Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant   = 0.015%), but that G330D is not (MAF  = 3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca2+ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca2+ uptake and Aβ levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca2+ and Aβ accumulation. While these results strengthen the notion that CALHM1 influences Aβ metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.CALHM1 is a plasma membrane voltage-gated Ca Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to the most common form of dementia in elderly people. Histological studies of the AD brain have revealed pathological changes triggered by two classical lesions, the senile plaques and neurofibrillary tangles Calcium homeostasis modulator 1 (CALHM1) 2+-permeable ion channel regulated by voltage and extracellular Ca2+ (Ca2+o) levels 2+ (Ca2+i) homeostasis and signaling, as well as Ca2+-dependent neuronal excitability CALHM1 (rs2986017) was associated in European cohorts with both AD risk and an earlier age-at-onset of AD CALHM1 per se has no significant impact on AD risk and is thus likely not a robust independent risk gene for AD CALHM1 with AD age-at-onset 2+-dependent pathway that suppresses extracellular Aβ accumulation in cell lines In vitro functional studies further demonstrated that the P86L variant – through a mechanism yet to be determined 2+ influx and Aβ repression We initially proposed that the naturally occurring P86L variant in CALHM1 coding regions in three independent series comprising a total of 284 EOAD patients and 326 controls. The authors found that the G330D and R154H variants were associated with a complete loss of CALHM1 control of Ca2+ influx in cell lines, suggesting that these variations interfere with CALHM1 function and may thus contribute to the risk of EOAD 2+ permeability by using electrophysiological recordings and Ca2+i measurements in CALHM1-expressing oocytes and mammalian cells, respectively. While there was no effect of the G330D mutation on CALHM1 function in any of our experimental approaches, we found that the R154H mutation resulted in a partial inhibition of CALHM1-dependent Ca2+ uptake in cell lines.In a recent study 2+ add-back” protocol 2+o and then adding it back at physiological concentration 2+ add-back protocols, including the modified version used by Rubio-Moscardo et al. In all conditions, we confirmed the robust and sustained repressing effect of CALHM1 activation on Aβ accumulation. In line with the effect of the two CALHM1 variants on CALHM1-mediated Ca2+ influx, we further demonstrate that the G330D variant has no functional effect on the control of Aβ levels by CALHM1, whereas the R154H variant is associated with a partial loss of CALHM1 function.In the context of characterizing CALHM1 function, Rubio-Moscardo et al. failed to observe an inhibition of Aβ accumulation by CALHM1 expression using a modified version of the originally described protocol of CALHM1 activation (hereafter referred to as the “CaWe interrogated the NHLBI Exome Sequencing Project (ESP) database to determine the frequency of the CALHM1 R154H and G330D variants (see Methods). The R154H variant  was found in a single individual across the entire ESP cohort   = 0.015%). In contrast, the G330D variation  was found in one individual of European-American ethnicity and in 151/4,255 individuals of African-American ethnicity (a 3.55% MAF in the latter population). Therefore, sequencing analysis of these large human cohorts show that CALHM1 R154H is a rare variation, but that CALHM1 G330D, with a MAF above 1%, is not.2+-permeable ion channel regulated by voltage and Ca2+o2+ influx in transfected mammalian cells 2+ permeability, we recorded whole cell currents from Xenopus oocytes injected with wild-type (WT)-CALHM1, R154H-CALHM1, and G330D-CALHM1 cRNA. In the presence of 2 mM Ca2+o, membrane depolarization of oocytes expressing WT-CALHM1 or the CALHM1 variants resulted in slowly-activating outward currents that deactivated upon stepping to hyperpolarized voltages (Heterologously expressed CALHM1 exhibits properties consistent with it being a Cavoltages . Using tvoltages . This in2+o resulted in a left shift in the apparent conductance-voltage (G-V) relationship for CALHM1, allowing channels to open at resting membrane potentials  of –75.9 mV in 0 mM Ca2+o to +60.8 mV in 2 mM Ca2+o  in solutions containing 0 or 2 mM Ca2+o with constant NaCl. The presence of Ca2+o resulted in a shift in Erev to more depolarized voltages for WT-CALHM1, R154H-CALHM1, and G330D-CALHM1  a 30 min Ca2+ omission, (ii) 1% FBS, and (iii) Ca2+ add-back secretion time periods that extended to 6 hr. In all tested conditions, Ca2+ add-back in CALHM1 expressing cells consistently inhibited the accumulation of extracellular Aβ  that would fail to translate into an effect on Aβ levels.In this context, we reassessed the effect of CALHM1 expression on Aβ levels not only in N2a cells, but also in HEK293 cells, and by using Calular Aβ . Specifiadd-back  and the add-back  did not add-back , with thadd-back  and at aadd-back . These d2+ add-back protocol, as in 2+ influx in mammalian cells  in cells transfected with WT or mutated CALHM1. No significant differences in BACE1 levels were observed in these conditions  channels. Our previous work has failed, however, to observe an effect of CALHM1 activation on SOC entry 2+ influx 2+ channels in excitable cells, such as the voltage-gated Ca2+ channels (VGCCs), was also without effect on CALHM1-mediated Ca2+ influx 2+ re-entry might be involved in this mechanism. The effect of the R154H mutation on CALHM1 is therefore likely to be complex and further investigation will be required to determine the precise mechanisms whereby this mutation interferes with CALHM1 function.Together our data show that, while R154H-CALHM1 exhibits normal channel properties at the plasma membrane , it impal uptake  and APP l uptake  and 4. TIn the present work, we used electrophysiology and cell biology approaches to show that: (i) CALHM1 G330D is a frequent variant with no functional consequences on CALHM1 channel properties and is therefore unlikely to contribute to the risk of EOAD; and (ii) CALHM1 R154H is a rare variant that causes a partial loss of CALHM1 function and increases Aβ levels in cell lines. These results motivate further investigation aimed at determining whether CALHM1 R154H, or other functional natural variants in CALHM1, is/are associated with EOAD.1–17 (6E10) antibody from Covance, and anti-BACE1 antibody from Abcam. Myc-tagged WT- and W114A-CALHM1 in pcDNA3.1 vectors, as well as WT-CALHM1 in the pBF oocyte expression vector, were described previously Anti-myc antibody (71D01) was from Cell Signaling Technology, anti-actin antibody from BD Transduction Laboratories, anti-Aβ695 (APP-HEK293) were provided by Dr. Luciano D'Adamio . N2a cells stably transfected with human APP695 (APP-N2a) were obtained from Dr. Gopal Thinakaran . HT-22 and APP-HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum , 2 mM L-glutamine, and penicillin/streptomycin (Life Technologies) at 37°C under 5% CO2. APP-N2a cells were maintained in DMEM/Opti-MEM (1∶1) supplemented with 10% FBS, 2 mM L-glutamine, and penicillin/streptomycin at 37°C under 5% CO2. Cells were transiently transfected with CALHM1 cDNAs using Lipofectamine 2000, as per the manufacturer's instructions (Life Technologies). Transfections were performed in complete culture medium for APP-N2a and APP-HEK293 cells, or in Opti-MEM for HT-22 cells. For HT-22 cells, Opti-MEM was replaced 5 hr post-transfection with complete culture medium. All cell lines were processed 24 hr after transfection.The cell lines used in this study were described previously 2+ add-back was performed as described previously 2+/Mg2+-free Hank's balanced salt solution (HBSS) supplemented with 20 mM HEPES buffer, 0.5 mM MgCl2, and 0.4 mM MgSO4. Ca2+ was then added back for the indicated periods of time to a final concentration of 1.4 mM in the absence or presence of 1% FBS.Ca2+i levels were measured using Fluo-4  as described previously CaCell extracts  were analyzed by SDS-PAGE using the antibodies listed above. Secreted total Aβ and sAPPα levels were analyzed by WB, as described previously Xenopus laevis were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Adult female Xenopus laevis obtained from Xenopus One were maintained in existing university facilities. To obtain oocytes, Xenopus laevis were first anaesthetized by immersion in 0.15% tricaine for 15 minutes. Once anaesthetized, frogs were placed supine, a 1.0 cm incision was made in the lower abdominal quadrant through which part of an ovary was raised and removed and then the incision was closed with 4–0 Reli sutures . Oocytes were defolliculated with type 2 collagenase . No Xenopus laevis frogs or embryos were sacrificed for this study.All procedures involving the use of Xenopus laevis oocytes with Xenopus connexin-38 antisense oligonucleotide . Injected oocytes were incubated for 1 day at 16°C in ND96 medium  and then injected with 50 nl of a 20 mM BAPTA, 10 mM Ca2+ solution at least 30 min before recording to prevent activation of endogenous Ca2+-activated Cl− currents. Data were acquired at 1 kHz using an OC-725C amplifier (Warner Instrument Corp.) with a 16-bit A/D converter (Instrutech ITC-16). Electrodes were made with TW100F-6 glass  using a model P-87 Sutter Instrument Co. micropipette puller and then filled with 3 M KCl.CALHM1 cRNAs were synthesized from MluI-linearized plasmids with SP6 RNA polymerase . CALHM1 cRNA (2 ng) was injected into stage IV–VI +, 100 Cl−, 2 K+, and 10 HEPES with 2 Ca2+ or 0.5 EGTA and 0.5 EDTA to create the divalent cation-free solution. To determine conductance-voltage relations in the presence and absence of Ca2+o, currents at −80 mV were measured following a series of voltage pulses, as described before +, 10 Cl− ±2 Ca2+. Instantaneous I–V recordings were analyzed to determine the reversal potential, as described before Standard bath solutions (pH 7.2) contained (in mM) 100 NaWe interrogated the NHLBI Exome Sequencing Project (ESP) release ESP6500SI-V2 using the Exome Variant Server


Cc, with residual polarity arising from unequal antiparallel packing. Temperature-dependent measurements showed that the highest SHG has been observed at 50 K and the lowest at 170 K.Based on the observation of second harmonic generation (SHG), the solid state structure of a Zn(II) complex has been reinterpreted in terms of the non-centrosymmetric space group  C2/c. This has been attributed to the unequal antiparallel packing of the metal complexes in the non-symmetric space group Cc or residual non-centrosymmetry in C2/c giving rise to polarizability leading to strong SHG. The enhancement of SHG by UV light has been attributed to the increase in non-centrosymmetry and hence polarity of packing due to strain induced in the crystals. The SHG signals measured from these crystals were as large as potassium dihydrogen phosphate crystals, KH2PO4 (KDP), and showed temperature dependence. The highest SHG efficiency was observed at 50 K. The SHG phenomenon was observed at broad wavelengths ranging from visible to below-red in these crystals.Second harmonic generation (SHG) is a well known non-linear optical phenomena which can be observed only in non-centrosymmetric crystals due to non-zero hyperpolarizability. In the current work we observed SHG from a Zn(II) complex which was originally thought to have crystallized in the centrosymmetric space group  For centrosymmetric crystals, based on the Kleinman approximation symmetry and centrosymmetry of crystals, χ(2) has no independent irreducible component of tensors, thus exhibiting no SHG effect  is a nonlinear optical phenomenon related to nonlinear electrical susceptibility χet al., 2014C2/c and the enhancement of surface defects by UV irradiation should be responsible for its observed behavior. The details of our investigations are presented and discussed below.Recently, we investigated the photosalient properties of centrosymmetric crystals of three Zn(II) complexes under UV light (Medishetty 2.2.1.3)2, sodium benzoate and 4-styrylpyridine (4spy) in a 1:2:1 molar ratio, the coordination metal complex [Zn2(benzoate)4(4spy)2] (1), was obtained . The asymmetric unit consists of half of the molecular formula where Zn(II) is present in square-pyramidal geometry and these Zn(II) atoms are bridged by four benzoate groups leading to the formation of a well known paddle wheel building unit. The axial sites of these units are coordinated by the N atoms of the 4spy ligand  studies indicate that there is no phase change observed in the temperature range 223–373 K.During the slow evaporation of a methanolic solution of Zn. The signal was coupled to a monochromator and the CCD, emission wavelength and intensity were measured using a CCD detector . Using the same set-up, the SHG emissions of KDP were also measured as a reference. Input laser energy-dependent SHG spectra from KDP and 1 are shown in Figs. 2a) and (b). Input laser energy-dependent SHG intensities of the crystals of KDP and 1 are shown in Fig. 2c), which clearly shows that the SHG intensity in 1 is two times higher than KDP. Slopes of the laser energy dependence are 1.8 and 1.7 for the crystals of KDP and 1, respectively. The scattered light on a metal mirror and the glass slide were also measured showing a negligible dark signal at 532 nm . These results support the fact that the emission is due to its non-linearity of 1. This has been confirmed from several crystals of 1 from several batches of crystallization. Although the absolute intensity of the SHG signals vary among crystals, the reproducibility of SHG behaviour in 1 has been demonstrated unequivocally. These crystals can also generate SHG in a range of near below-red wavelengths . During the SHG experiment, when the freshly prepared crystals were irradiated under UV light, the intensity of the SHG increased and slowly faded away on continued exposure to UV light as shown in Fig. 2d). The increase in intensity could be attributed to the strain created by UV irradiation . This observation is similar to previous reports for many non-centrosymmetric crystals  was refined close to 0.5 for the data in the range 90–298 K for various single crystals. Further, we have collected data at different temperatures and crystals showing different SHG intensities . However, we were unable to find any direct correlation between the SHG intensities measured and the Flack or other parameters. Furthermore, [Zn2(benzoate)4(2F-4spy)2]  and [Zn2(benzoate)4(3F-4spy)2]  were also found to be isomorphous and isostructural with 1 and exhibit photosalient properties similar to 1. On the contrary, these two crystals do not show any SHG properties, suggesting that they belong to the centrosymmetric space group C2/c unlike 1. Finally, it is worth mentioning that the SHG signal of 1 disappeared after grinding the single crystals into powder. Besides these, it was also found that 1 grown from ethanol has different morphology and did not show any SHG signal which implies that they belong to the centrosymmetric crystals like 2 and 3  property in the first place. A number of so-called ‘centrosymmetric crystals’ have been found to exhibit the SHG effect before  on the inhomogeneity created by the induced strain. The induced strain varies with temperature, as indicated from the temperature-dependent SHG measurements. By increasing the temperature from 10 to 50 K, a sharp increase in the SHG signal is followed by a slow decrease to its minimum at ∼ 175 K. A further increase in the temperature to 300 K is accompanied by a slow increase in the SHG signal. The underlying mechanism has been discussed.In summary, this communication reports so-called ‘centrosymmetric’ crystals of a Zn(II) complex, which is indicated surprisingly to have crystallized with a residual non-centrosymmetry in space group 3.3.1.−1. Elemental analysis  was carried out using an Elementar Vario Micro Cube instrument at the Elemental Analysis Lab, CMMAC, Department of Chemistry, National University of Singapore. Thermogravimetric analysis (TGA) was performed under N2 atmosphere with a heating rate of 5°C min−1 on a SDT 2960 Thermal analyzer. NMR spectra were recorded on a 300 MHz Bruker Avance 300 FT-NMR spectrometer by calibrating the residual solvent as the reference in DMSO-d6 solution. The powder X-ray diffraction (PXRD) patterns were recorded on a Siemens D5005 diffractometer with graphite monochromated Cu Kα radiation (λ = 1.54056 Å) at 298 K.Commercially available reagent-grade chemicals were used as received without any further purification unless mentioned. IR spectra were recorded on a FTS165 Bio-Rad FTIR spectrometer by using KBr pellets in the range 4000–400 cm3.2.SHG by the MOC crystals was measured with 6 ns laser pulses (850–1200 nm wavelength and 20 Hz repetition rate) emitted from a Q-switched Nd:YAG  laser  pumped OPO. The laser pulses were focused onto the sample on a glass plate with a spot size of 200 μm. The sample was kept in a cryostat whose temperature was varied for the measurement of temperature-dependent SHG signals. Optical density filters were used to change incident laser energy. The signal was collected using two collimation lenses and dispersed by a 750 mm monochromator (Hamamatsu R928) and the spectrum was recorded by a charged coupled device . During the measurement, a dichroic filter was used to block the pump beam. A UV lamp (375 nm) was used for UV enhanced SHG generation studies.3.3.3)2·6H20 , the sodium salt of benzoic acid  and 4spy  and dried at room temperature. Yield: 23.1 mg (60%). The elemental analysis (%): calculated for C27H21NO4Zn: C 66.33, H 4.33, N 2.87; found: C 65.94, H 3.91, N 2.83. 1H NMR : δ = 8.58 , 7.2–7.7 , 7.96 , 7.2–7.5 . IR : 1637, 1574, 1505, 1401, 1226, 1172, 1069, 1032, 965, 873, 839, 816, 715, 690, 538, 458.Colourless rod-like single crystals were obtained from slow evaporation of a methanol solution of Zn(NO3.4.Kα (λ = 0.71073 Å) radiation through a sealed tube (2.4 kW). An empirical absorption correction was applied to the data using the SADABS  K: C54H42N2O8Zn2, Mr = 977.64, monoclinic, Cc; a = 24.746 (2), b = 12.212 (1), c = 15.653 (1) Å, β = 109.192 (1)°, V = 4467.2 (6) Å3, Z = 4, ρcalc = 1.454 g cm−3, μ = 1.134 mm−1, GOF = 1.046, final R1 = 0.0323, wR2 = 0.0845 [for 8188 data I > 2σ(I)].10.1107/S2052252515002183/ed5004sup1.cifCrystal structure: contains datablock(s) global, e210, e219, e257, e254, e251. DOI: 10.1107/S2052252515002183/ed5004e210sup2.fcfStructure factors: contains datablock(s) E210-RT. DOI: 10.1107/S2052252515002183/ed5004e219sup3.fcfStructure factors: contains datablock(s) e219. DOI: 10.1107/S2052252515002183/ed5004e257sup4.fcfStructure factors: contains datablock(s) e257. DOI: 10.1107/S2052252515002183/ed5004e254sup5.fcfStructure factors: contains datablock(s) e254. DOI: 10.1107/S2052252515002183/ed5004e251sup6.fcfStructure factors: contains datablock(s) e251. DOI: 10.1107/S2052252515002183/ed5004sup7.pdfExtra tables and figures. DOI: 1031432, 1031433, 1031436, 1031435, 1031434CCDC references:


Although such elevations of Ca2+levels ([Ca2+]) also occur in the nucleus, the precise mechanism of nuclear [Ca2+] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca2+] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca2+ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca2+ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca2+ transients are slower than cytoplasmic Ca2+ transients, and chelating cytoplasmic Ca2+ abolished nuclear Ca2+ transients, suggesting that nuclear Ca2+ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca2+] compared to cytoplasmic [Ca2+]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca2+ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca2+ transient amplitude was significantly lower in myocytes lacking neuronal Ca2+ sensor-1 (NCS-1), a Ca2+ binding protein implicated in IP3R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP3R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca2+] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca2+ signal regulation.In cardiomyocytes, intracellular calcium (Ca In the heart, it is essential for muscle contraction, which is regulated by excitation-contraction (E-C) coupling ) also occur in subcellular compartments such as the nucleus [2+] is increased by receptor stimulation with growth factors, endothelin-1, and angiotensin II. This Ca2+ elevation is regulated by another Ca2+ release channel, inositol 1,4,5-trisphosphate receptor (IP3R), through activation of phospholipase C, followed by IP3 generation  changes elicited by different stimuli such as electrical and receptor stimulations. Several studies have investigated nuclear and cytoplasmic [Ca2+] regulation in various types of cardiomyocytes  are expressed as the percentage of fluorescence intensity (F) relative to basal fluorescence (F0) in each region . The respective regions of interests (ROI) were monitored within the nucleus and cytoplasm of the same cell.Cultured neonatal mouse ventricular myocytes (NMVMs) were infected with adenoviruses encoding G-GECO1 and NLS-R-GECO, which target the cytoplasm and nucleus, respectively, and two days after infection, Caeviously . Dishes Cultured NMVMs were subjected to immunofluorescence analysis using standard procedures. Briefly, cells were fixed with 10% neutral buffered formaldehyde, permeabilized and blocked, and incubated with primary antibodies at 4°C overnight, followed by incubation with fluorescent-conjugated secondary antibodies. The cells were then observed under an inverted microscope  attached to a confocal laser-scanning unit. Frozen sections obtained from mouse hearts were also subjected to a similar procedure without permeabilization. Images were acquired using the FV10-ASW imaging software at room temperature.In situ PLAs were performed to quantify protein-protein interactions between NCS-1 and IP3Rs in cells using the Duolink kit . Using this protocol, only when the two target proteins are in close proximity (<40 nm), high concentration of fluorescence amplified from each interacted molecule becomes visible as a distinct bright dot when viewed with a fluorescence microscope. Briefly, cultured NMVMs were fixed, permeabilized, and blocked. The cells were then incubated with primary antibodies against hemagglutinin  and IP3RI-III, followed by the appropriate secondary antibodies containing unique DNA strands . Anti-α-actinin antibody followed by Alexa Fluor 488-conjugated anti-mouse secondary antibody (Life Technologies) and 4′,6-diamidino-2-phenylindole (DAPI) solution  were added to visualize the myocytes and nuclei, respectively. The samples were evaluated by confocal microscopy. Images were analyzed by the ImageJ software (NIH) using the Cell Counter plug-in to count the number of PLA signals for each α-actinin-positive cell.3RI-III . 2-Aminoethoxydiphenyl borate, tetracaine, and human recombinant IGF-1 were purchased from Sigma-Aldrich. Xestospongin C was purchased from Wako Chemical Co. .We used mouse monoclonal antibodies against NCS-1 , LAP2 , SERCa2a , and rat monoclonal antibody against HA , as well as rabbit polyclonal IPt tests for 2-group-only analysis and by one-way analysis of variance (ANOVA) followed by Bonferroni-corrected t tests for multiple-group comparisons. A one-tailed P-value of <0.05 indicated statistical significance; group data are expressed as means ± standard error of the mean (SEM) or as representative traces.GraphPad Prism 4.0 was used for basic data analysis. Statistical comparisons were performed by unpaired Student’s 2+] in cultured NMVMs using GECOs  (both Ca2+ transients and Ca2+ oscillations) rather than cytoplasmic [Ca2+], and this response is largely mediated by IP3Rs  [2+/calmodulin-dependent protein kinase II (CaMKII)/histone deacetylase (HDAC) pathways [2+] elevation in the nucleus observed in this study would contribute to the gene transcription program that precedes cardiac hypertrophy  IP3R by interaction with NCS-1. Third, NCS-1 may maintain Ca2+ contents in the nuclear envelope by increasing the SR Ca2+ content, thereby increasing IP3R-mediated Ca2+ release into nucleoplasm.We observed that IGF-1-induced nuclear CaWT cells , indicat3 binds to IP3Rs located in the inner membrane of the nuclear envelope, leading to Ca2+ release into the nucleoplasm  within the cytoplasm and nucleoplasm in cardiomyocytes  regulation.There is an emerging concept in Camyocytes ,8. Ca2+ artments . Therefo nucleus , and it myocytes . Therefo2+ content of the nuclear envelope. We previously reported that NCS-1 increases the SR Ca2+ content via activation of IP3Rs followed by activation of CaMKII and SR Ca2+ pump activity in cardiomyocytes  in the nuclear envelope, leading to Ca2+ release from the nuclear envelope into the nucleoplasm via IP3Rs expressed in the inner membrane of the nuclear envelope. Further studies are required to discriminate the predominant mechanism that underlies NCS-1-mediated enhancement of nuclear Ca2+ signaling.Another possible mechanism of NCS-1 action is that it might increase the Camyocytes . Moreove+ stores . Therefo nucleus . Some of nucleus , where Ions (3Rs . Thus, N2+ indicators, we performed simultaneous measurements of nuclear and cytoplasmic [Ca2+] in cardiomyocytes. Our results suggest that distinct mechanisms exist for the regulation of nuclear [Ca2+] in response to electrical and receptor stimulations in the heart. Moreover, our results revealed a novel role for NCS-1 in the regulation of nuclear [Ca2+], mediated by IP3Rs in mouse cardiomyocytes. Receptor stimulation with IGF-1, enhances the interaction between NCS-1 and IP3Rs, and promotes nuclear [Ca2+] elevation. We have previously demonstrated that phenylephrine-induced cardiac hypertrophy was significantly attenuated in NCS-1 KO hearts concomitant with a reduced activation of both calcineurin/NFAT and CaMKII/HDAC pathways [2+] regulation may also be involved in cardiac hypertrophy through these pathways.Using two different kinds of Capathways . Therefo


The mystery is how a single signalling ion can lead to such diverse and specific changes in cell function. This is partly due to aspects of the Ca2 + signal itself, including its magnitude, duration, localisation and persistent or oscillatory nature. The transduction of the Ca2 + signal requires Ca2 + binding to various Ca2 + sensor proteins. The different properties of these sensors are important for differential signal processing and determine the physiological specificity of Ca2 + signalling pathways. A major factor underlying the specific roles of particular Ca2 + sensor proteins is the nature of their interaction with target proteins and how this mediates unique patterns of regulation. We review here recent progress from structural analyses and from functional analyses in model organisms that have begun to reveal the rules that underlie Ca2 + sensor protein specificity for target interaction. We discuss three case studies exemplifying different aspects of Ca2 + sensor/target interaction. This article is part of a special issue titled the 13th European Symposium on Calcium.Changes in the intracellular free calcium concentration ([Ca •2 + regulates multiple aspects of neuronal function over varying time scales.Ca•2 + signalling is determined by the properties of Ca2 + sensorsThe specificity of Ca•2 + sensors contributes to specificity.Differing properties of closely related Ca•2 + sensors determines specificity of target protein interactions.The structure of Ca•We discuss three case studies involving calmodulin, NCS proteins and CaBP proteins.•2 + sensors.The examples illustrate the structural–function relationships of Ca Changes in [Ca2 +]i can be local or global, highly transient, oscillatory or persistent. Localisation of [Ca2 +]i changes to particular neuronal compartments is important for the generation of specific neuronal responses. For example, certain stimuli strictly require nuclear rather than cytoplasmic changes in [Ca2 +]i2 +]i changes restricted to dendritic spines may play an important role in activity-dependent synaptic plasticity 2 +]i can be manifest in microseconds (neurotransmitter release), milliseconds (channel facilitation or inactivation) or over much longer time scales leading to changes in gene expression The intracellular free calcium concentration  in the same neurons are coupled through distinct signalling pathways to changes in gene expression 2 + signals are translated into changes in cellular function through various types of Ca2 + sensor proteins that in general terms detect increases in [Ca2 +]i by becoming loaded with Ca2 +, undergo a conformational change and then interact with and regulate various target proteins. The subtlety of neuronal Ca2 + signalling is underpinned by differential signal processing by these Ca2 + sensor proteins, which in turn determines the physiological specificity of Ca2 + signalling pathways.Clearly Ca2 + sensor proteins 2 +-sensor for fast (microsecond) neurotransmitter release 2 +-binding motif 2 + sensor protein calmodulin 2 +/calmodulin-dependent protein kinase II 2 + sensors expressed in neurons include the calcium-binding protein (CaBP) family Neurons express a large number of Ca2 +-signals is affected by multiple aspects of the properties of Ca2 + sensors, and we suggest that they have a key role in determining signalling specificity. Factors that influence differential Ca2 + signalling include varied expression levels of sensors between neuronal cell types 2 +-binding 2 +-sensors to signalling specificity requires analysis of both the Ca2 +-sensors and their target interactions at a structural level 2 + signalling in neurons have begun to emerge in recent years but still remain to be fully understood. In this review, we will present three case studies to illustrate current understanding of the molecular and structural basis of the contribution of the properties of Ca2 + sensors to differential neuronal signal processing.The differential processing of neuronal Ca22 + regulation of phototransduction. One other subfamily, consisting of the visinin-like protein (VILIP) 1–3, neurocalcin δ and hippocalcin has less well-defined functions. Hippocalcin, as suggested by its name, has a very restricted pattern of expression mainly in hippocampal neurons where it shows dynamic membrane association  1–3 have specialised roles in the CaCaenorhabditis elegans as a consequence of its restricted neuronal expression DrosophilaSaccharomyces cerevisiae NCS-1 (Frq1) is essential for survival as a consequence of its requirement for activation of the phosphatidylinositol-4-kinase Pik1 2 +-dependent exocytosis NCS-1 appears to be expressed in most neuronal cell types 2 + sensors particularly calmodulin in vitro binding assays, and so their biological importance is unclear. It is possible that those binding partners that are also calmodulin targets in vitroNCS-1 has many known interacting partners The other clinically important interaction is with the interleukin 1 receptor accessory protein-like 1 protein (IL1RAPL1), which appears to be specific for NCS-1 2 + sensors and more particularly other members of the NCS protein family which have their own specific targets? The NCS proteins show differences in membrane-association with some such as recoverin and hippocalcin showing transient membrane-association through their Ca2 +/myristoyl switch mechanism 2 +/myristoyl switch 2 + binding, the NCS proteins undergo a conformational change that exposes a hydrophobic groove within which one or two helices from target proteins can bind. Once Ca2 + loaded the NCS, proteins undergo relatively limited structural changes on target ligand binding unlike the more dramatic changes seen in calmodulin conformation on ligand binding (described below).One key question is, what determines the specificity of target recognition for NCS-1 over other CaKey hydrophobic amino acids within the hydrophobic groove of the NCS proteins that are conserved across the whole NCS family are directly involved in the target interactions in multiple NCS proteins . DespiteC. elegansThe structural studies on NCS protein/target interaction have been based on the use of short peptides derived from the target protein. These studies have provided a consistent picture in which conserved hydrophobic residues are crucial. Nevertheless, it is important to assess the significance of the structural data using a physiological model. This would, for example, allow a test of requirement for the key residues in the hydrophobic groove and also the requirement for the C-terminal tail for NCS-1 function. This was approached using an assay for temperature-dependent locomotion in Drosophila frequenins. In Drosophila species, a gene duplication event generated two genes encoding closely similar proteins known as Frq1  of CaBP7 was solved by solution NMR 3 receptor in the docked complex CaBP7 and CaBP8  or Ca2 +-dependent inhibition (CDI) on a millisecond timescale. The speed of this regulation is explained by the response of the channels being due to nanodomains of Ca2 + near the mouth of the channel 2 + and progress on the characterisation of the mechanisms particularly those involving calmodulin 2 + sensors has also been the subject of a recent review 2 + sensor required for CDI of L-type (CaV1.2) Ca2 + channels 2 +-free (apo) calmodulin was pre-bound to the pore-forming α1 channel subunit and that this was mediated through the so-called IQ domain  channels calmodulin is required for both CDI and CDF with the N-lobe of calmodulin involved in CDI and the C-lobe in CDF V2.1 1 subunit  neurons was used to probe the general role for interaction of Ca2 + sensors in synaptic plasticity. In this study, it was shown that mutation of the IQ (IM in Cav2.1) motif or the CBD that would prevent CDF or CDI reduced facilitation or short-term synaptic depression, respectively Many of the studies on the regulation of CDF or CDI of VGCCs by CaV1.2 V1.3 channels as a consequence of CaBP4 binding to regions other than the IQ domain One problem with the originally proposed competitive model of action for CaBPs in which they need to compete endogenous calmodulin from the IQ domain is that calmodulin is expressed in brain at considerably higher levels than CaBPs. CaBP1 has been shown, however, to interact with an additional site in the N-terminus of CaV1.4 V1.2 channels v1.3 channels Findings consistent with an important role for CaBP4 in the regulation of phototransduction have come from the study of a CaBP4 mouse knock-out and also the presence of mutations in human CaBP4 2 + sensors exert their Ca2 +-dependent effects on VGCCs has been a subject of intensive study. Key issues to be resolved include (1) how can the two lobes of calmodulin have divergent functions when both bind to the IQ domain, (2) why does the C-lobe dominate in CDI in CaV1.2 channels but the N-lobe does so in CaV2 channels, (3) how can binding of calmodulin to an intracellular region of the channel α1 subunit affect the gating properties of the channel pore and (4) how can additional Ca2 + sensors that mediate differing effects do so particularly in the presence of calmodulin tightly bound to the IQ domain 2 +-bound calmodulin with an IQ-like peptide from CaV1.2 (V2.1, CaV2.2 or CaV2.3 channels in an anti-parallel manner giving a possible explanation for the reverse role of the N- and C-lobes in CDI of the two classes of channels 1 subunit involving the Ca2 +-loaded N-lobe may be important The structural basis for how calmodulin or other Cam CaV1.2 . These iV1.2 channels. This has demonstrated that both lobes of CaBP1 are functionally important with the C-lobe anchoring to the channel subunit as does the C-lobe of calmodulin. The N-lobe and also the inter-lobe linker specific for the CaBPs are required for modification of channel function and this could contribute to the different effects on the channel of CaBP1 compared to calmodulin 1 subunit may be important, but this will require further structural characterisation. There is currently no published data on the structural bases for the interactions of other Ca2 + sensors with VGCCs.A full structural characterisation of the interaction of CaBP1 with VGCCs is not available but one analysis has used chimeras with calmodulin and specific mutants to dissect the requirements for the regulation of Ca52 + sensors which is determined by fundamental structural characteristics of the proteins. Calmodulin is the most promiscuous with many target proteins. It consists of two independent lobes joined by a flexible linker that allows calmodulin to wrap around and interact with target proteins in multiple different ways using mutually induced fit to ensure high affinity interaction One aspect that emerges from consideration of the three case studies above is the variation in level of specificity versus promiscuity of the Ca2 + binding, Ca2 + sensors undergo a conformational change that allows them to bind ligands and may then undergo further conformational changes. This in turn will allow them to regulate the function of the target proteins. Most of the available structural characterisation has provided information on the binding mechanism and specificity but so far has provided little information on how the activity of the target protein is regulated. This is particularly notable in the case of VGCCs where even different lobes of calmodulin can have different functional effects after binding to a site on the channel α1 subunit some distance from the channel pore. For VGCC regulation, we need further information on the structural basis of interactions of Ca2 + sensors outside of the IQ domain, with the rest of the C-terminus, with sites in the N-terminus of the α1 subunit and also potentially with other subunits of the holo-channel. The NCS proteins such as NCS-1 have a smaller range of targets and differ from calmodulin in having two lobes that have a very rigid inter-relationship severely reducing the conformational flexibility between the two lobes. Target binding is due to the exposure of a hydrophobic groove the size and surrounding residues of which in conjunction with a variable and more flexible C-terminal tail determine which ligands can bind. A completely un-investigated aspect of NCS-target interaction is how NCS protein binding influences target conformation and structure and how this translates to function. Again, structural insights in this regard are limited by the complexes characterised to date containing only short peptides derived from the target protein. Further progress in this field will, therefore, require characterisation of Ca2 + sensors complexes with larger, ideally full-length, target proteins in order to contribute to an understanding of the mechanistic basis of differential signal processing in response to Ca2 + signals.On Ca


A test experiment has shown that the number of ultracold neutrons (UCN) of one polarization state, transmitted through a 100 μm Al foil when placed in a 5 T magnetic field, is greater by 3.8 times. The increased transmission is due to the higher velocity of the UCN passing through the foil. There are two possible ways to obtain polarized UCN for the electric dipole moment (EDM) experiment: either one uses magnetized ferromagnetic layers on thin foils  or one uWe propose to use a superconducting (SC) solenoid polarizer on the fill lines of the EDM spectrometer. The advantages of such a choice are: 1) the possibility to obtain fully polarized UCN as compared to about 85 % polarization using magnetized foils; 2) the possibility to place the vacuum separation foil in the high magnetic field region.Separation foils are needed between the UCN source volume containing the solid deuterium and the EDM volume with the high voltage. A test experiment has recently shown that the number of UCN of one polarization state transmitted through a 100 μm Al foil when placed in a 5 T magnetic field is greater by 3.8 times. The increased transmission is due to the higher velocity of the UCN passing through the foil.The superconducting solenoids will be equipped with ARMCO return yokes in order to suppress the stray fields that might influence the EDM measurements. Due to the fact that the magnetic field of the SC magnets, once switched on, will be very stable over typical measurement times, its influence on the EDM experiment is only static and can be compensated for. In our test experiment on foil transmission at the Institut Max von Laue–Paul Langevin (ILL), the possibility to carry out the present RAL-Sussex-ILL EDM experiment together with an even unshielded SC solenoid nearby (about 4 m distance) has been demonstrated.As it is shown in the calculation, a cylindrical magnetic shield made of ARMCO with a diameter of 700 mm and a thickness of 100 mm can suppress the magnetic field in the EDM spectrometer down to 50 μT.The scheme of the experiment, which was recently performed at ILL, Grenoble, is shown in When the shutter 2 was opened, UCN were counted in the UCN detector. Various cases were studied: with and without an Al foil, with and without a magnetic field. All four possible cases were studied with the UCN absorber at various positions in order to obtain the energy dependence of the transmission.Thus the usage of a SC solenoid allows one to obtain polarized UCN and to increase the density of polarized UCN by factor of 3.8. In the cases when polarization of UCN is not needed in the experiment, the factor of the UCN density increase is about two times. It is important also for the experiments with unpolarized UCN.


Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury. Mitochondrial function is crucial for energy provision and health in higher eukaryotes, particularly in excitable cells, such as neurons, and in skeletal and cardiac muscle. Bioenergetic homeostasis, which ensures that energy supply has the capacity to meet energy demand, is key to normal cell and tissue health. Calcium signaling plays a central role in both communicating increased energy demand and in driving mechanisms that increase energy supply.2+](c) signaling plays a critical role in cell death. Mitochondria engage in an intricate and complex dialogue with calcium signals. The capacity of mitochondria to accumulate calcium was established many years ago through the work of the pioneers of mitochondrial biology, including Lehninger, Carafoli, and Attardi.et al.Calcium is a universal secondary messenger, playing a central role in a remarkably wide range of cellular processes, including muscular contraction, fertilization, synaptic transmission, cell migration and motility, and cell proliferation.2+](c). The other pathway, mediated by the mitochondrial calcium uniporter (MCU), requires a rise in matrix Ca2+ concentration ([Ca2+](m)). The two pathways operate in concert to increase oxidative phosphorylation in response to cellular calcium signals. Thus, a rise in [Ca2+](m) concentration drives the allosteric activation of the three rate‐limiting enzymes of the tricarboxylic acid cycle: pyruvate, isocitrate, and α‐ketoglutarate dehydrogenase.Vmax of pyruvate dehydrogenase, isocitrate dehydrogenase, and α‐ketoglutarate dehydrogenase is enhanced upon Ca2+ binding, upregulating the production of reduced nicotinamide adenine dinucleotide (NADH) and resulting in a subsequent increase in mitochondrial membrane potential (Δψm), followed by an increase in adenosine triphosphate (ATP) production. This was demonstrated directly in sensory neurons, where a calcium‐dependent increase in NADH fluorescence was observed upon stimulationCalcium signals directly drive an increase in mitochondrial bioenergetic efficiency through two major pathways Fig. , includi2+](c) acts as a positive effector of Ca2+‐binding mitochondrial carriers (CaMCs), including aspartate/glutamate carriers (AGCs) and ATP‐Mg/Pi carriers (SCaMCs). Aralar and citrin are mammalian AGCs involved in the malate–aspartate NADH shuttle, which promotes the transfer of NADH from the cytosol to the mitochondria, thus providing reducing power for ATP generation via oxidative phosphorylation while also keeping the cytosolic NADH/NAD+ ratio low, favoring generation of pyruvate and promoting mitochondrial substrate supply. Cells overexpressing aralar and citrin showed a larger increase in mitochondrial ATP levels upon calcium stimulation compared to control cells, while mutants lacking Ca2+‐binding sites failed to show a significant energetic response to a calcium signal.2+ for mitochondrial Pi in response to hormone‐induced calcium signaling.2+](m) and [Ca2+](c). Interestingly, these also operate over different concentration ranges: the S0.5 for the activation of aralar by calcium is approximately 300 nM,A rise in (m) measured as a function of extramitochondrial Ca2+ exhibits a sigmoidal relationship, with a threshold at approximately 2–3 μMMitochondrial calcium uptake depends on the electrochemical potential of Ca2+](c) signals to the mitochondrial matrix with little effect on resting [Ca2+](m).Although mitochondrial calcium uptake was first documented in the 1960s, the MCU was not identified until 2011.in vivo may be important in response to exercise.Interestingly, it is possible to create viable mice in which the MCU is globally knocked out (MCU‐KO)  has been found to act as a dominant negative subunit of the MCU channel. The MCU/MCUb ratio appears to vary across different tissues, potentially providing a molecular mechanism to regulate the efficiency of mitochondrial calcium uptake.2+](c) (<3 μM) but allows cooperative activation of the channel as [Ca2+](c) exceeds the threshold  display a striking mitochondrial phenotype; their mitochondria are largely fragmented and calcium loaded, even at basal unstimulated cytosolic calcium concentrations Fig. . The sig2+ inevitably leads to activation of efflux pathways, most notably the Na+/Ca2+ exchange. This increase will raise matrix Na+, which will in turn activate mitochondrial Na+/H+ exchange, bringing protons back into the matrix through a route that bypasses the ATP synthase, consuming proton motive force to balance calcium influx at the expense of ATP synthesis. In patients, it seems probable that the continuous transfer of Ca2+ into the matrix even at resting [Ca2+](c) is sufficient to drive a futile cycle where calcium is brought into the matrix and (indirectly) exchanged for protons, undermining the bioenergetic capacity of the cell. These observations illuminate the physiological role of MICU1, which serves as a signal‐to‐noise discriminator for mitochondrial calcium signaling and protects mitochondria from energetically wasteful basal calcium uptake.The identification of neuromuscular disease associated with impaired mitochondrial calcium handling highlights an aspect of mitochondrial calcium signaling that tends to be ignored, which is that mitochondrial calcium accumulation carries an energetic cost. An increase in matrix Cam, mitochondrial swelling due to the osmotic influx of water, and induction of ATP hydrolysis by the FoF1 ATPase. Mitochondrial swelling can cause rupture of the OMM, releasing proapoptotic factors, such as cytochrome c, apoptosis‐inducing factor, and procaspases from the intermembrane space. It has been suggested that a reversible low‐conductance state of the mPTP might play a physiological role in calcium signaling,Pathological and excessive mitochondrial calcium uptake is a major trigger for cell death and is mediated by the mPTP. Opening of the mPTP has been implicated in cell death associated with ischemia/reperfusion injury,2+](m), as well as by oxidative stress, increased phosphate levels, loss of Δψm, and ATP depletion, which can lower the Ca2+ threshold required to trigger pore opening. Importantly, pore opening is limited by cyclosporine A (CsA), an immunosuppressant drug in widespread clinical use. Therefore, the mPTP represents an exciting pharmacological target with translational potential. A modulator of the mPTP was identified by studies that demonstrated that the archetypal mPTP inhibitor, CsA, worked through the inhibition of a matrix peptidyl‐prolyl cis–trans isomerase (PPIase), cyclophilin D (CypD).2+](m) and oxidative stress. Nonetheless, mPTP opening was not completely abolished and could be provoked by a higher [Ca2+](m).Pore opening is primarily triggered by increased (i)). The rise in [Na+](i) is exacerbated by slowed or inactive Na+/K+ ATPase pumps as ATP is depleted and also stimulates the NCX to reverse, increasing intracellular calcium concentration ([Ca2+](i)).2+](i) in cells within an infarct, which is coupled with the depletion of ATP and adenine nucleotides, and a progressive rise in inorganic phosphate. These changes will ultimately culminate in necrotic—and minor peripheral apoptotic—cell death, if ischemia continues uninterrupted.Restriction of oxygen and nutrient delivery during ischemia leads to progressive changes in cell physiology that establish conditions that favor opening of the mPTP at reperfusion. Thus, a switch to anaerobic metabolism causes intracellular acidosis.Although the ischemic period of ischemia/reperfusion injury induces significant intracellular changes, the transitory phase of starvation primarily sets the scene for mitochondrial dysfunction and does not by itself necessarily drive irreversible injury. Although primed, mPTP opening is prevented due to the low intracellular pH during ischemia,2+](c) may be further stoked by the generation of free radical species , which stimulate calcium release from sarcoplasmic stores.At reperfusion, the reintroduction of oxygen restores respiration, immediately restoring the mitochondrial membrane potential and so driving rapid mitochondrial calcium uptake. The rise in [CaPrevention of mPTP opening is an important potential therapeutic target for the protection and preservation of cells, especially in nonproliferating cardiomyocytes and neurons. As with many complex cellular processes, numerous methods have been used to broach the chain of events that eventually lead to mPTP opening following ischemia/reperfusion injury. Primarily, these procedures have spanned preinjury priming events, from remoteOpening of the mPTP at reperfusion is a response to mitochondrial calcium overload in combination with high Pi, low ATP, and oxidative stress Fig. . Recent These experiments strongly suggest several potential novel therapeutic targets in the pathway to ROS generation that may help prevent mPTP opening and cell death following reperfusion. The field of mitochondrial biology faces the real prospect of combining strategies to salvage cardiac tissue—and likely tissues in other organ systems where the evidence is currently less developed—at risk for injury from ischemia.The authors declare no conflicts of interest.


BJPsych Bulletin is piloting an author mentoring scheme. Mentors will help clinicians and aspiring academics develop articles from a pedestrian manuscript to one that will hopefully provoke important debate and aid changes in current practices. The scheme will run on a trial basis for approximately 12 months and will then be reviewed. Mentoring has been found to have an important effect of research output including publication and grant success; the hope is that this new initiative at the BJPsych Bulletin will result in such dividends to all involved.The publishing world is changing rapidly. Innovations include the move to open access, the rise of social media and the transition to digitalisation. In the light of these developments and with ever-increasing pressures on early career psychiatrists and trainees to publish papers in journals with a recognised pedigree, the  Fast-evolving developments include a growing pressure to publish open access, the transition to digitalisation and the rise of social media. And although industry experts are unable to predict the ultimate appearance of the academic journal landscape, they are all in agreement that it will continue to evolve for quite some time.BJPsych Bulletin has gained a reputation for providing a platform for nascent researchers to begin publishing their work. And so, to help scientists in the mental health field navigate the scientific publishing industry and compete in the career market, the BJPsych Bulletin is now piloting a mentoring scheme for authors that have submitted papers to the journal.In addition to these innovations and changes there are ever-increasing pressures on early career psychiatrists, trainees and other budding academics to publish in well-recognised journals: such an achievement might provide the necessary edge to succeed at a job interview or in a grant application. The 2 Primarily, mentoring has been seen as an aide for budding academics keen to develop their research portfolio, but a systematic review of mentoring in academic medicine concluded that it was an important influence on personal development, career guidance, career choice and productivity. Furthermore, it was found that mentoring had an important effect on research output, including publication and grant success.3 Although the provision of hard evidence demonstrating clear links between mentoring and important outcomes is limited, surveys of mentees have found that mentored students rate their overall well-being as higher.2Mentoring has a long history as a key component providing assistance to junior staff looking for career progression. It was developed in the USA in the 1970s within large private sector organisations. Formal mentoring schemes for medical students and early career doctors began to be used in the late 1990s.4 The aim of the BJPsych Bulletin mentoring scheme is to provide input to articles with potential that are in need of revising before reaching a sufficient standard whereby they can be sent out for review. Feedback will focus on the study design and statistical analyses as well as writing tips including how to contextualise the paper in light of the current scientific thinking, both in the UK and internationally. In addition, mentors will advise about how to maximise the article's impact through the use of search-engine friendly titles, abstracts and keywords. The remit of the papers appropriate for the BJPsych Bulletin will be unchanged: service provision and development, teaching and education, legal, technological and cultural advancements relating to treatment and delivery of care, case reports and miscellaneous articles on governmental policy.The success of mentoring schemes depend on a variety of factors including the characteristics of the mentee (e.g. younger age), experience and skills of the mentor and the quality of the relationship between those involved.BJPsych Bulletin – with top-level teaching skills and bags of motivation!Mentors will help clinicians and aspiring academics understand the difference between a solid but pedestrian manuscript and one that will provoke important debate and aid the development of current practices. Mentors will be experienced researchers or academics – in some cases members of the editorial board of the The idea is that mentors and mentees will agree a predetermined timetable of tasks and activities. Although there will be no formal contract, it is important to avoid acrimonious disputes which sometimes arise if a paper undergoes multiple revisions and reviews; understandably, the more time invested and the more changes made, the more some authors become convinced of the value of their work. To minimise this possibility, the mentor will have the final word over the whole process and if at any stage it appears that the article will not reach the standard required for reviewing, it will be up to the mentor to decide when to draw stumps.With the understanding that time is a precious commodity to all healthcare professionals, it is envisaged that the average mentoring process for a paper, from start to finish, would require a maximum of about 12 hours over a period of up to 6 months: this estimate comes from a belief that no paper needing more than six revisions each taking about 2 hours will have reached the threshold for inclusion in the scheme.http://submit-pb.rcpsych.org/) along with a cover letter requesting entrance into the scheme. In addition, authors of articles already submitted to the BJPsych Bulletin but which the Editor or a member of the Editorial Board determines would benefit from mentoring will be contacted with an offer. If the paper is eventually published, credit will be given to the mentor by an acknowledgement added to the end of the article.Authors wishing to have papers considered for mentoring should submit them through the website (The scheme will run initially on a trial basis for the first 12 months and will be subject to regular informal reviews. These evaluations will be assessed by the journal's Editorial Board at the end of the year to decide the feasibility of continuing the scheme. We are very excited about this new initiative to help trainees with publication and we invite you to take part.


Individual cells responded to acetylcholine with a concentration-dependent increase in Ca2+ signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca2+ signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.—Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca In this noisy chemical environment, concentrations of each activator change almost continuously, and the endothelium detects the alterations and evokes a vascular response. The detection and signaling systems involved are robust to random fluctuations (noise) that obscure the signals, and yet the cells are sensitive and able to discriminate very small changes in agonist concentration  that arrive ntration . The end2+ concentration to coordinate the endothelium’s control of vascular tone. Ca2+ acts as a highly localized subcellular messenger and a multicellular communicator with wide reach . The endothelium regulates the contractile response of the carotid artery  lens. The miniature fluorescence endoscope permitted Ca2+ signaling to be measured from inside the lumen of intact pressurized arteries while the vessel is in a physiologic configuration and at normal intraluminal pressure. The endoscope allows ∼200 endothelial cells to be imaged with subcellular resolution and has a high depth of field (141 µm) so that focus is maintained across the curved endothelial layer of the pressurized artery.To address how the endothelium detects agonist and coordinates Ca2+ waves that are transmitted distances scaling with stimulus intensity. The clusters provide a coincidence detection system for noise rejection. The results show that the endothelium acts as a distributed sensing system with the response of each cell and cluster part of a collective decision on concentration; heterogenous cell sensitivity, clustering, and signal propagation provide an integrative mechanism for noise filtering and sensing agonists over wide concentrations ranges.We show that agonist sensing is carried out by cells with various fixed concentration sensitivities that operate over a narrow concentration range. By merging multiple standard concentration responses, from various cells working over various predefined concentration dependencies, a sensitivity range for agonist sensing that is effective over at least 3 orders of magnitude of concentration is achieved. Cells of comparable sensitivity cluster (multicellular macrodomains) to provide agonist concentration sensory spaces. Simultaneously activating several cells in the cluster generate Ca2+ indicator by perfusing the lumen with a Ca2+ indicator loading solution for 30 min at 37°C. After loading, arteries were removed from one of the cannulae and remounted on a custom microendoscope fluorescence imaging system to visualize the endothelium and maintain the arteries at physiologic pressures  Act 1986, United Kingdom]. The arteries were mounted onto stainless steel (22 gauge) cannulae in a custom vessel bath, and the endothelium was selectively loaded with a fluorescent Caet. al.  encased in a protective stainless steel sheath  , was coupled to the GRIN using a standard microscope objective  . The laser.org/) . The mager.org/) .2+ signaling was induced by 100 µM extraluminally applied acetylcholine (ACh). The concentration of ACh at the lumen of the intact pressurized artery is likely to be ∼1000-fold less than the extraluminal bath ACh concentration . In experiments designed to analyze the concentration dependence of ACh-induced endothelial Ca2+ signaling, viable arteries were stimulated with various concentrations of externally applied ACh. In other experiments (described in the text), viable arteries were treated with pharmacologic inhibitors and incubated for 20 min, unless otherwise indicated. In these experiments, incubation was performed after control responses had been obtained. All responses were studied in paired experiments, and are expressed relative to control .After loading and mounting, to confirm endothelial viability, endothelial Cae.g., caged inositol trisphosphate (IP3)]. In these experiments, Ca2+ signaling was examined in cut-open en face endothelial preparations, in which arteries were surgically opened and pinned out on a Sylgard block. Arteries were loaded with Ca2+ indicator loading solution or, for caged IP3 experiments, with Ca2+ indicator loading solution containing a membrane permeant caged IP3  for 30 min at 37°C. Sylgard blocks were then placed face down on a 0 grade thickness microscope coverslips and the endothelium imaged using a wide-field epifluorescence microscope . Steel pins (200 µm) were used as spacers to ensure the endothelium did not contact the coverslip. The Ca2+ indicator was excited with 488 nm wide-field epifluorescence illumination provided by a monochomator  and fluorescence emission was collected by the objective lens  and transmitted to a cooled, back-illuminated electron-multiplying charge-coupled device camera   . Photolyescribed –32, usin2+ signaling was induced by application of 1 µM ACh. In experiments designed to analyze the concentration dependence of ACh-induced endothelial Ca2+ signaling in en face preparations, arteries were stimulated with various concentrations of ACh. In other experiments, silicone blocks were removed from the imaging chamber and arteries were preincubated with 500 µM Gap27 for 60 min at 37°C. Subsequently, arteries were placed back on the microscope and Ca2+ responses were recorded in the same field of cells. Responses were studied in paired experiments and are expressed relative to control .After loading and mounting, endothelial viability was confirmed by stimulating endothelial CaSurgically opened arteries pinned to Sylgard blocks were fixed in 10% neutral buffered formalin for 15 min at room temperature. Arteries were then washed 3 times in glycine solution, washed with PBS, permeabilized with Triton-X 100 (0.2% in PBS) for 15 min, washed again 3 times in PBS, and incubated with PBS containing 2% bovine serum albumin for 15 min, and then washed 3 times with PBS containing 0.05% Tween. Arteries were then incubated with a rabbit anti-muscarinic acetylcholine receptor (AChR) antibody  in PBS containing 2% bovine serum albumin for 1 h. After extensive washing in PBS containing 0.05% Tween, arteries were incubated with an Alexa Fluor 555-conjugated donkey anti-rabbit antibody  in PBS containing 2% bovine serum albumin for 1 h and then washed extensively in PBS containing 0.05% Tween. All processing steps and incubations were performed at room temperature. Arteries were then incubated in PBS containing 0.5 mg/ml of DAPI before being placed visualized on the same inverted microscope described above. Negative controls were performed in the absence of primary antibody.2+ imaging and AChM3 receptor antibody staining were visualized in the same artery, endothelial Ca2+ signals were first evoked by bath application of ACh  and imaged as described above. Arteries were then removed from the microscope and stained for AChM3 receptor distribution and nuclear distribution. Arterial segments were then placed back on the microscope stage for visualization of the same region of endothelium in which Ca2+ signals were recorded. Images of Ca2+ activity and AChM3 receptor distribution were aligned using images of nuclear staining (DAPI). AChM3 receptor distribution was quantified, in endothelial regions that either responded or did not respond to 30 nM ACh, by summing background corrected AChM3 receptor staining intensity in the regions and dividing by the area of the region. For comparison across experiments, regional measurements were normalized against total staining in the total area of each of the images.In experiments where Ca2+ imaging data were processed using a largely automated data processing procedure. First, regions of interest (ROIs) corresponding to individual endothelial cells were generated using a custom, semiautomated image processing segmentation procedure . In paired experiments, ROIs from control experiments were applied to response images. In concentration response experiments, the cell masks generated from maximal ACh concentrations were applied to all data sets. ROI alignment was manually checked for all datasets, and, in the event of movement between data sets were aligned using an automated alignment plug-in in ImageJ   files. Fluorescence signals were then expressed as baseline corrected values (F/F0), calculated by dividing the raw signals by the average value of a 50-frame period preceding ACh-evoked Ca2+ activity. For an illustration of total Ca2+ activity, individual F/F0 traces were aligned with respect to their peak rate of change. The program automatically calculated baseline values of F/F0, peak amplitudes and the time of peak rate of change for each signal and stored them as .csv files. These files were then imported into Origin 9.1  for calculation of peak changes in fluorescence intensity (ΔF/F0), and for plotting using custom analysis scripts. An increase in cytoplasmic Ca2+ concentration ([Ca2+]c) was inferred when the peak F/F0 rose by more than 3 sd of baseline noise. As the choice of threshold affects the balance between detection rate and false positives, and depends on cellular loading and illumination/detection efficiency, baseline noise was calculated for individual cells over a 25 frame period preceding the onset of the rise in the F/F0 ratio. Curves were fitted to normalized concentration response data using GraphPad Prism 6.0 . The maxima and minima of the curves were constrained to unity and 0, respectively. Calculated curve-fit parameters (EC50) are presented with 95% confidence intervals.Endothelial CaMD, USA) . Raw ima2+ indicator loading solution consisted of the cell permeant acetoxymethyl ester form of Oregon Green 488 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate , with a final concentration of 0.04% Pluronic F-127 and of 0.96% DMSO, in physiological salt solution. Drugs were all obtained from Sigma-Aldrich  except for the following: OGB-1/AM and Pluronic F-127 were obtained from Invitrogen, caged IP3 DMNB from SiChem . The physiologic salt solution consisted of (mM): NaCl (145), KCl (4.7), 3-(N-morpholino)propane-sulphonic acid (2.0), NaH2PO4 (1.2) glucose (5.0), EDTA (0.02), MgCl2 (1.17), CaCl2 (2.0), pH adjusted to 7.4 with NaOH. Glycine solution consisted of MilliQ H2O containing 100 mM glycine (pH adjusted to 7.4 with NaOH). The PBS consisted of (mM): sodium chloride (137.0), potassium chloride (3.0) and disodium hydrogen phosphate (8.0), and potassium dihydrogen phosphate (1.5).The Casem. One-way nested ANOVA (with Tukey’s post hoc test as appropriate) was used for comparisons between groups. Biologic replicate  was treated as a random effect. A value of P < 0.05 was considered significant.Summarized data are expressed as means ± 2+ occurred infrequently (0.97% of cells from 154 separate imaging experiments). These spontaneous Ca2+ transients remained as solitary signaling events confined to particular cells and did not propagate even though substantial in magnitude ; 60 mmHg Supplemental Movie 1)] evoked rises in [Ca2+]c in the majority of cells in the field (2+ oscillations (uniform rises throughout the cell) and propagating Ca2+ waves  occurred in various cells . The Ca2+ rises evoked by ACh originated from an IP3-sensitive Ca2+ store. The Ca2+ increase persisted in a Ca2+-free bathing solution but was blocked by the sarco-endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid and the IP3 receptor blocker 2-aminoethoxydiphenyl borate . The approach calculates the outline of each cell by using the activity of the cells themselves to facilitate identification . Each region of measurement was verified in each experiment. Using this approach, each cell’s Ca2+ response was rapidly extracted. However, the temporal spread of responses was particularly problematic at low ACh concentrations  in a custom Python program. The aligned data illustrated the total Ca2+ response with greater clarity . As the agonist concentration increased, additional cells were recruited in a concentration-dependent manner . Second, after recruitment, the amplitude of [Ca2+]c response within each cell also increased in a concentration-dependent manner   D–F. Howeionship) D, E. Theionship) G. Each cntration E, thoughntration G. These 2+ signals, cells were split into the most sensitive 50% (red) and least sensitive 50% (green) . For each of the high-sensitivity and low-sensitivity cells, the number of high-sensitivity and low-sensitivity neighboring cells within 32 pixels were measured separately  A–C. Fromparately . In bothparately  that occas waves D. The teet. al. . After the initiating ensemble activated, Ca2+ rises expanded to neighboring cells , which were often arranged in strips following the length of the artery [also see Huang et. al. ], activang cells E. In theng cells E–G, all ng cells F, G. Theecreased , and the2+ signaling evoked by ACh and the distribution of AChRM3 receptors was examined in the same artery. Those regions most sensitive to a half-maximal effective concentration of ACh (30 nM) had a significantly (P < 0.05) increased number of AChM3 receptors (The variation in sensitivity to ACh in different regions of the endothelium may be explained by variations in the density of the ACh receptor (AChR) population. Regions most sensitive to ACh had a higher density of the muscarinic AChR M3 . Subsequeceptors .2+ waves arising from the initiator ensemble appeared to preferentially travel along the longitudinal axis of the vessel when initially activated by ACh. However, as the paths of Ca2+ waves from different initiating sites crossed, annihilation occurred on collision and the initial large-scale Ca2+ waves decoupled into complex spatiotemporal patterns of Ca2+ signaling . Although Ca2+ signaling appeared disordered, the pattern of initial wave progression was consistent on repeated activations . When the artery was activated consecutively, the average amplitude of the Ca2+ signal in all cells was 0.99 ± 0.04% (n = 7) of control. The reproducible nature of the disordered wave appears to suggest a signal may be encoded in the pattern.Ca2+ waves decoupled into multiple spatially restricted events, the Ca2+ changes did not subsequently entrain and synchronize, to become a uniform oscillation throughout the endothelium, or co-ordinate to move in a particular direction. However, waves still appeared to progress within cells, and close inspection revealed small groupings of cells that appeared to remain linked by the apparent transmission of the oscillatory Ca2+ waves between cells  and 18β-glycyrrhetinic acid  were used in separate experiments. Each blocker substantially reduced the response to ACh (2+ waves. Prolonged exposure to either 18β-glycyrrhetinic acid (100 µM) or CBX (100 µM) blocked all response to ACh after 40 min, and the drug itself caused a slow increase in [Ca2+]c that was followed by a decline to below resting values after approximately 2 h (2+ waves were not inhibited by suramin (100 µM) or apyrase (4 U/ml), which suggests that Panx1 and regenerative ATP release are unlikely contributors to wave progression  of neighboring cells  increased the bandwidth of concentration sensitivity and enabled collective endothelial responses to both low- and high-intensity stimuli. Cells with comparable sensitivity clustered in restricted spatial domains. This clustering acted as a noise rejection system by acting as a “coincidence detector.” Simultaneously activating several cells in the clusters triggered Ca2+ signals that transmitted to neighboring cells in a manner that scaled with stimulus intensity  are reconciled and achieved is not well understood. Our results from large areas (∼200 cells) of endothelium in intact, pressurized arteries show the endothelium achieves high sensitivity, robustness to noise, and wide concentration range detection by integration of populationwide heterogeneous responses. Individual cells contribute only a small aspect of the overall features of detection. Each cell responded to chemical activation (ACh) with concentration-dependent increases in Ca2+ transients in the population  or only low resolution to changes in concentration may be available. Another possibility is that graded responses within each cell may scale with agonist concentration to provide the concentration-dependent response . The endothelium combines elements of each proposal. Although each cell responds with an analog concentration-dependent response, the sensitivity difference in various cells overcomes the limited single-cell bandwidth to provide a detection capability at low and high agonist concentration. Differences in agonist-sensitivity in cells also generates a concentration dependence to the number of active cells and, as a result, provide a significant number of binary or digital (discrete) combinations that may contribute to the overall response.In other cell types, several proposals have been made to explain high sensitivity to low agonist concentration and maintained sensitivity and responsiveness at high agonist concentrations. Osteoclasts use several different purinergic receptor subtypes, each with various affinities, to provide responses to ATP over a concentration range from 1 nM to 1 mM . Howeverpulation  scale wipulation  . The question arises, why are spontaneous events not transmitted when activated Ca2+ rises are not? One possibility is that the wave does not reflect cell–cell communication but rather sequential activation of cell with various ACh sensitivity. Indeed, after the initial wave on ACh application, the coordinated wavelike behavior is lost rapidly and the links between cells decoupled giving rise to asynchronous Ca2+ changes in various cells . Asynchronous Ca2+ oscillations and Ca2+ waves have been previously seen in endothelial tubes and sheets  signals to regulate several vascular activities including smooth muscle contraction and proliferation. The sensing systems for extracellular activators are likely to vary through the vascular system  and between larger arteries and capillaries and with mechanical stimuli like pulsatile pressure. The results presented in intact arteries at a constant pressure suggest the endothelium detects extracellular activators by acting as a distributed sensing system that is organized into separate clusters of functionally coupled detector cells (macrodomains). The macrodomains act as linked relay elements of a communicating network, and excitation patterns are connected to the stimulus concentration. Although each endothelial cell is a detector, cells are set with a fixed and limited sensitivity to activators. The overall response is derived from the merged responses of many different cells with various agonist sensitivities to achieve a consensus on agonist concentration. Cells are able to detect small fluctuations in activator concentration around basal levels because cells of comparable sensitivity are clustered to provide a coincidence detection system. The extent of signal propagation depends on how many neighboring cells are activated simultaneously. After activation, propagation of signals provides a means to communicate sensed information across physical scales relevant to the artery via IP3 or Ca2+ waves. The propagating intercellular waves show complex progression patterns and appear chaotic initially because of the multiple directions of progression and annihilation when waves collide. Yet the pattern is repeatable on successive applications of ACh, which suggests an encoded signal. The complex interaction of bioactive molecules with spatial domains and cell–cell communication suggests that electrically nonexcitable endothelial cells function together as an artery-lining macroscopic network that enables scalability in highly sensitive responses.The wide control the endothelium exerts over cardiovascular structure and function requires endothelial cells to sense stimuli effectively, to have a wide sensitivity range to activators, and to communicate efficiently both with itself and other cell types. The endothelium is exposed to hundreds of chemical modulators and detects autocrine and paracrine signals with exquisite sensitivity. These extracellular activators are transduced to intracellular (Ca


Maintenance of Ca2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca2+ levels is a well-established indicator of cell stress.The quality of the oocyte and maintenance of this quality during n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca2+ levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide ), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP.Supernumerary human oocytes  age 34.1 ± 0.6 years, 2+ increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU  to 91.8 ± 14.0 AU  after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca2+ . Glibenclamide (100 µM) also inhibited the increase in Ca2+  induced an increase in intracellular Ca2+ that was inhibited by glibenclamide  but not by pinacidil .Intracellular levels of Ca2+ in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca2+ homeostasis and, furthermore, the altered Ca2+ homeostasis might have been associated solely with the protracted incubation.Owing to clinical and ethical considerations, it was not possible to monitor Cain vitro conditions is associated with intracellular increase in Ca2+, which can be counteracted by drugs targeting KATP channels. As Ca2+ homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that KATP channel openers and blockers should be tested as drugs for improving success rates of ART.These results show that maintenance of oocytes under University of Dundee, MRC , NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest. Approximately 1 in 7 couples of reproductive age are classed as infertile, equating to ∼72 million people globally. The primary treatment is assisted reproductive technology (ART) consisting of IVF and ICSI. However, despite the rapid developments in ART over the last decade, current success rates remain both sub-optimal and variable. For example, in the European Union the average IVF pregnancy rate is 29.2% per aspiration—ranging from 21.5 to 48.1% .in vitro processing in the ART laboratory is of critical importance to successful embryo development and a healthy live birth  and in vitro ageing of metaphase II (MII) eggs in mice induces impairment of calcium homeostasis and poor subsequent embryo development   channels are K+-selective channels gated by intracellular ATP. As such, they are suggested to be a link between intracellular metabolic conditions and cellular membrane excitability. In tissues where they are expressed, KATP channels play a crucial physiological role, including a role in insulin secretion, appetite control, smooth muscle tone and others  Control (in the absence of any compound expected to influence intracellular Ca2+), (ii) Dimethyl sulfoxide ), (iii) Pinacidil , (iv) Glibenclamide , (v) DNP , (vi) Pinacidil and DNP (both at 100 µM) and (vii) Glibenclamide and DNP (both at 100 µMs). Unless indicated differently, cells in each experimental group were monitored continuously for 2 h. In a separate series of experiments, oocytes were loaded with Fluo-3 at 37°C. These oocytes were also used to make comparison between those exposed to IVF or ICSI. Pinacidil, glibenclamide, DNP and DMSO were purchased from Sigma-Aldrich, Gillingham, UK. There was no statistically significant difference in basal levels of intracellular Ca2+ between experimental groups  oocytes. Oocytes were collected from 41 healthy women (average age 34.1. ± 0.6 years) undergoing assisted reproduction treatment at Ninewells Assisted Conception Unit, Dundee, UK [HFEA centre # 0004]. The main reasons for infertility in donors were unexplained (38%) and male factor (29%), while only 3% had endometriosis, a condition that could potentially affect Ca2+-sensitive fluorescent probe, Fluo-3AM . Afterwards, cells were superfused with collection solution  with and without compounds targeting KATP channels (described in previous section) and imaged using laser confocal microscopy coupled to an inverted microscope  with a ×10  oil-immersion objective lens at 37°C. The intensity of fluorescence of whole oocytes on the equatorial plane was measured. Microscope was calibrated by green calibration slide before each experiment. Intensity of fluorescence was described in arbitrary units (AU) covering a range from 0 to 60 000 AU. Ca2+ levels and cell morphology were imaged every 10 min for 2 h using an Argon/UV laser (excitation 488 nm/emission 520 nm). Images were analysed using Leica Application Suite AF Lite software (Leica). The parameters of image acquisition were similar for all examined cells. All compounds were purchased from Sigma-Aldrich. All obtained results were normalized in respect to the intensity of fluorescence at time point 0, that was considered to be 100%.MII oocytes for all experimental groups, apart from controls loaded at 37°C, were loaded (for 60 min) at room temperature with the esterified form of the Cat-test, where appropriate. P < 0.05 was considered statistically significant. Statistical analysis was performed using SigmaPlot 12 .Data are presented as mean ± SEM. Mean values were compared using analysis of variance (ANOVA), ANOVA on ranks and the paired or unpaired 2+ levels were captured every 10 min for 2 h. The increase in intensity of fluorescence reached statistical significance after 30 min , n = 19, P = 0.007; Fig. n = 19; P < 0.001 compared with time 0; Fig. 2+ under in vitro conditions , no statistically significant changes in intracellular Ca2+ dynamics in human oocytes when exposed to severe metabolic stress. Intracellular Ca2+ was significantly increased in the presence of 100 mM DNP , DNP (100 mM) did not increase the intracellular level of Ca2+ Fig. . On the 2+ does occur in human oocytes when exposed to in vitro conditions and that this can be reduced and/or prevented by drugs targeting KATP channels.This study demonstrates that an increase in intracellular Cain vitro. Although in handling and manipulation of the oocytes in the laboratory, we aim to minimize external stresses, data from work on oocytes of experimental animals and some other cell types indicate that cellular stress could occur  after collection, so we do not have information about Ca2+ dynamics prior to this period.IVF and ICSI are standard interventions aimed to treat infertility. In order to carry out these techniques, it is necessary to remove an oocyte from its natural environment (the follicle) and prepare it for further procedures ld occur . Due to 2+ during monitoring of MII human oocytes for 2 h in in vitro conditions. This is the first report to suggest that Ca2+ homeostasis is challenged when oocytes are incubated under routine in vitro conditions, although this might be associated solely with protracted incubation. During incubation of oocytes, we have maintained conditions similar to physiological ones. The only exception was loading of cells with Ca2+-sensitive dye at room temperature. It has been previously reported that drops in temperature cause depolymerization of microtubules with increased risk of segregation errors and aneuploidy. Depolymerization of microtubules is influenced by Ca2+  DNP has no a major effect on human oocytes or (ii) that human oocytes had passed a threshold of in vitro stress. In pig oocytes, exposure to in vitro conditions alone does not affect intracellular levels of Ca2+, while exposure to DNP significantly increases intracellular Ca2+ (unpublished data) suggesting that DNP induces significant stress in mammalian oocytes , NHS Tayside, Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. Funding to pay the Open Access publication charges for this article was provided by MRC.None declared.