N-Methyl-d-aspartate receptors (NMDARs) are Ca2+-permeable glutamate receptors that play a critical role in synaptic plasticity and promoting cell survival. However, overactive NMDARs can trigger cell death signalling pathways and have been implicated in substantia nigra pars compacta (SNc) pathology in Parkinson's disease. Calcium ion influx through NMDARs recruits Ca2+-dependent proteins that can regulate NMDAR activity. The surface density of NMDARs can also be regulated dynamically in response to receptor activity via Ca2+-independent mechanisms. We have investigated the activity-dependent regulation of NMDARs in SNc dopaminergic neurones. Repeated whole-cell agonist applications resulted in a decline in the amplitude of NMDAR currents (current run-down) that was use dependent and not readily reversible. Run-down was reduced by increasing intracellular Ca2+ buffering or by reducing Ca2+ influx but did not appear to be mediated by the same regulatory proteins that cause Ca2+-dependent run-down in hippocampal neurones. The NMDAR current run-down may be mediated in part by a Ca2+-independent mechanism, because intracellular dialysis with a dynamin-inhibitory peptide reduced run-down, suggesting a role for clathrin-mediated endocytosis in the regulation of the surface density of receptors. Synaptic NMDARs were also subject to current run-down during repeated low-frequency synaptic stimulation in a Ca2+-dependent but dynamin-independent manner. Thus, we report, for the first time, regulation of NMDARs in SNc dopaminergic neurones by changes in intracellular Ca2+ at both synaptic and extrasynaptic sites and provide evidence for activity-dependent changes in receptor trafficking. These mechanisms may contribute to intracellular Ca2+ homeostasis in dopaminergic neurones by limiting Ca2+ influx through the NMDAR. Overactivity of NMDARs and resultant elevated intracellular [Ca2+] can lead to neuronal excitotoxicity : sucrose, 100; NaCl, 62.5; KCl, 2.5; CaCl2, 1; MgCl2, 4; NaHCO3, 25; NaH2PO4, 1.25; 0.1 mM kynurenic acid and glucose, 25; of pH 7.4 when saturated with 95% O2 and 5% CO2. Coronal or horizontal brain slices (300 μm thick) were prepared using a vibrating microslicer . Slices containing the midbrain substantia nigra region were kept in oxygenated solution containing (mm): NaCl, 125; KCl, 2.5; CaCl2, 1; MgCl2, 4; NaHCO3, 25; NaH2PO4, 1.25; and glucose, 25 for 1–6 h before use.Sprague–Dawley or Wistar rats  were deeply anaesthetized with isofluorane or halothane and/or decapitated in accordance with the UK Animals (Scientific Procedures) Act 1986 and Local Ethical Committee approval. One hundred and fifteen rats were used in this study. The brain was removed into ice-cold oxygenated slicing solution containing (m2 and with bicuculline (10 μm), strychnine (10 μm) and tetrodotoxin  at room temperature (22–24°C). Synaptic currents were recorded from horizontal slices in 6,7-dinitro-quinoxaline-2,3-dione , picrotoxin (50 μm) and with 0.1 or 1.3 mm MgCl2 at 30 ± 2°C. Patch pipettes were made from borosilicate glass to a final resistance of 2–6 MΩ when filled with solution containing (mm): CsCl, 140; Hepes, 10; NaCl, 10; CaCl2, 0.5; MgCl2, 0.5–1.0; and CsOH, 29.53; pH 7.2, with Na2ATP, 2; NaGTP, 0.3 ; and Ca2+ chelators (EGTA or BAPTA) or other additions as specified in the text. Free Ca2+ was calculated using WinMAXC32.exe (http://maxchelator.stanford.edu/downloads.htm).Slices were placed in a recording chamber on the stage of an upright microscope with Nomarski differential interference contrast optics. During whole-cell current recordings, coronal slices were continuously bathed in the same solution used for storing the slices, without added MgClIh) of more than 50 pA in response to a voltage step from −60 to −110 mV , identified based on the presence of a time-dependent, hyperpolarization-activated inward current  and glycine  by switching to agonist solution for 15 s using a solenoid valve system  to regulate solution flow to a theta-glass applicator placed ∼1 mm above the surface of the slice. These responses had 10–90% rise times of 3–5 s , filtered at 2 kHz and digitized at 10 kHz using a Micro 1401 interface . Cell membrane capacitance and series resistance were estimated using the series resistance compensation circuit of the Axopatch 200B (series resistance was compensated by 40–80%) and checked throughout the experiment for stability. Whole-cell NMDA currents were evoked by adding NMDA (200 μhttp://spider.science.strath.ac.uk/PhysPharm/showPage.php?pageName=software_ses). Synaptic currents were acquired and analysed using Spike2 (version 4) software . To quantify run-down in responses to consecutive agonist applications in the different conditions tested, the decrease in peak INMDA between the first and nth NMDA application was measured. To quantify run-down of synaptic NMDAR responses over comparable time scales to agonist-evoked responses, nine responses to synaptic stimulation (given every 10 s) were averaged (e.g. ‘time 0 s’ is the average of nine responses from 0 to 90 s) and the decrease in mean peak NMDA-EPSC amplitude between the first and fifth time point at 400 s was measured. Data are expressed as means ± SEM; the ‘n’ values refer to the number of slices. For all data sets, normality was tested using the Kolmogorov–Smirnoff test (GraphPad Prism version 4). For comparisons of two groups of data, Student's t test was used (with Welch's correction if variance between the groups was different) and for more than two groups of data ANOVA followed by Bonferroni multiple comparisons test was used.Whole-cell NMDA currents were acquired and analysed using WinEDR version 2.5.9 software  was applied repeatedly every 100 s. As illustrated in Fig. 1A, in dopaminergic neurones voltage clamped to −60 mV and with 10 mm BAPTA in the pipette solution, following an initial application of NMDA, there was a subsequent decline in peak current with each successive application, which we define here as current ‘run-down’. The first response to NMDA (tI0) had an average amplitude of −4334 ± 296 pA, while the fifth response (tI400) was significantly less and averaged −2558 ± 235 pA . The tI400/tI0 ratio, used to quantify the extent of run-down, was 0.59 ± 0.03 . To determine whether the observed run-down was activity dependent, in a separate group of neurones NMDA was applied every 400 s . In these cells, tI400 was not significantly different from tI0 , with a ratio of 1.17 ± 0.09. Significantly more run-down was seen at tI400 when NMDA was applied every 100 s than when applied every 400 s , suggesting that repeated receptor activation or current through the receptor channel leads to run-down.The NMDAR-mediated whole-cell responses show run-down with repeated applications of NMDA to SNc dopaminergic neurones (Suárez F and G) to determine whether recovery from run-down would occur. There was a small but significant decrease in current amplitude at 700 s (−2721 ± 491 pA) compared with 400 s , indicating that the run-down persists and is not readily reversible in these whole-cell recording conditions.Modifications to NMDARs that may mediate current run-down in dopaminergic neurones, such as changes in receptor phosphorylation or association of regulatory proteins, can be transient; therefore, we tested the ability of NMDARs in dopaminergic neurones to recover from run-down. Following five repeated applications of NMDA every 100 s, a pause of 300 s was allowed  was compared with a low concentration of slow Ca2+ buffer . Changing the Ca2+ chelator in the intracellular solution from 10 mm BAPTA  to 0.6 mm EGTA  had no effect on the initial current amplitude  but resulted in more run-down (tI400/tI0) in dopaminergic neurones voltage clamped to −60 mV .Calcium-dependent run-down is a well-established form of NMDAR regulation . Interestingly, run-down was not significantly different between BAPTA  and EGTA , suggesting that run-down of the non-GluN2B component is not Ca2+ dependent.To determine whether this Ca2+ that could induce current run-down are that which has fluxed through the channel pore or that which has been released from intracellular stores (e.g. by Ca2+-induced Ca2+ release). Extracellular Ca2+ was replaced with equimolar Ba2+ . With 10 mm BAPTA in the pipette, following substitution of extracellular Ca2+ with Ba2+ there was still significant run-down  but significantly less than that seen with 1 mm extracellular Ca2+ .Potential sources of Ca2+ moving into the cell was reduced by voltage clamping the neurone at +40 mV. At +40 mV (with 10 mm BAPTA in the pipette solution), there was no significant change in peak current between the first and fifth response  and the tI400/tI0 ratio  was significantly different from that seen at −60 mV . Thus, the amount of current through active NMDARs in dopaminergic neurones is regulated by intracellular Ca2+ and is sensitive to the amount of Ca2+ influx through the NMDAR channel.The driving force for Ca2+ can also affect the amplitude of NMDA EPSCs over time, because synaptic NMDARs are also regulated in a Ca2+-dependent manner  had an average amplitude of 50.2 ± 7.8 pA (10 mm BAPTA) or 59.6 ± 10.3 pA (0.6 mm EGTA). The NMDA EPSCs showed a small but significant degree of run-down at +40 mV . Run-down was more marked at −50 mV . Run-down of NMDA EPSCs was not significantly different from run-down of whole-cell responses to NMDA at +40 mV  or −60 mV  with 10 mm BAPTA in the pipette. However, synaptic NMDARs showed no sensitivity to intracellular Ca2+ buffering; at −50 mV, with 0.6 mm EGTA, initial NMDA-EPSC amplitude was −54.2 ± 7.1 pA and tI400/tI0 was 0.67 ± 0.08 , not significantly different from tI400/tI0 for 10 mm BAPTA at −50 mV .Changes in intracellular Cam; minimal incubation time, 30 min). Run-down was not significantly different following incubation in the presence of ifenprodil .To determine whether NMDA-EPSC run-down at −50 mV was subunit dependent, the experiments were repeated following incubation with the GluN2B-preferring antagonist, ifenprodil  with that in GABAergic neurones  with the two levels of intracellular Ca2+ buffering. There was no significant difference in the initial amplitudes in GABAergic neurones compared with dopaminergic neurones with either BAPTA or EGTA . However, there was significantly more whole-cell NMDAR current run-down in GABAergic neurones  with BAPTA , but not with EGTA in the intracellular solution, compared with dopaminergic neurones (ANOVA with Bonferroni multiple comparisons test). Run-down of synaptic NMDARs in response to glutamate release at −50 mV was not significantly different between the two neuronal populations with either chelator (ANOVA with Bonferroni multiple comparisons test). Run-down of whole-cell and synaptic NMDARs in dopaminergic versus GABAergic neurones is summarized in Fig. 4C.Dopaminergic neurones of the substantia nigra are more vulnerable to cell death than neighbouring GABAergic neurones in Parkinson's disease  binding to the GluN1 subunit C-terminal domain can induce Ca2+-dependent inactivation of NMDARs , or a control peptide with several amino acid substitutions  included in the pipette solution. With 0.6 mm EGTA in the pipette solution (to optimize activation of Ca2+–CaM), there was no difference in the extent of whole-cell run-down between inhibitory  and control peptide . To ensure that slow dialysis was not preventing the peptide from reaching its target, the experiments were repeated but with a 20 min wait in whole-cell recording mode prior to the first NMDA application. Again, no significant differences were observed (Table S1).It is well established in hippocampal neurones that Ca2+-dependent protein phosphatase that regulates NMDAR function  in the pipette solution with 0.6 mm EGTA had no significant effect on current run-down when compared with 0.6 mm EGTA alone (Table S1).Calcineurin is a Cam). Phalloidin was unable to reduce run-down (Table S1). These results do not support a role for CaM, calcineurin or actin depolymerization in mediating Ca2+-dependent run-down in substantia nigra dopaminergic neurones and suggest that different Ca2+-dependent NMDAR regulatory mechanisms operate compared with those reported in hippocampal neurones.The NMDA current run-down in hippocampal neurones in culture involves actin depolymerization . To test whether this run-down might be dynamin dependent, a dynamin-inhibitory peptide  was included in the intracellular solution, along with 10 mm BAPTA. Recordings of whole-cell NMDAR currents at +40 mV using a control peptide  showed significant run-down , but no significant run-down was observed when using dynamin-inhibitory peptide . A significant difference in run-down was observed between control and dynamin peptides .In hippocampal neurones, agonist binding can lead to activity-dependent changes in NMDAR trafficking, whereby receptors are internalized . Although intracellular calcium dynamics are likely to be different between whole-cell and synaptic receptor activation protocols, these results suggest that during localized activation, synaptic NMDARs undergo less internalization by clathrin-mediated (dynamin-dependent) endocytosis than their extrasynaptic counterparts.The NMDARs are internalized via clathrin-coated pits, which in hippocampal neurones are located peri-or extrasynaptically . However, when no agonist was applied between the initial and test responses in the same conditions  the tI400/tI0 was significantly greater , suggesting that agonist binding might trigger some run-down independently of ionic flux.Agonist binding can lead to modulation of the receptor response even in the absence of ion flux through the channel  between receptor activation at tI0 and tI400 was used as a priming conditioning stimulus in an attempt to promote a more robust internalization of NMDARs in dopaminergic neurones . As a control, the glutamate binding site competitive NMDAR antagonist AP5 (30 μm) was applied instead of glycine . When glycine was used as the conditioning stimulus, the tI400/tI0 ratio  was not significantly different from that seen in the presence of AP5 . These results suggest that glycine priming was unable to cause an immediate decline in INMDA upon NMDA application, a finding that is in agreement with the results of Nong et al.  or glycine as the conditioning stimulus . The tI800/tI400 was 0.49 ± 0.09 with control (AP5) and 0.48 ± 0.05 with glycine . These results indicate that, unlike the situation in hippocampal neurones  than with 10 mm BAPTA . EGTA and BAPTA have a similar binding affinity for Ca2+, but markedly different binding kinetics, with BAPTA being able to sequester Ca2+ in the region of two orders of magnitude faster than EGTA  NMDAR inactivation (defined as the decline in peak current observed during a single application of agonist) in hippocampal neurones and in transfected HEK 293 cells .The persistence of a small degree of whole-cell current run-down in conditions where Caet al. et al. et al. et al. et al. et al. et al. Activity-dependent changes in NMDAR trafficking in SNc dopaminergic neurones were further substantiated by the reduction in current run-down during intracellular dialysis with a dynamin-inhibitory peptide, suggesting that run-down is in part due to removal of NMDARs from the neuronal membrane. This is consistent with previous findings that GluN2B-containing receptors are internalized by clathrin-mediated endocytosis , but run-down was significantly reduced by clamping neurones to +40 mV, which reduces the driving force for Ca2+ influx. The lack of sensitivity of synaptic NMDAR current run-down to the Ca2+ chelators, BAPTA versus EGTA, in contrast to whole-cell NMDAR current run-down, may be due to the mode of NMDAR activation (rapid and brief versus slower and more prolonged) or due to a much closer coupling of the Ca2+-dependent mechanism of run-down to synaptic NMDARs, such that even the fast chelator BAPTA is not able to sequester the rapid Ca2+ influx effectively through the synaptic NMDARs.In the data presented here, NMDA synaptic current run-down was observed in response to repeated low-frequency synaptic stimulation (0.1 Hz) in neurones voltage clamped to −50 mV Fig. . This waet al. et al. et al. et al. et al. et al. et al. et al. In cultured hippocampal neurons, trafficking of synaptic and extrasynaptic NMDARs is differentially regulated, with synaptic receptors being less sensitive to internalization than their extrasynaptic counterparts  may contribute to substantia nigra pars compacta (SNc) dopaminergic neurone dysfunction in Parkinson's disease.Calcium ion influx through 2+ influx and partly dependent on clathrin-mediated endocytosis.Responses of NMDARs in dopaminergic neurones showed use-dependent run-down that was not readily reversible and was partly dependent on Ca2+ at both synaptic and extrasynaptic sites and provide evidence for activity-dependent changes in receptor trafficking.Thus, we report regulation of NMDARs in SNc dopaminergic neurones by intracellular Ca


Urocortins are human homologues of urotensin I, a fish corticotropin-releasing-factor- (CRF-) like peptide secreted from the urophysis. There are three urocortins: urocortin 1, urocortin 2, and urocortin 3 in mammals. We have shown that urocortin 1 and urocortin 3 are endogenously synthesized in the myocardial cells of human heart and may act on CRF type 2 receptor (CRFR2) expressed in the heart. Expression levels of urocortin 1 in the heart and plasma urocortin 1 levels are elevated in patients with heart failure. Recent studies have shown that urocortins have various biological actions in the cardiovascular system, such as a vasodilator action, a positive inotropic action, a cardioprotective action against ischemia/reperfusion injury, and suppressive actions against the renin angiotensin system and the sympathetic nervous system. Urocortins and CRFR2 may therefore be a potential therapeutic target for cardiovascular diseases, such as congestive heart failure, hypertension, and myocardial infarction. Actions of the corticotropin-releasing factor (CRF) are mediated by two types of G-protein coupled receptors: CRF type 1 receptor (CRFR1) . CRFR1 m Urotensins are peptide hormones secreted from fish urophysis, the neuroendocrine organ located in the caudal spinal cord , 13. Uro Furthermore, Ucn 2  and Ucn 3  were discovered by searching the public genome databases and shown to be specific agonists for CRFR2 –17. Ucn  Thus, the CRF family consists of CRF, Ucn 1, Ucn 2 (SRP), and Ucn 3 (SCP) as well as fish urotensin I and frog sauvagine. Urocortins (Ucns) were shown to be expressed in various tissues and cells of the human, such as brain, pituitary, gastrointestinal tract, ovary, placenta, synovial tissue, lymphocytes, and skin –28. We have shown expression of Ucn 1, Ucn 3, and CRF receptors in the human heart obtained at autopsy , 30.α mRNA was expressed in four chambers of all cases studied. A weak band for CRFR1 mRNA was detected in the left atria of patients 2 and 3, in the left ventricles of patients 2 and 4, and in the right ventricle of patient 4. Although CRFR1 may be present in the heart, CRFR1 mRNA was detected in the heart of limited cases due to the low expression levels in most cases. CRFR2β mRNA expression was observed in the left atrium in all cases and in the right atrium of only one out of four cases studied (patient 2). Thus, the major CRF receptor subtype expressed in all four chambers of the human heart was CRFR2α. Moreover, RT-PCR analysis showed expression of Ucn 3 mRNA in the cerebral cortex, hypothalamus, pituitary, ventricles and atria of heart, and kidney  PCR analysis demonstrated that Ucn 1 mRNA was expressed in the right atrium, right ventricle, left atrium, and left ventricle in all cases studied  29]. Ho. Ho29]. d kidney  30]. RN. RNα mRN Immunocytochemistry showed that Ucn 1 and CRF receptor immunoreactivities were detected in the great majority of myocardia of human heart . Positiv Specific radioimmunoassay for Ucn 1 and Ucn 3 could detect respective immunoreactivity in the human heart. The antisera against Ucn 1 and Ucn 3 were raised in rabbits , 30. The The highest concentrations of immunoreactive- (IR-) Ucn 1 were found in the left ventricle , followed by the right ventricle (1.26 ± 0.3 pmol/g wet weight), the left atrium (1.21 ± 0.2 pmol/g wet weight), and the right atrium (0.92 ± 0.1 pmol/g wet weight) . IR-Ucn  Ucn 1 mRNA expression was shown in cultured rat cardiac myocytes and nonmyocytes . IR-Ucn 125I] antisauvagine 30 showed the presence of high affinity of CRFR2 receptor in human left ventricle with a Kd of 0.21 ± 0.03 nM. IR-Ucn 1 and IR-Ucn 3 were shown to be present in human plasma  , 36, but By contrast, the predicted amino acid sequence of human Ucn 2 (SRP) precursor lacks a consensus proteolytic cleavage site that would allow for C-terminal processing of the peptide . It has  Expression of Ucn 1 was shown to be elevated in the heart tissues obtained from patients with heart diseases , 38. Nis Furthermore, plasma levels of Ucn 1 are elevated in patients with chronic heart failure –41. Ng eβ mRNA expression was remarkably depressed in the left ventricle of DOCA-salt spontaneously hypertensive rats (SHRs) compared with control Wistar-Kyoto (WKY) rats, whereas Ucn 1 mRNA expression was elevated in DOCA-salt SHR [β mRNA expression in the heart was decreased by lipopolysaccharide, corticosterone, and physical restraint in rats [β mRNA expression in A7R5 aortic smooth muscle cells was decreased by inflammatory cytokines, interleukin- (IL-) 1β, IL-6, and tumor necrosis factor α (TNF-α) [α and TNF-α downregulated CRFR2 expression in the mouse heart whereas Ucn 1 decreased CRFR2 expression in cultured cardiomyocytes [ Elevated plasma Ucn 1 in heart failure patients may be mainly derived from diseased heart. However, the plasma levels may be too low to exert biological actions on myocardium or vascular tissues as mentioned in the previous section. Ucn 1 in the diseased heart may therefore act locally on the myocardium as an autocrine or paracrine factor and affect the myocardial function in heart failure. CRFR2salt SHR . CRFR2β  in rats . Moreove (TNF-α) . Coste emyocytes . They spα, and oxidative stress [α, IL-1β, and IL-6 are elevated in plasma, circulating leukocytes, and tissues of failing myocardium in patients with chronic heart failure. Expression of Ucn 1 mRNA was also increased after heat shock at 42°C in cultured rat cardiac myocytes [ In addition to the cardiovascular and endocrine systems and endocrine tumors , 44–47, e stress . Inflamme stress . Levels myocytes . Ucn 1 e Hypoxia induces expression of genes for various vasoactive peptides such as endothelin-1  and adre There have been no reports on altered expression of Ucn 2 or Ucn 3 in diseased heart. The relation of Ucn 3 with inflammation may not be so marked as that of Ucn 1. In the colon mucosa, Ucn 3 was expressed in myenteric and submucosal nervous plexus, blood vessels, smooth muscle layers, and enterochromaffin cells but hardly detected lamina propria inflammatory cells in which Ucn 1 was abundantly expressed . MoreoveCRFR2 is abundantly expressed in the vasculature and all Ucns exert potent vasodilator actions , 60–62. ATP channel (ATP-sensitive inwardly rectifying potassium channel Kir 6.1) in cardiac myocytes [ATP channel is believed to be involved in the protective effects. Townsend et al. showed that Ucn 1 protected heart with reperfusion injury by inhibiting the mitochondrial permeability transition pore opening, possibly via a protein kinase C-mediated reduction in oxidative stress [All three Ucns prevented cardiomyocytes from ischemia/hypoxia-induced cell death and apoptosis via a MARK-dependent pathway , 63. Ucnmyocytes . The mite stress .Barry et al. have recently reported that infusion of Ucn 1 or Ucn 2 before the onset of reperfusion resulted in the differential regulation of 66 and 141 genes, respectively, the majority of which have not been described previously . FunctioPre- and postconditioning are powerful endogenous adaptive response of the organism whereby different stimuli enhance the tolerance against various types of stress. Cserepes et al. showed that preconditioning with Ucn 1 induced a powerful cell protective effect, which was comparable with that of adenosine and ischemia, using isolated neonatal rat ventricular myocytes subjected to ischemia and reperfusion . Furtherμg Ucn 2 intravenously over 1 h) induced increases in cardiac output and left ventricular ejection fraction with falls in systemic vascular resistance and cardiac work in eight male patients with heart failure  [ The research group of Dr. Richards in New Zealand performed a series of experiments on cardiovascular effects of Ucns in sheep as well as in human subjects –80. Admi2+/calmodulin-dependent protein kinase II-dependent manner. However, this enhancement was accompanied by Ca2+-dependent arrhythmogenic effects mediated by protein kinase A and calmodulin-dependent protein kinase II [Effects of Ucn 2 were also shown in mice and rats with heart failure. Bale et al. showed that Ucn 2 treatment augmented heart rate, exhibited potent inotropic and lusitropic actions on the left ventricle, and induced a downward shift of the diastolic pressure-volume relation in mice. Ucn 2 treatment also reduced systemic arterial pressure associated with a lowering of systemic vascular resistance . These einase II . By continase II . In addition to heart failure, Ucn 2 has been shown to be effective for hypertension. Dieteterle et al. studied effects of acute and chronic administration of Ucn 2 in hypertensive salt-sensitive Dahl rats and showed that Ucn 2 had immediate and sustained blood pressure-lowering effects without affecting heart rate . Ucn 2 a Vasoconstrictor peptides, such as angiotensin II, endothelin-1, and urotensin II, promote the progression of cardiovascular and renal diseases and their complications –87. Angi Ucns are cardiovascular peptides which are endogenously synthesized in the heart and vasculature and may play protective roles against various types of cardiovascular stress, such as hypoxia. Ucns and Ucn-related drugs may therefore have therapeutic potential for the treatment of cardiovascular diseases, such as congestive heart failure, hypertension, and myocardial infarction. Further studies are required to clarify whether Ucns have any merits as drugs for cardiovascular diseases over other drugs for cardiovascular diseases, including ANP, and which Ucn is the most effective for this purpose among three Ucns.


The cell cycle was analyzed by flow cytometry. For probing the mechanisms, the content of NO in supernatant and cGMP level in VSMCs was measured by nitric oxide kit and cGMP radio-immunity kit, respectively; the expressions of protooncogene c-fos and endothelial NO synthase (eNOS) mRNA in the VSMCs were detected by real-time RT-PCR; the intracellular free calcium concentration ([Ca2+]i) was detected with Fura-2/AM-loaded VSMCs. Comparing with that in normal group, Rg1 180 mg·L−1 did not change the absorbance of MTT and cell percent of G0/G1, G2/M, and S phase in normal cells (P > 0.05). Contrarily, PDGF-BB could increase the absorbance of MTT (P < 0.01) and the percent of the S phase cells but decrease the G0/G1 phase cell percent in the cell cycle, accompanied with an upregulating c-fos mRNA expression (P < 0.01), which was reversed by additions of Rg1. Rg1 administration could also significantly increase the NO content in supernatant and the cGMP level in VSMCs, as well as the eNOS mRNA expression in the cells, in comparison of that in the group treated with PDGF-BB alone (P < 0.01). Furthermore, Rg1 caused a further increase in the elevated [Ca2+]i induced by PDGF-BB. It was concluded that Rg1 could inhibit the VSMC proliferation induced by PDGF-BB through restricting the G0/G1 phase to S-phase progression in cell cycle. The mechanisms may be related to the upregulation of eNOS mRNA and the increase of the formation of NO and cGMP.Ginsenoside Rg1 (Rg1) has been reported to suppress the proliferation of vascular smooth muscle cells (VSMCs). This study aimed to observe the role of nitric oxide (NO) in Rg1-antiproliferative effect. VSMCs from the thoracic aorta of SD rats were cultured by tissue explant method, and the effect of Rg1 (20 mg·L The [Ca2+]i was calculated by the formula described before [2+)]i = 224 × [(R − Rmin)/(Rmax − R)] × FD/FS, where FD and FS are the fluorescence proportionality coefficients obtained at 380 nm under Rmin and Rmax conditions, respectively. The number 224 is the KD value of Fura-2/AM. To observe the possible influence of Rg1 on the elevated [Ca2+)]i induced by PDGF-BB, Rg1 was added before the addition of this growth factor, and the fluorescence intensity was detected at 5 min after PDGF-BB addition.The fluorescence value from 1 mL cell suspension was measured by a RF-5000 dual-wavelength spectrofluorometer  with excitation wavelengths at 340/380 nm and emission wavelength at 480 nm. The ratio (d before  [Ca2+)]it-test (two-tails) with the SPSS 13.0 software , and significance was set at P < 0.05.All data are presented as mean ± S.E.M. and were analyzed by the one-way ANOVA followed by Student's −1 did not change the MTT absorbance, while the addition of PDGF-BB could significantly increase the absorbance in MTT assay (P < 0.01), which tended to be inhibited by an addition of Rg1 20 mg·L−1 (P > 0.05), and was remarkably inhibited by Rg1 60 mg·L−1and 180 mg·L−1 (P < 0.05 and P < 0.01), suggesting the antiproliferating effect of Rg1 on VSMCs  administration of Rg1  at 30 min after PDGF-BB addition could markedly decrease the S-phase cell percent and upgrade the G0/G1 phase percent in the cell cycle. The data suggested that Rg1 could inhibit the VSMC proliferation induced by PDGF-BB through restricting the G0/G1-phase to S-phase progression in cell cycle.The results shown in P < 0.01). On the other hand, addition of PDGF-BB decreased the expression of eNOS mRNA by about 67%, and Rg1 could reverse the decreasing eNOS expression in a concentration-dependent manner (P < 0.01)  .P < 0.01), the addition of the low concentration of Rg1 (20 mg·L−1) tended to increase the NO content (P > 0.05), and the higher concentrations of Rg1  could significantly elevate the content of NO (P < 0.05 or P < 0.01). Consistent with the changes of NO content in supernatant, the cGMP level in VSMCs was also decreased by PDGF-BB (P < 0.01) and was markedly increased by additions of all three concentrations of Rg1 (P < 0.05 or P < 0.01) (As shown in  < 0.01) .2+]i levels of VSMCs were detected at 3 minutes after the addition of PDGF-BB or at 33 minutes after Rg1-administration. In our experimental conditions, the basal [Ca2+]i was about 120 nmol/L in normal VSMCs; it tended to increase by Rg1 180 mg·L1− (P > 0.05), and was elevated to 182 nmol/L by the addition of PDGF-BB 25 μg·L−1(P < 0.05). When Rg1 was administered at 30 minutes before PDGF-BB addition, it could cause a further increase in the elevated [Ca2+]i induced by this growth factor in a dose-dependent manner  and sodium nitroprusside, inhibit VSMC proliferation by cGMP-dependent i [2+ influx in the neurons suffered from hypoxic-ischemic injury [2+ within seconds to minutes [2+]i level of VSMCs at 3 minutes after addition of PDGF-BB. Surprisingly, the result showed that in our experimental conditions, Rg1 (administered at 30 minutes before PDGF-BB addition) could cause a further increase in the elevated [Ca2+]i induced by PDGF-BB. Obviously, the increased [Ca2+]i could not be attributed to NO formation, and might be the direct action of Rg1 to Ca2+ influx or Ca2+ release from the endoplasmic reticulum. We conjectured that the NO formation-promoting effect of Rg1 might be not strong enough to reduce the [Ca2+]i level of VSMCs at the Ca2+-detection time(3 minutes after PDGF-BB addition), and the antiproliferative effect of Rg1 via promoting NO-formation perhaps occurred when larger amount of eNOS expression had been restored. On the other hand, some investigators reported that the Ca2+-increasing agents including Ca2+ ionophore A23187, ionomycin, cyclopiazonic acid, and di-tert-butylhydroquinone had little effect on proliferation of VSMCs, and they thought that an increase in [Ca2+]i per se does not appear to be important in VSMC replication i , and Rg1c injury . Because minutes , we detelication . What isIt is concluded that one of the mechanisms for Rg1 inhibition on VSMC proliferation is related to the up-regulation of eNOS mRNA and increases the formation of NO and cGMP.


Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca2+ elevations. We investigated the eugenol Ca2+ signature in further detail and show that exponentially growing cells exhibit Ca2+ elevation resulting exclusively from the influx of Ca2+ across the plasma membrane whereas in stationary growth phase cells Ca2+ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca2+ elevation. Ca2+ channel deletion yeast mutants were used to identify the pathways mediating Ca2+ influx; intracellular Ca2+ release was mediated by the vacuolar Ca2+ channel, Yvc1p, whereas the Ca2+ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca2+ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca2+ elevations. Taken together, these results indicate that a cch1p-mediated Ca2+ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast,  Fungi are emerging as major causes of human infections, particularly amongst a growing population of immunocompromised hosts, which is having significant economic and social impacts 2+ (Ca2+cyt) elevation in the model yeast, Saccharomyces cerevisiae. The authors specifically focussed on carvacrol and showed that this phenolic compound disrupted ion (Ca2+ and H+) homeostasis and induced transcriptional changes indicative of Ca2+ stress, raising the possibility that the antifungal activity of carvacrol depended, at least in part, on a toxic elevation of Ca2+. However, despite these new insights, it remains unclear if the Ca2+cyt elevation induced by these plant phenolic compounds represents an antifungal activity or if it forms part of signalling response to protect against the fungicidal activity.Plant essential oils have been widely documented to possess broad spectrum antifungal properties and are generally recognised as safe for human and animal consumption 2+ homeostasis of S. cerevisiae to gain insights into the mechanisms mediating its antifungal activity. We focussed on the role of the Ca2+ channels, Cch1p, Mid1p and Yvc1p and show that eugenol-induced Ca2+cyt elevations are dependent on Cch1p-mediated Ca2+ influx. Furthermore, in contrast to that proposed for carvacrol, eugenol-induced Ca2+cyt elevations do not appear to serve as a cytotoxic Ca2+ burst but instead are part of a signalling pathway which protects yeast against eugenol stress.In the present study, we monitored aequorin luminescence to investigate the effects of eugenol on Camid1Δ and cch1Δ mutants were derived from the parental Saccharomyces cerevisiae strain JK9-3da  by replacing the MID1 and CCH1 genes by a KanMX cassette yvc1Δ mutant was derived from the parental strain S. cerevisiae strain BY4742  by replacing YVC1 gene by a KanMX cassette (EUROSCARF strain Y11863). Unless otherwise stated, yeast strains were cultured at 30°C in standard synthetic complete media  or, for strains transformed with pEVP11/AEQ, SCM minus the addition of leucine (SCM-leu). All growth media contained 2% (w/v) glucose.Single and double S. cerevisiae strains expressing apoaequorin were grown overnight in SCM-leu in a shaking (150 rpm) incubator to optical density at 600 nm (OD600) of 8 (1×108 cells/ml). OD600 was determined after 1∶8 dilution of culture in water. To obtain cells in mid-logarithmic growth phase, 0.5 ml of stationary phase cells from overnight cultures were sub-cultured into 10 ml of fresh SCM-leu to give an OD600 of 0.8 and incubated at 30°C, shaking at 150 rpm for 4 to 5 hours until OD600 between 2.4 and 3.2 was reached. Cells were pelleted using a microcentrifuge and resuspended in fresh SCM-leu to an OD600 of 8.Yeast strains were transformed with pEVP11/AEQ  as previously described 8 cells/ml) transformed with pEVP11/AEQ were incubated with 0.5 µl of 0.5 mM coelentrazine  in absolute methanol for 20 minutes in order to reconstitute functional aequorin with in the cells. After incubation, the base line luminescence was recorded every 0.2 seconds for 40 seconds (unless otherwise stated) using a digital chemiluminometer . At 40 seconds, 200 µl of eugenol containing media was carefully added using a 1 ml syringe connected to a hypodermic needle. Luminescence (expressed in arbitrary units (AU) per 0.2 seconds) was measured for up to eight minutes after which cells were lysed with 1.6 M CaCl2 in 20% (v/v) ethanol to determine total (summed) luminescence. Comparing total luminescence indicated that aequorin production was similar in the strains used in the present study (data not shown), however, as previously reported by 2+cyt elevations. Samples were treated with coelentrazine sequentially maintaining a constant time of incubation before addition of eugenol. Eugenol (Sigma-Aldrich) was in liquid form (density 1.06 g/ml) and was made to 100× stocks in absolute ethanol and stored at 4°C. Eugenol was added to samples at indicated concentrations (containing 1% ethanol) in either SCM-leu (which contained 2% w/v glucose) or EGTA buffer .Luminometry measurements were conducted as previously described 600 of 8. 0.25 ml of cells were added to 5 ml of SCM (containing varying concentrations of eugenol and 1% ethanol) to a final OD600 of approximately 0.5 (6.6×106 cells/ml). Cells were then incubated at 30°C shaking at 150 rpm. Controls contained 1% ethanol (solvent). At the time points indicated, aliquots were removed and OD600 determined.Growth assays in liquid culture were conducted as follows: yeast strains were cultured overnight to OD600 of 8, centrifuged, washed in 10 ml of sterile water and resuspended to 0.5×108 cells/ml. Following 10-fold serial dilutions of each yeast suspension using sterile water, 5 µl drops were spotted on to SCM media (with varying concentrations of eugenol and 1% ethanol) containing 2% agar. Plates were incubated at 30°C.For drop assays, yeast strains were grown overnight at 30°C in SCM to OD2+cyt elevations in S. cerevisiae. CCH1 and MID1 have been shown to encode subunits of a high affinity Ca2+ channel located in the yeast plasma membrane 2+ influx across the plasma membrane using Ca2+ channel mutant yeast strains transformed with pEVP11/AEQ 2+-sensitive bioluminescent protein aequorin. It has been recently shown that Ca2+ channel activity is dependent on the metabolic state of yeast cells 2+-dependent luminescence was monitored in cells in mid-logarithmic growth phase and in stationary growth phase.Rao et al. 2+-dependent luminescence in exponentially growing JK9-3da cells in response to increasing concentrations of extracellular eugenol. At concentrations greater than 0.4 mg/ml, eugenol induced a biphasic elevation in Ca2+cyt: a rapid transient increase in Ca2+cyt is immediately elicited by eugenol followed by a more sustained increase in Ca2+cyt which lasted several minutes, peaking at approximately 220 seconds. Elevations in Ca2+cyt were difficult to resolve at concentrations less than 0.6 mg/ml, when recording luminescence every 0.2 seconds. However increasing the period over which the luminescence counts were integrated from 0.2 seconds to 2 seconds improved resolution and Ca2+cyt elevations following addition of 0.4 mg/ml eugenol were consistently observed (n = 5) while increases in luminescence following addition of 0.2 mg/ml eugenol were not apparent , thymol (0.5 mg/ml) and eugenol (0.5 mg/ml) however, their recordings were limited to 80 seconds duration and therefore the biphasic nature of the response observed in the present study would not have been detected.apparent  inset. A2+ in the eugenol response, 2+cyt following the application of 0.6 mg/ml eugenol in presence (i.e. SCM which contains approximately 0.1 mM Ca2+) and absence  of extracellular Ca2+. In the absence of extracellular Ca2+ no significant increase in Ca2+cyt was detectable in the yeast strains tested indicating that the eugenol-induced Ca2+cyt elevations resulted exclusively from an influx of Ca2+ across the plasma membrane. Interestingly, the eugenol-induced increase in Ca2+cyt in yeast mutants devoid of cch1p  responses to 0.6 mg/ml eugenol are shown in 2+, the eugenol-induced Ca2+cyt elevations in the parental strain (JK9-3da) were reduced by approximately 65% indicating that, in contrast to cells in mid-logarithmic growth phase, eugenol induces Ca2+ influx from both extracellular and intracellular sources  release of intracellular Ca2+ following addition of eugenol  pathways to maintain intracellular Ca2+.Previous reports have highlighted that Case cells ; specifind above . We inve sources . However1Δ cells . These r eugenol  indicati strains  was comp2+ release, the yvc1Δ mutant and the isogenic parental strain (BY4742) were transformed with pEVP11/AEQ. Yvc1p is a TRP-like Ca2+ release channel 2+ influx from the vacuole in response to osmotic shock 2+cyt elevations in yvc1Δ and BY4742  cells in stationary growth phase. Surprisingly, the magnitude of the eugenol-induced Ca2+cyt elevations in BY4742 cells was similar in both the absence and presence of extracellular Ca2+  but was absent in the yvc1Δ mutant  has been previously reported in logarithmically growing yeast cells 2+cyt elevation in response to amiodarone are reduced in stationary phase cells compared to that exhibited in actively growing cells The differences in the Ca2+ influx and Ca2+cyt elevation are necessary for eugenol antifungal activity or form part of a tolerance mechanism we examined the growth of cch1Δ and mid1Δ single and cch1Δmid1Δ double mutants in increasing concentrations of eugenol (cch1Δand cch1Δmid1Δ strains in liquid SCM (characterised by doubling times of 7.87 and 5.63 hours respectively in the presence of 0.2 mg/ml eugenol) was more sensitive to eugenol relative to that for the isogenic parental strain (doubling time of 2.63 hours in 0.2 mg/ml eugenol). Although the growth of the mid1Δ strain (doubling time of 4.26 hours in 0.2 mg/ml eugenol) was marginally more sensitive to eugenol compared to the parental strain, inhibition of mid1Δ growth was less than that exhibited by yeast strains devoid of Cch1p. The hypersensitivity of the cch1Δ and cch1Δmid1Δ mutant strains to eugenol could be also clearly demonstrated in drop assays on solid SCM  in animal cells 2O2) induces Ca2+ elevations in yeast S. cerevisae which appears to regulate Cch1p-mediated Ca2+ influx In the present study, actively growing cells are inhibited by eugenol  and henc2+cyt is unlikely to represent a toxic Ca2+ burst resulting in cell death. For example, there is no correlation between the inhibition of growth in different yeast strains and the magnitude of Ca2+cyt increase induced by eugenol. It is also noteworthy that the concentration of eugenol required to inhibit yeast growth (0.2 to 0.4 mg/ml) overlapped with modest increases in Ca2+cyt  and were below that required to induce large Ca2+cyt elevations (which are more likely to represent a toxic burst of Ca2+).It is also noteworthy that the elevation of Ca2+ burst 2+ influx does not appear to involve Cch1p or Mid1p 2+ influx results from Ca2+ channels (of unknown molecular identity) which are activated by amidorane-induced membrane hyperpolarisation 2+ stress and Ca2+-mediated cell death. It would therefore be interesting to test the sensitivity of cch1Δ and mid1Δ mutant growth to carvacrol.The antifungal mechanism of eugenol appears to be distinct to that reported for amiodarone, azoles and carvacrol. Amiodarone toxicity in yeast has been extensively studied and most lines of evidence points towards a drug-induced calcium influx which constitutes a toxic Ca2+ signal) conferring eugenol tolerance in yeast. It will also be interesting to identify the channel(s) mediating Cch1p-independent Ca2+ influx in response to eugenol and determine if these channels contribute to eugenol tolerance. This will improve our understanding of eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.Future studies should focus on elucidating the signalling pathway (downstream of the Ca


Searchable encryption technique enables the users to securely store and search their documents over the remote semitrusted server, which is especially suitable for protecting sensitive data in the cloud. However, various settings (based on symmetric or asymmetric encryption) and functionalities  are often realized by different methods with different searchable structures that are generally not compatible with each other, which limits the scope of application and hinders the functional extensions. We prove that asymmetric searchable structure could be converted to symmetric structure, and functions could be modeled separately apartfrom the core searchable structure. Based on this observation, we propose a layered searchable encryption (LSE) scheme, which provides compatibility, flexibility, and security for various settings and functionalities. In this scheme, the outputs of the core searchable component based on either symmetric or asymmetric setting are converted to some uniform mappings, which are then transmitted to loosely coupled functional components to further filter the results. In such a way, all functional components could directly support both symmetric and asymmetric settings. Based on LSE, we propose two representative and novel constructions for ranked keyword query (previously only available in symmetric scheme) and range query (previously only available in asymmetric scheme). Cloud storage provides an elastic, highly available, easily accessible, and cheap repository to users to store and use their data, and such a convenient way attracts more and more people. In many cases, the users require their sensitive data, such as business documents, to be secure against any adversary or even the cloud provider, and therefore all data must be encrypted before sending to the server . HoweverSearchable encryption technique provides a solution to such problem. It enables the users to encrypt their sensitive data and store it to the remote server, while retaining the ability to search by keywords. While searching, the user sends to the server a secret token (a transformation of the queried keywords); then the server uses the token to search over the encrypted data and returns the matched documents. During the process, the server does not know what the queried keywords and the document contents are, and therefore the privacy is guaranteed.Many searchable encryption schemes have been proposed with various settings and functionalities. For symmetric searchable encryption schemes, the user encrypts, searches, and decrypts the documents using his/her private symmetric key. For asymmetric searchable encryption schemes, the data sender encrypts the documents using the user's public key, and the user searches and decrypts the documents using the private key. Beyond the basic keyword matching, many functions are also added to either symmetric or asymmetric setting, such as range query, phrase query, and fuzzy keyword query.However, these functions are often realized by different methods with different searchable structures which are generally not compatible with each other. For example, the asymmetric encryption scheme introduced in  realizedLayered searchable encryption (LSE) scheme aims to provide compatibility, flexibility, and security for various settings and functionalities. In this new framework, keywords are firstly transformed to tokens that are filtered by the core searchable component (symmetric or asymmetric setting), and then the tokens are dynamically converted to uniform mappings which are transmitted to many stand-alone functional components  to further filter the results. Since all functional components are independent of each other and the interfaces are common, the functions are compatible with each other and directly support both symmetric and asymmetric settings, and adding or deleting a function is quite simple since each function is loosely coupled with the core searchable component. Furthermore, LSE supports combined query. For example, the query “SELECT ∗ WHERE keywords = “cloud, storage, encryption” AND “security classification > 5” ORDERED BY “keyword:cloud””  is a combination of three functional components: basic query, range query, and ranked keyword query .Furthermore, this framework is similar to the data stream processing architecture , where fIn this paper, our contributions are the following. (1) We propose a novel framework for designing searchable encryption scheme called layered searchable encryption (LSE), which enables combined query and provides compatibility, flexibility, and security for various settings and functionalities. The new framework consists of a core searchable component with a symmetric/asymmetric converter, many functional components, and a common interface with new security model. (2) We propose a concrete construction for LSE that could theoretically combine all possible functionalities which are proposed in the recent years, and prove its semantic security for the interface. (3) As a complement for the prior works, we formally define two new security models for ranked keyword query and range query, called semantic security against chosen ranked keyword attack (CRKA) and chosen range attack (CRA) respectively, which provide integral security models for cryptographic analysis. (4) Based on LSE, we propose two representative and novel constructions for ranked keyword query component (previously only available in symmetric scheme) and range query component (previously only available in asymmetric scheme) and prove them semantically secure under the new security models.The rest of the paper is organized as follows. Searchable encryption schemes are designed to help the users to securely search over the encrypted data by keywords. The first scheme was introduced in  by Song k relevant results to the user. In  = eij* . As to v*, 𝒮 randomly selects v* = vi* ∈ V*.The simulator C, L, v) from . Since the encryption key K is kept secret from the adversary, the interface-CPA-security directly guarantees that C* is indistinguishable from C. It also guarantees that v* is indistinguishable from v. Upon receiving v = vi ∈ V or v* = vi* ∈ V*, the adversary 𝒜 could invoke Filter or Filter to obtain  or . POPF-CCA security guarantees that the set  is indistinguishable from ; that is, the adversary is unable to distinguish the result of OPE from the result of a random order-preserving function. Therefore, L is indistinguishable from L*.We claim that no polynomial-size distinguisher could distinguish  is within a range. The SQL query format is “WHERE ‘Pa(w) = 1 if a > w where P is a predicate) could be used, and the functional structure could record all possible predicates and provide predicate test using a bloom filter.Although OPE could be applied here to support range query (similar to ranked keyword query), we propose another solution to demonstrate that how to apply the methods used in asymmetric setting to LSE. In , the auts is a member of a set S = . The set S is coded as an array B of m bits. Initially, all array bits are set to 0. The filter uses r independent hash functions h1,…, hr where each hi : {0,1}* →  for 1 ≤ i ≤ r. For each element sk ∈ S where 1 ≤ k ≤ n, set the bits at positions h1(sk),…, hr(sk) to 1. Note that, a location could be set to 1 multiple times. To determine if s ∈ S, just check whether the positions h1(s),…, hr(s) in B are all 1. If any bit is 0, then s ∉ S. Otherwise, we say s ∈ S with high probability (the probability could be adjusted by parameters until acceptable).A bloom filter  is a spad such as the cryptographic hash of the pathname, and write x >  to denote x > y1,…, x > yn for simplicity.In addition, we write d is labeled by some numbers. Here we only consider a single label a. Therefore, the aim of range query is to enable the user to submit a number w to search for the documents that satisfying the SQL-like query such as “WHERE “a > w””. We consider the five basic range query operators “>, ≥, <, ≤, = .” The other operators such as “∈” could be naturally derived from the basic operators.For range query, the document N discrete numbers A = , where a1 < a2 < ⋯<aN. Then we set five shared virtual documents d1′ = , d2′ = , d3′ = , d4′ = , and d5′ =  for all user's documents. The virtual document could be encrypted by LSE' core as a normal document. Therefore, for any keyword such as “>ai” where 1 ≤ i ≤ N − 1, there always exists a mapping vi.We consider the whole range to be a sequence of a ∈ A for a user's document satisfying ai−1 < a < ai+1 (or a = ai) could be represented as 2N + 1 keywords d = , and these keywords are stored in a bloom filter B. Suppose the user queries a keyword “>w,” where a1 < w < aN; then the query is transmitted to the bloom filter to test if “ > w” ∈B.Based on the notion of virtual document, a label c1, c2 labeled 5,10, respectively. Then the transformed sets are B1 =  and B2 = . If the user submits >7, then only B2 matches the query, which is the same result as direct comparisons since 5≯7 and 10 > 7, and then c2 is returned. Similarly, the query “>3” will match both documents, and c1, c2 are returned.For example (we only consider the operator “>” here for simplicity), suppose we have two documents A =  denote the domain of the label, and setup the bloom filter with r independent hash functions h1,…, hr. The identifier d or the ciphertext c. The concrete construction is presented in Now we construct the secure version of the aforementioned scheme. Let The size of the bloom filter could be dramatically reduced if the domain is bucketized  for examd1, d2 that are labeled with two numbers a1, a2, respectively, the challenger flips a coin b and encrypts . The adversary is allowed to adaptively query p keywords W = , where each wi ∈ W that  > wi. Note that querying wi that a1 > wi,  a2≯wi is not allowed since the document is immediately distinguished (only the document with a1 > wi is matched and returned). We propose the notion of chosen range attack (CRA) and formally define the security model for semantic security as follows.For simplicity without loss of generality, we only consider the operator “>” here, and the other operators are the same. Informally speaking, the functional component must guarantee that the adversary is unable to guess the queried range as well as the range in the ciphertext, and the basic game works as follows. Given two documents k ∈ ℕ be the security parameter. We consider the following probabilistic experiments, where 𝒜 is an adversary and 𝒮 is a simulator.  𝒜Σ,(k): the challenger runs Gen(1k) to generate the key K. The adversary 𝒜 generates a document d and the labeled number a and receives the encrypted document c and the functional structure L. 𝒜 is allowed to adaptively query p keywords W = . For each query “>wi,” 𝒜 receives a mapping vi from the challenger. Finally, 𝒜 returns a bit b that is output by the experiment.Real 𝒜,𝒮Σ,(k): given the document size |d|, the cardinality of the range N, and the size of the mapping |v|, 𝒮 generates c*, L*, and V* = , and then sends the results to 𝒜. Finally, 𝒜 returns a bit b that is output by the experiment.SimLet Σ be the functional component for ranked keyword query. Let 𝒜, there exists a PPT simulator 𝒮 such thatGen.We say that the functional component is CRA-secure, if for all PPT adversaries If LSE is interface-CPA-secure, then the ranked keyword query component is CRA-secure.𝒮 generates c*, L* and V* =  as follows. As to c*, 𝒮 generates a random string of size |d|. As to L*, 𝒮 generates a random string N + 1 distinct and random strings T = . For each ti ∈ T, 𝒮 computes r codewords y1*,…, yr* into a bloom filter B*. Let L* = B*. As to V*, for each vi* ∈ V*, 𝒮 randomly selects a distinct tj ∈ T maps to vi*, such that vi* = tj. Note that, if vx* = vy* for some locations 1 ≤ x, y ≤ p, the mapping is the same.The simulator c, L, V) from . Since the encryption key K is kept secret from the adversary, the interface-CPA-security directly guarantees that c* is indistinguishable from c. It also guarantees that each vi ∈ V is indistinguishable from the random string vi* such that V is indistinguishable from V*. Therefore, the locations  of vi in bloom filter B is indistinguishable from the locations  of vi* in bloom filter B*. Therefore, r · (2N + 1) locations in B are indistinguishable from r · (2N + 1) locations in B*. Thus, L is indistinguishable from L*.We claim that no polynomial-size distinguisher could distinguish  computation complexity for each document. Based on LSE, this functionality could be realized using bloom filter (as demonstrated in range query scheme) which recording biword or more words based on Partial Phrase Indexes [O(1) computation complexity . fied. In , the autfied. In . It allo Indexes . As a reFuzzy Keyword Search. It refers to a functionality that the user submit a fragment of a keyword  and the server could search for the documents with all possible keywords that are closed to the fragment. In [ment. In , the autment. In . By usinSimilarity Query. It refers to a functionality that the server could return to the user some documents containing keywords which are similar to the queried keyword. In both [ In both , 19, theSubset Query. It refers to a functionality that the server could test if the queried message is a subset of the values in the searchable fields. For example, let S be a set that contains multiple e-mail addresses. If the user search for some encrypted mails containing Alice's e-mail a, then the server must have the ability to test if a ∈ S without knowing any other information. A solution was also introduced in [duced in . SimilarThe algorithms of ranked keyword query component and range query component are coded in C++ programming language and the server is a Pentium Dual-Core E5300 PC with 2.6 GHz CPU. Each document is fixed to 10 KB with random words chosen from a dictionary, and the query is also some random keywords (random numbers). For bloom filter used in range query component, the number of hash functions is set to 8. The time costs of the filter algorithms are shown in n denote the number of documents. For ranked keyword query, the main operations are retrieving the relevance scores from the secure table managed by indirect addressing technique (O(n) search complexity) and selecting the top-k scores (O(n) computation complexity). For range query, the main operation is computing 8 hash values (O(n) computation complexity). Note that the current document will be passed if any position in bloom filter is 0. Therefore, not all eight hash functions are executed all the time. The figure demonstrates that, even for a single server, the algorithms are both efficient. Note that, since the functional components are loosely coupled with each other, they could be deployed to different servers. For example, two core components (Core), two ranked keyword query components (Rank), and three range query components (Range) could be executed as a data-flow boxes as shown in Let Layered searchable encryption scheme provides a new way of thinking the relationship among the searchable structure, functionality and security. It separates the functionalities apart from the core searchable structure without loss of security. Therefore, the loose coupling property provides compatibility for symmetric and asymmetric settings and it also provides flexibility for adding or deleting various functionalities. Furthermore, following the popular boxes and arrows paradigm, the loose coupling property makes the scheme more suitable for distributed and parallel computing environment.


Here, we report GECIs with improved neuronal Ca2+ signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (Fmax/Fmin = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca2+ imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca2+ responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.Imaging the activities of individual neurons with genetically encoded Ca Recently, Ca2+ imaging using green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) has been introduced as an alternative to using chemically synthesized fluorescent Ca2+ indicators Understanding brain function requires techniques for monitoring the spatio-temporal activity patterns of individual neurons and synapses. A promising approach for this purpose is Ca2+ signals in response to presynaptic single spikes at the single-synapse level.In this study, we developed high-sensitivity and fast-responsivity GECIs, termed G-CaMP6 and G-CaMP8, by mutagenizing existing G-CaMPs. These novel indicators allow us to reliably monitor neural spikes with larger fluorescence signals and higher temporal resolution than G-CaMP3, a recently reported variant of G-CaMP2 Fmax/Fmin = 9.03±0.06, n = 3) than G-CaMP2 Fmax/Fmin = 4.8)  than sfG-CaMP2  but also a high affinity for Ca2+   2+ sensitivity is based on the intramolecular interaction between the CaM domain and the M13 domain of MLCK. As expected, G-CaMP6, a variant of G-CaMP5.09 bearing an M36L substitution in the CaM domain  than G-CaMP5.09 or the previously reported G-CaMP2 variants G-CaMP-HS Fmax/Fmin = 11.4±0.11, n = 3) was not significantly different from that of G-CaMP3  (2+ absorbance peak (498 nm) was red-shifted by ∼10 nm  was ∼3-fold greater than that of G-CaMP6, even though this variant showed a lower Ca2+ affinity  than G-CaMP6  was between those of G-CaMP6 and G-CaMP7 and similar to that of G-CaMP3  (F/F) (2+ affinities (F/F) of G-CaMP6, G-CaMP7 and G-CaMP8 was greater than that of G-CaMP3. The baseline fluorescence of G-CaMP7 and G-CaMP8, unlike G-CaMP6 and other variants, was lower than that of G-CaMP3.In an effort to create a superior GECI, we first introduced mutations from “superfast GFP” n = 4.8) . For furn = 4.8) . sfG-CaMfG-CaMP2 . SubsequfG-CaMP2 , which s, n = 3) . G-CaMP5, n = 3) . Next, wM domain , showed , n = 3) . The spey ∼10 nm . Next wey ∼10 nm . The dyn G-CaMP6 . By perf G-CaMP6 . It was , n = 3) . The spe, n = 3) . To asse, n = 3)  and the 3) (F/F)  of each finities , the sig2+ imaging were performed on G-CaMP-expressing neurons 24–48 h after electroporation. The baseline fluorescence of the neurons expressing G-CaMP6 , unless otherwise specified. G-CaMP6 and G-CaMP8 responded to single spikes with 100% probability. The ΔF/F amplitudes of Ca2+ transients evoked by single spikes were 17.4±3.5%, 27.9±4.5% and 37.8±5.2%, and the signal-to-noise ratios (SNRs) were 8.0±1.5, 18.3±1.5 and 16.4±3.5 for G-CaMP3, G-CaMP6 and G-CaMP8, respectively (n = 7 each). The signal amplitudes grew almost linearly as the spike number increased . On the other hand, the signal decay of G-CaMP6 and G-CaMP8 was significantly faster than that of G-CaMP3   . The rapsitivity  of G-CaMsitivity .in utero electroporation, as previously described The detectability of G-CaMPs was also evaluated in pyramidal neurons in acute cortical slices. The expression of G-CaMPs in the mouse brain was driven by 2+ transients: they are small and short near physiological temperature because Ca2+ is removed quickly by strong Ca2+-pumping activity F/F value of G-CaMP6 was ∼40% lower at 37°C than at 25–28°C  than at 25–28°C   .By contrast, it was confirmed that the expression of G-CaMP6 does not affect the electrophysiological properties  of hippocampal neurons .C. elegans and successfully recorded spontaneous Ca2+ transients in A-type cholinergic motoneurons of freely moving L1 worms. The peak responses (ΔR/R) of G-CaMP6 during locomotion  were 1.6-fold greater than those of G-CaMP3    and S2.Vm = 18.5±4.8 mV) triggered small fluorescence responses  in a limited number of spines (48.6±6.3%) in the striatum lucidum, with a low response rate in the active spines (57.6±13.8%)  in virtually all of the spines in the imaged region including the striatum lucidum and the striatum radiatum, with a 100% activity rate  . In contity rate .2+ activity was imaged in spines in slices cultured for 8 and 29 days. After 29 days in vitro (Div), the amplitudes of spine Ca2+ transients in response to supra-threshold stimulation were not significantly different from those at 8 Div . These results confirmed that the expression of G-CaMP6-actin in spines remained stable after at least 4 weeks of culture  and S205N (in G-CaMP7 and G-CaMP8) and I47F (in G-CaMP8) mutations in the circularly permutated EGFP domain]. Based on the G-CaMP2 structure, the residue Ser-205 is located in the β-strand of the circularly permuted EGFP domain (corresponding to the tenth β-strand in EGFP) and facing the inside of the chromophore Fmax/Fmin = 32.7±1.5) than G-CaMP3 (Fmax/Fmin = 12.3±0.4), is the variant which responds with the greatest signals among G-CaMP5s to maximal stimulation when expressed in cultured neurons. Indeed G-CaMP5G is reported to show ∼70% greater signals (ΔF/F) than G-CaMP3 in response to 1–5 spike trains, but its SNR is not improved with respect to that of G-CaMP3 Fmax/Fmin = 37.5±3.6) is similar to that of G-CaMP5G, shows ∼100% greater signals than G-CaMP3 in terms of both ΔF/F and SNR (Kd = 189±5.0 nM) 2+ signals, similar to G-CaMP6 (Kd = 158±4.0 nM)(F/F and SNR) than G-CaMP3 in response to 1–5 spike trains Kd = 307±12 nM) and signal amplitudes (Fmax/Fmin = 17.4±1.2) among G-CaMP5s, but is reported as the preferred variant over G-CaMP5G and G-CaMP5K for use in worm and zebrafish Recently, Akerboom et al. have reported new series of GECIs termed G-CaMP5s  and SNR  and ∼2-f and SNR . On the 8±4.0 nM). G-CaMP52+ transients in response to single spikes in pyramidal neurons in hippocampal slices at 25–28°C. However, it is still unknown whether these G-CaMPs exhibit similar performance in vivo. As shown in 2+ and the sensitivity of Ca2+ indicators are temperature dependent. Indeed, it has been reported that GECI fluorescence is less intense in vivo compared to in vitro2+ transients of the newly-developed G-CaMPs ranged between 400 and 450 ms, which is shorter than that of G-CaMP3 2+ indicators contribute to discrete fast individual spikes in burst-spike trains with a temporal resolution of up to 15 Hz. To our knowledge, G-CaMP6 is the most suitable GECI currently available for detecting and isolating fast individual spikes in spike trains.The detection of neuronal activity patterns with single-spike resolution is required to elucidate neural network dynamics. We demonstrated that G-CaMP6 and G-CaMP8 faithfully detected Ca2+ increase in individual spines through the activation of voltage-sensitive Ca2+ channels and/or NMDA receptors. In previous studies, spine Ca2+ activity was imaged with synthetic indicators, such as Oregon Green BAPTA-1 2+ signals within spines but failed to detect synaptically evoked Ca2+ activity, presumably because of the low Ca2+sensitivity of G-CaMP2. In this study, we demonstrated that G-CaMP6-actin is the first GECI that allows the visualization of Ca2+ signals in response to synaptic stimulation at the single-spine level. Although the exact mechanisms of spine Ca2+ signals remain unknown, it seems likely that sub-threshold stimulation triggers Ca2+ transients through postsynaptic NMDA receptors 2+ transients at 100% of the spines by opening of voltage-gated Ca2+ channels through backpropagation of action potentials. In principle, we should be able to visualize spine responses to evaluate long-term plasticity, which is thought to be an elementary component of learning and memory. We expect that these novel G-CaMP technologies, together with advanced imaging systems Excitatory synaptic activity induces a transient CaB vector (Invitrogen) containing a T7 promoter, as described in utero electroporation, cDNAs encoding G-CaMPs and mCherry (Clontech) were subcloned into a pCAGGS vector containing a CAG promoter  C. elegans, cDNAs encoding G-CaMPs were subcloned into a pFX_EGFPT vector containing the unc-4 promoter Complementary DNAs (cDNAs) encoding sfG-CaMP2, sfG-CaMP2.02, G-CaMP5.09 and G-CaMP6 were synthesized by mutagenizing the cDNA encoding the prototype GECI, G-CaMP2 E. coli KRX (Promega) transformed with pRSETB-G-CaMP was grown at 37°C, and protein expression was induced by adding 0.1% rhamnose and incubating for an additional 5 h at 20°C. The indicator proteins with N-terminal histidine tags were purified, dialyzed against KM buffer containing (in mM) 100 KCl and 20 MOPS (pH 7.5) and used for in vitro characterization Fmax/Fmin, where Fmax is the fluorescence intensity at saturating [Ca2+], and Fmin is the fluorescence intensity at nominally zero [Ca2+] with 1 mM EGTA. The Ca2+ titration experiments were performed at pH 7.2 with 10 mM solutions of K2H2EGTA and Ca2EGTA from the Ca2+ Calibration Kit #1 (Invitrogen), as previously reported 2, 1 MgCl2, 10 glucose and 5 HEPES (pH 7.4), and after reading the baseline fluorescence, 100 µM ATP was bath-applied for 1 min. The images were analyzed using AquaCosmos version 2.0 software (Hamamatsu). The transient increase in fluorescence (ΔF/F) was calculated after subtracting the background fluorescence.HeLa cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and transfected with plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s manual. Fluorescence images of cells expressing G-CaMPs were acquired with a fluorescence microscope  equipped with a CCD camera , as previously described Punc-4::G-CaMP6) or G-CaMP3 (Punc-4::G-CaMP3) was co-injected with the plasmid carrying DsRed-Express-1 (Punc-4::DsRed-Express-1) jqEx97 (G-CaMP6) strain and the jqEx216 (G-CaMP3) strain were used in this study. Ca2+ imaging was performed in worms on a 1.5% agar pad placed on a glass slide . L1 animals were placed in M9 buffer R/R) of G-CaMP6 or G-CaMP3 against DsRed-Express-1 were calculated as (R1–R0)/R0, where R1 is the fluorescence ratio at any time point and R0 is the baseline fluorescence ratio.The expression plasmid carrying G-CaMP6 , 25% horse serum (Cell Culture Laboratory) and antibiotics. The medium was changed every 3.5 days. On days 3–5 in vitro, G-CaMPs and mCherry under the control of the CMV promoter were introduced into the neurons via targeted single-cell electroporation All experiments were performed with the approval of the animal experiment ethics committee at the University of Tokyo  and according to the University of Tokyo guidelines for the care and use of laboratory animals. Hippocampal slices from postnatal day 7 Wistar/ST rats (SLC) were prepared, as previously described 3, 1.4 NaH2PO4, 2.5 KCl, 7.0 MgSO4, 1.0 CaCl2, 0.5 ascorbic acid and 222 sucrose, bubbled with 95% O2 and 5% CO2. Coronal cerebral hemispheric slices (400-µm thick) were cut using a Vibratome 3000 (Vibratome).Day-14-pregnant ICR mice (CLEA Japan) were deeply anesthetized, and their intrauterine embryos were removed surgically, as previously described 3, 3.3 KCl, 1.24 KH2PO4, 1.0 MgSO4, 1.0 CaCl2 and 10 glucose, bubbled with 95% O2 and 5% CO2. All recordings were performed at room temperature (24–28°C), unless otherwise specified. Patch-clamp recordings were collected from hippocampal CA3 pyramidal neurons using a MultiClamp 700B amplifier and a Digidata 1440A digitizer controlled by pCLAMP10 software (Molecular Devices). Epifluorescence microscopy was used to select cells showing stable mCherry expression with a fluorescence intensity ranging from 103 to 125 (arbitrary units). Borosilicate glass pipettes (5–7 MΩ) were filled with a solution containing (in mM) 135 K-gluconate, 4 KCl, 10 HEPES, 10 phosphocreatine-Na2, 0.3 Na2-GTP and 4 Mg-ATP (pH 7.2). The signals were low-pass filtered at 1–2 kHz and digitized at 20–100 kHz. Data were discarded if the access resistance changed by more than 20% during the experiment. Spikes were evoked by current injections , and electrical stimulation was applied using a constant voltage-isolated stimulator and a glass pipette filled with aCSF (Nihon Kohden). The electrodes were placed in the dentate hilus to stimulate the mossy fiber pathways. For sub-threshold stimulation, the stimulation intensity was adjusted so that <50% of the spines exhibited Ca2+ transients . For supra-threshold stimulations, the intensity was raised to more than 200 µA so that almost all neurons generated action potentials. For the Ca2+ imaging, the G-CaMPs were excited at 488 nm with a laser diode  and visualized using a 507-nm long-pass emission filter. Images were captured at 50–500 fps using a Nipkow-disk confocal scanner unit , a cooled CCD camera , an upright microscope  and a water-immersion objective . The cell bodies of the neurons were carefully identified by eye to locate regions of interest (ROIs). Cells with labeled nuclei were excluded from further analysis because both Ca2+ homeostasis and G-CaMP function are impaired in these cells F/F = (tF – F0)/F0, where tF is the fluorescence intensity at time t, and F0 is the baseline averaged for 2 s before time t. The maximum ΔF/F within 1 s after action potential initiation was used a measure of the peak amplitude of the Ca2+ transient. The SNR was defined as the average spike signal amplitude divided by the standard deviation of the fluorescence intensity in the trace. Data were collected from >3 consecutive trials. The rise time τ1/2 was measured as the time between the onset of the spike initiation and the half-peak response. The decay time τ1/2 was measured as the time of half decay of a single exponential fit of the recovery from the peak response to the baseline. For spine Ca2+ imaging, response rates were measured in the spines that exhibited at least one Ca2+ transient during imaging period (i.e. active spines). The rates were defined as the ratio of the number of Ca2+ transients to the total trial of electrical stimulation.Hippocampal slices were mounted in a recording chamber and perfused at a rate of 1.5–3 ml/min with aCSF containing (in mM) 127 NaCl, 26 NaHCOMovie S1L1 larvae (jqEx97) co-expressing G-CaMP6 and DsRed-Express-1 in the DA neurons during locomotion (corresponds to). Green, red and transmitted-light images were overlaid. Images were taken at 15 fps.(AVI)Click here for additional data file.Movie S2L1 larvae (jqEx216) co-expressing G-CaMP3 and DsRed-Express-1 in the DA neurons during locomotion (corresponds to). Green, red and transmitted-light images were overlaid. Images were taken at 15 fps. A worm was kept in the imaging field by manual adjustment of the x-y stage.(AVI)Click here for additional data file.


This study investigates whether protein kinase G (PKG), protein kinase A (PKA) and protein kinase C (PKC) are involved in the regulatory mechanisms of store-operated channel (SOC) in pulmonary arteries.Pulmonary artery smooth muscle cells (PASMCs) were enzymatically dissociated from rat intralobar pulmonary arteries. Whole cell, cell-attached and inside-out patch-clamp electrophysiology were used to monitor SOCs in isolated PASMCs.2+-ATPase inhibitor cyclopiazonic acid  initiated a whole cell current that was reduced by the SOC blocker SKF-96365 (10 μM). Subsequent work using both cell-attached and whole cell configurations revealed that the PKG and PKA inhibitors, KT5823 (3 μM) and H-89 (10 μM), also stimulated SOC activity; this augmentation was attenuated by the SOC blockers SKF-96365 (10 μM) and Ni2+ (0.1 mM). Finally using the inside-out configuration, the PKC activator phorbol 12-myristate 13-acetate  was confirmed to modestly stimulate SOC activity although this augmentation appeared to be more substantial following the application of 10 μM inositol 1,4,5-triphosphate P3).Initially the CaSOC activity in PASMCs was stimulated by the inhibition of PKG and PKA and the activation of PKC. Our findings suggest that the SOC could be a substrate of these protein kinases, which therefore would regulate the intracellular concentration of calcium and pulmonary arteriopathy via SOC. Many agonists increase [Ca2+]i by mobilizing intracellular Ca2+ stores, such as the sarcoplasmic (SR) or endoplasmic (ER) reticulum. To maintain Ca2+ signaling, these intracellular Ca2+ stores must be refilled and as such many agonists are thought to activate specialized plasma membrane channels termed store-operated channels (SOCs) -1H-imidazole HCl  i), an event intimately tied to both contraction and proliferation in PASMCs. Such alterations could in turn contribute to the constriction and medial hypertrophy that commonly underlies pulmonary arterial hypertension (PAH).The guanylate cyclase-cyclic GMP-protein kinase G signaling pathway plays a pivotal role in several physiological processes including vascular tone development and cell cycle progression. Broadly speaking, studies have shown that activation of this NO-dependent signaling pathway characteristically attenuates SOC activity in smooth muscle -21. Thisted mode ,25. On t-1 cells , nor did oocytes , pancrea oocytes , or T ly oocytes . Regulat oocytes . Indeed, oocytes ,32. With oocytes ,31. Pete oocytes  demonstr of SOCs . In this3 and/or the depletion of PIP2 i: intracellular calcium; CCE: capacitative Ca2+ entry; CIF: calcium influx factor; CPA: cyclopiazonic acid; ICRAC: Ca2+ release-activated Ca2+ current; iPLA2: Ca2+-independent phospholipase A2; InsP3: inositol 1,4,5-triphosphate; PASMC: pulmonary artery smooth muscle cell; PKA: protein kinase A; PKC: protein kinase C; PKG: protein kinase G; PMA: phorbol 12-myristate 13-acetate; SERCA: sarco-endoplasmic reticulum Ca2+-ATPase pump; SOC: store-operated channel; SOCE: store-operated Ca2+ entry; TRPC3: canonical transient receptor potential isoform 3[CaThe authors declare that they have no competing interests.ISC performed the experiments and drafted the manuscript. ZKD and IJC provided the ideas and participated in the design and coordination of this study, and helped to draft the manuscript. DGW and BNW designed and directed the experiments, interpreted the data and polished the paper to meet the scientific content. All authors read and approved the final manuscript.