As Ca2+ is an essential messenger in cellular signaling, a sustained increase in Ca2+ concentration has dramatic consequences on cellular behavior, eventually leading to cell death. However, host cells have adapted mechanisms to protect against Ca2+ intoxication, such as Ca2+ efflux and membrane repair. The final outcome depends upon the nature and concentration of the toxin and on the cell type. This review highlights the repercussions of Ca2+ overload on the induction of cell death, repair mechanisms, cellular adhesive properties, and the inflammatory response.Bacterial pore-forming toxins induce a rapid and massive increase in cytosolic Ca Bacterial pore-forming toxins (PFTs) are the most frequently encountered virulence factors among bacterial pathogens ,2,3,4. T2+ concentration within host cells. The various scenarios described after PFT intoxication depend mainly on PFT identity and its local concentration, but also on the host cell type . For all these effects, Ca2+ is a central player in PFT-induced toxicity and downregulation of its cytosolic concentration is critical for cell fate.Once bound to a target cell receptor, PFTs form a pore in the plasma membrane, using a complex multistep process. The first step is toxin oligomerization. Oligomerization may occur once the toxins are inserted into the membrane or otherwise bound to the cell’s surface, forming a prepore. The prepore then inserts into the plasma membrane, forming a ring-shaped pore, which alters the target cell’s integrity . Pore fo2+, like the endoplasmic reticulum (ER) and lysosomes . However, multiphasic kinetics may also be observed, either because of rapid opening/closing of the pore or because forming pores are progressively eliminated by the host cell’s repair mechanisms  from Streptococcus pyogenes, and Staphylococcus aureus hemolysin A (Hla) [2+ from the ER by two different mechanisms successively: (i) a transient Ca2+ release from inositol P3-sensitive stores which involves G-proteins and phospholipase C, and (ii) a delayed and sustained release, the activation mechanisms of which remain to be determined [Cactivated . Release A (Hla) , induce termined .Listeria monocytogenes induces Ca2+ release from the ER via the G protein-phospholipase C-inositol P3 pathway, as well as a second wave of Ca2+ release involving damage to intracellular stores (ER and lysosomes) [2+–independent. Interestingly, organelle damage is reversible and does not result in cell death. This is an unconventional but efficient way to deliver Ca2+ in the cytosol, because of the high Ca2+ content of the ER.Similarly, listeriolysin (LLO) from sosomes) . The mecPasteurella haemolytica leukotoxin (LKT) induces increased cytosolic Ca2+ by activating voltage-gated channels in the plasma membrane via a G-protein-coupled mechanism involving activation of phospholipases A2 and C [Finally, A2 and C ,10,The mechanism of G-protein activation by PFTs remains undetermined. It is tempting to speculate that PFTs interact with a G-protein coupled receptor at the cell surface, which is the common way for G-protein activation. Alternatively, the transmembrane pore formed by PFTs may interact directly with G-proteins in the cytosol, without the need of a specific receptor.S. aureus leukotoxins , which increase Ca2+ levels by triggering its release from lysosomes followed by a second release from endoplasmic reticulum. This in turn stimulates the activation of store-operated Ca2+-channels in the plasma membrane, a process normally used when intracellular organelles are discharged of Ca2+ [2+ release from lysosomal stores through the two-pore Ca2+ channels [Other examples of PFTs with specific modes of action are  of Ca2+ . The ini of Ca2+ . CD38 ischannels ,26.Bordetella pertussis ACT, through its adenylate cyclase properties, activates non-voltage-dependent Ca2+ channels with L-type characteristics. This process involves ACT-induced cAMP production and subsequent protein kinase A activation [Finally, tivation .2+ entry exploited by bacterial PFTs, several toxins can also use other pathways to increase cytosolic Ca2+ concentration. It is yet unknown whether Ca2+ channel opening occurs before or simultaneously with pore formation.Thus, although passive influx through the pore is the most widespread mode of Ca2+ is maintained at low levels in the cytosol because it is an important second messenger activating several signaling pathways. Ca2+ can interact with and activate a number of cytoplasmic proteins [2+. Extrusion of cytosolic Ca2+ can be carried out by plasma membrane pumps  [2+ can also be efficiently sequestered in the ER thanks to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) [2+ uniporter (MCU) [2+ entry rapidly overloads the cytosol and exceeds the capacity of internal stores and the capability of Ca2+ pumps. If no membrane repair mechanism is activated, ion imbalance eventually triggers osmotic rupture of the plasma membrane or cell death by another pathway (see below).In general, Caproteins  that areproteins ,48, calpproteins ,37,61, pproteins , phosphoproteins , and calproteins . Sustaine; PMCA) , and wase; PMCA) . Large a (SERCA)  or into er (MCU) , which e2+ reported in the literature. Other major effects of pore formation by PFTs, for which the link with Ca2+ has not been established, are not dealt with here.In the following sections, we will focus on the main consequences of sustained PFT-dependent increase in cytosolic Ca2+ exporting systems, when the repair mechanisms are overwhelmed, the cellular ion imbalance reaches a point-of-no-return and cells engage in an irreversible process of cell death. Several excellent reviews have recently been published on this topic [Host cells have adapted to PFT injury by creating several mechanisms to eliminate pores that efficiently combat the dramatic effects of cell perforation by PFTs. The repair mechanism used depends upon the nature and number of pores, and on the cell type. As for the Cais topic ,65,66; hStreptococcus pneumoniae pneumolysin (PLY), Clostridium perfringens perfringolysin (PFO), and Streptococcus intermedius intermedilysin (ILY)) forming large pores (30 nm in diameter) that are surprisingly more efficiently eliminated by host cells than small-pore forming toxins.Membrane repair mechanisms have been studied for cholesterol-binding PFTs  the endosomal sorting complex required for transport (ESCRT) machinery, which drives microvesicle shedding [The primary mechanism of pore clearance involves the externalization of microvesicles  containing PFT pores ,68,69,70shedding ,72.The other process described is the endocytic model, whereby a portion of pore-containing membrane is internalized and then targeted to the lysosome for degradation ,73. ThisBoth processes of membrane repair (ectocytosis or endocytosis) require RAB-5 and RAB-11, two important regulators of vesicle trafficking .2+ influx sufficient for repair mechanism activation, however this hypothesis is contradicted by Ca2+ imaging data showing massive influx when cells are incubated with small-pore forming toxins [2+ channel opening or pore stability in the plasma membrane.As mentioned above, PFTs forming large pores trigger repair mechanisms much more efficiently than small-pore toxins. The reasons for this difference in triggering capacity remain elusive. One obvious hypothesis would be that small pores do not promote a Cag toxins ,49,50. A+-dependent manner [+ efflux, this repair mechanism may also be true for most PFT pores.Interestingly, ACT controls the path and kinetics of endocytic removal of toxin pores in a Kt manner . As many2+-independent repair mechanisms have also been described, such as p38 and JNK MAP kinase activation [Other Cativation ,76 and ativation . The con2+-dependent death programs  and cytochrome c, owing to Ca2+-induced opening of the mitochondrial permeability transition pore. Both proteins are known proapoptotic factors: AIF is translocated to the nucleus where it causes DNA degradation and chromatin condensation; cytochrome c activates the caspase cascade leading to DNA fragmentation [2+ can also trigger caspase-dependent or independent apoptosis as well as necrosis [C. perfringens enterotoxin (CPE) elicits apoptosis at low doses and necrosis at high doses, both pathways being caspase- and calpain-dependent [2+, is also involved in CPE-dependent apoptosis and necrosis processes [2+-dependent manner, which triggers apoptotic signaling [In some examples ,34,59,79entation . Indepennecrosis ,81. All ignaling . This meC. perfringens epsilon toxin (ET) stimulates the release of cytochrome c and mitochondrial–nuclear translocation of AIF, leading to chromatin condensation and nuclear shrinkage. However, ET fails to induce DNA fragmentation and thus to achieve apoptosis; cell death being then executed by necrosis [Similarly, necrosis . A possinecrosis .C. septicum, Ca2+ influx induces a mechanism of programmed necrosis involving calpain activation, release of cathepsins from lysosomes and increased reactive oxygen species (ROS) levels produced by deregulation of mitochondrial activity [2+-sensor proteins.In the case of α-toxin from activity . Thus, P2+-dependent necroptosis in pneumocytes [Serratia marcescens hemolysin (ShlA)- and PLY-induced necroptosis were independent of TNFR or TLR activation, but required RIP1, RIP3, and MLKL. This was confirmed in vivo in mice deficient either in RIP3 or in MLKL that exhibited increased survival when challenged with S. marcescens.Finally, two PFTs, ShlA and PLY, can trigger Caumocytes . Necroptumocytes . In geneumocytes . Signaliumocytes  showed tTaken together, PFTs can act on several pathways, probably in combination, to provoke cell death. It is noteworthy to point out the central role of mitochondria in this context. Still, much work has to be done to fully elucidate this critical effect of PFTs in order to identify the missing links in these pathways. Most of these findings result from investigations in one cell type, while most PFTs intoxicate several. As death programs are cell type-dependent, it would be important to extend these investigations to other cells and tissues. ,34,55,59Although not precisely demonstrated, PFTs are probably present at sublytic concentrations in vivo . HoweverCadherins, located in adherens junctions and required for intercellular adhesion, were recently shown to be targeted by PFTs. PFT-triggered cadherin cleavage was first described by Inoshima et al. after incubation of cells with Hla . The mec2+ is required for Hla-dependent cadherin cleavage by ADAM10, suggesting that the Hla-ADAM10 interaction alone cannot activate ADAM10; indeed, Ca2+ influx generated by pore formation is involved in this process. A similar effect was confirmed for PLY, which binds cholesterol rather than ADAM10 [ADAM10 is the cellular receptor for Hla. Extracellular Can ADAM10 ,85,86.Pseudomonas aeruginosa (ExlA) and S. marcescens (ShlA) were also shown to induce rapid E- and VE-cadherin cleavage through ADAM10 activation, even though ADAM10 is not a cellular receptor for these PFTs [2+ [2+, calmodulin releases pro-ADAM10, which is in turn activated by furin and transported to the plasma membrane where it induces cadherin shedding , whiA. hydrophila aerolysin via Ca2+- and myosin light chain kinase (MLCK)-dependent pathways [Interestingly, tight junctions, which control barrier permeability, are also disrupted by pathways  , whose activity is enhanced by Ca2+ binding [In addition to the mechanisms presented above, several intracellular signaling molecules are stimulated by PFTs, such as phospholipase A binding , and ear binding . The fin2+ is a very important communicator in cell signaling and drives important cellular functions, its manipulation by bacterial PFTs has profound consequences on cell behavior and homeostasis.Because Ca2+ concentrations can have dramatic consequences, including tissue destruction and bacterial dissemination, or more subtle effects, such as bacterial internalization or thrombosis. However, PFT-induced cell rupture and cytokine production are also alarming signals engaging a strong immune response that counteract the infection.Increased cytosolic CaAs indicated above, PFTs from various bacteria may induce diverse—sometimes opposing—effects, however the mechanisms activated by these toxins have not been systematically investigated for all PFTs and much work remains to be done to obtain a general view of the action of PFTs in various infection scenarios.


Hydrogen sulfide (H2S) is a physiological gaseous messenger that is proposed to act as an oxygen sensor in CBs, although this concept remains controversial. In the present study we have used the H2S scavenger and vitamin B12 analog hydroxycobalamin (Cbl) as a new tool to investigate the involvement of endogenous H2S in CB oxygen sensing. We observed that the slow-release sulfide donor GYY4137 elicited catecholamine release from isolated whole carotid bodies, and that Cbl prevented this response. Cbl also abolished the rise in [Ca2+]i evoked by 50 µM NaHS in enzymatically dispersed CB glomus cells. Moreover, Cbl markedly inhibited the catecholamine release and [Ca2+]i rise caused by hypoxia in isolated CBs and dispersed glomus cells, respectively, whereas it did not alter these responses when they were evoked by high [K+]e. The L-type Ca2+ channel blocker nifedipine slightly inhibited the rise in CB chemoreceptor cells [Ca2+]i elicited by sulfide, whilst causing a somewhat larger attenuation of the hypoxia-induced Ca2+ signal. We conclude that Cbl is a useful and specific tool for studying the function of H2S in cells. Based on its effects on the CB chemoreceptor cells we propose that endogenous H2S is an amplifier of the hypoxic transduction cascade which acts mainly by stimulating non-L-type Ca2+ channels.Carotid body (CB) chemoreceptor cells sense arterial blood PO Activation of chemoreceptor cells by either hypoxia or acidosis results in an increase of the ongoing basal normoxic/normohydric release of neurotransmitters leading to an increase in the action potential frequency in the CSN. Activity of the CSN is integrated in the brainstem to generate cardiorespiratory responses which act to restore blood gases to normal levels i elicited by NaHS, inhibited the hypoxia-induced Ca2+ response ~70%, and left intact the response elicited by high K+. We conclude that endogenous H2S has a specific physiological action in enhancing hypoxic responses in chemoreceptor cells; this may involve mainly the stimulation of T-type voltage-gated Ca2+ channels. Some preliminary data from this study were presented at the XIXth meeting of the International Society for Arterial Chemoreception held in Leeds in July 2014 i. A subsequent pulse of high K+ (35 mM) to assess the responsiveness of the cells to depolarization induced the expected rise in [Ca2+]i.2+ signal obtained from 74 cells subjected to this protocol reveals that neither Cbl itself nor 50 mM NaHS applied in the presence of Cbl had a significant effect on [Ca2+]i. 2+ response elicited by 50 μM NaHS. 2–95% N2; peak hypoxia in the recording chamber ≈ 10 mmHg; [N2(I)] in 2(II)]). After an additional normoxic recovery period in the absence of Cbl, a final hypoxic stimulus ([N2(III)]) was then applied. It is evident that Cbl reversibly inhibited the hypoxia-induced Ca2+ signal by ~70%. In an identical group of experiments using 35 mM K+ as a stimulus in 45 chemoreceptor cells . This was consistent with the well-established role of Ca2+ influx via voltage-gated channels in the hypoxic response [2+]i caused by NaHS was also due to Ca2+ influx. As shown in 2+ influx response to hypoxia, measured in 24 cells, was blocked by the L-type voltage-gated Ca2+ channel blocker nifedipine (2 µM). Whereas two successive hypoxia-induced Ca2+ transients were similar in size under control conditions (2+]i elicited by 50 µM NaHS tended to increase during the second exposure to this sulfide donor (51 chemoreceptor cells). This increase was significantly smaller when 2 µM nifedipine was present during the second response  on the increase in chemoreceptor cell [Ca2+]i caused by sulfide (47 chemoreceptor cells). As shown in As it has been proposed that sulfide-induced inhibition of BK sensing , we also3H-CA release, and this response was abolished by 300 µM Cbl; (2) Cbl diminished the hypoxia-induced release of 3H-CA by 75–100%, and eliminated and reduced the intracellular Ca2+ transients elicited by 50 µM NaHS and hypoxia, respectively, by ~70%; (3) in contrast, neither the release of 3H-CA nor the Ca2+ transients elicited by high external K+ were affected by Cbl; (4) nifedipine caused a relatively small inhibition in the rise in CB chemoreceptor cells [Ca2+] elicited by sulfide whilst attenuating the Ca2+ signal caused by hypoxia to a greater extent; (5) IBTx had no effect on the Ca2+ transients elicited by NaHS in chemoreceptor cells. The physiological forms of vitamin B12 in mammalian cells are methylcobalamin and 5′-deoxyadenosyl-cobalamin, with hydroxycobalamin being one of the most abundant forms of vitamin B12 in the diet. Cyanocobalamin and hydroxycobalamin are the forms of vitamin B12 used most commonly for therapy. The cellular uptake of vitamin B12 from the blood depends on its binding to the plasma protein transcobalamin II (TC II); this complex binds to a membrane receptor (TCbIR/CD320), which is then endocytosed i was mostly dependent on activation of N/P/Q type Ca2+ channels via a PKC-dependent mechanism [2+]i in CB chemoreceptor cells  the hydrogen sulfide donors NaHS and GYY4137 stimulated TCbIR/CD3, which iobalamin . This upTCbIR/CD3, which ito TC II . Howeverobalamin . Of morelatelets . The abilatelets  in whichlatelets ,25 usingeticulum , whilst or cells A, and Cbchannels ) or thatlar Ca2+ , as is t2+]i see B Thus, w3H-CA release, which at the high concentration (200 µM) of NaHS we used in this experiment was probably due to the block of cytochrome C oxidase [2+] induced in CB cells by 50 µM extracellular NaHS , we cal oxidase . Physiolytoplasm  probably3H-CA release suggests that endogenous sulfide contributes to the setting of the functional activity of chemoreceptor cells under normoxic conditions, the data presented in 3H-CA release induced by three levels of hypoxia , in each case by >75%.Whereas the effect of Cbl on basal 3H-CA release to completely subside following the re-imposition of normoxia. The elevated release of catecholamine during this period was blocked if Cbl was present but was not significantly reduced if Cbl had been present during hypoxia but was removed when normoxia was restored  virtually abolished the hypoxia-induced secretory response i elicited by NaHS to nifedipine suggests that sulfide is stimulating the opening of both L- and T-type Ca2+ channels, with a predominant effect on the latter. T-type Ca2+ channels, which are not blocked by nifedipine, activate and inactivate at more negative potentials than do L-type channels. They therefore demonstrate a window current which develops with relatively small depolarizations, such as those associated with O2 sensing in the CBs e from 5 to 25 mM K+ would shift the K+ equilibrium potential by about +39 mV (assuming an intracellular [K+] of 130 mM). Thus, whereas T-channels could be active at the resting potential, such that their stimulation by basally generated sulfide causes low-level CB activation, they are likely to inactivate rapidly during high K+ depolarization, thereby accounting for the lack of effect of Cbl, and therefore sulfide, on catecholamine release by high K+.The partial sensitivity of the rise in i and consequent CB stimulation by hypoxia is due to a sulfide-mediated activation of T-type Ca2+ channels.The results of Ca channels is important in CB O2 sensing i triggered by hypoxia, but to date this seems not to have been examined. We therefore assessed whether the BKCa channel blocker IBTx would suppress the effect of 50 µM NaHS on [Ca2+]i and found, as shown in 2+]i rise in these cells, these results do not support the concept that the involvement of sulfide in O2 sensing requires BKCa channel inhibition.There is evidence that hypoxia-induced inhibition of both TASK and BK sensing ,52. Alth sensing ,15,16, s sensing ,53. An i2 sensing in CB chemoreceptor cells and is capable of regulating their activity, both under basal conditions and during natural hypoxic stimulation. Our evidence supports the possibility that this contribution is due to the opening of T-type Ca2+ channels, as demonstrated previously by Makarenko and colleagues  during hypoxia, and there are also worries about the marked rise in plasma homocysteine which occurs in CSE knockout mice , Cbl woulleagues . Thus, e


There is an error in affiliation 2 for author Raúl Guillermo Enríquez-Habib. The correct affiliation 2 is: Departamento de Química Orgánica, Instituto de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico.


However, the role of MDM2 -309T>G (rs2279744) polymorphism in AML remains unclear. We have performed this study to examine the association of MDM2 -309T>G with AML in an Iranian population. Genetic factors play a substantial role in acute myeloid leukemia (AML) etiology. Overexpression of the N MDM2 -309T>G polymorphism in 73 cases diagnosed with AML and 80 healthy controls by tetra-primer amplification refractory mutation system (ARMS) PCR assay. Odds ratios (OR) and 95% confidence intervals (CI) were calculated on the risk genotypes and alleles. We have examined the association of MDM2 -309T>G polymorphism in patients were 32.9%, 23.2% and 43.9%, while in controls were 86.2%, 7.5% and 6.3%, respectively. Moreover, Frequency of mutant allele (G) was 55.6% in cases with AML and 10.0% in controls. The mutant homozygote genotype (GG) was associated with an increased susceptibility to AML . The TT, GG and GG genotypes of MDM2 -309T>G polymorphism was significantly associated with increased risk of AML in the Iranian population. Thus, the MDM2 -309T>G polymorphism might be useful genetic susceptibility factors in the pathogenesis of AML.Our results showed that the  Acute myeloid leukemia (AML) is a severe and life threatening malignancy which occurs in all ages but is more common in the elderly with a median age at diagnosis of approximately 70 years . AML is a clonal disease characterised by a variety of hematopoietic malignancies in the bone marrow, presenting impaired differentiation, deregulated proliferation and a survival advantage of myeloid progenitor cells . AML has an incidence of three to five cases per 100,000 population in the developed with a poor clinical outcome . In the United States, there are approximately 20,000 new cases of AML diagnosed per year, with death rate close to 50% . The etiology of AML is generally unknown .Mouse Double Minute 2 (MDM2) gene is amplified and/or overexpressed in leukemia and constitutes one of a number of ways the p53 pathway is disrupted in development of these malignancies .It is clear that the environmental factors such as previous treatment with chemotherapeutic agents as well as preceding haematological malignancies, such as myeloproliferative disease and myelodysplastic syndromes increase the risk of later developing AML . Chromosomal rearrangements have been detected in almost half of AML cases . A thorough examination of genetic variation is necessary for an exactly understanding etiology and development of AML. AML is also speculated to be associated with mutation in genes that plays an important role in tumours suppressor . As well as, the  MDM-2 (MIM#164785) gene encodes a 90-kDa protein that binds specifically to the p53 tumor suppressor and directly inhibiting the transcriptional activity of P53 . Moreover, MDM2 functions as an E3 ubiquitin ligase is responsible for the ubiquitination and proteolytic degradation of p53 . It is known that MDM2 has two promoters, a basal promoter (P1) and an alternative promoter (P2) starting in the intron 1. The P1 promoter is a constitutive promoter that does not affect the expression levels of MDM2. The P2 promoter is a p53-dependent intronic promoter containing two adjacent p53 binding elements . P2 may affect the expression levels of MDM2. The human MDM2 gene is mapped on chromosome 12q14.3-15 and contains 11 exons. The MDM2 gene contains several polymorphisms, which MDM2 -309T>G polymorphism (rs2279744) is most studies SNPs in various tumors and located on the within the intronic p53-responsive promoter of the MDM2 (P2). The MDM2 -309T>G polymorphism has been associated with the increased affinity of the transcriptional activator Sp1, resulting in higher levels of MDM2 mRNA and protein .TheMDM2 -309T>G polymorphism might be increased risk of AML. But the results are inconsistent and inconclusive. In order to get more precision results for the MDM2 -309T>G polymorphism and the risk of AML, we performed this case-control study among an Iranian population.It has been showed that the ParticipantsThis study was formally approved by the Ethics Committee of Ashkzar Universities of Medical Science and a written informed consent was obtained from each cases and controls. All procedures were performed in accordance with the approved guidelines. We recruited 73 patients diagnosed with de novo AML and 80 age and sex matched healthy controls between 2015 and 2016 from Shahid Sadoughi hospital at Yazd city. The diagnosis of AML was established according to the customary FAB criteria, and was based on bone marrow morphology and cytochemical staining.The sample size in cases was estimate by using the following formula:GenotypingMDM2 -309T>G polymorphism was genotyped using tetra-primer amplification refractory mutation system (tetra-ARMS) PCR method. The primers sequences were shown in Genomic DNA was extracted from 0.2 ml fresh peripheral blood samples using a DNA kit  according to the manufacturer’s protocols and stored at −20°C until used for genotyping. Extracted DNA samples had a final concentration ranging from 100-200 ng/µL. The Statistical analysisMDM2 -309T>G polymorphism to the Hardy-Weinberg Equilibrium (HWE) in healthy subjects. The allele and genotype frequencies of MDM2 -309T>G polymorphism between cases and controls were analyzed by using Chi-square (χ2) test. Odds ratios (OR) and 95% confidence intervals (CI) were calculated on the risk genotypes and alleles. P-values were two-tailed and a value of less than 0.05 was considered statistically significant.Statistical analysis was performed by using the using Statistical Package for Social Science (SPSS) program version 18. We used a Chi-square goodness-of-fit test to examine the adherence of the The demographic and clinical characteristics of patients with AML and controls are shown in MDM2 -309T>G polymorphism are summarized in MDM2 -309T>G polymorphism in the controls did not in agreement with the Hardy-Weinberg equilibrium (p≤0.001), and the Minor allele frequencies (MAF) in healthy subjects was 10%.The genotype frequencies of MDM2 -309T>G polymorphism in cases with AML were 32.9%, 23.2% and 43.9%, respectively. While, in the control group those genotypes frequencies were 86.2%, 7.5% and 6.3%, respectively. There was a significant difference in homozygote mutant genotype (GG) frequency between cases and controls. Analysis showed that the homozygote mutant genotype (GG) was significantly associated with increased risk of AML . Frequency of mutant allele (G) was 55.6% in AML and 10.0% in healthy controls. There was a significant differences between cases and controls regarding the mutant allele (G) frequency (p=0.001). Analysis showed that the mutant allele (G) was significantly associated with increased risk of AML  classification: the AML-M2 was the predominant FAB subtype (24.6%) followed by M3 (20.5%), M1 and M4 (15.1% each), M6 (8.2%), M0 and M5 (6.8%), M5 (7.2%), and M7 (2.7%). However, there were no significant differences between GG, GT and TT genotypes of ML cases .MDM2 binds to the transactivation domain of the p53 protein and inhibits p53’s transcriptional activity . MDM2 overexpression can cause dysfunction and inactivation of TP53, which promotes tumor progression. Since the first publication on MDM2 -309T>G polymorphism in 2004, several studies have been evaluated the association of MDM2 -309T>G polymorphism with development of various malignancies . To date, a few studies have been evaluated the association MDM2 -309T>G polymorphism with AML risk . However, those studies results about association of MDM2 -309T>G polymorphism with development of AML are inconclusive.MDM2 -309T>G polymorphism with AML risk in an Iranian population. To our best knowledge, this is the first study on the relationship of MDM2 -309T>G polymorphism with AML risk in Iranian and also Middle East populations. Our results showed that the MDM2 -309T>G polymorphism was significantly associated with increased risk of AML in the Iranian population. The biological mechanisms of this susceptibility may be as follows: the G allele of MDM2 -309T>G polymorphism could significantly increase the affinity of MDM2 to transcriptional activator SPl, resulting in increased mRNA transcription and protein expression of MDM2. Thus, MDM2 directly inhibits the p53 tumor suppressor pathway and increasing risk of AML in people with mutant allele (G).In the current study we have evaluated the association of MDM2-309T>G polymorphism and p53 codon 72 polymorphisms with risk of AML in a Chinese population. They have revealed that the mutant allele (G) of MDM2-309T>G polymorphism was associated with increased risk of AML. However, their results failed to show the association of MDM2-309T>G polymorphism and p53 codon 72 polymorphisms with age of onset and other clinical parameters of patients. In 2015, Cingeetham et al., in case-control study of 223 de novo AML cases and 304 age and sex matched healthy controls have evaluated the association of MDM2-309T>G polymorphism with susceptibility to AML by tetra-primer amplification refractory mutation system (ARMS)-PCR method in an Indian population. There results showed that the MDM2-309T>G polymorphism was significantly associated with increased risk of AML in the Indian population. Moreover, their results revealed that the MDM2-309T>G polymorphism might be serve as a good prognostic indicator for AML . In the same year, Falk et al., (2015) have examined the frequency and impact of TP53 and MDM2-309T>G polymorphism on treatment outcome and overall survival (OS) in 189 AML patients in Swedish people. They have found that the MDM2-309T>G polymorphism might be useful for prognostication, risk stratification, and selection of AML cases most likely to benefit from new drugs targeting the p53 signaling pathway. Ebid et al., (2012) have performed a study to evaluate the prevalence of P53, P21Ser31Arg and MDM2-309T>G polymorphism s in AML and to compare it to apparently normal healthy controls for assessment of impact on risk in an Egyptian population. The study results supported that these genes polymorphism, especially the combination of 21Ser31Arg/ MDM2-309T>G polymorphisms might be genetic susceptibility to AML. According to their results, the G allele of MDM2-309T>G polymorphism was itself without affect. However, it showed a synergistic effect with P21 Ser/Arg polymorphism in development of AML. Additionally, they have found that the P53 Arg72Pro polymorphism was not significantly associated with increased risk of AML among the Egyptian population. In 2010, Barnette et al., (2004) have assessed the relationship of MDM2-309T>G polymorphism with AML risk among 575 de novo acute cases treated on three Children’s Oncology Group protocols in USA. The study showed that the MDM2-309T>G polymorphism was associated with increased risk of pediatric AML, but does not impact overall response to therapy.In 2009, Xiong et al., (2009) for first have examined the association of MDM2-309T>G polymorphism and susceptibility to leukemia. Their results showed that individuals carrying the mutant allele (G) had increased susceptibility to leukemia compared to individuals who carrying the wild allele (T) . Moreover, they have showed that the individuals with the homozygous mutant genotype (GG) had 1.47 times the risk of leukemia compared to individuals with the wild homozygote (TT) and heterozygote (TG) genotypes. In another meta-analysis, He et al., (2015) have the association of MDM2-309T>G polymorphism with prognosis of leukemia. In the meta-analysis, they have selected eleven studies with a total of 2,478 patients accrued. Their pooled results showed that cases with homozygote mutant genotype (GG) had a significantly increased risk of developing leukemia . However, they have found that patients with heterozygote (GT) not significantly associated with increased risk of leukemia . In addition, their pooled data showed that the MDM2-309T>G polymorphism did not significantly associated with patient survival .Ou et al., (2015) in a meta-analysis of ten case-control studies with 1889 cases and 5707 controls have evaluated the association between MDM2 -309T>G polymorphism with increased risk of AML in the Iranian population. Thus, the MDM2 -309T>G polymorphism might be useful genetic susceptibility factors in the pathogenesis of AML. However, our results are still needed to obtain more precise results by further well-designed case-control studies with larger sample sizes and in different ethnicities.In summary, the current study results support the association of


However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III) on [Ca2+]i and pHi and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+]i) and intracellular pH (pHi) in Sf9 cells were determined. R-III induced a significant dose-dependent  increase in [Ca2+]i and pHi of Sf9 cells in presence of Ca2+-containing solution (Hanks) and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+]i, because completely treating Sf9 cells with CdCl2 (5 mM), a Ca2+ channels blocker, R-III (100 μg/mL) induced a transient elevation of [Ca2+]i in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pHi showed similar changes with that of [Ca2+]i on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+]i, cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.Many studies on intracellular calcium ([Ca Rhododendron molle and determined as the main insecticidal ingredient in the plant i) is a highly versatile intracellular second messenger and signal transducer in both excitable and non-excitable cells. It is involved in many functions in proliferative cells, including gene expression, protein synthesis, cell secretion, motility, metabolism, cell-cycle progression and apoptosis cell death [2+]i concentration is maintained at 10–100 nM, but sustained Ca2+ release from intracellular Ca2+ stores, Ca2+ influx through receptor- or voltage-dependent Ca2+ channels or blockage of re-uptake can perturb [Ca2+]i homeostasis [2+]i which may lead to cellular physiological changes such as cell arrest or cell death i are closely linked. In effect, pHi has been described as being able to affect intracellular Ca2+ homeostasis and contribute to the length, magnitude, and frequency of the Ca2+ signal through the modulation of voltage-dependent or -independent plasma membrane Ca2+ channels and/or through regulation of the mobilization of Ca2+ from internal stores i level, cell cycle and cell proliferation, and the possible linkage between changes of intracellular Ca2+ and that of pHi in Sf9 cells induced by R-III, all of which are helpful to explore some new clues for the further study on insecticidal mechanism of R-III.Although many studies on i, Sf9 cells were exposed to Hanks, a buffer solution containing Ca2+. Ca2+ influx took place in Sf9 cells stimulated by R-III, which elicited a significant increase of [Ca2+]i. As shown in 2+]i by 29.9%, 38.28%, 64.21% and 111.78%, after the stimulation with 1, 10, 100 and 200 μg/mL of R-III respectively, showing a dose-dependent fashion. Under these conditions, we observed a similar change of pHi with that of [Ca2+]i. As shown in i by 22.27%, 37.13%, 69.17% and 89.58% after stimulation with 1, 10, 100 and 200 μg/mL of R-III respectively, also in a dose-dependent fashion. The increase of pHi was only 5.7% in control. In order to further investigate the effects of R-III on [Ca2+]i in Sf9 cells, we checked the time-dependent changes of Ca2+ fluorescence signals in Sf9 cells induced by R-III. As shown in 2+]i in cells. A transient elevation of [Ca2+]i characterized by a fluorescence intensity increase followed by a recovery to basal level was observed in Sf9 cells stimulated with low concentration of R-III (1 μg/mL)  -b, sugge2+]i, which reached to a high steady state [2+]i may derive from extra cellular Ca2+influx through calcium channels or transporters i declining sharply in a index fashion to a steady state lower than the basal level  which gives rise to [Ca2+]i efflux. Since R-III was a botanic pesticide showing significant toxicity to many kinds of insect, the second stimulation of R-III possibly produced toxic effect on Sf9 cells causing [Ca2+]i efflux, in agreement with viewpoint of [2+]i efflux exchanged for H+ influx through Ca2+-ATPase in PM i and pHi, Sf9 cells were exposed to Dhanks, a Ca2+-free buffer solution, and recoded the change of fluorescence intensity in Sf9 cells stimulated with R-III (100 μg/mL) at 130 s. Comparing to the control with only a slightly decrease of [Ca2+]i (3.52%) , [Ca2+]i sharply decreased by 24.64% after the stimulation of R-III  to the Ca2+-free buffer solution at 500 s , and sustained decrease (61.02%) was observed even if addition of CaCl2 at 130 s after stimulation . The results in this assay indicated that R-III not only induced [Ca2+]i in Sf9 cells decline through Ca2+ efflux but also elicited the intracellular acidification, possibly through H+ entry in exchange for Ca2+ extrusion by the Ca2+-ATPase in cell PM i homeostasis are associated with induction of apoptotic i resulting from the entry of extra cellular Ca2+. In recent years, some reports show that calcium signal is a key component of the molecular switch mechanism in cell division cycle [2+]i participates in regulating key steps in the cell cycle such as reentry of quiescent cells into proliferation and the transition through the G1/S, G2/M, and the metaphase/anaphase boundaries [3R-induced calcium transients [2+]i homeostasis such as increase of [Ca2+]i level in response to external stimulation may interfere with cells division cycle, resulting in cell cycle arrest [2+]i induced by R-III [2+]i, cell cycle arrest, cell proliferation inhibition in Sf9 cells induced by R-III. Moreover, we tentatively hypothesize that disturbance of [Ca2+]i homeostasis in Sf9 cells induced by R-III may result in cell cycle arrest, which finally causes inhibition of insect cells proliferation or even cell death (including apoptopic cell death). This dual negative effect would significantly decrease the absolute number of cells, and finally induce remarkable decrease of survival cell number in R-III treatment.Caessenger . For exapoptotic  or cell poptotic . An expe i and pHi in Sf9 cells incubated with CdCl2  showed a slight decrease (4.52% and 3.03% respectively). After treatment with R-III, [Ca2+]i and pHi rose by 235% and 40.32% respectively. As indicated in dynamic change of trace  to incubate with Sf9 cells for 10 min to block the Ca2+ channels completely, and then stimulated with R-III (100 g/mL), both [Ca2+]i and pHi decreased sharply by rate of 33.85% and 48.74% respectively , synthesized in response to external stimulation, induces the release of Ca2+ from intracellular stores i to a level far lower than basal level occurred in this study   presented an markedly increase of [Ca2+]i by rate of 79.43% in the first stimulation of R-III for 200 s, and the second stimulation of R-III induced [Ca2+]i decrease by 40.35% , diluted to their final concentration in phosphate buffer solution  and stored at −20 °C until used. All other chemicals were from standard commercial sources and reagent grade or the highest purity.The Rhodojaponin-III (98%) was isolated by using HPLC from the flowers of Sf9 cells were obtained from State Key Laboratory of Biocontrol, Sun Yat-sen University, and were cultured in Grace’s medium  supplemented with 10% fetal bovine serum (heat-inactivated) and 1% penicillin/streptomycin. Cells were cultured at 27 °C and subcultured every 3 days. Confluent cells with >95% viability (tested with Trypan blue exclusion) were used in all experiments.4 cells per well). When cells were adherent, at concentrations of 1, 10,100 and 200 μg/mL was added to treated cells for serial times . Cells that cultured with 0.1% DMSO at the same times were used as control. After harvested, survival cell number for each time point was counted using Trypan blue exclusion test with a standard haemocytometer. Growth curve was made after analyzing the data.Sf9 cells were seeded onto 24-well plates (2 × 102 plastic tissue culture flask (2 × 105 cells per flask). When the density was 1 × 106 cells/mL, cells were treated with R-III at concentrations of 1, 10, 100 and 200 μg/mL for serial times . After harvested, cells were re-suspended and washed twice with phosphate buffer solution (PBS) . After that, cells were fixed in cold 70% ethanol, and stored below −20 °C over night. Prior to analysis, ethanol was removed and fixed cells were washed twice with PBS (pH 7.2). Cells were re-suspended in PI solution  and incubated for 30 min at room temperature. After that cells were processed in a FACSCalibur . At least 2.0 × 104 cells were counted in each assay. The fraction of the total cell population presented in G2/M phase was obtained from DNA histograms using Cell Quest and Modfit Software . All cytometry experiments were performed on cells in log phase of growth. Cells that cultured with 0.1% DMSO at the same times were used as control.Sf9 cells were seeded onto a 25 cm2+]i measurements from Sf9 cells were performed with the fluorescent Ca2+ indicator fluo-3/AM. Cells were cultured in 35-mm polystyrenetissue culture dishes  and washed twice with PBS, than loaded with 1 uM membrane-permeant acetoxymethyl ester of the dye (fluo-3/AM) for 45min at 37 °C. After dye loading, cells were washed twice with Dhanks solution . The pre-treatment measurement of pHi was carried out same as [Ca2+]i measurements, except that the final concentration of dye solution were 10 μM (Snarf1/AM).Cell treatment: i and pHi in various cells. It can be excited by an argon ion laser at 488 nm, and its emitted fluorescence (at wavelengths 520 nm) increases with increasing [Ca2+]i i [2+]i change could be shown by relative change of fluorescence intensity.Fluorescence measurement: Fluo-3/AM and Snarf1/AM is one of the most suitable Ca [Ca2+]i  or pHi.  [Ca2+]i , [Ca2+]iData analysis was carried out using SAS software (SAS Institute Inc.) and Microsoft Excel software. Differences between the treatments were determined by Tukey’s multiple range tests (P < 0.05 being considered significant).2+]i homeostasis by inducing [Ca2+]i influx or efflux in Sf9 cells in the presence of Ca2+-containing or Ca2+-free buffer solution. In these conditions, pHi showed proportional changes with that of [Ca2+]i on the whole. According to the results and discussion in this paper, we supposed that there was a certain linkage for change of intracellular calcium, cell cycle arrest, cell proliferation inhibition in Sf9 cells induced R-III and that cytosolic alkalinization or acidification shifts are associated with changes of [Ca2+]i level in Sf9 cells induced R-III.R-III displayed strong inhibitory activity on the proliferation of Sf9 cells, and interfered with the Sf9 cell division cycle and arrested them in G2/M. In addition, R-III perturbed [Ca


While this mechanism is important in controlling the direction of long-term plasticity, it is still unknown whether it can operate at all excitatory synapses converging onto FS cells or at a set of synapses receiving a particular input. Using a combination of patch-clamp recordings and two-photon Ca2+ imaging in acute mouse hippocampal slices with computational simulations, here we compared the generation of supralinear post-CaTs between apical and basal dendrites of FS cells. We found that, similar to basal dendrites, apical post-CaTs summated supralinearly and relied mainly on the activation of the CP-AMPARs, with a variable contribution of other Ca2+ sources, such as NMDA receptors, L-type voltage-gated Ca2+-channels and Ca2+ release. In addition, supralinear post-CaTs generated in apical dendrites had a slower decay time and a larger cumulative charge than those in basal, and were associated with a stronger level of somatic depolarization. The model predicted that modulation of ryanodine receptors and of the Ca2+ extrusion mechanisms, such as the Na+/Ca2+-exchanger and SERCA pump, had a major impact on the magnitude of supralinear post-CaTs. These data reveal that supralinear Ca2+ summation is a common mechanism of Ca2+ signaling at CP-AMPAR-containing synapses. Shaped in a location-specific manner through modulation of ryanodine receptors and Ca2+ extrusion mechanisms, CP-AMPAR/CICR signaling is suitable for synapse-specific bidirectional modification of incoming inputs in the absence of active dendritic conductances.In fast-spiking (FS), parvalbumin-expressing interneurons of the CA1 hippocampus, activation of the GluA2-lacking Ca In cortical networks, GABAergic inhibitory interneurons play a crucial role in the development and maturation of neural circuits, regulation of synaptic plasticity and rhythmogenesis Cossart, . These c2+ -permeable alpha-amino-3-hydroxi-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (CP-AMPARs) provide fast postsynaptic Ca2+ influx that is associated with the induction of distinct forms of synaptic plasticity in interneurons  have been involved in the formation of highly localized dendritic Ca2+ microdomains during basal synaptic activity  under basal levels of synaptic activity in interneurons at rest  via ryanodine receptors (RyRs) and generation of supralinear Ca2+ signals in dendritic microdomains  FS cells, including basket and bistratified cells, switch from LTP to LTD following transition from small to supralinear Ca2+ signals via CP-AMPARs in basal dendrites. Whether the apical dendrites of these cells may demonstrate supralinear Ca2+ signals triggered upon CP-AMPAR-dependent CICR remains unknown. To understand the input-specific organization of dendritic Ca2+ signaling in FS interneurons, here we compared the summation of postsynaptic Ca2+ transients (post-CaTs) between apical and basal dendrites, and further explored the common mechanisms that may shape the supralinear post-CaTs in dendritic compartments of FS cells. Our data indicate that CP-AMPAR-mediated Ca2+ influx is a common mechanism operating in FS interneuron dendrites, regardless of the input-specific innervation patterns. We also predict that the subcellular distribution and functional state of Ca2+ sources and Ca2+ extrusion mechanisms will have a major impact on supralinear Ca2+ dynamics and, accordingly, on the direction of synaptic plasticity in CP-AMPAR-expressing interneurons.Interestingly, the hippocampal CA1 n = 26) were used in this study. This age range is prior to the onset of puberty, and the results obtained from both sexes were combined.All experiments were conducted in accordance with the Canadian Council on Animal Care in Science and with approval of the Laval University Animal Protection Committee. CD1 mice . Transverse hippocampal slices (300 μm thick) were cut with a Vibratome , transferred to heated (35°C) oxygenated recovery solution containing (mM) 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 3 MgSO4, 1 CaCl2, and 10 glucose , and allowed to cool down to room temperature in 30 min. Slices were allowed to recover for at least 1 h before experiments.Animals were anesthetized with isoflurane and decapitated. The brain was rapidly removed into ice-cold, oxygenated “cutting” solution containing (in mM) 220 or 250 sucrose, 2 KCl, 1.25 NaHstratum oriens/alveus were identified with the aid of an infrared-scanning Dodt gradient contrast , 0.5 Oregon green-488-BAPTA-5N-hexapotassium salt , and 0.2–0.3% biocytin . Current clamp recordings were performed at −65 to −85 mV. Data were low-pass filtered at 2 KHz, digitized at 10 KHz and stored on a computer using a data acquisition board  and Clampex 10.3 software .During experiments, slices were perfused continuously (2.5 ml/min) with artificial cerebro-spinal fluid (ACSF) at 30–33°C. CA1 interneurons of t Figure  or an instratum radiatum (RAD) or oriens/alveus (O/A) was achieved using a bipolar stimulating electrode made from the borosilicate theta-glass capillaries , filled with ACSF containing 5–10 μM Alexa Fluor 594 and positioned ~10 μm from the dye-filled dendrite. The electrode was connected to a constant current isolation unit . Synaptic responses were evoked by a burst of 3 stimuli .Local synaptic stimulation in the After obtaining the whole-cell configuration, 20–30 min were allowed for intracellular diffusion of the fluorescent dye. Imaging was performed using a two-photon laser scanning microscope  with a mode-locked Ti:Sapphire laser  operated at 800 nm wavelength, 80 MHz pulse repeat, 140 fs pulse width. A long-range water-immersion objective  was used. Fluorescence was detected through external photomultiplier tubes, and images were acquired using the Leica LAS software . Fluorescence signals were collected by scanning a line along the dendrite of interest . Fluorescence transients were measured at 50–200 μm from the soma. The image focus of the line was carefully checked and occasionally adjusted for possible drift.2+-transients and associated voltage responses were examined in presence of GABAA and GABAB receptor antagonists gabazine (1 μM) and CGP55845 (2 μM), respectively. In some experiments, the NMDA receptor antagonist DL-AP5 (100 μM), the SERCA antagonist CPA (30 μM) and the L-type VGCC blocker nifedipine (10 μM), or nimodipine (10 μM) were added to the extracellular solution. All compounds were prepared as stock solutions, frozen at −20°C, and diluted on the day of experiment.Postsynaptic Ca2+ measurements and electrophysiological recordings were analyzed using the Leica LAS software, Clampfit 10.3 and Igor Pro . Fluorescence changes were quantified as increases in green OGB-5N fluorescence from the baseline normalized to the red Alexa-594 fluorescence: ΔG/R = (G − G0)/R or normalized to the baseline green fluorescence: ΔF/F = (F − F0)/F0. Data from dendritic branches was binned and analyzed into segments of 5 μm. Original raw traces from fluorescence experiments were filtered with a Gaussian filter and transformed to changes in Ca2+ concentration using the expression , KD is the affinity of the Ca2+ indicator (25.8 μM for the OGB-5N dye), fmax is the maximum fluorescence, Rf = fmax/ fmin is the dynamic range of the dye :The resting CaR and G0 are the baseline levels of red and green fluorescence, respectively. These values were determined experimentally. Decay kinetics of Ca2+ transients were fitted using single exponential fitting algorithms of Igor Pro.where Rm, prox = 8 kΩ cm2 to distal Rm, dist = 70 kΩ cm2. The membrane capacitance Cm = 0.93 μF cm−2 and axial resistance Ri = 172 Ωcm were uniform throughout the cell. The reversal potential of the leak current was set to −65 mV. Model neuron's dendrites also contained uniformly distributed delayed-rectifier type K+ channels with a maximal conductance of 250 S/m2. Soma contained transient Na+, delayed-rectifier type K+ and leak channels with maximal conductances To simulate the electrical activity of a PV+ FS cell, we used a detailed compartmental model based on the morphological reconstruction of PV+ cells  equationj = Ca, Na, K, NAMPA is the number of open AMPA receptors, pCa/pNa = 1.59 . Considering that both the full current through CP-AMPARs and its Ca2+-carried fraction are negligible for voltages higher than 0 mV and are quasilinear functions of V for voltages lower than 0 mV, we reduced the model of the CP-AMPAR current to a Hodgkin-Huxley formalism:where where ε is a proportionality coefficient calculated from the GHK theory: ε = 0.077 (7.7%) and consistent with experimental findings of 4–12% . To convert synaptic current into Cagsyn is the peak synaptic conductance, p is the fraction of CP-AMPAR current carried by Ca2+ ions, L is the effective length of the dendritic domain in which Ca2+ concentration change takes place, r is the dendritic radius. Such a strong dependence of CP-AMPAR mediated Ca2+ signals on dendritic membrane potential was observed experimentally in cortical FS cells  to compute the Ca2+, x is the coordinate along the dendrite ; the fourth term represents exponential Ca2+ decay and accounts for the actions of the Na+- Ca2+ exchangers and Ca2+ membrane pumps; the last term represents synaptic input. The second term Bm is the flux due to Ca2+ binding to the dye (mobile Ca2+ buffer):where 2+-bound dye concentration [CaMB] obeyswhere the CaMB]tot = 500 μM, diffusion coefficient of the dye 2+-bound dye concentration and the Ca2+ concentration holds and, thus, fluorescence measurements with OGB-5N do not significantly overestimate the spatial extent of the evoked Ca2+ events.The OGB-5N properties are as follows: total dye concentration  and [caff] denote ryanodine and caffeine concentrations, respectively. Values of α2+ flux to the endoplasmic reticulum (ER) created by SERCA pumps is modeled with a second-order Hill function:An inward CaVs = 50 nM ms−1, Ks = 900 nM. The resulting equation for the cytosolic Ca2+ concentration c readsInitial guesses for pumping parameters were set to Isyn represents Ca2+ influx through synaptic receptors, the linear relaxation term approximates fluxes through membrane Ca2+ pumps, Na+-Ca2+ exchangers and binding reactions with the indicator dye (hence τc is the effective intracellular Ca2+ decay time), ce is Ca2+ concentration in the ER. In some simulations, the effect of Na+-Ca2+ exchangers was modeled separately with a more detailed voltage-dependent expression  of biocytin-filled interneurons were acquired using a Leica SP5 imaging system equipped with a 488 nm Argon laser. The morphological identification of recorded interneurons was achieved by the analysis of their axonal arborization.Slices with recorded cells filled with biocytin were fixed overnight with 4% paraformaldehyde at 4°C. To reveal biocytin, slices were permeabilized with 0.5% Triton X-100 and incubated with streptavidin-conjugated Alexa Fluor 488  overnight at 4°C. Sections were mounted in Dako fluorescence medium  and confocal n represents the number of cells studied . As the effect of pharmacological agents could differ between neighboring dendritic segments, the total number of analyzed dendritic segments per cell is indicated for pharmacological experiments in addition to the number of cells recorded. Statistical significance was evaluated with Student's paired or unpaired t-test for data that presented a normal distribution, and with the Mann–Whitney test (unpaired) or Wilcoxon signed-rank test (paired) for other data. Normality was tested using the Shapiro-Wilk test .All data are presented as means ± SEM. The 2+ dynamics along the dendritic tree of hippocampal CA1 FS interneurons, we performed whole-cell current clamp recordings in combination with two-photon dendritic Ca2+-imaging  in dendritic sub-regions receiving anatomically and functionally distinct excitatory inputs  vs. those within str. oriens/alveus . Dendritic excitatory synapses were activated locally using bipolar stimulation through a theta-glass microelectrode positioned near the dendrite of interest  evoked by minimal electrical stimulation in apical and basal dendrites had similar amplitude, but the signal's area under the curve (cumulative charge) was higher in apical dendrites, consistent with a slower, longer-lasting CaT than in basal dendrites , and in dendrites having a similar diameter . In line with previous findings in basal dendrites . Supralinear post-CaTs also had similar amplitude in apical and basal dendrites , indicating that dendritic site-specific mechanisms may shape the summation of post-CaTs in individual postsynaptic microdomains.To compare the rules of summation of post-CaTs between apical and basal dendrites, we gradually raised the stimulation intensity up to 200% of minimal stimulation. For both inputs, the recordings were obtained in distal dendrites at similar distance to the soma , consistent with previous ultrastructural data on the highly variable NMDAR distribution in dendrites of PV+ cells  of supralinear post-CaTs, pointing to the role of Ca2+-release in dendritic Ca2+ amplification , consistent with the inhibition of ER uptake  in apical dendrites was also highly variable. In 5 dendritic segments from 2 cells, application of CPA resulted in a significant decrease  to the supralinear post-CaTs in apical vs. basal dendrites , we found that generation of these signals is facilitated in thin dendrites  below which supralinear post-CaTs cannot be generated . For the optimal model parameter values, the system behaved linearly as soon as Ca2+ concentration stayed below the non-linearity threshold. The Ca2+ flux depends quasilinearly on the Ca2+ concentration, thus subthreshold Ca2+ dynamics may be approximated by a simple exponential relaxation process with a different effective Ca2+ decay time constant. The effective time constant found by least-squares fitting to Ca2+ flux vs. concentration dependence appeared to be in the range of 50 ± 15 ms. The linear relaxation approximation allowed analytical estimation of the minimal number of synapses activated to generate a supralinear post-CaT  and membrane Ca2+-pumps  parameters in shaping the time course and magnitude of supralinear Ca2+ elevations. The model predicted that thin dendrites will be particularly prone to generation of supralinear post-CaTs, and that the number of the spatiotemporally clustered synapses required for such event to occur will depend on the CP-AMPAR/NMDAR ratio, with NMDARs decreasing the number of synapses necessary for supralinear Ca2+ integration. These data are in line with theoretical and experimental observations obtained in principal neurons, indicating that the synapse location, clustering, and composition are the leading factors in dendritic input integration  and functional -channels , the mechanism through which the ER triggers Ca2+ influx following the store depletion, may contribute to Ca2+ signaling in neurons. While there is evidence that SOCE is involved in neuronal function (reviewed in Majewski and Kuznicki, 2+ conductance of SOCE-associated channels makes them unlikely contributors to post-CaTs. However, following a massive release of Ca2+, the implication of these channels may become more important. Interestingly, SOCE is linked to L-type VGCC inhibition (Park et al., 2+ non-linearities in our experiments.In addition, it has been suggested that mitochondria may be involved in CICR in certain conditions (Ichas et al., 2+ signaling has been implicated in the pathophysiology of several brain diseases. For example, the expression of CP-AMPARs increases following induction of experimental seizures (Rajasekaran et al., 2+ signaling regulates depression-like behavior in chronic neuropathic pain (Goffer et al., 2+ signaling in dendrites of interneurons in combination with other factors may be involved in abnormal cellular and network computations. From this perspective, the identification of the CP-AMPAR interacting partners, and how these affect neuronal computations, will likely open new therapeutic avenues toward the prevention and treatment of several devastating disorders.More broadly, CP-AMPAR-mediated CaOC, TG, and LT performed experiments. IL performed computational simulations. OC and LT analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AS declared a shared affiliation, with no collaboration, with one of the authors, IL, to the handling editor at time of review.


A growing body of evidence highlights the importance of competent mentoring in academic research. We describe the development, implementation, and evaluation of four regional 2-day intensive workshops to train mid- and senior-level investigators conducting public health, clinical, and basic science research across multiple academic institutions in low- and middle-income countries (LMICs) on tools and techniques of effective mentoring. Sponsored by the Fogarty International Center, workshops included didactic presentations, interactive discussions, and small-group problem-based learning and were conducted in Lima, Peru; Mombasa, Kenya; Bangalore, India; and Johannesburg, South Africa, from 2013 to 2016. Mid- or senior-level faculty from multiple academic institutions within each region applied and were selected. Thirty faculty from 12 South America–based institutions, 29 faculty from eight East Africa–based institutions, 37 faculty from 14 South Asia–based institutions, and 36 faculty from 13 Africa-based institutions participated, with diverse representation across disciplines, gender, and academic rank. Discussions and evaluations revealed important comparisons and contrasts in the practice of mentoring, and specific barriers and facilitators to mentoring within each cultural and regional context. Specific regional issues related to hierarchy, the post-colonial legacy, and diversity arose as challenges to mentoring in different parts of the world. Common barriers included a lack of a culture of mentoring, time constraints, lack of formal training, and a lack of recognition for mentoring. These workshops provided valuable training, were among the first of their kind, were well-attended, rated highly, and provided concepts and a structure for the development and strengthening of formal mentoring programs across LMIC institutions. Despite a burgeoning understanding that faculty can benefit from mentoring training,13 there are only a limited number of formal mentorship training programs14 for academic researchers in low- and middle-income country (LMIC) settings that offer faculty the opportunity to develop skills and incorporate best practices.The importance of effective mentorship in the development, success, and retention of trainees and early career investigators in academic research settings is increasingly recognized.13 inadequate mentoring may be a “modifiable risk factor” in the disparate patterns of academic progress observed for early stage investigators in LMIC settings. Robust mentorship training programs geared specifically toward training mentors in the skills and practices that cultivate effective mentoring relationships with early stage investigators could improve the depth and success of the LMIC global health academic workforce. However, there is a dearth of empirical data on the relative efficacy of mentor training methods. Although graduate and health profession programs often provide some content related to overall teaching skills, most graduates are expected to perform mentoring activities eventually without any formal training in the area. These mentors often perform the role ad hoc or may mimic  mentors they have interacted with in their own careers, with mixed results in terms of effectiveness and mentee-reported satisfaction with the mentoring relationship.17 Experiential and/or practical mentoring approaches may improve mentorship skills, especially in resource-limited settings. However, theory-based programs designed to provide systematic training in mentoring and reinforce standard approaches that can be tailored to individual needs, although limited, have been highly effective.18As there is good evidence that trained mentors, as opposed to untrained mentors, are more effective in their interaction with, and support of their mentees,5 and senior faculty and academic leaders at academic institutions in Latin America, East Africa, South Asia, and Southern Africa. Investigators from multiple academic institutions in regions proximal to the hosting academic institution applied and were accepted to the workshops. This article describes the results of each workshop on the practices and facilitators of, and barriers to, mentoring; action plans by individual participants; evaluations and follow-up actions to establish local mentoring programs in each region; and post-workshop surveys to evaluate the impact of the trainings on local mentorship practices and programs.The purpose of this article is to describe the development, implementation, and evaluation of a mentorship workshop to train mid- and senior-level investigators conducting public health, clinical, and basic science research across multiple academic institutions in LMICs to be more effective mentors. The programs were sponsored by the six-funded Fogarty International Center Global Health Program for Fellows and Scholars and were developed collaboratively between United States–based investigators with expertise in mentoringThe Fogarty International Center at the National Institutes of Health (NIH) supports six United States—based university consortia to provide mentored global health research training opportunities in LMICs through the Global Health Program for Fellows and Scholars. The six consortia are focused at Harvard University, Cambridge, MA; University of California Global Health Institute (UCGHI) based at the University of California, San Francisco (UCSF), the University of California, Los Angeles, the University of California, San Diego, and the University of California, Davis; University of North Carolina, Chapel Hill; University of California, Berkeley; University of Washington, Seattle, WA; and Vanderbilt University, Memphis, TN. The GloCal Health Fellowship Consortium at UCGHI, which supports fellows from all 10 University of California campuses, initiated the “Mentoring the Mentors” training workshops in 2013 to train faculty across all international sites supported by the Global Health Program. All six of the consortia contributed resources and faculty to one or more of the four “Mentoring the Mentors” workshops held in Lima, Peru, in May 2013; Mombasa, Kenya, in June 2013; Bangalore, India, in November 2014; and Johannesburg, South Africa, in March 2016.Each “Mentoring the Mentors” workshop in the four LMICs hosted faculty participants from a number of neighboring academic institutions within the region. Each program was developed collaboratively between local faculty and faculty leadership at the host institution and neighboring institutions, with input from UCSF-based faculty on standard program content. Each training program was designed to cover overarching principles of mentorship through a series of didactic trainings, along with a number of small group sessions and large group discussions focusing on the cultural context of mentoring, along with specific barriers and facilitators of mentoring, within each region.21 For instance, the UCSF Center for AIDS Research had established a strong mentoring program in the years before the development of this initiative which had already launched initiatives to mentor early career investigators in HIV research21 and provide mentoring training programs for HIV researchers across the United States.20 In addition, the UCSF-based Clinical and Translational Science Institute (CTSI) launched a robust Mentor Development Program (MDP) in 2006,19 where faculty receive intensive training over a year in mentorship techniques and are provided with tools to enhance mentee success. This program helped define a set of core competencies for mentors in clinical and translational research.22 An interim evaluation of the MDP program demonstrated a sustained impact on mentoring skills, techniques, and focus.12One impetus of the GloCal Health Fellowship Consortium and faculty based at UCSF to help lead this initiative stemmed from strong mentoring initiatives and “Mentoring the Mentor” training programs already being conducted at UCSF through other NIH-funded initiatives.Each training program was developed iteratively over 6–8 months via conference calls between UCSF-based faculty, faculty based at other Global Health Program consortia in the United States, and faculty leadership at the host and neighboring international institutions. The LMIC leadership included deans or department chairs of the hosting institutions, along with Division/Section Heads and key investigators involved in mentoring initiatives. A call for applicants from institutions in LMICs associated with each consortia was made approximately 3 months before each workshop and applicants submitted a curriculum vitae and a paragraph summarizing their interest in mentoring training. Each regional workshop was restricted to around 30 participants to keep the format intimate and interactive, with preference given to mid-level and senior faculty at institutions from neighboring countries who were currently mentoring junior investigators and were in positions to help develop mentoring capacity at the institutional level.Each 2-day workshop included formal presentations, interactive discussions, and small-group problem-based learning activities. Because each workshop had been developed iteratively over time with intimate participation by local faculty leaders, each workshop was unique in its tailored content, although some core principles of mentoring were covered uniformly. The workshops included skill-based learning in a number of topics including: setting goals and expectations for the mentor–mentee relationship; developing a mentoring philosophy; enhancing communication strategies, and identifying and resolving challenges; time management strategies for mentors and mentees; navigation of work-life balance in a research career; sharing online tools and resources developed by the UCSF CTSI’s Mentoring Program; strategies to enhance institutional commitment to mentoring programs; how to mentor mentees in grant writing and manuscript preparation; mentor and mentee evaluation and bidirectional feedback strategies; barriers and facilitators to mentoring at the global and local level; addressing diversity in the mentor–mentee relationship and challenges for mentees from diverse backgrounds in each setting  in academic institutions; and developing a framework and action plan for launching or strengthening mentoring programs at each represented institution. Each program was launched by a keynote speech, usually from a recognized senior leader from the hosting institution in each region.Because of the importance of peer-to-peer mentoring support, each program ended with a “Mentor Consultation Clinic,” which involved organization of the participants into small groups (five to seven mentors each). Each small group was presented with a strict structure for the “clinic” in which one person presented a mentoring challenge (2–3 minutes), followed by an information-gathering/question period from the group (5 minutes), ultimately leading to recommendations from the group for addressing the challenge (5 minutes). Mentors in each clinic were provided with each other’s contact information and groups were encouraged to continue informal peer-to-peer mentoring relationships after each program had ended.Before each workshop, a pre-meeting survey was sent out to assess the academic rank of each participant; experience in mentoring; and current number and training level of mentees. Additional data provided by the workshops came from the small group activities in each workshop where participants identified barriers to mentoring at the individual, institutional, and global levels and proposed possible solutions. Finally, a Mentor Action Plan (MAP) was completed by each participant at the end of the workshop. The MAP solicited each participant’s self-reported individual strengths and areas of improvements as a mentor, learning points from that workshop’s activities, and action plans for implementing recommendations and techniques covered in the workshop in their individual mentoring activities going forward. Copies of the MAPs were retained for qualitative analysis. Members of the project team who were independent of those who designed and implemented the program conducted thematic analysis of the MAPs as presented in Results. The thematic analysis presented in During the fifth year of the program, GloCal hosted a senior-level technical advisory meeting on global health mentoring at LMIC institutions before the Consortium of Universities for Global Health conference in April 2017. Low- and middle-income countries leaders of the four “Mentoring the Mentors” workshops, along with the principal investigators from each of the six Fogarty International Center Global Health Programs for Fellows and Scholars, along with staff from the NIH FIC, gathered to discuss and plan how the program can continue to best support the development, sustainability, and productivity of strong mentorship programs for global health researchers across LMIC institutions. Before this advisory meeting, a survey was sent out to participants in all four “Mentoring the Mentors” training programs to elicit further feedback on the content of the training programs and assess the impact of the training programs on subsequent mentoring efforts and establishment of mentoring programs at participating LMIC institutions.Thirty faculty from 12 Latin America–based institutions participated in the 2013 workshop held in Lima, Peru; 29 faculty from eight East Africa–based institutions participated in the 2013 workshop held in Mombasa, Kenya; 37 faculty from 14 South Asia–based institutions participated in the 2014 workshop held in Bangalore, India; and 36 faculty from 13 Africa-based institutions participated in the 2016 workshop held in Johannesburg, South Africa. Of the 132 mentors trained in the four workshops from 2013 to 2016, 29% were at the full-professor level at their institutions; 21% were associate professors; 18% were assistant professors; 32% were instructors or in another academic series; and 24% of participants were section heads, department chairs, or held other major leadership positions at their respective institutions. Participants of the four workshops were 55% female and each participant had a median of four primary mentees (range 0–12 mentees).Barriers identified for effective mentoring by various faculty independent of region  were a lA variety of solutions, applicable to different regional contexts to different degrees, solu were proposed to improve the effectiveness of the mentor–mentee relationship. These included the implementation of a formalized structure of the mentoring process as developed by the UCSF CTSI program and taught in each workshop. Faculty participants stated that they planned to establish a regular meeting schedule with their mentees and implement individual development plans (IDPs) to be filled out at least annually by mentees to identify expectations and goals, and monitor progress and troubleshoot impasses. Faculty planned to provide greater clarity in delineating expectations for their mentees, including establishment of meeting agendas and follow-up procedures following regular meetings. Many faculty planned to approach their division chiefs, department chairs, or deans to suggest mentoring training programs such as this workshop at their institutions and to aid in the establishment of a greater culture of mentoring, including funding for mentoring efforts and adding mentoring to the criteria list for academic rank promotion. Participants all cited a need for more formalized mentoring workshops such as those provided by these workshops at their institutions, and formal mentoring programs to both structure and evaluate the progress of mentoring relationships over time.Finally, During the fifth year of the program and after the final workshop described here, GloCal hosted a senior-level technical advisory meeting on global health mentoring focused on LMIC institutions in Virginia in April 2017. With representation from the leadership of all six consortia, the NIH FIC, and LMIC faculty leaders from the four “Mentoring the Mentors” workshops, the success of the workshops in launching local mentoring initiatives were discussed. At many of the participating institutions, deans and department chairs had committed to establishing mentoring programs, although many of them were still nascent. The group discussed the need for ongoing FIC presence in LMICs to encourage the development of local mentoring capacity and structured programs.The 47 respondents to the pre-meeting surveys were from 19 LMICs . They reported medians of 18 years of experience in research and 10 years in mentoring trainees. Nineteen were institutional leaders . Only 16 of the 47 respondents had attended one of the four regional workshops. Of these respondents, 11 had shared their mentorship training experience with colleagues in their institutions, six had been able to set up mentorship training workshops or other training opportunities in mentoring within their institutions, and 11 had had opportunities to meet with other mid-level and senior mentors to discuss mentoring.All surveyed individuals were asked whether their institutions have any mentorship programs; 23 of 40 respondents (57.5%) indicated that their institutions did, with 19 of the 23 programs focused on mentees and four geared toward mentor training. Only 11 of the programs (48%) included any evaluation components. There was general support for, and ideas offered, on the possibility of regional mentor training programs to assist institutions that may not be able to launch them on their own. Factors considered key to successful regional programs included clear support from one’s own institution, especially with regard to sustainability, and funding.The survey ascertained whether there was a hierarchical relationship between mentor and mentee at the respondents’ institutions, and how much they thought a hierarchical relationship prohibits effective mentoring. Both questions offered Likert scales where 1 = not hierarchical or no impact, and 10 = extremely hierarchical and high impact. Median responses to both questions were five, including both presence and impact of hierarchical cultures. The final question asked whether mentor training programs could reduce the institution’s hierarchical mentoring culture. Of 34 responses, 19 (55%) said yes, 14 (42%) said maybe or not sure, and only one (3%) said no, implying some degree of optimism for change.In free-text comments entered about challenges and barriers respondents faced in institutionalizing mentorship training, most cited competing time commitments or simply insufficient time. Some individuals also cited lack of sufficient numbers of trained mentors and of mentorship training programs, and lack of prioritization of mentor training. Additional barriers included siloing or lack of communication/coordination among specialties and research groups, and lack of institutional mentorship cultures or mandates from institutional leaders. By contrast, however, several respondents indicated that their leaders were quite receptive to instituting mentorship training. Additional challenges included lack of funding for mentorship training and lack of follow-up training to sustain mentorship skills.Success stories launched by the NIH FIC mentoring initiatives were shared by 10 workshop attendees, including growth of institutional support and establishment of several new institutional mentorship training programs, and initiation of peer mentorship networks, regular mentor–mentee meetings, and IDPs. One newly established mentorship training model in Latin America was reported as being expanded as a national mandate for research training, nested within a required training program in the responsible conduct of research.This article describes robust regional “Mentoring the Mentors” workshops designed to train mid-level and senior faculty in LMICs on techniques and tools to improve mentoring practices for early career investigators and other trainees. The four workshops were conducted from 2013 until 2016 in Lima, Peru; Mombasa, Kenya; Bangalore, India; and Johannesburg, South Africa, and trained mostly mid-level and senior faculty, including department chairs and deans, actively mentoring trainees in global health research from surrounding countries. Each program was co-led and tailored by local experts and faculty leaders from each host institution and surrounding institutions who facilitated each workshop in collaboration with faculty leaders from the United States—based FIC consortia.23 Moreover, the discussion in the four workshops suggested a series of mentoring tools that can be applied and standardized across institutions.24There were a number of shared barriers to mentoring across all institutions, including a lack of a culture of mentoring, time constraints , lack of formal training and a lack of recognition (in terms of either remuneration or advancement in academic rank) for mentoring. A number of creative and robust solutions to the barriers associated with mentoring at LMICs were proposed by the 132 participants in the four workshops, focusing at both the institutional and individual levels. The most consistent solution proposed to effective mentoring was the establishment of sustainable mentoring training programs such as the ones described in this article, dissemination of the training materials associated with these workshops, and the formalization and recognition of the mentoring process within academic institutions to inculcate a culture of mentoring. In the context of newly developed mentorship programs, formal and well-structured evaluation of such programs will be necessary.A number of contextual barriers to mentoring were raised in the four workshops. Although these contextual barriers suggest a number of solutions that can only be enacted locally with support from institutional leadership, these barriers require global health researchers from high-income countries to be sensitive to the local context, and ensure equitable partnerships based on mutual respect and collegiality. Finally, a post-workshop survey administered to both workshop attendees and faculty leadership at LMIC institutions associated with the FIC training programs offered hope and optimism in growing both a mentoring culture and culturally specific mentoring programs to enhance the success of global health researchers and advance health around the world.


The results on Ca2+ homeostasis were obtained adding exogenous Se in the form of SeO42- to sheep lymphomonocytes cultured in vitro. In particular, Se strongly attenuated 1mM H2O2-induced alteration of intracellular [Ca2+]C as well as the entry of extracellular Ca2+ into the cells with comparable EC50 values for sodium selenate accounting to 1.72 and 2.28 mM, respectively. In an ex vivo trial, it was observed that Ca2+ homeostasis can effectively be rescued in sheep lymphomonocytes exposed in vivo to a Se concentration of approximately 1.9 mM, that was achieved by feeding sheep with olive leaves previously sprayed with 500 mg/plant Na-selenate. Thus the results obtained suggest that the mode of action of selenium markedly influenced Ca2+-related signaling events. Furthermore, results clearly reveal that the protective effect of Se on Ca2+ homeostasis under oxidative challenge can be clearly and effectively achieved through an appropriate dietary regimen obtained also in a circular economy logic using pruning of olive trees treated to reduce tree drought stress.Selenium (Se) is an essential element in human and animal diets, based upon a widespread range of beneficial effects that are primarily due to its antioxidant properties. While Se can be associated to anti-cancer and anti-diabetic activities, reproductive efficiency, and enhancement of the immune system, the mechanistic details of the corresponding biological processes are still largely elusive. To avoid deficiencies and increase bioavailability, Se it is generally supplied to livestock through Se-supplemented feeds or forage plants fertilized with inorganic Se. While the relationship between Ca Selenium (Se) is a trace chemical used as a dietary supplement in humans and animals. Initially regarded as a toxic molecule, it was subsequently shown to play a role in a number of biological processes across a variety of different experimental models , 2. In tIn ruminants Se deficiency has been mainly associated with nutritional muscular dystrophy (“white muscle disease”) (BIB) . It was A number of studies have demonstrated that Se is involved in the physiological response to heat stress , immune 2 and primary amino groups c concentrations [2+]c (nM). In this regard, we employed a “Ca2+ add-back” approach to study the effects of H2O2 on the depletion of intracellular Ca2+ stores as well as Ca2+ entry into the cells. “Ca2+ add-back" implies an experimental procedure conducted in two steps: initially, Ca2+ is not included in the incubation medium; subsequently, CaCl2 is added to challenge the cellular system in order to obtain information about Ca2+ entry in cells c in sheep lymphomonocytes subject to oxidative stress by hydrogen peroxide.Levels of cytosolic calcium [Ca2+]c levels in sheep lymphomonocytes were in the 4–5 nM range however, in the presence of hydrogen peroxide (H2O2), we observed a rapid(100s) spike in [Ca2+]c followed by a quick return to baseline values. Sharp rises in calcium levels were also found when the tissue culture medium was supplemented with increasing doses of H2O2, as shown in Basal c was found to correlate with the amount of H2O2 used to challenge the cells  of SeO42-. Under these conditions, [Ca2+]c was not subject to changes.Sheep lymphomonocytes labelled with FURA-2 were washed, transferred to Ca42-, were subsequently challenged with 1.0 mM H2O2, the impact of oxidative stress appeared markedly reduced in relation to [Ca2+]c and, more emphatically, Ca2+ entry , clearly displayed levels of cytosolic calcium that increased with increasing doses of H2O2. However, this trend was undoubtedly counteracted by 1.6 or 3.3 μM SeO42- dispensed in the tissue culture medium. On the other hand, under the same experimental conditions, C3 lymphomonocytes previously exposed in vivo to approximately 1.9 μM Se  can cause irreparable damage to cellular components and eventually disrupt the integrity of the cell membrane. As an oxidant, hydrogen peroxide can induce both apoptosis and necrosis of cells as a consequence of aberrant Caar space .2+ homeostasis could be rescued by Se, which was either added to the lymphomonocytes as an exogenous reagent, SeO42-, or appropriately included in the animals’ diet in order to physiologically impact lymphomonocytes.In this work, we employed hydrogen peroxide to experimentally induce oxidative stress in sheep lymphomonocytes and, consequently, assess whether marked alterations of Ca2+- stores depletion in relation to Ca2+ entry from the outer space, we employed a "Ca2+ add-back" protocol based on the supplementation of 1mM CaCl2 in the tissue culture medium in order to enhance the detection of mechanisms related to an inflow of exogenous ion or, alternatively, a store-operated release of intracellular Ca2+ [To better discriminate the implication of Calar Ca2+ .2O2 elicit a dose-dependent increase of cytosolic Ca2+ under Ca2+ free conditions as well as a result of CaCl2 supplementation in the incubation medium, thereby suggesting that H2O2 can effect both Ca2+ mobilization from intracellular stores as well as its entry through the cell membrane. These observations essentially recapitulate what others had reported in different cell types [2O2in vitro with comparable EC50 values , exhibited identical patterns of altered, H2O2-induced, Ca2+ homeostasis, thereby suggesting that any protecting activity shown by Se-treated leaves did not imply any contribution from other anti-oxidants contained in food such as, for example, natural phenols and polyphenols.The results indicate that, in sheep lymphomonocytes, Hll types , 53. How0 values , suggest2+ homeostasis prompted oxidative stress.Thus, these observations point to a protecting impact of Se against deregulation of CaConsidering that treatment with Na-selenate is used to reduce the stress caused by drought in the olive tree , 41, in 2+ homeostasis, thus elucidating, mechanistically, its beneficial role as an anti-oxidant. Thus, given the vital role of Ca2+ homeostasis in a plethora of physiological events [In conclusion, we show that the dietary intake of Se can largely offset the effects of oxidative stress on Cal events , 2, and l events  and earll events , this pa


Within the cellular signaling network, the accurate decoding of diverse Ca2+ signal is a fundamental molecular event. Calcium-dependent protein kinases (CDPKs), identified commonly in plants, are a kind of vital regulatory protein deciphering calcium signals triggered by various developmental and environmental stimuli. This review chiefly introduces Ca2+ distribution in plant cells, the classification of Arabidopsis thaliana CDPKs (AtCDPKs), the identification of the Ca2+-AtCDPK signal transduction mechanism and AtCDPKs’ functions involved in plant growth regulation and abiotic stress responses. The review presents a comprehensive overview of AtCDPKs and may contribute to the research of CDPKs in other plants.As a ubiquitous secondary messenger in plant signaling systems, calcium ions (Ca Among them, Ca2+ is the most important secondary messenger . Co. CoA. th2+-dependent manner and prompts kinase activity . Th. ThN-terEF lobe) . The covEF lobe) .2+, a Ca2+ ion loads into the EF-lobes and triggers the conformational shift of CaM-LD. Meanwhile, the EF-lobes adhere to the junction region to release the auto-inhibitor. The kinase domain is then exposed and phosphorylated by an uncharacterized cellular kinase  composed of 12 amino acids confers the Ca2+-binding activity. The negatively charged amino acids in the loop are in charge of the binding . In . In 2+-b loading . Notably loading .2+ signals are sensed by CDPK and various sensors, the signals are decoded into global signaling events and induce the numerous corresponding responses in plants via diverse targets and pathways involved. The first plant CDPK was reported in Pisum sativum in 1984 ,622+, H+cpk17cpk34 displayed a normal morphology, its PT growth rate and pollen transmission efficiency was seriously impaired compared to wild-type plants . U. U62]. U2+ imaging assay indicated that CPK32 over-expression disrupted the Ca2+ homeostasis; cyclic nucleotide-gated channels (CNGCs), a kind of voltage-gated ion channel, were supposed to be involved in this process. CNGC18 was later identified as an interactive partner via yeast two-hybrid assay. CPK32 can activate CNGC18-mediated Ca2+ in Xanopus Oocytes. Over-expression of CNGC18 produces a similar phenotype to that triggered by CPK32 over-expression. A synergistic effect in the depolarization of PT growth was also observed when the two proteins were co-expressed. CPK32-CNGC18 may regulate the PT growth by increasing Ca2+ accumulation [The most recent reported pathway in PT is the CPK32-cyclic nucleotide-gated channel (CNGC) 18 pathway A 55]. Z. Z55]. ZArabidopsis and other plants, and the most important component is FLOWERING LOCUS T (FT). FT is a 20 kD protein acting as a mobile floral molecule which is also termed as florigen  pyrimidin-4-amine or 3MBiP) with the engineered mutant CPK10 (M141G) circumvents embryo lethality and enables conditional analyses of cpk10cpk30cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. CPK10, CPK30 and CPK32 were then found to play a central role in controlling of primary transcription by the RNA sequencing. In an in vivo assay, it was possible to phosphorylate conserved NLP, a key transcription factor of primary nitrate responses with Ca2+ dependence . A. AArabidn plants . When 0.pendence . More bi2+ signals have been reported to promote stomatal closure through the inhibition of inward K+ channels and the activation of anion channels. The first pioneering progress in K+ transport is uncovered in Vicia faba guard cells by Luan et al. . HS. HSAtCDPArabidopsis [CPK4 and CPK11 showed greatly enhanced ABA-sensitivity, whereas only negligible tolerance to salt stress was observed. Two ABA responsive transcription factors, ABF1 and ABF4, are phosphorylated by CPK4 and CPK11 in vitro, suggesting that the two kinases regulate Ca2+-dependent ABA signaling through these transcription factors [CPK12 is ubiquitously expressed in Arabidopsis and localizes to both cytosol and nucleus [CPK12 RNAi lines exhibit ABA hypersensitivity in seed germination and post-germination growth [CPK4, CPK11 and CPK12 are located in the same branch in subgroup I . CPK4 anbidopsis . Single  factors . CPK12 i nucleus . CPK12 Rn growth ,85. Furtn growth . Interesn growth .CPK3 and CPK6 showed that the ABA-induced current of the slow-type anion channel was significantly inhibited  manner /CPK23 (weakly dependent), SLAC1 and ABI1, was explored [CPK21 and CPK23 were found to act as negative regulators in enotypes ,89. Bioc) manner . As openexplored .CPK32 is an ABA signaling component that positively modulates the ABA-responsive gene expression via ABF4  physically interacts with CPK33. In an in vitro kinase assay, THI1 repressed CPK33 auto-phosphorylation activity, which is consistent with the phenotypes of the THI1 over-expressing lines. THI1 might negatively regulate CPK33 activity in ABA-induced guard cell signaling [CPK33 plays a negative role in stomatal closure and slow anion currents . Under dignaling .CPK8 in abiotic stresses [2O2 to water and oxygen was obtained by yeast two-hybrid assay. CPK8 is verified to be able to interact and phosphorylate CAT3 via a series of biochemical methods. Both cpk8 and cat3 plants were more sensitive to drought stress than wild-type plants, and ABA and Ca2+ inhibition of inward K+ currents were diminished in guard cells of those plants. CPK8 over-expressing lines showed enhanced tolerance to drought stress. It is suggested that CPK8 functions in ABA-mediated stomatal movement in response to drought stress through the regulation of CAT3 [To date, there has been only one report about the role of stresses . Using t of CAT3 .2+-CDPK signaling network participates in numerous activities in the plants. Diverse Ca2+ signatures are generated, then recognized and decoded by specific Ca2+sensors, and huge physiological and biochemical responses will be triggered in plants. Although much progress has been made in this field, how complex Ca2+ signatures in plant cells are generated to diverse stimuli at the very beginning is still largely unknown. Post-transcriptional modifications of CDPKs in plant cells, including autophosphorylation, myristoylation and palmitoylation, are greatly involved in their subcellular localization and their function. The relationship and the detailed mechanism between these modifications and the localization remain to be further elucidated.As CDPKs are evolutionarily conserved from algae to land plants, the Ca2+-dependence, based upon the contained EF-hands. Although most CDPKs are conserved in amino acid sequences, their EF-hands differ to varying degrees. Some EF-motifs are degenerated or lost, resulting in changes in Ca2+ dependence or sensitivities. The Ca2+ perception capability of CDPKs might also be influenced by the specific cellular circumstances and the surrounding regulators, as different Ca2+ signatures might involve many specific cellular factors. The interactive protein effectors and other surrounding regulators may also participate in Ca2+ perception or Ca2+ loading of CDPKs.CDPKs have the feature of Ca2+ perception or signaling transduction in a wider range.Despite the fact that tremendous progress in CDPK functional characterization has been made in the past decades by genetic, biochemical and physiological methods, only limited downstream targets were identified, including ion channels, biochemical enzymes and transcriptional factors involved in hormone or abiotic signaling. This might be attributed to the redundancies in CDPK genes hampering the phenotyping, as well as the interaction nature, which is transient, instantaneous and dynamic. Further research is likely to extend the identification and functional characterization of some interactive regulators and discover new phosphorylated substrates involved in the Ca