding of panel images below corresponds with line colours in bulk F/F0 signal above. Tausim-2 and Tausim-3 had the same time course, thus Tausim-3 is plotted with blue crosses over the red curve representing Tausim-2.With a given RyR cluster distribution, four different triggering patterns were applied and executed to examine the effect of variations in stochastic trigger release on [Ca(EPS)Click here for additional data file.S5 Fig2+]i with corresponding 30 ms snapshot at the bottom when no Fluo-4 was present in the model.Top curve shows time course of [Ca(EPS)Click here for additional data file.S6 Fig0 signal at t = 30 ms with the default simulation parameters. (B) shows the F/F0 signal when freely diffusing Calmodulin and ATP are introduced as buffering species for Ca2+. The right-hand figure shows the distribution of calmodulin-bound Ca2+, CaMCa. The image shows that although the heterogeneity in the F/F0 signal may be reduced with calmodulin and ATP buffering, the heterogeneity of structure now introduces heterogeneity in buffered Ca2+. (C) shows the F/F0 and CaMCa signals are still heterogeneous when Ca2+ dependent RyR gating kinetics was introduced.The top profile (A) shows the distribution of the F/F(EPS)Click here for additional data file.S7 Fig0 at t = 30 ms with 4 to 6 RyR clusters un-activated, using the original model of Ca2+ release  shows a comparison of F/F(EPS)Click here for additional data file.S8 FigThis figure demonstrates the envelope-based hypothesis test to validate the sufficiency of proposed models of RyR cluster distributions. Black curves represent the measured F (top left), G (top right), K (bottom left) and PCF (bottom right) functions from the segmented RyR cluster distribution of cell 1 at the first z-disc. The shaded regions are the envelopes of possible values for each function if the RyR cluster distribution were modeled as completely spatially random see . The mea(EPS)Click here for additional data file.S9 Fig(EPS)Click here for additional data file.S10 Fig(EPS)Click here for additional data file.S11 FigThe measured F, G, K and PCF curves for cell 1 fit within the envelopes simulated by the proposed RyR cluster simulation parameters see . The hyp(EPS)Click here for additional data file.S12 FigThe envelopes for G and K show that RyR clusters simulated on Cell 4, using RyR distribution properties from Cell 1 also fit the RyR cluster distribution in Cell 4. The figure is representative of the other combinations of cells in which the same tests were conducted.(EPS)Click here for additional data file.S13 FigThe envelopes in this figure were generated using the nearest-neighborhood distribution properties and the number of clusters, without the myofibril radial distance distribution. The measured curves from the experimental data still fell within the model envelopes.(EPS)Click here for additional data file.S14 FigThe envelope for the G-function was generated by only enforcing a minimum distance between RyR cluster centroids that was equivalent to the confocal resolution. The model envelope does not fit the observed G-function.(EPS)Click here for additional data file.S1 Movie(MOV)Click here for additional data file.S2 Movie(MOV)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 TableThe cross-sectional area and depth are the dimensions of the field of view within the yellow rectangular windows in (DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableParameters were obtained from ,38,49,50(DOCX)Click here for additional data file.