Cmpt–dl1Abc, Cmpt) labeled by a tremendous increase in body weight along with signs of muscle weakness, easier fatigability, decreased Orai1 expression and store operated calcium entry (SOCE). Here, on the one hand, Cmpt fibers were reconstructed with venus-Orai1 but this failed to restore SOCE. On the other hand, the endogenous Orai1 was silenced in fibers from wild type C57Bl6 mice which resulted in ∼70% of Orai1 being silenced in whole muscle homogenates as confirmed by Western blot, accompanied by an inhibitory effect on the voltage dependence of SR calcium release that manifested in a slight shift toward more positive potential values. This maneuver completely hampered SOCE. Our observations are consistent with the idea that Orai1 channels are present in distinct pools responsible for either a rapid refilling of the SR terminal cisternae connected to each voltage-activated calcium transient, or a slow SOCE associated with an overall depletion of calcium in the SR lumen. Furthermore, when Cmpt cells were loaded with the mitochondrial membrane potential sensitive dye TMRE, fiber segments with depolarized mitochondria were identified covering on average 26.5 ± 1.5% of the fiber area. These defective areas were located around the neuromuscular junction and displayed significantly smaller calcium transients. The ultrastructural analysis of the Cmpt fibers revealed changes in the mitochondrial morphology. In addition, the mitochondrial calcium uptake during repetitive stimulation was higher in the Cmpt fibers. Our results favor the idea that reduced function and/or expression of SOCE partners (in this study Orai1) and mitochondrial defects could play an important role in muscle weakness and degeneration associated with certain pathologies, perhaps including loss of function of the neuromuscular junction and aging.In mice a naturally occurring 12-bp deletion in the myostatin gene is considered responsible for the compact phenotype (Mstn The very low resting myoplasmic Ca2+ concentration from the nanomolar (nM) range rises rapidly into the micromolar (μM) range through the mechanism termed “excitation-contraction (EC) coupling” during which the action potential triggers Ca2+ release from the intracellular store (sarcoplasmic reticulum (SR)) mediated by coupling between the voltage sensor dihydropyridine receptor (DHPR) in the transverse tubule membrane and the ryanodine receptor (RyR1), Ca2+ release channel embedded in the SR membrane  during which Ca2+ enters into the cytoplasm from the extracellular space to replenish the depleted intracellular stores. SOCE is coordinated by two key proteins: stromal interaction molecule 1 (STIM1) and Orai1. The former is the luminal Ca2+ level sensor in the SR membrane, while the latter is the Ca2+-release activated calcium (CRAC) channel located in the surface membrane ; In our former studies a mouse strain with a naturally occurring mutation in the myostatin gene i was calculated as ΔF/F0, where ΔF is the fluorescence intensity change and was calculated over the cell, whereas F0 is the background fluorescence and was calculated in a region next to the cell. The ratio of P2/P1 over time was then calculated and plotted.All calcium measurements were carried out on single skeletal muscle fibers from the FDB muscles of the mouse. SOCE measurement was performed similarly, as in our earlier report . Brieflyexternal solution and voltage clamped . Changes in cytosolic Ca2+ were recorded, following the application of depolarizing voltages and simultaneously imaged using a confocal microscope . Fibers were dialyzed through the patch pipette with the rhod-2 or fluo-8 and 10 mM EGTA containing internal solution and the application of depolarizing pulses was started 15–20 min following the seal formation to assure enough loading time. The ambient temperature was set to 20–22°C. The holding potential was −80 mV and the pipette resistance varied between 2 and 4 MΩ. Correction for linear capacitive currents was performed by analog compensation.The experimental design was similar to the one described in our earlier study , 2020. BThe voltage dependence of activation was described by Boltzmann function:50 and limiting logarithmic slope 1/k.to derive the transition voltage V2+ transients were analyzed by an in-house custom-made program taking into account the dissociation constant for rhod-2 (Kd rhod–2 = 1.58 μM). The SR Ca2+ content was calculated taking into account the SERCA pump activity, the calcium binding to intracellular buffers as troponin-C, EGTA, parvalbumin and the dye as reported earlier  were performed as follows: a line was drawn parallel to the longitudinal axis of the fiber and the fluorescence was calculated at the peaks ) and at troughs . Relative mitochondrial Ca2+ uptake expressed as Fmito was calculated as FI–band – FA–band and then normalized to the baseline fluorescence.Activity-dependent changes in mitochondrial Ca0 values were obtained and plotted at close to maximal depolarizations. Single exponential functions were used to fit the time course of the changes of the maximal F/F0 values during the series of tetanic depolarizing pulses as described earlier  single exponential functions were used to fit the time course of the changes of the maximal F/F0:Images of rhod-2, fluo-8, TMRE and Alexa Fluor 488 α-bungarotoxin fluorescence were acquired with a laser scanning confocal microscope  equipped with a 20x air and a 40x oil objectives. Excitation of rhod-2 was done 543 nm, with emission collected above 550 nm with a long pass filter whereas for fluo-8 the excitation wavelength was 488 nm, the emission was at 520 nm. Line-scan image recordings were synchronized with the application of the depolarizing pulses via pClamp 11.0  and the protocol used is always indicated on the figure. The time resolution was 0.5 ms per line whereas the spatial resolution was 0.24 μm/line. Peak F/F earlier , 2020. P0 is the difference between the total and the remaining SR calcium content following the 8th tetanic pulse.This then enabled the estimation of SR calcium content as described in our earlier report . In the The Western blot technique was applied to detect expression levels of Orai1. Protein samples were prepared from whole FDB muscle homogenates as well as homogenates of isolated single FDB muscle fibers using standard methods of BCA Protein Assay Kit . Protein samples were subjected to SDS-PAGE using 10% precast gels . 20 μg protein per lane was loaded in each case and separated proteins were transferred to 0.2 nitrocellulose membranes (Bio-Rad Laboratories). For immunological detection of Orai1 mouse monoclonal anti-Orai1  was used in 1:500 dilution as primary antibody. As for secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG  was used in a 1:1000 dilution and this was visualized by chemiluminescence reaction  using LAS-3000 Intelligent Dark Box . For immunolabelling experiments on Cmpt fibers, we marked the endogenous Orai1 protein with Alexa-Fluor Plus 488 secondary antibody (Invitrogen cat. No. A11001) Densitometry of the signals was performed by using ImageJ software (NIH) and optical densities were calculated as normalized to control samples. To prove equal loading, membranes were re-probed with anti–β-actin antibody  or vinculin  and visualized as described above.The plasmid venus-Orai1 was kindly provided by R. Dirksen . The GFP tagged Orai1 shRNA and scrambled shRNA cassettes in pGFP-V-RS vector were purchased from Origene Technologies (cat. No. TG510804). The plasmids were amplified using bacterial cultures grown in the presence of selective antibiotics . Plasmid DNA preparations were obtained using Endofree Plasmid Kits (Qiagen Inc).in situ with fixative solution  (FDB muscles were fixed  buffer) . Small bKreb’s solution (in mM): 135 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 10 Hepes, 10 glucose, 10 NaHCO3; pH 7.2.Normal Tyrode’s solution (in mM): 137 NaCl, 5.4 KCl, 1.8 mM CaCl2, 0.5 MgCl2, 11.8 HEPES; 1 gL–1 glucose; pH 7.4.2+ free Tyrode’s solution:Ca same as above except 5 mM EGTA was added and no CaCl2 was present.cocktail” consisted of 0.4mM 4-CmC, 0.004mM thapsigargin, and 0.05 mM N-benzyl-p-toluene sulphonamide.The “External solution (in mM): 140 TEA-CH3SO3, 1 CaCl2, 3.5 MgCl2, 10 Hepes, 1 4-AP, 0.5 CdCl2, 0.3 LaCl3, 0.001 TTX (citrate), and 0.05 BTS (N-benzyl-p-toluene sulphonamide). pH was adjusted to 7.2 with TEA-OH and osmolality was adjusted to 320 mOsm with TEA methanesulfonate.Internal solution (in mM): 110 N-methylglucamine, 110 L-glutamic acid, 10 EGTA, 10 Tris, 10 glucose, 5 Na2 ATP, 5 PC Tris, 0.5 rhod-2, 3.56 CaCl2, and 7.4 mM MgCl2 were added for a nominal 1 mM [Mg2+] and 100 nM [Ca2+]. pH was set to 7.2 with NaOH and osmolality to 320 mOsm with N-methylglucamine.Fluorescent dyes  were purchased from Invitrogen . Fluo-8 tripotassium salt was from AAT Bioquest (cat. no. 21087). All other chemicals were purchased from Sigma-Aldrich .t-test for two independent populations was used for calculating statistical significance (p < 0.05).Pooled data were expressed and plotted as mean ± standard error of the mean (SEM). Student’s two-tailed in vivo electroporation. Five days after the procedure the rate of transfection efficiency was confirmed in FDB fibers by the green fluorescence of the venus-Orai construct  tagged with GFP against Orai1 was introduced into FDB muscles via electroporation. Whole FDB muscles and ∼30 individual fibers identified via their green fluorescence from four different animals were collected and analyzed by Western blot. The representative immunoblot in 2+), which was exchanged manually to a Ca2+-free Tyrode’s solution (0 mM Ca2+) while continuously imaging the fiber using a confocal microscope with the appropriate laser line. Application of a cocktail  lead to a rapid increase in dye fluorescence due to Ca2+ release via RyR1s from the SR (marked as P1). After washout and following the re-addition of 1.8 mM Ca2+ concentration in the external solution, a slowly activated secondary increase in fluorescence was observed after the SR store depletion as a result of Ca2+ influx via activated Orai1 channels  normalized to RyR1-mediated Ca2+ transient induced by the depleting-cocktail (P1) revealed a slight increase of SOCE in Cmpt animals reconstructed with venus-Orai1  reflects the actual SOCE in that particular fiber, P2/P1 would normalize the SOCE to SR depletion making comparisons across fibers more appropriate.We continued the functional studies by examining the possibility of altered coupling between the DHPR and RyR1 in single FDB fibers combining whole cell patch clamp and confocal imaging. 2+-release activation in WT silenced fibers was found to be slightly shifted to more negative potentials as underlined by the V50 of release, however, this proved to be not different statistically.0 values obtained at maximal depolarizing pulses (+30 mV) are presented in 0 in WT+SCR (dark red) and silenced cells (green) we found slightly increased but statistically unaltered peak values (2.65 ± 0.38 vs. 3.38 ± 0.41) which suggests that the global voltage activated cytosolic Ca2+-transients were not affected by the lack of Orai1.2+ content during repetitive stimulation in different fibers. The amount of calcium released decreased by the removal flux was used to calculate the decline in SR Ca2+ content, which covers the uptake of Ca2+ into the SR by the SERCA pump  is precisely related to the flux (K1) through Orai1 channels. The data points from Earlier a simple model including three compartments  calculated as (FI–band – FA–band)/FA–band was compared in WT and Cmpt FDB fibers at rest and following tetanic stimulation. The fluorescence was averaged over the spatial domain along a line (white trace) placed in parallel with the fiber’s orientation. Typical traces are shown in mito values were already higher at rest in the mutant when compared to WT  and significantly increased following the first and the fifth tetanic stimulation . The pooled data  and defective (d) area  smaller calcium transients were observed in regions displaying loss of mitochondrial membrane potential  area . Figure  regions  and a cootential . On averp < 0.05 .2+ stores, not only during repetitive pronlonged tetanic stimulation, but also following fast activation  to the levels measured in WT fibers, while in fibers, where Orai1 has been silenced, no significant alterations of Ca2+ fluxes have been detected. This phenomenon could be explained by the assumption of two pools of Orai1 channels . One pool is located within the triad in a pre-assembled formation with STIM1L and is responsible for the rapid activation of SOCE in the nanodomain of the junctional membrane, adjacent to the RyR1s, during an action potential (phasic SOCE). Because of their occurrence in pairs with STIM1, we refer to this pool as “mated” Orai1 channels. The other pool is distributed along the t-tubule membrane randomly  increased significantly as compared to untreated Cmpt fibers, denoting an increased number of “mated” Orai1 channels. Acute depletion of Orai1 channels in wild type muscles eventuates in a different response, indicating that the “solitary” pool of Orai1 has been concerned. In these fibers slow SOCE has been abolished almost completely which might reflect to a reduced “solitary” Orai1 pool, however, the K1 parameter has been found to be similar as in WT, that is, the “mated” Orai1 pool has been untouched.On the other hand, reconstructing the Cmpt fibers with venus-Orai1 resettle the Caes place , but in 2+ entry pathway  mitochondria support SOCE activity through their ability to buffer and release Ca2+ , and ii)of ICRAC . Althougof ICRAC . This co2+ signaling  production . While tignaling . ROS provia RyR1 . The rel2+] and thereby become depolarized  , corruptolarized . Filadi The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Care Committee of the University of Debrecen.in vitro calcium experiments. NB did the Western Blot experiments. GK did the electron microscopy. MS, PS, ZS, ÁA, and NB analyzed the data. MS and PS did the statistical analysis. All authors contributed to discussion, laboratory support, and reviewed/edited the manuscript.MS, BD, IB, PS, and LC wrote the manuscript, contributed to discussion and reviewed and edited the manuscript. MS, ZS, and BD did The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nicotine induces a concentration-dependent increase in [Ca2+]i. The elimination of Ca2+ from extracellular fluid or intracellular stores demonstrated that the nicotine-induced [Ca2+]i elevation was due to extracellular influx and intracellular mobilization. The effects of tubocurarine on nicotine-induced [Ca2+]i elevation and current suggest that intracellular mobilization is caused by plasma membrane-permeating nicotine. The inhibition of α3 *, α5 *, α7 nAChR and voltage-gated Ca2+ channels by using siRNAs and selective antagonists revealed the involvement of these nAChR subunits and channels in nicotine-induced [Ca2+]i elevation. To distinguish and characterize the α3 * and α5 * nAChR-mediated Ca2+ influx, we measured the [Ca2+]i elevation induced by nonmembrane-permeating acetylcholine when muscarinic receptors, α7nAChR and Ca2+ channels were blocked. Under this condition, the [Ca2+]i elevation was significantly inhibited with a 48-h treatment of dibutyryl cAMP, which was accompanied by the downregulation of α3 and β4 mRNA. These findings suggest that α3 * and α5 * nAChR-mediated Ca2+ influx is possibly regulated by cAMP at the transcriptional level.The pathway from the medial habenular nucleus to the interpeduncular nucleus, in which nicotinic acetylcholine receptor (nAChR) including the α3 and α5 subunits (α3 * and α5 * nAChRs) are expressed, is implicated in nicotine dependence. We investigated whether α3 * and α5 * nAChRs are regulated by cAMP using SH-SY5Y cells to clarify the significance of these receptors in nicotine dependence. We analyzed the nicotine-induced elevation of intracellular Ca The influx of Ca2+ triggers a variety of intracellular signaling events and the release of neurotransmitters from nerve terminals i) elevation because both α3 * and α5 * nAChRs have calcium ion permeability [2+]i elevation and the cAMP-mediated regulation of these nAChRs using SHSY-5Y cells, in which both α3 * and α5 * nAChRs were endogenously expressed i was determined by setting the fluorescence intensity to 100% before administration of nicotine or acetylcholine  and determined the average fluorescence change in these regions. The time-dependent change in intracellular calcium ion i elevation in SH-SY5Y cells after nicotine administration. As shown in 2+]i was observed when nicotine at concentrations between 100 μM and 500 μM was administered.We investigated the [Ca2+ involved in the nicotine-induced intracellular [Ca2+]i elevation observed in SH-SY5Y cells. First, we examined whether nicotine (500 μM)-induced intracellular [Ca2+]i elevation occurs when extracellular Ca2+ was eliminated. As indicated by the light blue line in 2+ did not remarkably affect the nicotine-induced intracellular [Ca2+]i elevation, suggesting that nicotine-induced intracellular [Ca2+]i elevation may also be critical for intracellular Ca2+ mobilization.We examined the origin of Ca2+]i levels were observed when thapsigargin was administered in the presence of extracellular Ca2+. In this state, when intracellular Ca2+ stores were depleted, nicotine (500 μM) induced an increase in [Ca2+]i, which is thought to be the result of extracellular Ca2+ influx. The black line indicates that a nicotine-induced [Ca2+]i elevation was also observed when the buffer was administered alone as a vehicle. Then, as indicated by the purple line in 2+-free buffer was used to eliminate the extracellular Ca2+, and thapsigargin (5 μM) was administered to deplete the intracellular Ca2+ stores. Depletion of intracellular and extracellular Ca2+ completely abolished the nicotine (500 μM)-induced [Ca2+]i increase , an endoplasmic reticulum calcium pump inhibitor. As shown in the purple line in 2+]i elevation was mediated by nAChRs, we examined the effects of the nonselective nAChR antagonist tubocurarine. We first examined effects of tubocurarine on nicotine-induced currents using whole-cell recording. As shown in To elucidate whether nicotine-induced intracellular [Ca2+]i elevation. As shown in 2+]i elevation, which suggests that [Ca2+]i elevation is largely mediated by nAChRs expressed in plasma membrane. Unexpectedly, the small, but apparent [Ca2+]i elevation was still remained in the presence of tubocurarine (2 +]i elevation.Next we examined effects of tubocurarine on the nicotine (250 μM)-induced [Cacurarine . Because2+]i elevation compared with the control in siRNA-transfected cells. The small interference RNA for the α3 nAChR subunit tended to decrease the nicotine-induced [Ca2+]i elevation, but not significantly. Since β4 nAChR functions as a core subunit of α3 * and α5 * nAChRs, these results suggest the involvement of β4-containing nAChRs, such as α3β4 and α5β4 nAChR, in nicotine-induced [Ca2+]i elevation. This idea is supported by the finding showing that α-conotoxin AuIB (10 μM), an α3β4 nAChR-specific inhibitor, significantly inhibited the increase in nicotine (500μM)-induced [Ca2+]i level , a specific α7 nAChR inhibitor, tended to inhibit nicotine (250 μM)-induced [Ca2+]i elevation but not significantly -induced [Ca2+]i elevation. As shown in 2+]i elevation, simultaneous administration of both inhibitors significantly inhibited the nicotine-induced [Ca2+]i elevation -induced [Ca2+]i elevation in the presence of atropine (1 μM). As shown in 2+]i. As shown in 2+]i increase. These results suggest that the voltage-gated Ca2+ channel and α7nAChR are involved in the acetylcholine-induced [Ca2+]i elevation in the presence of atropine.We next examined whether voltage-gated Ca2+]i elevation in SHSY-5Y cells, we suggest the following hypotheses -induced [Ca2+]i elevation in the presence of atropine (1 μM), MLA (5 μM), nifedipine (5 μM) and ω-conotoxin GVIA (1 μM). We designated the solution containing atropine (1 μM), MLA (5 μM), nifedipine (5 μM) and ω-conotoxin GVIA (1 μM) an inhibitor mix. We consider acetylcholine-induced [Ca2+]i elevation in the presence of this inhibitor mix as an α3 * and α5 * nAChR-mediated outcome of Ca2+ influx.In contrast to nicotine, nonmembrane-permeable acetylcholine induces [Ca2+]i elevation mediated via α3 * and α5 * nAChRs in the plasma membrane were investigated upon short-term (15 min) or long-term (48 h) treatment of SH-SY5Y cells with dbcAMP (1 mM), a membrane-permeable cAMP analog. As shown in 2+]i elevation induced by acetylcholine (10 μM) and the inhibitor mix, whereas 1 mM dbcAMP treatment for 48 h significantly inhibited the increase in[Ca2+]i level.The effects of cAMP on [Ca2+]i increase, we analyzed the mRNA expression of the α3, α5 and β4 subunits of the nAChRs, which are expressed in SH-SY5Y cells, using real-time PCR. As shown in 2+ through the α3 * β4 * nicotinic receptor.To investigate the mechanism by which the 48-h treatment with 1 mM dbcAMP significantly inhibited the acetylcholine and inhibitor mix-induced [Ca2+]i elevation in SH-SY5Y cells in a concentration-dependent manner , the main intracellular Ca2+ store. It has been reported that α7nAChR expressed in microglia activates PLC and releases Ca2+ from IP3 receptors in the endoplasmic reticulum i elevation induced by acetylcholine and inhibitor mix has not been quantitatively demonstrated. Further studies and methodological breakthroughs are needed. Another limitation is that the concentration of nicotine used in this experiment was relatively high. It is debatable whether the phenomena observed in this study reflect the responses of nicotine under physiological conditions. However, our previous studies have shown that isolated neurons require relatively high concentrations of nicotine to produce nicotine-induced responses Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this article entitled “Component of nicotine-induced intracellular calcium elevation mediated though �3- and �5-containing nicotinic acetylcholine receptors are regulated by cyclic AMP in SH-SY 5Y cells”, Takahashi and colleagues have evaluated the mechanistic aspects of regulation of intracellular Ca2+ ([Ca2+]i) by nicotine acetylcholine receptors (nAChRs). The authors tested nicotine and acetylcholine dependent elevation of [Ca2+]i levels as well as effect of cAMP using SHSY-5Y cell line. The authors observed nicotine and acetylcholine induced increase in [Ca2+]i levels, that nicotine dependent Ca2+ elevation was a result of both extracellular and intracellular mobilization of Ca2+ ions and that dibutyryl cAMP (dbcAMP), a cAMP analog, could suppress transcription of α3 and β4 subunits of nAChR.In general, the results shown agree with the conclusions drawn and data has been presented in a clear and easily understandable manner. However, the authors need to address the issues described below:1) In section 2-3, effect of tubocurarine was tested on [Ca2+]i levels of cells treated with 250 µM nicotine. A complete inhibition of inward currents was observed while [Ca2+]i was significantly reduced but not complete. Could the authors comment on why 250 µM nicotine was used rather than 500 µM. This is important since (a) in Section 2-2 , to check the source of [Ca2+]i the authors used 500 µM nicotine and (b) effect of 500 µM nicotine on [Ca2+]i  was much more pronounced compared to 250 µM. Verifying the effect of 500 µM nicotine is therefore important, especially on the current.2) Based on Fig. 1A, expression of α7 in SHSY-5Y cells is significantly lower than α3 and α5, which raises the question whether this cell line is an ideal system for studying the involvement of α7nAChR in regulating [Ca2+]i levels.Grammatical errors / typos:1) Typo in the title of the article “though” should be replaced by “through”.2) Page 12, Line 4 – Fig. 4E has been mistakenly typed as Fig. 2EReviewer #2: Takahashi et al. investigate the role of nicotine and specific nicotinic acetylcholine receptor (nAChRs) subunits on extracellular and intracellular calcium mobilization. Nicotine is involved in signaling in the central nervous system and is known to mediate addictive behavior. Thus, it is important to study the underlying effects of nicotine in the CNS using a cell culture model (SH-SY5Y).The authors used various molecules that release intracellular calcium stores, including agonists and antagonists of nAChRs, muscarinic AChRs and voltage-gated calcium channels (VGCCs). The authors ultimately identified that the alpha-7 subunit of nAChR was important in mediated calcium mobilization. The authors proposed that nicotine affects cellular calcium response at the plasma membrane level through calcium influx through VGCCs. They also suggested that nicotine, being membrane-permeable, can induce intracellular calcium release through an unknown mechanism based on their results.Overall, the strength of this study is the thoughtful and logical approaches they have implemented to answer their questions. Appropriate controls were included in all experiments. The authors also addressed the limitations of the study and discussed their results appropriately.Issues that need to be addressed to improve the manuscript:Page 3, Line 23: The authors should define “Gs” before using the abbreviation here or elsewhere.Page 5, Line 24: For real-time qPCR using the Livak method (2^-deltadelta Ct), the authors should follow the MIQE (Minimum Information required for publication of Q-PCR Experiments) guidelines , Clin Chem, 55:611-622) by providing the primer efficiencies and R-squared of the PCR.Page 7, Line 13: The authors should indicate how many independent trials were done, and how many cells were voltage-clamped to arrive with the data they presented on Fig. 3A.Pages 24-28, Figure Legends: The authors should consistently indicate or mention the independent trials or cell numbers if applicable. For example, on figure 1, the legend does not provide experimental trials for the left panel of Fig. 1B, but they show on the right panel n=6 for 100 uM, n=4 for 250 uM, n=4 for 500 uM). It is important to mention the trials every time especially that they mentioned that the data are represented as means +/- SEM.Also, the same issue apply in other figures where the authors showed %fluorescence or trace, but they did not mention on the Fig Legend the experimental trials done.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  29 Sep 2020Overall revisionWe have added two authors, Takayuki Yoshida and Kana Harada, who did the experiments for revising manuscript.Reply to reviewer #1We appreciate the comments that lead to the improvement of this manuscript.1) In section 2-3, effect of tubocurarine was tested on [Ca2+]i levels of cells treated with 250 µM nicotine. A complete inhibition of inward currents was observed while [Ca2+]i was significantly reduced but not complete. Could the authors comment on why 250 µM nicotine was used rather than 500 µM. This is important since (a) in Section 2-2 , to check the source of [Ca2+]i the authors used 500 µM nicotine and (b) effect of 500 µM nicotine on [Ca2+]i  was much more pronounced compared to 250 µM. Verifying the effect of 500 µM nicotine is therefore important, especially on the current.According to the reviewer’s comment, we investigated the effects of tubocurarine on the currents induced by 500 µM nicotine. As shown in revised figure 3A, 100 µM tubocurarine almost completely suppressed the current induced by 500 µM nicotine as well as the currents induced by 250 µM nicotine as shown in a former figure 3A. In addition, quantitative analysis of tubocurarine effects was added as Figure 3B. A former figure 3A showing the effect of tubocurarine on the 250 µM nicotine-induced current has been presented as a supplemental figure 1.We have observed nicotine-induced calcium elevation at concentrations up to 500 µM. Generally, pharmacological studies using inhibitors and siRNAs are usually performed at intermediate concentrations (preferably at ED50 concentrations) rather than at the highest concentrations tested. Therefore, in this study, we examine the effects of various inhibitors and siRNAs on nicotine-induced calcium elevation at the concentration of 250 µM. However, in the experiments shown in Figure 2, we had to observe a significant nicotine-induced calcium elevation under various conditions, so we used 500 µM nicotine, at the highest concentration of nicotine tested.The description in the main text has been changed in accordance with the change in Figure 3 as follows.Material methods section; page 7, line 16- page 8, line 1, page 8, line6-7 Results section; page 10, line18-24Figure legends; page 25, line 11-20 2) Based on Fig. 1A, expression of α7 in SHSY-5Y cells is significantly lower than α3 and α5, which raises the question whether this cell line is an ideal system for studying the involvement of α7nAChR in regulating [Ca2+]i levels.In this study, the mRNA expression of each nAChR in SHSY5Y cells was examined only once. Therefore, three new cases were examined and the results was presented as a new Figure 1A. Again, α7 expression appears to be lower than α3 and α5 expressions in the graph of new figure 1A. However, the efficiency of the primers to detect each nAChR subunit and the efficiency of the cDNA synthesis of each nAChR subunit in the process of synthesizing cDNA from mRNA are not same between each subunit. Therefore, we cannot simply judge from this graph that the expression of α7 is lower than those of α3 and α5.A PubMed search yielded 88 papers investigating nicotine-induced calcium elevation via the α7 nAChR in SHSY-5Y cells. This fact does not suggest that SHSY-5Y cells are not ideal for studying nicotine-induced calcium elevation via the α7 nAChR.Grammatical errors / typos:1) Typo in the title of the article “though” should be replaced by “through”.2) Page 12, Line 4 – Fig. 4E has been mistakenly typed as Fig. 2EWe corrected these errors in the revised manuscript.Reply to reviewer #2We appreciate the comments that lead to the improvement of this manuscript.-Page 3, Line 23: The authors should define “Gs” before using the abbreviation here or elsewhere.We have changed the description “Gs” to “Gs (stimulatory G protein)”. - Page 5, Line 24: For real-time qPCR using the Livak method (2^-deltadelta Ct), the authors should follow the MIQE (Minimum Information required for publication of Q-PCR Experiments) guidelines , Clin Chem, 55:611-622) by providing the primer efficiencies and R-squared of the PCR.We noticed that the delta Ct method was used in this study, rather than the 2^-deltadelta Ct method, to assess mRNA expression, so we have changed the description to reflect that. Information for the primer efficiencies and R-squared of the PCR have been also described in revised manuscript. And, we re-performed the RT-PCR studies three times because the experiment was done only once in a former manuscript. The new results have been presented as figure 1A.- Page 7, Line 13: The authors should indicate how many independent trials were done, and how many cells were voltage-clamped to arrive with the data they presented on Fig. 3A.We newly investigated the effect of tubocurarine on the current induced by 500 µM nicotine. We performed 8 independent experiments. The descriptions were changed accordingly. Material methods section; page 7, line 16- page 8, line 1, page 8, line6-7 Results section; page 10, line18-24Figure legends; page 25, line 11-20 - Pages 24-28, Figure Legends: The authors should consistently indicate or mention the independent trials or cell numbers if applicable. For example, on figure 1, the legend does not provide experimental trials for the left panel of Fig. 1B, but they show on the right panel n=6 for 100 µM, n=4 for 250 µM, n=4 for 500 µM). It is important to mention the trials every time especially that they mentioned that the data are represented as means +/- SEM.In accordance with the reviewer's suggestion, we have added a description concerning the number of experiments and the representation of the data.  With regard to data representation, we have already described them as “Data represent the means ± standard error”.-Also, the same issue applies in other figures where the authors showed %fluorescence or trace, but they did not mention on the Fig Legend the experimental trials done.Concerning the trace using %fluorescence, we have already described the information in materials methods and figure legends section as bellow. The time-dependent change in intracellular calcium ion [Ca2+]i was determined by setting the fluorescence intensity to 100% before administration of nicotine or acetylcholine. The fluorescence before nicotine administration was assumed to be 100%, and the change in [Ca2+]i after application of nicotine at each concentration is shown AttachmentReply to reviewer.docxSubmitted filename: Click here for additional data file. 2 Nov 2020Component of nicotine-induced intracellular calcium elevation mediated through a3- and a5-containing nicotinic acetylcholine receptors are regulated by cyclic AMP　in SH-SY 5Y cells.PONE-D-20-22474R1Dear Dr. Sakai,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #3: There are no further concern.Authors have improved their manuscript according to previous comments by reviewers.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoYes: Abdul Qadir SyedReviewer #3:  16 Nov 2020PONE-D-20-22474R1 Component of nicotine-induced intracellular calcium elevation mediated through α3- and α5-containing nicotinic acetylcholine receptors are regulated by cyclic AMP　in SH-SY 5Y cells. Dear Dr. Sakai:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! 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MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling—RRAGA, MLST8, WDR24 and LAMTOR2—as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the  Lysine-specific demethylase 1  has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML), as well as in some solid malignancies . Indeed LSD1 knockdown (KD) or LSD1 pharmacologic inhibition promotes differentiation of, in particular, AML cells with chromosomal translocations targeting MLL  dem demMLL gCDC7 and KCNQ4, there was no overlap of the 44 genes with the 369 genes whose sgRNA representation was depleted in both day 18 samples vs. the day 0 sample, suggesting that LSD1 inhibition created novel genetic dependencies rather than exacerbating existing ones. Given our recent finding that inhibitors of LSD1 promote myeloid differentiation in MLL-translocated AML through disruption of the protein:protein interaction of the transcription repressor GFI1 with LSD1 , 1716, 1We also explored transcriptional changes using GSEA and the Molecular Signatures Database hallmark gene set collection, each of which conveys a specific biological state or process and displays coherent expression . Immune-MLL-translocated AML cells from seven separate individuals  translocation , RAD001 (5 mg/kg), OG-98 (3 mg/kg) or a combination of RAD001 (5 mg/kg) and OG-98 (3 mg/kg) for 5 days. OG-98 (2-((trans-2-(4-(benzyloxy)phenyl)cyclopropyl)amino)-1-(4-methylpiperazin-1-yl)ethanone)  Fig.  is an orne) Fig.  and a plne) Fig. . It is ane) Fig.  but withp < 0.05 by one-way ANOVA, mean fold change in expression >1.5 in at least one of the six pairwise comparisons) , there was a significant increase in cell surface expression of the differentiation markers CD11b and CD86 in the combination treated vs. all other cohorts, and in expression of CD86 in OG-98 vs. vehicle-treated mice  Table . Hierarcs) Table  revealeds) Table . GSEA uss) Table  17] dem demp < 0lar Fig. . Leadinglar Fig.  revealedlar Fig. . Expresslar Fig. . As for lar Fig. , immune-lar Fig. ; similarlar Fig. . Thus thlls Fig. , and thenot IRF8 . Indeed ice Fig. . Thus in6 per mouse) and 3 weeks later treated animals with drugs for 4 weeks . The RAD001/OG-98 combination was well tolerated: at the end of treatment there was no significant difference in weight, haemoglobin, total white cell count or neutrophil count   Fig. . Based o51) Fig.  and the 51) Fig. . To eval51) Fig. , S7A. Fl51) Fig. . The clo51) Fig. , and tha51) Fig. . The con51) Fig. .Fig. 4CoMLL-leukaemias we evaluated OG-86 and RAD001 in primary AML cells of additional genotypes in stromal co-culture  in acute promyelocytic leukaemia, are essential components of therapeutic regimens. Induction of differentiation is often coupled with loss of self-renewal in AML; novel combinations that promote differentiation may therefore also target leukaemic stem cells.The choice of which therapeutic combinations to test in clinical trials is often empiric. Genetic screening approaches such as CRISPR hold out promise for more rational decisions. Using a CRISPR-based screening approach with confirmatory KD, pharmacologic and amino acid deprivation experiments we find that JARID2, core polycomb repressive complex 2 components. By contrast, our genetic screen revealed a strong depletion signal for mTORC1 pathway genes.Prior studies have reported additive or synergistic anti-leukaemic activity of drugs in combination with LSD1 inhibition. For example, ATRA in combination with tranylcypromine or related derivatives confers enhanced inhibition of growth, upregulation of differentiation markers or leukaemia cell engraftment in AML cell models , 22, 23,MLL-translocated AML, perhaps following intensive chemotherapy and remission induction, with the goal of inducing differentiation of minimal residual disease may prove beneficial.mTOR signalling is elevated in AML and consequences include inhibition of autophagy and altered cell growth . ClinicaMLL-translocated AML cells reduces activity of GFI1 through physical disruption of the LSD1/CoREST complex with GFI1 on chromatin [Rptor, which codes for the substrate-guiding subunit of the mTORC1 complex, mice develop a modest pancytopenia with increased monocytoid cells in bone marrow and spleen [Rptor knockout phenotype will be key to understanding the synergy between LSD1 and mTORC1 inhibition in MLL-translocated and other AML cells. Of note, we did observe reduced phospho-ERK but not phospho-MEK signalling in AML cells treated with the combination raising a question as to whether the synergy arises at the level of the lysosome where scaffolding of MEK and ERK occurs and where too mTORC1 is located. Additional investigations will be revealing.LSD1 inhibition in hromatin . GFI1 ishromatin . The althromatin . Howeverhromatin . It is id spleen  suggestiSupplemental informationSupplementary Tables


Background: The immunomodulatory enzyme, indoleamine 2,3-dioxygenase (IDO) facilitates tryptophan catabolism at the rate-limiting step of the kynurenine (Kyn) pathway. IDO expression and elevations in Kyn metabolites are associated with immunosuppressive tumor microenvironment including T cell proliferative arrest and generation of regulatory T cells (Tregs) which can favor tumor progression. However, the extent of the role of IDO in acute myeloid leukemia (AML) is currently ill-defined. This study reviews the role of IDO-driven Treg function in AML and evaluates the current body of evidence implicating IDO in AML pathogenesis.Method: Studies related to IDO in AML were identified through a systematic review of PubMed and Scopus. Data extracted described sample analysis, IDO expression, IDO in prognosis, techniques used in Treg phenotypic studies, and the effect of IDO inhibitors.Results: Twenty studies were included in the systematic review. Expression of IDO was identified in a range of cells in AML, both inducible and constitutive. Seven studies indicated an association between elevated expression and poor clinical prognosis. Six studies suggested a positive correlation between IDO expression and Treg induction, with FoxP3 being the prominent Treg phenotypic marker. Of eight studies investigating IDO inhibition, some reported reductions in Treg frequency and enhanced effector T cell proliferation.Conclusion: This review highlights that IDO expression in AML is associated with poor prognosis and measurement of IDO and its Kyn metabolites may offer utility as prospective prognostic markers. Pharmacological inhibition of IDO using novel drugs may hold promise for the treatment of AML. Acute myeloid leukemia (AML) is a hematological malignancy which interferes with the normal haematopoietic production of blood cells. It is characterized by the accumulation and expansion of immature myeloid cells (blasts) within the bone marrow that proliferate rapidly and inhibit differentiation of the normal progenitor cells . AML is The “two-hit” model of AML leukaemogenesis proposes that two mutation classes drive the disease, those that augment proliferation and cell survival  and those which disrupt haemopoietic differentiation and apoptosis  . Recent The heterogeneity of immune response between patients has made it difficult to characterize the landscape of AML tumor microenvironment. Several components within the tumor microenvironment interact to reprogram tumor initiation, progression, and response to therapies. Malignancies like AML deploy a plethora of immune escape mechanisms, including evasion of immunosurveillance systems, whereby cancer cells are able to avoid destruction . Such es+ T cell proliferation, favoring tumor progression  is a haem-containing, immunomodulatory enzyme that catalyzes the oxidative cleavage of the indole ring of the essential amino acid tryptophan (Trp), producing immunoregulatory metabolites, collectively known as kynurenines [Kyn(s)] . IDO wasgression .Defining the tumor microenvironment and immune escape mechanisms of AML will support the development of new therapeutic strategies. IDO is of particular interest in this regard as studies report IDO expression in both bone marrow and in peripheral blood AML blasts, but the role it plays in disease progression remains unclear . ResearcA systematic review of literature was performed to identify the role of IDO AML progression. The review was conducted according to the guidelines outlined in the PRISMA statement  17)..17).A systematic search of PubMed (performed in October 2020) was conducted to identify appropriate studies. The following keyword combinations were used:“Indoleamine 2,3-dioxygenase” and “AML”“Indoleamine 2,3-dioxygenase” and “Acute Myeloid Leukemia”“1-Methyltryptophan” and “Acute Myeloid Leukemia”“1-Methyltryptophan” and “AML”“1-MT” and “Acute Myeloid Leukemia”“1-MT” and “AML”“IDO” and “Acute Myeloid Leukemia”“Tryptophan” and “Acute Myeloid Leukemia”An additional search was conducted in the Scopus database using the keywords “Indoleamine 2,3-dioxygenase” and “AML”. Reference lists of identified papers were also scrutinized to identify additional sources.Inclusion criteria for the systematic review were: Studies based on human cells and/or cell lines; Studies that assessed the role of IDO in AML; Studies published in English.Exclusion criteria for the review were: Review/Systematic review articles, case studies, letters to the editors, books, and abstracts; Studies focusing on other cancer types, such as chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) etc.; Studies primarily based on animal models of AML and AML studies that did not investigate IDO.Studies satisfying the inclusion criteria were independently assessed and a data extraction was conducted to obtain the following data: Authors, journal, publication year, sample size, sample type, sample analysis method, IDO expression, role of IDO in prognosis, Treg markers identified, role of Tregs in AML, and the effect of IDO inhibitors. Due to the heterogenous nature of findings and variations in study designs, number of samples and methods employed for quantitation of IDO, meta-analysis of the results was not feasible.n = 28), manuscripts that did not tackle the role of IDO in AML (n = 20), those with unavailable abstracts or only abstracts were available (n = 20), review articles (n = 19), books (n = 2), those based on case studies alone (n = 3), and those focused on other types of cancer aside from AML (n = 1). A flow diagram detailing the screening process used to determine the studies suitable for inclusion in the systematic review is shown in Of 190 papers identified through PubMed and Scopus search, 108 met the inclusion criteria once duplicates and non-English papers were removed. When full texts were screened, a final 20 studies were found to be suitable for inclusion in the study. The reasons for manuscript exclusions were those that had no mention of both IDO and AML  with poor prognosis , 28, 30.Fewer IDO-positive patients achieved CR than IDO-negative patients in three of the studies reviewed , 27, 28.Five of the studies investigating the effect of IDO and its consequent elevation of Kyn levels also identified key prognostic markers for patient outcomes , 27–29. The link between IDO expression and Treg abundance was addressed in eight studies , 35, 36.via an IDO mediated mechanism in the presence of PGE2 , providing more reliable statistical data. Accordingly, FAB subtypes should not be used to classify the likelihood of IDO expression.In this review, a controversy was also highlighted regarding IDO expression relating to FAB subtypes. Some of the studies suggested that higher IDO expression is observed in the FAB-M5 subtype , 30, 31.AML mortality rates remain high despite attempts to refine treatment and develop more targeted techniques. Prognostic markers stratifying disease progression, such as the FLT3 mutation, have thus far only had limited effects on patient outcomes. Among the reviewed articles, seven studies assessed IDO expression prognosis , 28, 30.bonafide Tregs  chemotherapy was conducted in AML patients (NCT02835729) which has yet to report any findings . ResultsThe present review is subject to limitations as only English papers could be included and a meta-analysis was not suitable due to the heterogeneity of data. It is also acknowledged that many of the papers came from the same authors and research groups/institutions therefore findings potentially could be subject to bias. The papers included in this review investigated IDO with no delineation of its isoforms or focused entirely on IDO1. It must be noted that IDO has two isoforms, IDO1 and IDO2, which exhibit high sequence homology but exhibit kinetic, functional and expression differences, with IDO2 expression limited to subsets including antigen-presenting cells . Current+CD25+CD127low/−) markers may be required to delineate the contribution of bonafide Treg within the tumor microenvironment where IDO activity is functionally relevant. Furthermore, the role of other similar immunosuppressive enzymes, such as Tryptophan 2,3-dioxygenase may offer novel insights into the contribution of immunomodulatory enzymatic cascade in AML pathogenesis and prognosis.To the best of our knowledge, this is the first systematic review highlighting the importance of IDO in AML progression and prognosis. IDO can be expressed in various cell types involved in AML pathogenesis and this expression can be either inducible or constitutive, differing between patients. The review identified the potential of IDO and Kyn levels as prospective prognostic markers as IDO expression correlates with worse patient outcomes. However, more robust CD4 Treg (CD4The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GW performed the search, screening and data extraction, interpreted the results, and wrote the manuscript. PK contributed to the results interpretation and writing the manuscript. LD designed the study, checked the accuracy of the data extraction, and contributed to the results interpretation and writing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The increased Ca2+ is associated with the most severe symptom of sickle cell disease (SCD), the vaso-occlusive crisis (VOC). The Ca2+ entry pathway received the name of Psickle but its molecular identity remains only partly resolved. We aimed to map the involved Ca2+ signaling to provide putative pharmacological targets for treatment. (2) Methods: The main technique applied was Ca2+ imaging of RBCs from healthy donors, SCD patients and a number of transgenic mouse models in comparison to wild-type mice. Life-cell Ca2+ imaging was applied to monitor responses to pharmacological targeting of the elements of signaling cascades. Infection as a trigger of VOC was imitated by stimulation of RBCs with lysophosphatidic acid (LPA). These measurements were complemented with biochemical assays. (3) Results: Ca2+ entry into SCD RBCs in response to LPA stimulation exceeded that of healthy donors. LPA receptor 4 levels were increased in SCD RBCs. Their activation was followed by the activation of Gi protein, which in turn triggered opening of TRPC6 and CaV2.1 channels via a protein kinase Cα and a MAP kinase pathway, respectively. (4) Conclusions: We found a new Ca2+ signaling cascade that is increased in SCD patients and identified new pharmacological targets that might be promising in addressing the most severe symptom of SCD, the VOC.(1) Background: It is known that sickle cells contain a higher amount of Ca However, the deoxygenation of HbS alone is insufficient to explain the erratic nature of the crises [2+-activated K+ channel, KCNN4, KCa3.1, hSK4) activation and the following K+ loss increases the mean cellular hemoglobin concentration (MCHC) and promotes HbS aggregate formation. Furthermore, Ca2+ overload promotes phosphatidylserine externalization already in healthy RBCs [2+ may promote the perturbation of microcirculation with participation of even non-deoxygenated cells, independent of dehydration. This would increase RBC transit time in poorly oxygenated vascular areas and further increase the risks for VOC.In sickle cell disease (SCD) patients’ red blood cells (RBCs), the Cathy RBCs ,2. This thy RBCs , and as thy RBCs . Thus, t2+ increase was named Psickle due to its conductive nature but without any knowledge about its molecular identity [sickle [2+ entry pathway, which is elevated in SCD RBCs, was shown to be the NMDA receptor [2+ and this Ca2+ entry pathway is upregulated in RBCs of SCD patients. LPA-induced Ca2+ entry into RBCs was previously shown and believed to be channel mediated [The Caidentity ,9. Howev [sickle ,11. One receptor ,13. A rereceptor ,15. Heremediated ,17.V2.1 channel [2+ influx [2+ influx was phenomenologically characterized [V2.1 channel is rather controversial [2+ entry mechanisms in healthy and SCD RBCs.Whereas pharmacological studies pointed to the Ca channel ,18, patc+ influx ,20. Althcterized ,22,23, vcterized ,24, whiccterized . Howeveroversial . TherefoExperiments with human RBCs were authorized by the ‘Ärztekammer des Saarlandes’ under registration number 132/08 for healthy donors and by the Canton Zürich ethics committee under registration number KEK ZH NR 2010-0237 as well as by the regional ethics committee CPP Sud/Ouest Outre Mer III, Bordeaux under registration number 2010-A00244-35 for SCD patients. In all cases, blood donors provided their written informed consent to participate in this study. This consent procedure was approved by the respective ethics committees under the above-mentioned study registration numbers. For the experiments, we used RBCs from healthy adult donors and non-transfused HbSS SCD patients. Blood was drawn from a vein into heparinized containers and used within 30 h of the withdrawal.Experiments with mouse blood were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the State Office for Health and Consumer Protection (Permit Number: C1-2.4.3.4). All efforts were made to minimize suffering. Blood samples were collected from the cheeks of the mice by lancet puncture and were collected into heparinized Eppendorf tubes.g for 3 min. The buffy coat and plasma were discarded, and the remaining RBCs were washed three times with Tyrode solution (Tyrode) containing the following components (in mM): 135 NaCl, 5.4 KCl, 10 glucose, 1 MgCl2, 1.8 CaCl2 and 10 HEPES. The pH was adjusted to 7.35 using NaOH. For imaging the cells were loaded with Fluo-4 AM  at a concentration of 5 μM for 1 h at 37 °C. Then, the cells were washed three times with Tyrode.The following procedure was followed identically for RBCs from human and mouse. RBCs were isolated via centrifugation at 5000× 2+ kinetics in individual cells treated with hormones and pharmaceuticals (see below). Fluo-4-loaded cells were plated onto coverslips. We waited 15 min for cell sedimentation and dye de-esterification. Fluorescence was measured on the stage of an inverted microscope  equipped with a 60× Plan Apo 1.4 objective. A video imaging device  was attached to the microscope and contained a monochromator (Polychrome IV), a camera (Imago), the imaging control unit and acquisition software (TILLvision V4.0) for Figures 1, 3, 4, 6 and 8. The system was upgraded to Polychrome V, a sCMOS camera  and the software  before the experiments presented in Figures 5, 7 and 9 were carried out. Fluo-4-loaded cells were excited at 480 nm, and the resulting fluorescence images (using a 505 nm long pass dichroic mirror and a 535/40 bandpass filter) were collected every 5 s for 15 min. A gravity-driven local perfusion system was utilized to quickly exchange solutions in the field of view. All measurements were performed at room temperature (~22 °C). The images were analyzed in ImageJ , and the traces were further processed by IGORpro software .Life-cell imaging was performed to monitor intracellular Ca2O. U0126 as well as Wortmannin (both Sigma-Aldrich) were dissolved at 10 mM in DMSO . Gö6976  was dissolved at 1 mM in DMSO and ω-agatoxin-TK  at 100 µM in H2O. For AlF4−, 30 µM AlCl3 and 10 mM NaF were mixed. FK506  was dissolved at 20 mM in DMSO and Cyclosporin A  at 1 mM in ethanol.All activators and inhibitors were of analytical grade and freshly prepared from frozen stock solution. The stock solution of LPA was 5 mM dissolved in phosphate-buffered saline . Pertussis toxin  was dissolved at a concentration of 250 µg/mL in H® No 4, Cod. 1004125, Schleicher & Schuell BioScience, Dassel, Germany) was placed on the bottom of the syringe barrel. The cellulose slurry was carefully poured inside the syringe and the cellulose allowed to settle evenly. The excess Tyrode was allowed to flow through, exposing the surface of the cellulose bed. At this point, 1 mL of blood sample was carefully layered on top of the cellulose. When all the blood had entered the cellulose bed, Tyrode was added to the filter, and the fraction of filtered RBCs was collected. The filtered cells were concentrated by centrifugation and washed two more times with Tyrode. HeLa cells used as positive controls were handled as previously described [For Western blots, RBCs were filtered to make sure samples were not contaminated by mononuclear cells . Brieflyescribed .g for 10 min. The cell lysate was separated on 12% polyacrylamide gels. The gel electrophoresis was followed by transfer to nitrocellulose transfer membranes . Blots were incubated with rabbit polyclonal anti-GAPDH , anti-LPAR 1 (1:2000), anti-LPAR 2 (1:1000), anti-LPAR 3 , anti-LPAR 4 (1:1000), anti-LPAR 5 .Cells were lysed by EP extraction buffer  supplemented with 0.5% Triton X-100, 20 mM DTT, complete protease inhibitor cocktail and Phospho Stop  on ice for 30 min. The whole-cell lysates were then centrifuged at 5000× Blots were washed with phosphate-buffered saline with 0.1% tween-20 (PBS-T) and incubated with the horseradish peroxidase (HRP)-conjugated antibodies (1:5000). Enhanced chemiluminescence (ECL) was used for signal detection.g for 5 min. After blocking with 5% bovine serum albumin (BSA) in Tyrode for 20 min, cells were incubated with the first antibody, such as LPAR1-5 (1:50), in blocking buffer (5% BSA in Tyrode) for 4 h at room temperature while gently shaking. After washing in Tyrode3 times, cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG  in Tyrode for 2 h. Confocal recordings were performed on a Leica TSC SP5II microscope  using a 63× oil immersion objective with a numerical aperture of 1.4 (HCX PL APO) as described recently [For LPA receptor immunostainings, RBCs were fixed with 2% PFA plus 0.0075% glutaraldehyde in Tyrode for 30 min. Then cells were permeabilized with 0.3% Triton X-100 in Tyrode for 10 min, followed by centrifugation at 3700× recently . Fluores®Phospho-Erk1 (T202/Y204)/Erk2 (T185/Y187) ELISA Kit was used to detect MAPK phosphorylation, following the manufacturer’s protocol. In brief, RBCs from healthy donors and SCD patients were treated with Tyrode (control) or 5 µM LPA for 15 min. After RBC lysis using the cell lysate buffer provided in the kit, 100 μL sample or positive control  to Erk1/2 was placed on a coated microplate. The cell lysates were incubated for 2.5 h at room temperature while shaking. After washing the plates 4 times with the wash solution provided by the kit, they were incubated with 100 μL detection antibody anti Erk1 (T202/Y204)/Erk2 (T185/Y187) for 1 h while shaking. Then 100 μL of prepared 1x HRP-conjugated anti-rabbit IgG was added to each well and incubated for 1 h at room temperature while shaking. After washing another 3 times, 100 μL of TMB one-step substrate reagent was added to each well and incubated for 30 min at room temperature in the dark while shaking. Then 50 μL of stop solution (Item I) was added to each well and read immediately at 450 nm in a PT-FUJI-SCANNER.For the ELISA phosphorylation assay, the RayBioSingle-cell traces were analyzed as previously described in detail . All datp < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p > 0.05 (ns) for not significant. All statistical tests were performed in Prism, GraphPad, La Jolla, CA, USA.According to the normality test and the grouping conditions for testing significance, Student’s t-test, a Mann–Whitney test, an ordinary one-way ANOVA or a Kruskal–Wallis test with Tukey’s or Dunn multiple comparison test was performed. The level of statistical significance was indicated as 2+ entry in RBCs of healthy donors and SCD patients  Ca2+ influx contributes to the entire picture of the Ca2+ signaling (see Discussion below).Based on LPA stimulation of single RBCs and the previously introduced fluorescence microscopy analysis method ,30, we cpatients . In Figu2+ increase in both healthy and SCD RBCs is caused by the application of LPA, it was a logical step to look at the abundance of LPA receptors in RBCs. Since the Ca4− [2+ even exceeding the LPA stimulation. Following the literature, we probed the LPA stimulation in RBCs of a Gα11 knock-out mouse line [i utilizing the specific inhibitor PTX [2+ entry in mouse RBCs  channels C4 and C5 [2+ entry of TRPC4/5−/− RBCs compared to WT RBCs. Because TRPC6 has been reported to contribute to the increase in residual Ca2+ influx in RBC [−/− mice [2+ entry in TRPC6−/− RBCs is suppressed, but still significantly increased compared to control conditions , the relative amount of Gö6976-inhibited Ca2+ entry is identical compared to wild-type mouse conditions   B. FK506 s Gö6976 B, supporor FK506 B, supporuse RBCs C. In bot2+ entry, we focused on a MAP kinase (MAPK) pathway which was reported to be abundant in RBCs [In search of the cause of the remaining amount of Ca in RBCs . Therefo2+ channel that is regulated by this signaling pathway is expected to be ω-agatoxin-TK sensitive [1A subunit, this channel was proposed to be CaV2.1 [2+-imaging assay of LPA stimulation with and without pre-incubation of ω-agatoxin-TK on RBCs of WT mice and TRPC6−/− mice , which leads to RBC hyperpolarization from −10 mV to approximately −70 mV [The results presented in  in RBCs . On firsy −70 mV . We recey −70 mV . The maiV2.1 is phosphorylated. Both channels contain numerous putative phosphorylation sites. Although we kept both options in the scheme of The data presented in V2.1, inhibition of the Gárdos channel should have an impact on LPA-induced Ca2+ entry. The result of this experiment is depicted in 2+ level when the LPA stimulation is performed in the presence of 500 nM of the Gárdos channel inhibitor Charybdotoxin (ChTX). To complement the approach presented in 2+ entry by the pharmacological interventions in wild-type RBCs, while in TRPC6−/− RBCs, Ca2+ entry is completely abolished. In human RBCs, Ca2+ entry upon incubation with the blocking agents  but slightly increases. Therefore, a small fractional Ca2+ entry by a mechanosensitive channel such as Piezo1 could be the Ca2+ source enabling both branches of the signaling cascade.Both in vivo as well as in our in vitro experiments, RBCs experience a certain shear stress. In vivo, this is almost obvious and there it results in a measurable Caincrease  most proincrease ,53,54. I2+ entry could be prevented under both additional conditions tested, supporting the hypothesis that a mechanosensitive channel such as Piezo1 provides fractional Ca2+ entry, enabling the signaling cascades as outlined in To test this hypothesis, we performed experiments on human RBCs, where the 5 µM LPA were applied (i) without perfusion (less exact time point of application) and (ii) in the presence of 2 µM of the mechanosensitive channel inhibitor GsMTx-4 . The res2+-induced Ca2+ release or store-operated Ca2+ channels do not exist in RBCs. Furthermore, it is worthwhile to mention that mature mammalian RBCs do not contain translational machinery, i.e., they have the set of proteins from when the erythroblasts enucleated and the RBCs were released into the circulation. Depending on the aging process of the particular proteins, young and old RBCs contain different sets of proteins [2+-imaging experiments, are more sensitive than molecular biological approaches.When judging the results, we like to point out that RBCs are very special cells and although much of our current knowledge of membrane transport originates from RBCs when they have been ‘the’ model cell for membrane transport during the last century , they laproteins , i.e., rproteins  and 45 dproteins . Additioproteins ,62, and proteins . Therefo2+ uptake evoked by LPA stimulation and well described in healthy RBCs [2+ influx are so far unknown, we aimed at elucidating the various steps in the signaling cascade in healthy RBCs and compared it with pathological RBCs. By using Western blot and immunocytochemistry, we first probed for LPA receptors and found that whereas the same isoforms  were expressed in both populations, LPA receptor type 4 was augmented more than 3-fold in SCD RBCs  comparethy RBCs ), and si2+ channels in focus as pharmaceutical targets for the treatment of one of the most severe symptoms of SCD, i.e., VOC. This holds particular true, as Ca2+ has been identified as a trigger for RBC self-aggregation [The LPA signaling pathway and the consecutive activation of channels as well as the discovery of NMDA receptors in RBCs  place Caregation ,22,67 anregation ,69,70.2+ signal. This variety of direct players is supplemented by a presumably varying contribution of indirect modulators, particularly the plasma membrane Ca2+ ATPase (PMCA) [2+ [2+ which enters the RBCs, and its activity will depend on the metabolic status of the RBC, which can vary from cell to cell.Recently, direct channel activation of LPA in RBC was discussed ,6. Howeve (PMCA) , which iMCA) [2+  and may i protein is well documented in RBCs [i–PKCα interaction in RBCs. However, for platelets, such a signaling step has been previously reported [2+ influx within minutes. Direct interaction (phosphorylation) of TRPC6 by PKC is still controversial [Although the abundance of Gα in RBCs ,75, thisreported . Data onreported ,78, whicoversial ,79,80. Hoversial —similar i has already been described in the context of platelets [2+ entry in TRPC6−/− RBCs using ω-agatoxin-TK (V2.1 in the proposed signaling cascade. Although the modulation of voltage-activated Ca2+ channels by G protein-induced signaling has been reported [2+ channels have been shown [+-conducting Gárdos channel was blocked, LPA-induced Ca2+ entry was also diminished (V2.1 channel inhibitor ω-agatoxin-TK (Activation of a PI3 kinase/MAP kinase pathway by Gαlatelets ,83. The toxin-TK B providereported , the abuen shown ,86,87, oen shown , and recen shown . In the minished B—to a sitoxin-TK C. We hav2+ signaling cascade that is increased in RBCs of SCD patients in the first place by an increased occurrence of the LPA receptor 4. However, since Gi protein signaling triggers an entire signaling cascade, we provide a set of new pharmacological targets that might be promising in addressing the most severe symptom of SCD, i.e., VOC.We found a new Ca


More recent advances provide a strong basis to the emerging view that hereditary cardiac arrhythmia syndromes are accompanied by maladaptive remodeling of Ca2+ homeostasis which substantially increases arrhythmic risk. This brief review will focus on functional changes in elements of Ca2+ handling machinery in cardiomyocytes that occur secondary to genetic mutations associated with catecholaminergic polymorphic ventricular tachycardia, and long QT syndrome.Sudden cardiac death due to malignant ventricular arrhythmias remains the major cause of mortality in the postindustrial world. Defective intracellular Ca Enhancement of Na+ conductance increases depolarization and induces rapid accumulation of Na+ in the cell, and consequently drives Ca2+ “overload” via inhibition of forward mode NCX1. This can result in increased amplitude of Ca2+ release during systole and generation of spontaneous Ca2+ waves during diastole. However, genetic mutations modifying the same ion fluxes often produce minimal changes in net intracellular Ca2+ cycling under basal conditions both in human patients and animal models. Furthermore, even mutations in components of SR Ca2+ release machinery are relatively well tolerated and manifestation in the form of deadly arrhythmias is a rare, primarily occurring under stress  during the Ca2+ transient directly or indirectly forces the cessation of RyR2 cluster activity, eliciting the termination of SR Ca2+ release . As a result, RyR2-mediated SR Ca2+ leak must be sufficiently large to reduce Ca2+ transient amplitude. Notably, enhancement of RyR2 activity is the most common finding throughout the whole spectrum of acquired cardiac diseases including heart failure (HF), myocardial infarct (MI), diabetic cardiomyopathy, and age-related cardiac dysfunction  and spontaneous Ca2+ waves, similar to the effects in mouse ventricular myocytes with pressure-overload induced HF  despite the profound effect on APD. If this is the case, prolonged LTCC-mediated Ca2+ influx during the long AP is sufficient to increase SR Ca2+ content in LQT3. Indeed, mouse models of LQT8 (Timothy Syndrome) linked to LTCC gain-of-function mutations in CACNA1C also show increase in SR Ca2+ content and increased frequency of spontaneous Ca2+ waves in ventricular myocytes [Scn5a ΔQKP LQT3 model, RyR2 phosphorylation remained unchanged and no evidence of enhanced activity of the channel was presented despite an increase in spontaneous Ca2+ waves [2+ transients exhibited longer time-to-peak, suggesting subcellular dyadic structural remodeling: a hallmark of HF.LQT3 is associated with sed INaL , 41. Unloduction . Increasduced HF . Experimus waves . Interesrylation . Furthermyocytes . Remarka2+ waves , which i2+ cycling patterns between mice and larger animals, mechanisms of secondary remodeling uncovered in mice may differ greatly than those in humans. In small rodents, an increase in stimulation frequency decreases Ca2+ transient amplitude, e.g., a negative staircase. In rabbits or humans, increased stimulation frequency increases Ca2+ transients [2+ at slower rates due to higher NCX1 activity and lower SERCa activity in large animals and humans vs mice. Therefore, given that arrhythmia episodes in human LQT3 patients are prevalent during slower heart rates and assuming that spontaneous Ca2+ release is a key element of trigger [2+ influx, increased SR Ca2+ load, increased Ca2+ transient amplitude, increased contraction, and importantly, arrhythmogenic EADs at very slow pacing rates in the absence of β-adrenergic stimulation. At higher stimulation rates, presence of β-agonist was necessary for EADs induction.Notably, to our knowledge, a large animal model of hereditary LQT3 is yet to be created. Given substantial differences in Caansients . Accordi trigger , there i trigger  showed e+ and Na+ conductance, the list of mutations in genes directly involved in Ca2+ handling that manifest as LQTS continues to grow. Calm1, Calm2, Calm3, TRDN, and TECRL (LQT14–18) are recent additions to the CACNA1C gain-of-function mutations associated with LQT8 [2+ handling. Furthermore, the key roles of secondary to initial insult Ca2+ remodeling become widely recognized in other forms of hereditary arrhythmias including Arrhythmogenic Right Ventricular Hypertrophy [The list of genes associated with LQTS is rapidly expanding. Although most of these genes encode proteins that regulate Kith LQT8 , 41, 58 ertrophy  and Brugertrophy .2+ homeostasis. This remodeling, in an attempt to provide long-term preservation of basic contractile cardiac function, ultimately exacerbates arrhythmic potential under certain conditions such as stress. Notably, since the main goal is the same, i.e., preservation of contractile function, remodeling pathways evoked by arrhythmogenic mutations in genes encoding proteins involved in Ca2+ transport, cell electrical activity, or structural elements that underlie cardiac conduction often converge, bearing resemblance of those in structural heart disease and each other –derived cardiomyocytes, this experimental platform needs further development to ensure the highest maturation degree of subcellular structure, metabolic, and signaling cascades, given their key roles in revealing arrhythmogenic phenotype . Finally


When novice nurse educators enter academia, they are expected to demonstrate and implement knowledge in the clinical and classroom environment. However, when one enters academia without proper guidance and support, these expectations create lack of role models. Although mentorship has proved to make the transition easier, there is a lack of mentoring in most nursing schools and/or departments at higher education institutions in South Africa because of scarcity of mentoring programmes for novice nurse educators.The aim of this study was to explore and describe the experiences and mentoring needs of novice  nurse educators at a public nursing college in the Eastern Cape, to make recommendations for the mentoring of novice nurse educators.Urban and rural public nursing college campuses and sub-campuses in the Eastern Cape.Qualitative research approach and exploratory, descriptive, contextual and phenomenological designs were used. Sampling was purposive, data were collected by using semi-structured individual interviews and analysed using Tesch’s method.Five themes emerged from this study. Findings indicated that novice nurse educators experienced lack of theoretical and clinical mentoring and lack of orientation and resources. Participants also provided recommendations to optimise the experience and performance of novice nurse educators.Lack of mentoring causes difficult transition by novice nurse educators from the nursing role into the nurse educator role. The implementation of the recommendations on mentoring of novice nurse educators would optimise the experience and performance of the novice nurse educators, thus enhance their smooth transition into academia. Mentoring is the guidance and support provided to a junior or inexperienced personnel member by a professional, experienced person and can be both formal (assigned) and informal (chosen). The mentor acts as a teacher, friend or guide and is actively involved in the empowerment and professional development of the junior member .What are or were your mentoring needs as a novice nurse educator?What recommendations would you make to optimise the experience and performance of the novice nurse educators in their first year of teaching at a nursing college?Data analysis entails categorising, ordering, manipulating and summarising the data and describing it in meaningful terms .Qualitative research is based on trustworthiness rather than reliability and validity , prior to the assessment and evaluation of students, were not moderated by the supervisors.Participants shared that the supervisors would sanction their work without reviewing it and that when they implemented these assessments, errors would sometimes be identified by the students or by the participants themselves. Furthermore, participants sensed that the supervisors denied them feedback, as if they were setting them up for failure.Guidance and support in the usage of other assessment instruments such as specification tables, mark schedule compilation and the type of template to use were not received from the supervisors, which made it all more difficult for the novice nurse educator. One verbatim quotation illustrates this:I]n fact she was not even checking my work because I wouldn’t say she was checking my work if just going, doesn’t even read the question paper if I’m submitting for test, if I’m submitting a lesson plan she doesn’t even check for it and she doesn’t even make some comments on the question paper and she doesn’t even put her signature on my question paper showing that she has seen it, moderated it, find any corrections or whatever.’ ‘[An individualised inventory of faculty strengths and weaknesses based on the core competencies for nurse educators should be initiated. Efforts can then be undertaken to reduce the stress and anxiety of new faculty by acknowledging their strengths and supporting their weaknesses to fulfil the nurse educator role ‘[I]magine when you write a test, (an exclamation for acknowledging a mistake of saying you instead of they), when they write a test, you have to mark hundred scripts, in the mean, in the mean time you not focusing that you need also to do (i) [the] accompaniment, which is, is kind of eh … not ok.’ ‘ felt that I needed somebody who can sit with me and go through the policies, specifically policies related to academics.’ ‘t frustrates a lot because there was no communication, yes I realise later on that there was a year plan, but eh eh eh at the beginning I couldn’t eh gel much.’ ‘ur letter of our appointment came very … late, at least after a year when we’ve been fighting.’ ‘[A]fter two months, It’s when I only received the, what is it? The job descriptions.’ ‘uring that period of two months I was just lying around, sometimes I was asked to help lecturers who would be going to (saying the name of the town), then I, I would eh work as a driver, in that case because almost, almost every week I was asked to drive people to the meetings, to (saying the name of the town) and back.’ ‘he resources are not enough. We don’t have all the resources to assist those students, even myself alone, my own resources, we don’t have computers, I have to consult hospitals so that I can borrow one of the resources that I can use to help the students.’ ‘es there was a library but there were not enough books and the most books in our library were too were too old, so it was making things difficult for us especially when we are doing our lesson plans.’ ‘here should be structured orientation programme that should be followed by the, by the senior nurse educators to assist the new lecturers.’ ‘ recommend that we have a mentoring programme in the college and then we … support the new nurse educators.’ ‘[The National League for the Nursing Board of Governors in Brown :20 asserNovice nurse educators identified gaps in the college management’s communication and implementation of institutional policies and procedures and recommended that there be more uniformity in these areas to optimise the experience and performance of novice nurse educators in their first year of teaching at the college. One such proposal from a participant is:M]anagement needs also to improve on their side, so that they talk one thing and, and they mean what they say, they mustn’t in other because irrespective one is in Transkei and one is in other side, the treatment must be the same, you, things must not be based on discretions, if we talk policies, things must be, must be applied according to policies, not depending on, on discretions because discretions are the ones that create problems.’ ‘[According to Huber ‘[T]here should be a person who is assigned to monitor quality or anyone who would be an overseen that it is it.’ ‘[I] would recommend that eh the paper work eh be … minimised.’ ‘[The significance of standardisation of clinical procedures is to avoid unwarranted variation in treatment, and to improve patient care and caregiver accountability as it is the establishment of standards and protocols for caregivers to follow when treating patients Grant :1.Quality assurance activities must be coordinated, and quality assurance itself must be an ongoing process; it should not be limited by time and should not be a trial and error or once off exercise (Meyer et al. Legally, an electronic copy of a document can serve the same purpose as a hard copy, as long as the quality of the scanned copy is good enough to read and is a complete copy of the document. Paperwork can pile up quickly, requiring extra filing cabinets, file room space and employee time to file and organise. The amount of paperwork in an HR office can be easily reduced by using computer technology to scan and store electronic copies of documents Winston :1.The following are the limitations of this study:Data were collected from novice nurse educators at a public nursing college. The experiences and mentoring needs of novice nurse educators at private universities were not explored in this study; therefore, the research context may prohibit generalisation.A broader range of views may have been elicited had the study sample included more sub-campuses as the college under study has more sub-campuses than main campuses.Based on the research findings and the limitations provided above, the following recommendations for nursing practice, nursing education and nursing research were suggested by the researcher.The following recommendation is proposed for nursing practice:The findings of this research study could be disseminated to the clinical practice environments where the students of the college under study are allocated for clinical exposure. This would inform the clinical nurses and clinical managers of the mentoring needs of novice nurse educators related to clinical practice.The following recommendations are proposed for nursing education:The findings of this study could be disseminated by the college managers to the senior nurse educators to inform them of the experiences and mentoring needs of novice nurse educators. The suggestions made by the novice nurse educators regarding their mentoring would facilitate an easier transition to the academic environment.The recommendations for the mentoring programme of novice nurse educators could be disseminated to all public nursing colleges in the country for them to assess, contextualise and implement as there is a recognised lack of mentoring programmes for newly appointed nurse educators at schools and/or departments of nursing at higher education institutions in South Africa.The development and implementation of a structured and comprehensive mentoring programme.The following recommendations are proposed for nursing research:A similar study could be conducted at the private universities to obtain a complete picture of the experiences and mentoring needs of novice nurse educators at nursing education institutions in the Eastern Cape.A study could be conducted at the college under study for the evaluation of the effectiveness of the mentoring programme after implementation.In South Africa there are many nursing colleges and/or departments of nursing at higher education institutions that are lacking mentoring programmes for newly appointed nurse educators. The latter is one of the causes of difficult transition by nurse educators from the nursing role to the nurse educator role because of the complex demands and expectations of the core roles of nursing education.The study explored and described the experiences and mentoring needs of novice nurse educators at a public nursing college in the Eastern Cape regarding core roles of nurse educators, to make recommendations for mentoring of novice nurse educators. The implementation of the recommendations on mentoring of novice nurse educators would optimise the experience and performance of the novice nurse educators, thus enhance their smooth transition into academia.


Although such fluctuations can be evoked by neuronal activity, rhythmic astrocytic Ca2+ oscillations may also spontaneously arise. Experimental studies hint that these spontaneous astrocytic Ca2+ oscillations may lie behind different kinds of emerging neuronal synchronized activities, like epileptogenic bursts or slow-wave rhythms. Despite the potential importance of spontaneous Ca2+ oscillations in astrocytes, the mechanism by which they develop is poorly understood. Using simple 3D synapse models and kinetic data of astrocytic Glu transporters (EAATs) and the Na+/Ca2+ exchanger (NCX), we have previously shown that NCX activity alone can generate markedly stable, spontaneous Ca2+ oscillation in the astrocytic leaflet microdomain. Here, we extend that model by incorporating experimentally determined real 3D geometries of 208 excitatory synapses reconstructed from publicly available ultra-resolution electron microscopy datasets. Our simulations predict that the surface/volume ratio (SVR) of peri-synaptic astrocytic processes prominently dictates whether NCX-mediated spontaneous Ca2+ oscillations emerge. We also show that increased levels of intracellular astrocytic Na+ concentration facilitate the appearance of Ca2+ fluctuations. These results further support the principal role of the dynamical reshaping of astrocyte processes in the generation of intrinsic Ca2+ oscillations and their spreading over larger astrocytic compartments.Fluctuations of cytosolic Ca This way, a fraction of the ECS in the synaptic environment was increased from 11.2 ± 3.0% to 18.2 ± 3.3% that is closer to physiological values  and the postsynaptic dendritic spine was calculated by counting the number of surface pixels of boutons and spines having close contact with astrocytes. The surface/volume ratio (SVR) was determined by dividing the number of surface pixels counted according to the above method by the number of all pixels belonging to astrocyte processes.+]e = 140 mM; [K+]e = 3 mM; [Ca2+]e = 2 mM) as well as astrocytic [K+]i (130 mM) and [Glu]i (3 mM) were kept constant during the simulation, while [Glu]e (0.3 μM), [Na+]i (15 mM) and [Ca2+]i (100 nM) were allowed to change due to Glu release, intracellular Ca2+ diffusion and activation of EAATs and NCX  at the synapse centroid as determined by Kasthuri et al. . The dif2+ ions were updated by moving them with normally distributed random distances around their mean square displacement values. If a particle moved outside of the sample volume, it was removed from the available pool, except if [Glu]e, [Na+]i or [Ca2+]i dropped below the baseline level, in which case it was moved back to its previous position. Particles moving out of their compartment  were also placed back to their previous position. (2) Transition states of EAAT and NCX molecules were determined according to their rate constants and dynamic rate constants based on the current intra- and extracellular concentrations of relevant ions  Glutamate molecules bound to the extracellularly faced EAAT were removed from the available pool until they were released back by reverse operation of the transporter. Ca2+ ions bound to the intracellularly faced NCX were removed from the available pool until they were released back by reverse operation of the transporter.Each time steps (1 μs) was comprised of the following functions: (1) position of extracellular glutamate molecules and intracellular Caant ions . In the http://downloadables.ttk.hu/heja/Front_CellNeurosci2021. Synapses were visualized using Cinema4D.All simulations were done in Matlab using custom-written scriptsp < 0.05.Data are shown as mean ± SEM and were analyzed with one-way analysis of variances . Statistical significance was considered at 2+ oscillations in real astrocyte processes, we used the saturated reconstruction of a 1,500 μm3 volume of mouse neocortex  and NCX were simulated as previously described  simulating baseline Ca2+ fluctuations when only NCX was allowed to operate and no synaptic glutamate release occurred; and (2) simulating Ca2+ fluctuations according to our original conditions, releasing 5,000 glutamate molecule after 10 ms of baseline activity and letting EAATs function. The powers of the 100–500 Hz range of the Ca2+ concentration signals were compared in the 12–21 ms period. In some synapses, glutamate release significantly increased the 100–500 Hz power. As an example, 100–500 Hz power increased from −23.85 ± 0.20 dB to −22.72 ± 0.27 dB due to synaptic glutamate release  in a synapse with high SVR . Therefore, we investigated whether synapses characterized by different SVR of the surrounding astrocytes may respond differently to Glu release. Resolution of Glu release-induced changes in the power of astrocytic Ca2+ fluctuations by astrocyte SVRs, however, still did not reveal a significant effect of Glu release  changes in astrocytic Na+ concentration may significantly affect NCX-mediated Ca2+ fluctuations. Changing astrocytic Na+ concentration from the original 15 mM to 10 or 20 mM, indeed, markedly altered Ca2+ oscillatory power  Ca2+ signals in tiny processes  or mitochondrial permeability transition pores  depend on SVR (Wu et al., 2+ waves as predicted by the dynamically rich repertoire of distinct Ca2+-dependent Ca2+ release dynamics (Matrosov et al., 2+ fluctuations. Indeed, the shape of the slow Ca2+ wave with fast Ca2+ fluctuations (Savtchenko et al., 2+ fluctuations locally. It is to mention, that fast astrocytic Ca2+ signaling with mean onset time as rapid as that of neurons is not unprecedented (Kékesi et al., 2+ fluctuations on the evolution of cellular- and network-scale Ca2+ oscillations necessitates further studies, that include models describing downstream Ca2+ stores and Ca2+ buffers (Savtchenko et al., 2+ dynamics in multiple, neighboring synapses contacted by the same astrocyte. We may also conjecture that the structural plasticity of astrocytic processes may serve as a de novo signal generator, independently of its role in regulating glutamate spillover, K+ buffering, or other indirect forms of modulation of neuronal activity (Henneberger et al., It is evident from our simulations that the appearance of fast Cahttp://downloadables.ttk.hu/heja/Front_Cell_Neurosci_2021. Matlab scripts used to process the data can be downloaded at https://github.com/hejalaszlo/Astrocyte-leaflet-simulation.Raw data is available to download at LH: conceptualization, data curation, formal analysis, funding acquisition, methodology, software, supervision, validation, visualization, roles/writing—original draft, writing—review and editing. ZS and MP: data curation, methodology, validation, roles/writing—original draft, writing—review and editing. JK: conceptualization, investigation, methodology, supervision, roles/writing—original draft, writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.