Selective pharmacological inhibition of specific arms of the β2AR signaling network revealed the differential contribution of Gs-, Gi- and Gβγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca2+ in the impedance response and led to the discovery of a novel β2AR-promoted Ca2+ mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of β-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity.The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the β One considerable challenge in obtaining a full description of ligand activity at a given GPCR is the lack of knowledge about the complete signaling repertoire of most receptors. In addition, monitoring multiple signaling pathways can be time and resource consuming and involves the use of different assay formats with different sensitivity and dynamic ranges that can lead to spurious conclusions.An integrative approach that could capture the global signaling profile of a ligand in a single assay would therefore greatly facilitate the identification of biased ligands and enable their classification into pharmacologically relevant categories. Besides the potential impact on the drug discovery process, such an approach could also provide greater insight into how the pluridimensionality of GPCR signaling is integrated into an overall cellular response. A string of recent studies has explored the use of such integrative assays to study GPCR pharmacology using label-free, cell-based technologies developed to monitor real-time changes in higher-order cellular events such as morphology, viability, adhesion and mass distribution 2-adrenergic receptor (β2AR) as a model, we dissected the impedance responses from individual ligand-receptor pairs and associated the activation of specific signaling events with distinct features of the impedance curves. This dissection of the impedance responses led to the identification of a novel β2AR-mediated Ca2+ signaling event, highlighting the strength of such an integrative readout of cellular response to reveal biological processes even at a well-characterized target. Finally, we performed an unsupervised clustering analysis that permitted a rapid and systematic classification of compounds based on the impedance signatures obtained in both heterologous and primary cell cultures, demonstrating that impedance responses represent signatures that are predictive of the global signaling profiles of β-adrenergic ligands previously shown to display functional selectivity In the present study, we sought to determine if impedance signatures represent an integrative measure of the receptor signaling repertoire that can be used to differentiate among ligands with distinct signaling profiles. Using the β2AR (6HisHA-β2AR-HEK293S) following treatment with the prototypical β2AR-selective agonist isoproterenol (ISO). As shown in 2AR-specific since the response was blocked by the selective β2AR antagonist ICI118,551 (ICI) but was not affected by the β1AR-selective antagonist CGP20712A . In addition, the concentration-response curve describing the maximum impedance response elicited by ISO was progressively right-shifted with increasing concentrations of ICI, indicating a competitive antagonism of the response by β2AR blockade . Further supporting the β2AR specificity of the response, we observed a qualitatively similar ICI-sensitive impedance response in the parental HEK293S cell line, which endogenously express low amounts of β2AR . Although the impedance response maintains the same general shape, a decrease in the magnitude of the positive component of the impedance response and a larger transient negative phase was observed.We first sought to determine if impedance measurements could provide a composite representation of the multiple signaling events elicited upon receptor activation. To assess this possibility, impedance measurements were made in HEK293S cells stably overexpressing the human β2AR-HEK293S cell line to obtain a more robust response to receptor stimulation. The distinct features of the response  were differentially modulated by increasing concentrations of ISO, suggesting the contribution of distinct signaling events to discrete components of the impedance response. For instance, concentration-response curves describing either the maximum impedance response , ISO promoted a concentration-dependent increase in the magnitude of the minimum . These results further support the above data that the signaling events contributimg to the transient negative phase are fully activated with fewer stimulated receptors. As the stimulus strength (i.e. agonist concentration) or receptor number is increased, the impedance response becomes more positive, likely due to the engagement of another signaling event with a weaker stimulus-coupling. Altogether, these results suggest that the impedance response obtained reflects the integration of multiple signaling events which may be individually represented in the various components of the response.Further analyses of the impedance responses were performed in the 6HisHA-β2AR activation is known to elicit at least two distinct signaling events in HEK293S cells: cAMP accumulation via adenylyl cyclase (AC) stimulation and activation of the ERK1/2 MAPK pathway 2AR-HEK293S cells were pre-treated, prior to ISO stimulation, with the AC inhibitor SQ22536 to inhibit cAMP production or the MEK inhibitor U0126 to block ERK1/2 activation. Interestingly, both of these inhibitors led to similar decreases in the slope of the slow ascending phase and the maximum impedance response without affecting the transient negative phase or the rapid ascending phase . SQ22536 and U0126 were found to be pathway-specific since they blocked the ISO-stimulated accumulation of cAMP and activation of ERK1/2, respectively, and had no significant effects on the other signaling pathway , suggesting the contribution of additional Gs-dependent signaling events other than cAMP accumulation to the impedance response. Selective inhibition of Gi-dependent signaling with pertussis toxin (PTX) led to changes in the impedance response that were different from those caused by CTX , which only partially blocked ERK1/2 activation. Although the differences observed between acute pharmacological inhibition of cAMP production and ERK1/2 activation versus chronic inhibition of Gs and Gi suggest the contribution of alternative G protein-dependent signaling pathways to the impedance response, we cannot exclude the possibility that the chronic treatment with CTX and PTX may affect other downstream components involved in the response.From the above data, it is clear that cAMP production and MAPK activation are not sufficient to explain the entire ISO impedance response since a considerable residual signal is observed when both of these pathways are inhibited. To further explore the source of this residual response, we examined the role of the G proteins themselves. The βFigure S4C) indicating the existence of additional Gβγ-dependent signaling beyond ERK1/2.Next, we assessed the contribution of Gβγ subunits to the impedance response using the pharmacological inhibitor, gallein, which selectively inhibits interactions between Gβγ and effector molecules Altogether, these data demonstrate that a variety of G protein-dependent signaling events are integrated in the ISO-stimulated impedance response, including the activation of canonical second messenger pathways (i.e. cAMP and ERK1/2) and additional G protein-dependent events not yet identified.2AR would generate distinct impedance signatures. Impedance responses were obtained for a series of β-adrenergic ligands , which we confirmed by complete linkage hierarchical clustering After demonstrating that impedance responses represent the integration of multiple signaling events elicited upon receptor activation, we hypothesized that compounds with different signaling profiles at the βands  and S5 at To determine the relationship between ligand signaling profiles with the compound classes obtained in the above analysis of impedance signatures, we assayed each of the ligands for their ability to stimulate cAMP accumulation and ERK1/2 activation in conditions identical to those used for the impedance measurements  , reflecting a fundamental difference in the nature of the transient negative phase between Group I/II ligands and Group III/IV ligands. Indeed, Group I and II ligands have a shorter-lived transient negative phase than would be predicted given their amplitudes ( inset), suggesting that these compounds may selectively engage a signaling pathway that accelerates the rapid ascending phase thus shortening the duration of the transient negative phase. Given that many GPCRs are capable of activating pathways leading to an increase in intracellular [Ca2+], we assessed whether a β2AR-mediated Ca2+ response could be responsible for accelerating this phase. Consistent with this hypothesis, only Group I and II ligands elicited a significant increase in intracellular [Ca2+], with ISO and epinephrine (Group I) eliciting a much stronger Ca2+ response than salbutamol and salmeterol (Group II) . This β2AR-promoted increase in [Ca2+] was sensitive to the inositol trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB) and the intracellular Ca2+ chelator BAPTA-AM  Figure 52+] , which will lead to an increase in intracellular Ca2+ by preventing the reuptake of Ca2+, thus emptying the intracellular stores. Since the intracellular Ca2+ stores are already empty, ISO does not elicit an additional Ca2+ release following TG inhibition . Yet, in contrast with what was observed with 2-APB and BAPTA-AM, and despite the absence of a de novo Ca2+ release, the TG treatment does not inhibit the rapid ascending phase of the impedance response . In fact, it modestly potentiated the maximal response while slightly reducing the slope of the slow ascending phase. Taken together, these data support the notion that it is the intracellular [Ca2+] itself, more than the de novo Ca2+ release, that is the prime determinant of the ascending phases. The fundamental role of Ca2+ in the impedance response is further demonstrated by the effect of the Ca2+ ionophore A23187 on the impedance responses . As was the case in β2AR-expressing HEK293S cells, ISO induced a response that was completely abolished upon pre-treatment with the β2AR-selective antagonist ICI , salbutamol (Group II) and labetalol (Group III) were observed. Propranolol (Group IV) and ICI (Group V) generated distinct signatures from these other compounds, but could not be distinguished from each other in VSMCs  that indirectly affect the impedance response. Our analysis also provided evidence that both cAMP and ERK1/2 pathways converge on a common downstream effector that mediates their contribution to the impedance response, although the identity of this target remains unknown.Analysis of the β2AR stimulation was found to be extremely sensitive to inhibition of intracellular Ca2+ mobilization. Although the β2AR has previously been shown to increase intracellular [Ca2+] through modulation of L-type voltage-gated Ca2+ channels 3-gated release of Ca2+ from intracellular stores. Ca2+ is a well-established regulator of actin cytoskeleton dynamics 2AR stimulation and the impedance changes observed. Although we found this de novo Ca2+ release to contribute to the rapid ascending phase of Groups I and II ligands, the impedance responses of ligands that do not themselves induce a Ca2+ mobilization are also sensitive to a disruption of intracellular Ca2+ homeostasis, stressing the importance of Ca2+ in the cellular mechanisms that initiate an impedance response. We propose that the normal resting concentration of intracellular Ca2+ is necessary and sufficient to permit the cytoskeleton to respond to subsequent signaling events, such as cAMP production or ERK1/2 activation, and that ligands that induce an additional increase in intracellular [Ca2+] further sensitize the cell to these signaling inputs leading to an enhancement of the impedance response. Thus, the overall impedance signature of a given ligand truly represents the integration of the signaling pathways activated.The impedance responses generated following βs- and Gi-dependent signaling were integrated in the overall β2AR-promoted response. Moreover, the fact that the signatures obtained in VSMCs were dramatically different from those observed in 6HisHA-β2AR-HEK293S cells highlights the cell-type dependency of the impedance response, which likely reflects differences in the relative abundance of signaling molecules and/or mediators underlying the changes in cell shape and adhesion that give rise to the impedance responses. These observations highlight the inherent complexity of signaling systems and their measurement and demonstrate the diversity of cellular responses that can be generated by the stimulation of a given GPCR in different cell contexts. The ability to detect and dissect these complex cellular responses, as demonstrated in the present study, provides information concerning the full signaling repertoire of a receptor that can be more useful in drug discovery than the attempt to ascribe a single signaling pathway to a receptor.The contribution of multiple signaling events to the overall impedance response challenges some of the efforts that have been made to reduce such integrated responses to the engagement of a single G protein 2AR, demonstrating the existence of at least five groups of compounds with distinct impedance signatures. Previous work assessing the signaling profiles of β-adrenergic ligands towards cAMP and ERK1/2 pathways alone using endpoint-specific assays proposed the existence of 3 distinct compound classes The fact that multiple signaling events are integrated in the impedance response makes it an appealing platform to classify compounds based on their signaling repertoire. Clustering of the impedance responses generated by a series of β-adrenergic ligands revealed an unprecedented level of ligand texture at the βAlthough cell-based, integrative assays have begun to be employed in some screening applications Gallein was obtained from Tocris, U0126 from Cayman, and bucindolol was a generous gift from Dr. Michael Bristow . Cell culture reagents were from Wisent Incorporated. Other reagents were obtained from Sigma-Aldrich unless stated otherwise.2AR construct was generated as follows: The 6HisHA-β2AR gene was amplified by PCR from the previously described vector pCDNA3RSV-6HisHA-β2ARBsrGI and NheI restriction sites, respectively. The amplified 6HisHA-β2AR sequence was digested with BsrGI/NheI and then subcloned in a BsrGI/NheI-digested pIREShygro3 vector . The 6HisHA-β2AR-HEK392S polyclonal cell line was generated by transfecting the pIREShygro_6HisHA-β2AR construct in HEK293S cells 2AR-HEK293S stable cell line was found to express 1.07±0.14 pmol of 6HisHA-β2AR per mg of membrane protein as determined by a 125I-CYP radioligand binding assay. Cells were routinely grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. For Ca2+ assays and impedance assays in the presence of the MEK dominant negative mutant, 6HisHA-β2AR HEK293S cells were transiently transfected with the obelin biosensor The pIREShygro_6HisHA-βFigure S9). All data presented in figures represent means of at least three independent experiments.The xCELLigence system (Roche Applied Science) was employed to measure changes in cellular impedance following ligand stimulation Cells were plated, grown in 96-well plates and treated with ligand as described for the cellular impedance assay. cAMP accumulation was measured using the HTFR-cAMP dynamic kit (Cisbio). After 10 minutes of ligand stimulation, cells were lysed on ice in the lysis buffer provided by the manufacturer and immediately snap-froze at −80°C for at least 4 hours. After thawing on ice, 10 µl per well of lysate was transferred to a 384-well plate , and cAMP measurements were performed as per manufacturer's instructions.Cells were plated, grown in 96-well plates and treated with ligand as described for the cellular impedance assay. ERK1/2 activation was measured using the ERK1/2 AlphaScreen Surefire kit (Perkin Elmer). Cells were lysed on ice after 2 minutes of ligand stimulation in the lysis buffer provided by the manufacturer and placed immediately at −20°C overnight. After thawing on ice, 5 µl were transferred to 384-well ProxiPlates (Perkin Elmer) and ERK1/2 activation was measured as per manufacturer's instructions.2, 140 mM NaCl, 2.7 mM KCl, 900 µM MgCl2, 37 µM NaH2PO4, 5.6 mM d-glucose, 8.3 mM NaHCO3 and 12.6 mM Hepes, pH 7.5) and cells were incubated with 5 µM coelanterazine H  for 2 hours. Intracellular Ca2+ concentration is reflected by the magnitude of bioluminescence emitted by the obelin biosensor and data are expressed as relative luminescence units (RLU). Ligands were injected as indicated and bioluminescence was measured for 1 minute per condition using the SpectraMax L Microplate reader (Molecular Devices). Baseline readings were obtained for 5 seconds prior to ligand addition and subtracted from post-injection readings to obtain a ligand-induced Ca2+ response. Peak Ca2+ responses represent the maximum response in the largest peak obtained following ligand addition. Peaks were defined to have a minimum width of ten adjacent points.Cells were plated, grown in 96-well microtitre plates and transfected as described above. Forty-eight hours after transfection, growth medium was replaced with Tyrode's solution , GraphPad Prism 5  or MATLAB . To quantify the slope of the rapid ascending phase, a line was drawn from two data points after the minimum  to maximum response, and the impedance response was bisected at the point farthest from that line. Then, a second line was drawn from two data points after minimum response to the first bisection point, and the impedance response was bisected again. Slope was calculated as the ratio between the difference in impedance and the difference in time between two data points after the minimum response and the second bisection point.A between two curves i and j was estimated asi,tI is the impedance response of curve i at time t.Differences in ligand impedance signatures were determined by calculating the area between their impedance curves. The area Clustering of the data was assessed using the visual assessment of tendency method (VAT) For complete linkage hierarchical clustering, clustering trees were cut at 0.8 times the median of all pairwise dissimilarities.Figure S12AR-specific.ISO-promoted impedance response is β (A) Impedance response upon ISO stimulation following 1 hour pre-treatment with the β2AR-selective antagonist ICI118,551 (ICI) or the β1AR-selective antagonist CGP20712A (10 µM each). (B) Concentration-response curves of ISO-promoted maximum impedance response following pre-treatment with increasing concentrations of ICI118,551. Data represent the means from three independent experiments (+/− SEM for B).(TIF)Click here for additional data file.Figure S22AR-HEK293S cells.Signaling and impedance responses in parental HEK293S and 6HisHA-β (A) Comparison of the impedance responses observed in parental HEK293S and 6HisHA-β2AR-HEK293S stable cell lines following treatment with ISO (1 µM). (B) Impedance responses upon ISO stimulation in the presence or absence of a 1 hour pre-treatment with the β2AR-selective antagonist ICI118,551  in the parental HEK293S cells, demonstrating that the response observed in these cells resulted from activation of endogenous β2AR.  The ISO-promoted cAMP production (C) and ERK1/2 activation (D) observed in the parental HEK293S expressing low levels of endogenous β2AR was potentiated by the overexpression of human β2AR in 6HisHA-β2AR-HEK293S cells. Accumulation of cAMP was detected using the EPAC cAMP biosensor , pp. 297–307). Data represent means of at least three independent experiments. Statistical significance of the differences between the conditions for panels C and D were assessed using Student's paired t-test. ** P<0.01, * P<0.05.(TIF)Click here for additional data file.Figure S3Involvement of the MEK/ERK1/2 pathway in the ISO-promoted impedance response. Cells were transfected or not (Mock) with the MEK1-K97A-Flag dominant negative mutant 48 hours prior the impedance measurements and treated with ISO (1 µM). Data represent means of three independent experiments.(TIF)Click here for additional data file.Figure S4s, Gβi and Gβγ vs inhibition of cAMP and ERK1/2 pathways on the impedance response.Comparison of the direct inhibition of Gα (A) Comparison of cholera toxin (CTX) and SQ22536 pre-treatments on the ISO-promoted impedance response (B) Comparison of pertussis toxin (PTX) and U0126 pre-treatments on the ISO-promoted impedance response (C) Comparison of gallein  and U0126 pre-treatments on the ISO-promoted impedance response (response  and 3A. response  and 3A. response  and 3D.(TIF)Click here for additional data file.Figure S5Statistical analysis of the variability of impedance responses. (A–E) Repeated impedance responses were obtained for ligands from each of the five groups of compounds. In each case, 3–7 independent measurements were made and mean impedance values (CI) were determined for each time-point. The mean impedance responses are shown in solid, colored lines whereas the dotted black lines represent the standard error of the mean (SEM) for each time-point.(TIF)Click here for additional data file.Figure S6Visual assessment of clustering tendency of -adrenergic ligand impedance signatures. Gray levels represent the extent of dissimilarity between ligands as determined by area between the curves, ranging from black  to white (largest difference). Ligands are ordered based on similarities in the area between their curves. Ligands with similar impedance signatures  are visualized as dark clusters along the diagonal. See (TIF)Click here for additional data file.Figure S72+ response is β2AR-specific.ISO-induced Ca 6HisHA-β2AR-HEK293S were pre-treated or not with the β2AR-selective antagonist ICI118,551 (100 nM) for 1 hour before stimulation with 1 µM ISO. Data represent means of three independent experiments (± SEM).(TIF)Click here for additional data file.Figure S82+ and impedance responses.Effect of thapsigargin on ISO-induced Ca ISO-induced Ca2+ response (A) and impedance response (B) upon pre-treatment or not with thapsigargin  for 30 minutes. Data represent means (+/− SEM for A) from at least three independent experiments.(TIF)Click here for additional data file.Figure S9Normalization and baseline-correction of impedance responses. (1) A measurement of cell index is made in the presence of growth medium prior to cell seeding to determine the background cell index in each well, which is subtracted from the cell index values generated by cell attachment. Cells are grown for 16–20 hours before ligand treatment. (2) Cell index values are obtained immediately following ligand treatment every 20 seconds for a total time of at least 100 minutes. (3) Cell index values are normalized by dividing by the cell index at the time of ligand addition and (4) baseline-corrected by subtracting the cell index obtained in vehicle-treated conditions. See (TIF)Click here for additional data file.


The 1000 Functional Connectomes Project is a collection of resting-state fMRI datasets from more than 1000 subjects acquired in more than 30 independent studies from around the globe. This large, heterogeneous sample of resting-state data offers the unique opportunity to study the consistencies of resting-state networks at both subject and study level. In extension to the seminal paper by Biswal et al. , where a Since the seminal report by Biswal et al. , low-frea priori selection of seed regions rendering seed-based functional connectivity an inherently parametric approach.In the most general terms, the concept of brain networks is based on the measure of functional connectivity, defined as temporal coherence between the low-frequency (< 0.1 Hz) BOLD signal of spatially remote brain regions —or, more specifically, spatial ICA, as opposed to temporal ICA—that has emerged as the most successful method to identify spatially independent brain networks, as witnessed by a large number of influential studies . To avoid the most important sources of heterogeneity as well as complications due to non-independence, subjects with more than one run in the dataset were excluded from the analysis. The final sample consisted of 1000 subjects  and the power ratio  are computed for each of the group components identified  , and 3 show consistent activity mainly located in voxels outside the gray matter . Component C.01 corresponds mainly to the occipital lobe. Component C.02 includes the posterior cingulate cortex and precuneus. Component C.03 shows activation in ventral medial prefrontal, posterior cingulate, and lateral parietal cortex as well as hippocampus and, to a lesser extent, the inferior temporal lobe. Component C.06 is situated in ventral and dorsal medial prefrontal cortex, posterior cingulate cortex and, to a lesser extent, lateral parietal cortex. Thus, these two components correspond to regions commonly identified as part of the default-mode network, with component C.03 more focused in the posterior, and component C.06 in the anterior parts. C.04 is centered on the posterior cingulate cortex and precuneus, with co-activations in the dorsolateral prefrontal cortex. Components C.07 and C.09 are strongly lateralized, situated in the ventral and dorsal lateral prefrontal cortex, lateral parietal cortex and superior temporal lobe—predominantly right for component C.07, and left for component C.09—as well as the respective contralateral part of the cerebellum. Component C.10 and C.12 encompass dorsal parietal, precentral, as well as occipitotemporal (BA 37) areas, with C.10 being more focused on the ventral parts and C.12 more strongly involved in the dorsal parts of these areas, in particular the precentral areas. Component C.08 covers the pre- and postcentral gyri and can be described as a sensory-motor network, C.11 is focused on the anterior cingulate cortex, with co-activations in the dorsolateral prefrotal, orbitofrontal as well as posterior cingulate cortex. Finally, components C.13 and C.14 are located on the temporal lobes.Gray matter networks, designated C.01 to C.16 in descending order of their voxelwise sum of Clustering results of the networks are presented as a dendrogram in Figure The spectral characteristics of the networks indicate that the artifact components have lower power ratio between high and low frequencies as well as lower dynamic range , other components appear only in the single-study results of about half of the studies included in the 1000 Functional Connectomes dataset . Still, even the most consistent group components do not exhibit uniformly high spatial correlation with their matching components or fail to bidirectionally match with a component from each set of single-study components. Component C.08, for instance, has a partner-matched component in every single study, yet spatial correlations with its matched components are as low as 0.12 for the dataset Ann Arbor b, 0.39 for Milwaukee a and 0.46 for München. Conversely, there is a generally low consistency of some studies with all group results . On the other extreme, there are some group components which could not be unambiguously matched to only one component in a given study despite there being a component with high spatial correlation. This is an indication that there might be a second equally well matching component in this study's dataset, probably due to a division of the network into subcomponents.The components can thus be divided in three categories. First, there are components with high consistency at both single-subject and study level; these include C.01, C.03, C.04, and C.07–C.11. The second group of components can be characterized as those least consistent at both levels, notably C.12–C.16. As a third group, some components show differences in these two metrics: C.02 is about as consistent as other gray matter components at single-subject level, but can be found in only half of the single-study samples, C.05 and C.06 are among the less consistent components at single-subject level, but show average consistency at study-level. Figure Finally, single-subject results show systematic variation of the number of components, identified by MELODIC using the LAP criterion, depending on the study of origin of the individual-subject dataset , with the posterior of the two (C.03) being one of the most consistent networks identified, appearing in 94% of single study results, and the anterior (C.06) being found in 79% of the study samples. The anterior default mode component, however, exhibits lower spatial correlation both between the group component map and study-level components as well as between the group component map and single-subject component maps. This supports the hypothesis of a functional segregation of the default-mode network , the algorithm is less likely to detect components that are not present in every subject, for example a cerebellar component if the cerebellum is not wholly within the field of view of all studies included in the analysis. This is corroborated by Biswal et al.  who, usiAnother confounding effect could be the influence of motion on the component map estimation, as highlighted by Power et al. , which cThis work presents the largest exploratory fMRI study to date, including 1000 single subjects simultaneously, made possible due to new computational methods implemented in an R framework (Boubela et al., Siegfried Kasper has received grant/research support from Eli Lilly, Lundbeck, Bristol-Myers Squibb, GlaxoSmithKline, Organon, Sepracor, and Servier; has served as a consultant or on advisory boards for AstraZeneca, Bristol-Myers Squibb, GlaxoSmithKline, Eli Lilly, Lundbeck, MSD, Pfizer, Organon, Schwabe, Sepracor, Servier, Janssen, and Novartis; and has served on speakers' bureaus for AstraZeneca, BMS, Angelini, Eli Lily, Lundbeck, Schwabe, Sepracor, Servier, Pfizer, Pierre Fabre, and Janssen. All other authors declare that their research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fully comprehending the brain as an information-processing organ will involve analysis of both the physical and algorithmic components of the system. The physical substrates comprising the brain, including neurophysiology, ion gradients, neurotransmitter release, and the molecules involved in action potential firing have been studied since Hodgkin and Huxley developed the squid giant axon model in 1952 Hodgkin et al. . Mapping2+ reporters that fluoresce in the presence of high Ca2+ concentration. During an action potential of a neuron, Ca2+ is released into the cytoplasm and the Ca2+ is quickly returned to its compartments in the endoplasmic reticulum and extracellular space as the neuron restores resting potential. Thus a Ca2+ “bursts” marks a firing neuron. The genetically encoded reporters are derived from fusing the Ca2+-binding domain from calmodulin, another peptide domain, and a circularly permuted green fluorescent protein (GFP) whose fluorescence properties are modulated by the Ca2+ sensing interaction. These are termed GFP CalModulin proteins (GCaMP), which have been modified and optimized for Ca2+ responsiveness and fluorescence properties , and pulsed, rather than continuously illuminated, to the sample in such a manner that the spectral components of the pulse overlap in time and space only at the focus region with near diffraction-limited axial  confinement. With this technique, they achieved a wide-field excitation area of 60 μm diameter with 1.9 μm axial confinement. Imaging a depth of 30 μm, they were able to essentially detect neuronal Ca2+ dynamics of a 9.6 × 107 μm3 box is captured in 1.6 s.The light-sheet microscopy technique described in Ahrens et al.  allows fC. elegans and the sculpted light technique is the ability to demonstrate neuronal response to chemical stimulus with high time resolution and that all 302 neurons and 8000 synaptic connections have been mapped  Initiative (Insel et al., Erick T. Tatro is supported by the US National Institutes of Health grants R01MH94159, R03DA033849, P50DA026306, and R21DA036423.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oryctolagus cuniculus), classical genetic studies have identified five alleles at the Extension locus: ED (dominant black), ES , E , eJ and e . Sequencing almost the complete coding sequence (CDS) of the rabbit MC1R gene, we recently identified two in-frame deletions associated with dominant black  and recessive red  coat colours. It remained to characterize the eJallele whose phenotypic effect is similar to the Orange and Sex-linked yellow loci of cat and Syrian hamster.In the domestic rabbit (eJ) was present in homozygous state in Japanese, Rhinelander and Dutch tricolour rabbits only . Rabbits with eJ/eJ genotype were non fixed at the non-agouti mutation we previously identified in the ASIP gene. Segregation in F1 and F2 families confirmed the order of dominance already determined by classical genetic experiments with a possible dose effect evident comparing eJ/eJ and eJ/e animals. MC1R mRNA was expressed in black hair skin regions only.We sequenced the whole CDS in 25 rabbits of different coat colours including 10 Japanese and 10 Rhinelander (tricolour) rabbits and identified another 6 bp-in frame deletion flanked by a G > A transition in 5'  that was present in all animals with Japanese brindling coat colour and pattern. These mutations eliminate two amino acids in the first transmembrane domain and, in addition, cause an amino acid substitution at position 44 of the wild type sequence. Genotyping 371 rabbits of 31 breeds with different coat colour this allele (MC1R transcription may be regulated epigenetically in rabbits with the Japanese brindling phenotype. Further studies are needed to clarify this issue.The c. allele may be responsible for a MC1R variant determining eumelanin production in the black areas. However, the mechanism determining the presence of both red and black hairs in the same animal seems more complex. Expression analyses of the c. allele suggest that  Extension and Agouti are the main loci that affect the production and relative amount of these two melanin types in the melanocytes  allele, confirming the results of classical genetic studies  allele over the e allele and that there was a dose effect of the new mutation that was in heterozygous condition in the F1 animals. A second F1 family was created by crossing the same Rhinelander buck with a Japanese doe obtaining 9 F1 rabbits. Four of them had the same coat colour pattern of the buck and 5 were like the doe. In this family, the tricolour or Japanese brindling phenotypes were determined by the segregation of a dominant English spotting allele (heterozygous in the Rhinelander father), like in the previous family  MC1R allele and of the English spotting locus allele. Of the nine F2 rabbits, 4 were completely black , 3 were black and white , 2 had a tricolour phenotype, confirming the segregation of the Japanese brindling coat colour together with homozygosity for the c. allele in these latter rabbits (data not shown).Three Fs family ,33,35,42ant buck ,33,35,42MC1R transcripts including the c.125_130del6 mutation (see Methods). In all skin samples with black hair a transcript with expected size was identified whereas no amplification was obtained from skins of both red or white hair regions  cDNA fragment was amplified from all skin samples   just after moulting. Skin samples were collected from regions with different hair colours . Total RNA was isolated, retrotranscribed and amplified to evaluate  the presence of  Figures  and 3B. eJ allele at the rabbit Extension series. This is the third in-frame MC1R mutation associated with a coat colour in rabbit, making this species quite unique. One MC1R allele determined by a nonframeshift deletion associated only with melanism has been observed in each of other four species   composite mutation is associated with the ea fowl) ,26,30.ED or ES), that eliminates two amino acids in the second transmembrane domain), may determine a constitutive activation of the MC1R protein, that in turn, drives eumelanin production. It seems that the eJ in-frame mutation that eliminates two amino acids in the first transmembrane domain (including an amino acid substitution at position 44) could cause the melanic phenotype of the black skin areas by either determining constitutive activation of the receptor or increasing the affinity to αMSH. Otherwise, the presence of black spots or black hairs in Rhinelander and Japanese rabbits could not be explained. The black of the Japanese and tricolour rabbits has been also suggested to be dominant by the pioneering studies of Castle  composite mutation or by the same composite mutation, may determine the Japanese brindling phenotype in rabbits. To identify a promoter polymorphism we sequenced about 1 kb of the MC1R gene 5'-flanking region in two Japanese and in two Checkered Giant rabbits but we did not find any sequence difference (data not shown). Regulation of MC1R expression might be epigenetically determined as already reported for other genes  (eJ) alleles have been indicated as Δ6DS, Δ30, Δ6J, respectively. The MC1R genotypes of the parental animals is reported together with the genotype and coat colours of the F1 rabbits. All parental animals were homozygous for the nonagouti mutation identified in the ASIP gene [Dilute locus allele (d) determining the blue coat colour [SIP gene . The Chet colour ,33.Click here for fileGAPDH cDNA fragment amplified from retrotranscribed RNA extracted from skin specimens. Lines A and B: from black hair skin regions of a Rhinelander rabbit. Lines C and D: from red hair skin regions of a Rhinelander rabbit. Line E: control genomic DNA. Line F: DNA ladder.Click here for fileJ/Δ30 (A) and Δ6J/Δ6J (B) genotypesTricolour rabbits with Δ6. Rabbits with these two genotypes differ in terms of extension of black regions.Click here for filePrimers and PCR conditions. Primer sequences, PCR conditions and use of the reported primer pairs.Click here for file


The kinetic parameters of transient changes in Zn2+ and Ca2+ under electrical stimulation and the spatiotemporal properties of Zn2+ and Ca2+ sparks in resting cells are found to be normal in the treated diabetic group. Biochemical analysis demonstrated that the NAC treatment also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10 µM ZnCl2 exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB, can be activated if the cells are exposed directly to Zn2+. We thus for the first time report that an enhancement of antioxidant defence in diabetics via directly targeting heart seems to prevent diastolic dysfunction due to modulation of RyR2 macromolecular-complex thereby leading to normalized [Ca2+]i and [Zn2+]iin cardiomyocytes.We examined whether cellular antioxidant-defence enhancement preserves diastolic dysfunction via regulation of both diastolic intracellular free Zn Diabetic cardiomyopathy was first recognized by Rubler et al.  in diabe2+ i) is measured to be less than one nanomolar under physiological conditions i and [Ca2+]i were measured from Fura-2 loaded (4 μM Fura-2 AM) cardiomyocytes at room temperature as described previously i level are given in N-acetyl cysteine (NAC)-treated diabetic groups with the control group. As can be seen in 2+ chelator TPEN (50 μM) caused a rapid decrease in fluorescence ratio (ΔF340/380) to a level lower than that of initial value, verifying that the initial observed fluorescence ratio is attributable to intracellular basal free Zn2+ level. This decrease is bigger in the diabetic group compared to that of the control group while it is almost the same level in the NAC-treated diabetic group and the control group as well. As can be seen in the same bar graphs, the fluorescence change with respect to the basal level of intracellular free Ca2+ is significantly higher in the diabetic cardiomyocytes compared to those of both the control and NAC-treated diabetic groups. The present data demonstrate that both increased intracellular basal free Zn2+ and Ca2+ levels are dependent on increased oxidative stress and decreased antioxidant-defence system in the hyperglycemic rats.To identify and compare basal intracellular free Zn2+ and Ca2+ of diabetic rat cardiomyocytes, we incubated cardiomyocytes from diabetic rats with NAC  before loading the cells with Fura-2 AM. As can be seen from 2+ and Ca2+. These groups of experiments confirm also that diabetes can induce significant increases not only in basal Ca2+ level but also in basal Zn2+ level, at most, due to increased reactive oxygen species, ROS.To test directly whether increased oxidative stress can induce simultaneous increases in basal levels of both intracellular free Zn2+ releases (named as Zn2+ sparks) in resting quiescent cardiomyocytes isolated from 3-month-old male rats and loaded with a Zn2+-specific fluorescence dye, FluZin-3, which can be visualized in a similar manner to known Ca2+ sparks [2+ release similar to Ca2+ release, either Zn2+ or Ca2+ sparks, to increased basal levels of both Zn2+ and Ca2+ (mentioned in previous section) and altered Zn2+/Ca2+ contents of SR  were examined and monitored the differences in parameters of these sparks among the diabetic and control groups. Representative line-scan images of of both type of sparks from control (CON group), diabetic (DM group), and N-acetyl cysteine (NAC)-treated diabetic (DM + NAC group) rats  are displayed in F/F0 at the peak of either Zn2+ or Ca2+ sparks (2+/Ca2+-spark frequencies (N-acetyl cysteine (NAC)-treatment of the diabetic rats (DM + NAC group) for 4 weeks significantly prevented diabetes-induced all changes in both Zn2+ and Ca2+ sparks parameters.Recently, we have demonstrated that there are local tiny Zn+ sparks . The con2+ and Ca2+-sparks were obtained in cardiomyocytes isolated from diabetic rats after 1 h incubation with 1 mM NAC at 37°C (+NAC group).Of note, as shown in Figures 2+ and Ca2+ changes in isolated cardiomyocytes, we performed some additional experiments to monitor intracellular transient changes of either intracellular free Zn2+ or Ca2+ elicited by electrical-field stimulation. 2+ and Ca2+ transients elicited in control, diabetic, and N-acetyl cysteine (NAC)-treated (4 weeks) diabetic rat cardiomyocytes as well as NAC-incubated diabetic cardiomyocytes for 1 h with 1 mM NAC at 37°C. The averaged peak fluorescence amplitudes of both FluoZin-3 and Fluo-3 as F/F0 were significantly smaller in diabetic cells than in control cells (50) of transient changes of FluoZin-3 were found to be similar between diabetic and control groups as well as NAC-treated or NAC-incubated diabetics  under hyperglycemia [It has been previously shown that the mechanisms underlying the dysfunction of RyR2 in diabetes include, in part, a hyperphosphorylation of RyR2 due to both high phosphorylation levels of both protein kinase A (PKA) and Caglycemia . The phoFor an assessment of a direct action of antioxidant NAC, we first incubated freshly isolated cardiomyocytes from diabetic rats with 1 mM NAC for 1 h at 37°C and then performed the similar Western blot analysis for pRyR2, RyR2, FKBP12.6. Incubation of diabetic cardiomyocytes with 1 mM NAC significantly declined hyperphosphorylation level in RyR2, while there was no effect of its depressed protein level , left. S2+ distribution is linked to redox metabolism despite Zn2+ itself not being redox-active and generally Zn2+-proteins being redox-inert [2+ through the modulation of the redox status of intracellular protein thiols, in here, we aimed to examine the effects of external ZnCl2 incubation (10 μM) of heart homogenates from both control and diabetic groups rats for 20–30 min at 37°C on members of RyR2-macromolecular complex. As can be seen from Figures μM (data not shown) or 10 μM ZnCl2 incubation of left ventricular heart homogenates from both group rats. However, we measured significantly increased phosphorylation levels of RyR2, PKA, and CaMKII with these two ZnCl2 incubations in a concentration-dependent manner. The last data further support the hypothesis that Zn2+ disbalance results in a signaling disbalance caused by a local surplus of Zn2+ interfering with cellular signaling networks.The intracellular free Znox-inert , 40. TheκB is protective or detrimental for heart function although its important role in cardiac pathology [κB signaling that involves multiple components and regulation at several steps. Furthermore, NF-κB is a pleiotropic transcription factor that receives signals from multiple pathways including different important modulators of cardiac remodeling. To asses a possible direct role of intracellular Zn2+ on NF-κB, we tested the effect of external ZnCl2 incubation (10 μM) of heart homogenates from both control and diabetic groups rats for 20–30 min at 37°C, similar to previous section. As can be seen from Figures κB was not affected with 10 μM ZnCl2 incubation of left ventricular heart homogenates from both control and diabetic group rats. However, the activation levels of NF-κB in both group rat heart homogenates were significantly increased (~3-fold in both groups) after ZnCl2 incubations. This last data further supports the hypothesis that although cardiac remodeling is associated with increased oxidative stress, inflammation, and activation of hormonal systems under hyperglycemia, increased intracellular ZnCl2-mediated NF-κB activation seems to be a distinct fact among the others under high Zn2+ exposure of heart.There are multiple studies with different conclusions regarding whether NF-athology , 42 has N-acetyl cysteine (NAC) effect in STZ-induced diabetes in rats reports a significant role of cellular antioxidant-defence enhancement on preservation of diastolic dysfunction via regulation of not only diastolic Zn2+ but also diastolic Ca2+ due to a prevention of RyR2-leak in diabetic rat heart. At first, we confirmed a defective intracellular Zn2+ signaling, besides previously shown Ca2+ signaling [2+ transients as well as markedly increased diastolic levels of both Zn2+ and Ca2+ since all these alterations could be prevented with either NAC treatment of diabetic rats or NAC-treatment of diabetic cardiomyocytes. Our electron microscopy data further corroborated these findings fully. Furthermore, we clearly established that these defects in diabetic cardiomyocytes could be attributed to anomalous RyR2 behavior, as revealed by the spatiotemporal properties of both Zn2+ and Ca2+ sparks that especially exhibited slower kinetics and higher frequencies. The reduced amount of RyR2 and FKBP12.6 levels as well as a marked hyperphosphorylation of RyR2 could be responsible for most of these observations, in part, due to a disbalanced ratio of oxidants/antioxidant-defence. Moreover, the initial findings of this study indicate that supplementation with NAC improves the general status of diabetic rats, with an effect on body weight gain and hyperglycemia, protecting diastolic function, and helping to maintain baseline myocardial mechanics.This study on ignaling , in part2+ and Ca2+ regulation in diabetic cardiomyocytes without any effect on high blood glucose level and restores normal macromolecular complex composition and function of the RyR2 channels in diabetic rat hearts. There is associated restoration of myocardial diastolic function and reverse structural remodeling of the left ventricle in diabetic rats with N-acetyl cysteine, NAC treatment for 4 weeks. Furthermore, we clearly established that these defects in diabetic cardiomyocytes could be attributed to anomalous RyR2 behavior, as revealed by the spatiotemporal properties of both Zn2+ and Ca2+ sparks that especially exhibited slower kinetics and higher frequencies. These alterations can coincide perfectly with the behaviour of leaky RyR2 under strong oxidative/nitrosative stress. Indeed, it is known that both acute and chronic hyperglycemia trigger several biochemical and electrophysiological changes resulting in impaired cardiac contractile function [2+ release [2+ leak in cardiomyocytes has also been shown under various diseased heart models induced in animals [2+ level in cardiomyocytes due to Zn2+ release from intracellular stores by reactive, ROS/RNS [2+ level in the heart. A group of supporting data has been previously shown by our group, in which we demonstrated that inhibiting SR-Ca2+ release or increasing Ca2+ load in a low Na+ solution suppressed or increased Zn2+ movements, respectively. Furthermore, we also showed that mitochondrial inhibitors significantly reduced Zn2+ transients as well as Zn2+ sparks. In addition, either oxidation by H2O2 or changing to acidic pH inhibited the Ca2+-dependent Zn2+ release. Taken into consideration the previous ones with the present data, we proposed that Zn2+ release results, in part, from Zn2+ displacement by Ca2+ ions from metalloproteins binding sites whose availability depends on pH and redox status of cardiomyocytes, or rather that Ca2+ triggers ROS production inducing changes in metal binding properties of metallothioneins and other redox-active proteins as well as more Zn2+ release from SR due to defective RyR2 [2+ alters several enzymatic activities and leads to RyR2 phosphorylation as seen in pathological conditions. Moreover, another supporting data to this study has been given by Kamalov et al. [2+ level is increased by 70% in cardiomyocytes from male diabetic rats being parallel to an unbalanced oxidant-status/antioxidant capacity in the same heart preparations, and by over 200% in aldosteronism. Coordinated changes in the basal intracellular free levels of both Ca2+ and Zn2+ have recently been also reported by our group under experimental conditions (intracellular Zn2+ overload or intracellular Ca2+ decrease), as well as during a single beat (transients or elementary events) in the cells [The present study shows that systemic antioxidant treatment of diabetic rats preserves changes in both Znfunction –5, 43. Ffunction  while hifunction , and nit release . Moreove animals –24. Anot ROS/RNS . Moreove ROS/RNS , 44, proive RyR2 , 28, 45.v et al. . Their dhe cells .in vivo or in vitro conditions, may improve cardiac muscle function by reversing a maladaptive defect in Zn2+ signaling besides already known Ca2+ signaling, being parallel or individual, in cardiac myocytes of diabetic rats.Taken together, these data suggest that one of the beneficial effects of NAC treatment, under 2+ caused marked phosphorylation in RyR2 while there was no effect on the protein levels of both RyR2 and an accessory protein of RyR2 macromolecular complex, FKBP12.6, as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. These data are further supported, in part, with the fact that a Zn2+-binding protein calsequestrin resides in the SR and can bind up to 200 mol Zn2+ per mol, independently of binding up to 50 mol Ca2+ per mol calsequestrin. It is known as an association of Zn2+ with over 300 enzymes, where it can interact strongly with electronegative sulfur, nitrogen, and oxygen moieties in multiple coordination forms, serving catalytic and structural roles in maintaining active peptide conformations [2+ is most known for its ability to bind and stabilize proteins involved in gene regulation in domains called Zn2+-fingers, Zn2+-clusters, and Zn2+-twists [2+ plays critical roles in the redox signaling pathway and maintains the normal structure and physiology of various cell types while it can be toxic to cardiomyocytes [2+ is a vital element for mammalian in a certain range, some triggers such as increased ROS/RNS, ischemia, and infarction lead to release of Zn2+ from proteins and cause myocardial damage [Notably, in here, we have shown that exogenously applied Znrmations . In addi+-twists . Therefomyocytes , 49, 50.l damage , 51–54.2+ may compete with or substitute for metal ions crucial for the activity of signaling proteins. In addition to this hypothesis, it has been shown that Zn2+ has multiple functional effects on Ca2+/calmodulin-dependent protein kinase II (CaMKII) and modulates RyR1 binding to sarcoplasmic reticulum vesicles in skeletal muscle biphasically [2+ signaling can easily interfere with that of Ca2+ signaling in cardiomyocytes, particularly under pathological conditions, underlying, in part, cardiac dysfunction. Moreover, supporting this last statement, Zn2+ is known to induce CaMKII autophosphorylation, inhibit protein tyrosine phosphatases [2+ channels in mammalian cells [2+ signals is the modulation of protein phosphorylation, it is a strong possibility that both activated/phosphorylated CaMKII and PKA due to increased intracellular free Zn2+ turn into a hyperphosphorylated RyR2 under high extracellular Zn2+ exposure. Therefore, increased intracellular free Zn2+, most probably a contribution of increased intracellular free Ca2+, under increased oxidative stress together with depressed antioxidant-defence in the cells via hyperglycemia induces important defects in excitation-contraction coupling of cardiomyocytes. This statement is further supported with our previous observation related to coordinated changes in the basal intracellular levels of both free Ca2+ and Zn2+ under experimental conditions, as well as during a single beat in isolated cardiomyocytes [Therefore, it can be hypothesized that Znasically , 56. Accphatases –57 and van cells , and to an cells  while dian cells . In heremyocytes .2+ may be the modulation of cell signaling cascades, especially those involving protein phosphorylation, and considering also that intracellular Zn2+ can rise quickly and can influence Ca2+ regulation [2+ in cardiomyocytes suggests its availability as second messengers for gene expression [2+ could activate gene expression in GH3 cells due to modification of expression of Zn2+ regulating proteins by changes in intracellular free Zn2+ cellular systems [2+-fingers of Zn2+-finger transcription factors by Zn2+ availability [2+ may serve as a second messenger for transcription factor activation, and therefore, gene expression in response to stress. Accordingly, our present data showed that intracellular free Zn2+ can directly and markedly phosphorylate a transcription factor NF-κB in cardiomyocytes, of note much more range in hyperglycemia cells. Of particular interest in cardiomyocytes, our data suggest that Zn2+ or Zn2+ carrying metalloproteins may be released from internal stores during increased oxidative/nitrosative stress and thereby affect transcription/translation pathways [Thus, a major physiological role for Zngulation , 58, thepression , 61. In pression  showed t systems . In addilability , it can pathways .2+ and Ca2+ leaks leading to normalization of basal levels of intracellular free Ca2+ and Zn2+ in the heart. This is nicely in line with an early report performed on a rodent heart with hereditary muscular dystrophy, demonstrating accompany of intracellular Ca2+ overloading and oxidative stress with increased intracellular free Zn2+ [2+/Ca2+ ratio in cardiomyocytes and mitochondria must be preserved to combat oxidative stress. Thus, the increased cytosolic and mitochondrial free Zn2+ level are coupled to the induction of oxidative stress, while antioxidant effects result from the rise in cytosolic and mitochondrial free Zn2+ levels accompanied by a simultaneous activation of metal response element transcription factor-1 and its induction of such antioxidants as metallothionein-1 and glutathione peroxidase. However, with a long-term treatment, by maintaining both a cellular redox-status and intracellular Zn2+ level near to control level, antioxidant NAC prevents the subsequent alterations in intracellular Ca2+ homeostasis including both Zn2+/Ca2+ releases by RyR2, which leads to major defects in cardiac activity.Taken into consideration all present and also previously published data, in here, we report that an enhancement of antioxidant defence in diabetics, directly targeting heart, seems to prevent diastolic dysfunction, being associated with normalization of RyR2 macromolecular-complex, and thereby prevention of both Znree Zn2+ . Besidesree Zn2+  suggeste


Spontaneous T cell responses generated against a variety of solid malignancies are often subverted by immune evasion mechanisms active in the tumor microenvironment. In contrast, the mechanisms that regulate T cell activation versus tolerance to hematopoietic malignancies, such as acute myeloid leukemia (AML), have not been well-characterized. Our recent work in a murine AML model has demonstrated that following a systemic introduction of leukemia cells, T cells specific for leukemia-derived antigens underwent abortive proliferation and were deleted from the host. This deletional tolerance in mice with established AML was reversible upon administration of an agonistic anti-CD40 antibody to activate host dendritic cells (DCs), and argued that these cells may play a dominant role in tolerance induction to AML. Investigation of the DCs populations which engulfed AML cells in vivo, and which were likely promoting T cell tolerance, led to the critical observation that AML cells were phagocytosed exclusively by CD11c+CD8α+ DCs (CD8α+ DCs). CD8α+, but not CD8α- DCs purified from mice following an intravenous inoculation of AML cells, were able to cross-present leukemia-derived antigens to T cells in vitro, providing strong evidence that CD8α+ DC generate T cell tolerance to AML. Ongoing work utilizing mice deficient in particular DC subsets is focused on identifying a functional link between CD8α+ DCs and T cell tolerance. Additionally, the receptors expressed selectively on CD8α+ DCs which facilitate phagocytosis and cross-presentation of leukemia derived antigens are under investigation.


RyR2) mutations are implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT) thought to result from altered myocyte Ca2+ homeostasis reflecting inappropriate “leakiness” of RyR2-Ca2+ release channels arising from increases in their basal activity, alterations in their phosphorylation, or defective interactions with other molecules or ions. The latter include calstabin, calsequestrin-2, Mg2+, and extraluminal or intraluminal Ca2+. Recent clinical studies additionally associate RyR2 abnormalities with atrial arrhythmias including atrial tachycardia (AT), fibrillation (AF), and standstill, and sinus node dysfunction (SND). Some RyR2 mutations associated with CPVT in mouse models also show such arrhythmias that similarly correlate with altered Ca2+ homeostasis. Some examples show evidence for increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2. A homozygotic RyR2-P2328S variant demonstrates potential arrhythmic substrate resulting from reduced conduction velocity (CV) in addition to delayed afterdepolarizations (DADs) and ectopic action potential (AP) firing. Finally, one model with an increased RyR2 activity in the sino-atrial node (SAN) shows decreased automaticity in the presence of Ca2+-dependent decreases in ICa, L and diastolic sarcoplasmic reticular (SR) Ca2+ depletion.Ryanodine receptor type 2 ( RyR2) gene    in turn producing the delayed afterdepolarisation (DADs) thought to be the major cause of RyR2-associated CPVT , fibrillation (AF), and standstill as well as sinus node dysfunction (SND)  is one of the most malignant cardiac channelopathies. It is characterized by episodic, life-threatening, arrhythmias provoked by stress and emotion in individuals with otherwise structurally normal hearts  clusters are confined to the cell peripheries in contrast to their dense distribution throughout the more extensive ventricular tubular system  resulting in early afterdepolarisation (EAD) or SR Ca2+ release resulting in DAD phenomena, and may trigger re-entrant activity or drive atrial rhythm at rapid rate resulting in fibrillatory conduction. However, reentry requires a vulnerable substrate, likely determined by the balance of conduction velocity (CV) and refractory period  (NM-001035)  for CPVT showed AT/AF both during exercise and on ambulatory electrocardiographic (ECG) recording  and cardiac structure  phosphorylation of RyR2. Conversely, pharmacological inhibition of CaMKII prevented this AF inducibility. So did genetic inhibition of CaMKII-mediated RyR2 phosphorylation in RyR2-S2814A mice studied in a vagotonic AF model. Atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF similarly showed increased CaMKII phosphorylation of RyR2. These findings implicate a RyR2-dependent Ca2+ release resulting from increased CaMKII activity in susceptibility to AF  and atrial effective refractory periods (AERP) max], despite similar effective refractory periods, AP durations and AP amplitudes.The ) Figure . These fl Figure  , CPVT is associated with high incidences of SAN dysrhythmia in the form of reduced SAN automaticity resulting in basal bradycardia, sinus pauses, and impaired chronotropic responses to β-adrenergic stimulation  density in whole-cell patch-clamped RyR2-R4496C SAN cells. Isoproterenol dramatically increased the frequency of Ca2+ sparks and Ca2+ waves by ~5 and ~10-fold .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acute urticarial lesions may display central clearing with ecchymotic or haemorrhagic hue, often misdiagnosed as erythema multiforme, serum-sickness-like reactions, or urticarial vasculitis. We report a case of acute annular urticaria with unusual presentation occurring in a 20-month-old child to emphasize the distinctive morphologic manifestations in a single disease. Clinicians who care for children should be able to differentiate acute urticaria from its clinical mimics. A directed history and physical examination can reliably orientate necessary diagnostic testing and allow for appropriate treatment. Acute annular urticaria, an acute urticarial hypersensitivity syndrome, is a morphologic subtype of urticaria characterized by the acute onset of blanchable annular, arcuate, and polycyclic erythematous wheals that subsequently fade within hours. However, lesions may display central clearing with ecchymotic or haemorrhagic hue, often misdiagnosed as erythema multiforme, serum-sickness-like reactions, or urticarial vasculitis . We repoA 20-month-old boy was hospitalised in our ward with the presence of erythematous-purpuric lesions, roundish in shape, not itchy, 1 to 5 cm in diameter, localized on the trunk, the neck, and upper limbs . His hisAcute annular urticaria is reportedly more common in children 4 months to 4 years of age . The diaAcute annular urticaria is underrecognized as a result of the paucity of reported cases in the literature mainly due to similarities between distinct clinical entities, such as erythema multiform or serum-sickness-like reactions. It has been our experience that erythema multiforme is incorrectly diagnosed in many patients with an urticarial drug eruption because the edema from the urticaria can result in some surrounding pallor and some central pallor or duskiness, which can mimic a type of targetoid pattern. An important distinction is the fleeting duration of the lesions of urticaria, which usually last minutes to hours as opposed to the fixed lesions of erythema multiform and serum-sickness-like reactions, which typically last from days to weeks. Erythema multiform represents a self-limiting cutaneous cytotoxic hypersensitivity reaction mostly due to Herpes simplex virus and generally requires symptomatic treatment only.Serum sickness-like reactions are a recognizable reaction to a drug. It has occurred most frequently following cefaclor, but may occur with other cephalosporins, penicillins, or other drugs. Prior treatment with the drug is not necessary. The acute onset in a child of inflammatory, red papulonodules that spread out into annular, urticarial plaques with dusky centers is characteristic. The onset is usually 7–10 days after the causative drug was begun. There are no true target lesions as with erythema multiforme, and the lesions may expand but do not have the day-to-day fluctuation seen in urticaria. The child often has joint pains and fever. Lymphadenopathy and renal involvement usually do not occur, in contrast to true serum sickness. The causative medication should be stopped. Corticosteroids can be dramatically helpful, if needed. Eruption in urticarial vasculitis is characterised by hives with a dusky purpuric centre and usually resolves with postinflammatory dyspigmentation. Although drugs are sometimes implicated in urticarial vasculitis, the condition is most often idiopathic; other causes include infections, autoimmune processes, and neoplastic processes. When distinguishing between urticarial vasculitis and urticarial drug eruption on the basis of clinical clues proves difficult, a complete blood cell count, sedimentation rate, urinalysis, and skin biopsy can be helpful. The treatment of this condition includes discontinuation of the offending agent, administration of systemic antihistamines, and administration of a 2- to 3-week course of systemic corticosteroids for more severe symptomatic cases.Clinicians who care for children should be able to differentiate acute urticaria from its clinical mimics. A directed history and physical examination can reliably orientate necessary diagnostic testing and allow for appropriate treatment.


One of the mechanisms responsible for this ability is believed to be the rapid mode (RaM) of Ca2+ uptake. Despite the existence of many models of mitochondrial Ca2+ dynamics, very few consider RaM as a potential mechanism that regulates mitochondrial Ca2+ dynamics. To fill this gap, a novel mathematical model of the RaM mechanism is developed herein. The model is able to simulate the available experimental data of rapid Ca2+ uptake in isolated mitochondria from both chicken heart and rat liver tissues with good fidelity. The mechanism is based on Ca2+ binding to an external trigger site(s) and initiating a brief transient of high Ca2+ conductivity. It then quickly switches to an inhibited, zero-conductive state until the external Ca2+ level is dropped below a critical value (∼100–150 nM). RaM's Ca2+- and time-dependent properties make it a unique Ca2+ transporter that may be an important means by which mitochondria take up Ca2+in situ and help enable mitochondria to decode cytosolic Ca2+ signals. Integrating the developed RaM model into existing models of mitochondrial Ca2+ dynamics will help elucidate the physiological role that this unique mechanism plays in mitochondrial Ca2+-homeostasis and bioenergetics.Mitochondria possess a remarkable ability to rapidly accumulate and sequester Ca The general consensus is that a highly-selective, high-conductance Ca2+ channel, the Ca2+ uniporter (CU), accommodates Ca2+ entry while the combined action of an nH+/Ca2+ and an nNa+/Ca2+ exchanger facilitate Ca2+ removal from the mitochondrial matrix 2+ transport mechanisms to include the rapid mode (RaM) of Ca2+ uptake and the mitochondrial ryanodine receptor 2+ transport related proteins, the molecular identity of only two transporters, nH+/Ca2+ (letm1) and nNa+/Ca2+ (NCLX), have been recently discovered 2+ channel identified in recent patch clamp experiments 2+ transport molecular machinery, and even more so, its role in Ca2+-induced mitochondrial dysfunction.Calcium plays many key roles with respect to mitochondrial bioenergetics 2+ signals consisting of either low impulse, long duration or high impulse, short duration transients 2+-sensitive pathways that exist in mitochondria. In fact, mitochondria themselves are able to modulate and shape these cytosolic Ca2+ transients due to their uptake and sequestration capabilities 2+ uptake pathway, the CU, is only operational when Ca2+ rises in the µM range in the presence of physiological concentrations of Mg2+2+ varies from 10 to 90 µM as Mg2+ levels rise from 0 to 5 mM 2+ would appear to prevent the CU from facilitating any significant Ca2+ uptake when Ca2+ is in the physiological nM range. Thus, it seems that the RaM mechanism may be a component of the physiological response to upstream Ca2+ signaling. Supporting this view, many groups have observed mitochondria in situ exhibit RaM-like behavior when exposed to nM levels of Ca2+Mitochondria are faced with a multitude of cytosolic Cain situ, the mitochondrial network contains contact points with the sarcoplasmic reticulum known as micro-domains 2+] rises in these micro-domains above 10 µM during the peak of the cytosolic Ca2+ transient. This helps explain the rapid rise in mitochondrial Ca2+ observed in situ; however, it fails to explain how isolated mitochondria are able to rapidly take up Ca2+ in the sub-micromolar range 2+ pulses resulting in a more efficient maintenance of NADH/NAD levels in rat hepatocyte mitochondria 2+ signal transduction and bioenergetics. The response to each pulse resulted in a rapid increase in matrix Ca2+ via a RaM-like process. Thus, it is likely that both, micro-domains and RaM, help modulate mitochondrial bioenergetics and Ca2+-buffering in a tissue dependent manner.It must be stressed that 2+, the traditional CU has been the primary Ca2+ uptake mechanism encoded in mitochondria-related mathematical models 2+ oscillations 2+ and bioenergetics remains speculative. Considering the relative importance of Ca2+ with respect to mitochondrial metabolism and integrity, a more accurate model of Ca2+ sequestration including the RaM uptake pathway would help drive innovative therapies targeted at Ca2+-related mitochondrial dysfunction. Also, the manner in which mitochondria decode the cytosolic Ca2+ signals may prove pivotal in understanding the regulatory impact that Ca2+ possesses on cellular and mitochondrial processes.Despite the large body of evidence suggesting that mitochondria are able to rapidly take up and buffer exogenous Ca2+ uptake in isolated mitochondria from both liver and heart tissues with good fidelity 2+ plays in mitochondrial bioenergetics using mathematical modeling.Presented herein is a simple 4-state model that is capable of capturing the salient features of RaM. The model reproduces the available experimental data of rapid Ca2+ affinity, Ca2+ cooperativity, Ca2+ binding constant for state 1I and state O-1I transition rate. When more data becomes available, it may be possible to identify these parameters with more confidence.The model parameters that were estimated for the 4-state RaM model in order to reproduce the experimental data 2+ uptake profiles for a sustained Ca2+ pulse  and inhibition (3C and 3D) profiles for heart and liver RaM demonstrate the major differences between the two RaM types. Although the steady state values for state R (4A and 4B) are identical, the time constants (4C and 4D) is dramatically different between heart and liver RaM. When [Ca2+] exceeds the OK, the time-dependent activation rates are identical; however, when [Ca2+] drops below the IK, the time-dependent recovery rates varies considerably between heart and liver RaM. For example, heart RaM takes up 90 seconds to recovery while liver RaM recovers in less than 1 second. Equations 4 and 1b were used to generate the surface plots for the recovery and inhibition profiles, respectively. Equations 5 and 6 were used to plot the time constants and the steady state values for state R, respectively.2+ uptake profiles were probed using Ca2+ pulses of varying amplitudes and frequencies. For details regarding the methodologies employed, see the 2+ uptake facilitated by a gradual increase in buffer [Ca2+]  when begun, Ca2+ pulse experiments, it's quite clear from the Ca2+ uptake versus time profile that, the Ca2+ uptake for liver RaM is quite flat for [Ca2+] below 300 nM, but a small “leak” is present for the heart RaM for [Ca2+] as low as 200 nM. Therefore, in order to simulate this leak phenomenon for heart RaM, the rate constant −1 but needed to be below 0.01 s−1 to simulate the liver response . Thus it appears likely that this small leak phenomenon for heart RaM is a manifestation of CU activity at low Ca2+. If the data is adjusted for this observation, −5 s−1. This value is well within the given confidence interval shown in 2+] above 196 nM while it takes more than 224 nM in heart tissue. This difference is very small, approximately 25 nM, and may be too small to experimentally confirm considering the uncertainty in the interpulse heights.Although the 4-state RaM models for the heart and liver are structurally identical, there were differences between the two parameter sets used to simulate their respective data sets. This is evident after inspection of the available experimental data onse see . It's in2+- and time-dependencies. This concept is explored deeper in the 2+ pulse yielded an uptake of Ca2+ of approximately 0.08 nmol/mg by liver RaM as in seen in 2+ pulse with an interpulse duration of less than 1 second resulted in an uptake of Ca2+ of 0.09 nmol/mg by heart RaM as seen in 2+ transients Buntinas et al. 2+ dynamics was developed. This basic model consisted of Ca2+ influx via RaM and the CU and efflux via the Ca2+/H+ exchanger. This approach was favored over a more encompassing endeavor because far fewer adjustable parameters were required; moreover, the experimental data was qualitative in nature and prevented any quantitative analysis. The model was driven by Ca2+ pulses generated with square waves, and despite the very simple dynamical model used, the simulations were able to capture the matrix Ca2+ trends very well. Namely, the simulation results were able to demonstrate that i) at high amplitude and frequency Ca2+ pulses, the mitochondrion was unable to respond fast enough due to the Ca2+-induced inhibition of the RaM complex and ii) pulses delivered at a lower amplitude and frequency produced a more robust response that enabled the mitochondrion to sequester more Ca2+. It follows that a more sophisticated modeling effort must follow and demonstrate how mitochondria respond to rapid changes in extra-mitochondrial Ca2+ when more quantitative data becomes available.Corroboration is a necessary step in order to affirm the underlying hypotheses used to derive any model. This was done by the simulating experimental data presented by Gunter et al. 2+ influx through RaM is very large, the mitochondria could have overcome the buffering capacity of the buffer for the low concentration Ca2+ pulses and dropped the [Ca2+] below the reported value. This would underestimate RaM's capacity for Ca2+ uptake and hinder model fitting to the reported experimental data. Despite any issues with the data, the underlying premise used to construct the model could be incorrect; however, considering that the cycling mechanism was able to produce all of the major features presented by the data, this scenario is unlikely.The degree of control required to assay RaM kinetics at a quantitative level is very demanding. It is highly likely that no matter how skillful the experimenter or precise the experiment, endogenous uncertainty may dominate the measured response and influence the parameter estimation. For example, the quantitative difference between liver and heart RaM could be attributed to the differences in their native environments. Perhaps cardiolipin or cardiac specific mitochondrial proteins are responsible for differences in rate constants between the heart and liver RaM parameters via direct interaction with the RaM complex 2+-dependencies of RaM dynamics for both liver and heart tissue very well, two data points shown in 2+ uptake at “high calcium” which corresponds to approximately 140 nM Ca2+ according to the figure caption. However, in 2+ is sufficient for the RaM mechanism to fully reset and allow a full Ca2+ uptake cycle. It is unclear how 140 nM Ca2+ can trigger an activation cycle when the mechanism is still in the resting state at 180 nM. As another example, a single pulse of 484 nM Ca2+ shown in 2+ uptake; however, in the same data set, a 421 and 400 nM Ca2+ pulse results in 0.1 nmol/mg of Ca2+ uptake as shown in 2+ does not fully inhibit the RaM mechanism and prevent further Ca2+ uptake for a second pulse shown in 2+ cooperativity constant is less than 60. If this value is increased, the model can simulate the Ca2+ uptake at 180 nM exceptionally well (not shown); however, the fits to the liver data set are compromised since both models share this parameter. A very similar, but opposite, reason exists why the model cannot reproduce the Ca2+ uptake corresponding to 200 nM in 2+ cooperativity constant less than 14 is necessary to fit this point. Perhaps, the RaM complex exhibits some sort of adaptivity property and/or a tissue-dependent degree of Ca2+ cooperativity which would help explain these apparent behavioral inconsistencies. However, until more quantitative data examining the intriguing properties of the RaM complex is collected, this possibility remains merely speculative. Regardless, the model is adequately able to capture the trends observed in both data sets.Mathematical models are informative abstractions of a physical reality, and their development and subsequent predictive capabilities are strongly dependent on the availability of experimental data, more so than the underlying theoretical constructs in which they are based. Although the 4-state RaM model presented herein is able to reproduce the experimental data regarding the time- and Ca2+ uptake 2+ uptake when RaM is in the open state is determined by the electrophoretic driving force alone (see below). One additional possible voltage-dependent mechanism is that the transition from one state to another is facilitated by the presence of a membrane potential. Unfortunately, the available experimental data do not allow testing of this hypothesis. Therefore, the model was constructed assuming that there was no requirement for an energy input; the transition rates were assumed to be voltage-independent. Future experiments will need to be carried out to facilitate an exploratory endeavor regarding these two different mechanisms. Considering the large body of evidence that the RaM is a precursor state of the CU The model was constructed assuming that there was no requirement for some form of exogenous energy input in order to cycle. However, some of the transition rates for the state model may in fact be voltage-dependent. This would bias the mechanism to cycle forward only in the presence of a sufficiently polarized membrane potential. In fact, it has been shown that the uncoupler, CCCP, abolishes any Ca2+ transport expression (equation 7) warrants some discussion. Although we assume a simple, centered Eyring energy barrier, many other variants, such as a trapezoidal energy barrier or Goldman-like energy barrier, can fit the experimental data equally well. This is due to our assumption that ΔΨ was constant throughout the experimental conditions. Considering that the experimental protocols used saturating substrate to energize the mitochondria, this assumption is justified. During a Ca2+ pulse, the ΔΨ would be expected to depolarize a few mVs; however, unless [Ca2+] rises above 10 s of µM, the ETS capacity would maintain a steady ΔΨ. Therefore, in order to maintain identical fits to the data with a different voltage-dependence for the Ca2+ transport equation, only the activities (RaMX) would need to be adjusted. It's quite possible that the voltage-dependence of the Ca2+ transport via RaM is similar to that found for the CU 2+ transport expression (equation 7), the underlying Ca2+-dependence and cycling mechanism remain valid.The functional form of the voltage-dependence of the Ca2+-binding protein stored in the SR, also contains many aspartic and glutamic acid residues and is capable of binding up to 40–50 Ca2+ ions per molecule 2+ rich environments), it's possible that p32 may play a role in Ca2+ storage in the matrix or Ca2+ transport across the mitochondrial inner membrane. In the CU model proposed by Starkov, several p32 trimers are recruited in the inner membrane to form the CU 2+ ions to associate with p32 to form a functional oligomer that is capable of transporting Ca2+ down its electrochemical gradient across the mitochondrial inner membrane. Also, since the proposed mechanism is dependent on Ca2+ binding to the aspartic and glutamic acid residues, the pH dependence on Ca2+ uptake via the uniporter measured by Moreau et al. 2+ uptake model. Based on Starkov's original hypothesis, a plausible mechanism capable of explaining much of the observed RaM-like properties is depicted in 2+-dependent activation cycle and transient burst of Ca2+ uptake implied from the available experimental data may be a result of the physical, “on demand” construction of the CU. In fact, we have successfully fit recent experimental data on matrix Ca2+ uptake when the buffer Ca2+ was in the sub-micromolar range The unique characteristics of RaM reveal some potential insight into the molecular identity of this obscure entity. One relatively overlooked potential molecular component of RaM is the rather conserved eukaryotic protein, p32/gC1qR 2+ signaling in a variety of tissues 2+ dynamics, the RaM mechanism may be involved with the underlying events that comprise the global Ca2+ response. In fact, Gunter et al. originally proposed that the RaM mechanism is controlled by a stochastic process 2+ regime. Thus, a stochastic approach describing the RaM mechanism may serve as the natural evolution of the model.In recent years, stochastic models have become increasingly prevalent when describing various biological phenomena 2+ uptake present in cardiac, liver and brain mitochondria. The model simulations are consistent with the available experimental data, and the model presents a unique opportunity to study the information encoded within the cytosolic Ca2+ transients presented to mitochondria in situ. We postulate that the frequency response of mitochondria is regulated by RaM and is critical to the translation of the cytosolic Ca2+ signal with respect to the cells energy supply and demand. We feel that the RaM model will serve as a means in which to further explore the role Ca2+ plays in mitochondrial bioenergetics in situ using mathematical modeling. Integrating this model into existing models of mitochondrial Ca2+ dynamics will help elucidate the physiological role this unique mechanism plays in mitochondrial Ca2+-homeostasis and bioenergetics.In conclusion, we have presented a basic model of the recently uncovered RaM mechanism for Ca2+-dependent dynamics of RaM in a mathematical formulation. Therefore, the models' ability to reproduce the data describing these dependences will be discussed herein. All calculations were performed with MATLAB®  on an Intel® Xeon® CPU(W3565 at 3.0 GHz) with 12 GB RAM. The results obtained were displayed using MATLAB®.A quick overview of the available experimental data will first be discussed followed by the derivation of the 4-state RaM model. Although RaM is shown to be differentially regulated by a variety of effectors such as adenine nucleotides, spermine and ruthenium red, the primary goal of this work was to capture the time- and Ca+ was included in the experimental medium to inhibit the Na+-dependent Ca2+ extrusion pathway. A dual pipetter system was constructed that was capable of delivering repeatable Ca2+ pulses via the application of various Ca2+-EGTA/EDTA buffers 2+ pulse waveform used in the experimental setup is presented in 2+ uptake via RaM was quantified using radiolabeling, fluorescence and atomic absorption techniques, and in some cases, corrections were made to compensate for the Ca2+ egress via the Na+-independent Ca2+ efflux pathway. The time- and Ca2+-dependences of RaM facilitated Ca2+ uptake was quantified by adjusting the pulsatile Ca2+ input delivered to the mitochondrial suspension. The pulses were adjusted to either be rapid in succession, high in amplitude, possess high interpulse Ca2+ concentrations or a mixture of either  to initiate at very brief transient of high Ca2+-conductance that is indicative of RaM. After approximately 30 ms, the mechanism switches from this highly conductive state into an inhibited state. The mechanism is then locked into this inhibited state until the external Ca2+ is lowered below ∼100–150 nM. While the external Ca2+ lies below this value, the mechanism enters the resting state which takes up to 60–90 seconds for heart 2+ uptake trends is pictured in 2+ binds to the external site(s) at least two orders of magnitude faster than the transition rates from the open/rest to inhibited states.The RaM model is developed here to reproduce the experimentally observed rapid CaThe RaM mechanism was modeled to adhere to the laws of thermodynamics. For example, the cyclical model obeys microscopic reversibility. The principle of microscopic reversibility, or detailed balance, has its origin in thermodynamics. For cyclical processes at equilibrium, the flow through each state in one direction must be equal to the flow in the opposite direction; otherwise, there would be no true equilibrium, and the system would require some form of energy input . Currently, there is no direct experimental evidence supporting the requirement of some form of energy input in order to cycle the RaM mechanism; therefore, the RaM model was constructed with microscopic reversibility strictly enforced. This removed a single adjustable parameter for each independent cycle from the optimization. If determined that the RaM mechanism is driven by some form of an energy input, the model can easily be refined to include such a dependence.2+ binding to the rest state (R), RaM enters the open state (O), and Ca2+ rapidly flows down its electrochemical gradient. Once open, RaM quickly closes and enters an inhibited state (1I and 2I) until [Ca2+] is decreased below a threshold value. Applying the rapid equilibrium assumption for states R and O and 1I and 2I, the dynamical equations for the 4-state RaM model can be reduced to the following deterministic ordinary differential-algebraic equationRO represents the fraction of RaM in the rest and open states, 1I2I is the fraction in the inhibited states, 1I to O, 2I to R, O to 1I, R to 2I, Caf is the fraction of state RO with Ca2+ bound and Cag is the fraction of state 1I2I with Ca2+ bound. The relationship between the rest and open states as a function of Ca2+ is expressed asOK is the threshold concentration that triggers the cycling mechanism and n is the Hill coefficient representing the steepness of the profile for the fraction of bound versus free RaM. Therefore, the amount of RaM in the rest or open states can be computed using IK is the threshold concentration locking the cycling mechanism in the inhibited state, and n is the Hill coefficient representing the steepness of the profile for the fraction of bound versus free RaM for the inhibited states. The fraction of inhibited RaM without and with Ca2+ bound are computed as similar to the fraction of rest and open RaM states where To derive the 4-state RaM model shown in 2+] is constant. However, in all the experimental protocols 2+] was held fixed. Thus, the non-linear state model simply becomes a linear state model, which can be integrated to obtain an analytical solution:τ is the time constant for transitions between RO and 1I2I at a given [Ca2+], ∞RO is the steady state value of RO at a given [Ca2+] and 0RO is the initial value of state RO. The time constant and the steady state value of RO at a given [Ca2+] are calculated usingThe non-linear state model presented in equation 1 must be solved numerically unless the external [Ca2+], the Ca2+ flux entering the mitochondrial matrix facilitated by RaM is computed using equation 7, which is a modified form of the ion transport equation assuming a single, centered Eyring energy barrier and applying the Goldman constant field assumption 2+ across the inner mitochondrial membrane. In the presence of a physiological ΔΨ, the Ca2+ flux leaving the matrix via RaM is negligible. Therefore, to simplify model development and analysis, only the flux entering the matrix was considered. With these assumptions, the unidirectional, Ca2+ transport rate via RaM is computed asRaMX is the activity of RaM, O is the fraction of RaM in the open state, Caz is the valence of Ca2+ (+2 unitless), F is Faraday's constant (96.480 J·mol−1·mV−1), R is the universal gas constant (8.314 J· mol−1·K−1), T is the temperature (298 K) and CaK is the RaM affinity for Ca2+. Since ΔΨ was not reported in the data sets With the dynamics of the 4-state RaM model known at any given x is the matrix free Ca2+ concentration, [Ca2+]e is the buffer free Ca2+ concentration, RaMJ is the rate of Ca2+ influx via RaM computed with the liver parameters found in O since the external Ca2+ was not fixed, CHEk is a first order rate constant approximating Ca2+ release via the CHE, [Ca2+]x,min is the basal matrix [Ca2+], CUJ is the rate of Ca2+ influx via the CU, Caβ is the buffering capacity of the mitochondrial matrix, pulseJ is a square wave simulating the Ca2+ release from the UV laser flash, washoutk is the first order rate constant approximating Ca2+ diffusion away from the site of photodegredation and [Ca2+]e,min is the basal buffer [Ca2+]. In the experimental protocol, Gunter et al. 2+: the experiment with the higher frequency used a pulse duration of 5.5 ms while the experiment with the lower frequency used a pulse duration of only 0.5 ms. Also, they reported that the 0.5 ms UV pulse gave a Ca2+ pulse with a peak of 400–500 nM 2+ pulse with a peak of approximately 5 µM. These peak values were used when simulating the dynamical system presented in equation 12 and shown in 2+ exchanged between the buffer and mitochondrion was assumed to be negligible with respect to the buffer Ca2+. Thus, the mitochondrial uptake and release fluxes are not included in equation 12b. The rate of Ca2+ influx via the CU was approximated using the following expression taken from CUX is the approximate CU activity and CUK is the CU affinity for Ca2+. The parameter values for the simplified dynamical system is located in The experimental system described above was reproduced in vivo-like Ca2+ pulse train is then explored, and it is shown that a bimodal 4-state RaM model, where a small fraction is liver-like while the majority is heart-like, would be a far more relevant mode of Ca2+ uptake without compromising mitochondrial integrity.In the File S15-STATE RAM MODEL.(DOC)Click here for additional data file.