Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].In the heart, genetic or acquired mishandling of diastolic [Ca In heal [Ca2+]i .2+ handling is regarded as one hallmark of disease  RyR2 hyper-phosphorylation by PKA because of a chronic hyper-adrenergic state in heart failure  involving exchange protein directly activated by cAMP (Epac2) and nitric oxide synthase 1  Rynthase 1 , and  by reducing RyR2-mediated Ca2+ spark activity and prohibited arrhythmia but not the progression of heart failure in mice.  explained by the increased Ca2+ leak through the exogenously expressed, spontaneously opening RyR2 channels; if a drug compound would inhibit this spontaneous RyR2 activity, the ER Ca2+ signals monitored by the calcium indicator R-CEPIA1er will show an increased signal . Notably, 10 of the new small chemical compounds significantly increased ER Ca2+ signals resulting in an S36-comparable or apparently greater effect (orange and green bars). Out of these, the new compound GM1869  and stimulated quickly with a high concentration of caffeine (10 mM) to empty the intracellular ER Ca2+ store and, consequently, allow an indirect estimation of the SR Ca2+ content  also significantly decreased the Ca2+ spark frequency in cardiomyocytes  each for GM1869, dantrolene, and S36 . Segments (1–2 µm) for analysis were chosen to avoid interference with wave propagation. Here, SR Ca2+ uptake is represented as the decay of the Ca2+ wave from the half-maximal peak amplitude to baseline obtained by a fit with a mono-exponential function giving the time constant (τ). All fits described the mono-exponential decay (r2 > 0.93) well, thereby characterizing the activity of SERCA2a Ca2+ pumps and indicating no contamination by other processes . 2+ uptake in permeabilized cardiomyocytes. Dantrolene significantly slowed down the kinetics of Ca2+ uptake showing that this drug may inhibit SERCA2a activity  and 2+ uptake (relative changes to control). Each in WT and RyR2R2474S/+ cardiomyocytes, dantrolene significantly reduced the Ca2+ spark frequency to a similar degree  and ther degree , whereasficantly . Interes4S/+ CMs . Whereas+ uptake , GM1869 4S/+ CMs . These d2+ transients in intact adult murine ventricular CMs to get insights in the pharmacokinetic properties of the drugs. For this purpose, cells were loaded with the intracellular Ca2+ indicator Fluo-4 AM (5 µM) and paced five times at 1 Hz every 2 min after drug application. Ca2+ transients were analyzed by mono-exponential functions, each with respect to the rise time (from baseline to peak) mainly reflecting RyR2-mediated Ca2+ release and the decay time  mainly reflecting SERCA2a activity. 2+ transients each for the control and S36  matured for at least 60 d. Again, Ca2+ transients were analyzed by mono-exponential functions to characterize the intracellular Ca2+ release and decay kinetics. 2+ transients of untreated control and S36  and 40 pM (R2474S+/−) for S36 (+/−) for GM1869  and 71 pM (R2474S+/−) for GM1869  that acts simultaneously by both decreasing SR Case model  comparedse model  that mayfunction .2+ cycling, particularly SR Ca2+ leak, is a phenomenon observed in a number of heart disease phenotypes , calmodulin (CaM), and calsequestrin (CASQ2). At a systemic level of regulation, the association of FKBP12.6 with the tetrameric macromolecular RyR2 channel complex is especially thought to serve as a potential therapeutic strategy against heart failure  and the immature status of iPSC-CM   was shown to prevent fatal cardiac arrhythmias and to reduce SR CaS/+ mice . In cryoS/+ mice . FurtherS/+ mice . The RY1S/+ mice . In detaS/+ mice  of RyR2 S/+ mice . Overallaffeine) .2+ spark FWHM and the pronounced amelioration of the Ca2+ spark frequency in RyR2R2474S/+ CMs by GM1869 suggest a shift of RyR2 cluster ensembles towards a lower Ca2+ spark fidelity. However, this concept has to be clarified by future studies using mathematical homology modeling as described previously  and were approved by local authorities . The mouse line used for the study carried the RyR2–R2474S knock-in mutation  and was www.endotherm-lsm.com). The synthesis of the tested compounds is described in the Supplemental Data 1  were performed as described . Changes in ER calcium concentrations [Ca2+]ER were measured on a multi-well plate reader TECAN Spark 20 M at 37°C. The 1,000 × stock solutions of the test compounds were prepared in DMSO. After the culture medium was removed, 100 µl of Tyrode’s solution containing 2 mM CaCl2 was added to the cells. Fluorescence was evoked by 560 nm excitation wavelength and collected in a bottom-read mode at 610 nm. The time courses were recorded in each well. Data were recorded every 10 s, exposure: 20 flashes, excitation bandwidth: 20 nm, emission bandwidth: 20 nm. Test compounds or vehicle  were applied at 90 s after start of the assay at the indicated concentrations. The ratio values of the averaged fluorescence intensities F/F0 before and after drug application were taken for analysis.Measurements on Caescribed . These c2+ concentrations were measured on a multi-well plate reader TECAN Spark 20 M using FLIPR Calcium 6 Assay Kit (Molecular Devices LLC) at 37°C. HL-1 cells were loaded with the FLIPR Calcium 6 dye for 2 h at 37°C, together with drugs or vehicle  at the indicated concentrations. Fluorescence intensities  were continuously monitored in each well for 1 min. Caffeine  was injected at 10 s. The differences in intensities between the caffeine-induced maximal Ca2+ signal and baseline, ΔFCaff, were taken for analysis. The dose response curves were calculated using the software package GraphPad Prism version 8.3.1. All data are presented as mean ± SD from eight independent experiments. Y=Bottom + (Top-Bottom)/(1 + 10^((LogEC50-X)*HillSlope)) was used where HillSlope describes the steepness of the curve, Top and Bottom are plateaus in the units of the Y axis.HL-1 cells  were cultured in black-walled, clear-bottom 96-well micro-plates (Corning) after coating with fibronectin according to the manufacturer’s instructions. Cytosolic Ca+/− iPSC cell line that was kindly provided by L Cyganek . In brief, differentiation was initiated at 80–90% confluence in Geltrex-coated plates with a cardio differentiation medium composed of RPMI 1640 with Glutamax and HEPES (Thermo Fisher Scientific), 0.5 mg/ml human recombinant albumin, and 0.2 mg/ml L-ascorbic acid 2-phosphate and sequential treatment with 4 μM CHIR99021 (Merck Millipore) for 48 h and then 5 μM IWP2 (Merck Millipore) for 48 h. The medium was changed to RPMI 1640 with Glutamax, HEPES, and 2% B27 (Thermo Fisher Scientific) at day 8. Metabolic CM selection was performed using RPMI 1640 without glucose (Thermo Fisher Scientific), 0.5 mg/ml human recombinant albumin, 0.2 mg/ml L-ascorbic acid 2-phosphate, and 4 mM lactate (Sigma-Aldrich) for 5 d. Afterwards, iPSC-CMs were cultured at least to day 60 for further maturation and then used in the experiments. All experiments on cardiomyocytes were performed at room temperature.The human iPSC line is WT1. Bld2 (GOEi014-A.2) was differentiated into iPSC-vCMs as described  and also2 5.5, and adenosine Tri-phosphate di-Na+ (di-Na+ ATP) 5, KH2PO4 5, phosphocreatine-diNa+ 5, phosphocreatine-diTris 10, and creatine phosphokinase 10 U/ml. pH was adjusted to 7.2 with NaOH. After adding 0.03 mM Ca2+, the free [Ca2+] was estimated to 80 nM (Maxchelator). After 2 min, the supernatant was replaced with the relaxing solution containing saponin (40 μg/ml) for 1 min. Cells were then washed three times with the relaxing solution. The solution was replaced by the relaxing solution containing 10 µM Fluo-4 pentapotassium salt (Thermo Fisher Scientific). About 5% of the remaining cardiomyocytes exhibited spontaneous contractions and Ca2+ waves at a constant frequency for at least 5 min.Ventricular cardiomyocytes were isolated from mice as described previously . Isolate2+ waves at frequencies below 4 waves/10 s. These cells were attributed to have experienced a mild permeabilization procedure, that is, the cell membrane was sufficiently permeabilized to allow the membrane-impermeable Ca2+ indicator to enter the cell but the intracellular membranes and contractile structures were not affected, as evidenced by Ca2+ release from intracellular stores and cell shortening, respectively. All measurements were performed under control conditions and after 2 min incubation with relaxing solution containing either vehicle  or the indicated drug concentration. Drugs were added cumulatively if appropriate. Intact cardiomyocytes were loaded with Fluo-4 AM  for 45 min at room temperature and imaged as described above. Electrical field stimulation was performed as described (Cardiomyocytes were imaged using an LSM 880 inverted microscope equipped with a 63x oil immersion objective (Zeiss). Fluo-4 was excited at 488 nm with a krypton/argon laser. The fluorescent emission was collected through a long-pass filter (>515 nm). All images were acquired digitally in line-scan mode  with 6,000 lines/image. The scan line was placed in parallel to the longitudinal axis of brick-shaped cardiomyocytes with clear cross striations that were solid attached to the cover slip. Images were recorded only from Fluo-4 loaded permeabilized cells that show spontaneous contractions and corresponding Caescribed .http://rsb.info.nih.gov/nih-image) equipped with the SparkMaster plugin , duration was expressed as full duration or taken from the FDHM, and width was expressed as full width or taken from FWHM.Images were analyzed using ImageJ  is reported to be different between RyR+/+ and RyRR2474S/+ channel clusters, especially with the observation of ember sparks in cardiomyocytes from mice expressing the RyRR2474S/+ mutation . Ca2+ decay was obtained in a 1–2 µm part of the wave (10–20 pixels in width) before and after drug application to exclude the influence of wave propagation but to reduce the noise in the recording. Because of permeabilization procedure, activity of sodium–calcium exchanger (NCX) was not expected to contaminate our signals. The obtained Ca2+ decay was fitted by a mono-exponential function from 50% of the peak maximum to baseline resulting in the time constant τ as an indicator of decay velocity with r2 > 0.93.Spontaneous Cat test (two groups), by Mann–Whitney’s test , or by one-way ANOVA followed by a Dunnet’s post-hoc test (multiple groups). P < 0.05 was considered as statistically significant. No hierarchical clustering of the datasets was observed after analysis according to Statistical analysis was performed using Prism software (GraphPad Software Inc). Data are presented as means ± SEM or means ± SD. Numbers indicate the number of analyzed experiments and the number of cells isolated from at least three different animals. Statistically significant differences were determined either by paired or unpaired


However, all available data to support this hypothesis have been collected from “in vitro” experiments using high concentrations of exogenous Aβ. It is still unknown whether Aβ channels can be formed by endogenous Aβ in AD animal models. Here, we report an unexpected finding of the spontaneous Ca2+ oscillations in aged 3xTg AD mice but not in age-matched wild-type mice. These spontaneous Ca2+ oscillations are sensitive to extracellular Ca2+, ZnCl2, and the Aβ channel blocker Anle138b, suggesting that these spontaneous Ca2+ oscillations in aged 3xTg AD mice are mediated by endogenous Aβ-formed channels.Alzheimer’s disease (AD), the leading cause of dementia, is characterized by the accumulation of beta-amyloid peptides (Aβ). However, whether Aβ itself is a key toxic agent in AD pathogenesis and the precise mechanism of Aβ-elicited neurotoxicity are still debated. Emerging evidence demonstrates that the Aβ channel/pore hypothesis could explain Aβ toxicity, because Aβ oligomers are able to disrupt membranes and cause edge-conductivity pores that may disrupt cell Ca  Although numerous studies have laid the foundation for this hypothesis, the predominant evidence is indirect and limited to in vitro system tests. Ideally, if one could record ionic currents under patch-clamp conditions on a cell, isolated from relevant regions in AD animal models, after all the native ion channels on such a hypothetical cell were blocked without altering the physiology, one could expect empirical evidence that would be direct, widely accepted and most useful to the understanding of AD.15 Based on this type of experimental setup, we provide direct evidence of an endogenous Aβ channel/pore in aged 3xTg AD mice.Alzheimer’s disease (AD), the leading cause of dementia, is characterized by the accumulation of beta-amyloid peptides (Aβ) in senile plaques in the brain of affected patients.2+ signal transduction mechanisms, because it has been possible to directly obtain considerable insight into intracellular Ca2+ handling under both normal and pathological conditions.16 Unlike nerve and endocrine, as well as muscle cells, exocrine cells such as pancreatic acinar cells are non-excitable cells and do not possess voltage-gated Ca2+ channels, and the cytosolic Ca2+ signals governing pancreatic acinar secretion are primarily generated by release of Ca2+ from intracellular stores, principally the endoplasmic reticulum (ER).2+ oscillatory signals (monitored by changes in the Ca2+-activated Cl− current) through the G protein-IP3 pathway. Moreover, additional factors work together to keep the intracellular Ca2+ homeostasis in acinar cells, such as the plasma membrane Ca2+-activated ATPase, sarco(endo) plasmic reticulum Ca2+ activated ATPase and store-operated channels.The pancreatic acinar cell is a classical cell model, for studies of Ca20. AD can cause the Aβ deposition in the pancreases of mice overexpressing human APP, suggesting that Aβ deposits may occur in organs other than the brain.20 Here, we show that Aβ deposits can be detected in pancreatic acinar cells in aged 3xTg AD model mice. Furthermore, unexpected spontaneous Ca2+ oscillations were found in such AD mouse acinar cells. Since mouse pancreatic acinar cells do not express voltage-gated Ca2+ channels, we hypothesize that the spontaneous Ca2+ oscillations mediated through endogenous Aβ-formed Ca2+-permeable channels in these aged AD mice.As a highly innervated organ, the pancreas shares a number of striking pathophysiologic similarities in Aβ deposition comparing to that in brain21 This AD mouse carries transgenes encoding mutants of presenilin-1 , amyloid precursor protein , and tau (P301L). These mutant genes lead to rapid accumulation of amyloid in mice and premature dysfunction of synaptic transmission and long-term potentiation.22 All mice were kept in groups under constant standard conditions of temperature and humidity, with ad libitum access to food and water, and on a 12-h light/dark cycle. All experimental procedures using mice conform to the Medical Animal Care and Welfare Committee and were approved by the Laboratory Animal Ethics Committee of Shantou University Medical College.C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology company. 3xTg AD mice, 12–24-mo-old, were a gift from Prof. Diling Chen , who has published a paper using 3xTg AD mice.Single isolated mouse pancreatic cells were prepared as previously described.2+-activated Cl− currents to monitor intracellular Ca2+ signaling oscillations. When the recording pipette was filled with K+-containing pipette solution, the resistance was 3–4 MΩ. After a GΩ seal was formed between the cell membrane and the pipette, the Aβ perforated whole-cell conformation was formed as judged by gradual (5–30 min) reduction of the access resistance (to <60 MΩ). Whole-cell recording mode was achieved by brief suction. The cells were held at −30 mV and the series resistance was not compensated. Transmembrane currents were recorded with a patch clamp amplifier .As previously reported,m): 140 NaCl, 4.7 KCl, 1.2 MgCl2, 1 CaCl2, 1.13 MgCl2, 10 glucose, and 10 HEPES, pH 7.3 adjusted using Tris-base. A Ca2+-free solution was prepared by replacing Ca2+ with Na+ (142 m m NaCl and 0 m m Ca2+) and adding 1 m m EGTA. The pipette solution for whole-cell recordings contained (in m m): 140 KCl, 0.24 EGTA, 1.13 MgCl2, 5 Na2ATP, 10 glucose, and 10 HEPES, pH 7.2. ACh, 2APB, and atropine sulfate used in this study were purchased from Sigma . Ruthenium red was purchased from Wako Chemical, and CdCl2 and ZnCl2 were purchased from Macklin . For external drug application, a “U-tube” rapid application system was employed. Data were filtered at 2 kHz, acquired at 5 kHz, and digitized online . All data were displayed and stored on a computer.The standard extracellular solution contained  mice of the same age were anesthetized, and the brain and pancreas were fixed by 4% paraformaldehyde perfusion through the heart. Paraffin-embedded blocks were prepared by sequential dehydration in graded ethanol before embedding, and tissues were serially sectioned to a thickness of 4 μm and mounted on pre-coated Poly-l-lysine glass slides. Sections were first pre-incubated with potassium permanganate solution and oxalic acid, then placed in 3% sodium borohydride solution for 5 min. Staining was performed using filtered 0.05% Thioflavin S (Sigma) in 50% ethanol for 30 min in the dark, and differentiated with two changes of 80% ethanol for 10 s. This was followed by 3 washes with large volumes of distilled water and an incubation step in 5× PBS buffer at 4°C for 30 min. Finally, slides were briefly rinsed in PBS and covered with coverslips using Vectashield Hard Set mounting media with DAPI (Vector). Slides were allowed to set in the dark at 4°C and imaged immediately thereafter. Fluorescence images were acquired on a Zeiss LSM800 confocal microscope.To identify the Aβ accumulation, the thioflavin S staining was used, samples were prepared as described.1–42 (oAβ), a 1 m m Aβ solution was prepared by dissolving as-synthesized Aβ1–42 powder (DGpeptides Co.) in 1,1,1,3,3,3-hexafluoroisopropanol, and blow drying the liquid with nitrogen to form a peptide film at the bottom of the centrifuge tube. The Aβ1–42 peptide membrane was resuspended by adding DMSO and sonicated under sterile conditions for 10 min before use, then diluted to a 100 μm stock solution in PBS at 4°C. For the application of monomers, the 100 μm stork solution was directly diluted to the required concentration with standard extracellular solution. For the preparation of oligomers, the 100 μm stork solution was vortexed (15 s), centrifuged, and transferred to a 4°C freezer for 24 h. To avoid fibril formation, all samples were used within one day.For the oligomerization of Aβ2+ oscillations from 3xTg AD mice for approximately 2 min  and were compared to the changes induced by drugs exposure. Data are presented as mean ± SEM. One-way ANOVA was employed to compare the time to first calcium spike generation at different concentrations of oAβ. Differences with P < 0.05 were considered significant.The net charge of the current was obtained by dividing the current area by the cell membrane capacitance (Cm) over a certain time . Before values were measured from the baseline of spontaneous Ca2+ oscillations can be induced by different stimulations, such as ACh, CCK, InsP3, or Ca2+ ion.28 However, by using patch-clamp whole-cell recordings in freshly dissociated pancreatic acinar cells from aged 3xTg AD mice, we found unexpected spontaneous Ca2+ oscillations -induced Ca2+ oscillation responses , the spontaneous Ca2+ oscillations exhibited much lower oscillational frequency but longer oscillatory spike duration (n = 7 cells and n = 7). Student’s t-test analysis showed that the difference in both oscillatory frequencies and duration between the spontaneous Ca2+ oscillations in pancreatic acinar cells from aged 3xTg AD mice and the ACh (10 n m)-induced Ca2+ oscillations in pancreatic acinar cells from age-matched WT mice were highly significant (P < 0.001).Usually, in mouse pancreatic acinar cells, Callations . In 16 as tested . In 5 WTllations , suggest2+ oscillations, we performed 3 experiments. First, we removed extracellular Ca2+ by replacing standard extracellular solution with an extracellular Ca2+-free solution containing 1 m m EGTA. In 8 cells tested, we found that the removal of extracellular Ca2+ reversibly abolished these oscillations  eliminated spontaneous Ca2+ oscillations (n = 7). Finally, we tested the effects of an amyloid (Aβ) channel blocker, Anle138b, and found that Anle138b (100 n m) completely inhibited the spontaneous Ca2+ oscillations (n = 11), suggesting that the spontaneous Ca2+ oscillations in aged AD acinar cells are elicited by extracellular Ca2+ efflux into cell through the Aβ-formed channels. Statistical analysis  induced reduction of spontaneous Ca2+ oscillations are highly significant (P < 0.0001).To explore the nature of these spontaneous Callations , suggestllations , n = 7. llations , n = 11,analysis  demonstr2+-signal pathways (CICR and/or IICR) mediated the spontaneous Ca2+ oscillations. As shown in m) inhibited the spontaneous Ca2+ oscillations. To test whether the IICR participates in the spontaneous Ca2+ oscillations, we performed 2 experiments. First, we tested the effects of 2APB, both InsP3 receptor and store-operated Ca2+ channel blocker, and found that 100 μm 2APB failed to block the spontaneous Ca2+ oscillations (n = 7). Second, we activated the IICR pathway, by application of ACh (10 n m), and showed that the ACh-induced Ca2+ oscillations can be blocked by 10 μm atropine (n = 6), but cannot be blocked by 100 n m Anle138b (n = 5). These results suggest that the spontaneous Ca2+ oscillations in acinar cells from aged 3xTg AD mice are mediated by extracellular Ca2+ influx into cytosol through the Aβ-formed channels, subsequently triggering the CICR signal pathway.In these experiments, we further identified which intracellular Callations , n = 7. atropine , n = 6, Anle138b , n = 5. 29 There are also extensive amyloid deposits in the pancreas of 8-mo-old APP/PS1 transgenic mice.30 In addition, transgenic mice overexpressing both amyloid beta-protein and perlecan in pancreatic acinar cells.32 We detected Aβ deposition, in the pancreas of 3xTg AD mice, using thioflavin staining. Brain tissue  of 3xTg AD mice was used as a positive control. Hippocampal sections of 12-mo-old 3xTg AD mice showed large numbers of Aβ deposits  to induce Ca+ oscillations by using perforated patch recording  on the time required for oAβ channel formation. Either ketone co-application (oAβ and ketone were added into peptide solution together) or ketone pre-treatment  significantly prolonged the formation of oAβ channels, based on perforated whole-cell configuration measurements (m oAβ could induce Ca2+ oscillations like the spontaneous oscillations that we observed in aged APP mice. 2+ oscillations before perfusion of oAβ (n = 4), but after bath perfusion of 500 n m oAβ for 30 min, Ca2+ oscillations occurred (n = 10) in a time and oAβ concentration-dependent manners .Data presented thus far indicate that pancreatic acinar cells of aged 3xTg AD mice display spontaneous Ca WT mice . First, ecording . After eurements . Furtherurements . Neitherurements  and elecurements . Then, woccurred , n = 10  manners . ImportaAnle138b , n = 8.2+ oscillations, we performed similar pharmacological tests as for the spontaneous Ca2+ oscillations found from the aged 3xTg AD mice (m) induced Ca2+ oscillations (n = 6), and the removal of extracellular Ca2+ (by Ca2+-free extracellular solution containing 1 m m EGTA) reversibly stopped the oscillations (n = 8). Like spontaneous Ca2+ oscillations found in the pancreatic acinar cells from the aged 3XTg AD mice, the Aβ-induced Ca2+ oscillations were sensitive to 3 m m ZnCl2 (n = 6) and 100 μm ruthenium red (n = 6), but were not sensitive to 100 μm 2APB (n = 6). In 6 cells tested, we found that bath-applied atropine did not affect the oAβ-induced Ca2+ oscillations (n = 4). These results suggest that the oAβ-induced Ca2+ oscillations are mediated through the Aβ channels.To evaluate the nature of the exogenous Aβ-induced Ca AD mice . As showllations , n = 6, llations , n = 8.  m ZnCl2 , n = 6 anium red , n = 6,  μm 2APB , n = 6. llations , n = 4. 2+ oscillations (see m) could induce typical Ca2+ oscillations (n = 6). The ACh-induced Ca2+ oscillations were sensitive to the muscarinic receptor blocker, atropine (n = 4), but were not sensitive to either removal of extracellular Ca2+ (n = 6) or treatment with the Aβ channel blocker Anle138b (n = 5). These results suggest that the trigger and pharmacology of ACh-induced Ca2+ oscillations are different from oAβ-induced Ca2+ oscillations.In these experiments, we compared the oAβ-induced Caions see  and the llations , n = 6. atropine , n = 4, lar Ca2+ , n = 6 oAnle138b , n = 5. 2+ oscillations in aged 3xTg AD mice but not in age-matched WT mice. These spontaneous Ca2+ oscillations are sensitive to extracellular Ca2+ and high concentrations of ZnCl2, and more importantly can be eliminated by Anle138b, an Aβ-formed channel blocker, suggesting that these spontaneous Ca2+ oscillations in aged 3xTg AD mice are mediated by Ca2+ influx through endogenous Aβ-formed channels. To confirm this, we mimicked Aβ-formed channels in freshly isolated pancreatic acinar cells from adult (6-mo-old) WT mice, and used patch-clamp recording to show that oAβ in the recording electrode (500 n m) can perforate the membrane and form a pore, and bath-perfusion of exogenous 500 n m oAβcan induce Ca2+ oscillations that exhibit properties similar to the spontaneous Ca2+ oscillations observed in aged 3xTg AD mice. To our knowledge, this is the first evidence of the Aβ-formed channels by endogenous Aβ in aged 3xTg AD mice.The major and novel finding in this study is the unexpected spontaneous Ca2+ homeostasis, leading to neuronal damage and death.9 In 1992, Hardy and Higgins reported that Aβ can eliminate neuronal calcium homeostasis, thus making neurons more vulnerable to environmental damage.34 In 1993, Arispe et al. used electrophysiological techniques to show that Aβ1–40 can combine with the planar lipid bilayer to further form a cation-selective channel, thereby demonstrating the ability of Aβ to form channels and in turn induce an imbalance of Ca2+ homeostasis, which may underlie Aβ neurotoxicity.35 Although this hypothesis sounds very interesting and is likely an important mechanism of Aβ toxicity, the evidence supporting this hypothesis was collected using artificial membranes or cell culture at a nascent stage.10,15 and (2) the existence of Aβ channels has not been shown in AD animal models. These knowledge gaps significantly diminish enthusiasm for this hypothesis. In this study, we unexpectedly found the spontaneous Ca2+ oscillations in pancreatic acinar cells isolated from aged 3xTg AD AD model mice, but not from age-matched WT mice. This had surprised us because there are no classical voltage-gated Ca2+ channels expressed in pancreatic acinar cells. Then, the question was how do these Ca2+ ions enter the cell? Considering that the pancreas shares a number of striking pathophysiologic similarities in Aβ deposition compared to that in brain, and AD also can cause Aβ deposition in the mouse pancreas upon overexpression of human APP, suggesting that Aβ deposits may occur in other organs than the brain.20 Therefore, we hypothesized that Aβ channels may mediate these spontaneous Ca2+ oscillations in aged 3xTg AD mice. We designed 4 experiments to test this hypothesis. First, we excluded the possibility that the spontaneous Ca2+ oscillations were mediated through classical ACh receptor—G-protein—IP3–Ca2+ (IICR) pathway by showing that atropine had no effect on the spontaneous oscillations  channels, and these are vitally important for continuous Ca2+ signaling as the intracellular (ER) store is not infinite. Direct electrophysiological recordings of the CRAC current in mouse pancreatic acinar cells have been reported,41 in which, it is shown directly that the CRAC current is acutely (and reversibly) blocked by 100 µm 2APB, the exact same concentration that is shown in 2+ oscillations in the 3xTg AD mice, strongly suggesting that the spontaneous Ca2+ oscillations are not mediated through the CRAC channels.To our knowledge, this is the first evidence that the Aβ formed channels from endogenous oAβ in an aged AD model. Aβ accumulates in pancreatic tissues and pancreatic acinar cells are not expressed in classical voltage-gated Ca2+ signal transduction mechanisms because it enables one to directly obtain considerable insight into intracellular Ca2+ handling under both normal and pathological conditions.42 Unlike nerve and endocrine cells, as well as muscle cells, exocrine cells such as pancreatic acinar cells are non-excitable and do not possess voltage-gated Ca2+ channels, and the cytosolic Ca2+ signals governing pancreatic acinar secretion are primarily generated by the release of Ca2+ from intracellular stores, principally the ER2+ oscillatory signals through a G protein-IP3 pathway  on Aβ-induced neuronal toxicity and neurodegeneration, and also on cognitive deficits and learning and memory behavioral impairment in AD models. These experiments are ongoing.In this study, we unexpectedly found spontaneous Ca


The new study by Fedotova et al.1 systematically compared Ca2+ activity induced by locomotion in neurons and astrocytes of awake, behaving mice. A main strength of this study lies in the detailed analysis of Ca2+ dynamics for the two cell types, in consideration of the coupled activity of neurons and astrocytes during spontaneous locomotion. The authors used recombinant adeno-associated viral (AAV) vectors to express the genetically encoded Ca2+ sensor GCaMP6f selectively in either astrocytes or excitatory neurons  in the somatosensory cortex. The mice were first trained to accommodate head-fixation to a mobile home cage device mounted on a microscope stage, with detection of locomotion onset by tracking software, thus enabling an accurate temporal correlation of locomotion onset with Ca2+ dynamics. The authors found that astrocytic Ca2+ rose upon initiation of movement, with first appearance in the distal processes, followed by increases of magnitude in the cell bodies. Unlike the astrocytes, neurons displayed Ca2+ activity during rest, with more rapid onset of Ca2+ elevation by locomotion. Thus, neurons and astrocytes show distinct temporal/spatial dynamics of intracellular Ca2+ during the initiation of voluntary movement.A modest literature links the onset of locomotion with increased intracellular calcium  release from locus coeruleus projections is the initiator for astrocytic cytoplasmic Ca2+ increases upon locomotion; agonism at alpha-1 adrenergic receptors leads to the delayed mobilization of intra-astrocytic Ca2+ stores. Conversely, alpha-1 receptor antagonists such as prazosin block not only locomotion-induced astrocytic Ca2+ increases, but also >90% of spontaneous astrocytic Ca2+ events, thus positing norepinephrinergic (noradrenergic) transmission as the primary driver of cortical astrocytic Ca2+ signaling.4 The alpha-1 receptor is a Gq-linked G protein-coupled receptor (GPCR) with abundant expression in astrocytes and cortical neurons,5 although alpha-1 receptors induce large-amplitude cytosolic Ca2+ surges predominantly in astrocytes.6Perhaps the most novel part of the study is in the comparison of Cacomotion . In that2+ response upon contingent locomotor initiation as indicating a refractory period due to Ca2+ mobilization from the endoplasmic reticulum. We note that GPCR activation also entails a refractory period that is determined by a desensitization/re-sensitization mechanism involving the heterotrimeric G protein complex.8 Upon GPCR activation, the Gα and Gβγ G protein complex subunits are detached from the membrane-bound receptor. Gα initiates the downstream signaling cascade, whereas Gβγ promotes the recruitment of GPCR kinases (GRKs) that phosphorylate the C-terminal tail of the GPCR to prevent G protein binding. The phosphatase PP2A eventually dephosphorylates the C-terminal, thus reinstating the responsiveness of the GPCR. Moreover, a recent study demonstrated that distinct GRKs bind to Gαq, the initiator of the Gq pathway, to drive rapid desensitization.9 Regardless of the various mechanisms, minute-scale refractory periods of locomotion-induced astrocytic Ca2+ signaling are a signature of GPCR activation. For instance, repeated activation of the optogenetic GPCR Optoα1AR shows a similarly attenuated response between the first and the second activations at an interval of 60 s.10The authors interpreted their observation of attenuated astrocytic Ca2+ increases are involved in the initiation and command of the skeletal muscle activity. On the other hand, in their analysis, the astrocytic Ca2+ responses were clearly delayed, first appearing seconds after the onset of locomotion. Prior studies have suggested that the delayed astrocytic Ca2+ increases upon locomotion promote local blood flow, energy metabolism, K+ buffering, and the termination of neuronal signaling. Now that genetically encoded fluorescent biosensors and opto-/chemo-genetic actuators have come of age, we anticipate that future studies shall address the functional consequence of astrocytic Ca2+ signaling at various spatio-temporal scales. The detailed analysis now presented in Fedotova et al. brings us a step closer to deciphering the fundamentally different types of signaling in astrocytes and cortical neurons, and how each cell type contributes to optimizing motor-related brain activity such as the initiation of voluntary locomotion.The authors proposed that the neuronal Ca


Tagetes erecta is a plant of great cultural value, and recent reports have suggested its cytotoxic effects in tumor cells. Our objective was to evaluate the antitumor activity of Tagetes erecta extract in a lung carcinoma model. Hydroalcoholic extracts were obtained from fresh flowers and leaves of T. erecta; both extracts did not exert toxicity on Artemia salina. We observed cytotoxic effects induced by the floral extract in Lewis lung carcinoma (LLC) and breast tumor cell line (MCF7), but not by the leaf extract. In vivo, a xenograft lung carcinoma model was performed with LLC cells implanted on C57BL/6 mice, which showed that the floral extract reduced tumor growth and improved the effect of etoposide. Microscopic analysis of tumors showed a reduction in mitoses and an increase in necrotic areas with the extract and the etoposide. The main phytochemical compounds found are 2,3-dihydro-benzofuran, octadecanoic acid, benzenacetic acid, oleic acid, linoleic acid, and acetic acid. We conclude that the hydroalcoholic extract of T. erecta flowers has cytotoxic effects in lung carcinoma cells and enhances the effect of etoposide.Among all cancers, lung cancer is the one with the highest mortality rate, and it also has limited therapeutics. Antitumor agents based on medicinal plants have gained importance as a source of bioactive substances.  According to the 2020 estimates by GLOBOLCAN, cancer is the leading cause of death worldwide; every year, 19.3 million new cases are recorded, and it takes the lives of approximately 10 million people around the world. Among all cancers, lung cancer has the highest mortality rate in the general population, with an estimated 1.8 million deaths per year [Currently, the available drugs for cancer treatment have limited therapeutic potential due to high toxicity, inefficiency, and high cost. Considering these facts, the development of treatments without these disadvantages is necessary. Therefore, the identification and synthesis of new efficient and less toxic anticancer agents remain an important and challenging task for cancer treatment. For this reason, natural products have become increasingly relevant in the pharmaceutical field, and traditional herbalism has been a pioneering specialty in biomedical sciences. The search for effective plant-derived anticancer agents has gained momentum in recent years. Conventional plants are a valuable source of new cytotoxic agents and play a critical role in health due to their wide range of biological and pharmacological effects and lack of toxicity ,4,5,6.Tagetes erecta, is an annual, dicotyledonous herbal species that measures between 60 cm and 1 m in height, with aromatic characteristics. Its stems have small grooves, and it has opposite leaves with indentations with serrated edges [Tagetes erecta has been used as a muscle stimulant and relaxant [Tagetes erecta on cancer by evaluating its impact on several tumor cell lines such as B16F10 (murine melanoma), HT29 (human colon carcinoma), MCF-7 (human breast adenocarcinoma), HeLa , HepG2 (human hepatocellular liver carcinoma), MO59J, U343 and U251 (human glioblastoma) [Tagetes erecta extracts inhibit tyrosinase and elastase in the H-460 (lung cancer) and Caco-2 (colon cancer) cell lines [T. erecta flowers and leaves are hexadecanoic acid, linoleic acid, quinic acid, coumaran, β-Stigmasterol, Limonene, (Z)-β-Ocimene, Terpinolene, (Z)-Ocimenone, (E)—Ocimenone and β-Caryophyllene [T. erecta’s cytotoxic effect in lung cancer cells and its antitumor effects in animal models. Therefore, the present research aims to evaluate the cytotoxic effect of T. erecta’s extract on the Lewis lung carcinoma cell line, as well as in a syngenic murine lung cancer model.Aztec Margaroid, scientifically named ed edges . In addirelaxant , as an arelaxant . Differelastoma) . Moreovell lines . The actphyllene . HoweverTagetes erecta and assessed their toxicity level using the Artemia salina model  cell lines. Both extracts were observed to exert a cytotoxic effect, but the leaf extract was less effective and that of the floral extract was comparable to an etoposide, which is an inhibitor of topoisomerase useful in lung cancer treatment ; we inclTo evaluate the selectivity of the extract in the lung cancer cell line, both extracts were tested in MCF-7 cells C, a breaTo evaluate whether the extract, in addition to being cytotoxic, had antiproliferative activity, the crystal violet method was used, where cell proliferation was evaluated after 24 h of treatment with different concentrations of both extracts. In Tagetes erecta extract, etoposide, and the combination.The tumors were visible from day 6 post implantation, as shown with arrowhead in T. erecta extract were detected, which indicate that the administration of the T. erecta extract has a greater effect in slowing tumor growth compared to etoposide. Furthermore, the combination group (T. erecta extract + etoposide) had similar results to those of the extract, including a slight tendency to further decrease tumor volume compared to the group treated with T. erecta, indicating that the compounds present in the extract do not inhibit the effect of etoposide. In fact, the extract enhances the effect of etoposide, indicating that Tagetes erecta extract could have synergistic or additive effects with conventional therapy.At the end of the treatment (day 20 post implantation), it was observed that the 3 treated groups were able to delay tumor growth; significant differences between the group treated with etoposide and the group treated with the In the control group with the greatest tumor growth, a drastic decrease in tumor weight is observed because of the deterioration of the health of the mice D, while T. erecta floral extract does not modify its metabolism.To evaluate the metabolic control of the animals, glucose was evaluated at three points of experimental kinetics: on day 1 of implantation, day 10 post implantation, and the day of sacrifice (day 20). The sample was taken with a small cut in the tail of the mice using a commercial glucometer. As can be seen in T. erecta extract, etoposide, and the combination significantly decreased cell proliferation. Furthermore, the combination of T. erecta extract with etoposide decreases proliferation more effectively than the individual administration of etoposide and the extract.To assess whether the treatments decreased tumor proliferation, a mitotic count was performed via observing the histological sections under an optical microscope at 40× magnification. The mitotic count has a directly proportional relationship to tumor proliferation. In the following illustration, the mitoses found in the representative fields of the tumors extracted from treated and untreated mice are pointed with black arrow heads A. In theTagetes erecta extract had a greater increase of 40 mm2 in the necrotic areas, compared to the 20 mm2 obtained with the etoposide. There are significant differences between the groups treated with the extract, suggesting that tumor viability of the mice that received treatment with Tagetes erecta is lower than that of the mice treated only with etoposide.The stained histological sections were observed under an inverted microscope, where photographs of 50 fields per sample were taken. These images were analyzed with the Image J program to quantify the necrotic areas of the tumors. In Tagetes erecta , octadecanoic acid, benzenacetic acid, oleic acid, linoleic acid, acetic acid, methyl β-d-galactopyranoside, n-butyric acid, methyl β-l-arabinopyranoside, and n-hexadecanoic acid.The results of GC–MS analysis revealed the presence of various bioactive components in the flowers of s erecta . The majT. erecta extracts have been evaluated in different cell lines in vitro. For example, Oliveira et al. [T. erecta extracts on five tumor cell lines and compared it with the effects of the extracts of other species. They found that the extract that had an effect in all the cell lines evaluated was T. erecta; the extract of flowers was also used and evaluated in the MCF-7 cells, where it had a cytotoxic effect of 30% [T. erecta extract did not have significant results in cytotoxicity evaluations. Other researchers, used the non-tumor cell line MDCK (Madin–Darby canine liver) and during their evaluations, no cytotoxic effects were observed at 24 and 48 h of treatment, which coincides with our in vitro results using the non-tumor cell line COS-1 [Different types of a et al.  evaluatet of 30% , a resulArtemia salina instead of a model in higher animals; we did not observe signs of toxicity in the nauplii even after adding high concentrations of the extracts, which coincides with the results reported by Cui et al. [T. erecta extract is safe.We used the toxicity assay with i et al. , where tT. erecta extracts in mice. Cui et al. [T. erecta essential oil in an acute model of induced gastric cancer. They found that the extract reverses the damage induced by cancer and prevents metastasis in other organs, so they concluded that the administration of essential oil can be used for the prevention and treatment of gastric cancer [T. erecta, as in our experiment, and found that the administration of T. erecta maintains and increases the weight of the mice, in addition to reducing the size of the extracted tumors; this is similar to our result of tumor reduction in the tumors of mice that were treated.There are two reports of in vivo models of the administration of i et al.  evaluatec cancer . In addic cancer  carried T. erecta extract, which is associated with a decrease in cell proliferation and explains the decrease in tumor volume. The sum of these two evaluations allowed us to see the effect of the treatments on tumor viability, which refers to active and developing cells that can damage healthy tissue. The groups treated with Tagetes erecta and in combination with etoposide significantly decreased cell viability compared to the two controls used, obtaining more promising results than the approved drug used as a reference.In addition, the mitotic count decreases in the groups treated with The phytochemical description of the extract developed by Barhoi et al.  has compT. erecta [Many of the main compounds, even in isolation, have activities related to cancer treatment, ranging from the preventive effects of acetic acid, which can inhibit the generation of oxaloacetate , to the . erecta ; these eIn addition, some of these compounds are found in other plant species that have anticancer activities, such as coumaran , which has anticancer activity in cancers such as prostate, kidney, breast, laryngeal, lung, colon, leukemia, and malignant melanoma . It is kT. erecta had in the different stages and evaluations of this study. The evaluations carried out demonstrate the cytotoxic and anticancer potential of the T. erecta flower extract in vivo and in vitro evaluations.It is also known that n-butyric acid, present in our extract, is capable of inhibiting cell proliferation in colorectal cancer. In addition, Forte et al.  evaluateFresh plants of TE were purchased from a local nursery in Zacatecas, Mexico, in November 2021. A taxonomist of the University Autonomous of Aguascalientes Mexico confirmed the identity of the plant material (Registry number TEUAA/nov21). Fresh flowers and leaves were mixed with 40% of ethanol/water and liquefied using an electric blender  to prepare the extract. Maceration remained for five days at room temperature. The solvent was changed twice, and the extracts were mixed, filtered, and placed in a rotatory evaporator  to concentrate it. The concentrated extract was lyophilized , and the dry powder was used in the biological assays.The TE flower extract was analyzed using gas chromatography coupled to mass (GS-MS) using an Agilent Technologies 5975C VL MSD, Santa Clara, CA, USA. The silica column used was Agilent 19091s-433E HP5MS, Santa Clara, CA, USA . The gas used was Helium, with a constant flow of 2.0 mL/min. Manual injection volume was 0.1 µL (Separation ratio: 33.4:1). The injector and ion source temperatures were set at 260 °C. The mass spectrum was taken at intervals of 2.5 min; the mass range was from 30 to 500 Da. Fragmentation was compared to the computer library .Artemia salina cysts were used; 250 mg of them were incubated in 350 mL of water with 34 g/L of salinity at room temperature with lighting. The cysts were kept in suspension with continuous light aeration for 24 h until their hatching. The hatched cysts (nauplii) selected for the test were healthy and without malformations. A total of 10 nauplii were used per replicate; while 35 g/L of saline solution was used as the negative control, quillaja standard was used as the toxic control. These organisms are sensitive during their first 24–36 h of life; during this time, a series of concentrations of both TE extracts  were added. The experiment was then allowed to run for 24 h to make a count of the dead and alive Artemias.2.Lewis lung carcinoma cells (LLC1 and ATCC CRL-1642), human breast carcinoma (MCF-7 and ATCC HTB-22), and monkey kidney fibroblasts (COS-1 and ATCC CRL-1650), obtained from the organization American Type Culture Collection (ATCC), Washington, DC, USA were cultured with a DMEM medium supplemented with 10% fetal bovine serum  and antibiotic (streptomycin-penicillin) at 37 °C with 5% COT. erecta extracts. As a positive control, etoposide 50 µM  was used, which is a first-line drug used to treat lung carcinoma whose mechanism of action is to inhibit cellular topoisomerase. The cells were incubated for 24 h; then, a 10 µL sample was taken and mixed with 10 µL of trypan blue . The resulting product was placed in a Neubauer chamber to proceed with the counting of the living (refractive) and dead (dyed blue) cells under an optical microscope in 40× objective.The cells were seeded in 24-well dishes for the treatments with T. erecta extracts or etoposide (50 µM) were incubated for 24 h in a 24-well plate. Then, the culture medium was removed to separate the unbound cells, and the plate was washed 3 times with sterile PBS , fixed with absolute methanol, and refrigerated at 4 °C for 20 min. Then, 300 µL of crystal violet (Sigma-Aldrich) solution was added, and it was incubated for 1 h at 4 °C. Next, the cells were washed with sterile PBS and 300 µL of acetic acid was added as a cellular lysis solution, 100 µL of each well in triplicate in a 96-well plate. They were then analyzed in a microplate reader  at an absorbance of 600 nm.The cells treated with C57BL/6 mice of both sexes, from 45 days of age, weighing between 25 and 30 g, were kept in conditions of room temperature (22 °C), in a cycle of 12 h light/12 h darkness, and acclimatized for one week before handling. The animals were fed pellets with food and water ad libitum.6 LLC cells/animal) using an ultrathin 1 mL syringe as previously reported [Tagetes erecta flower extract. The combination treatment group was administrated with 9 mg/Kg of etoposide 3 times a week and with 400 mg/Kg/day of extraction. The tumor volume of all experimental groups was followed from day 5 post implantation with an electronic vernier to calculate tumor volume (V) using the Attia and Weiss formula: V = 0.4 (Long diameter × Short diameter). Mice bearing LLC tumors were randomly divided among the experimental groups, treated, and sacrificed after 20 days of implantation using a CO2 chamber. Observe Lewis lung carcinoma (LLC) cells were implanted subcutaneously into the back of 15 mice .Mice were weighed daily from the day of implantation (day 1) until the day of sacrifice, and changes in body weight were reported for each day. Three glucose measurements were taken—on the day of implantation, on day 10, and on the day of sacrifice—with an Accu-checkBiopsies of the excised tumors were taken; to subsequently fix them in a 10% formalin solution, the samples were submerged for a period of 48 h in the solution, and then the tissue was embedded in paraffin. Once the paraffin block was obtained, cuts were carried out in a microtome, and the cuts obtained were spread and adhered to slides; then, they were deposited onto the surface of the liquid contained in a container called a “flotation bath”. In the staining procedure, hematoxylin eosin was added; in this staining process, the nuclei are stained with hematoxylin, and then eosin is responsible for coloring the cytoplasm. The nuclei stain appears to be purple, while the cytoplasm and extracellular material stain appear in various shades of pink.For the analysis of mitotic cells, 100 fields were considered in the total samples per group, and an optical microscope with 40× magnification was used. An inverted microscope was used for the analysis of necrotic areas; they were photographed and analyzed with the Image J program. In total, 50 fields per sample were considered.p < 0.05.To demonstrate differences between more than two groups, a one-way Analysis of Variance (ANOVA) and Dunnett’s post hoc were used. Recording of tumor volume during days was analyzed using repeated measures two-way ANOVA with Sidak’s or Tukey post-hoc. Results were expressed as mean ± standard error deviation or standard deviation as indicated in the legends of the figures. The data were considered statistically different when T. erecta flowers and leaves has a cytotoxic effect on Lewis lung carcinoma cells. Particularly, the flower extract showed high effectiveness in lung cancer cells, and interestingly, had selectivity to cancer cell lines without effects on non-cancer cells. The present study indicates that the T. erecta flower extract inhibits tumor progression and increases its effectiveness in combination with etoposide. The results contribute to the evidence that the bioactive compounds present in the hydroalcoholic extract of T. erecta have therapeutic potential against lung cancer. The compounds found in the floral extract to which the antitumor effect is attributed are coumaran, octadecanoic acid, benzeneacetic acid, oleic acid, linoleic acid, acetic acid, methyl-β-d-galactopyranoside, n-butyric acid and n-hexadecanoic acid.The hydroalcoholic extract of


In this study, we explored in area postrema NSCs the presence and roles of a subset of Ca2+ channels, the store-operated Ca2+ channels (SOCs) that have the capacity to transduce extracellular signals into Ca2+ signals. Our data show that NSCs derived from the area postrema express TRPC1 and Orai1, known to form SOCs, as well as their activator STIM1. Ca2+ imaging indicated that NSCs exhibit store-operated Ca2+ entries (SOCEs). Pharmacological blockade of SOCEs with SKF-96365, YM-58483  or GSK-7975A resulted in decreased NSC proliferation and self-renewal, indicating a major role for SOCs in maintaining NSC activity within the area postrema. Furthermore, our results show that leptin, an adipose tissue-derived hormone whose ability to control energy homeostasis is dependent on the area postrema, decreased SOCEs and reduced self-renewal of NSCs in the area postrema. As aberrant SOC function has been linked to an increasing number of diseases, including brain disorders, our study opens new perspectives for NSCs in brain pathophysiology.Neural stem cells (NSCs) persist in specific brain germinative niches and sustain neurogenesis throughout life in adult mammals. In addition to the two major stem cell niches in the subventricular zone and the hippocampal dentate gyrus, the area postrema located in the brainstem has been identified as a neurogenic zone as well. NSCs are regulated by signals from the microenvironment that adjust stem cell response to the needs of the organism. Evidence accumulated over the past decade indicates that Ca In vitro, area postrema NSCs have the capacity to proliferate and give rise to neurons and glial cells  reside in defined niches in the adult brain where they produce new neurons and glial cells throughout life . In addial cells . In vivonditions  and are nditions , as expenditions . As a manditions , and amynditions  while stnditions .2+, as an intracellular second messenger, controls a remarkable diversity of biological processes occurring throughout life, including genesis and homeostasis of organs and tissues . SOCs are so named because they are activated by the emptying of intracellular Ca2+ stores of the endoplasmic reticulum (ER) . IP3 interacts with its receptors in the ER membrane and induces ER Ca2+ release. The subsequent transient decrease in ER Ca2+ content is sensed by STIM1, which oligomerizes and activates SOCs, thereby inducing a Ca2+ influx from the extracellular compartment , not only provides Ca2+ to refill the ER, but also, importantly, drives a wide set of biological processes by elevating the cytosolic Ca2+ concentration for minutes to hours. This Ca2+ signal enables the recruitment of cellular effectors and Ca2+-dependent signaling cascades.Ca tissues . One of lum (ER) . SOCs arpartment . This Ca2+ levels  C57BL/6J mice . Animals were housed in animal care facilities . Experimental procedures were carried out in accordance with the guidelines of the French Ministry of Agriculture and the European Communities Council Directive and validated by the Regional Ethical Committee (Authorization no. 02184.01).SKF-96365 and YM-58483 were purchased from Sigma Aldrich  and dissolved in water and DMSO, respectively. GSK-7975A from Aobious  was dissolved in DMSO. Leptin was purchased from R&D Systems  and dissolved in water. Cell culture media and growth factors were from Invitrogen .–1 EGF and 10 ng.ml–1 FGF2. Neurospheres were grown in suspension and obtained after 1 week in vitro  containing 0.1% (volume/volume) Tween 20 (TBS-Tween), immunoblots were probed overnight at 4°C in TBS-Tween with 3% (weight/volume) fat milk with either rabbit polyclonal anti-Orai1 , or mouse monoclonal anti-TRPC1 , or mouse monoclonal anti-STIM1 , or rabbit polyclonal anti-actin . Membranes were then incubated for 1 h at 4°C with anti-mouse or anti-rabbit immunoglobulin-HRP-linked  before detection of the bound antibodies using the ECL enhanced chemiluminescence system . Results were analyzed with GeneGnome XRQ .For western blotting analysis, area postrema neurospheres were lysed in Laemmli loading buffer (Sigma Aldrich) and heat denatured at 95°C for 5 min, in parallel with subventricular zone neurospheres used as positive controls . All samImmunostaining was performed on the one hand, on area postrema neurospheres thrown on glass slides with a cytospin and fixed in methanol at −20°C, and on the other hand, on adult mouse brain sections. For brain sections, mice were deeply anaesthetized and transcardially perfused with 0.9% NaCl solution followed by phosphate-buffered 4% paraformaldehyde solution at 4°C during 10 min. Brains were removed, post-fixed for 1 h at 4°C, and cryoprotected in phosphate-buffered 30% sucrose solution at 4°C overnight before cutting 20 μm-thick sections using a cryostat (Leica 2500).–1) or a mouse monoclonal anti-SOX2  antibody along with one of the following antibodies: rabbit polyclonal anti-Orai1 , or mouse monoclonal anti-TRPC1 , or mouse monoclonal anti-STIM1 . Preparations were then revealed with the appropriate Alexa fluor 555 or Alexa fluor 488 conjugated antibodies . DAPI was used to label nuclei. Preparations were then analyzed with an FV-1000 spectral confocal station installed on an inverted microscope IX-81 (Olympus). The emitted fluorescence was detected by spectral detection channels between 425–475 nm, 500–530 nm, and 550–625 nm, for UV, green and red fluorescence, respectively.For immunostaining, preparations were permeabilized and non-specific binding sites were blocked prior to incubation with a goat polyclonal anti-SOX2 . Assays were performed in standard external buffer solution  supplemented with 1.8 mmol.l–1 Ca2+ or 0.1 mmol.l–1 EGTA for the Ca2+- free solution. Cells were perfused with the buffer solution containing 0.1 mmol.l–1 EGTA (Ca2+-free solution) and SOCE was triggered with thapsigargin . For experiments with leptin, area postrema cells were incubated with 6.25 nmol.l–1 leptin during 30 min at 37°C prior to intracellular Ca2+ measurements. Fluorescence images were recorded using a lambda 421 coupled to an Olympus IX73 inverted microscope and a Zyla sCMOS 4.2 PLUS camera. Consecutive excitation of Fura-2AM at 340 nm and 380 nm was undertaken every 2 s, and emission fluorescence was collected at 505 nm at 37°C using the Metafluor software. The 340/380 nm fluorescence ratio was measured over time in selected regions of interest (ROI) on several cells whose numbers are given in 2+ re-addition .Neurospheres were dissociated and 30,000 cells were plated as single cells at on fibronectin-coated glass coverslips. After 2 h, the cells were incubated for 30 min at 37°C with 3 μmol.l–1 in poly-L-Lysine-coated 96 well plates. The cells were treated for 24 h with SKF-96365, YM-58483 or GSK-7975A used at the concentrations ranging, respectively, from 10 nmol.l–1 to 10 μmol.l–1, 100 nmol.l–1 to 10 μmol.l–1 or 1 μmol.l–1 to 30 μmol.l–1, as indicated in Cell proliferation was determined by a bromodeoxyuridine (BrdU) incorporation assay. To this aim, area postrema neurospheres were dissociated as single cells and plated at the concentration of 30,000 cells.ml–1), YM-58483 (5 μmol.l–1), GSK-7975A (20 μmol.l–1) or leptin (6.25 nmol.l–1). Controls were performed with culture medium alone (for SKF-96365 and for leptin) or supplemented with DMSO . Primary neurospheres were allowed to grow during 1 week and then counted under a microscope. For self-renewal assays, area postrema primary neurospheres were collected, dissociated as single cells, and reseeded in culture medium to obtain secondary neurospheres.The area postrema was dissected from mouse brains, dissociated, and seeded at 10,000 cells per ml in 24-well plates in cell culture medium supplemented with any of the following treatments: SKF-96365 . The statistical significance level was set for p-values < 0.05 and represented on the figures by: *p < 0.05, **p < 0.01, ***p < 0.001.Statistical significance of differences was examined by one-way analysis of variance (ANOVA) followed by the Bonferroni’s 2+ sensor STIM1 at the ER membrane and can mobilize also TRPC1 which will amplify the Ca2+ entry. Western blot . 2+ solution induces an increase in intracellular Ca2+ concentration in area postrema NSCs due to inhibition of Ca2+ uptake and a passive leak of Ca2+ from the ER. The ER store-depletion is sensed by STIM1 and secondarily activates SOCs at the plasma membrane. Although SOCs open within a few tenths of a second after Ca2+ release from the endoplasmic reticulum, no Ca2+ can enter the cell as long as the cell is maintained in a Ca2+-free buffer solution. After Ca2+ release from ER was over, Ca2+ was added to the extracellular medium and permitted a Ca2+ influx into the cell  and 46% for YM-58483  compared to control (DMSO). These results indicate that SOC inhibitors decreased SOCE, confirming the presence of functional SOCs in area postrema cells  or presence of the SOC inhibitors SKF-96365, YM-58483, and GSK-7975A  or presence (dashed line) of leptin. Quantification of the initial slope and maximum of the traces reveals that leptin significantly reduces SOCEs of 54 and 15%, respectively, compared to control .CB: collection, assembly, analysis and interpretation of data on RT-PCR, and self-renewal assays. ET and ND: collection, assembly, and interpretation on calcium imaging. PA: collection, assembly, analysis and interpretation of immunostaining data, and manuscript writing. TH: design, analysis, and interpretation of western blot data. LC: collection and analysis of cell proliferation data. BC: analysis and interpretation of calcium imaging data, and manuscript writing. EM and VC: conception and design, financial support, collection, assembly, analysis and interpretation of data, and manuscript writing. All authors approved the manuscript.


The Emerging perspectives on calcium signaling in plant-microbe interactions suggest that this complex interplay could be harnessed to improve plant resistance against pathogenic microbes. We present here an overview of current understanding in calcium signatures during plant-microbe interaction so as to imbibe a future direction of research.The process of plant immune response is orchestrated by intracellular signaling molecules. Since plants are devoid of a humoral system, they develop extensive mechanism of pathogen recognition, signal perception, and intricate cell signaling for their protection from biotic and abiotic stresses. The pathogenic attack induces calcium ion accumulation in the plant cells, resulting in calcium signatures that regulate the synthesis of proteins of defense system. These calcium signatures induct different calcium dependent proteins such as calmodulins (CaMs), calcineurin B-like proteins (CBLs), calcium-dependent protein kinases (CDPKs) and other signaling molecules to orchestrate the complex defense signaling. Using advanced biotechnological tools, the role of Ca Besides these, Ca2+ functions as a secondary messenger in cell to cell signaling, and disruptions in its levels occur during biotic or abiotic stress responses , cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP)  to produce diacylglycerol and InsP3. The InsP3 subsequently interacts with InsP3 receptors located on the endoplasmic reticulum, inducing the discharge of Ca2+ into the cytosol (2+ levels and increased resistance against Pseudomonas syringae (2+ from acidic compartments such as vacuoles and lysosomes by activating ryanodine receptors (RyRs) and two-pore channels (TPCs), respectively  . InsP3 i cytosol . The ovesyringae . On the ectively . For exast cells .2+. Calcium flow occurs through the cell wall, vacuoles, apoplast and different cellular organelles  in plants. The CDPKs target different defense-responsive proteins and bring about resistance response . Additioganelles . Figure2+ into the cytoplasm of plant cells  protein. CaM binds to calcium ions in a calcium-dependent manner, leading to a conformational change that allows it to interact with downstream effectors. CDPKs are a group of calcium-dependent protein kinases involved in several physiological processes, including stress response, development and hormone signaling . CBL-intFactors like greenhouse conditions, extreme temperatures, drought stress, and chelation can hinder calcium uptake, leading to symptoms like black spots, bushy morphology, and stunted growth . Therefo2+) into host cells, which activates various defense responses. Calcium signaling pathways can also play a role in mediating communication and coordination between microbes, as quorum sensing systems can involve calcium signaling -Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI). These pathways have different Ca2+ signatures. The PAMPs are recognized by Pattern Recognition Receptors (PRRs) triggering PTI, which induces a rapid and brief influx of Ca2+ from extracellular sources. This event activates downstream signaling pathways resulting in the expression of defense-related genes, ROS production and callose deposition. ETI is initiated when plants perceive specific pathogen effectors that disrupt host defenses. This recognition results in sustained elevations of Ca2+ and the activation of downstream signaling pathways, transcription factor activation, mitogen activated protein kinase cascade and hypersensitive response  or damage associated molecular patterns by plant pattern recognition receptors (PRRs) . Notably2+ concentration. PRRs can directly activate calcium-permeable channels. For example, upon activation, FLS2 interacts with Arabidopsis-autoinhibted Ca2+ ATPase 8 (ACA8) and ATPase10, which acts as Ca2+ pumps situated in plasma membrane , such as cAMP and cGMP, to regulate Ca2+ levels and transduce different signaling events , two pore channels 1 (TPC1), glutamate receptors (GLRs) and annexins. In g events .Arabidopsis have been identified to play significant roles in the immune response against pathogens. CNGC2 and CNGC4 were found to trigger ROS generation and ETI response upon recognition of flg22, a bacterial PAMP  and genes involved in the biosynthesis of plant hormone salicylic acid (SID2) (Plant GLR-type Captations . Respons soybean . An immud (SID2) . The res2+-dependent or -independent manner. They play essential roles in regulating various cellular processes in plants, including responses to abiotic and biotic stresses as well as developmental processes , into the cell. This increase in intracellular calcium concentration serves as a crucial second messenger, initiating a cascade of signaling events that regulate various cellular processes. In plants, mechanosensitive channels have been extensively studied, particularly in Arabidopsis , contain EF-hand motifs, which are common calcium-binding structural domains found in calcium sensor proteins. In CMLs, the EF-hand motifs are typically present in pairs, and each motif consists of approximately 30 amino acids, forming a helix-loop-helix structure. The functional significance of these EF-hand motifs in CMLs lies in their calcium-binding properties and their role as calcium sensors in plant cells . The CalArabidopsis thaliana alone, there are 50 members of the CML family, each of which may have a specific role in plant physiology due to their varying expression patterns in response to developmental stages, tissues, and environmental stimuli. Recent research has highlighted the critical role of CMLs in plant immunity, where they act as essential Ca2+ sensors involved in defense mechanisms against microbial pathogens and herbivores. Numerous studies have demonstrated the importance of individual CaM/CML gene expression in plant defense responses to various pathogens. Gain-of-function experiments have demonstrated that overexpression of specific CMLs, such as soybean CMLs, can boost plant tolerance to a broad range of pathogens. For example, overexpressing soybean CMLs in tobacco results in increased resistance to various pathogens, while overexpression of a typical CaM does not have the same effect, suggesting that CMLs are selectively activated in response to pathogen invasion.Plants possess a diverse range of CaM-like proteins (CMLs) that are distinct from typical CaMs. In P. syringae by overexpressing the APR134 ortholog from Arabidopsis CML43. Studies have shown crucial roles in regulating plant defense against various pathogens in Arabidopsis are played by calcium binding proteins such as CML8, CML9, CML24 and CML41. The hypersensitive response is impaired and nitric oxide production is reduced upon recognition pf pathogen-associated molecular patterns when CML24 is knocked out. Plant defense against different strains of Pseudomonas syringae is positively regulated by CML9 and CML8 and CML8 functions mainly through salicylic acid (SA)-dependent pathways. Furthermore, defense against P. syringae is positively regulated by CML41 through the facilitation of plasmodesmata closure in response to bacterial flagellin  does not  leads to higher tolerance against the oomycete pathogen Phytophthora capsici, as this CML was found to exert negative control on the activation of PR genes.Loss-of-function genetic studies have provided valuable insights into the roles of different CMLs in plant immunity. The reduction in expression of pathogen-induced CaM isoforms, specifically NtCaM1 and NtCaM13, had distinct effects on disease resistance in tobacco . Silenciognition . CML8 anc stress . Recent 2+ decoders, CDPKs integrate both Ca2+ sensing and downstream signal propagation capabilities into a single module. In angiosperms, the number of CDPK members is around 30, and they are organized into four distinct groups based on their family architecture. CDPKs or calcium dependent protein kinase are diverse group of serine/threonine protein kinase found in plants. They play crucial roles in regulating various aspects of plant growth development and responses to abiotic and biotic stresses. CDPKs consists of three main structural components: a variable N-terminal, a central kinase domain, and an activation domain. It typically consists of an auto-inhibitory pseudo-substrate linked to a CaM like domain containing four EF- hands and acts as calcium sensor. When calcium ions bind to the EF-hands, a conformation change occurs, relieving the auto-inhibition and enabling kinase activation. This allows the kinase to become active and initiate downstream phosphorylation events, which can lead to various cellular responses such as gene expression, ion fluxes, and protein degradation  in regulating guard cell S-type anion- and Ca2+-permeable channels, which are crucial for stomatal closure. The research demonstrates that CDPK5/CPK6 plays a pivotal role in ABA-mediated signaling pathways, leading to the activation of downstream defense responses in guard cells. This example sheds light on the specific functions of CDPKs in tomato and highlights their significant contribution to enhancing plant defense against pathogens.CDPKs play a crucial role in plant defense signaling against various biotic and abiotic stresses. Unlike other Caradation . The rolPseudomonas syringae is collectively contributed by CDPK4, CDPK5, CDPK6 and CDPK11, while AtRBOHD, the primary reactive oxygen species producing enzymes involved in immunity, can be phosphorylated by CDPK5, CDPK6, CDPK11 and CDPK4  revealed 37 CIPKs, indicating their abundance in plants  and CBL-interacting protein kinases (CIPKs) have emerged as a new class of plant calcium sensors that play a crucial role in response to both biotic and abiotic stresses . Arabidon plants .Puccinia striiformis f. sp. tritici (Pst)  .Capsicum annum L.), nine CaCBLs and twenty-six CaCIPKs were induced under stress conditions. CIPK1 specifically participates in biotic stress responses in C. annum, as shown by the increased sensitivity of cacipk1 mutant lines to the fungal pathogen Phytophthora capsici. Conversely, CaCIPK1-OE lines exhibit heightened defense activity, accompanied by H2O2 accumulation and cell death (Triticum aestivum), TaCBL4 has been shown to be involved in defense responses against Puccinia striiformis f. sp. tritici infection. The upregulation of TaCBL4 following infection and increased susceptibility in the loss-of-TaCBL4 function mutants suggest its role in the defense mechanism. TaCBL4 interacts with TaCIPK5, indicating their joint participation in positive defense mechanisms . TaNH2, a counterpart of Arabidopsis NPR3/4, has been shown to play a crucial role in salicylic acid (SA) signaling and collaborate with TaCIPK10 in the defense response by interacting and phosphorylating it  and observed a constitutively enhanced Ca2+ in cytosol. Since the Ca2+ signals are very localized and are influenced by other cellular and sub-cellular factors, they pioneered to use a whole plant system to explore calcium connections during plant-pathogen interaction. Botrytis–induced kinase 1 (BIK1) is transcribed in plants upon infection by Botrytis and defends plants from biotrophic and necrotrophic pathogens by regulating salicylic acid (SA) synthesis in plant cell . It is also known to increase ROS generation by modulating the calcium concentration owing to site specific phosphorylation of NADPH oxidase RbohD (2+ indicators (GECIs) to investigate the oscillatory nature of flg22‐induced cytosolic Ca2+ signals at single cell level in the guard cells of the leaves of Arabidopsis thaliana. They observed that the flg22 could elicit oscillations in single cell and accordingly, all the oscillations occurring in all the cells contribute to the total oscillation of the whole plant. The pathogenic and mutualistic fungi too establish their relation with plants by altering Ca2+ efflux or influx. The cytoplasmic free calcium ([Ca2+]cyt) transiently increased to about 1 μM in transgenic Parsley expressing apoaequorin, upon treatment with Pep-13, an oligopeptide elicitor obtained from Phytophthora sojae. This peaked concentration of ([Ca2+]cyt), later returned to normal level of about 300 nM. This biphasic variation in calcium concentration was observed to be associated with oxidative burst and phytoalexin production (Nicotiana plumbaginifolia expressing apoaequorin proteins have been studied for changes in ([Ca2+]cyt), when treated with cryptogein and oligogalacturonides, the elicitors of defense mechanism in plants. The elicitor treated cells showed Ca2+ influx and released calcium from internal stores  had different kinetics of Ca2+ spiking. The strain Rm1021 elicited a robust Ca2+ spiking after an initial lag phase of 10–15 min, while no Ca2+ spiking was detected in the strain Rm2011. They also observed in their study that the calcium spiking triggered by bacteria is no different than the calcium spiking observed in response to purified nodulation factors. Therefore, it was concluded that the calcium spiking is a nod gene-dependent host response. The availability of genetically encoded Ca2+ probes facilitated the study of Ca2+ concentrations at the cell and organ level during symbiotic interaction of plants with microbes. 2+ oscillations induced by them. The two symbiotic pathways required both DMI1 and DMI2 for functional symbiosis and for Ca2+ oscillations. The results also indicate that Ca2+ oscillations are an essential and necessary process for rhizobial or mycorrhizal colonization of plant roots. It is notable that Ca2+ oscillations induced by symbiotic fungus differ in its amplitude and periodicity from the Ca2+ oscillations induced by Nod-factors. Medicago plants to evaluate Ca2+ responses in nucleus of mutant lines defective for NFP, DMI1, and DMI2. These three genes function upstream of Ca2+ spiking in Nodulation factor (NF) mediated signal transduction. The defective mutant of DMI3, which encodes for CCaMK was also studied. When treated with NF elicitors, no change in Ca2+ concentration in nucleus of nfp, dmi1, and dmi2 mutants was observed. However, the dmi3 mutant showed an immediate surge in nuclear calcium spiking. Using a nucleoplasmin-tagged cameleon (NupYC2.1) coupled with mathematical modeling and time lapse imaging, they concluded that the initial spiking in Ca2+ originates in the vicinity of both sides of the nuclear envelope.The endophytic microbes, usually fungal or bacterial, inhabit the internal tissues of plants but without causing disease. The endophytes reside in the seeds and along with the process of germination, they promote healthy growth of the plant. The endophytes are present in nearly all the plants. In some cases, they may have originated from soil, which upon successful interaction in the rhizosphere, colonize the plants internally to the benefit of the host. Likewise, the endophytes become an integral component of plants. These endophytes are beneficial to plants in multiparous ways like they enhance nutrients uptake by plant roots; strengthen the defense mechanism of plants; alleviate stress induced damages; modulate plant development and keep a check on the weed growth. 2+ signals. In rhizobia-legume symbiosis, Ca2+ signals are necessary for recognition and infection process for root nodule formation  symbiosis as they investigated the mutualistic associations of plant roots and fungi of the Glomeromycota phylum. The hyphae enter the root cells and forms arbuscules, wherein nutrient exchange between the plant and the fungus takes place. The Ca2+ signals regulate this nutrient exchange and is involved in the establishment of the plant-fungus interface.2+ spiking response. This spiking in Ca2+ are recognized by the plant-specific CCaMK that contains a kinase domain and three Ca2+-binding EF hands. The activation of CCaMK is determined by free Ca2+ or activated calmodulin, while variations in the Ca2+ signatures determine whether the Ca2+ information is decoded into symbiosis or nodule formation. The autophosphorylation site of CCaMK is responsible for nodule formation, indicating that it acts as a regulatory switch.In mycorrhizal symbiosis, calcium signaling is essential for the recognition and coordination of the mutualistic association between plant roots and mycorrhizal fungi to establish a successful association. Flavonoid compounds released from plant roots signal compatible fungal species, while chitin oligosaccharides secreted by the fungal hyphae stimulate calcium signaling in plant cells. Calcium signaling activates downstream signaling pathways that lead to the formation of a symbiotic interface, where the exchange of nutrients between the plant and the fungus can occur. The nuclei in plants respond to rhizobacteria and AM fungi through the induction of a Ca2+) occur, which indicate that host cells perceive the signaling molecules diffused by fungi. The Ca2+ signal is induced by these diffusible molecules and there appears a biphasic Ca2+ trace, which is characterized by a sudden surge in (Ca2+) cyt and a small transitionary increase thereafter, that dissipates in about 30 minutes. This Ca2+ response is essential for the formation of a polarized cytoplasmic assembly called the perpetration apparatus (PPA), which is required for successful colonization of the plant root by the fungi.During the establishment of a symbiotic relationship between plants and mycorrhizal fungi, transient changes in cytosolic calcium  leads to downstream events that promote root hair deformation and initiate infection thread formation  such as volicitin or linolenoyl-L-glutamine have been demonstrated to cause changes in cytosolic Ca2+ levels. Studies with lima bean leaves and soybean cell cultures have demonstrated that Ca2+ signaling is activated by Spodoptera littoralis larvae bites and certain components present in their regurgitate, like linolenoyl-L-Glutamine and volicitin  is a luminescent calcium-sensitive protein, isolated from the bioluminescent crystal jelly, Aequorea victoria and is the pioneering GECI used to study Ca2+ signaling in plants. The AEQ luminesces upon binding with Ca2+ ions and thereby serves as a reporter for any change in Ca2+ ions concentration, brought about by touch, cold stress or pathogenic stress . Furthermore, the researchers generated Tacipk14 mutant plants via CRISPR/Cas9 technology that showed higher resistance to Pst without any significant difference in crop yield in comparison to control plants. These results demonstrate the potential of using CRISPR/Cas9 technology to develop disease-resistant wheat varieties by targeting the genes involved in various calcium signatures  using Barley stripe mosaic virus (BSMV) has been employed to study the function of various genes, including those encoding Cyclic nucleotide-gated channels (CNGCs) during plant-pathogen interactions. 2+ signaling elements, which have been extensively studied in plants. The diversity of Ca2+ transport elements in plants reflects their evolutionary origins, functional diversification, and roles in stress response. In order to fulfil the intricate and myriad functions of the Ca2+ transport system, different types of Ca2+ transport elements are present in plants , in rice as well as in drought stress in tobacco (Botryosphaeria dothidea-infected apple leaves revealed differential expression of MdCNGC genes in response to the pathogen (2+ signaling elements play crucial roles in regulating the resistance to pathogens in plants. For instance, a study by Ralstonia solanacearum. The research found that numerous genes involved in calcium signaling were associated with plant defense responses. Previous studies have typically investigated bacteria and host plants separately; however, meta-transcriptomic analysis, provides a more comprehensive information on the simultaneous transcriptional state of a variety of microorganisms. Bacillus subtilis MBI600, a biocontrol agent. They found that an upregulation of expression of genes involved in plant growth promotion, stress response, and defense-related pathways were upregulated in the treated plants. The metabolomic analysis revealed changes in the levels of metabolites involved in primary and secondary metabolism, including amino acids, organic acids, and phenolic compounds. These results suggest that the Bacillus subtilis MBI600-mediated growth promotion and enhanced defense response in cucumber plants may be attributed to the activation of signaling pathways and the biosynthesis of defense-related metabolites. Proteomics studies have also been utilized for identification of proteins involved in calcium signaling pathways in plant-microbe interactions. For instance, a proteomic study discovered calcium-binding proteins and ion transporters as vital components of calcium signaling in the interactions between soybean roots and Bradyrhizobium japonicum (The use of innovative “omics” techniques, combined with remarkable advancements in modern molecular biology, has empowered scientists to uncover the intricate interactions between stress signaling and response in plants. Genome-wide studies have helped to identify genes encoding Can plants . In silit stress . Also, C tobacco . Similarpathogen . These saponicum .The intricate involvement of calcium signaling in plant-microbe interactions presents opportunities for biotechnological applications aimed at enhancing plant growth and improving crop yields. Calcium-based fertilizers have been proposed as potential tools for promoting symbiotic interactions between plants and beneficial microbes, such as endophytes, mycorrhizal fungi and rhizobia. Additionally, the manipulation of calcium signaling pathways in plants through genetic engineering or chemical treatments could be utilized to confer resistance against microbial pathogens or improve stress tolerance.in vivo. Another challenge is the specificity of calcium signaling responses, which must be finely tuned to ensure appropriate responses to different microbes and environmental conditions. Understanding the mechanisms of specificity and cross-talk between different signaling pathways will be essential for developing strategies for manipulating calcium signaling in plants.Despite the potential benefits of harnessing calcium signaling in plant-microbe interactions, there are several challenges that must be overcome. One major hurdle is the complex nature of calcium signaling pathways, which involves a large number of components and regulatory mechanisms. The identification and characterization of key components and their interactions will require further research and development of new techniques for studying calcium signaling dynamics New research avenues are emerging to provide deep insights into the intricacies of calcium signaling in plant-microbe interactions. For example, advances in imaging techniques and biosensors are enabling the visualization and measurement of calcium signals in real-time, providing a detailed understanding of the spatiotemporal dynamics of calcium connections in plants and microbes. Furthermore, the identification and characterization of novel components of calcium signaling, such as ion channels and transporters, will help to further unravel the complex mechanisms of calcium signaling in plant-microbe interactions. The omics approaches, such as transcriptomics and proteomics, may provide a system-level understanding of calcium signatures in plants and microbes.2+ binding and Ca2+ decoding proteins are involved in the process of Ca2+ dependent signaling, mutation studies for identification of genes responsible for Ca2+ surge is recommended as a promising method to unravel this complexity. The study of calcium signaling mechanisms in plant-microbe interactions has significant implications for biotechnological applications, particularly in the development of calcium-based fertilizers to enhance symbiotic interactions. However, there are still key challenges to overcome in understanding the intricacies of calcium signaling in plant-microbe interactions, particularly in the development of novel techniques for studying calcium signaling in plants and microbes. The precise manipulation of Ca2+ signals may be harnessed to obtain climate smart crops with enhanced stress tolerance. The generation and dissipation of Ca2+ could be controlled using reverse genetic approaches. Overall, the unravelling of calcium signaling mechanisms in plant-microbe interactions provides exciting research avenues for future investigations.This review highlights the crucial role of calcium signaling in plant-microbe interactions. Calcium signaling pathways in plants involve the release of calcium ions, transporters, and sensors, which play a critical role in plant responses to microbial pathogens and beneficial microbes. The modulation of calcium signaling by microbial effectors, particularly in pathogenic interactions, has been extensively studied. Additionally, calcium signaling has been identified as a crucial mediator in plant-microbe symbiosis, particularly in the establishment of arbuscular mycorrhizal and rhizobia interactions. Since a large number of CaNN and AR conceived and conceptualized the idea. NN, AR, PN, RP, GP, BC, and DK contributed to writing, editing, reviewing and finalizing the article. All authors contributed to the article and approved the submitted version.