A receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABAB receptors is still unknown. Preliminary evidence showed that GABAA receptor stimulation can induce an increase in endothelial Ca2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca2+]i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca2+ release from neutral and acidic Ca2+ stores, respectively, and sustained by store-operated Ca2+ entry. GABAA and GABAB receptors were both required to trigger the endothelial Ca2+ response. Unexpectedly, we found that the GABAA receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptors requires functional GABAA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABA Of note, recent studies have revealed that GABAA receptors may trigger a metabotropic  signaling pathway that leads to InsP3-induced Ca2+ release from the ER [2+]i regulates both angiogenesis i [q-protein coupled receptors (GqPCRs) and stimulate PLCβ to trigger InsP3-dependent Ca2+ release from the ER. This initial Ca2+ peak can be supported by lysosomal Ca2+ mobilization through two-pore channels 1 and 2  and is maintained over time by SOCE activation [2+]i in hCMEC/D3 cells in a flux-independent mode by interacting with metabotropic glutamate receptors [2+ signals in hCMEC/D3 cells. We found that both GABAA and GABAB receptors are expressed and that GABA triggers a biphasic increase in [Ca2+]i. Pharmacological manipulation revealed that GABA-induced intracellular Ca2+ release was mediated by ER Ca2+ mobilization through InsP3Rs and lysosomal Ca2+ discharge via TPC1-2, whereas Ca2+ entry was mediated by SOCE. However, GABAA receptors did not mediate any detectable inward current, but they rather signaled in a flux-independent manner to cause intracellular Ca2+ release. Conversely, the selective stimulation of GABAB receptors induced the expected metabotropic Ca2+ signal that has been described in other cell types. Intriguingly, we provide evidence that GABAA and GABAB receptors interact to elicit a full Ca2+ response to GABA in hCMEC/D3 cells. These data provide the first elucidation of the signaling pathways whereby GABA can increase the [Ca2+]i in cerebrovascular endothelial cells.The hCMEC/D3 cell line represents the most suitable in vitro model of human cerebral microvascular endothelial cells ,30 and h2+]i ,31. For 2+]i , adenosi2+]i ,34, glut2+]i , and his2+]i  bind to tivation ,31. Intreceptors . Herein,2 and grown in tissue culture flasks coated with 0.1 mg/mL rat tail collagen type 1, in the following medium: EBM-2 medium  supplemented with 5% fetal bovine serum, 1% penicillin–streptomycin, 1.4 μM hydrocortisone, 5 μg/mL ascorbic acid, 1/100 chemically defined lipid concentrate , 10 mM HEPES and 1 ng/mL basic fibroblast growth factor. The cells were cultured at 37 °C, 5% CO2 saturated humidity.Human cerebral microvascular endothelial cells (hCMEC/D3) were obtained from the Institut National de la Santé et de la Recherche Médicale . hCMEC/D3 cells cultured between passage 25 and 35 were used. As described in , the cel2, 1 MgCl2, 10 glucose, 10 HEPES. In Ca2+-free solution (0Ca2+), Ca2+ was substituted with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of PSS as measured with an osmometer  was 300–310 mOsm/L.Physiological salt solution (PSS) had the following composition (in mM): 150 NaCl, 6 KCl, 1.5 CaCl2+ imaging set-up that we have described elsewhere i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm [Ratio (F340/F380)]. An increase in [Ca2+]i causes an increase in the ratio i into a transient Ca2+ signal. The subsequent restitution of Ca2+ to the perfusate induced a second elevation in [Ca2+]i, which was due to extracellular Ca2+ entry . As discussed elsewhere ,632+ relA receptors initiate the Ca2+ response to GABA in mouse cerebrovascular endothelial cells i in both neurons i that closely resembled the Ca2+ response to GABA (100 µM). There was no significant difference in the percentage of responding cells (2+ response to GABA was significantly (p < 0.0001) higher as compared to both muscimol and baclofen (2+ response to the combined application of muscimol (15 µM) and baclofen (100 µM) was also significantly (p < 0.0001) lower as compared to GABA  and acidic (lysosomes) Ca2+ stores and maintained over time by SOCE. It has long been known that GABAB receptors are coupled to InsP3-induced ER Ca2+ mobilization and SOCE activation [A receptors could signal an increase in [Ca2+]i in a flux-independent manner i via Gi/o proteins and lend further support to the evidence that GABAB receptors are also crucial to the onset of GABAA-receptor-mediated Ca2+ signals.To further confirm this hypothesis, we pretreated hCMEC/D3 cells with pertussis toxin (PTx), which blocks the heterotrimeric Gtivation ,12,22. FA and GABAB receptors mediate the Ca2+ response to the inhibitory neurotransmitter GABA in the widely employed human cerebrovascular endothelial cell line, hCMEC/D3. We further show that GABAA receptors operate in a metabotropic, i.e., flux-independent, mode, to signal the downstream increase in [Ca2+]i via the metabotropic GABAB receptors. In line with this evidence, GABAB receptors require functional GABAA receptors to elevate the [Ca2+]i in hCMEC/D3 cells. Unravelling the molecular mechanisms involved in endothelial GABA signaling will contribute to gather further insights into the mechanisms whereby endothelial GABAergic signaling regulates the NVU.Herein, we provided the first evidence that both GABAA receptor agonist, muscimol, demonstrated specific binding sites in cerebral arterioles i during GABAergic signaling [B receptors have recently been shown to cause a transient increase in [Ca2+]i in HAECs, but it is still unknown whether they are able to regulate the [Ca2+]i also in cerebrovascular endothelial cells. We found that GABA evoked a biphasic Ca2+ response over a wide concentration range in hCMEC/D3 cells. The Ca2+ response to GABA was already detectable as concentrations as low as 1 pM and achieved the peak at 1 µM. The amplitude of the initial Ca2+ peak progressively decreased with further increases in agonist concentration. This dose–response relationship is quite different from the sigmoidal curve that has been described for GABA-evoked intracellular Ca2+ signals in neurons and astrocytes [2+ response is exclusively mediated by the metabotropic GABAB receptors [2+ response to GABA is triggered by both GABAA and GABAB receptors could explain the peculiar profile of the dose–response relationship obtained in hCMEC/D3 cells. These preliminary observations confirmed that intracellular Ca2+ signaling could be instrumental for GABA to control endothelial cell functions [2+ imaging recordings showed that 100 µM GABA elicits a robust biphasic elevation in [Ca2+]i in hCMEC/D3 cells, and this concentration was used to unveil the underlying signaling pathways. Notably, GABA-induced intracellular Ca2+ waves have been observed in many cell types that do not belong to the NVU, including HAECs i, as previously reported in cortical neurons i whereby GABA regulates multiple endothelium-dependent functions in brain microvessels is mediated by the ionotropic GABAA receptors i [2+ occurring in these cells [2+]i in the absence of extracellular Ca2+.Although the metabotropic GABAh within ,10,11 anh within ,64,71,72eceptors ,20. GABA [Ca2+]i . A potense cells . If thisA and GABAB receptors mediate the Ca2+ response to GABA. In accord, GABA-evoked intracellular Ca2+ signals were significantly reduced by inhibiting both the ionotropic GABAA receptors with gabazine and the metabotropic GABAB receptors with saclofen or CGP35348. Moreover, muscimol and baclofen, which, respectively, activate GABAA and GABAB receptors, also elicited a biphasic Ca2+ response in hCMEC/D3 cells. The following pieces of evidence support the notion that the ionotropic GABAA receptor may signal the increase in [Ca2+]i in a flux-independent  mode. First, whole-cell patch-clamp recordings showed that neither GABA nor muscimol evoked a sizeable membrane current in hCMEC/D3 cells. Second, the Ca2+ response to muscimol was sensitive to gabazine. Third, muscimol induced a robust Ca2+ signal also under 0Ca2+ conditions, which demonstrates that GABAA receptors are able to mobilize the endogenous Ca2+ pool and thereby can operate in a metabotropic manner [2+]i in a flux-independent manner by interacting with mGluR1 and mGluR5 i in human cerebrovascular endothelial cells. The Ca2+ response to GABA is triggered by InsP3-induced ER Ca2+ release and NAADP-dependent lysosomal Ca2+ mobilization, whereas it is mainly maintained by SOCE (A and GABAB receptors support GABA-evoked intracellular Ca2+ signals (A receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptor stimulation requires functional GABAA receptors. Endothelial Ca2+ signals finely tune a myriad of vascular functions, including those regulated by GABA, such as angiogenesis and CBF control via NO release. Therefore, this study sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the NVU.Herein, we unveiled the complex signaling pathway whereby the inhibitory neurotransmitter GABA can induce an increase in [Ca by SOCE . Both GA signals . The ion


Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers. Methods: A two-microelectrode voltage clamp technique was used to test the effect of Croc fiber suspensions on outward chloride currents evoked by step membrane depolarization. Calcium imaging experiments were also performed to investigate the variation of ‘resting’ oocyte [Ca2+]i following asbestos exposure. Results: The increase in chloride current after asbestos treatment, was sensitive to [Ca2+]e, and to specific blockers of TMEM16A Ca2+-activated chloride channels, MONNA and Ani9. Furthermore, asbestos treatment elevated the ‘resting’ [Ca2+]i likelihood by increasing the cell membrane permeability to Ca2 in favor of a tonic activation of TMEME16A channels. Western blot analysis confirmed that TMEME16A protein was endogenously present in the oocyte cell membrane and absorbed by Croc. Conclusion: the TMEM16A channels endogenously expressed by Xenopus oocytes are targets for asbestos fibers and represent a powerful tool for asbestos–membrane interaction studies. Interestingly, TMEM16A channels are highly expressed in many types of tumors, including some asbestos-related cancers, suggesting them, for the first time, as a possible early target of crocidolite-mediated tumorigenic effects on target cell membranes.Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in  Xenopus oocytes as a suitable model for studying in detail, the interaction between asbestos fibers and biological cell membranes at the electrophysiological and morphological level i levels were calculated offline from the ratio images (340/380) of tested oocytes as mean values of the fluorescence intensity in cell regions of interest. For each experimental group, the mean fluorescence value was then expressed relative to the mean fluorescence value measured in appropriate control oocytes from the same frog donor and expressed as %.For intracellular CaXenopus oocyte cell membranes was performed by modification of the method of Clémençon et al. (2014) e replacement , as well as for those recorded in the Ctrl oocytes of the same batch . The replacement also reduced the effect of Croc on Rm, which now became similar to that of Ctrl cells , a transient outward current was clearly visible on stepping to a positive potential, in both untreated and Croc-treated cells , while at positive membrane potential, the percentage was significantly different; e.g., at + 40 mV in normal Ringer solution, the increase was 175 ± 51 % more than Ctrl . Despite the donor’s variability, the Rm tended to decrease following Croc-exposure, even more than that in normal Ringer solution . The Croc-sensitive currents recorded in high Ca2+ , although this was slightly more negative than that recorded in Ringer .The next series of experiments were aimed at further characterizing the ed cells a. The cunditions b 5]. Ho. HoCroc-igh Ca2+ e also reN′-(2-methoxybenzylidene) acetohydrazide), another blocker for TMEM16A channels [2+]e in Ctrl and Croc-treated oocytes and MONNA-sensitive currents obtained from their subtractions are shown in m  with Ani9 (2-(4-chloro-2-methylphenoxy)-channels . The I–VCroc-sensitive chloride current as well as the effect of Croc on Rm, were enhanced, most likely by an increased Ca2+ influx across the cell membrane. To further investigate such a hypothesis, a set of [Ca2+]i measurements was conducted by using a Ca2+ imaging approach. Briefly, oocytes were microinjected with Fura 2 and the [Ca2+]i levels were measured as mean 340/380 fluorescence values . In accordance with what has previously been reported in the literature, preparatory experiments revealed that oocyte incubation in Ca11 did not significantly alter the [Ca2+]i with respect to oocytes bathed in normal Ringer  and the percentage increased to 31.83 ± 1.77 % when the oocyte exposure was carried out in Ca11 .These results clearly suggested that in Ca11, the 2+ membrane permeability of the oocyte is usually very low, especially at rest, and mainly carried by voltage-gated calcium channels (VOCC) e from 1.8 to 11 mM and by stepping the voltages of the membrane from a more hyperpolarized level Vh (−80 mV) and the blockage of the Croc-sensitive currents by the specific TMEM16A channel antagonists MONNA and Ani9.In addition to the effect of asbestos fiber exposure on the passive membrane properties  [h (−80 mV or −100 mV) as well by increasing the [Ca2+]e e ,29. Bothchannels . 2+]i following step depolarization: Ca2+ released from internal stores  or the Ca2+ influx from the extracellular space. Here, we found that in Ca2+-free Ringer solution, the [Ca2+]i of Croc-treated cells was similar to Ctrl cells. On the other hand, in normal condition, the Croc exposure increased the [Ca2+]i with respect to non-treated cells. This finding supports the Ca2+ influx from the extracellular space as a predominant source of Ca2+ induced by the asbestos fibers.At least two “sources” could be the responsible for any variation of the [Ca2+ influx and thus the “resting” [Ca2+]i  and TMEM16A activation in Xenopus oocytes; 1) by increasing the Ca2+ influx through VOCC; 2) by forming membrane “pores” permeable to Ca2+ [2+ currents from some unknown leak channels [2+]i and thus induce a tonic activation of the TMEM16A channel activity, altering in this way the passive membrane properties of the cells. One more possibility must be also mentioned. Many proteins, with different structures, molecular weight and isoelectric point are adsorbed with a specific profile by specific asbestos fibers, suggesting a specific interaction i variation observed during asbestos exposure and the activation of TMEM16A channels. Asbestos fibers can also affect cell function both directly via ROS production, induction of membrane lesions [Xenopus oocytes will be addressed in a future dedicated research program.Taking advantage of the g cancer . Our fin lesions  and foll lesions ,47. Acti lesions . Therefo


Mounting evidence have indicated that long noncoding RNA (lncRNA) muscleblind like splicing regulator 1 antisense RNA 1 (MBNL1-AS1) play a crucial regulatory role in cardiovascular disease, myocardial infarction (MI) included. In this research, we sought to probe into the biological function and potential mechanism of MBNL1-AS1 in MI.  Cardiomyocytes were treated under hypoxic conditions for 0–12 h. Functional assays including CCK-8 and flow cytometry were performed to assess hypoxia-stimulated cardiomyocyte viability and apoptosis, respectively. Moreover, bioinformatics analysis and mechanical assays were conducted to reveal the competitive endogenous RNA (ceRNA) mechanism of MBNL1-AS1.  The upregulation of MBNL1-AS1 was found in hypoxia-stimulated cardiomyocytes. Functionally, the downregulation of MBNL1-AS1 dramatically promoted hypoxia-induced cardiomyocyte viability and inhibited apoptosis. Mechanistically, miR-132-3p bound to MBNL1-AS1 in hypoxia-induced cardiomyocytes, and miR-132-3p directly targeted RAB14, member RAS oncogene family (RAB14) and calmodulin binding transcription activator 1 (CAMTA1). Furthermore, MBNL1-AS1 upregulates the expression of RAB14 and CAMTA1 in hypoxia-stimulated cardiomyocytes via targeting miR-132-3p.  The current study revealed the critical role of the MBNL1-AS1/miR-132-3p/RAB14/CAMTA1 axis in MI, indicating MBNL1-AS1 as an innovative therapeutic target for MI. As a kind of ischemic heart disease, myocardial infarction (MI) is accompanied with high morbidity and mortality , 2. ExisNoncoding RNAs (ncRNAs), a type of RNAs lack of the potential of protein-coding, were initially recognized as “junk DNAs” , 6. IncrlncRNA MBNL1-AS1 has been widely documented to be implicated in multiple cancers, such as nonsmall cell lung cancer , bladderIn this research, to reveal the function of MBNL1-AS1 in MI, we constructed an in vitro model of MI in H9c2 cells treated with hypoxia. Moreover, we explored the interaction between MBNL1-AS1 and miRNA as well as the downstream genes. Our study might provide a promising prospect for MI treatment.2 at 37°C. To mimic MI, H9c2 cells were maintained in a hypoxia incubator containing 1% O2, 5% CO2, and 94% N2.The rat embryonic ventricular cardiomyocyte H9c2 and human embryonic kidney cell (HEK293T) were procured from American Type Culture Collection. Both H9c2 and HEK293T cells were maintained in DMEM  containing 10% fetal bovine serum with 5% COμl CCK-8 solution procured from Dojindo  in 96-well plates. The absorbance was measured at 450 nm.Three independent experiments were performed. Cardiomyocytes were cultured with 10 The experiment was performed thrice independently. The FITC-annexin V/PI detection kit procured from Biosea Biotechnology  was applied as per the user guide. Cardiomyocytes were collected and resuspended in 6-well plates, and then dyed with FITC-annexin V and PI, and assessed by the cytometry procured from Beckman Coulter .Three independent experiments were performed. Total protein was extracted via RIPA lysis buffer procured from Santa Cruz Biotechnology . After the proteins were assessed by SDS-PAGE  and transferred to PVDF , 5% nonfat milk  were applied for membrane blocking. Next, the membranes were incubated with primary antibodies including Bax , Cleaved caspase-3 , total caspase-3 , Bcl-2 , RAB14 , CAMTA1 , and GAPDH . After washing, the secondary antibodies procured form Abcam were incubated. The blots were detected using the chemiluminescence detection kit .ΔΔCt− method, with GAPDH or U6 as the internal control.Three experiments were performed independently. Total RNA was extracted from hypoxia-induced cardiomyocytes using the Trizol reagent procured from Life Technologies Corporation . After the application of First Strand cDNA Synthesis Kit , qPCR was implemented using Takara SYBR® PrimeScript™ PCR kit . Calculation of gene expression was performed using 2Three experiments were performed independently. This assay was carried out via a Nuclear and Cytoplasmic Extraction Reagent . Fractionated RNAs were detected by RT-qPCR analysis.Three experiments were performed independently. Ribo™ Fluorescent In Situ Hybridization Kit procured from RiboBio  was applied as per the user guide. The MBNL1-AS1 probe was purchased by RiboBio. DAPI  was applied for nuclear staining. Images of both H9c2 and H9c2/Hypoxia cells were captured by confocal microscope .Three experiments were performed independently. Specific shRNAs for MBNL1-AS1, RAB14, and CAMTA1 were obtained by GenePharma  with the sh/NC being negative control. miR-132-3p mimics, miR-132-3p inhibitor, and the negative controls (NC mimics and NC inhibitor) were also supplied by GenePharma. Cell transfection was performed using lipofectamine 3000 reagent (Life Technologies Corporation), and the transfection efficiency was verified by RT-qPCR.Three independent experiments were performed. Maltose-binding protein (MBP)-affinity purification was employed to evaluate miRNAs that are related to MBNL1-AS1. Cells were treated with MS2-tagged MBNL1-AS1 to acquire miRNAs related to MBNL1-AS1. The RIP assay was conducted as previously described . The abuThree experiments were performed independently. The fragment of RAB14 3′-UTR or CAMTA1 3′ UTR or MBNL1-AS1 containing the miR-132-3p binding site were inserted into pmirGLO dual luciferase vector . The luciferase reporter plasmids were cotransfected into hypoxia-induced cardiomyocytes or HEK293T cells with miR-132-3p mimics. The luciferase activity of wild-type MBNL1-AS1/RAB14/CAMTA1  and their mutant types  was measured by Dual Luciferase Reporter Assay System procured from Promega.t test or one-way analysis of variance (ANOVA). Statistical differences were considered significant when P < 0.05.Data of experimental results was presented as mean ± standard deviation and processed by Graphpad Prism 8 software . Statistical analyses were implemented with SPSS 19.0 software . Statistical differences were tested by Student's Firstly, H9c2 cells were subjected to the exposure of hypoxia for 0–12 h, and the viability of H9c2 cells was declined in a time dependent manner . Since cTo seek the functional impacts of MBNL1-AS1 in hypoxia-induced H9c2 cells, two specific shRNAs targeting MBNL1-AS1 were transfected into cells to silence MBNL1-AS1 expression. Then, RT-qPCR analysis verified the successful transfection . The reshttp://carolina.imis.athena-innovation.gr) tool, we predicted 11 miRNAs harboring binding sites of MBNL1-AS1 with the threshold value > 0.95 (http://starbase.sysu.edu.cn) and presented in Given that MBNL1-AS1 was preferentially located in cell cytoplasm, we conjectured that MBNL1-AS1 might work as a ceRNA to sponge specific miRNA in MI progression. Based on DIANA  targeted by miR-132-3p were sifted out . Then weWe further upregulated RAB14 and CAMTA1 expression , and theA large amount of lncRNAs have been identified to be associated with multiple kinds of cardiovascular diseases, MI included. lncRNA CAIF represses autophagy and improves MI via p53-mediated myocardin transcription . lncRNA Recent studies have revealed that lncRNAs interact with miRNAs and exert important functions in the development of diseases. Dysregulation of miRNAs was extensively reported in the progression of MI. miRNA-145 represses MI-induced apoptosis via Akt3/mTOR signaling pathway . miRNA-4To sum up, we demonstrated that lncRNA MBNL1-AS1 promoted hypoxia-induced MI in H9c2 cells via the regulation of miR-132-3p/RAB14/CAMTA1 axis. Our study might provide an innovative target for the treatment of MI. However, our study also exist some limitations. First, our study did not explore the upregulation mechanism of MBNL1-AS1 in hypoxia-treated H9c2 cells. Moreover, our study did not investigate whether MBNL1-AS1 is involved in MI via the modulation of certain pathways. All of these deficiencies will be improved in the near future.


A 14-year-old boy was admitted for the evaluation of progressive torticollis accompanied by cervicothoracic scoliosis. During the preoperative drape of the patient for the release of the sternocleidomastoid muscle under general anesthesia, his wrist and ankle were observed to have severe flexion contracture. The body temperature was 37.1 °C. To treat MH, the patient was administered a bolus of dantrolene intravenously (1.5 mg/kg) and sodium bicarbonate. After a few minutes, muscle rigidity, tachycardia, and EtCO2 all resolved. Next-generation panel sequencing for hereditary myopathy identified a novel RYR1 heterozygous missense variant , which mapped to the MH2 domain of the protein, a hot spot for MH mutations. Ex vivo RYR1-mediated intracellular Ca2+ release testing in B lymphocytes showed hypersensitive Ca2+ responses to isoflurane and caffeine, resulting in an abnormal Ca2+ release only in the proband, not in his family members. Our findings expand the clinical and pathological spectra of information associated with MH with multi-minicore myopathy.Malignant hyperthermia (MH), a rare autosomal dominant pharmacogenetic disorder of skeletal muscle calcium regulation, is triggered by sevoflurane in susceptible individuals. We report a Korean having MH with multi-minicore myopathy functionally supported by RYR1-mediated intracellular Ca RYR1, CACNA1S, or STAC3 genes in molecular genetic testing i) release response to isoflurane and caffeine can differentiate between MH-susceptible individuals and normal controls [2+]i, single floating B lymphocytes were attached to Matrigel matrix -coated dishes. The culture medium was replaced with Dulbecco’s Modified Eagle Medium (DMEM) containing the membrane-permeable Ca2+ indicator Fluo-4 AM . [Ca2+]i and measured with a confocal microscope . Calculations were performed using an equation by Tsien et al. [2+]i = Kd (F − Fmin)/(Fmax − F), where Kd was 335 nM for Fluo 4 and F was the observed fluorescence level. Each tracing was calibrated for maximal intensity (Fmax) by adding ionomycin (8 μM) and for minimal intensity (Fmin) by adding 50 mM EGTA at the end of each measurement. To measure sarcoplasmic reticulum (SR) Ca2+ release, SR Ca2+ release was measured by the method with modifications described previously I release testing in B lymphocytes was performed. B lymphocytes isolated from the proband showed hypersensitive Ca2+ responses to isoflurane and caffeine, resulting in an abnormal Ca2+ release only in the proband, but not in his family members. As a result, two independent ex vivo studies all show the release of Ca2+ in response to the RYR1 agonist  and his family members . Furthermore, molecular diagnosis revealed the deleterious effect of this rare RYR1 variant classified as PS3_Supporting by modified ACMG-AMP criteria. These results suggest that enhanced Ca2+ responses are associated with mutations in the RYR1 gene in MH-susceptible individuals. Although the criteria for MH diagnosis have not been established and further comparative studies are required, RYR1-mediated intracellular Ca2+ release testing in B lymphocytes might have promise as an adjunct to in vitro muscle biopsies.Currently, there are two diagnostic approaches for patients potentially at increased risk of developing an MH reaction. These include molecular genetic testing and ex vivo muscle contracture testing, the in vitro contracture test (IVCT)  and the RYR1 and CACNA1S as two major MH-causative genes, are huge, containing numerous exons. Mutations in these two genes account for 50–70% of the known MH cases. A comprehensive analysis by traditional Sanger sequencing is challenging because it is costly, time-consuming, and labor-intensive, without even considering other possible genes in the genome [On the other hand, e genome ,10. Nexte genome ,42. Moree genome ,44.RYR1 p.Ser2300Pro variant classified as PS3_Supporting by modified ACMG-AMP criteria, leading to MH accompanied by multi-minicore myopathy. Our findings expand the clinical and pathological spectra of information associated with MH with multi-minicore myopathy. Ex vivo functional studies such as RYR1-mediated intracellular Ca2+ release testing in B lymphocytes may support the damaging effect of the rare RYR1 variant.In conclusion, we reported a novel heterozygous


Furthermore, newcomer learning adaptability amplified the influence of career mentoring on newcomer occupational self-efficacy, as well as the impact of psychosocial mentoring on newcomer social self-efficacy. Our study extended the mentoring and socialization literature and provided significant practical implications for managers on how to arrange tailored mentoring to facilitate newcomer socialization.Drawing on the social cognitive career theory, this study proposed an integrative framework to uncover how and when different types of mentoring accelerate newcomer’s socialization in corresponding domains. We tested this relational model with time-lagged, multisource survey data collected from 157 newcomers and 88 supervisors. The results indicated that career mentoring facilitated newcomer task mastery, task performance, and job satisfaction by improving newcomer occupational self-efficacy, whereas psychosocial mentoring promoted newcomer job satisfaction and social integration  Organizational socialization is the process by which individuals acquire the essential knowledge and social skills to perform their organizational roles . The adeMentoring refers to “a process for the informal transmission of knowledge, social capital, and psychosocial support perceived by the recipients as relevant to work, career, or professional development” , p. 731.To date, numerous scholars have bridged the socialization and mentoring literature, and most of them focus on revealing mentoring functions in facilitating socialization . NeverthThe social cognitive career theory posits that contextual factors of supports or barriers can facilitate or inhibit employee career goals through taking part in the formulation of their person-cognitive variables . In the Furthermore, it has been well documented that learning can be regarded as the main process leading to effective mentoring and socialization as it especially determines whether inexperienced mentees acquire knowledge and skills and whether newcomers are successfully socialized . CombiniOur research contributes to mentoring and socialization literature from the following three aspects. First, our study extends Self-efficacy refers to individuals’ beliefs in their capabilities to produce given attainments by exercising influence over events associated with their lives . Given tThe social cognitive career theory stresses that contextual supports could promote employees’ certain learning experiences and further affect their self-efficacy ; it thusIn addition, previous research on self-efficacy has also highlighted that mentoring played a crucial role in building employee self-efficacy beliefs through vicarious learning and verbal persuasion . SpecifiHypothesis 1: Newcomer career mentoring is positively related to occupational self-efficacy.Hypothesis 2: Newcomer psychosocial mentoring is positively related to social self-efficacy.The social cognitive career theory proposes that individuals’ self-efficacy further affects their choices or goals to conduct particular career-related activities . That isTo be specific, we contend that occupational self-efficacy is crucial for leading to newcomers’ favorable task-related outcomes , where task mastery reflects the extent to which one’s capability to successfully fulfill job demands  and taskAs such, we further postulate newcomers’ self-efficacy in the social interaction context as a critical antecedent to aspects of their social outcomes . In particular, social integration refers to the extent to which one’s feeling of attachment and inclusion in the current organization , indicatHypothesis 3: Newcomer occupational self-efficacy is positively related to (a) task mastery, (b) task performance, and (c) job satisfaction.Hypothesis 4: Newcomer social self-efficacy is positively related to (a) job satisfaction and (b) social integration.We further suggest that receiving career mentoring, by improving newcomers’ social cognitions of occupations may promote their occupational self-efficacy beliefs, which, in turn, could enhance their task mastery, task performance, and job satisfaction. Simultaneously, we also argue that newcomers’ social self-efficacy may act as a mediating mechanism, transmitting the positive effects of psychosocial mentoring on newcomers’ job satisfaction and social integration. Taken together, we advance the following hypotheses:Hypothesis 5: Newcomer occupational self-efficacy mediates the relationship between career mentoring and (a) task mastery, (b) task performance, and (c) job satisfaction.Hypothesis 6: Newcomer social self-efficacy mediates the relationship between psychosocial mentoring and (a) job satisfaction and (b) social integration.The social cognitive career theory emphasized that employees’ learning experience in career development is influenced by not only environmental factors but also employee characteristics . That isDrawing on social cognitive career theory, learning adaptability can be regarded as an important personal characteristic related to the formation of employee personal cognition factors since it can directly affect the employee’s learning experience . As suchMoreover, previous research on learning adaptability also provides supports for the inferences mentioned before. For example, Hypothesis 7: Newcomer learning adaptability strengthens the positive effect of career mentoring on occupational self-efficacy.Hypothesis 8: Newcomer learning adaptability strengthens the positive effect of psychosocial mentoring on social self-efficacy.Our sample consisted of newcomers (employees who are within the first 6 months of joining their current company) and their supervisors from two high-tech companies located in the northern part of China, one specializing in household appliance manufacturing and the other in battery manufacturing. These participants were employed in different departments, including R&D, engineering, production, operations, marketing, and manufacturing. During the preliminary interviews with several newcomers and supervisors, we found that mentoring was prevalent in both companies, especially for newcomers, which was comprehensive and varied. Most newcomers indicated that such mentoring facilitated them to integrate into the current organization. Thus, we believe that these companies are appropriate contexts to test the associations between different forms of mentoring and newcomer socialization.Before the formal survey began, under the assistance of the human resources department, we sent an email to newcomers introducing the purpose and procedures of our investigation and inquiring about their intention to participate. Finally, we received 287 positive responses and further included them and their supervisors as participants, matching each of them with a four-digit code. To minimize the potential common method bias and relieve participantse human resources department, we sent an email to newcom. We maintained a 1-month interval between each wave of data collection. At time 1, newcomers were asked to report career and psychosocial mentoring, learning adaptability, and demographics. At time 2, newcomers were instructed to report their occupational and social self-efficacy, social exchange relationship with supervisor, and perceived supervisor support. At time 3, we asked newcomers to self-report their task mastery and job satisfaction, while their supervisors were required to rate newcomers’ task performance and social integration, with each supervisor rating an average of 1.78 newcomers.SD = 2.63). The average organizational tenure and previous working experience of newcomers were 2.77 months (SD = 1.45) and 9.66 months (SD = 11.31), respectively.Finally, 157 newcomers and 88 supervisors of the total participants returned surveys with completed and matched data, representing an overall response rate of 55.70%. Of the valid sampled newcomers, 61.15% were men, and 83.44% had attained a bachelornse rate of 55.70%. Of the job satisfaction, years old , unless otherwise specified. Following the translation-back-translation procedure suggested by Career and psychosocial mentoring. Career and psychosocial mentoring were measured with a 14-item scale developed by Occupational and social self-efficacy. Occupational and social self-efficacy were measured using items from the social and emotional self-efficacy scale developed by Task mastery. Task mastery was measured with the five-item performance proficiency scale developed by Task performance. Task performance was captured with a three-item scale developed by Job satisfaction. Job satisfaction was measured with a three-item scale developed by Social integration. Social integration was measured with a three-item scale developed by Learning adaptability. Learning adaptability was measured using five items (α = 0.942) from the Control variables. Consistent with prior socialization and mentoring research, this study included newcomers’ gender, age, education, organizational tenure, previous working experience, and company as control variables. In addition, 2/df = 1.152, p < 0.001; CFI = 0.981, TLI = 0.977, RMSEA = 0.031, and SRMR = 0.050, than all the alternative models. Thus, the results indicated discriminant validity for our focal variables.Following the suggestion proposed by B = 0.342, SE = 0.104, 95% CI = ), whereas newcomer psychosocial mentoring significantly predicted social self-efficacy , as such, hypotheses 1 and 2 were both supported. Newcomer occupational self-efficacy was significantly and positively related to task mastery , task performance , and job satisfaction , thereby supporting hypotheses 3a, 3b, and 3c. Likewise, significant positive associations existed between newcomer social self-efficacy and job satisfaction , as well as social integration , which provide supports for hypotheses 4a and 4b. Furthermore, the results of the bootstrapping test revealed that the indirect effects of newcomer career mentoring on task-related outcomes through occupational self-efficacy were significantly positive , supporting hypotheses 5a, 5b, and 5c. Similarly, the positive indirect effects of newcomer psychosocial mentoring on social outcomes via social self-efficacy were significant , supporting hypotheses 6a and 6b.We performed the structural equation modeling using the maximum-likelihood estimation method in Mplus 8.3 to calculate standardized path coefficients. As shown in B = 0.266, SE = 0.052, 95% CI = ), as well as the significant interaction effect of newcomer psychosocial mentoring and learning adaptability on social self-efficacy . Moreover, the results of a simple slope test plotted in B = 0.622, SE = 0.105, 95% CI = ); for those with lower learning adaptability (–1 SD below the mean), such association was weaker but still significant . Likewise, B = 0.652, SE = 0.121, 95% CI = ) when newcomers have higher learning adaptability, whereas the effect was weaker and not significant  when newcomers’ learning adaptability was low. Accordingly, hypotheses 7 and 8 were supported.Then, the results also demonstrated the significant interaction effect of newcomer career mentoring and learning adaptability on occupational self-efficacy  as the boundary condition for the linkage between mentoring and socialization outcomes. Future empirical studies could consider alternative contextual explanations to further expand the mentoring literature. For instance, mentoring scholars have demonstrated that not all mentorships are constructive. The effectiveness of mentorship varies according to relationship quality and mentees’ trust in mentors . MenteesDrawing on the social cognitive career theory, our study uncovered the differential influences of different types of mentoring on boosting newcomers’ organizational socialization by introducing their self-efficacy in the corresponding domain as the potential mechanisms. Moreover, the findings in our study contribute to the existing literature by revealing that the extent to which different types of mentoring stimulate newcomers’ specific self-efficacy was moderated by their learning adaptability. Altogether, these findings provided a comprehensive picture for scholars and managers to understand the specialized roles of mentoring on newcomer adjustment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Review Board of Beijing Jiaotong University. The patients/participants provided their written informed consent to participate in this study.HD: conceptualization, methodology, investigation, data curation, and writing—original draft preparation. WenbW: investigation and writing—reviewing and editing. YZ, ZY, and HX: writing—reviewing and editing. WenW: data curation. All authors have read and agreed to the submitted version of the manuscript.


We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract’s ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimer’s disease (AD) by promoting uncontrolled elevation of cytosolic Ca An ase (AD) ,5,6.2+ to rise to a toxic level [3) production, triggering intracellular Ca2+ fluxes leading to a disruption of intracellular Ca2+ homeostasis [3 overproduction triggered by Aβ42 oligomers may significantly contribute to Ca2+ signaling disruptions and neurotoxicity in AD-affected neurons. While there is a widespread agreement on the toxic properties of Aβ42 oligomers, most of this knowledge comes from the use of synthetic Aβ peptides, aggregated in vitro using distinct protocols, generating ambiguity whether endogenous Aβ oligomers may exert similar effects on neurons [2+ signaling as observed in neurons of AD-affected brains. Here, we investigate the Ca2+-dependent toxicity of selected brain extracts, chosen from postmortem brain samples that had been previously characterized by immunohistochemistry and found to contain high level of OC-positive oligomers. Fluorescence Ca2+ imaging experiments were performed using seven different samples of soluble brain extracts: three from normal brain  and four from AD-affected samples  [Xenopus oocytes loaded with Ca2+ sensitive dye and their ability to trigger cytosolic Ca2+ signal was tested. Injections of brain extracts from normal patients evoked little or no detectable fluorescent Ca2+ signals. On the contrary, intracellular injection of extracts from AD brains consistently triggered cytosolic Ca2+ elevations. These Ca2+ signals consisted of both local events and global Ca2+ elevations, and showed strong similarity with those evoked after intracellular injection of synthetic Aβ42 oligomers and IP3 [3 receptor inhibitor) and OC-antiserum support the ability of endogenous Aβ aggregates contained in the AD brains extracts to trigger cytosolic Ca2+ release by uncontrolled activation of IP3Rs [3 and Ca2+ and the resulting disruption of the normal cell bioenergetics. Overall, our results support the notion that early intracellular rise in Aβ aggregates seen in AD-affected neurons may inflict neuronal cytotoxicity by disrupting intracellular Ca2+ homeostasis and cellular bioenergetics.Previous results from our lab support the notion that intracellular, as well as extracellular, interactions of Aβ42 oligomers with cell membranes promote cytosolic Caic level ,8,9. Phaeostasis ,9,10,11. neurons ,13,14. TAD3-B11) . Experim and IP3 . Blockagof IP3Rs ,17,18. NXenopus oocytes as a model cell preparation, as their large size enables intracellular microinjection of samples [2+ about 3 μM) to a final intracellular concentration of ~40 μM. Oocytes were then placed animal hemisphere down in a chamber whose base was formed by a fresh, ethanol-washed microscope cover glass , and bathed in a Ca2+-free Ringer’s solution . A gravity-fed superfusion system allowed exchange of the Ringer’s solution. Oocytes were imaged at room temperature by wide-field fluorescence microscopy using an Olympus inverted microscope (IX 71)  of the change in fluorescence at a given region of interest (ΔF) relative to the resting fluorescence at that region before stimulation (Fo). Mean values of Fo were obtained by averaging over several frames before stimulation using MetaMorph (Molecular Devices). Fluorescent traces were exported to Microcal Origin version 6.0  for analysis and graphing. Microinjection of 10 nl of brain extract samples into oocytes was performed using a Drummond nanoinjector mounted on a hydraulic micromanipulator. A glass pipette (tip diameter of 8–10 μm) was filled with samples and inserted vertically down through the entire oocyte to a pre-established position with the tip positioned 6 to 8 μm inward from the plasma membrane and centered within the image field.We used  samples . Experim (IX 71) A equippeFrozen brain tissue was obtained from the UCI Alzheimer Disease Research Center (ADRC). The cognitive status of subjects enrolled in the ADRC was assessed with mini mental status exam (MMSE). As a standard protocol for ADRC autopsy cases, Braak & Braak neurofibrillary tangle and plaque staging was evaluated . Our obj3, pH 7.4, supplemented with protease inhibitor cocktail (Roche). The samples were ultracentrifuged at 100,000× g for 1 h at 4 °C. The PBS soluble fraction was collected, aliquoted and stored at −80 °C for future testing.Frozen tissues from the B11 area or trans-entorhinal cortex (TEC) were processed as previously described . BrieflyFor dot blot analysis, 2 μg of samples was spotted in duplicate onto nitrocellulose membrane and allowed to air dry. After blocking the non-reactive sites with 10% nonfat dry milk in low-Tween TBS-T, , the blots were incubated overnight at 4 °C in primary antibody at the following dilutions: OC 1:10,000, A11 1:2000, 6E10  1:2000. The blots were then incubated with goat-α-rabbit (for OC and A11) and goat-α-mouse HRP conjugated secondary antibody , washed three times for 5 min, followed by ECL detection. Stock solution for OC was 0.4 μg/ml in PBS solution.2+ dynamics and mitochondrial function, and numerical methods are described in detail in Equations modeling cytosolic Ca2+ rise observed in neurons during aging has been linked to the action of specific oligomeric forms of Aβs.Of relevant interest in understanding the role of soluble Aβ (Aβs) oligomers in the etiology of AD is to uncover the prevailing type of aggregates that more closely associate with the development of the symptoms and the associated molecular mechanisms . PreviouIn this work we selected samples of postmortem brain extracts from three normal and three AD-affected patients which were screened for their affinity to selected antibodies for different types of Aβ aggregates ,5. Figur2+ mobilization from intracellular stores, we used fluorescent Ca2+ imaging in Xenopus oocyte as a model system. The experiments described here followed similar approaches we previously applied to investigate the Ca2+ toxicity of synthetic Aβ42 oligomers. The stock solution of brain extracts used in this work were estimated to contain up to 1 μg/ml of Aβ in PBS solution. Intracellular injection of 10 nl samples at this concentration triggered fast and robust fluorescent responses. Further, we observed that injection of 10 nl of lower concentrations, down to 0.3 μg/ml dilutions, retained the ability to consistently trigger Ca2+ signaling (data not shown).To examine the ability of human brain extracts to trigger Ca2+ mobilization of different spatiotemporal patterns, ranging from repetitive local brief events such as blips and puffs to global repetitive Ca2+ waves covering the entire image field. Conversely, injection of 10 nl of brain extracts from normal individuals failed to promote either local or global Ca2+ fluxes. In 2+-dependent fluorescent signals evoked by injection of different brain extracts.Intracellular microinjection of 10 nl samples from AD brain extracts evoked potent cytosolic Ca2+-fluorescent signal evoked in response to intracellular injection of 10 nl of AD3-TEC sample at the time indicated by the arrow in the line-scan. Below is the corresponding line-scan (kymograph) image, showing temporal evolution of the fluorescent signal originated by measuring changes in fluorescence intensity along a single line scan (25 μm long and 5μm wide) properly positioned above the centroid of the fluorescent signal. The panel displays the signal evolution over time as a pseudo-colored representation where warmer colors indicate increase in fluorescence ratio intensities. Fluorescent transients with different amplitudes and duration are evident along the line-scan, reminiscent of the hierarchical organization of IP3-mediated Ca2+ signaling. In 2+ signaling. We applied similar approach for screening all the trials performed with active responses classified as fluorescent responses consisting of at two separated sites with repetitive transients at each site.In 2+ dependent fluorescent transients observed after injection, we used the conformation-specific OC antiserum to neutralize the activity of endogenous Aβs oligomers. To this aim, we performed a series of experiments where samples from AD brains were pre-incubated for 24 h with OC-antiserum prior to injection. In parallel, equivalent aliquots of corresponding samples were incubated without addition of antibody. In To confirm that the soluble Aβ oligomers contained in the AD-affected samples were responsible for the CaWe then performed parallel experiment using the corresponding sample which had been previously incubated with OC antiserum. Top-right trace in 3-mediated Ca2+ signaling suggests that intracellular AD brain extracts promote the liberation of Ca2+ from the ER stores via opening of IP3Rs. Moreover, following a similar approach, we have previously reported that intracellular injection of synthetic Aβ42 oligomers into Xenopus oocytes triggers cytosolic Ca2+ release through a mechanism involving IP3 overproduction. In 2+ events triggered by AD brain extracts  positioned above the puff`s footprint in the video record, with time running left to right along the x-axis. Increasing fluorescent signals are represented by warmer colors as indicated by the color bar and by increasing height of each pixel. Below, the left plot is generated by the overlapping of 10 randomly selected traces aligned to the initial rise of the fluorescent trace. Inset is generated by averaging all the puffs traces. Center and right plots in In 3 (10 nM).The top image displays three fluorescent transients occurring at the same location, closely resembling the local transients observed when synthetic and endogenous Aβ oligomers are injected. Overlapping of the puff-like profiles is shown in left plot and inset, shows the resulting averaged intensity profile. Corresponding distribution of the average events duration and amplitudes are shown in the center and right plots, respectively.2+ events triggered by brain extract from AD patient are similar to those generated by the synthetic Aβ42 oligomers and by IP3, and involve the opening of IP3Rs [Overall, this comparative investigation supports our hypothesis that the elementary Caof IP3Rs ,23.3Rs on the Ca2+ fluxes triggered by endogenous Aβ oligomers [3Rs in Xenopus oocytes [To further strength our hypothesis, we examined the effect of caffeine, known to act as a reversible antagonist of IPligomers . Moreove oocytes ,24.2+ fluorescent transient triggered when AD3-TEC was injected alone. Confirming the reversibility of caffeine’s effect on the IP3Rs, the Ca2+ response after injection of 10 nl of AD3-TEc was completely restored after extensive washing-of caffeine from the bathing solution  to increase the adhesion of oocytes to the coverslip, ensuring recordings at the same exact location during application and after the washing of caffeine from the bathing solution. In 3 generated and the resulting concentration of Ca2+ released from intracellular stores in response to intracellular injection of brain extracts from normal and AD-affected brains, we fitted the whole-cell model discussed in the To quantify the concentration of IP2+ concentration in response to brain extracts injection. The time-traces for Ca2+ and IP3 concentrations are integrated to estimate the total Ca2+ and IP3 concentrations generated by brain extracts from control brain and three different AD brains, and are shown in 3 generated and consequently higher Ca2+ released from intracellular stores due to extracts from AD patients as compared to extracts from control brains.Observed fluorescence changes (black) are shown for comparison. The blue curves represent the estimated change in cytosolic Ca2+ rises due to brain extracts. Specifically, we feed the Ca2+ traces estimated above to a model for mitochondrial bioenergetics to estimate the changes in mitochondrial Ca2+ concentration, cell’s ATP, and ROS levels as detailed in the Next, we used our model to estimate the bioenergetic cost of the aberrant Ca2+ concentration, ATP and ROS obtained from fits to three experiments are shown A summary of changes in mitochondrial Ca2+ to a poisonous level, with consequent impairment of normal cellular functioning and final death [2+-dependent fluorescent imaging we had previously proposed two distinct mechanisms by which Aβ42 affects intracellular Ca2+ homeostasis. One, by forming self-gating Ca2+-permeable pores allowing uncontrolled influx of extracellular Ca2+ into the cytosol; and a second mechanism involving the release of Ca2+ from intracellular stores through the opening of IP3R channels [2+ release upon intracellular injection, closely mirroring the effect of synthetic Aβ42 oligomers in analogous experiments. The samples consist of homogenate extracts from postmortem brain tissues from frontal cortex of donors with clinical and pathological details shown in A great deal of progress has been made in recent years to better understand the involvement of Aβ in the progression of Alzheimer’s disease ,26,27. Aal death ,28. Usinchannels ,31,32,33channels . Here, w2+ fluxes ranging from local events to global events involving large areas in the image field. Conversely, 10 nl N2-B11 sample from control brain, failed to trigger any significant response similarly to the effect of 10 nl injection of PBS . Considering the amplitudes and temporal evolution of these Ca2+ transient we speculate that over time, interaction of Aβ42 oligomers with the ER membrane may trigger formation of Aβ42 pores in the ER membrane causing the Ca2+ leak described by those authors [In comparable conditions, i.e., pre-treatment with caffeine and intracellular delivery of Aβ42 oligomers, similar result was reported using SH-SY5Y cells a model system . Further authors .2+ mobilization by the brain extract is further strengthened by the ability of the conformation-specific antiserum OC to specifically decrease or completely neutralized the ability of the brain extracts to trigger Ca2+ fluxes  constructs to promote either intra or extracellularly expression of oligomeric Aβ42 [a2+ rise . Interesg events .2+ rise triggered by synthetic Aβ42 oligomers may involve disruption of normal mitochondrial functioning [3 generated and the corresponding concentration of Ca2+ released through IP3Rs caused by AD-affected brain extracts, we fed the resulting time-traces of Ca2+ concentration to our computational model for cell bioenergetics to assess the ATP and ROS produced. Our simulations show a significant drop in ATP and rise in ROS due to extracts from AD-affected brains, mostly of short duration. However, we believe that over extended periods, the pathological Ca2+ signals and consequently the low ATP and high ROS levels would make the cell progressively vulnerable, eventually leading to the observed synaptic dysfunction and cell’s demise.We have previously proposed that the downstream effect of cytosolic Cactioning . Here, a2+ signaling on neurotransmitter release [2+ release from the ER increases the probability of neurotransmitter release in AD-affected neurons. Moreover, over very short timescales (30–60 ms), the model exhibits activity-dependent and enhanced short-term plasticity in AD, indicating neuronal hyperactivity. Similar to previous observations in AD animal models, pathological Ca2+ signaling increases depression and stimulus desynchronization causing affected synapses to operate unreliably [In line with this interpretation are the results from our recent study using a biophysical model of hippocampal synapses to investigate the effect of aberrant Ca release . The modreliably .2+ without directly interacting with the mitochondrial membrane. Furthermore, the model ignores the contribution of ryanodine receptors to cytosolic Ca2+ concentration and assumes that Aβ oligomers only cause the release of Ca2+ from the ER through IP3Rs. While this assumption is valid for Xenopus oocyte, ryanodine receptors are shown to play a key role in Ca2+ signaling disruptions in AD-affected neurons [2+ signaling must be taken into consideration while interpreting the results from our model.We remark that while our bioenergetics model has been developed over many years, is based on extensive data, and has been successfully applied to different cell types including oocytes  and mamm neurons ,47. Thus2+ homeostasis and consequently in cell bioenergetics as plausible mechanisms for the toxicity of endogenous Aβ oligomers in AD affected brains.Nevertheless, we believe that our experimental observations and model simulations make a strong case for disruptions in intracellular Ca


Sarcoptes scabiei, Psoroptes cuniculi, Cheyletiella parasitovorax, and Notoedres cati. Body mange and ear mange are the most common forms of this disease in rabbits. Animals can get mite infestation through direct contact with infected animals or contaminated fomites. This infestation is characterized by zoonotic nature and public health burden. The skin affection is characterized by pruritus, alopecia, severe cachexia, and sometimes death. Infestation is diagnosed mainly by skin scraping and microscopic examination. Control measures mainly depend on the use of different types of systemic and topical acaricides and the use of natural products and supportive elements. Vaccine is not commercially available and is still under investigation. Accordingly, this review article was designed to shed the light on the mange disease in rabbits in terms of mite's infestation and susceptibility, clinical manifestations, zoonosis, diagnosis, and control strategies.Mange in rabbits is a very important parasitic disease causing high losses. The disease is caused mainly by  Rabbits have been regarded as key livestock, which are increasingly being raised in many countries worldwide . These aSarcoptes scabiei var. cuniculi, Psoroptes cuniculi, and Cheyletiella parasitovorax are the causes of mange in rabbits. Moreover, Notoedres cati cuniculi burrowing mite has been detected in rabbits [Psoroptes ovis has been identified in rabbits with ear lesions [Mange in rabbits is an emerging and highly contagious disease caused by different burrowing and non-burrowing mite species protozoon . Mange a rabbits , 7, 8. H lesions –11. In 2 lesions . Mange i lesions , 14. Thi lesions , 16 , heated in a water path at 60–80°C for 15 min, and then centrifuged at 1500–2000 rpm for 5 min. The supernatant is removed, some drops of sediment are placed on a glass slide, and permanent mounts of the parasite should be prepared [Sarcoptic scabiei is round with short legs, having a long unjointed stalk with a sucker on the front pair of legs. The dorsal body surface of sarcoptic mite has a thick and chitinous wall with large spines. The anus is terminal, and the dorsum possesses scales, cones, and bladelike setae [The main method of mite laboratory diagnosis is the skin scrapings of lesion edges in suspected animals . A lesioprepared . Mite spprepared , 58. Somke setae .Sarcoptes-infested rabbits showed altered antioxidant systems to states of oxidative stress . Blood aSarcoptes scabiei [Serological tests such as enzyme-linked immunosorbent assay are used to detect the antibodies against  scabiei , 65.Since conventional diagnostic techniques for mite diagnosis have less than 50% accuracy , confirmMany strategies have been applied to control mange in rabbits  includinEffective treatment of mange in rabbits could be achieved through using some specific parasiticides or acaricides. Organophosphorus compounds (diazinon), synthetic pyrethroids (deltamethrin and permethrin), and macrolactones (ivermectin derivatives) are the most commonly used acaricides. Avermectin derivative group includes ivermectin , 69–71, Sarcoptes scabiei in rabbits. Administration of ivermectin at a dose of 0.2–0.4 mg/kg body weight (b/wt) once every 2 weeks for 2-3 times [μg/kg·b/wt for 3 weeks at weekly intervals [Psoroptes cuniculi and Sarcoptes scabiei in rabbits. Besides, Isingla et al. [Sarcoptes scabiei-infested rabbits showed recovery after 4 successive ivermectin treatments at a dose of 200 μg/kg·b/wt at weekly intervals with supportive therapy using pheniramine maleate [μg/kg·b/wt) combined with chlorpheniramine maleate (0.4 mg/kg·b/wt) and topical liquid povidone-iodine twice a day helped in the complete cure of Notoedres cati var. cuniculi-infested rabbits. The authors suggested prolonged availability of ivermectin with a residual effect against mites in rabbits. Complete cure from mite infestation after 7 days of ivermectin treatment is suggestive of the availability of sufficient drug concentration to kill any larvae hatching from the eggs. Ivermectin may cause the release of free radicals, which results in cytotoxic effect on the parasite [Ivermectin opens the glutamate-gated and gamma amino-butyric acid-gated chloride channels, which block the transmission of signals from the central interneurons to peripheral neurons of mite. Thus, the resistance of muscle's membrane is decreased with hyper-polarization of cells, and the parasite shows paralysis and finally dies . The dru-3 times , 0.2–0.4-3 times , and 400ntervals , and 700ntervals  was verya et al.  reported maleate . Nearly  maleate  who demoparasite .Sarcoptes scabiei to ivermectin has been developed [However, ivermectin may show some disadvantages such as the genotoxic and cytotoxic effects . Moreoveeveloped  along wieveloped . This dreveloped  demonstreveloped . Moreoveeveloped .Psoroptes cuniculi at a dose of 6 or 18 mg/kg·b/wt once or twice at 28-day intervals [Sarcoptes scabiei var. cuniculi at doses of 8–14 mg/kg·b/wt once or twice, 30 days apart [Cheyletiella species, selamectin has been efficiently applied once at a dose of 12 mg/kg·b/wt [Sarcoptes scabiei var. cuniculi, Psoroptes cuniculi, and Cheyletiella species for at least 58 days after treatment.Selamectin could be used effectively for the treatment of ntervals , or at antervals . Moreoveys apart . Regardi/kg·b/wt  or at do/kg·b/wt . Moonarm/kg·b/wt  revealedPsoroptes species in rabbits with promising results [Psoroptes ovis were regressed after 14 days of treatment with doramectin injection [μg/kg·b/wt along for 3 days was very efficient against Notoedric mange in rabbits [It has been found that doramectin, a genetically modified avermectin, shows a greater half-life time in plasma than that of ivermectin . After p results . Ear lesnjection . A singl rabbits . The eff rabbits .Psoroptes cuniculi in rabbits without adverse reactions [A topical mixture of imidacloprid and moxidectin was a practical and well-tolerated means of eradication of eactions . Rabbitseactions .Carbaryl, one of the carbamate groups, is a broad-spectrum insecticide . It actsDiluted benzyl benzoate is used as a topical treatment of scabies to decrease the severity of skin irritation . A mixtuTopical sulfurated lime is a mixture of polysulfides of plant origin, which has been used for controlling skin disease of ectoparasite origin via weekly application with dilution of 1 : 16 or 1 : 32 .in vitro study showed that 5% of permethrin demonstrated 100% effectiveness against mite within 12 hr, while 5% of deltamethrin killed 28% and 32% of mites [Psoroptes cuniculi. Cyfluthrin proved to have an efficient pesticidal effect in the elimination of Sarcoptes scabiei from the environment [Synthetic pyrethroids such as deltamethrin, permethrin, and cyfluthrin were used for long time as efficient acaricides. Permethrin (5%) is recommended by the Centers for Disease Control and Prevention as the first-line topical therapy for scabies . Permethof mites . Anotherof mites  showed tironment . IncreasSome oils such as liquid paraffin may be efficient in mange treatment . Oils maμg/kg·b/wt, fipronil spray in intervals of 15 days along with oral chlorpheniramine maleate (1 mg/kg·b/wt once in a day), and topical application of 5% of iodine for 5 days were the effective incomplete recovery protocol of rabbits from Sarcoptes scabiei after one month of treatment [Fipronil and milbemycin oxime are topical compounds, which have been applied via pouring them on lesions for 2–4 successive weekly doses . In addireatment .This treatment is easily applicable, is relatively cheap, has no adverse effects, reduces the possibility of using systemic acaricides, and shows low incidence of resistance and accumulation of edible tissue's residues.The development of drug resistance, presence of tissue residues, and adverse effects on the health and productivity of rabbits prompted and necessitated the search efforts to discover non-conventional and innovative effective alternative control therapies against mange.μg/kg·body weight) or 10% of propolis ointment induced the reduction in signs and pathological lesions, absence of adult mites and developmental stages, and improvement of some biochemical parameters [Propolis (bee glue) has various biological and pharmacological activities due to the presence of flavonoids, aromatic acids, and esters . It coulrameters .Bacillus (B) thuringiensis strains that may have specific insecticidal efficacy [B. thuringiensis [B. thuringiensis protein extracts are regarded to be potential biological control of mange in rabbits. The mode of action of B. thuringiensis against mites is not well defined; however, alterations in the intestinal columnar epithelial cells after mite infestation may be attributed to the activation of B. thuringiensis protoxins such as Cry1A. Cry1A acts through a specific “Receptor A,” which is aminopeptidase-N that has been purified from brush border membranes of intestinal epithelial cells of Lepidoptera [There are about 60,000 efficacy . It has ngiensis . Dunstanngiensis  concludeidoptera .Sarcoptes scabiei-infested rabbits without any side effects on the liver and kidney functions or the semen characteristics [Phytobiotics such as plant extracts , plant eeristics .Sarcoptes scabiei [Psoroptes cuniculi-infested rabbits [Supportive treatment using vitamins with other therapeutic medicaments could improve the clinical recovery of rabbits against  scabiei . Some vi rabbits , 62.Sarcoptes scabiei could induce a degree of protective immunity in different species of animals [Sarcoptes scabiei tropomyosin allergen showed reduction in lesions' size; however, the vaccine did not completely treat sarcoptic mange [Sarcoptes scabiei glutathione S-transferase [Sarcoptes scabiei infestation. Tarigan [λ15 and Ssλ20ΔB3) that were used as recombinant proteins were not effective as a vaccine against Sarcoptes scabiei in rabbits. Although the vaccination with both antigens induced high levels of humoral immunoglobulins (Ig)G and IgE and reduced mite density, this vaccine showed no clinical improvement in the skin lesion scores. Developed recombinant proteins containing purified rSs chitinase-like proteins (CLP)5 and rSs CLP12 subunit cocktail vaccine against Sarcoptes scabiei in rabbits induced a strong immune response and significantly decreased the parasite load in the host [It has been shown that the application of acaricides for controlling skin affections has some disadvantages such as the development of resistance and toxicities to the environment, feed, and humans , 133. Ma animals –136. Acc animals . Unfortu animals . Some stic mange . In addiic mange , and Sarnsferase  were use Tarigan  explaine Tarigan  demonstrthe host , 65, 143The rabbit farming industry is subject to several challenges that make it difficult to manage. One of the main constraints that still encounters rabbit farming is the mange disease. Many strategies have been applied to control mange in rabbits. The application of restricted hygienic measures surrounding the animals is crucial to prevent such infestation. Humans also must take all precautions during handling rabbits to avoid zoonotic transmission. Searching for new types of acaricides or even developing vaccines should be taken into consideration in future research work.


Ca-L) and reverse-mode current of the sodium–calcium exchanger (NCX). Adrenomedullin reduces spontaneous PV activity and PV diastolic tension by reducing ICa-L and NCX current and thus may be useful for treating atrial tachyarrhythmia.Adrenomedullin, a peptide with vasodilatory, natriuretic, and diuretic effects, may be a novel agent for treating heart failure. Heart failure is associated with an increased risk of atrial fibrillation (AF), but the effects of adrenomedullin on atrial arrhythmogenesis remain unclear. This study investigated whether adrenomedullin modulates the electrophysiology of the atria (AF substrate) or pulmonary vein  arrhythmogenesis. Conventional microelectrode or whole-cell patch clamps were used to study the effects of adrenomedullin  on the electrical activity, mechanical response, and ionic currents of isolated rabbit PV and sinoatrial node tissue preparations and single PV cardiomyocytes. At 30 and 100 pg/mL, adrenomedullin significantly reduced the spontaneous beating rate of the PVs from 2.0 ± 0.4 to 1.3 ± 0.5 and 1.1 ± 0.5 Hz (reductions of 32.9% ± 7.1% and 44.9 ± 8.4%), respectively, and reduced PV diastolic tension by 12.8% ± 4.1% and 14.5% ± 4.1%, respectively. By contrast, adrenomedullin did not affect sinoatrial node beating. In the presence of L-NAME , adrenomedullin (30 pg/mL) did not affect the spontaneous beating rate or diastolic tension of the PVs. In the single-cell experiments, adrenomedullin (30 pg/mL) significantly reduced the L-type calcium current (I Adrenomedullin is a 52-amino acid peptide of the calcitonin gene-related peptide family. Adrenomedullin has several cardiovascular effects  ,2,3,4,5 Ca-L in the myocytes of rats with septic shock through the PKA signaling pathway [HF is a major risk factor for atrial fibrillation (AF), and AF is the most common sustained arrhythmia in patients with HF, with an average prevalence of 25% . HF incr pathway . Becausen = 5; As depicted in 50  did not alter LA resting membrane potential (RMP) or APD90. Adrenomedullin at 100 pg/mL reduced RA APD20 and APD50  reduced LA action potentials duration (APD)50 , and adrnd APD50 , but adrLA or RA .Ca-L and sodium–calcium exchanger (NCX) in PV cardiomyocytes. We found that adrenomedullin (30 pg/mL) significantly reduced the ICa-L in PV cardiomyocytes, resulting in a 37.6% ± 4.7% decrease in peak ICa-L (elicited from −40 to +10 mV). However, adrenomedullin increased the forward- and reverse-mode NCX currents  incubation followed by marked decreases in cell shortening and Ca2+ transients [2+ and ICa-L in rabbit ventricular myocytes [Ca-L in PV cardiomyocytes, and such reduction may contribute to its effect on spontaneous PV activity. We also observed that adrenomedullin significantly shortened the APD20 and APD50 but not the APD90 of the LA and RA. These results suggest that the shortening of the APD20 and APD50 of the atria is mainly attributable to the effects of adrenomedullin on ICa-L. Our findings suggest that adrenomedullin reduces AF risk through its inhibition of PV, LA, and RA arrhythmogenesis.Adrenomedullin can induce an initial increase in cell shortening and Caansients ,27. In amyocytes . In thisWe observed that 100 pg/mL adrenomedullin only mildly reduced spontaneous SAN activity. These mild effects on the SAN may prevent bradycardia during adrenomedullin administration, which is a critical factor in atrial arrhythmogenesis, because the loss of SAN overdrive modulation of subsidiary pacemaker cells may facilitate the generation of ectopic arrhythmias. However, the mechanisms underlying the different responses of the PVs and SAN to adrenomedullin are unclear and may be caused by different characteristics underlying spontaneous SAN and PV activity because pacemaker current plays a major role in SAN activity but contributes little to PV activity . MoreoveGuide for the Care and Use of Laboratory Animals published by the United States National Research Council Institute for Laboratory Animal Research . Male New Zealand rabbits (average age of 6 months and weighing 2.5–3.0 kg) were anesthetized with inhalational isoflurane (2.0–2.5%) from a precision vaporizer for 10 min. The adequacy of anesthesia was confirmed by a lack of corneal reflexes or motor response to the painful stimulus of a scalpel tip. The SAN was dissected from the junction of the superior vena cava and RA. PV tissues were dissected from the LA at the LA–PV junction and from the lungs at the end of the PV myocardial sleeve. The LA–PV-junction end of the tissue preparations was attached with needles at the bottom of a tissue bath, and the other  end was connected to a Grass FT03C force transducer  by silk thread. As described previously [2) were separated from the LA and RA appendages, respectively. The LA, RA, PV, and SAN tissues were perfused at a constant rate (3 mL/min) with Tyrode solution containing NaCl (137 mmol/L), KCl (4 mmol/L), NaHCO3 (15 mmol/L), NaH2PO4 (0.5 mmol/L), MgCl2 (0.5 mmol/L), CaCl2 (2.7 mmol/L), and glucose (11 mmol/L) and then saturated with a gas mixture containing 97% O2 and 3% CO2. The LA, RA, PV, and SAN tissues were treated before and after the acute administration of various concentrations  of adrenomedullin with or without 100 μM L-NAME (an NO synthesis inhibitor) for analysis of the electrophysiological effects of adrenomedullin.All experiments were conducted in accordance with the eviously , the LA 90, APD50, and APD20, respectively.The transmembrane action potentials of the LA, RA, PV, and SAN tissues were recorded using machine-pulled glass capillary microelectrodes filled with 3 mol/L KCl, connected to a World Precision Instruments Duo 773 Electrometer  under tension of 150 mg. The temperature was maintained at 37 °C, and the tissue preparations were allowed to equilibrate for 1 h before the electrophysiological and pharmacological evaluations. Electrical and mechanical events were displayed on a Gould 4072 Oscilloscope  and simultaneously recorded with a Gould TA11 Recorder as described previously . The APASingle PV cardiomyocytes were isolated from the rabbits (2.0–3.0 kg) as described previously . A wholeCa-L was measured for 300 ms at a frequency of 0.1 Hz as the inward current during depolarization from a holding potential of −50 mV with test potentials increasing from −40 to +60 mV with 10 mV steps. Measurement was conducted using a perforated patch clamp with an external solution that contained tetraethylammonium chloride (137 mmol/L), CsCl (5.4 mmol/L), MgCl2 (0.5 mmol/L), CaCl2 (1.8 mmol/L), HEPES (10 mmol/L), and glucose (10 mmol/L), which was adjusted to pH 7.4 with CsOH. Micropipettes were filled with a solution containing CsCl (130 mmol/L), MgCl2 (1 mmol/L), MgATP (5 mmol/L), HEPES (10 mmol/L), Na-guanosine triphosphate (0.1 mmol/L), and Na2 phosphocreatine (5 mmol/L), which was adjusted to pH 7.2 with CsOH.I2 (2 mM), MgCl2 (1 mM), HEPES (5 mM), and glucose (10 mM) at pH 7.4 as well as strophanthidin (10 μM), nitrendipine (10 μM), and niflumic acid (100 μM).The NCX current was elicited through the administration of test pulses of between −100 and +100 mV from a holding potential of −40 mV for 300 ms at a frequency of 0.1 Hz. The amplitudes of the NCX current were measured using 10 mM nickel-sensitive currents. The external solution consisted of NaCl (140 mM), CaClt test or one-way repeated-measures analysis of variance with a Bonferroni post hoc test was employed to compare variable values before and after the administration of adrenomedullin and L-NAME. A p value < 0.05 was considered statistically significant.All continuous variables are expressed as means ± standard deviations. A paired Ca-L and NCX current. Adrenomedullin administration represents a potential novel strategy for management of atrial arrhythmogenesis.Adrenomedullin may activate NO signaling and exert antiarrhythmic effects by reducing spontaneous PV activity through mechanoelectrical feedback, reducing I


Here, we conducted a systematic review of the in vitro effects of cardiotonic steroids on Ca2+ in the brain of rats and mice. Methods: The review was carried out using the PubMed, Virtual Health Library, and EMBASE databases (between 12 June 2020 and 30 June 2020) and followed the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). Results: in total, 829 references were identified in the electronic databases; however, only 20 articles were considered, on the basis of the inclusion criteria. The studies demonstrated the effects of ouabain on Ca2+ signaling in synaptosomes, brain slices, and cultures of rat and mouse cells. In addition to the well-known cytotoxic effects of high doses of ouabain, resulting from indirect stimulation of the reverse mode of the Na+/Ca2+ exchanger and increased intracellular Ca2+, other effects have been reported. Ouabain-mediated Ca2+ signaling was able to act increasing cholinergic, noradrenergic and glutamatergic neurotransmission. Furthermore, ouabain significantly increased intracellular signaling molecules such as InsPs, IP3 and cAMP. Moreover treatment with low doses of ouabain stimulated myelin basic protein synthesis. Ouabain-induced intracellular Ca2+ increase may promote the activation of important cell signaling pathways involved in cellular homeostasis and function. Thus, the study of the application of ouabain in low doses being promising for application in neurological diseases.The NaSystematic Review Registration:https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020204498, identifier CRD42020204498. It is responsible for the active movement of Na+ and K+ against their electrochemical gradients at the expenses of ATP hydrolysis, and supports the maintenance of the cellular osmotic balance, membrane potential and the secondary active transport of substrates and neurotransmitters  with 10 transmembrane domains. This subunit harbors the binding sites for ions , ATP, and selective ligands collectively known as cardiotonic steroids, as well as for several other regulators  and it comprises around 65 amino acids. FXYD2 is not required for enzymatic activity but regulates Na+/K+-ATPase affinity for cations  , the latter by inducing a lower/reverse activity of the Na+/Ca2+ exchanger that colocalizes with the Na+/K+-ATPase  . Its inhhimperi) ; this dihimperi) . Ouabain+-ATPase , , and that cardiotonic steroids, more specifically ouabain, may be endogenous modulators of the Na+/K+-ATPase, we conducted a systematic review of the in vitro effect of cardiotonic steroids on cytoplasmic Ca2+ in rat and mouse brain, which are the most used experimental animal models. Therefore, favoring the perception of the relationship between ouabain-Na+/K+-ATPase and homeostasis Ca2+ signaling in the CNS in aging and neurodegeneration.Different cell functions such as growth, proliferation, differentiation and membrane excitability are regulated by Ca2+ , . The following question guided the selection of articles and the development of this review: “What are the effects of cardiotonic steroids on calcium signaling in the central nervous system of rodents?” This question was structured according to the acronym PECO  and the eligibility criteria are described in This study followed the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), and the protocol was registered on the PROSPERO platform . A systematic review was performed by searching PubMed, Virtual Health Library, and EMBASE databases (between 12 June 2020 and 30 June 2020) using the following search terms:  AND  AND  do not apply to or are not determined for biochemical studies of the kind here reviewed, and no standard quality assessment tool exists for t et al. . QuestioA total of 829 references were identified from electronic databases. Once duplicate entries (125) had been removed, the references were further evaluated for inclusion based on the title and/or abstract. Potentially 59 relevant articles were included in the next stage for full-text evaluation. Of these, it was not possible to gain access to 6 and 3 studies were excluded since they failed to satisfy the inclusion criteria established by the PECO. Finally, 20 studies were included in the analysis .n = 10; 50%), followed by Germany and Japan , Brazil, Canada, Denmark, and Hungary . Two trials were conducted in more than one country (10%).The publication period of the 20 eligible studies ranged from 1970 to 2020, and they were published in the English language. Most of the studies were conducted in the United States  were categorized into Tier 1, 13 (65%) were categorized into Tier 2, and 2 (10%) were categorized into Tier 3 based on their quality ratings across domains . These rAlthough no exclusion criteria were applied for the cardiotonic steroid studied in this systematic review, when the PECO criteria were applied, it was observed that all included studies used ouabain.They employed different methodological strategies: six studies focused on synaptosomes, eight used brain slices, and six employed cell cultures. It was possible to group the 20 articles included in this study, on the basis of their main characteristics, which are summarized in 2+ concentrations  mice, was not affected by 1 mm ouabain , tetrodotoxin (TTX), and ruthenium red. Despite being multitarget drugs, the first two agents inhibit voltage-gated Na+ channels, and ruthenium red inhibits Ca2+ transportation, since it competes for the Ca2+ binding site in many proteins .Blaustein and Wiesmann  and SwanRobinson  noted thproteins . On the proteins , when co−8 −5 M × 10−4 M) release of acetylcholine from synaptosomes regardless of the presence or absence of extracellular Ca2+, but such effect was impaired when the protein kinase C (PKC) and ryanodine receptor blocker TMB‐8 was coincubated with ouabain, suggesting the importance of intracellular Ca2+.Moreover, acetylcholine release was not detected upon ouabain treatment, despite the degree of depolarization being comparable to those of other depolarizing agents that induce neurotransmitter exocytosis. Satoh and Nakazato , showed 2+ concentrations, and all observed an increase of it  accumulation  in freshly prepared hippocampal and neocortical slices. Interestingly, although ouabain produced a significant elevation of in [Ca2+ ]i, Ca2+ by itself was shown not to be responsible for SD  (−4 M)  , and the) (−4 M) . It is i [Ca2+]i -  was added to the incubation medium together with ouabain. APH also prevented cells from swelling, mitochondrial deenergization, neuronal death. Furthermore, in primary cortical cultures, the neuronal apoptotic and necrotic death associated with Na+/K+-ATPase inhibition, caused by the application of 1 M × 10−4 M ouabain, was consistent with the intracellular depletion of K+ and the accumulation of Ca2+ and Na+, In addition, exposure for 20 h to ouabain induced DNA fragmentation  showed that addition of 1 M × 10entation . In the  ouabain .−4 M) had a calcium dependent effect on [Ca2+]i levels, leading to enhanced acetylcholine release. This effect of ouabain on acetylcholine release was dose and time dependent, achieving the maximum value after 30 min and was not inhibited by the addition of 1 µM tetrodotoxin (TTX). However, the effect of ouabain was suppressed with the addition of BAPTA-AM  showed that ouabain  failed to significantly change [Na+]i levels, but ouabain treatment significantly increased [Ca2+]i and stimulated myelin basic protein synthesis  cultures with ouabain  receptors in neuro–glial cell cultures of the cerebellum (Excitotoxicity is a term used to indicate the death of nerve cells by glutamate (Glu) as well as other amino acids, resulting in neurodegenerative diseases , Olney,, increasactivity ,  levels, similar to NMDA treatment, which was reversed by the NMDA antagonist MK-801 , discarding the involvement of TTX-sensitive Na+ channels. However, the effect of ouabain was suppressed with the addition of BAPTA-AM, suggesting the involvement of intracellular calcium stores. The authors suggested that in cholinergic neurons the ouabain induced increase in [Na+]i results in intracellular calcium alterations inducing an increase in [Ca2+]i, causing a release of acetylcholine independent of Ca2+  ethylenediamine (TPEN) eliminated ouabain-SD, implying that Zn2+ entry and mitochondrial dysfunction may play a critical role in the Ouabain-induced SD mechanism (Another phenomenon observed on the effect of the micromolar application of ouabain was the Spreading depression (SD), which is a wave of profound depolarization that propagates throughout the brain tissue after traumatic or vascular brain insults . Interesechanism . Thus, t+], stimulating an increase in intracellular Ca2+transient in oligodendrocytes, due to the reversion of the Na+/Ca2+ exchanger, which led to increased synthesis of myelin basic protein (MBP) , changed the Na+/Ca2+ exchanger-mediated [Ca2+]i, modulating MBP synthesis in OLCs (−7 M) also increased Ca2+ transient frequency. Additionally, this work showed that the inhibition of ryanodine receptor type 3 with 10 µM ryanodine also blocked Ca2+ transient.Different studies have demonstrated that neuronal activity promotes an elevation in extracellular i levels, but ouabain treatment significantly increased [Ca2+]i and stimulated MPB synthesis  or strophantidin effect (an equipotent inhibitor), and this may suggest that this outcome is modulated by cell signaling mechanisms besides the classic inhibitory effect. A similar effect was found in the study by Myles et al. , and triggering slow Ca2+ oscillations and the activation of NF-kB; this ultimately leads to the proliferation of these cells  in the nervous system, which has been shown to be concentration dependent. Structural modifications of cardiotonic steroids may be useful for the generation of new agents that are less toxic and with neuroprotective action.Ca