TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca2+]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca2+]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence.Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca The molecular basis of the basal tone generated by internal anal sphincters (IAS) is largely unknown. Here, the authors show that the tone arises from a global rise in intracellular Ca2+ in smooth muscle cells via a Ryanodine receptor-TMEM16A-L-type Ca2+ channel-MLC kinase pathway, suggesting a potential therapy for IAS motility disorders. The human body, and those of other mammals, contains up to 50 sphincters, ring-shaped structures encircling an opening or passage in hollow organs such as the intestine and the bladder. These sphincters control the entrance of material into, or the release of contents from, these organs, and participate in a variety of biological functions essential for homeostasisin vitro and in vivo experiments have indicated that the basal tone of IAS is independent of extrinsic nerve and hormone stimulation2+/calmodulin-dependent MLC kinase (MLCK) and Ca2+-independent MLC phosphatase (MLCP). MLCP consists of three subunits including a regulatory 110–130 kDa subunit, called the myosin-targeting subunit of MLCP (MYPT1), which anchors MLCP to p-RLC. MLCP can be phosphorylated by activation of small GTPase RhoA and Rho-associated, coiled-coil containing serine/threonine kinase (ROCK)Thr696-MYPT1 may be responsible for the basal tone in IAS  located at the end of the gastrointestinal tract, has served as a prototypical model to understand basal tone genesis in sphincters. A significant number of IAS refs , 6, 7.2+-mediated MLCK activation is required for the IAS basal tone. We find that the basal tone in IAS from MLCK knockout mice is essentially abolished and these mice give rise to larger faeces, a sign of impaired faecal continence. By directly examining Ca2+ signals and ion channel activity, we further find that Ca2+-releasing ryanodine receptors/channels (RyRs), TMEM16A Ca2+-activated Cl− (ClCa) channels and L-type voltage-dependent Ca2+ channels (VDCCs) form a module which generates a global rise in Ca2+, and that pharmacologically altering any one of the three channels can severely impair IAS basal tone (to the same degree as MLCK deletion). Moreover, genetic deletion of TMEM16A in IAS smooth muscle cells (IAS-SMCs) severely impairs both the RyR-mediated Ca2+ rise and the basal tone, and results in wider and longer faeces. Our results hence demonstrate that MLCK activation by a RyR-TMEM16A ClCa channel-L-type VDCC signalling cascade in the IAS-SMCs is required for basal tone formation and maintenance, and is essential for faecal continence.In this study, we use smooth-muscle-specific MYPT1 knockout mice to directly test this hypothesis. We find that the basal tone of IAS from the knockout mice is the same as that from wild-type mice. We, therefore, also test a new hypothesis that Can=12) and their littermate controls , neither was the amplitude of the tone   . Our genMlckSMKO)MlckSMKO mice was reduced by 70–98%. For the phenotypic analysis, other than where noted, only the IAS tissue in which MLCK was decreased by >95% was used : 0.29 ±0.02 g, P<0.0001 by two-tailed Student's t-test; MlckSMKO mice increased from 5.4±0.1 to 11.3±0.8 mm , and the diameter increased from 2.1±0.1 to 2.7±0.1 mm   . These rMlckSMKO mice was due to a decrease in regulatory light-chain (RLC) phosphorylation, we measured p-RLC in MlckSMKO mice and their controls. As shown in MlckSMKO was not detectable during the same time frame.To determine whether the suppression of the IAS basal tone in MlckSMKO mice developed much weaker contraction  (MlckSMKO mice showed a significantly lower level of RLC phosphorylation at different time points on stimulation with KCl or bethanechol (It is known that MLCK is required for phasic smooth muscle contraction induced by contractile agonistsctively) . Moreovehanechol .MlckSMKO mice were detected  at resting Ca2+ concentration ([Ca2+]i), we hypothesized that RyRs could be a critical component of this essential Ca2+ signal. To examine this possibility, we investigated the effects of (RyR inhibitor) ryanodine and (RyR agonist) caffeine on IAS basal tone. As shown in P<0.05 by paired two-tailed Student's t-test; 92+ currents, which can be blocked by nifedipine, a specific L-type VDCC blocker  niflocker 18. The aboCCs refs , 21, 22.sal tone . Niflumisal tone , similarCa channel-VDCC module controls the basal tone of IAS, we next studied the nature of the Ca2+ signalling generated by activation of these channels in IAS-SMCs. Since RyRs generate spontaneous Ca2+ sparks in a variety of smooth muscle cells26272+ events may directly regulate IAS smooth muscle contraction. To test this possibility, we examined the relationship between Ca2+ sparks and cell length using isolated single IAS-SMCs. We found that freshly isolated IAS-SMCs generated spontaneous localized Ca2+ events  increased their activity by almost eightfold (n=10) while ryanodine at 100 μM suppressed them by 62%±22 (n=4)  . Interes lengths . Only atortening . This cacaffeine . These rCa channels and L-type VDCCs form a signalling module to control the basal tone  than in the presence of extracellular Ca2+ ), indicating that in addition to Ca2+ release from ryanodine-sensitive Ca2+ stores, caffeine also induced Ca2+ influx. To determine the potential roles of the ClCa channel and L-type VDCCs in this Ca2+ influx we compared the caffeine-induced Ca2+ rise in control cells with the rise produced when modulators of ClCa channels or L-type VDCCs were present. inh-A01 (10 μM) and nifedipine (1 μM) inhibited the caffeine-induced increase in global [Ca2+]i by 33, 35 and 74.0%, respectively. Eact (1μM) and FPL64176 (1 μM) enhanced the caffeine-induced rise in [Ca2+]i by 28 and 20%, respectively. These results demonstrated that activation of RyRs can turn on ClCa channels and L-type VDCCs, resulting in a global rise in [Ca2+]i.Since RyRs, Clsal tone , and a RIAS-SMCs , we aske2+]i can activate ClCa channels, we examined the relationship between Ca2+ signals and ClCa currents by simultaneously measuring [Ca2+]i with high- speed imaging and membrane currents with the patch-clamp technique2+ sparks activate ClCa channels to generate spontaneous transient inward currents (STICs)2+ sparks did not associate with any detectable currents when the membrane was clamped at voltages more negative than the reversal potential for Cl− (n=5), indicating they do not activate STICs. We then assessed whether an increase in global [Ca2+]i could elicit ClCa currents. To raise global [Ca2+]i, we stimulated the cells with caffeine (10 mM) and recorded the membrane current at different voltages. As shown in Ca channels, because at the reversal potential for Cl− , the same cell failed to generate current in response to the same caffeine stimulation. At +40 mV, the cell produced a markedly outward current, as would be predicted if this current was a Cl− current. Finally, caffeine raised the global [Ca2+]i to the same level at three different holding potentials  from α-smooth muscle actin-GFP mice produced similar currents in response to caffeine  detected three types of Ryrs with the dominant expression of Ryr1  were used as the KO mice, while Tmem16aflox/+, SMA-Cre littermates were used as control mice (CTR). Birth of Tmem16aSMKO and CTR pups occurred in the expected Mendelian ratio. Tmem16aSMKO mice were fertile, viable and lacked apparent developmental defects. Western blot demonstrated that TMEM16A in the KO IAS tissues was significantly decreased to ∼20% of the control levels , while in the Tmem16a−/− IAS-SMCs, the same concentration of caffeine yielded essentially no ClCa current  at +100 mV and showed an outward rectification and time-dependent activation at positive potentials  at +100 mV, and the other 28 cells had markedly reduced currents with 6.31±0.30 pA pF−1 (n=28) at the same potential . These results indicate that TMEM16A is required for ClCa currents in IAS-SMCs, and its deficiency impairs IAS basal tone formation and faecal continence.We further examined potential functional changes in IAS as a result of TMEM16A deletion in IAS-SMCs. We first studied the effect of TMEM16A modulators on caffeine-induced Ca release . We nextMKO mice . We founCa channels and L-type VDCCs in IAS-SMCs form a signalling module to regulate global [Ca2+]i, which activates MLCK and sets IAS basal tone , a potential endogenous ligand for RyR, or a stretch-gated ion channel. In this regard, it is worth mentioning that stretch can activate the non-selective cation channel TRPV4, which is known to activate Ca2+ sparks in vascular smooth muscle2+ levels in both the cytosol and the internal Ca2+ store can auto-regulate RyR activity28462+ in the cytosol inhibits L-type VDCCs and ClCa channels482+]i in these cells could be oscillating during basal tone development and maintenance. It would be interesting to examine whether an oscillating [Ca2+]i leads to a low level of RLC phosphorylation during tone generation as revealed in In our model, calcium release from the opening of RyRs in the sarcoplasmic reticulum of IAS-SMCs could be an initial signal for the basal tone generation and maintenance. The increased calcium then activates TMEM16A Cl mV refs , 44, an Ca channel genes32Out of two identified Cl2+ release is intact in IAS-SMCs from TMEM16A knockout mice 51et al. 2014 who also elegantly demonstrated its role in the blood vessel smooth muscleIn the TMEM16A deficient mice, the tone was decreased by 51% compared with littermate controls. What accounts for this remaining tone? One possibility is that Cre in a small subset of IAS-SMCs has an insufficient efficiency, resulting in an incomplete deletion of the TMEM16A in these cells. Another possibility could be that activation of RyRs alone  is sufficient to generate a certain amount of IAS tone. This is supported by our finding that caffeine-induced Caout mice . Finallyout mice . In someCa channels near the submucosal space to the entire tissue. This is certainly possible as gap junctions are expressed abundantly in IAS cells  and Mypt1SMKO  mice, TMEM16A CTR  and Tmem16aSMKO  mice at 6–10 weeks were decapitated. MLCK CTR  and MlckSMKO  mice were decapitated 20 days after tamoxifen injection. MYPT1 CTR, Mypt1SMKO, MLCK CTR and MlckSMKO mice were in a C57BL/6 background. TMEM16A CTR and Tmem16aSMKO mutant mice were in a mixed C57BL/6 and Sv/129 background. Both genders of the mice were used equally. The anal canal and an adjacent region of the rectum were quickly removed and transferred to ice-cold and oxygenated Krebs physiological buffer (KPS) which was comprised of (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3 and 11.10 glucose. Strained skeletal muscle fibres, mucosal layer and other extraneous tissues  were carefully dissected away, whereas the anal canal was left intact. The IAS was identified as a thickened circular smooth muscle situated at the lowermost part of anal canal, and the strips of IAS smooth muscle (∼1 × 6 mm) were prepared for experiments.C57BL/6, MYPT1 CTR , or University of Massachusetts Medical School, Massachusetts, USA.2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3 and 11.10 glucose was used to achieve membrane depolarization. Bethanechol (100 μM) was used to induce agonist-induced contraction in IAS. For RyR inhibitor ryanodine experiments, IAS strips were pretreated with 100 μM ryanodine for 5 min before giving the initial stretch. For RyR agonist caffeine experiments, 3 mM caffeine was added into the bath 8 min after the initial stretch. Both ryanodine and caffeine were present through the experiments after administration. For L-type VDCC modulators (nifedipine and FPL64176) and ClCa channel modulators , experiments were performed in the presence of 1 μM atropine and 10 μM guanethidine to eliminate the possible effects of cholinergic and sympathetic nerves on the basal tone.Isolated strips of IAS smooth muscle were transferred to 8 ml muscle baths containing ice-cold oxygenated KPS. One end of the smooth-muscle strip was anchored to the bottom of the muscle bath. The other end of the smooth-muscle strip was connected to a force transducer  and isometric tension was measured by a PowerLab (AD Instruments) recording device. A wire myograph  was also used for force measurements. Each smooth-muscle strip was equilibrated for 60 min followed by a 0.5-g load8Tmem16a locus including Exon 12 was retrieved from a 129/sv bacterial artificial chromosome clone  by a retrieval vector containing two homologous arms. Exon 12 which encodes a partial transcript in the second trans-member domain was floxed by 2 loxP sites, and an frt-Neo-frt cassette was inserted as a positive selection marker : 135 NaCl, 6 KCl, 5 MgCl2, 0.1 CaCl2, 0.2 EDTA, 10 Hepes and 10 glucose (pH 7.3). The tissue was then incubated in the dissociation medium containing 30 unit per ml papain, 1 mM DTT and 0.5 mg ml−1 BSA, at 35 °C for 30 min. The tissue was then transferred to a dissociation medium containing 3 unit per ml collagenase F and 0.5 mg ml−1 BSA, and incubated at 35 °C for another 15 min to produce isolated IAS cells. Finally, the tissue was agitated with a fire polished wide-bore glass pipette to release the cells.Mice as listed above from 6 to 10 weeks of age of both genders were decapitated or euthanized with COg, 4 °C to remove cell debris. Protein concentration was measured with a bicinchoinic acid protein assay kit (Pierce). The proteins were denatured at 95 °C for 5 min with sample buffer and reducing agent . General SDS-page processes were followed. The blots were visualized by using the enhanced chemiluminescence method, the Super Signal West Dura substrate (PIERCE) and MaxiSignal Western Solution (SUDGEN) were used. All the primary antibodies used in western blot assay are listed in IAS smooth muscle strips were homogenated in 120 μl lysis buffer , pH=8). Then the homogenization was incubated on ice for 15 min, and centrifugation at 3,000Anal tubes were isolated and fixed in ice-cold acetone for 10 min, then washed with PBS overnight at 4 °C and rewashed for 4 h with a change of PBS per hour. Cryosections with a 10-μm thickness were used. The non-specific binding of primary antibodies was blocked by incubation with PBS containing 1% BSA and 5% non-immune goat serum for 1 h. Incubation was carried out overnight at 4 °C with a rabbit polyclonal antibody to TMEM16A  diluted 1:200, together with a rat anti-c-Kit antibody  or mouse monoclonal Myh11 antibody . After washing in PBST, sections were incubated with a Alexa Fluor 555-conjugated goat anti-Rabbit antibody (Sigma) diluted 1:250 and a FITC-conjugated goat anti-rat antibody (Invitrogen) diluted 1:250 or a Alexa Fluor 488–conjugated goat anti-mouse antibody  for 1 h. 4,6-diamidino-2-phenylindole was used for nuclear staining. Immunoreactivity was evaluated using a FV1000 confocal laser scanning microscope system (Olympus).2, 1 MgCl2, 10 Hepes and 5.6 glucose; pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM): 137 CsCl, 1 MgCl2, 10 Hepes, 3 Na2ATP; pH adjusted to 7.3 with CsOH. Amphotericin B was freshly made and added to the pipette solution at a final concentration of 200 μg ml−1. Whole-cell currents were recorded when cells were held at the designated potentials and the currents were low-pass filtered using the Axopatch 1D amplifier (200 Hz cutoff) and then digitally sampled at 1 kHz and stored for analysis. Caffeine was applied to the cells via a puffing pipette placed ∼100 μm from the cells under the control of a picospritzer.Membrane currents were recorded using the perforated whole-cell patch recording configuration. The extracellular solution contained (in mM): 130 NaCl, 5.5 TEA-Cl, 2.2 CaCl2+-induced currents, conventional whole-cell patch clamp was carried out. The extracellular solution contained (in mM) 144.5 NaCl, 5.5 TEA-Cl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 mannitol and 10 Hepes; pH was adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 130 CsCl, 10 EGTA, 1 MgCl2, 8 CaCl2, 10 Hepes, 1 MgATP; pH was adjusted to 7.3 with NaOH. Whole-cell currents were recorded in response to 1 s voltage pulses from −80 to +100 mV in 10 mV increments followed by 700 ms pulses to −60 mV, in freshly isolated IAS-SMCs. Holding potential was 0 mV. Currents were sampled at 20 kHz using the Axon MultiClamp 700B amplifier (Molecular Devices) and then low-pass filtered at 2 kHz.For recording of 600 nM Ca2, 1 MgCl2, 10 Hepes, 10 glucose; pH was adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 130 CsCl, 4 MgCl2, 10 Hepes, 10 EGTA, 5 Na2ATP; pH was adjusted to 7.2 with CsOH. Cells were held at −80 mV, whole-cell currents were recorded in response to 200 ms voltage pulses from −80 to +50 mV in 10 mV increments. Freshly isolated IAS-SMCs were incubated in extracelluar solution with 1 μM Nifedipine or vehicle (0.1% EtOH) during recording. Currents were sampled at 20 kHz using the Axon MultiClamp 700B amplifier (Molecular Devices) and then low-pass filtered at 2 kHz.For recording L-type VDCCs in IAS-SMCs, conventional whole-cell patch clamp was carried with extracellular solution containing (in mM) 130 TEA-Cl, 10 CaCl2+ indicator and a custom-built wide- field, high-speed digital imaging system2+ indicator was monitored at wavelengths >510 nm. Ca2+ sparks were measured as the conventional fluorescence ratio (ΔF/F0) within a restricted volume as described previouslyFluorescent images were obtained using fluo-3 as the CaThe IAS and rectal smooth muscle (4 mm above IAS) from mice were carefully isolated, quickly removed from connective tissue and then frozen in liquid nitrogen. The total RNA was isolated using the TRIZOL (Invitrogen) method according to the manufacturer's guidelines, and cDNA was synthesized. Primers were synthesized by Invitrogen, and their sequences are available in t-test, or two-way analysis of variance (ANOVA) for significant differences. The significance levels were indicated as follows: NS P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.Data are presented as the mean±s.e.m. Differences between groups were determined by Student's How to cite this article: Zhang, C.-H. et al. The molecular basis of the genesis of basal tone in internal anal sphincter. Nat. Commun. 7:11358 doi: 10.1038/ncomms11358 (2016).Supplementary Figures 1-17, Supplementary Tables 1-2 and Supplementary References


Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.The emergence of human stem cell–derived cardiomyocyte (hSCCM)–based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO  to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling “fingerprint” in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca However, there are acknowledged issues regarding their hybrid embryonic-adult proteomic and transcriptomic signatures, cellular heterogeneity postdifferentiation, intracellular microarchitecture and signaling organization, electrophysiological profiles, and pharmacological sensitivities.Cardiovascular (CV) drug development urgently needs enhanced screening systems that enable improved early stage assessments of drug hazard and more accurately predict in vivo drug toxicity and clinical efficacy.2The incorporation of hSCCM-based assays into the drug discovery toolkit now warrants the development of improved systems that can better interrogate the rich information that these new cellular models have the potential to yield. Moreover, the shift toward phenotypic profiling strategies, which evaluate drug bioactivities in the context of a deep understanding of cellular signaling networks, requires methodological innovation beyond the transposition of conventional optical and electrophysiological technologies onto these new cell systems.2+) signaling network underpins virtually every biological phenomenon,6 and thus understanding the spatiotemporal organization of Ca2+ signals in functionally coupled hSCCMs, under normal and drug-exposed conditions, could potentially reveal the mechanistic bases of drug-evoked phenotypic modulation and cellular (dys)function.79 Previous studies in single mammalian cells showed that subtle changes in Ca2+ signaling dynamics, which did not perturb steady-state Ca2+ homeostasis, had profound consequences for cell phenotype.1012 In this study, we extend these approaches to profile Ca2+ signal organization in spontaneously contractile, functionally coupled hSCCMs. We describe the development and proof-of-concept validation of a novel system  that enables a detailed interrogation of the spatiotemporal patterning of Ca2+ signals in hSCCMs and the exploration of the mechanisms of drug action. We discuss the potential utility of this system in the drug safety screening landscape and how, in combination with other contemporary screening approaches, SALVO may enable a better understanding of the mechanisms underlying CV drug cytotoxicity and lead to the improved prediction of drug hazard in humans.The calcium . Cells were thawed and seeded into 7-mm2 chambers created by the adherence of silicon gaskets  on glass-bottomed culture chambers  that had been precoated with Matrigel , Franklin Lakes, NJ, USA diluted 1:30 (v/v) in Knockout DMEM . Cells were seeded as per the manufacturer’s instructions at a density of 2500 cells per mm2 surface area  in antibiotic-free RPMI 1640 (15 µL) supplemented with B27 (1:50 [v/v] dilution) (RPMI/B27) (Life Technologies) and maintained at 37 °C in a humidified 5% CO2 environment. Cells were allowed to adhere to the coverslip for 2 h before the dish was filled with RPMI/B27 (2 mL). Medium was exchanged every 48 h.Cytiva hSCCMs were derived from monolayer differentiation of an H7 hESC cell line that had been expanded under feeder-free conditions and then subjected to a proprietary differentiation protocol .2HPO4 [10], NaH2PO4 [2], pH 7.4) for 10 min at room temperature (RT) and then washed three times with PBS prior to permeabilization . Nonspecific antibody interactions were blocked by incubation in horse serum  before cells were incubated with mouse anti–troponin-T   overnight at 4 °C. Cells were washed with PBS (3 × 5 min) before being incubated with Alexa Fluor 546 anti–mouse IgG  for 1 h at RT in the dark. Following washing with PBS (3 × 5 min), cell nuclei were counterstained with DAPI  prior to further washing in PBS (2 × 1 min) and mounting under Prolong Gold (Life Technologies). Cells were imaged using a confocal microscope , and assessments of TnT positivity and cellular alignment were made using image analysis .Cells were fixed  using argon laser excitation (488 nm) and a confocal microscope . Cells were maintained at 37 °C throughout experiments. Images were recorded every 100 ms at a 512 × 512–pixel resolution. The application of caffeine  was used to trigger sarcoplasmic reticulum (SR) Ca2+ release and thereby estimate the intra-SR Ca2+ store load.Cells were incubated with fluo-4 AM (5 µM) (Life Technologies) for 1 h at 37°C before coverslips were flooded with RPMI/B27 (2 mL). Fluo-4 Ca2+ signals,14 we developed SALVO to decode the spatiotemporal patterning of Ca2+ oscillations within individual cells and across multicellular populations. SALVO outputs 30 parameters that describe Ca2+ signal organization,15 but for the purposes of this study, we focused on five parameters: oscillation rate  and four other parameters that quantify the amplitude and temporal patterning of Ca2+ oscillation: (1) amplitude heterogeneity index (AHI) and (2) temporal heterogeneity index (THI), statistical assessments of signal amplitude and temporal variability within single cells, respectively; (3) intertransient noise (ITN), defined as the Ca2+ signal variability occurring between Ca2+ oscillations and that extends the use of signal variability (SV) to measure point-by-point differences in very low-amplitude Ca2+ signals12; and (4) synchronization, an index of the temporal coincidence of Ca2+ oscillation maxima occurring across cells in the population. The calculation of these parameters is described in Supplementary Figure S1. SALVO is implemented using a Python-based computer program.15 The detection of signal maxima and minima over a 30-s period in data obtained from 6 to 20 cells in each instance was performed using SALVO’s autodetection algorithms (based on spline detect or threshold detection methods) or by manual assignation of the start, peak, and end of each Ca2+ spike. SALVO outputs were archived and interrogated using a custom-built SQL-based database system .Taking our lead from Uhlen’s method of using spectral analysis to investigate the organization of Ca2+ signals in fluo-4–loaded cells prior to the addition of drug (control) were established as described above. Cells were then sequentially exposed to increasing concentrations of cardioactive drugs, selected to represent those categories assigned by Redfern and colleagues16 as category 1 , category 2 , category 3 , and category 5 . Valdecoxib, a COX-2–selective nonsteroidal anti-inflammatory drug (NSAID) that was initially approved for use in the treatment of osteoarthritis and rheumatoid arthritis but was subsequently withdrawn from the market due to adverse CV effects,19 was not included in the assessment by Redfern et al.16 but was included here. We purchased rofecoxib (Vioxx) from Sequoia Research Products , but the supplied compound exhibited inconsistent bioactivity in our assay system.Baseline assessments of Ca2+ signals were acquired for 30 s. Using this protocol, the same population of cells was imaged throughout the entire drug addition sequence.Stock solutions of drugs (10 mM) in ultrapure water or tissue-culture grade DMSO were diluted in RPMI/B27, and the concentration of each drug under test was sequentially increased. After each addition and following a period of equilibration (approximately 90 s), fluo-4–dependent Ca1-blocker); celecoxib (COX-2 inhibitor), and aconitine (non–QT-prolonging proarrhythmic). We also included two drugs that have mechanisms of actions distinct from those above: verapamil  and ranolazine (late INa current inhibitor). In all experiments, operators were blinded to the identities of the drugs under test throughout all phases of postexperimental data analysis.To validate the system, we used a second set of compounds at maximal effective concentrations in our assay (1–30 µM): category 1, amiodarone (class III antiarrhythmic); category 2, terodiline (QT-prolonging proarrhythmic); category 5, metoprolol  or NT-containing drugs maintained at 37 °C using a heated jacket.Cells in 35-mm glass-bottomed culture dishes containing a superfusion insert  were mounted on the stage of an inverted microscope  and were superfused with normal Tyrode (NT) solution  in current-clamp (IC) mode using a CV-7B headstage and MultiClamp 700B amplifier controlled by MultiClamp software . Data were digitized and acquired using a Digidata 1322a card and pClamp software, respectively (Molecular Devices). Action potentials were sampled at a rate of 20 kHz and low pass (Bessel) filtered at 10 kHz.Cells were whole-cell clamped with 3- to 4-MΩ patches and an intrapipette solution  recordings from blebbistatin-immobilized cells were taken under control (no-drug) conditions (1 min) and following the switch to drug-containing NT. APD90, corrected for AP cycle length using Fridericia’s method (APD90(corr) = APD90/cycle length0.3), was calculated from five consecutive APs following a 3-min exposure of the cells to drug.Spontaneous cell contracture was terminated using blebbistatin , an agent that inhibits myofilament shortening and thereby negates patch-clamp recording instability but preserves normal Cat test  or the Mann-Whitney test  was used. All statistical analysis was performed using Prism 6.0 . All data are given as mean ± standard error (SE). A p value of 0.05 was considered statistically significant.Hierarchical clustering of SALVO outputs was performed using the uncentered correlation similarity metric and centroid clustering method in Cluster 3.0  with output visualization using TreeView . Data sets were tested for normality using the D’Agostino-Pearson algorithm, and normally distributed data were compared using analysis of variance with intergroup comparisons performed using Bonferroni’s post hoc test. Nonnormally distributed data were compared using the Kruskal-Wallis test with Dunn’s post hoc test. For comparing only two groups of data, two-tailed Student ,21 all Cytiva CMs possessed the requisite cellular machinery to support caffeine-induced Ca2+ release even by day 2 postseeding . However, the amplitude of caffeine-induced Ca2+ release, an index of the functional Ca2+ storage capacity of the SR, was augmented at day 4 and remained unchanged through day 7 . CMs between days 4 and 7 also exhibited a faster rate of Ca2+ sequestration/extrusion after caffeine-induced Ca2+ release .The relative axial alignment of TnT-positive cardiomyocytes (CMs) was low on days 2 and 3 postseeding but progressively increased to a maximum at day 5 with no further change to day 7 , yet were incompletely aligned .Our data point to the culture-dependent alignment of CMs, the intracellular reorganization of TnT, and the improved functional capacity of the Ca2+ signaling changes observed at day 4 coincided with the onset of spontaneous Ca2+ oscillations  and synchronized contraction (Suppl. Movies 1–3). Between days 4 and 7, the proportion of cells exhibiting spontaneous Ca2+ release remained static , but the Ca2+ oscillations became qualitatively . Notably, there was a marked decrease in AHI and THI  and a reduction in ITN, indicative of less variability in low-amplitude Ca2+ signals occurring between large Ca2+ transients . It has been reported that hSCCMs plated at “low density” (500–1200 cells/mm2) exhibit markers of cellular hypertrophy and electrical remodeling.22 Although our mean cell densities were below this threshold, the alignment of cells in culture leads to regional heterogeneity, with some regions at very high densities and other areas that contain few cells (see 2) on Cytiva CM phenotype.The morphological and CaSuppl. Fig. S3) existed only for a short period. Beyond day 7, the proportion of TnT-positive cells progressively decreased, and by day 14, there was a pronounced functional deterioration in Ca2+ signaling . At day 21, the few cells that remained adherent  had mostly reverted to fibroblastic-like morphologies with a loss of TnT striation and organization . This short window meant that we performed all subsequent experiments on CMs between days 6 and 8. We did not pursue strategies reported to prolong hSCCM functionality , and it is likely that such protocols could improve the functional longevity of Cytiva in culture.The optimal functional state of Cytiva CMs . Sotalol, a class III antiarrhythmic that has been reported to have QT-prolonging effects at therapeutic plasma concentrations exceeding 10 µM, caused an isolated change in AHI at concentrations >1 µM but did not reduce the extent of intercellular synchronization (Suppl. Table S2). Concentration-response profiling revealed that drugs with recognized proarrhythmic hazards resulting from hERG-blocking activities  or via other “non-hERG” mechanisms  evoked large effect sizes in multiple parameters of Ca2+ handling (Suppl. Table S2). These data confirmed that the perturbation of cellular Ca2+ signals in vitro is a hallmark feature of drugs with known cardiovascular risk in humans irrespective of whether their proarrhythmic mechanism of action involves IKr/hERG blockade. Notably, SALVO quantified the Ca2+-disrupting effects of valdecoxib, a drug that had hitherto not been considered to exert direct effects on CMs using conventional electrophysiological assessments. Our data are consistent with those of Pyrko and colleagues,24 who reported the elevation of cytoplasmic Ca2+ within seconds of the addition of 2,5-dimethyl-celecoxib, an analogue valdecoxib, to glioblastoma cells. These findings add weight to the concern that contemporary electrophysiological measurements do not adequately resolve potential CV risk associated with some drugs, especially those that do not directly modulate cellular Na+ and K+ ion handling. Illustrating this point, impedance measurements in hSCCM failed to identify the potential hazard associated with rofecoxib (Vioxx),25 an analogue of valdecoxib that was withdrawn because of unacceptable CV risk.19 We believe that SALVO has the potential to occupy this void in the contemporary drug screening landscape.The disruption of the spatiotemporal patterning of Car/hERG blocking activities. However, flecainide and valdecoxib, drugs that markedly perturbed Ca2+ signals . We used a fluorescence-activated cell sorting (FACS)–based strategy to enrich CMs, but the small population of viable yet morphologically abnormal CMs that were obtained offset any improvement in Ca2+ signal organization observed in post-FACS CMs .Cytiva are supplied as a heterogeneous mix of CMs and non-CMs. It is therefore plausible that effects of drugs on contaminant non-CMs could influence the measured effect on CMs  was in very close agreement with the known proarrhythmogenic liabilities of these drugs in humans26 . Moreover, SALVO exhibited a superior correlation with established CV drug risk in humans compared with electrophysiological readouts . Thus, for the reasons outlined above, we propose that SALVO will improve early stage assessments in CV drug hazard liabilities. However, the extrapolation of in vitro cell-based toxicity outputs to risk prediction in clinical scenarios is problematic,2 and it is accepted that, at present, no single approach can accurately translate in vitro readouts to drug effects in humans. Consequently, SALVO outputs should be appropriately integrated with information obtained from other platforms .The five-parameter SALVO output (27 used an impedance-derived index of arrhythmic beating in hSCCMs to derive a predicted proarrhythmic score (PPS) that took into account the therapeutically relevant total concentration of drug in human plasma (defined as Ceff). More recently, Clements and Thomas13 used a similar approach to generate a predictive risk score (PRS) based on their measurement of field potential duration (FPD) in Cytiva. In keeping with these studies, we adjusted the five-parameter SALVO output to account for the free plasma concentration of the clinically effective drug (ETPCunbound) (Suppl. Tables S1 and S2). Our resultant PPS (ETPCunbound/EC20)  described a rank order of thioridazine > cisapride > valdecoxib that was entirely consistent with the SALVO toxicity scores . The erroneous values for these two drugs, which were also noted by Clements and Thomas,13 result from skew introduced by very high and low ETPCunbound values  (Suppl. Table S1). Consequently, our data highlight the significant potential for the misleading categorization of “risk” following the calculation of a PPS or PRS . These data strongly suggest that such simple indices are unlikely to accurately predict in vivo risk/hazard. However, by studying a larger set of compounds with known risk, together with consideration of other factors that influence in vivo drug responses, our future understanding of the relationship between in vitro assay outputs and CV risk in humans should be improved.There is also the issue regarding the relevance of drug potency determined in vitro to clinical dosing in humans. Guo and colleagues2+ channel blocker, reduced the rate of Ca2+ oscillation at very high concentrations of drug (>10 µM) but otherwise had no measurable effect on cellular Ca2+ handling. Ranolazine, an inhibitor of the late INa that is not associated with elevated CV hazard,28 reduced intercellular synchronization against a background of unchanged Ca2+ cycling. Consistent with their different mechanisms of action, verapamil and ranolazine were separated in the resultant dendrogram and did not cluster with the other compounds tested in this study . Hierarchical cluster analysis (HCA) revealed that, as anticipated, drugs with comparable mechanisms of action clustered together—for example, hERG blockers (terodiline with astemizole), non-hERG blockers (aconitine with flecainide), COX-2 inhibition , class III antiarrhythmics , and β-blockers (nadolol with metoprolol)  but have negligible impact on the cellular electrophysiological profile. We also demonstrate the utility of SALVO to discriminate drugs based on their mechanism of action. The present work used only a small number of compounds of known proarrhythmia risk, and clearly, a systematic evaluation of a much larger panel of compounds is now warranted.This study reports the development and proof-of-concept validation of SALVO, a new system to quantify drug-induced (dys)organization of intra- and intercellular Ca2 gaskets and with the potential to use fewer cells still—suggests that this system is amenable to scaling to higher throughput formats . Moreover, the system could be configured toward “multiplexing” ion-sensitive fluorescent probes or in combination with electrophysiological platforms.30 Outside of the predictive toxicology arena, such detailed assessments of cellular Ca2+ signaling may also be useful in quality control (QC) processes, cellular phenotyping, and profiling batch-to-batch variability during the manufacture/differentiation process. It is also noteworthy that SALVO analysis could be applied to any oscillatory biological signal. To this end, it is of interest to explore SALVO in the context of other cell systems  and to the analysis of other cellular signals that change on different timescales .The comparative assay miniaturization reported here—17,500 cells seeded in 7-mm


Here, structure-based mutant analyses of the Methanococcus jannaschii Na+/Ca2+ exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na+or Ca2+ binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct “noncatalytic” residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.In analogy with many other proteins, Na Inverted twofold symmetry of repeated structural elements, which evolved via gene duplication and fusion events, represents a common motif among multi-helical proteins including enzymes, receptors, pumps, transporters, and channels. This structural set-up is particularly frequent in transporters12334567+/Ca2+ exchanger (NCX) proteins extrude Ca2+ from the cell52+ homeostasis7+:1Ca2+)+ and Ca2+ ions are transported in separate steps114-fold in the turnover rates of the transport cycle1314152+ levels713m at opposite sides of the membrane781316+ or Ca2+ ions, it has remained unclear whether the ligand-free NCX_Mj is already asymmetric in the absence of ions or ligand binding shifts the equilibrium in favor of the outward-facing conformation.Naext, Smid, Sint, and SCa), where twelve residues are involved in ion coordinationint and Sext sites have high selectivity for Na+, whereas Smid and SCa lack selectivity for ion binding. The ion pocket of NCX_Mj encompasses highly conserved α1 and α2 repeats with inverted topology, where twelve ion-coordinating residues  and two helix-breaking signature sequences (GTSLPE) form the ion-pocket181920ext, Smid, and Sint are occupied by 3Na+ ions, where E54, E213, D240, and N81 coordinate one Na+ ion at Smid, and 1Ca2+ occupies SCamid  with inverted twofold symmetry12567s SCamid  and thusThe present work was undertaken to evaluate the mechanistic role of inverted pair residues within the ion pocket of NCX_Mj by exploring especially suited kinetic analyses  of overe13232426The present HDX-MS and kinetic studies revealed that the asymmetric preorganization of inverted pair residues in the ligand-free ion pocket predefines the catalytic reorganization of key ion-coordinating residues, where distinct “noncatalytic” residues stabilize the outward-facing conformation.+ or Ca2+ —it also permits quantitative evaluation of local changes in backbone flexibility upon ion binding at respective sites. Highly purified preparations of NCX_Mj protein were used in all these experiments. MS identification of peptic peptides resulted in 26.7% coverage   . In cont of TM8) .+-free samples, Na+ was replaced by choline and for Ca2+-free samples, EDTA was added. Interestingly, in the absence of Na+ and Ca2+ ions, there are marked differences in the local backbone dynamics at key ion-coordinating residues in apo-NCX_Mj , in contrast with a much higher deuterium uptake observed in region 54–59 , is much more rigid than the backbone of the ion-coordinating residues on TM7 .For Nao-NCX_Mj . For exaon 54–59 . This dron 54–59 , which c+ (100 mM) or Ca2+ (2 mM). In general, the exchange rate is higher for the apo form than for either the Na+ or Ca2+ bound forms . This behavior may be related to the presence of Na+ ligands A47, T50, and S51 in Sint and the presence of Ca2+ ligands T50, S51, and E54 in SCa, thus, stabilizing the backbone conformation in this region upon ion binding. Assuming the occupation of Smid by Na+ + binding at Smid will result in a major decrease in deuterium uptake for peptide 238–245 encompassing D240  is much more dynamic in both the apo and bound forms and ligand binding results in less protection. This may represent the functional asymmetry of NCX_MjThe two GTSLPE signature sequences on TM2 and TM7 (residues 49–54 and 208–213), although being “symmetry-related”, exhibit marked differences in HDX kinetics . The α1-+i or Ca2+i-dependent 45Ca2+ -uptake were measured in right-side-oriented isolated vesicles derived from the plasma membranes of E. coli cells containing overexpressed NCX_Mj mutants13+/Ca2+ and Ca2+/Ca2+ exchange reactions at the ‘extracellular’ (K″m and V″max) and ‘cytosolic’ (K′m and V′max) sides can be measured by varying ion concentrations at the intravesicular and extravesicular compartments  reveals the asymmetric effects of alanine substitutions on the ion exchange activities, with RA values ranging from 2 to 32 , the S77/S236 pair has an extraordinarily high RA value for both the Na+/Ca2+ and Ca2+/Ca2+ exchanges, which is at least 10 times higher than the RA value imposed by any other pair residues  of bidirectional Ca2+ movements16int ≃ 0.15 observed for NCX_Mj represents the intrinsic asymmetry of bidirectional ion movements , whereas the same mutations have rather dramatic effects on the turnover rates (kcat = 0.01–0.8 s−1) of Ca2+/Ca2+ exchange . Collectively, the present data indicate that N73 and N232 control Kint by affecting the ground/occluded states of Ca2+ with very little (if any) effects on the transition state.Previous studies have shown that the K′ents see , suggesth mutant  and the e mutant . A strikexchange . In sharat value . These d+ at Sint and Sext, respectively, through their backbone carbonyls), all ion-coordinating side chains were replaced by alanine to evaluate the mutational effects on the kcat/Km values of the Na+/Ca2+ or the Ca2+/Ca2+ exchange reactions , S51 (Sint), T209 (Sext), and S77 (Sext) exhibit 10–25% of the WT kcat/Km values for both exchange reactions mut/(kcat/Km)wt]) , whereas E54 and E213 are major residues that stabilize the transition-state, each contributing ∆∆G≠app = 1.5–1.8 kcal/mol , suggesting that these polar residues also participate in stabilizing the transition state. Thus, the side chains of two negatively charged residues (E54 and E213) and of four uncharged (polar) residues  stabilize the Ca2+ transition state wt]) . Notablykcal/mol . Four ioon state .+ or Ca2+ coordination, although these residues are highly conserved among NCXs and other membrane proteins18m values of either ion-exchange reaction  and 208-GTSLPE-213 (TM7) are not directly involved in Nareaction . Althougm values . Thus, Gs of HDX  and ligas of HDX . Interesectively , therebyectively . Althougkcal/mol . This sukcat /Km) and on the intrinsic equilibrium of bidirectional Ca2+ movements (Kint = K′m/K″m) by assaying the Ca2+/Ca2+ exchange reaction in E. coli-derived cell-membrane vesicles containing overexpressed NCX_Mj proteins with a right-side-out orientation. In another set of experiments the backbone dynamics of purified NCX_Mj were investigated in the presence or absence of Na+ or Ca2+ ions using HDX-MS, with the goal of tracking the protein backbone dynamics in the ligand-free and ion-bound pocket of NCX_Mj.The present work was undertaken to analyze the structure-dynamic determinants of ion-transport catalysis and the intrinsic asymmetry of bidirectional movements in the NCX_Mj protein as related to the inverted twofold symmetry of ion-coordinating pair residues. For this purpose, structure-based kinetic analyses of mutants were performed by testing the effect of alanine mutations on the catalytic capacity , more dedicated research is required for elucidating the relevant mechanism.In general, HDX-MS is the technique of choice for investigating the structure-dynamic features of local backbone dynamics in apo- and ligand-bound proteins, although the outcome of valuable information depends very much on the sequence coverage in the mass spectrometry experiments. The observed sequence coverage (26.7%) of the NCX_Mj protein is quite low  compared+ or Ca2+  and intrinsic equilibrium (Kint) of bidirectional Ca2+-movements  the alanine substitutions affect the kcat/Km but not the Kint value  in these mutants  for “catalytic residues” , in conjunction with four polar residues, namely, S51, S77, T209, and D240  serves as a rigid anchor around which the catalytic residues E54 (SCa), E213 (SCa), protonated D240 (Smid), S51 (Sint), S77 (Sext), and T209 (Sext) stabilize the transition state  may largely predefine the positioning of catalytic residues in the transition state , whereas the other catalytic residues  may possess a higher degree of freedom for Ca2+ interaction in the transition state  and ligated between the NcoI and BamHI restriction sites of a pET-28a plasmid13DNA encoding the WT NCX_Mj was amplified by PCR from a E. coli BL21 (DE3) pLysS competent cells. Cells were grown, harvested, homogenized, and lysed by French-Press1313E. coli-derived cell-membrane vesicles was tested using an antibody against the 6xHis-tagExpression vectors were transformed into The ratio of NCX-Mj expression to the total vesicle protein was approximated by calculating the concentration of GFP by its absorbance at 488 nm (e.c. = 56000) and dividing it by the value determined by the Lowry assay. The GFP absorbance signal at 488 nm was isolated from vesicle noise using a correction factor determined by measuring non-expressing vesicles2+ affinity column. Protein was then desalted to eliminate imidazole with a buffer containing 2 mM DDM and digested overnight with TEV protease. Following a second passage through the Co2+ column (with a buffer containing 2 mM DDM) to eliminate the His-tag and TEV protease, the protein was purified on Superdex-200 pre-equilibrated with 0.5 mM DDM buffer. Concentrated (1–2.5 mg/ml) preparations of purified proteins  were stored at −80 °C in buffer containing 0.5 mM DDM.NCX_Mj was overexpressed and purified as outlined before1318+/Ca2+ and Ca2+/Ca2+ exchange reactions were assayed by measuring 45Ca2+-uptake in the E. coli-derived vesicles containing the overexpressed WT or mutants of NCX_Mj, as previously described1317+ (160 mM) or Ca2+ (250 μM)-loaded vesicles were rapidly diluted 25–50-fold at 35 °C in an assay medium containing 20 mM MOPS/Tris, pH 6.5, 100 mM KCl, and 5–2000 μM 45CaCl2. The 45Ca2+-uptake was quenched by cold buffer containing 10 mM EGTA and filtrated on GF/C filters, as outlined before1317m and Vmax values were measured in at least three independent experiments (data are presented as mean ± SEM) with GraFit 7.1 1013kcat was calculated as kcat = Vmax/[E]t, where [E]t was determined by the GFP assay (see above).The initial rates of the NaLC-MS/MS analyses were performed under H/D exchange compatible conditions and data analysis was performed with MStools+ or Ca2+) at a concentration of 40 μM. Deuteration was initiated by a 5-fold dilution of the protein samples (10 μL) with the same buffer in D2O (40 μL). The proteins were deuterated for 15 sec, or for 2 or 20 min at 4 °C. Back-exchange quench and digestion were performed using 50 μL of porcine pepsin (Sigma) solution at 0.23 mg/mL in 100 mM glycine-HCl, pH 2.5 at 4 °C for 2 min. The peptides were desalted for 6 min using a HPLC pump (Agilent Technologies) with 0.03% TFA in water, at a flow rate of 100 μL/min. The peptides were separated using another HPLC pump (Agilent Technologies) at 50 μL/min for 6 min with 15–50% gradient B , followed by 9 min at 50% B and 1 min at 100% B. The peptide masses were measured using an electrospray-TOF mass spectrometer (Agilent 6210) in the 300–1300 m/z range. Each deuteration experiment was conducted in triplicate. The Mass Hunter (Agilent Technologies) software was used for data acquisition. The HD Examiner software  was used for HDX-MS data processing. MSTools was used for visualization and presentation of the HDX-MS dataHDX-MS experiments were fully automated using a PAL autosampler . This controlled the start of the exchange and quench reactions, the proteolysis temperature (4 °C), the injection of the deuterated peptides, as well as management of the injection and washing valves; it also triggered the acquisition of the mass spectrometer and HPLC pumps. A Peltier-cooled box (4 °C) contained two Rheodyne automated valves, a desalting cartridge (peptide Opti-Trap Micro from Optimize Technologies) and a HPLC column . HDX MS reactions were carried out using different forms of WT NCX_Mj .


Contact electrification between two different materials is one of the oldest fields of study in solid-state physics. Here, we introduced an innovative system based on optical electric-field-induced second harmonic generation (EFI-SHG) technique that can directly monitor the dynamic performance of the contact electrification on the surface of polyimide film. After the contact, the EFI-SHG system visualized briefly three relaxations of the tribo-induced charges on the surface of a polyimide film, a fast relaxation within 3 min followed by two much slower relaxations, which were possibly related to different charge diffusion routes. The contact electrification under several special experimental conditions  was studied to demonstrate the high flexibility and material selectivity of the EFI-SHG. The EFI-SHG studies confirmed the motion of the water can remove the surface charge, while the appearance and the evaporation of a thin water layer cannot enhance the charge diffusion. We anticipate that this experimental technique will find a variety of applications in the field of contact electrification and the development of the recently invented triboelectric nano generator. The contact and the separation between two different materials lead to the charge transfer between two surfaces and this is so-called contact electrification or triboelectricity effect, which has been one of the oldest study fields for hundreds of years135679104A promising solution for completing these detailed studies of contact electrification is to develop an optical experimental method that allows electrostatic fields to be probed. In this paper, we demonstrate an innovative optical method for directly observing contact electrification and triboelectricity, which is an optical method based on optical second harmonic generation (SHG) techniques12121412141216t = 0. Note that square-root of the SHG signal intensity is in proportion to the electric field strength126 V/m. Hence, this relationship is available for the estimation of the tribo-generated electric field from the EFI-SHG observation.The polyimide sample with a Metal-Insulator-Metal (MIM) structure was prepared for studying the SHG generation under stable voltage source, as shown in We prepared a simple polyimide (Kapton) film as the tribo-material to work under the monitoring of the EFI-SHG system. The sample structure was shown in 19E ≈ 9 × 106 V/m (450 V) and the related surface charge density changing was Δσ ≈ 318 μC/m2.  and charge density is approximately 163 μC/m2. These residual charges can usually stay on the surface of polyimide for a quite long time . The existence of such residual charges may relate to the presence of some deep energy states on the surface, where the trapping force may balance the diffusion force from the concentration gradient103 s. After this relaxation, the SHG signal returned to 0.3 and showed very slow decrease after that. The SHG signal at this level is caused by the residual charges on the surface, which will experience an extremely long relaxation with a time constant on the order of 104 s (the third relaxation). This multi relaxation process of the tribo-charges has been confirmed by repeating the experiments with different samples.It is also interesting to find that the tribo-excited SHG signal always returns to a base line (0.3) after the second slow relaxation, as shown in 2 . It is important to note that a latest research based on a triboelectric nano generator using liquid electrode has reported a maximum charge density of 430 μC/m2 on the surface of tribo-excited Kapton film, which is in very good agreement with our measurementThe water applications in A comparison of traditional KPM and EFI-SHG is listed in 3 s and 104 s, respectively. We also designed several special experimental conditions  for studying the adaptability of contact electrification process. The results proved that only the motion of the water can remove the tribo-charges on the surface, while the appearance of water steam cannot change the charge diffusion process. The EFI-SHG system is a new approach to study the contact electrification process. Accordingly, we carefully analyzed the advantages and the defects of this method in comparison with the conventional KPM method. The experimental flexibility and material selectivity of the EFI-SHG system can allow many potential applications of this technique in the study field of contact electrification and in the development of electrification based energy devices.In conclusion, by applying the EFI-SHG measurements to the polyimide film, we systematically studied the contact electrification process happen between Al foil and polyimide film. The real-time observation from the EFI-SHG visualized a fast relaxation of tribo-induced charges within 3 min after the tribo-excitation and two slower relaxation processes with the time constants of 10Q-switched Nd:YAG laser. A p-polarized pulsed laser beam was focused onto the sample surface at an incident angle of 45°. We inserted two SH-cut filters to remove influence light signal, optical filters and a spectrometer was set in the path of the reflected laser, in order to allow only the second harmonic signals to pass through. The EFI-SHG light generated from the sample was finally detected using the photomultiplier tube (PMT) at the end of the spectrometer, and its intensity was recorded with a digital multimeter. It is important to note that the size of the laser spot is about 0.8 mm2 (see  mm2 see  and the E(ω) in the presence of electrostatic local electric field E(0) (The “0” means that it is static field), as can be seen in 24By laser irradiation, EFI-SHG signal is generated from polyimide film due to the coupling of electrons in molecules and electro-magnetic waves P(2ω) is the nonlinear polarization induced in the polyimide film, ε0 is the vacuum permittivity, and χ(3) is the third order nonlinear susceptibility tensor. E(0) is the electrostatic local field in the organic layers (we define as that positive electric field E(0) points from the Ag electrode to the top of polyimide film), and E(ω) is the electric field of incident laser beam. E(0) in the polyimide film. Here, E(0) is given by E = Qm/ε0ε, where charges Qm is the charge on top and bottom surface of the polyimide and ε is the relative permittivity of polyimide. The susceptibility tensor χ(3) in (3) among materials allows us to selectively probe electric field only in the polyimide film with an appropriate laser beam wavelength16where How to cite this article: Chen, X. et al. Direct probing of contact electrification by using optical second harmonic generation technique. Sci. Rep.5, 13019; doi: 10.1038/srep13019 (2015).


Background: Phosphatidylinositol 4,5-bisphosphate levels need to be replenished during calcium signaling, but how this is achieved is unclear.Results: Ca2+ entry through Orai1 channels contributes to the resynthesis of phosphatidylinositol 4,5-bisphosphate and, thereby, helps sustain cytoplasmic Ca2+ oscillations.Conclusion: Ca2+ influx sustains cytoplasmic Ca2+ oscillations by regulating phosphatidylinositol-4-phosphate 5-kinase.Significance: Store-operated Ca2+ influx is important in maintaining phosphatidylinositol 4,5-bisphosphate levels. 2+ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca2+ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca2+-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca2+ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li+, an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li+-treated cells, cytoplasmic Ca2+ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca2+ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca2+ oscillations, and these could not be rescued by inositol or PI4P. In Li+-treated cells, recovery of the cytoplasmic Ca2+ oscillations in the presence of inositol or PI4P was suppressed when Ca2+ influx through store-operated Ca2+ channels was inhibited. After rundown of the Ca2+ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca2+ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca2+ entry is needed to sustain cytoplasmic Ca2+ signaling following leukotriene receptor activation both by refilling the Ca2+ stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.Oscillations in cytoplasmic Ca Low lev plateau . Informaoduction , exocytooduction , and Ca2pression , 9 while2+]i require either oscillations in the levels of InsP3 or biphasic gating of the InsP3 receptor by cytoplasmic Ca2+ in the presence of a steady increase in InsP3. Regardless of the mechanism, two conditions need to be satisfied for repetitive oscillations in [Ca2+]i to occur. First, the Ca2+ content of the stores must be maintained at a level sufficient for Ca2+ release. This is necessary because, following each Ca2+ release event, a fraction of the mobilized Ca2+ is extruded from the cell by plasma membrane Ca2+ATPase pumps (2+ content would therefore drop below the level that supports InsP3-dependent Ca2+ release. Store refilling is accomplished through activation of store-operated CRAC channels in the plasma membrane that open following loss of Ca2+ from within the ER (2+ sensors and migrate toward the plasma membrane upon store depletion (reviewed in Ref. 2+]i is that PIP2 levels must be replenished following each Ca2+ spike to support production of the InsP3 that is needed for the next oscillation in [Ca2+]i. InsP3 has a lifetime of just a few seconds in the cytoplasm (+. Inositol combines with cytidine diphosphate diacylglycerol to form phosphatidylinositol (PI), which can then be phosphorylated by PI4 and PIP5 kinases to make PIP2.Oscillations in i that are supported by Ca2+ entry through CRAC channels , although the amplitude decreased gradually with time because of receptor desensitization . Pre-exposure to 10 μm La3+ accelerated the rundown of the oscillations in [Ca2+]i induced by LTC4 in 2 mm external Ca2+-containing solution (D). The rate of rundown in La3+ was similar to that seen in cells challenged with LTC4 in the absence of external Ca2+ . Similar results were obtained when 20 μm BTP2 was used to block CRAC channels instead .Activation of cysteinyl leukotriene type I receptors with LTCchannels . We repet 800 s, A, althoutization . To see  blocker  and inhi ∼300 nm , B and Csolution D. The raG and H; Orai1, I and J). Oscillations in [Ca2+]i evoked by LTC4 in the presence of external Ca2+ ran down rapidly after knockdown of either STIM1 or Orai1 (K), and the responses resembled those obtained when agonist was applied in the absence of external Ca2+ (L).To extend the pharmacological results, we knocked down either STIM1 or Orai1 using siRNA-based approaches. Although both proteins were robustly expressed in wild-type RBL-2H3 cells, knockdown significantly reduced expression . The effect of LiCl was concentration-dependent (B), and the dose-inhibition curve yielded an IC50 of 15 mm (C), close to the value reported for IMPase2  or ionomycin (E and F) in Ca2+-free external solution was unaffected by pretreatment with even high concentrations (50 mm) of Li+. Store-operated Ca2+ entry, induced by readmission of external Ca2+ to cells treated with thapsigargin, was also unaffected by Li+ (D). Pretreatment with Li+ also had no inhibitory effect on the transient oscillatory cytoplasmic Ca2+ signals evoked by LTC4 when agonist was applied in Ca2+-free solution . Collectively, these results rule out an action of Li+ on store Ca2+ content, store-operated Ca2+ entry, and agonist-induced InsP3 production.The extent of Casigargin D or ionoonomycin , E and Fd by Li+ D. Pretre2+ oscillations by LiCl in our experiments is broadly similar to the decline in InsP3 levels reported in Chinese hamster ovary cells following muscarinic receptor stimulation , in agreement with these earlier reports showing little effect of LiCl on the initial increase in InsP3 levels.The time course of rundown of cytoplasmic Camulation , 27. In solution , G and H2+ signals, we reduced external Na+ from 155 to 105 mm and then added 50 mm LiCl. The oscillations in [Ca2+]i evoked by LTC4 still ran down quickly (I). Therefore, changes in osmolarity are unlikely to explain the accelerated rundown of cytoplasmic Ca2+ oscillations in the presence of Li+.To test for an osmotic effect of LiCl on oscillatory Ca quickly I. Theref2+ flux that had subsided in the continuous presence of agonist. We therefore examined whether the presence of exogenous inositol could rescue the cytoplasmic Ca2+ oscillations in Li+-treated cells. Although oscillations in [Ca2+]i evoked by LTC4 ran down in the presence of LiCl (A and B), pre-exposure to 10 mm inositol for a few minutes prior to stimulation rescued the oscillations in [Ca2+]i . Interestingly, addition of inositol to control cells prior to stimulation with LTC4 in the presence of external Ca2+ resulted in an increase in the frequency of oscillations in [Ca2+]i after agonist was added (B and C).In the blowfly salivary gland, a seminal study by Fain and Berridge  has demo of LiCl , A and B2+]i in Li+-treated cells, Ca2+ entry through CRAC channels was still required to sustain the oscillatory response. Exposure to the CRAC channel blocker La3+ accelerated the rundown of cytoplasmic Ca2+ oscillations in Li+-treated cells despite the presence of inositol . Similar results were obtained when BTP2 was used to block the CRAC channels instead (E and F).Although inositol rescued oscillations in [Ca2+]i in Li+-treated cells when CRAC channels are blocked, despite the presence of inositol, could reflect a role for Ca2+ entry in the synthesis of PIP2 from inositol. Alternatively, loss of the oscillatory response might simply arise from compromised store refilling. To distinguish between these possibilities, we took advantage of a protocol that enables store refilling to occur but in the absence of Ca2+ entry through CRAC channels. Cytoplasmic Ca2+ oscillations can be sustained in the absence of external Ca2+ when Ca2+ extrusion across the plasma membrane is inhibited, for example with high (millimolar) concentrations of La3+ . Preincubation with LiCl led to rundown of the cytoplasmic Ca2+ oscillations (H). However, inositol no longer rescued the oscillatory response in Li+-treated cells . These results, therefore, reveal a major role for Ca2+ influx through CRAC channels in replenishment of the PIP2 pool from inositol.The rundown of oscillations in i G. Preincllations H. Howeve2+ influx through CRAC channels helps replenish PIP2 levels during cytoplasmic Ca2+ oscillations. This would mean that significant amounts of PIP2 are hydrolyzed following stimulation with LTC4 and that, therefore, phosphatidylinositol 4- and 5-kinases should be active during stimulation. A simple prediction is therefore that inhibition of these kinases should accelerate the rundown of the oscillations in [Ca2+]i because of the loss of PIP2 production. At relatively high concentrations, wortmannin inhibits phosphatidylinositol 4- kinase . The number of Ca2+ oscillations that were triggered by LTC4 in the presence of wortmannin  was similar to the number seen in Ca2+-free solution (L). A previous study reported that wortmannin inhibited store-operated Ca2+ entry through inhibition of phosphatidylinositol 4-kinase and, thereby, depletion of PIP2 (2+ influx was seen when wortmannin pretreatment was combined with exposure to the agonist methacholine to ensure PIP2 depletion. Pretreatment with wortmannin (20 μm) but in the absence of methacholine had no inhibitory effect on Ca2+ entry (2+ oscillations evoked by LTC4 in the presence of wortmannin could be rescued by exogenous PI4P (B and C), consistent with an action by wortmannin on phosphatidylinositol 4-kinase. Wortmannin also blocks phosphatidyl inositol 3-kinase. However, pretreatment with the PI 3-kinase inhibitor LY294002 did not mimic the inhibitory effect of wortmannin on oscillations evoked by LTC4 .The preceding results suggest that Ca- kinase , 30 and,solution L. A prev of PIP2 . In that2+ entry . These d2+ entry . Further2+]i evoked by LTC4. To test this more directly, we applied purified PI4P to Li+-treated cells to see whether this could prevent rundown of the cytoplasmic Ca2+ oscillations. In control cells not treated with LiCl, exposure to PI4P led to a small increase in the number of oscillations in [Ca2+]i induced by LTC4 (D). Pre-exposure to PI4P prevented the rundown of the oscillations in [Ca2+]i in Li+-treated cells (E). By contrast, the histone carrier for PI4P failed to rescue the oscillations in [Ca2+]i in Li+-treated cells (F). Blocking of CRAC channels with BTP2 prevented the rescue of the cytoplasmic Ca2+ oscillations by PI4P in Li+-treated cells (G), demonstrating that Ca2+ influx was still important for maintaining the response despite the presence of PI4P. Aggregate data for the various conditions are summarized in Fig. 4I. Importantly, as was the case with inositol (H), PI4P failed to rescue cytoplasmic Ca2+ oscillations evoked by LTC4 when agonist was applied to Li+-treated cells in Ca2+-free external solution containing 1 mm La3+ (H and I). Rescue of oscillations in [Ca2+]i in Li+-treated cells by PI4P therefore requires Ca2+ influx and places the Ca2+-dependent site downstream of PI4P production.The results described above with wortmannin suggest that PI4P production is important for sustaining oscillations in i evoked by LTC4 in the presence of external Ca2+ was abolished (H and J). Similar results were observed when PIP5 kinase 1γ was knocked down instead (I and J), suggesting redundancy between these isoforms. Knockdown of either PIP5 kinase isoform had only a modest effect on responses in Ca2+-free solution (K–M). Neither the store Ca2+ content, as measured by the response to thapsigargin in Ca2+-free solution, nor the rate of store-operated Ca2+ influx were affected by knockdown of either PIP5 kinase isoform (N and O).PI4P is converted to PIP, and 1γ , 34. RT-, and 1γ , A and Bhemistry , C and Drn blots , E and Fpression , C–F. Knce versa G. Followbolished , H and J instead , I and Jsolution , K–M. Ne isoform , N and O2+]i in Li+-treated cells challenged with LTC4 in the presence of external Ca2+ run down within a few minutes (A and B) but can be rescued by PI4P (E and I). To see whether this rescue required conversion of PI4P to PIP2, we knocked down either PIP5 kinase 1α or 1γ and examined whether this prevented PI4P from rescuing the cytoplasmic Ca2+ oscillations evoked by LTC4. In Li+-treated cells in which PIP5 kinase 1α or 1γ had been knocked down, oscillations in [Ca2+]i ran down within 5 min (A–C). However, under these conditions, application of PI4P no longer rescued the cytoplasmic Ca2+ oscillations (D–F).Oscillations in i evoked by LTC4 ran down considerably more quickly following talin1 knockdown (C and D). Neither store-operated Ca2+ influx, measured by stimulating cells with thapsigargin (E), nor Ca2+ release in the absence of Ca2+ influx (F) were impaired following talin1 knockdown. Pre-exposure to either inositol (G) or PI4P (H) did not rescue cytoplasmic Ca2+ oscillations to LTC4 following knockdown of talin1. Aggregate data from several such experiments are summarized in Fig. 7I.To identify a potential mechanism linking Caskeleton . There a5 kinase , 36. Tal calpain , providitectable A. Immunonockdown B. Oscillnockdown , C and Dsigargin E, nor Ca+ influx F were iminositol G or PI4P or PI4P H did not2+ signaling is affected by the presence of caveolin-1 and is disrupted by removal of cholesterol from the plasma membrane by methyl-β-cyclodextrin (MβCD), suggesting the leukotriene receptors signal within lipid raft domains . Oscillations in [Ca2+]i evoked by LTC4 were abolished by MβCD (B and C). However, ATP still elicited a large Ca2+ transient in the presence of MβCD (D).In RBL-1 cells, stimulation of P2Y receptors with ATP generates InsPeceptors . To test by MβCD A. Oscill by MβCD , B and C of MβCD D.2+ oscillations evoked by LTC4 had run down in Li+-treated cells, we applied ATP to see whether Ca2+ release could still be evoked. A robust Ca2+ transient occurred (E and G), revealing that P2Y receptors were able to couple to phospholipase C under conditions where cysLT1 receptors could not.After cytoplasmic Caoccurred , E and G2 resynthesis during the interval between LTC4 exposure and ATP stimulation, we pretreated cells with both LiCl and wortmannin. After oscillations in [Ca2+]i following challenge with LTC4 had run down (F), subsequent exposure to ATP elicited robust Ca2+ release (F and G).To ensure that there was no PIPrun down F, subseq release , F and G2+ entry through CRAC channels in regulating PIP2 replenishment and, therefore, in supporting oscillations in [Ca2+]i following physiological levels of cysLT1 receptor stimulation. A schematic summarizing our main findings and the sites of intervention is shown in Our findings reveal a role for Ca2+ oscillations evoked by LTC4 ran down in the presence of Li+, an inhibitor of inositol monophosphatases (2 resynthesis was required for maintaining the Ca2+ response. Direct application of either inositol or phosphatidylinositol 4-phosphate was able to rescue the oscillatory Ca2+ response in the presence of Li+, consistent with an action of Li+ on inositol monophosphatase. However, recovery of the oscillations in [Ca2+]i evoked by LTC4 was prevented by block of CRAC channel activity. Our results suggest that Ca2+ influx is required to replenish the PIP2 pool that is targeted by cysLT1 receptors. Phosphatidylinositol 4-phosphate is phosphorylated to PIP2 by three isoforms of the type I PIP5 kinase (2+ oscillations evoked by LTC4. The simplest explanation for our results is, therefore, that Ca2+ entry through CRAC channels stimulates PIP5 kinase α/γ to convert phosphatidylinositol 4-phosphate to PIP2 and, therefore, ensure adequate levels of the phospholipid for sustained cysLT1 receptor-dependent Ca2+ signaling. Type I PIP5 kinases are regulated by a variety of signals, including small G-proteins such as Rho, Rac, and Arf; phosphatidic acid; phosphorylation by tyrosine kinases; and the cytoskeletal protein talin 1 (2+ dependence, and it is therefore conceivable that Ca2+ entry modulates a regulatory pathway that serves to stimulate PIP5 kinase. Our data using an siRNA-based approach suggest a role for talin1, although how the protein is activated by Ca2+ influx and whether it is actively involved in supporting oscillations in [Ca2+]i or has more of a housekeeping role remains unclear.Cytoplasmic Caphatases , demonst5 kinase  that are5 kinase . RBL-2H3 talin 1 . Many of2+ influx might also stimulate PI transfer from the peripheral ER to the plasma membrane. Recent work has shown that extended synaptotagmins are activated by cytoplasmic Ca2+ and serve to bring the subplasmalemmal ER closer to the plasma membrane, where phosphatidylinositol transfer proteins, including Nir2, shuttle PI from the ER to the plasma membrane (2 replenishment under our conditions, our data suggest that the key step that is regulated by Ca2+ influx following cysLT1 receptor activation is the conversion of phosphatidylinositol 4-phosphate to PIP2 by PIP5 kinase, a reaction that takes place mainly in the plasma membrane.Camembrane . Althoug3-dependent cytoplasmic Ca2+ oscillations can be altered by varying the rate or extent of Ca2+ entry (4 can also be increased by supply of exogenous inositol or PI4P. This suggests that PIP2 availability is rate-limiting. Providing more precursor accelerates its replenishment, and this increases the frequency of oscillations in [Ca2+]i.The frequency of InsP2+ entry . We now 2 within the plasma membrane (2+]i evoked by cysLT1 receptor activation ran down quickly in the presence of Li+ and wortmannin, which inhibit inositol monophosphatases and PI4P kinases, respectively. Nevertheless, subsequent stimulation of P2Y receptors, which also couple to phospholipase Cβ, elicited Ca2+ release that was similar in size and kinetics to responses obtained in the absence of the inhibitors. Therefore, P2Y receptors access a PIP2 pool that is distinct from that utilized by cysLT1 receptors.Growing evidence points to the existence of local pools of PIPmembrane . Our fun2+ entry through CRAC channels in maintaining a plasma membrane pool of PIP2 that is accessible to cysLT1 receptors. Ca2+ influx, therefore, has two important roles in controlling cytoplasmic Ca2+ oscillations: maintaining PIP2 levels and refilling the intracellular Ca2+ stores. The former will be important in initiating each oscillation in [Ca2+]i, whereas the latter will help set the interspike interval. Both will affect spike frequency but through distinct mechanisms. Stimulation of PIP2 production constitutes an interesting autoregulatory mechanism through which CRAC channels will be able to sustain their activity by ensuring that there are sufficient PIP2 levels in the plasma membrane to produce InsP3 and, therefore, store depletion, which is required for the opening of the channels. How Ca2+ influx activates PIP5 kinase and whether this is driven by local Ca2+ entry requires further investigation.In summary, our findings identify a role for CaA. A. and A. B. P. designed the study. A. A. performed and analyzed the experiments. A. B. P. wrote the manuscript. Both authors approved the final version of the manuscript.


Their complete structural assignments were elucidated by comprehensive spectroscopic investigation. Quantum chemistry calculations were used in support of the structural determination of 1. The absolute configuration of 1 was determined by calculation of its optical rotation. Cladosporilactam A (1) was the first example of 7-oxabicyclic[6.3.0]lactam obtained from a natural source. Compound 1 exhibited promising cytotoxic activity against cervical cancer HeLa cell line with an IC50 value of 0.76 μM.One new bicyclic lactam, cladosporilactam A ( Cladosporium fungi have been known to produce various secondary metabolites with novel structures and interesting biological activities such as cytotoxic sporiolides A and B lactam. Compound 1 represents the first example of bicyclic lactam characterized with the bicyclic[6.3.0]lactam nucleus structure from a natural source. The carbon framework of 1 was similar to the linear chain amide product, fuscoatramide, isolated from the fungus Humicola fuscoatra 20D −50.1° ; UV (MeOH) λmax (log ε): 213 (2.89) nm; IR (KBr) maxν 2931, 2364, 1658, 1611, 1428, 1114 cm–1; 1H and 13C NMR data, see m/z 385.4 [2M + Na]+, 220.2 [M + K]+, 204.2 [M + Na]+, 182.2 [M + H]+   . 2). White, amorphous solid; [α]20D +18.7°  [lit. 11: +26.9° ].Cladospolide B (3). White, amorphous solid; [α]20D +32.0°  [lit. 12: +41.9° ].Dendrodolide A (4). Colorless oil; [α]20D +98.4°  [lit. 12: +122° ].Dendrodolide C (5). Colorless oil; [α]20D +85.8°  [lit. 12: +92.6° ].Dendrodolide M (6). White, amorphous solid; [α]20D +23.6°  [lit. 12: +10.7° ].Dendrodolide L (iso-Cladospolide B (7). White, amorphous solid; [α]20D −106.2°  [lit. 13: −90° ].1–7 against mouse lymphocytic leukemia P388 and human lung carcinoma A549 cell lines were evaluated using the SRB method lactam (