In this regard, the lamprey spinal central pattern generator (CPG) stands out as a well-characterized, conserved vertebrate model of a neural network (Smith et al., 2+ signaling in spinal neuron dendrites orchestrate oscillations that drive network behavior.Within neural networks, synchronization of activity is dependent upon the synaptic connectivity of embedded microcircuits and the intrinsic membrane properties of their constituent neurons. Synaptic integration, dendritic Ca Thus, to explore the idea that active dendritic properties are at the core of this behavior, we examine in detail the lamprey spinal network and draw from other areas of dendritic research to enhance our understanding of what occurs at the level of the dendritic synapse to generate behavior.Orchestration of neuronal activity within networks is integral to correct execution of behavior. Synchronization between groups of neurons is an organizational feature of many neural networks found in the central nervous systems of invertebrates , or even actual movement, to observed locomoting animals. Despite this similarity, it is crucial to understand how physiological patterns of synaptic input and intrinsic membrane electrodynamics generate rhythmic behaviors.Vertebrate locomotion is initiated and maintained by evolutionarily conserved serial pathways originating in the forebrain  of individual VHNs in isolated spinal cords to undergo repetitive oscillations that are in-phase with the ipsilateral VR of the corresponding hemi-segment  during NMDA-TTX oscillations causes the cell to become similarly trapped at a depolarized mV. Thus, Ca2+ is necessary to hyperpolarize the cell from the depolarized state. Ca2+ activates myriad Ca2+-dependent proteins. In particular, VHNs contain Ca2+-dependent K+ channels  and the entering Ca2+ activates KCa2 channels to cause an afterhyperpolarization  NMDAR activation depolarizes VHNs; (2) increasing NMDAR conductance by ejecting Mg2+ from the pore; (3) causing further depolarization and Ca2+ entry via the NMDAR as the mV plateaus; (4) which activates KCa2 channels to hyperpolarize the cell; and (5) ending the depolarized plateau to repolarize the cell where it can repeat the cycle  is unknown. Do many dendrites receive synchronous input from their various synaptic partners? Does input occur in discrete spatial locations? The location and timing of synaptic input is crucial for the transmission of potentials arriving at the soma, which will greatly influence neuronal output  to show synchronized increases in intracellular Ca2+ (Ca2+i). In contrast, local Ca2+ signaling domains  in dendrites can occur following neurotransmitter receptor activation , but also from VGCCs following depolarization from local synaptic potentials , is a large determinant for the extent of spread of Ca2+ because diffusion is restricted at the spine neck -driven VGCC activation in dendrites , NMDARs and metabotropic glutamate receptors (mGluRs) may be activated in the postsynaptic compartment. AMPARs are responsible for fast depolarization, and can locally activate nearby VGCCs to cause Ca2+ entry. Local depolarization, or depolarization induced from bAPs can alleviate Mg2+ block of the NMDAR, facilitating Ca2+ influx during concurrent and subsequent release of glutamate at that synapse    will profoundly impact cell firing rates, dendritic integration, and processing both in real-time during individual cycles of locomotor activity, but also in the long-term. The molecular complexing of Ca2+ sources to secondary effector proteins, like KCa2 in lamprey, will consequently impact spike-timing through activation of the mAHP , alone or within a cocktail of monoamines, can activate locomotion and fictive locomotion in many preparations  as well as from RS command neurons (Buchanan and Grillner, Ca2 channel activation.Thus, the serotonergic system in the spinal cord plays a crucial role in modulating the output of the spinal network. While these results, whether mediated by pre- (Schwartz et al., in vivo are not physiologically activated by a tonic and diffuse release of glutamate. Instead, the release of neurotransmitter and subsequent receptor binding is exquisitely targeted to discrete postsynaptic loci with temporal precision. The physiological activation of NMDARs in any circuit is almost entirely mediated by the synaptic release of glutamate. This will only occur at synapses, and only following presynaptic release of glutamate at those synapses. This constrains the activation of NMDARs spatially and temporally, as well as the KCa2 channels that are subsequently activated.In all vertebrates, 5-HT and glutamate applied exogenously can initiate and influence locomotor-like activity. While it is remarkable that systemic drug application can reliably produce ethologically relevant locomotor patterns in lamprey (Sigvardt et al., Xenopus larval tadpoles, there may be little specificity in anatomical connections early in development (Li et al., While pharmacological activation of the spinal network is presumably far from physiological, it has remained to be demonstrated just how distinct this artificial induction is from supraspinal control of descending command neurons and subsequent spinal CPG activation. It is important to note that generating rhythmic activity and appropriate phase coupling has many theoretical solutions (Wallén et al., in vivo is even less certain than glutamate because exogenous application of these modulators over an artificially and pharmacologically activated network merely compounds errors and cannot match physiological release. Indeed, monoamine cocktails with glutamate agonists evoke spinal network activity (Rossignol et al., Furthermore, our understanding of how monoamines in general and 5-HT in particular act 2+i while Ca2+ oscillations are synchronized throughout the dendrites of a single neuron (Bacskai et al., 2+ entry, whether NMDARs or VGCCs, may become obscured. This may allow KCa2 channels to couple to Ca2+ microdomains as opposed to nanodomains implied by their physiological BAPTA sensitivity. Indeed, in VHNs dialyzed with EGTA, oscillation plateau progressively lengthen immediately following whole-cell access, on a time course equivalent to the diffusion of dyes to the most distal dendrites (Alpert and Alford, 2+ buffering into the distal dendrites where Ca2+ oscillations are largest (Viana di Prisco and Alford, 2+ from binding KCa2 channels to cause the repolarization (Alpert and Alford, 2+ diffusing greater distances from its site of entry can, under certain circumstances, activate KCa2 channels, which then contribute to the repolarization. Nevertheless, even under these non-physiological conditions of bath applied NMDA, cells dialyzed with BAPTA displayed immediate, severely impaired repolarization (Alpert and Alford, Bath-applied NMDA leads to a large increase in baseline Ca2+ entry to bind KCa2 channels in Ca2+ microdomains may be an artifact of bath-applied NMDA and the robust increase in intracellular Ca2+, which may also cause Ca2+-induced Ca2+ release from internal stores. However, it was recently demonstrated that this KCa2 channel conductance is physiologically activated by synaptically driven NMDAR-mediated Ca2+ entry (Alpert and Alford, Ca2 channels, then it should follow that there is complementary patterning dendritic Ca2+ entry which drives the activation of KCa2 channels important for rhythm generation. However, we know very little about the spatiotemporal pattern of dendrite activation during behavior and how synaptic input is integrated in real time to impact cell output.The ability of NMDAR-induced Ca2+ dynamics in active networks are crucial to understanding how physiological patterns of synaptic input are integrated in real time to shape the cellular output and have only recently been investigated. With new advances in genetically encoded Ca2+ indicators (Muto et al., in vivo 2-photon microscopy, it is now becoming possible to “watch dendrites in action” and correlate their activity to sensory input and behavioral output (Dombeck et al., Several recent advances have made it possible to begin to assess how dendrites integrate incoming synaptic information within an active, behaving network. Dendritic spatiotemporal CaThe 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.


Phenethyl isothiocyanate (PEITC) is a cancer chemopreventive agent from cruciferous vegetables. Cholangiocarcinoma (CCA) is a chemo-resistant cancer with very poor prognosis. We evaluated the effects of PEITC on induction of apoptotic cell death in relation to cellular glutathione (GSH) and mitochondrial function of a CCA cell line, KKU-M214.Cytotoxic effects of PEITC on a CCA cell line, KKU-M214, and a reference cell line, Chang cells were evaluated. To delineate mechanisms of cell death, the following parameters were measured; GSH and superoxide levels as the oxidative status parameters, apoptosis related proteins levels using Western blotting. Cellular free calcium level and mitochondrial transmembrane potential were also measured.N-acetylcysteine (NAC) prevented redox stress in Chang but not in KKU-M214 cells. The loss of mitochondrial transmembrane potential was induced by PEITC concurrent with GSH stress, but was not a primary cause of cell death. The rapid increase of free calcium level in cytosol was associated with cell death in both cell lines. These events were prevented by NAC in Chang cells, but not in KKU-M214 cells.PEITC induced apoptotic cell death of both KKU-M214 and Chang cells. After PEITC treatment, both cells showed decrease of Bcl-xl and increase of Bax levels. While KKU-M214 cells released AIF, Chang cells released cytochrome c, with subsequent activation of caspase 3 and 9, upon PEITC treatment. PEITC induced superoxide formation in both cells, although it seemed not play a role in cell death. PEITC caused GSH redox stress in different ways in two cell types, because PEITC induced cell death KKU-M214 cells and Chang cells via increase of cellular calcium mobilization and activation of mitochondrial cell death pathway. The effects of PEITC on the redox stress was mediated via different ways in CCA and Chang cells because NAC could prevent redox stress in Chang cells, but not in KKU-M214 cells. The multiple effects of PEITC may be useful for the development of novel chemotherapy for CCA. Cholangiocarcinoma (CCA) is a malignancy originating from the bile ducts, usually adenocarcinomatous, and is the second common primary liver cancer . CCA is Ψm)  cells were used as the reference because our previous study showed that oxidative stress caused the suppression of glutathione (GSH) redox in this cell line .2 in air at 37°C as described previously  cells were both kindly provided by Prof. Banchob Sripa, Department of Pathology, Faculty of Medicine, Khon Kaen University. Cells were grown in Ham’s F12 medium supplemented with 12.5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid , 100 U/mL penicillin, 100 μg/mL streptomycin sulfate and 10% fetal calf serum and maintained under 5% COeviously . The celKKU-M214 and Chang liver cells were seeded onto 96-well culture plates at a density of 7,500 cells/well. After an overnight culture, the serum-free medium was replaced with the media containing PEITC, and the cells were cultured for various time intervals. The cytotoxicity was examined by the sulphorhodamine B (SRB) assay. In brief, cultured cells were washed once with phosphate-buffered saline (PBS), fixed with 100 μl of ice-cold trichloroacetic acid for 1 h, and stained with 50 μl of 0.4% SRB in 1% acetic acid for 30 min. The cells were then rinsed several times with 1% acetic acid, and protein-bound dye was dissolved with 200 μl of 10 mM Tris base solution. The absorbance was determined using a microplate reader with the filter wavelength of 540 nm.To determine cells undergone apoptosis, cultured cells were stained with fluorescent dyes as previously described with modifications ,21. In b4 cells were seeded in 96 black well plates and cultured overnight. Then, the medium was removed and the cells were washed with phosphate buffered saline (PBS). They were then treated with PEITC and 25 μM DHE with or without 2 mM N-acetyl-L-cysteine (NAC) or 0.5 mM 4-hydroxy-TEMPO (TEMPOL), in serum-free medium and kept in 5% CO2 atmosphere at 37°C for 90 min. The fluorescence intensity was read and quantified in a Gemini XPS fluorescent plate reader with the excitation and emission wavelength of 518 nm and 605 nm, respectively.Intracellular ROS generation was measured using a cell-permeable fluorescent probe, dihydroethidium (DHE). Briefly, 5 × 10Total glutathione was measured essentially according to Tietze’s method . GlutathIntracellular calcium level was measured using an assay kit . In brief, KKU-M214 and Chang cells were grown on 96 well plates at the density of 10,000 cells/well and treated with 3 and 10 μM PEITC with/without 2 mM of NAC for 1 h. Then, the media were removed and FluoForte® dye-loading solution was added according to the manufacturer’s instruction. The plate was incubated at 37°C for 30 min and the fluorescent staining was analyzed under a fluorescent microscope with the excitation and emission wavelengths of 485 nm and 535 nm, respectively. Images of the cultured cells were captured and the integrated optical density (IOD) of each image was analyzed by using the Image-Pro Plus (Media Cybernetics) software.Ψm), cells were seeded in 96 black well plates at the density of 10,000 cells/well and cultured overnight before treatment with various concentrations of PEITC for 3 and 24 h. The assay was performed according to the method described previously [Ψm was analyzed under a fluorescent microscope with the excitation wavelength of 485 nm and emission wavelength of 535 nm. JC-1 forms J-aggregates in healthy mitochondrial matrix, which can be visualized as red fluorescence. In depolarized mitochondria, JC-1 effluxes to the cytoplasm and exists as monomers with green fluorescence. The shift of red to green fluorescence is an indicative of the depolarization of ∆Ψm.The dissipation of the mitochondrial electrochemical potential gradient is known as an early event leading to apoptosis. To measure the change in mitochondrial transmembrane potential  and fluorogenic Ac-LEHD-AFC, a caspase 9 substrate (50 μM) or Z-IETD-AFC (EMD Millipore), a caspase 8 substrate (50 μM). Reaction mixtures were incubated for 8 h at 37°C in dark and fluorescent signals were read using the Gemini XPS fluorescent plate reader with the excitation and emission wavelengths of 400 and 505 nm, respectively.After treatment with PEITC for 3 and 6 h, the cultured cells were trypsinized, and adjusted to 10Cell lysates were prepared as above and mixed with the reaction buffer , EnzCheck® Caspase-3 Assay kit#1 (Molecular Probe). Reaction mixtures were incubated for 1 h at 30°C in dark and fluorescent signals were read using the Gemini XPS fluorescent plate reader with the excitation and emission wavelengths of 342 and 441 nm, respectively.p < 0.05.All the results were presented as the mean ± SEM. Statistical comparison between control and treated group was performed using Student’s t-test or One-way ANOVA with Student-Newmann-Keuls post-hoc test, where appropriate. The level of significance was set at 50 values were not different between 24 and 48 h of incubation (2.99 ± 0.87 and 3.25 ± 0.19 μM), respectively, for KKU-M214 . We examined also the role of antioxidants on GSH depletion and ROS formation. For this purpose, we used TEMPOL, a ROS scavenging agent, and NAC, a thiol modifier. As shown in Figure 2+ from some intracellular Ca2+ storages, particularly from the endoplasmic reticulum, resulting in the increase of cytosolic and mitochondrial Ca2+, which initiates cell death [2+ mobilization in KKU-M214 and Chang cells. As shown in Figures 2+ mobilization into cytosol within the first 1 hour of incubation, which was visualized by Ca2+ fluorescent probe in KKU-M214 and Chang cells. NAC, a thiol modifier, could not inhibit Ca2+ flux into cytosol in KKU-M214 cells  pores, we examined whether the opening of MPT was the primary effect of PEITC to induce cell death. The results show that cyclosporine (Cs) , a potent MPT inhibitor, could prevent the losses of ∆Ψm (data not shown), but could not prevent cell death in both cell types  in DU145 but not in LnCaP cells  or with Present study showed that PEITC could induce apoptosis of both KKU-M214 and Chang liver cell lines in association with the decreased Bcl-xl and increased Bax expressions. It is known that p53 plays an important role in physically and functionally interacting with Bcl-2 family members for their translocation to mitochondria . HoweverIn the present study, the cytotoxicity of PEITC was mediated via caspase-independent and caspase-dependent pathways for KKU-M214 and Chang cells, respectively. AIF is released from mitochondria and translocated to the nucleus where it fulfills the lethal function. Similar to cytochrome c, AIF play an important role in mitochondrial respiratory chain and is required for cell survival . HoweverOne of the prominent effects of PEITC is the induction of oxidative stress in cancer cells, which is characterized by ROS formation, GSH depletion and protein oxidation ,12. Our PEITC is known to induce ROS formation, which is assumed to be a main mechanism of cell killing action of PEITC on some tumor cells ,12,17. H2+ into cytosol, where endoplasmic reticulum is the important Ca2+ storage [2+ into cytosol shortly after PEITC treatment. The rapid increase of cytosolic Ca2+ may cause elevation of mitochondrial Ca2+ and decrease of Ca2+ in endoplasmic reticulum, and such imbalance of Ca2+ may trigger a variety of cascades leading to cell death. The lack of protective effect of NAC on cytosol Ca2+ flux in KKU-M214 cells suggests that PEITC may exert the effect on the release of Ca2+ by cellular stress other than the GSH redox system. On the other hand, in Chang cells, GSH redox disturbance may be primarily involved in cytosolic Ca2+ flux.Oxidative stress can trigger the mobilization of Ca storage . We obse2+ was accompanied with the rapid loss of ∆Ψm. The depolarization of ∆Ψm is resulted from the opening of MPT pores, which is formed in the inner membrane of mitochondria [Ψm. This may imply that the depolarization of ∆Ψm is probably an associated event of PEITC treatment, but is not a direct effect leading to cell death. Taken all these together, mitochondria may not be a primary target of the increase of cytosolic Ca2+ flux for initiation of cell death. Instead, the increased cytosol Ca2+ may initiate the death signals in the cytosol by activation of a variety of Ca2+-sensitive enzymes, such as calpain, leading to the cleavage and targeting of Bax to mitochondria [In the present study, an increase of cytosolic Cachondria . The opechondria  to activ2+ mobilization and activation of Bcl-2 proteins by PEITC remain to be elucidated. Since the precise mechanisms of PEITC-induced cell death of KKU-M214 cells remain unclear, further study is needed to explore novel mechanisms of the expression of cytotoxicity of PEITC.Our study revealed that PEITC induces up-regulation of Bax and down regulation of Bcl-xl. The formation of Bax pore and mitochondrial outer membrane permeabilization (MOMP) upon activation may be the critical event leading to the release of proapoptogenic proteins, cytochrome c and AIF, and ensuing cell death . The mec2+ flux, which leads to mitochondrial cell death mechanisms. Effect of PEITC on redox status of GSH may be not important for cell killing for CCA cells but it is important for maintaining cell functions in Chang cells. The different effect of PEITC on different cell types was clearly shown by the cytoprotection response to antioxidant, NAC. More study is needed using several CCA cell lines over the response to PEITC. Taken together, the present results highlighted different responses of the cells to PEITC, which may facilitate the new approaches in the study of PEITC for drug development for the treatment of CCA.In conclusion, the present results show that PEITC induced apoptosis of CCA cells and Chang cells. This process may involve induction of oxidative stress and triggering of CaCCA: Cholangiocarcinoma; Cs: Cyclosporine; GSH: Glutathione; GSSG: Glutathione disulfide; JC-1: 5,5′,6,′-tetrachloro- 1,1′,3,3′ tetraethylbenzimidazolyl carbocyanine iodide; JNK: c-Jun-N-terminal kinase; NAC: N-acetylcysteine; PEITC: Phenethyl isothiocyanate; ROS: Reactive oxygen species; SRB: Sulphorhodamine B; ∆Ψm: Mitochondrial transmembrane potential.The authors declare that they have no competing interests.VK designed the study. OT performed the experiments. VK and OT analyzed the data. VK, OT, LS, AP and UK interpreted the results. OT, VK, and LS wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/571/prepub


On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca2+ release specifically regulates distinct Angs-mediated angiogenic steps.Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca Angiogenesis is a complex remodeling process characterized by the sprouting of new blood vessels from preexisting ones, occurring mainly during embryonic development and in pathological processes such as cancer. During the early stages of this process, endothelial cells (ECs) are stimulated to form new capillaries by growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Angiopoietins (Angs) play a fundamental role in the subsequent maturation step, in which microvessels acquire a layer of mural cells, pericytes, and smooth muscle cells, which are critical for the development and maintenance of functional vasculature , 2. Expe2+) in the regulation of angiogenic processes. Ca2+ is a crucial point of intersection for many distinct molecular signaling pathways that promote and modulate angiogenesis. Specific Ca2+ signatures rely upon spatiotemporal variations in [Ca2+]i i by mobilizing Ca2+ from internal stores. The latter study demonstrated the direct role of NAADP in VEGF-induced Ca2+ mobilization from acidic organelles and its important involvement in the control of angiogenic processes i  and are ganelles , 30. We g in ECs  and haverocesses . To our Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Sales Ltd., cultured in EGM-2 Endothelial Cell Growth Medium-2 , + 100 mM penicillin/streptomycin (Sigma). Cells were maintained at 37°C in a humified 5% (vol/vol) incubator and grown to reach the confluence. Monolayers were used at passages 1 to 6.μM Fura-2-AM (Invitrogen) for 1 h at 37°C and then rinsed with Hanks' Balanced Salt Solution (HBSS Sigma) or Krebs-Henseleit-HEPES (KHH) buffer . Plates were placed into a culture chamber kept at 37°C controlled temperature on the stage of an inverted microfluorimeter (Nikon TE2000E) connected to a cooled CCD camera . Samples were illuminated alternately at 340 and 380 nm using a random access monochromator  and emission was detected using a 510 nm emission filter. Images were acquired (1 image/sec ratio) using MetaFluor software . Calibration of the signal was obtained at the end of each experiment by maximally increasing intracellular Ca2+-dependent Fura-2-AM fluorescence with 5 μM ionomycin  followed by recording minimal fluorescence in Ca2+-free medium. [Ca2+]i was calculated according to previously described formulas i-independent. To further assess whether Ca2+ signal is dispensable for Ang-2 to induce capillary-like formation, we performed the Matrigel assay in the presence of second messengers inhibitors as shown in Figures 3-mediated Ca2+ signaling in capillary-like formation in vitro.The formation of capillary-like structures process . Matrige in vitro and in vivo angiogenesis; conversely, Ang-2, a somehow context-dependent antagonist of Ang-1, promotes vessels destabilization favouring pericytes detachment [2+-dependent machinery activated by Angs which controls angiogenic processes, including migration and the ability to induce capillary-like structures in vitro. Our data suggest that Ca2+ signaling might be envisaged as a possible “signaling hub” for Angs and other angiogenic factors. Given the therapeutic potential of angiogenesis-blocking therapies based on the inhibition of specific signaling pathways, this concept could open the way to novel experimental approaches in this field.During the complex process of angiogenesis, a variety of growth factors and cytokines are upregulated and exert their functions through autocrine/paracrine signaling. Among these, Ang-1 and Ang-2 play important roles. Ang-1 does not stimulate ECs growth but rather promotes stabilization of vascular network and branching morphogenesis intachment . In the Densitometric Analysis of Western Blots: Densitometry was performed on scanned immunoblot images using the ImageJ analysis tool to obtain the intensity for each experimental band calculated by normalizing to the correspond control β-actin band.


Increasing evidence has implicated the dysregulation of STIM–ORAI and IP3Rs in tumorigenesis and tumor progression. By controlling the activities, structure, and/or expression levels of these Ca2+-transporting proteins, malignant cancer cells can hijack them to drive essential biological functions for tumor development. However, the molecular mechanisms underlying the participation of STIM–ORAI and IP3Rs in the biological behavior of cancer remain elusive. In this review, we summarize recent advances regarding STIM–ORAI and IP3Rs and discuss how they promote cell proliferation, apoptosis evasion, and cell migration through temporal and spatial rearrangements in certain types of malignant cells. An understanding of the essential roles of STIM–ORAI and IP3Rs may provide new pharmacologic targets that achieve a better therapeutic effect by inhibiting their actions in key intracellular signaling pathways.The stromal interaction molecule (STIM)-calcium release-activated calcium channel protein (ORAI) and inositol 1,4,5-trisphosphate receptors (IP Therefore, it is not surprising that any derangement of Ca2+ channels and/or receptors will contribute to the establishment of many life-threatening diseases, such as cardiopathy [2+ channels or receptors is characterized by the features of specific cancer subtypes, of which the most important is the protein complex consisting of the stromal interaction molecule (STIM), calcium release-activated calcium channel protein (ORAI), and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). STIM–ORAI is able to sense and respond to intracellular Ca2+ microenvironmental changes that occur during cancer development. This complex primarily mediates the Ca2+ influx, with STIM serving as the endoplasmic reticulum (ER) Ca2+ sensor and ORAI as the Ca2+-selective entry channel. Furthermore, the roles of IP3Rs are also discussed. There is emerging evidence that IP3Rs, which regulate the Ca2+ flux from the ER into the cytosol and mitochondria, play crucial roles in the apoptotic pathway; additionally, IP3Rs have been implicated in cellular senescence [2+ channels/transporters that are thought to be responsible for the development of cancers [3Rs, with a focus on exploring the mechanism underlying the hijacking of the Ca2+-transporting molecules STIM-ORAI and IP3Rs by malignant cancer cells that leads to tumor onset, growth, and metastasis. The concomitant interaction between STIM–ORAI and IP3Rs is also discussed. Understanding the molecular basis and pathologic transformations of Ca2+-transporting molecules in cancer cells will offer an opportunity for pharmacologic modulation and therapeutic intervention.Calcium signals are widespread and rigorously regulate the majority of fundamentally important physiologic processes ranging from cell proliferation to cell apoptosis . The pre+ stores . Any perdiopathy , heart fdiopathy , neurodediopathy , and candiopathy . Remarkasphate IP receptor cancers . Althoug2+ from either the entry of extracellular Ca2+ across the plasma membrane or the release of Ca2+ from internal calcium stores in the ER and sarcoplasmic reticulum (SR) [2+ channels that are located on the plasma membrane. Upon the stimulation, extracellular Ca2+ can enter the cytoplasm through Ca2+ channel transport. Moreover, the plasma membrane is responsible for refilling the internal Ca2+ stores when they are depleted. The primary Ca2+ entry pathway is store-operated Ca2+ entry (SOCE), which includes two key components: a sensitive sensor of calcium store depletion (STIM) and an effective channel that can facilitate calcium entry into the cell (ORAI) [External stimulation leads to an increase in cytoplasmic Calum (SR) . Both ofl (ORAI) –12.2+ store levels in the ER and control calcium refilling by forming connections with ORAI [2+-binding EF-hand , a hidden EF hand, and a sterile α-motif (SAM) domain. The cytosolic strand includes three putative coiled-coil  regions, calcium release-activated calcium (CRAC) modulatory domain (CAD) or a STIM-ORAI-activating region (SOAR), serine or proline-rich segments, and lysine-rich regions [2+-binding affinity of EF-SAM perfectly matches the detailed alteration of the Ca2+ concentration and enables the ER sensor protein to respond to changes in the Ca2+ concentration in ER. The Ca2+ depletion in the ER leads to the dissociation of Ca2+ from the EF hand, thereby destabilizing the entire EF-SAM entity. The CC regions and the serine/proline-rich region promote the oligomerization of STIM, thereby enabling its redistribution into multiple punctae and its localization at ER-plasma membrane junctions. The CC domain has been proven to control the exposure and oligomerization of the SOAR [2+ in the cytosol and ER [2+ levels in the human signaling proteome, whereas STIM1 seems to be more involved in the Ca2+ entry associated with more pronounced depletion [STIM, which is predominantly located in the ER, was identified using an RNA interference screen in Drosophila S2 cells; then, two mammalian orthologs (STIM1 and STIM2) were found . Both STith ORAI . STIM1 c regions . The lowthe SOAR . The strl and ER . Thus, Sepletion , 19.The ORAI gene encodes a family consisting of three proteins . Each of these proteins consists of 4 TM-spanning segments and 3 cytosolic strands, which include the N-terminus, the second loop con-necting TM2 and TM3, and the C-terminus . The ORA2+ stores in the ER and moves within the ER to ER-plasma membrane junctions. Then, STIM recruits ORAI to the ER-plasma membrane junctions, where the two proteins form a close contact. The formation of the active STIM–ORAI complex at the conformational gate of the SOCE channel allows Ca2+ entry [The primary intracellular hysiologic functions of STIM and ORAI are straightforward Fig. a. STIM d2+ entry .Fig. 1Di3R is the most ubiquitous intracellular Ca2+ channel, and its isoforms  have been identified in vertebrates [3R isoforms have distinct but overlapping expression patterns. IP3R1 is expressed in neuronal cells, IP3R2 is expressed in liver and muscle cells, and IP3R3 is expressed in most cultured cell types [3Rs (which exist as tetramers) has been determined; IP3Rs contain a binding site for IP3 in the N-terminal region, the channel domain, and the determinants for tetramer formation in the C-terminus [The IPtebrates . The majll types . The genterminus , 25.3Rs predominantly reside in the ER. IP3 is produced by phospholipase C and binds to IP3Rs to induce calcium release from the ER upon cell activation by endogenous or exogenous hormones, growth factors, or neurotransmitters. Notably, IP3-induced Ca2+ release is typically regulated by the Ca2+ concentration in the cytosol and ER. Furthermore, the activities of IP3Rs are biphasically regulated by cytosolic Ca2+. The concentration–response relationship is a typical bell-shaped curve, indicating that the IP3-mediated Ca2+ release is potentiated at a low Ca2+ concentration and inhibited at a higher concentration [2+ storage in the lumen of the ER also regulates IP3Rs, which can prevent excessive ER depletion at low levels of store filling [3R-mediated Ca2+ elevation regulates fundamental cellular functions, such as fertilization, cell cycle entry, cell division, metabolism, and transcription [3Rs is to decide the cell fate by controlling the mitochondrial Ca2+ elevation and mitochondrial metabolism. Cell survival or apoptosis is encoded in the frequency and amplitude of Ca2+ oscillations mediated by IP3Rs and decoded by different Ca2+-sensitive kinases or phosphatases that in turn regulate the target proteins. When IP3Rs transport appropriate amounts of Ca2+ from the ER to the mitochondria, they catalyze the conversation of pyruvate to acetyl-coenzyme A (CoA) to produce adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide 2′-phosphate (NADPH). The insufficient transport of Ca2+ to the mitochondria induces cellular autophagy. Conversely, when activated IP3Rs excessively transport Ca2+ from the ER to the mitochondria, the mitochondrial Ca2+ is overloaded, which induces a dissipation of the mitochondrial potential, the opening of the permeability transition pore, and the release of pro-apoptotic factors such as cytochrome c [3Rs play pivotal roles in the apoptotic process via controlling the cellular response to apoptotic signals and conferring oncogenic features to the cell [IPntration . The Ca2 filling . IP3R-mecription . An impochrome c ; this prthe cell .STIM and ORAI have been found to be abundantly expressed in human cancer tissues and multiple tumor cell lines. Abnormal spatial and temporal changes in these two proteins have been found to be involved in many aspects of tumorigenesis, including cancer cell proliferation, migration, and apoptosis resistance.STIM1 mRNA levels in tumors was significantly reduced compared with the control group. Additionally, STIM1 could also be used as a predictive marker for metastatic potential in patients with hepatocellular carcinoma [ORAI3 mRNA was increased in breast cancer tissues from the majority (76.9 %) of patients compared with healthy control tissues. Increased expression of ORAI3 in tumor tissues from 60 patients presenting non-small cell lung adenocarcinoma was also noted by Ay et al. [Increasing evidence has shown that STIM and ORAI are overexpressed in many types of malignant tumors, including breast cancer , glioblaarcinoma . The higarcinoma  demonstrarcinoma , who shoy et al. . Additioy et al.  showed tThe functions of STIM and ORAI in certain types of cancer have fascinated many investigators. The use of pharmacologic interference and small interfering RNA (siRNA)-mediated gene knockdown approaches to down-regulate STIM and ORAI at both the mRNA and protein levels inhibits tumor cell proliferation, promotes cell apoptosis, and reduces tumor size. These results revealed that STIM and ORAI promoted tumorigenesis and tumor progression through the following key events: elevated proliferation, enhanced migration, and increased resistance to apoptosis.A study of STIM1 indicated that the gene locus encoding STIM1 on chromosome 11p15 was deleted in human rhabdomyosarcoma and rhabdoid tumor cell lines . Ectopicc-Myc, which is a key proto-oncogene and is enhanced in most human cancers [1 phase. Ay et al. [Compared with STIM, the role of ORAI in tumorigenesis may be more explicit. The dysregulation of ORAI is affected by the activation of proto-oncogenes or the inactivation of tumor suppressors. Recently, compelling evidence has suggested that ORAI3 is closely related with  cancers . In thisy et al.  found thy et al.  also dem2+. Furthermore, the remodeled ORAI channels promoted cancer cell proliferation via activation of the transcription factor nuclear factor of activated T cells (NFAT), followed by the stimulation of cyclin D1 expression, which is a key rate-limiting controller of the G1/S phase transition. Faouzi et al. [Any structural remodeling and functional changes of ORAI3 may trigger a switch to a more aggressive cell phenotype. Dubois et al.  showed ti et al.  demonstr2+-dependent signaling process, and STIM–ORAI is hijacked by malignant cancer cells to drive the biological functions required for tumor development [2+ influx accelerates focal adhesion (FA) turnover through the constitutively active forms of the small GTPase RAC1 and the Ca2+-dependent proline-rich tyrosine kinase 2 (Pyk2) [2+ signaling enhances contractile forces by regulating the actomyosin reorganization. Actomyosin is a complex of actin filaments and non-muscle myosin II. The actomyosin generates cortical tension with the extracellular matrix or neighboring cells and transmits the contraction to FAs that move the cell body [2+ signaling exerted comprehensive and crucial functions to promote tumor cell migration by interacting with FA and actomyosin [2+-dependent transcription factor that plays a critical role in the tissue invasion of tumor cells by promoting the expression of autotaxin and cyclooxygenase 2 (COX2); these factors participate in the epithelial-to-mesenchymal transition [STIM and ORAI have also been found to affect the migration of cancer cells. Increasing evidence has shown that tumor migration can be viewed as a Caelopment . In otheelopment . Blockinelopment , breast elopment , glioblaelopment , pancreaelopment , and melelopment . The STI2 (Pyk2) . The eff2 (Pyk2) , 53. At ell body . These ftomyosin . Moreovetomyosin . NFAT isansition .3Rs in tumorigenesis and tumor metastasis. Over the last 20 years, IP3Rs have been regarded as key regulators that control cell death and survival in a variety of cellular systems. Interfering with the amount of IP3R-mediated Ca2+ transport from the ER to the mitochondria determines the susceptibility of cells to apoptotic stimulation. Because IP3Rs can promote senescence and/or apoptosis, the available evidence indicates that down-regulating IP3Rs or dampening their activities can decrease cellular sensitivity to apoptotic signaling, finally resulting in the survival of cells with oncogenic features. An in vitro study showed that knockdown of IP3R1 prevented apoptosis in bladder cancer cells and rendered them resistant to chemotherapeutics [3Rs might increase the sensitivity of cancer cells to cisplatin [3Rs are also hijacked by different proto-oncogenes to give rise to cells with oncogenic features [3Rs through its tetrahydrobiopterin (BH4) domain to inhibit their functions [3Rs hindered mitochondrial Ca2+ accumulation by abrogating Ca2+ transport from the ER to the outer mitochondrial membrane  cells [3Rs. The expression levels of IP3R3 in colon cells were directly related to tumor aggressiveness [3Rs may vary in different types of cancer, and many mechanisms are not fully understood.Growing attention has been paid to the special role of IPapeutics . Conversisplatin . As the features , such asfeatures , Bcl-2 ffeatures , 63, Baxfeatures , 65, andfeatures . Recentlunctions . The spaor cells . The Bcln the ER , 69. TheL) cells . HoweverL) cells  reportedsiveness . These r3 to IP3Rs releases intracellular Ca2+, leading to a reduction in the Ca2+ concentration in the lumen of ER, which in turn activates the STIM sensor to allow extracellular Ca2+ to refill the empty ER Ca2+ stores across the ORAI in the plasma membrane. In rapidly growing cancers, IP3Rs are blocked by a variety of anti-apoptosis proteins, resulting in Ca2+ overload in the ER. The harsh microenvironment perturbs the STIM-ORAI functions and induces the accumulation of misfolded proteins in the ER. This triggers an adaptation program referred to as “ER stress.” Chronic ER stress kills normal cells but can contribute to tumor cell dormancy, thereby permitting survival in the stressed environment until more favorable conditions are encountered. Overexpression of STIM could reverse ER stress, implying that Ca2+ overload restrains the STIM functions in cancer cells [2+ store content in the ER via promoting IP3R1 overexpression to suppress agonist-induced Ca2+ release and mitochondrial Ca2+ accumulation in cancer cells [3Rs are not completely understood, and further investigations are needed to elucidate the mechanisms by which cancer cells control the functions of STIM-ORAI and IP3Rs.The binding of IPer cells . Oncogener cells . Neverth3Rs in several types of human cancer have made them attractive drug targets for tumor therapy. Inhibiting ORAI1 by pharmacologic antagonists in cultured epithelial cells derived from ESCC patients impeded ESCC cell proliferation, invasion, and migration [2+ entry, inhibited the growth and metastasis of tumor cells after 1 week of treatment [Multiple roles of STIM–ORAI and IPigration . Importareatment , 77. No reatment , even 2 2+ signal channels have shown that STIM-ORAI and IP3Rs either do not differ or are overexpressed in tumor tissues compared to those in normal tissues. However, the roles of STIM-ORAI and IP3Rs may be over- or underestimated depending on the use of the pharmacologic inhibitors or siRNA-mediated gene knockdown approaches in cancer cells. Moreover, only a relatively limited amount of information concerning STIM-ORAI and IP3Rs is available to date due to their complicated and comprehensive functions in tumor cells. Despite a wealth of data describing their functions, the elucidation of their roles in cancer is still at the beginning stages. How STIM-ORAI and IP3Rs affect carcinogenesis in vivo, the relationship between these proteins and Ca2+ oscillations in cancer cells, and whether the participation of these proteins in the cancer procedure is a general mechanism need to be investigated.To date, “proof of principle” studies of the Ca


Epigenetic aberrations play a central role in the pathophysiology of acute myeloid leukemia (AML). It has been shown that molecular signatures based on DNA-methylation (DNAm) patterns can be used for classification of the disease. In this study, we followed the hypothesis that DNAm at a single CpG site might support risk stratification in AML.C1R) as best suited biomarker: patients with higher methylation at this CpG site (>27 % DNAm) reveal significantly longer overall survival . This finding was validated in an independent set of 62 DNAm profiles of cytogenetically normal AML patients (P = 0.009) and with a region-specific pyrosequencing assay in 84 AML samples (P = 0.012). DNAm of C1R correlated with genomic DNAm and gene expression patterns, whereas there was only moderate association with gene expression levels of C1R. These results indicate that DNAm of C1R is a biomarker reflecting chromatin reorganization rather than being of pathophysiological relevance per se. Notably, DNAm of C1R was associated with occurrence of specific genomic mutations that are traditionally used for risk stratification in AML. Furthermore, DNAm of C1R correlates also with overall survival in several other types of cancer, but the prognostic relevance was less pronounced than in AML.Using DNAm profiles of 194 patients from The Cancer Genome Atlas (TCGA), we identified a CpG site in complement component 1 subcomponent R  contains supplementary material, which is available to authorized users. It is usually based on many parameters including age, white blood cell count, cytogenetic abnormalities, and specific mutations . Epigenel sequel . This exP < 0.05; Fig. P < 0.05; Additional file C1R, cg08799922), revealed significant association with OS , corresponding to the median beta-value in the training set (TCGA). Again, there was a clear association with OS  [C1R catalyzes the initial event of the classical complement pathway, which is crucial for innate defense mechanisms against pathogens and altered-self cells [C1R and may interfere with a CCCTC-binding factor (CTCF) and cohesin-binding site . Furthermore, neighboring CpGs in the pyrosequencing assay revealed similar correlation with OS as well  was co-regulated with other genomic regions, we performed linear correlation of DNAm with all CpGs from the 450K BeadChip: 1448 CpGs correlated (Pearson’s R >0.5), and these were particularly located in homeobox genes , and the same trend was also observed in the other groups , low-grade glioma (P = 0.0002), skin cutaneous melanoma (P = 0.002), hepatocellular carcinoma (P = 0.026), and glioblastoma  of AML patients from The Cancer Genome Atlas (TCGA)  and a stBlood samples were taken from 40 healthy donors and 84 AML patients after written consent according to the guidelines of the ethics committee of the Medical Faculty of the RWTH Aachen (Permit Number: EK206/09). Patient characteristics are summarized in Additional file https://tcga-data.nci.nih.gov/tcga/) [http://www.ncbi.nlm.nih.gov/geo/) under the accession numbers GSE58477 [The data sets supporting the results of this article are available at The Cancer Genome Atlas portal (v/tcga/)  and at GGSE58477 , GSE4027GSE58477 , and GSEGSE58477 .


Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca2+ release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca2+] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (−220 mV). Conversely, with 20 µM cytoplasmic 4,4′-DTDP or buffering of the redox potential to an oxidising value (−180 mV), RyR2 activity increased with increasing luminal [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal [Ca2+] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca By contrast, the open probability of RyR2 channels from healthy heart tissue was increased to levels seen in RyR2 from failing hearts by the addition of 4,4′-DTDP  ATP or MgATP were added when required, then redox reagents were added; (3) luminal [Ca2+] was reduced to 0.1 mM and then increased in three steps to 1.5 mM. Channel activity was recorded for 4 min after each step, except with a luminal [Ca2+] of 0.1 mM, where recording was for 2 min in order to minimise calsequestrin (CSQ) dissociation  was 0.042±0.006 (±s.e.m.) at a luminal [Ca2+] of 1 mM and fell significantly to 0.016±0.003 when luminal [Ca2+] was lowered to 0.1 mM. There were significant increases in Po as luminal [Ca2+] increased, to a mean Po of 0.071±0.01 with 1.5 mM Ca2+ (Po was due to a decrease in the mean closed time (Tc), which, at 1 mM luminal Ca2+, was ∼50% of the value observed at 0.1 mM luminal Ca2+ (Fo), but no change in the mean open time  to 0.039±0.01 when luminal Ca2+ was initially lowered to 0.1 mM. Subsequently, an unexpected and significant decline in Po to ∼75% of the value recorded at 0.1 mM was observed as luminal [Ca2+] was increased to 1.5 mM  when luminal [Ca2+] was increased from 0.1 to 1.5 mM was not significantly different between that in the presence of 4,4′-DTDP  and its absence (4.25±1.2-fold).As expected, RyR2 activity increased significantly when 20 µM 4,4′-DTDP was added to the cytoplasmic solution with 1 mM luminal Camic Ca2+ . As in th 0.1 mM . Consistpen time . It is n,4′-DTDP  than in ,4′-DTDP , owing t2 into the solution   . The lenotential , and Po o 1.5 mM . Also, wo 1.5 mM . The chaen times .2+] when the redox potential was buffered with GSH∶GSSG in the presence of 1 mM Mg2+. The changes in channel gating are consistent with those seen under all other conditions, although occurring over a much lower range of Po values that might, in fact, approach levels of RyR2 activity in myocytes from normal hearts during diastole, when channel activity is likely to be relatively low.Therefore, we observed characteristic changes in RyR2 activity with increasing luminal [Ca2+] would influence the RyR2 response to luminal [Ca2+] under reducing cytoplasmic redox conditions in the presence of 5 mM MgATP. The mean Po values at each stage of the experiment were at least two orders of magnitude higher than with 1 µM cytoplasmic Ca2+. However, the changes in Po at each step with 10 µM cytoplasmic Ca2+  according to a sign test, because parameters in a critical number of individual channels changed in the same direction (either increased or decreased) with the particular treatment.It is recognised that channel gating varies between individual RyR channels independently of cytoplasmic  (2+] (10 µM), reducing cytoplasmic redox potential, Mg2+ and ATP, there were the same changes in Po as a function of luminal [Ca2+]  junctional gap between the transverse (T)-tubule and the sarcoplasmic reticulum membrane forms a microdomain within the cytoplasm. Not only is the gap narrow (∼10 nm) but it is crowded with many junctional proteins, including the huge cytoplasmic domain of RyR2, which effectively forms a two-dimensional crystal lattice , the dih2+] increases during diastole. However, the situation becomes detrimental if RyR2 is hypersensitive to activation in disorders such as catecholaminergic polymorphic ventricular tachycardia or if the redox potential is further increased and the degree of thiol modification is increased with NOX2 stimulation and excess ROS production as a result of chronic β adrenergic stimulation, as in heart failure  are likely closest to the in vivo values. Overall, our data reveal a basic response of RyR2 channels to changes in luminal [Ca2+] that are consistent with changes in Ca2+ release in intact cells, with the notable exception of the responses under reducing redox conditions. The data reveal a baseline response that can be modulated up or down by other endogenous RyR2 regulators.It is notable that, although rhythmia . Clearly2+] suggest that redox potential alters the inter- or intra-molecular factors that link the luminal [Ca2+] sensor or sensors to the channel-gating mechanism. This would not be surprising and is consistent with the established influence of thiol modification on channel gating  at ambient partial O2 pressure  (2 in solution can interact with NOX enzymes that might be associated with the sarcoplasmic reticulum vesicles (2O2), are generated in proportion to pO2 by NADPH oxidase 4 (NOX4) in the sarcoplasmic reticulum membrane. H2O2 is responsible for pO2-dependent RyR1 oxidation  . In addivesicles . ROS, suxidation . Therefosolution . Althoug2+ sensitivity of normal canine RyR2 channels at 0.02 and 2.0 mM is lost after exposure to 4,4′-DTDP  and also the association status of cardiac calsequestrin (CSQ2) and junctin. Both CSQ2 and junctin regulate RyR2 sensitivity to luminal [Ca2+] . All other chemicals were obtained from Sigma-Aldrich .Sarcoplasmic reticulum vesicles were prepared from sheep heart as described previously .2 and 10 mM tetraethylsulfamide (TES) pH 7.4, and the luminal (trans) solution containing 30 mM CsMS, 20 mM CsCl, 1 mM CaCl2 and 10 mM TES pH 7.4. Sarcoplasmic reticulum vesicles (∼50 µg) were added to the cis solution so that the cytoplasmic surface of the sarcoplasmic reticulum and RyR2 faced the cis solution after incorporation into the lipid bilayer. After channel incorporation, free cis Ca2+ was decreased to 1 µM with the addition of ∼1.32 mM BAPTA, and 200 mM CsMS was added to the trans solution to achieve symmetrical [Cs+]. Luminal [Ca2+] was reduced from 1 mM to 0.1 mM by perfusion, and was then increased in three steps to 1.5 mM by the addition of CaCl2. Free [Ca2+] in all solutions was determined using a Ca2+-selective electrode .Sarcoplasmic reticulum vesicles were incorporated into artificial planar bilayers as described previously , with thVcis−Vtrans. All experiments were performed at 23±2°C. Single-channel parameters were obtained using the Channel 2 programme  applied to 60 to 90 s of channel activity. The threshold levels for channel opening were set to exclude baseline noise at ∼20% of the maximum single-channel conductance and open probability (Po), mean open time (To), mean closed time (Tc) and open frequency (Fo) were measured. Data obtained at +40 mV and −40 mV were analysed separately. No voltage-dependent differences were evident and so data for +40 mV and −40 mV were combined in the final mean values presented in the Results.The techniques have been described previously . By convThe redox potential was manipulated with a GSH∶GSSG buffer . An oxid


A capacitor-based circuit model is proposed to explain the electrochemical delamination of two-dimensional materials from their native substrates where produced gas bubbles squeeze into the interface. The delamination is actually the electric breakdown of the capacitor formed between the solution and substrate. To facilitate the procedure, the backside of the ubstrate has to be shielded so that the capacitor breakdown voltage can be reached. The screening effect can be induced either by nonreactive ions around the electrode or, more effectively, by an undetachable insulator. This mechanism serves as a guideline for the surface science and applications involving the bubbling delamination. MoS2 is a semiconductor that can serve as the transistor channel material in post-silicon electronics. With modern technologies like chemical vapor deposition (CVD) ∙[OH−] = Kw where KW is the self-ionization constant of water cannot be reflected in this figure when [H+] and [OH−] are pushed towards the abscissa axis. Now let us look into the delamination efficiency when using reactions other than H2O electrolysis. The Kolbe electrolysis is decarboxylative dimerisation of two carboxylic acids (or carboxylate ions):3COONa + 2H2O = C2H6 + 2CO2 + 2NaOH + H2 (the effective ionic radii for COOH− is 1.6 Å) [3COONa is also taking part in the redox reaction, one may expect it to give a better delamination efficiency. Also, at the anode, there are two types of gases (C2H6 and CO2) with the total mole ratio to cathode H2 being 3:1. Nevertheless, in experiments we find that the delamination efficiency at the anode is very poor (200 mL CH3COONa solution at 0.25 mol/L), much worse than that for HNO3 under the same conditions. This is because the solution is dominated by CH3COO− which is reactive (unlike NO3−) and cannot offer a screening effect.Other transitional situations, e.g., mixture, of HNOs 1.6 Å) . At a fih-BN, MoS2, etc. also demand a film transfer technology. We believe the screening effect is quite general and should be considered in the electrochemical delamination of all 2D materials beyond graphene. The bubbling separation process and the associated screening effect not only provide an ideal platform for surface science studies involving 2D materials, but also lead to economic and environmental benefits when it comes to applications. The metal foil catalysts used for 2D material growth are entirely reusable, drastically reducing the cost on raw materials and the heavy metal pollution. In the For simplicity, the discussion above only involves the delamination of bare PMMA thin films. Actually, we have repeated the experiments on Pt-grown graphene samples (spin-coated with a thin PMMA layer) and exactly the same screening effects are observed . Similarf0 can be reached more easily. This shielding effect can be created by nonreactive ionic cloud adjacent to the electrode and/or, preferably, by an undetachable insulator on the back of the substrate. The proposed mechanism will be essential for the emerging 2D material surface science and industrial applications derived from the bubbling delamination technological route.In summary, we have developed a capacitor-based lumped circuit model to explain the mechanism of electrochemical delamination of 2D materials from their native substrates. The hydrogen or oxygen gas bubbles produced by water electrolysis are directed to the interface between the 2D materials and substrates and separate them. The delamination is in fact the electric breakdown of the capacitor formed between the solution and substrate with the thin polymeric support being the dielectric. We have discovered that if the substrate backside is screened against the active ions that are contributing to the detachment, the thin films are much easier and faster to exfoliate. In other words, the capacitor’s critical breakdown voltage V


The topic of how to prevent and reduce burnout has drawn great attention from researchers and practitioners in recent years. However, we know little about how mentoring as a form of social support exerts influence on employee burnout.This study aims to examine the contingency side of the mentoring-burnout relationship by addressing the exploratory question of whether individual differences in power distance and relationship quality play important roles in mentoring effectiveness in terms of reducing a protégé’s burnout level.A total of 210 employees from a technology communications company completed the survey questionnaire.(1) A protégés’ power distance moderates the negative relationship between mentoring and burnout in such a way that the relationship is stronger for protégés who are lower rather than higher in power distance; (2) mentor-protégé relationship quality moderates the negative relationship between mentoring and burnout in such a way that the relationship is stronger when the relationship quality is higher rather than lower.In sum, our results highlight the importance of studying the contingency side of mentoring effects on protégé burnout. Our findings suggest that the individuals’ different cultural values of power distance and mentor-protégé relationship quality are the boundary conditions for the mentoring-burnout relationship. We therefore suggest that research on mentoring-burnout will be advanced by considering the role of the moderating process. Burnout refers to a psychological response to chronic work stress combining emotional exhaustion, depersonalization and reduced professional efficacy , 2. GiveWorkplace informal mentoring refers to a developmentally oriented relationship voluntarily initiated and maintained between a less experienced employee (the protégé) and a more experienced employee (the mentor) where the goal is the personal and professional development of the protégé . Our stuPower distance, defined by Hofstede , is the Previous mentoring studies have pointed out that mentor-protégé relationship quality is important for mentorship effectiveness , 16. WheWe hypothesize that (1) protégés’ power distance moderates the negative relationship between mentoring and burnout in such a way that the relationship will be stronger for protégés who are lower rather than higher in power distance and (2) mentor-protégé relationship quality moderates the negative relationship between mentoring and burnout in such a way that the relationship is stronger when the relationship quality is higher rather than lower.Participants in the current study consist of 388 full-time employees of a high-tech communications company in a major city in northern China. There are three main reasons why we chose this company. First, it is a privately owned and operated firm, which generally means that the work environment is more flexible and less uniform than state-owned enterprises (SOEs) in China, leading to more unpredictable work patterns and more sources of variance regarding employees’ level of burnout e.g., , 18). Se. Se18]).Survey packets were distributed in a company-wide meeting. Surveys were completed on a voluntary basis. Each packet contained an information sheet explaining the objective of the survey, along with a consent form, the survey questionnaire and a return envelope with a self-sealing closure to protect the respondents' confidentiality. Participants were instructed to complete the survey and to bring it back to the upcoming meeting two weeks later. To protect participant confidentiality, they were instructed to seal the questionnaires in the envelopes provided after finishing their questionnaires. Two short messages were sent to the participants three days after the questionnaire was distributed and one day before the second meeting to encourage participants to complete the survey and to remind them to bring it with them. A box was placed outside the meeting venue, and the participants were reminded by one of the authors to put their completed and sealed questionnaire into the box before and after the meeting.SD = 7.51) and male (69.0%). Most participants held a bachelor’s degree (68.6%), with the remainder reporting a polytechnic diploma or associate degree (14.3%), a graduate degree (15.7%) or high school education (1.4%). The average company tenure was 8.23 years (SD = 6.57). A total of 62.9% were non-supervisory employees, 31.9% were first line supervisors and 5.2% were middle managers. The average number of informal mentors reported was 1.79 (SD = .74). The average mentorship duration for the referred mentoring relationship was 5.5 years (SD = 4.17). 73.8% of the mentors were male and 33.3% of the protégés had mentored others before.A total of 285 surveys were returned, with a response rate of 73.5%. After eliminating 43 incomplete questionnaires and 32 questionnaires that did not report any informal mentoring, 210 respondents remained and contributed to the sample of the present study. On average, protégés were 34.4 years old  screen participants to identify those who currently have informal mentors, 2) instruct those who have mentors to complete the questionnaire by filling in the following five sections, 3) instruct protégés who have more than one mentor to respond to the following five sections by referring to the most influential mentor and 4) guide non-protégés to ignore the following sections and return the questionnaire on the designated date.Whether an employee currently had an informal mentor was determined by two items preceded by the following definition based on past mentoring studies .A mentor is an experienced employee who serves as a role model and who provides direction, support and feedback regarding career and personal development. A mentor is also someone with influence and insight, who directly provides upward mobility and/or brings your accomplishments to the attention of people who have power in the company. A mentor can be your supervisor or anybody else in the company.Respondents were asked to indicate whether they are currently in an informal mentoring relationship. Those without an informal mentor were coded “0”. Others who had mentor(s) were also asked to give the number of informal mentors they currently had. Protégés who reported more than one mentor were instructed to complete the questionnaire by referring to the most influential mentor.career-related mentoring functions subscale consists of seven items . The psychological mentoring functions subscale contains 14 items . The Cronbach’s alpha for career and psychological mentoring functions were .89 and .94, respectively. The internal consistency reliability for the scale was .96.Noe’s  21-item Maslach Burnout Inventory – General survey (16 items) was used to measure protégés' burnout . This inWe used the six-item scale developed by Dorfman and Howell . ResponsMentor-protégé relationship quality was measured with a three-item scale developed by Allen and Eby . It has We included nine control variables for testing the hypotheses. In keeping with other mentoring research e.g., –31), we , we 31])We also controlled four mentorship status variables, as previous research has demonstrated that they could account for variance in mentoring received and/or mentoring outcomes e.g., , 33). Th. Th33]).First, although the variables included in the current study are theoretically distinctive, we conducted a confirmatory factor analysis using AMOS 17.0 to empirically demonstrate the distinctiveness of mentoring, burnout, power distance, and relationship quality. Second, preliminary analyses evaluating the descriptive statistics and correlations among study variables, and possible group differences in study variables based on demographic characteristics were performed. Next, the two moderation models, with power distance and relationship quality as moderators on the relations between informal mentoring and burnout, were tested using SPSS MODPROBE macro, developed by Hayes and Matthes  for estiTable t-tests demonstrated no gender difference on any study variable. One-Way ANOVA tests showed no education, position or tenure differences on any study variable, except for the effect of education difference on burnout. Post-hoc analysis indicated that people with undergraduate degree reported more job burnout than people with graduate degree .See Table p < .000, 95% CI = , whereas the association was negative for low power distance group, b = -.48, p < .001, 95% CI = .The first moderation analysis examined whether power distance moderated links between informal mentoring and burnout. Preacher et al.  demonstrp = .000, 95% CI = , whereas the association was negative but non-significant for high mentor-protégé relationship quality group, b = -.14, p = .073, 95% CI = .Similarly, the second mediation analysis examined whether mentor-protégé relationship quality moderated the relations between informal mentoring and job burnout. Applying the modprobe macros for moderation analysis, the conditional effect of mentor-protégé relationship quality was estimated at values of one standard deviation below the mean, the mean, and one standard deviation above the mean. Demographic and mentorship status variables were again controlled in the model. Results showed that mentor-protégé relationship quality moderated the links between informal mentoring and job burnout Table . SpecifiWhile research into mentoring has steadily grown, the contingencies under which mentoring may be related to protégé burnout remain largely unknown. To this end, we contribute to the emerging studies investigating individual differences and contextual factors in reactions to mentoring by testing whether the relationship between informal mentoring and burnout is moderated by protégés’ power distance and mentor-protégé relationship quality e.g., , 14). Th. Th14]).The results of this study provide important contributions to the literature on burnout and mentoring in three ways. First, Halbesleben and Burkley’s  review oSecond, we examined the contingency side of the mentoring-burnout relationship by addressing the exploratory question of whether individual differences of power distance played a moderating role in mentoring effectiveness. Whereas mentoring has made considerable progress in Western countries, a recent review of the mentoring literature shows that research conducted in other cultures has lagged behind . AlthougAdditionally, The reason we test our model in a Chinese setting is that the domain of organizational research is becoming more international, bringing into question the transportability of social science models from one society to another , and parThird, we made a contribution by examining the moderating influence of a contextual factor of mentor-protégé relationship quality on the burnout-reduction effects of mentoring. It indicates that having a mentor is not enough, the perceived quality is of great importance in mentoring effectiveness. The popular press tends to present mentoring as an essential ingredient for employee development and organizations are trying to promote various mentoring relationships; however, very little attention has been focused on the importance of and process of building a high quality mentor-protégé relationship , 49, 50.This study offers some practical implications for mentoring practice. Our work suggests mentoring as a strategy to reduce and prevent employees’ burnout. A further implication stems from the results of the moderation analysis. To ensure the maximum effects of mentoring provided to employees, organizations and mentors should pay extra attention to individual differences in power distance. Special attention should be paid to those with high power distance values. For those employees, organizations could provide some complementary mentoring programs such as peer mentoring . In addiDespite these findings, this study is not without limitations. First, given our cross-sectional research design, it is impossible to assert the causal relationship. Future research should incorporate longitudinal or experimental design to ascertain the causal basis of the relationship examined in this study . Second,In sum, our results highlight the importance of studying the contingency side of mentoring effects on protégé burnout. Our findings suggest that the individuals’ different cultural values of power distance and mentor-protégé relationship quality are the boundary conditions for the mentoring-burnout relationship. We therefore suggest that research on mentoring-burnout will be advanced by considering the role of the moderating process.


We investigated the role of Ca2+-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca2+ transients followed by sustained Ca2+ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca2+ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca2+ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca2+ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca2+ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively.Ca Pretreatment of cardiomyocytes with bafilomycin A1 completely blocked the ISO-induced [Ca2+]i increase  induced a sustained Ca2+ signal in the presence, but not in the absence, of extracellular Ca2+  Ca2+ stores by NAADP and cADPR, respectively.To corroborate above observations, we tested the effect of NAADP-AM, a cell-permeable NAADP analog , on Ca2+lar Ca2+ . Furthert intact . Taken t2+ influx pathway for the ISO–induced sustained Ca2+ signal, we studied the contribution of store-operated Ca2+ entry (SOCE). We used deaza-cADPR, a cell-permeable cADPR analog, to deplete Ca2+ from SR. Cells were incubated with 100 nM deaza-cADPR for 30 seconds in Ca2+-free medium and then Ca2+ added to the bath perfusion solution to assess the presence of SOCE, as described earlier [2+ stores by deaza-cADPR induced Ca2+ entry in the presence of extracellular Ca2+  ISO-induced cADPR production in cardiomyocytes after infection with scrambled or CD38 shRNA. *, P< 0.01 versus scrambled shRNA control. #, P < 0.01 versus scrambled shRNA+ ISO. Values are the mean ± SEM of three independent experiments.(A) Representative immunoblots with summary quantifications of CD38 protein expression in cardiomyocytes after infection with lentiviral particles expressing scrambled or CD38-specific short hairpin (shRNA). (B) Representative tracings of the Ca(PPTX)Click here for additional data file.S2 FigHeart were isolated and analyzed for protein expression before (Con) and following 7 days of ISO infusion (ISO).(PPTX)Click here for additional data file.S3 Fig(A) mRNA level of fibronectin and TGFβ1 (B) protein expression level of fibronectin and TGFβ1. Heart were isolated and analyzed for protein expression before (Con) and following 7 days of ISO infusion (ISO).(PPTX)Click here for additional data file.S1 Table(PPTX)Click here for additional data file.


During haemodialysis (HD) sessions, patients undergo alterations in the extracellular environment, mostly concerning plasma electrolyte concentrations, pH, and volume, together with a modification of sympathovagal balance. All these changes affect cardiac electrophysiology, possibly leading to an increased arrhythmic risk. Computational modeling may help to investigate the impact of HD-related changes on atrial electrophysiology. However, many different human atrial action potential (AP) models are currently available, all validated only with the standard electrolyte concentrations used in experiments. Therefore, they may respond in different ways to the same environmental changes. After an overview on how the computational approach has been used in the past to investigate the effect of HD therapy on cardiac electrophysiology, the aim of this work has been to assess the current state of the art in human atrial AP models, with respect to the HD context. All the published human atrial AP models have been considered and tested for electrolytes, volume changes, and different acetylcholine concentrations. Most of them proved to be reliable for single modifications, but all of them showed some drawbacks. Therefore, there is room for a new human atrial AP model, hopefully able to physiologically reproduce all the HD-related effects. At the moment, work is still in progress in this specific field. In the last fifteen years, the increasing interest towards atrial electrophysiology and atrial fibrillation (AF), together with a greater availability of experimental data, led to remarkable developments in human atrial action potential (AP) models –6. As a  in vivo, in human, the effects of sudden changes in plasma ionic concentrations and blood volume: in a few hours, patients undergo significant plasma electrolytes variations, together with a significant decrease in extracellular volume. In particular, the HD session causes removal of excess Na+ and water, the extent of which depends on the interdialytic weight gain of the patient. Plasmatic K+ concentration increases during the interdialytic interval, so that during all HD sessions its level must decrease, while Ca2+ variations might change depending on the dialysate Ca2+ concentration and its relationship with pre-HD plasma Ca2+ levels. o) is set to the standard value used in the perfusion bath during V-clamp experiments; that is, 5.4 mM. K+ increases during the interdialytic interval and is removed during the HD session: therefore, HD patients often show hyperkalaemia at the beginning of the therapy and hypokalaemia at the end. In order to explore both clinical conditions, we considered the range 3–9 mM. The lower [K+]o value (3 mM) has been set considering the experimental post-HD measurements available in literature  is actually a bit high compared to the pre-HD measurements available . The up.5 mM in , 5.3 ± 0.5 mM in ) but we  or more .+]o should be a hyperpolarization of the cell membrane, due to a different Nernst potential for K+ ions. In addition, a [K+]o decrease leads to a QT interval increase [+]o. Finally, while slowed cardiac tissue conductivity is a well-known effect of severe hyperkalemia, in the range of [K+]o concentrations usually measured in HD patients, a positive dependence of conduction velocity on [K+]o has been observed: this phenomenon is known as supernormal conduction o range.The main effect of a decrease in o is shown in +]o, the corresponding biomarkers have not been computed.A summary of the AP biomarkers for all the different o. As for the ERP, in this model it is always much longer than the corresponding APD90, even if it shows a similar dependence on [K+]o. In addition, probably due to the high stimulus amplitude needed to stimulate the GB model (as explained in the methods section), the ERP could not be computed for most of the [K+]o: when considering values below 4 mM or above 6 mM, the S2 AP peak was never lower than 80% of the corresponding S1, no matter how short the diastolic interval considered.As for the GB model  is set to the standard value used in the perfusion bath during V-clamp experiments, that is, 1.8 mM, which is quite high compared to the normal serum Ca2+ measured in vivo (1–1.3 mM), as discussed in detail in [2+ can either rise or decrease. Two previous simulation studies explored the effects of [Ca2+]o on cardiac electrophysiology, considering the range 1–3 mM o is shown in In all the models the extracellular Caetail in . During e 1–3 mM , 65. How09 mM in  and 1.0609 mM in . Therefo2+]o increase is a significant decrease of APD o has been recorded in ventricular cells, there are a few recordings confirming that the trend is the same for human atrial cells o , while  Figures  and neitures +]i .+]i is finely tuned by Ca2+: the raise of [Ca2+]o increases the outward Na+/Ca2+ exchanger current (INCX), but the corresponding increase of intracellular Ca2+ contrasts this effect. At the same time, the Na+/K+ pump counteracts [Na+]i variations, in both ways. As a result, [Na+]i concentration is not much sensitive to [Ca2+]o in most of the models  is set to the standard value used in the perfusion bath during V-clamp experiments, that is, 130 mM for CN, GB, and CZ, 140 mM for NG, MT, and KT, in agreement with the normal serum levels of 135–145 mM. Na+ variation during a regular HD session is usually quite small  and we ± 3.1 in , 68.90 and ERP slightly increase with [Na+]o in all the considered models except GB . It is worth noting that, in CZ and CN, [Na+]i regularly shows a stronger sensitivity to changes in extracellular concentrations.No significant differences were found in CaT biomarkers nor intracellular concentrations, apart from an increase in o or volume on cardiac cells to either confirm or deny these findings.Unfortunately there are no experimental data on the effect of changes in o = 3 mM . In the NG paper the authors explicitly say that the IKr conductance has been reduced to fit AP data and that this current has been assigned a very low density [+]o.We found a major problem in the NG, MT, and KT models: they all fail to repolarize and to produce physiological APs when o, it behaves nonphysiologically when [K+]o is lower than 4 mM: the RMP depolarizes instead of hyperpolarize and, as a consequence, the APD90 also goes in the opposite way (shortening) and dV/dtMAX dramatically decreases. Moreover, the excessive sensitivity to the amplitude of the stimulus current makes the computation of the ERP very unstable, leading to too long ERP values or no ERP at all. Since also in GB the repolarising K+ currents (both IKr and IKs) are quite small in amplitude, increasing their magnitude may improve the model stability for low [K+]o concentrations. Finally, the GB model responds poorly to [Ca2+]o changes too: the APD90 trend is opposite to what is observed in human atrial cells [2+]o, even displaying EADs for [Ca2+]o equal to 3 mM, and the intracellular Ca2+ transient is almost nonexistent when [Ca2+]o is lower than 1 mM. Therefore, the GB model turns out to be completely unsuitable to simulate the HD conditions.The GB model exhibits several shortcomings as well. First of all, although it produces a proper AP at all the tested o a possible solution should address a modification of the L-type Ca2+ current, increasing the Ca2+-dependent inactivation with respect to the voltage-dependent one, in order to reduce the overall current for higher Ca2+ levels, despite the increase in driving force. Indeed, Ca2+-dependent inactivation seems to be underestimated in many AP models [2+]o just by strengthening this mechanism o changes, at least from a qualitative point of view. It also reproduces well the “uncoupling” between APD and ERP variations when [K+]o is increased (APD slightly decreases whereas ERP increases): this was experimentally reported by Downar et al. o: we are not aware of any available experimental data to confirm/deny this observation, which could have relevant implications.The CNr et al.  when per+]o, while the opposite should happen [amp is insensitive to [Ca2+]o while in all the other models it increases with it, in agreement with experimental data reported in o and [Ca2+]o are the ones mostly affecting cellular electrophysiology [+]o and volume seem to have a minor impact. A qualitative summary of the expected variations in [K+]o and [Ca2+]o during HD and of the corresponding biomarker changes is shown in Tables As for the quantification of cardiac side-effects of HD therapy, overall simulation results confirm that changes in o and Ca+o are thud, suggesting a slower conductivity, also consistent with the increased vulnerability to arrhythmias, such as AF, due to an increased vagal activity [Simulation results of acetylcholine effect show a reduction of APD and ERP in all the models, together with a more hyperpolarised RMP, in agreement with experimental data and previous modeling studies , 69–73. activity , 70. HowOther HD-related effects  have not been addressed in our analysis and are left to further investigations.+/K+ pump and high levels of circulating Na+ pump inhibitor have been reported in uremic patients compared to individuals with normal renal function, by several investigators [Finally, it is worth remembering that HD patients are first of all uremic patients: this pathological condition  can also affect some aspects of cardiac cellular electrophysiology and should be incorporated into the models. As a relevant example, downregulation of the Natigators –80.* seems to be the more suitable human atrial model to analyse HD-related effects on atrial electrophysiology, though it is the oldest one and, therefore, it has a less detailed description of several cellular mechanisms.In conclusion, computational modeling of human atrial cells constitutes a very useful tool to investigate the electrophysiological changes occurring in patients undergoing HD therapy. Nevertheless, it is always important to select carefully the specific model to use, depending on the particular aspect of interest. Currently, CNTherefore, an additional model could be developed, trying to integrate and reconcile the knowledge of cellular and subcellular processes and their reactions to changes in the extracellular environment, taking into account the possible suggestions given above.In this respect, work is still in progress in this specific field.


In the present study we investigated the effects of carbachol and NO donor SNAP on Ca2+ signalling and vasomotor responses of arterioles and venules in intact urteric microvascular network in situ using confocal microscopy. Vasomotor responses of arterioles and venules induced by AVP correlated with the occurrence of Ca2+ oscillations in the myocytes and pericytes and were not abolished by the removal of Ca2+ from extracellular fluid. Carbachol-induced rise of intracellular Ca2+ in endothelium was accompanied by the termination of the Ca2+ oscillations in myocytes and pericytes. This carbachol-induced inhibitory effect on Ca2+ oscillations in myocytes and pericytes was reversed by ODQ, an inhibitor of soluble guanylyl cyclase (sGC) and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Ca2+ oscillations in myocytes and pericytes were also effectively blocked by NO donor SNAP. An Inhibitory effect of SNAP was markedly enhanced by zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase-5, and reversed by sGC inhibitor, ODQ and PKG inhibitor, Rp-8-pCPT-cGMPS. The cGMP analogue and selective PKG activator 8pCPT-cGMP also induced inhibition of the AVP-induced Ca2+ oscillations in myocytes and pericytes. SNAP had no effects on Ca2+ oscillations induced by caffeine in distributing arcade arterioles. Consequently, we conclude that NO- mediated inhibition of Ca2+ oscillations in myocytes and pericytes predominantly recruits the cGMP/PKG dependent pathway. The inhibitory effect of NO/cGMP/PKG cascade is associated with suppressed Ca2+ release from the SR of myocytes and pericytes selectively via the inositol triphosphate receptor (IP3R) channels.In ureteric microvessels the antagonistic relationship between Ca In myocytes and pericytes of ureteric microvessels the antagonistic relationship between Ca2+ signalling in endothelium and media cells is NO-mediated 2+ ([Ca2+]i) in ECs activates Ca2+/calmodulin-dependent constitutive endothelial NO synthase (eNOS) to generate NO, which diffuses to the adjacent myocytes or pericytes and binds to a prosthetic group on soluble gyanylyl cyclase (sGC). Stimulated sGC catalyses synthesis of the second messenger cGMP, which in turn activates cGMP-dependent PKG and/or other effector proteins, including ion channels, pumps, and phosphodiesterases (PDEs) 2+ signalling and tone was reported and contribution of cGMP-independent mechanisms was suggested 2+ oscillations in myocytes and pericytes of ureteric microvascular networks in situ in the absence and the presence of external Ca2+. To achieve this goal we used our unique methodology developed back in 2003 in situ, which allows to monitor simultaneously an intracellular [Ca2+] in myocytes, pericytes and endothelial cells and correlate it with vasomotor activity An endothelial cell (EC) Ca22.12 anaesthesia followed by cervical dislocation, in accordance with UK legislation. Whole ureters were dissected, carefully cleaned of connective and paraureteric adipose tissue using fine curved scissors and keeping the sharp edges away from the tissue to avoid physical damage of the ureteric bundles.The experiments were performed on Wistar rats of both sexes (3–4 months old). Rats were humanely killed using CO2.22+ measurements ureters were cut into small segments (4–5 mm long) and loaded with Fluo-4. Strips of ureter were placed in a plastic cuvette containing 1 ml of HEPES-buffered Krebs solution including 15 μM-fluo 4-AM dissolved in dimethyl sulphoxide premixed with Pluronic F127 . Loading was performed at 23 °C for 3 h with the cuvettes wrapped in black tape and rotated at 30 rpm. Tissue samples were then removed from the loading medium and placed in normal Krebs solution for at least 30 min to allow cleavage of fluo 4-AM to fluo 4 by intracellular esterases. Fluo-4 loaded segments of ureter were transferred to a custom-made perfusion chamber mounted on the stage of inverted Olympus microscope. Superfusion of the ureteric segments in the chamber was performed by applying a positive pressure valve controlled flow of solution via a 1 mm diameter tip attached to a 3-d mechanical manipulator  which allowed positioning the superfusion tip in a desired region of the chamber. Solution was removed by suction at the other end of the chamber. All experiments were performed at 30 °C. We used a Nipkow disc, confocal microscope in situ. Images were collected at 33–66 frames per second using a ×60 water objective (NA 1.20) for spatial resolution or dry  for a larger field of view. To measure elemental events and Ca2+ waves in myocytes and pericytes, tangential sections were used, whereas radial sections through the centre of the microvessel were used to measure Ca2+ events in myocytes, pericytes, endothelial cells and changes in vessel diameter. Mechanical activity of individual smooth muscle cells was tracked by putting the region of interest close to the edge of the contracting cell. It was possible to correlate Ca2+ signalling with contraction of individual myocytes and pericytes in both radial and tangential sections.For Ca2.34 1.2, CaCl2 2, glucose 11.5, and HEPES 11. The Ca2+-free solutions contained 2 mM EGTA. [Arg8]-Vasopressin acetate salt (AVP), Carbachol (CCh), Caffeine, Zaprinast (ZAP), 1H-Oxadiazoloquinoxalin-1-one (ODQ), Rp-8-pCPT-cGMPS sodium salt, 8pCPT-cGMP, S-nitroso-N-acetyl-DL-penicillamine (SNAP) were from Sigma. Fluo-4 acetoxymethyl ester was from Molecular Probes, Life Technologies, UK.Physiological saline of the following composition was used (mM): NaCl 120.4, KCl 5.9, MgSO8]-Vasopressin acetate salt, Carbachol, Caffeine, Rp-8-pCPT-cGMPS sodium salt, 8pCPT-cGMP were dissolved in water; S-nitroso-N-acetyl-DL-penicillamine (SNAP), 1H-Oxadiazoloquinoxalin-1-one (ODQ), Zaprinast in DMSO.i and vascular tone In addition, our data also indicate that NO had no effects on caffeine- induced Ca2+ signalling terminates Ca2+ oscillations in myocytes and pericytes of ureteric microvessels by activation of NO/cGMP/PKG signalling cascade, which results in an inhibition of IP3R- mediated Ca2+ mobilization from the SR.Finally, we conclude that endothelial cell CaThe authors confirm that there are no conflicts of interest.


In mammalian sperm the Ca2+-permeable plasma membrane ion channel CatSper is central to [Ca2+]i signalling, but there is good evidence that Ca2+ stored in intracellular organelles is also functionally important. Here we briefly review the current understanding of the diversity of Ca2+ stores and the mechanisms for the regulation of their activity. We then consider the evidence for the involvement of these stores in [Ca2+]i signalling in mammalian (primarily human) sperm, the agonists that may activate these stores and their role in control of sperm function. Finally we consider the evidence that membrane Ca2+ channels and stored Ca2+ may play discrete roles in the regulation of sperm activities and propose a mechanism by which these different components of the sperm Ca2+-signalling apparatus may interact to generate complex and spatially diverse [Ca2+]i signals.[Ca Large changes in [Ca2+]i can be achieved ‘instantaneously’ by flux of Ca2+ into the cytoplasm from the extracellular fluid or from storage organelles (primarily the endoplasmic reticulum) within the cell , the induction of acrosome reaction (AR) and the process of capacitation  and their possible roles in the regulation of function in the mature sperm cell -sensitive Ca2+ channel  and the ryanodine receptor (RyR)  and by the action of the putative second messenger cyclic-adenosine diphospho-ribose (cADPR)  a. The IP (cADPR) a. cADPR  (cADPR) . It is a the RyR .2+ mobilizing ability is nicotinic acid adenine dinucleotide phosphate  within the acidic organelles, these channels being first described in plants  and reactive nitrogen species (RNS), and this occurs mainly through modification of specific cysteine (cys) amino acid residues. Oxidation of these cys residues in RyRs occurs both by S-glutathionylation as well as S-nitrosylation by the second messenger nitric oxide  and naturally generated ROS cause sensitization of this channel, while higher concentrations of thimerosal inhibit channel activity , which is localized to the Golgi apparatus  and calreticulin-containing vesicles are situated  are regulated through phosphorylation by PKA, PKG and CamKII . For instance, exposure of human sperm to NO at levels equivalent to those produced by explants of reproductive tract lining mobilises stored Ca2+ and modifies flagellar activity  are synthesised in sperm and/or produced in response to stimulation. Sea urchin sperm contain significant levels of both cADPR and NAADP, which may contribute to oocyte activation (2+ stores and their regulation’) could not be detected by western blotting. In contrast, 2+-regulated and most active at acid pH whereas the human enzyme shows only weak Ca2+-regulation and activity is maximal at pH 7–8  and a store at the sperm neck (2+ channel of acidic Ca2+ storage organelles (2+ stores and their regulation’).Recent findings have supported the idea that NAADP is functional in human sperm. erm neck b. Consis2+]i signalling. They concluded that CatSper channel proteins were present in the differentiated sperm, but other Ca2+ signalling ‘tools’ including RyRs and CD38 were added to the freshly-ejaculated sperm upon mixing with prostasomes, by fusion with the membrane of the midpiece. They also examined the effects of stimulation with progesterone on [Ca2+]i and motility of sperm exposed to prostasomes and sperm that had been rapidly removed from semen to minimise mixing with prostasomes. Their data suggest that the generation of sustained [Ca2+]i signals  and consequent effects on motility may depend, at least partly, upon generation of cADPR by prostasome-derived enzymes. Interestingly, CD38-null mice proved to be fertile, but analysis showed that 20% of normal ADPR cyclase activity remained in prostasomes from these animals, indicating the presence of a non-CD38 ADPR-cyclase, potentially that described by 2+ store activity but their roles are not yet clear.2+ induces secondary Ca2+ influx through channels at the cell membrane  by the process of capacitative Ca2+ entry (CCE)  has been identified as the sensor molecule present in the membrane of the Ca2+ store. The intraluminal part of STIM includes a Ca2+-binding EF hand that detects depletion of stored Ca2+. STIM then redistributes, moving to a position adjacent to the plasma membrane where it activates channel proteins  family  but did not affect the response in the flagellum, where progesterone activates CatSper channels (2+]i transient in a subset of cells (In somatic cells mobilisation of stored Cary (CCE) a. CCE bo) family ). i response, including a transient and plateau, that was blocked by pre-treatment with the non-genomic VDR antagonist 1β,25(OH)2D3 but was insensitive to the nuclear VDR antagonist ZK159222 . Mitochondria may thus contribute to shaping of Ca2+ signals in sperm. However, the primary stimulus-regulated Ca2+ storage in this part of the sperm is in the RNE and/or a second, apparently separate group of calreticulin-containing vesicular structures, both of which are sited at the sperm neck region and cytoplasmic droplet  show pro-hook bends whereas those activated by store mobilisation  show anti-hook bends  elevate [Ca2+]i but have only minor stimulatory effects on the proportion of hyperactivated cells. Instead, these manipulations significantly increase penetration into viscous media  and mobilises stored Ca2+ channels contribute to Ca2+ influx in mature mammalian sperm. In mouse sperm null for CatSper1 and the K+ channel Slo3, only a small leak current was recorded even at high intracellular pH and strong depolarisation ethane, 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane , p,p′-dichlorodiphenyldichloroethylene and 4-methylbenzylidene camphor are potent agonists (2+ influx (such as 8-Br-AMP) have been shown directly to activate CatSper by binding at the extracellular surface  we have observed a late [Ca2+]i response at the neck region, often including multiple peaks  under capacitating conditions (3Rs and RyRs are sensitised by oxidative stress (2+ stores and their regulation’). We propose that CICR from the sperm neck Ca2+-store is regulated during capacitation, perhaps through the effects of oxidative stress on Ca2+ release channels  and regulated, particularly the significance of capacitation in Ca2+-store filling and in sensitising Ca2+ release mechanisms. Also, similarly to the important species differences in expression and function of sperm ion channels between human and mouse sperm (2+ release.The central role of [Ca