Ca2+ signals can either be spatially restricted  or propagate throughout the cell . Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of SOC. In this paper we discuss these issues within the framework of simple models of Ca2+ signal propagation. We also analyze the size distribution of local signals (“puffs”) observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca2+ concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca2+-clearing from the cytosol is key to determine whether IP3-mediated Ca2+ signals can display a SOC-like behavior or not.Many natural phenomena display “self-organized criticality” (SOC), (Bak et al.,  Whether the signal remains local or propagates throughout the cell depends on Ca2+ itself, since IP3R's become open upon Ca2+ and IP3R binding. Ca2+, in turn, plays a dual role, inducing channel opening at moderate concentrations and channel closing at higher ones  to reduce Ca2+ concentration. In any case, the local elevation of [Ca2+] that originates at an open channel eventually spreads out in space inducing new channel openings if it finds IP3-bound IP3R's on its way. This Calcium-Induced-Calcium-Release (CICR) is basic for Ca2+ signal propagation.Ca3R-clusters are not evenly distributed inside cells introduce a new factor that also modulates the ability of a signal to propagate or not: the mean inter-cluster distance, d. In Keizer et al. (2+) and the time it takes for Ca2+ to diffuse over the distance d. The existence of an inter-cluster separation can also lead to propagation failure even if some clusters are able to release Ca2+. In Pearson and Ponce-Dawson  IP3R clusters. In systems where the dynamics involves some type of propagation that could evnetually fail, as in the case of Ca2+ signals, the relevant concept is that of directed percolation. In particular, it has been observed that the stochastic fdf model of  to an inactive (non-percolating) state . The exponent, α, depends on the quantity whose dependence with p is being analyzed. The values that these exponents take on depend on the type of transition that takes place at p = po independently of the specificities of the system under study . The inhomogenous spatial distribution of IP3R's also shapes the kinetics of local signals such as puffs  to a situation for which very few IP3R's of the cluster opened (non-percolating states). To that end we introduced a very simple model with which we were able to reproduce the experimentally observed puff size distribution reported in Smith and Parker . This means a very strong inter-channel coupling due to CICR inside the cluster, but not strong enough so that all IP3R's with IP3-bound would become open during each puff. Actually, the experimental data seemed to be near the transition between the percolating (IP3-dominated) and non-percolating (for which Ca2+ is limiting) states. In this paper we investigate this aspect further by analyzing both new experimental data and a simple model that describes inter-cluster coupling. Again in this case the experiments seem to be close to a transition point.The fact that IPr et al. ; Pearsonr et al. ; Ponce Dr et al. , we intre-Dawson , we anale-Dawson . These se-Dawson . In factd Parker . In part2+ signals that propagate over different regions and eventually fail to advance. In fact, noise-induced spiral Ca2+ waves in astrocytes , caged InsP3 -(1-(2-Nitrophenyl)ethyl) Ester) and the exogenous calcium buffer EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Final intracellular concentrations of Fluo-4 and InsP3 were 36 μM and 9 μM respectively, assuming 1 μl cytosolic volume. The final concentration of EGTA was varied from 0 μl to 90 μM. Recordings were made at room temperature. All recordings were obtained with the scan line focused at the depth of the pigment granules in the animal hemisphere of the oocyte. Fluo-4 and InsP3 were from Molecular Probes Inc.; EGTA was from Sigma Aldrich.Experiments were performed on immature oocytes from Confocal linescan imaging was performed using a spectral confocal scanning microscope Olympus FluoView1000 that has a spectral scan unit connected to an inverted microscope IX81. Flash photolysis of the caged compound was made with a mercury lamp that comes with the microscope using the modification introduced in Barella et al., A, of a puff, the mean basal fluorescence at each spatial point, F0, was computed before delivery of the UV flash. The region occupied by a puff was identified manually. The puff amplitude, A, was then defined as the maximum value of ΔF/F0 (F − F0/F0) in the region, with F the fluorescence value at each point. We show in Figure F/F0 that goes from colder to warmer colors with increasing fluorescence. In this example two puffs appear at the same release site but at different times, the first one being more intense than the second one. In order to evaluate the area occupied by a puff in the spatio-temporal record provided by the image, a threshold criterion is applied. The mean fluorescence F and its standard deviation σ are calculated for the whole (ΔF/F0) image. Then all pixels with ΔF/F0 lower than 2Fm+10 σ are set equal to 0 and all pixels with fluorescence above that value are set equal to 1. After appliying the threshold, a binary image is obtained for wich the area of the “1 pixels” is calculated. We show in Figure All images were analyzed using routines written in MATLAB. Images were smoothed out with a filter prior to this analysis. To determine the amplitude, In order to verify that photobleaching was not affecting our data we grouped together puffs that occurred within the same 1 s-long interval along the experimental record and computed the mean and standard deviation of their amplitude. We show the results in Figure 2) defined by 1 − Rss/Tss where Rss is the residual sum of squares and Tss is the total sum of squares.Some of the data analysed were fitted with different distributions. To evaluate the goodness of those fits, we used the coefficient of determination . The dynamical phase starts by picking an available channel and assuming it is open. We then open all other available IP3R's within a distance rinf = 0.250 μm of the open IP3R. We iterate the scheme for every new open channel until there are no available IP3R's within rinf of an open channel that is still closed. We characterize the size of the event by the number of IP3R's that became open. Repeating the simulation of our local model for many realizations we produce puff size histograms as shown in other sections. This modeled “size” distribution can be compared with the puff amplitude distribution obtained in our experiments provided that all open IP3R contribute equally to the [Ca2+] at the cluster. Care must be taken in doing this since this approximation does not always hold  and inter-cluster distance (~2 μm). Under this assumption, taking into account that inter-channel coupling is due to the Ca2+ ions that flow through open IP3R's and that Ca2+ diffuses with an effective coefficient between neighboring cluster sites we assume that the contribution of a cluster with Noi open channels located at position ri to the Ca2+ concentration at another position, rj, is proportional to Noi/|ri − rj|. Then, since at the intra-cluster level we assume that all available IP3R's within a distance rinf of one open channel become open, at the level of how clusters activate one another we assume that a cluster with No open channels induces the “opening” of all other clusters with available channels that are within a distance rinf No of it. We also assume that, when a cluster is “open” (or active), all its available channels are open . In this scheme we do not add up the Ca2+ concentration coming from several open clusters to decide the opening of a new cluster. This simplification is valid as long as the influence of the closest open cluster is more important than that of the more distant ones. The simulations that we show in this paper were done considering a circular total area of radius R = 5 μm and rinf = 0.250 μm as before. For each realization we selected the number of clusters from a Poisson distribution with mean 25 to guarantee a mean inter-cluster distance of ~2 μm and then determined the number of available channels for each cluster from a Poisson distribution with mean 8. In this way, the fraction of clusters with available channels is similar to the fraction of available to total channels in the case of the intra-cluster simulations.In order to obtain the distribution of event sizes that involve the opening of IP2+  wave  is considered where a certain number of clusters was randomly distributed with uniform probability over the area. The number of clusters is drawn from a Poisson distribution with mean 400 in order to guarantee a mean inter-cluster distance of ~2 μm and the number of IP3-bound IP3R's per cluster is drawn from a Poisson distribution of mean 8, as in the previous model. These numbers do not change during the simulation. In order to include signal coupling, we do a time-dependent simulation in which signals are assumed to occur instantaneously and the clusters that participate of it are determined as in the previous intercluster model with some minor modifications. In between signals, the number of active IP3-bound IP3R's and the Ca2+ concentration change with time. Thus, there is an implicit time-scale separation with a fast dynamics during a burst of activity and a slower recovery between such bursts. On the other hand, in this new model we distinguish between active IP3-bound and inactive IP3-bound IP3R's. In particular, we assume that only active IP3-bound IP3R's can become open during a signal and that all those that participate of an event become inactive immediately after it. Each inactive IP3R remains inactive for a certain (random) time. Following  amount immediately after the occurrence of a signal per each IP3R that opened during the event. [Ca2+] subsequently decays exponentially with time towards [Cabasal] with rate 200/s. We add a spatially uniform amount to [Ca2+] for each open channel under the assumption that Ca2+ diffusion occurs fast enough so that [Ca2+] becomes approximately uniform in the vicinity of the clusters on a timescale that is much shorter than the mean separation time between events  where β is a unit conversion factor and r is the distance to the open channel. Given the values of I and D considered, it is Δ[Ca2+] ≈ 0.414 μ M μ m/r. Then, we assume that the total amount of ΔCa2+ contained in a cylinder of height h and radius R which encloses the ER's area of the simulation (π R2) can be approximated by 3R is open and gets uniformly distributed over the same volume immediately after the channel becomes closed. In this way, we estimate the fixed spatially uniform contribution to [Ca2+] of each open channel immediately after it becomes closed as 2 π h R 0.414 μ M μ m/(hπ R2) = 0.828 μ M μ m/R = 0.0414 μ M. Therefore, after a signal with No open channels we add No0.0414 μ M to [Ca2+]. As mentioned before, this concentration subsequently decays towards its basal value with a rate 200/s that has been estimated as D/1 μ m2 given that, for each open channel, Δ[Ca2+] is close to its basal value at a distance of the order of 1 μm. In this new model the initiation of a signal not only depends on the number of active IP3-bound IP3R's but also on [Ca2+]. In particular, we assume that the probability per unit time that a cluster with Nactive IP3R's become open starting a new signal is 0.225s−1*Nactive*[Ca2+]/Cabasal. In this way, this probability takes on the value estimated in  background Ca2+ at the time of the signal and rinf = 0.250 μm.The previous model of the intercluster dynamics treats every signal as independent of one another. There is evidence, however, that several local signals usually precede a Ca2+ accumulation described in the “Materials and Methods” section to generate distributions of Ca2+ signal sizes. As explained in that section, one of these models is used for local signals (puffs) that involve the opening of IP3R's within a cluster of ~400 nm sides. The other one models signals that can propagate between clusters that are ~2 μm apart from one another. Both models include a simplified description of the two main dynamical processes that shape the signals: IP3 and Ca2+ binding, treating each signal as independent of one another. In another section we describe the results obtained when Ca2+-mediated signal coupling is included in the intercluster model. Signal types strongly depend on IP3 and Ca2+ since IP3R's need to bind these two species to become open. In the experiments with which we compare the models the cells are loaded with caged IP3 that is eventually released by means of a UV flash. In this way, the experimentalist has control over the IP3 distribution. Therefore, the models assume that [IP3] is fixed. The dynamics of Ca2+, on the other hand, is entirely controlled by the cells. Thus, its concentration is very low at the beginning of the experiment and only after a latency a Ca2+ ion encounters an IP3-bound IP3R inducing its opening. Once that happens, the released Ca2+ diffuses away from the open channel and can induce the opening of nearby IP3R's with IP3 bound. The change in [Ca2+] due to one open channel is noticeable only in its vicinity. Therefore, the Ca2+-mediated inter-channel coupling depends on the distance. This feature is taken into account in both models in a simplified way. In the local model, we assume that each channel opens a nearby IP3-bound IP3R if it is within a fixed distance, rinf. In the model of global signals we assume that each cluster that is releasing Ca2+ through No open IP3R's can induce Ca2+ release from a nearby cluster if the latter is within a distance Norinf of the former.In this section we show the results of using the intra and intercluster Models without Ca3 dominated situation in which all the channels of the cluster that have IP3 bound when the event starts participate of the signal. This occurs when IP3-bound IP3R's are densely packed in the cluster, in which case the size distribution is Poisson. The other limiting case corresponds to events in which IP3-bound IP3R's are so far away from one another that the opening of one them is not likely to induce the opening of any other available IP3R in the cluster. In this case Ca2+-binding is the limiting factor for the propagation of the signal. Although there is not a clear phase transition, in Solovey and Ponce Dawson  channels of a cluster become open upon activation of the cluster, the mean numbers of IP3-bound IP3R's and of open channels during a release event, 〈No〉, in a cluster coincide. Having 〈No〉=8 gives 〈No〉 rinf =2 μ m for rinf = 0.250 μ m as in the intracluster model. For each open cluster, Norinf is to be compared with the distance to all the other clusters to decide on their activation. Therefore, the parameters are such that Norinf is, on average, equal to the mean inter-cluster distance. This is similar to the situation encountered for the intracluster dynamics model when using the parameters that gave the best fits to the experiments of . In fact, we observe that the event size distributions of Figures 2+-binding dominated and an IP3-binding dominated distribution. This is illustrated in Figure 3-bound IP3R's in a cluster. In Figure 3R's participate of each event.We show in Figure d Parker   were injected in the oocytes. The photorelease of the caged compound was then induced with a UV flash and the resulting fluorescence was measured. All images were obtained in the linescan mode which gives information on the fluorescence along a single line inside the oocyte. A variety of events were elicited in the experiments ranging from small puffs to waves. We only analyzed events that were localized in space and time which limits our experimental distributions. Puff sizes were characterized by their amplitude and  area which were computed as described in the “Materials and Methods” section. We present in this section the distributions of these two quantities derived from the experiments.In this section we present the experimental results obtained after analizing puffs from 13 different cells as described in the “Materials and Methods” section. In all the experiments 3 compounds  wave when cells are continuously subjected to IP3 release  and the Ca2+ concentration change with time. Thus, the model has an implicit time-scale separation with a fast dynamics during a burst of activity and a slower recovery between such bursts. IP3R's become inactive after having opened and go back to being active with a timescale of the order of 2 s. [Ca2+], on the other hand, increases after a signal depending on how many channels were open and then decays back to its basal value with a rate of the order of 200s−1. For more details on this new model we refer the reader to the “Materials and Methods” section.In the previous section experimental signal sizes of mainly local signals were characterized by two quantities: their amplitude and their spatio-temporal spread. These quantities not only depend on the number of IP3R's (receptors) that participated of each event. We show the distributions of these two quantities in Figures We show in Figure 3R's changed with the mean inter-cluster distance, dm, the mean time an IP3R remains inhibited, tinh, the rate at which cytosolic Ca2+ decays to its basal value, δCa and with the radius of influence of an open channel, rinf which were dm= 2 μm, tinh = 2.5s, δCa = 200/s and rinf = 0.250 μm in all the simulations of the previous section. We found that in almost all cases the distributions could be fitted by a power law (with exponents between 1 and 1.8) but over a range of event sizes that could be different depending on the parameter values. This is reasonable since the largest event size is strongly dependent on some of these parameters. For example, for large values of the intercluster distance, dm, or of the inhibitory time, tinh and for small values of the mean number of IP3-bound IP3R's per cluster, λ, the largest event size decreased significantly. Varying rinf between 0.01 μm and 0.1 μm did not produce any noticeable changes. While varying the cytosolic Ca2+ clearing rate, δCa, between 50/s and 100/s did not change the power law behavior of the signal size distribution, using smaller values (we probed 20/s ≤δCa≤ 30/s) produced a striking change. Namely, the event size distribution ceased to be long-tailed and looked more like Gaussian. We illustrate the results obtained in Figures 3R's that participate of the signals, No, as a function of a varying parameter when all the others are kept fixed at the values used in the previous section. We can observe in this Figure the trends we have just described with respect to how the mean number of participating (open) IP3R's, No, varies with dm, λ and tinh. We also observe that, when varying any of these 3 parameters the standard deviation of No decreases with decreasing No. We have also plotted No and σNo normalized to the initial number of active IP3R's in the simulation obtaining a similar behavior (data not shown). The behavior encountered for small vallues of δCa is quite different. Namely, the standard deviation is much smaller than for all the other sets of parameter values explored. Furthermore, the deviation, σNo, decreases with increasing 〈No 〉. This is the consequence of the distribution no longer being long-tailed. This can be observed in Figure 3R's that participate of the signals when all the parameters are kept at the values used in the previous section except for the cytosolic Ca2+ clearing rate for which we used δCa = 20/s (A), δCa = 25/s (B), δCa = 30/s (C) and δCa = 200/s (D). There we observe that for δCa large enough the distribution is well approximated by a power law. As δCa decreases, this power law behavior still holds for small enough No but a new “Gaussian-like component” seems to build in the large No region. Eventually, when δCa is small enough, the power law region disappears and we obtain a Gaussian distribution.In order to analyze the robustness of the power law behavior encountered in the previous section we simulated the model for other values of its various parameters. More specifically, we studied how the distributions of the number of event-participating clusters and IPNo and the number of IP3R-bound IP3R's that are active, Nact at any given time. We show a plot of these two numbers as a function of time for δCa = 200/s Figure Ca=20/s in Figure No < Nact for most events, for δCa=20/s all active receptors participate of each event (No = Nact). In this way, the distribution of event sizes coincides with that of active channels which, after a short transient, has a well defined mean value with a relatively small deviation around it. The event size distribution then looks Gaussian.In order to interpret this result, we compared the number of receptors that participate of each event, 2+ signals are ubiquitous across cell types. Among them, those that involve Ca2+ release from the ER through IP3 receptors (which are Ca2+ channels) play a relevant role. Apparently, activatable IP3R's are distributed in clusters on the surface of the ER. This uneven distribution together with the kinetic properties of the IP3R according to which the channel needs to bind Ca2+ and IP3 to become open give rise to a variety of signals that go from very localized ones (puffs) to global waves that propagate throughout the cell. Ca2+-mediated channel coupling due to CICR thus plays a key role to determine the spatial size over which the signal spreads. The sequence of channel openings during a signal is then similar to the processes involved in percolation. In particular, we used these ideas in Solovey and Ponce Dawson (3R's). In Solovey and Ponce Dawson (3-bound IP3R's of the cluster (percolating states) to a situation for which very few IP3R's of the cluster opened (non-percolating states). By fitting the model to the experimental results of Smith and Parker  areas in this cell type, on the other hand, was slightly different and could be fitted by a power law (goodness of fit) or a log-normal distribution (goodnes of fit). It is always difficult to distinguish power law and log-normal distributions . In particular, we determined that the distributions could still be fitted by a power law if we changed any of these parameters around the values used in the original simulations. This robust behavior could lead to the interpretation that our model and thus, the Ca2+ signals it describes, display SOC. Such a conclusion would not be surprising given that the system under study has the main “ingredients” with which a SOC state can be reached  and which is allowed to relax upon activation by decreasing this quantity . In any case, as already mentioned, it is always difficult to establish with certainty that a given distribution follows a power law or not. In the case of the experimental data that we analyzed the log-normal distribution gave a better fit. However, this could be due to the limited amount of data that we have. It is in fact quite difficult to distinguish a power law from a log-normal distribution  and one could argue that this could favor a power law over a log-normal distribution. From the point of view of the mechanisms that could lead to one or the other situation, our modeling results seem to favor the power law distribution as well. However, given that our model is only an approximation to the real problem, we cannot tell for sure that IP3-mediated Ca2+ signals are an example of SOC. We are certain, however, that the signal size distribution has a long tail and this could have physiological implications as we discuss later.In order to investigate the size distribution of signals that involve the opening of IPe Dawson  to inclue Dawson . Assumind Parker . This is2+ clearing time, δCa, was decreased by an order of magnitude we obtained a Gaussian-like distribution around a mean for both the number of IP3R's  failed to give a purely Gaussian distribution giving instead a mixture of a power law for small events and a Gaussian for large ones. Our experiments and those of Marchant and Parker  size distribution and in which ways this can be changed.Although further studies are necessary to verify our findings, the results obtained in this paper show under what circumstances intracellular CaThe 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.


Mentoring is important in the career development of novice and experienced nurses. With the anticipated shortage in nursing, it is important to explore factors such as mentoring that may contribute to career satisfaction and intent to stay in the profession. This study explored the effects of mentoring on career satisfaction and intent to stay in nursing, and the relationship between career satisfaction and intent to stay in nursing. It was conducted through a mailed survey of RNs 55 years or younger currently in practice, education, administration, or research. Career satisfaction was measured through the use of the newly developed Mariani Nursing Career Satisfaction Scale. Findings revealed no statistically significant effect of mentoring on career satisfaction and intent to stay in nursing. There was a statistically significant relationship between career satisfaction and intent to stay in nursing. The majority of nurses reported participating in a mentoring relationship. Although the findings related to mentoring, career satisfaction, and intent to stay were not statistically significant, there was a prevalence of mentoring in nursing, thus suggesting the need for future research to identify outcomes of mentoring. In addition, the study contributed a newly developed instrument to measure the concept of career satisfaction in nursing. Mentoring is important in the career development of both novice and experienced nurses in the areas of clinical practice, nursing education, administration, and research, as it supports the novice's need to feel satisfaction and success as a professional nurse and offers the experienced nurse an opportunity to contribute to the profession. This study explored the effect of mentoring on career satisfaction and intent to stay in the nursing profession, two critical elements in the retention of nurses in the profession.Despite an encouraging recent 5.7% increase in enrollments in baccalaureate nursing programs (American Association for Colleges of Nursing (AACN)) , it is aSeveral factors contribute to the shortage of nurses in the profession. National data indicate that the average age of nurses nationwide continues to increase. In 2008, the average age of nurses nationwide was 48 (HRSA) , and it The concept of mentoring is not new to nursing, as Florence Nightingale was known to have mentored many nurses in her day . StewartMentoring is a reciprocal, long-term relationship with an emotional commitment that exists between a novice (protégé) and experienced (mentor) nurse; mentoring implies a knowledge or competence gradient, in which the teaching-learning process contributes to a sharing of advice or expertise, role development, and formal and informal support to influence the career of the protégé , 18–23. With the predicted shortage and anticipated need for nurses in healthcare and academia in the future, it is more important than ever to explore career satisfaction, not just job satisfaction, for nurses. Career satisfaction is defined as the contentment that a nurse feels as a nursing professional in terms of intrinsic and extrinsic rewards . Career This study explored the influence of participation in a mentoring relationship on career satisfaction and on intent to stay in nursing, and the relationship between career satisfaction and intent to stay in nursing. Intent to stay in the nursing profession was defined as the nurse's plan in projected number of years to remain active in the nursing profession in practice, education, administration, or research. The following hypotheses were tested in this research study:registered nurses who participated in a mentoring relationship will have greater career satisfaction than those who did not participate in a mentoring relationship;registered nurses who participated in a mentoring relationship will report the intent to stay in the nursing profession longer than registered nurses who did not participate in a mentoring relationship;career satisfaction will be positively related to intent to stay in the nursing profession.For purposes of this study, the mentor was a nurse who was considered experienced, competent, or expert in the novice to expert continuum and more experienced than the novice nurse in terms of knowledge, skill, and competence. The protégé was the novice nurse in the mentoring relationship, in any area of the profession who was new to the area of practice, education, administration, or research, and lacked knowledge, experience, or competence in that setting. The mentor and protégé were self-identified on the demographic questionnaire. Novice to expert: Excellence in clinical practice [The theoretical works of Patricia Benner , 19 on npractice , 19, suppractice –22 TheorMentoring has been discussed in the nursing literature as early as the days of Florence Nightingale, but more recently since 1974 with the initial work of Kramer . The litVance and Olsen  and VancN = 100) were surveyed on their perceptions of mentoring experiences, expectations of mentoring relationships, organizational environment, career satisfaction, and career influence; 79% reported having a mentor at some time throughout their career, with a strong positive correlation  in response to a question regarding the extent to which a mentor influenced their career. The nurses who reported that mentors had a strong influence on their career believed that the most significant contributions of a mentor were offering feedback on performance, sharing expertise with the protégé, serving as a role model, and demonstrating a belief in the protégé. The findings revealed that one of the most crucial times for mentoring was early in a nurse's career and that organizational support was necessary.Boyle and James  found thYoder's  concept Stewart and Krueger's  concept This study used a combination of a descriptive comparative and correlational design. The phenomenon of interest in this study was mentoring, the independent variable was participating versus not participating in a mentoring relationship as a protégé or mentor, and the dependent variables were career satisfaction and intent to stay in nursing.The study was conducted through the use of a written survey. Professional nurse databases that were available for scholarly use or purchase from the State Boards of Nursing were used to access names and addresses of RNs. The target population included RNs aged 55 or below, who were currently employed in the practice, education, administration, or research setting. The age of 55 or less was selected because nurses who are over 55 may be considering retirement which could have skewed the results. Since the aging workforce in nursing is partly responsible for the predicted shortage, the aim of this study was to explore the intent of younger nurses to stay in nursing.In addition, the intent was to study nurses who were currently employed in nursing practice, education, administration, or research; therefore, the letter of explanation describing the study included current employment as a criterion for inclusion in the study. The demographic questionnaire also addressed the current employment setting of the study participants and their educational level.A survey was used in this study in an effort to reach a large number of nurses throughout the country; the disadvantage was that the rate of return for a survey or questionnaire is often low . ColoredTo obtain a national random sample throughout the United States, the country was divided into the nine geographic regions see  that wert-test and the Pearson Correlation, using a power of  .80 and a moderate effect size for use of  t-tests and Pearson's correlation. For this study, the total sample was 173, but the group size for those RNs who did not participate in a mentoring relationship was only 37; therefore, the actual power in this study was  .70 [The required sample size of 102 subjects was determined based on a power analysis for both the was  .70 . A demographic questionnaire and a newly developed, pilot-tested research instrument, the Mariani Nursing Career Satisfaction Scale (MNCSS), was used for data collection. The MNCSS developed by this researcher measured career satisfaction. The entire survey took approximately 20 minutes for the subjects to complete. The demographic questionnaire addressed the nurse's age, gender, highest educational level, current employment status, years in practice, education, administration, or research, participation in a mentoring relationship, intent to stay in nursing (in years), whether the participant would choose nursing again and would recommend nursing as a career. Although, there were several valid and reliable instruments, such as the Stamps and Piedmonte Index of Work Satisfaction , to measThe MNCSS is a semantic differential scale with 16-bipolar adjective pairs on which the participants rated their feelings about their nursing career that best represented their attitude toward the adjective. For the MNCSS, each adjective pair was scored on a seven-point scale using the lower number for the less positively worded adjective and the higher number for the most positively worded adjective. An example of some of the 16 adjective pairs is satisfied-dissatisfied, fulfilled-discouraged, stimulated-bored, content-discontent, rewarded-frustrated, meaningful-not meaningful, and successful-unsuccessful. The sum of the point values of responses to all 16 items yielded a total MNCSS score. The total scores could range from 16 to 112 with a midpoint of 64. The overall content validity index (CVI) of the MNCSS was  .84 for the 16-bipolar adjectives that were included as part of the instrument. Cronbach's alpha internal consistency reliability of the instrument was  .82 for the pilot study, and  .94 for the full study. Prior to data collection, IRB approval was obtained. The participants received the demographic questionnaire and the MNCSS with a letter of explanation including the intent of the study and the definitions of key terms. Consent was implied by the participant's return of the completed questionnaires. A stamped, return envelope was included to mail the questionnaire back to the researcher. The participants were given information as to how they could access results of the study, if desired.Of the total 722 surveys that were mailed and distributed, 196 surveys were returned, and 173 of the surveys were usable for the research study. Of the 23 surveys that were not used 6 were not included because the respondents exceeded the age of 55 years of age, 10 were not currently employed in practice, education, administration, or research, 6 were returned after the final due date, and 1 did not complete the career satisfaction scale. The statistical analyses were determined by the research hypotheses. For the MNCSS, a subject with more than two missing data bits was removed from the total sample. For two subjects who were missing a response to one item on the MNCSS, the missing data were replaced with the group mean . Frequency and percentage statistics were computed for the variables of gender, education level, and area of practice and are presented in N = 173), 78.6% reported participating in a mentoring relationship, 41% of these mentoring relationships were informal; 21.4% of the RNs had not participated in a mentoring relationship. From the total sample, 44.5% had been in the role of both mentor and protégé at some point in their career, not just a mentor or a protégé. The majority of RNs  responded that they would choose nursing as a career again, and 152 (87.9%) of these RNs responded that they would recommend nursing as a career to others  and nurses who did not participate in a mentoring relationship . The mean number of years for intent to stay (n = 167) was 18.51 years (SD = 8.38). For hypothesis 2, there was no statistically significant difference in the number of years nurses intended to stay for those who did or did not participate in a mentoring relationship. Additional details of the statistics for career satisfaction and intent to stay are presented in Tables r = .15) and statistically significant , however it was a weak correlation.For the total sample of 173 RNs, the overall mean for the MNCSS was 89.05 SD = 14.33) with a range of 46 to 112 . For hyp.33 with  Additional statistical analyses were computed using a one-way ANOVA to compare types of mentoring  on career satisfaction and intent to stay, and a one-way ANOVA compared the types of the roles in a mentoring relationship  on career satisfaction and intent to stay; however, there was no statistical significance found in any of the additional analyses.The differences in RNs' career satisfaction and intent to stay in the profession were not statistically significant for those who participated or did not participate in a mentoring relationship, regardless of whether it was a formal or informal relationship or if they were a mentor or protégé. Although 78.6% of the nurses surveyed indicated some type of participation in a mentoring relationship, the largest percentage of mentoring occurred informally. This suggests that nurses may create informal mentoring relationships whether or not there is an opportunity for a formal mentoring relationship. These findings are consistent with what has been reported for several decades in the mentoring literature in nursing, which points to the prevalence of mentoring relationships in nursing , 36–40. Overall, the RNs in the sample reported generally moderate to high career satisfaction, regardless of whether or not they participated in a mentoring relationship. Although participation in a mentoring relationship did not have a statistically significant influence on career satisfaction, the majority of nurses surveyed participated in these relationships, which suggests that the value of mentoring may lie not in career satisfaction, but in other outcomes that were not measured in this study.Although, there was limited literature on intent to stay in the profession, Krozek  indicater = .15).The findings of this research did not support the research hypotheses 1 and 2. It is possible that nurses who were not satisfied with their career or did not participate in a mentoring relationship or planned to leave their career in nursing may have elected not to participate in the study. In addition, the mean age plus the overall high satisfaction scores of the sample may have influenced the overall results of the study, and the MNCSS may not have been sensitive enough to detect a difference between the groups. The sample of nurses in this study, although consistent with the age reported in the literature at the time of the study, represented nurses who may be more than halfway through their career; therefore, the results of this study may not be representative of the younger population of nurses. For hypothesis 3, the correlation of career satisfaction to intent to stay in nursing was weak  was used to distribute the surveys; however, there was only a 27% overall return rate. While this was not extremely low, the rate of return did not reach the projected 60% that Dillman –44 suggeAnother limitation may have been attributable to the overall population that received the survey. The database lists for RN names and addresses were not as accessible as expected, and since the names were randomly selected from the lists, it was not possible to know in advance if those who received the survey actually met the inclusion criteria for the study of being currently employed in clinical practice, education, administration, or research despite having an active RN license and meeting the age criterion. In addition, the random search for names nationwide was a time intensive and financially expensive process.n = 136) and those who had not participated in a mentoring relationship (n = 37). It was difficult to achieve an adequate sample size for RNs who had not participated in a mentoring relationship. From the 722 surveys that were mailed, there was only a 5.12% rate of return for the group that had not participated in a mentoring relationship. This may have been attributable to the following reasons: RNs who were not in a mentoring relationship did not participate because they thought the survey pertained only to RNs who had been in a mentoring relationship; RNs who had not participated in a mentoring relationship are no longer in nursing; or there is more mentoring occurring in the profession than is typically thought. Although a mentoring relationship at any point in a nurse's career may have significance, the question on the survey pertaining to participation in a mentoring relationship did not ask when the mentoring relationship occurred; therefore, the mentoring relationship may have occurred recently or 20 or more years ago.Another limitation pertained to the unevenness of the survey response rate from those who had participated in a mentoring relationship (In addition, the survey question that asked the study participant about participation in a mentoring relationship was a self-assessment of their participation and role in a mentoring relationship. Although the distinctive definitions of a preceptor, a mentor, and a protégé were included in the letter of explanation, participants who responded positively to participating in an informal mentoring relationship may have been referring to a precepting relationship or role modeling, not necessarily a mentoring relationship in the truest form of the definition. And lastly, RNs who were dissatisfied with nursing as a career may not have been interested in participating in a research survey.There are implications for nursing related to this study's findings. With the predicted shortage of nurses, the implications of this study and the recommendations for future studies may have an effect on nursing practice, education, administration, research, and science. Although the results of this study were not statistically significant related to the outcomes of career satisfaction and intent to stay, 136 of the 173 participants indicated that they had participated in a mentoring relationship either formally or informally as a mentor or protégé at some point in their career. Nurses should continue to encourage mentoring relationships within the profession to foster some of the other positive outcomes of mentoring such as personal satisfaction, self-confidence and empowerment , and pro Although, this study did not yield statistically significant results supporting the outcomes of career satisfaction and intent to stay in the profession with a mentoring relationship, the presence of the phenomenon of mentoring was very apparent. Through this study, future research initiatives can be identified to explore the outcomes of mentoring. In addition, this study contributed to nursing science and research through the development of a new valid and reliable nursing research instrument, the MNCSS, to measure the concept of career satisfaction in nursing.This study demonstrates that mentoring is a prevalent phenomenon in nursing. Mentoring is a concept that is well-supported in the literature; research that continues to explore mentoring relationships in nursing can help demonstrate the positive outcomes of mentoring in nursing.This study explored whether participation in a mentoring relationship had a positive effect on career satisfaction and intent to stay in the nursing profession. Based on recent statistics in the literature, despite increasing nursing school enrollment, it is predicted that the nursing shortage will continue. This study did not demonstrate a statistically significant difference in career satisfaction and intent to stay between nurses who participated in a mentoring relationship and nurses who did not participate in a mentoring relationship. However, there was a statistically significant positive relationship between career satisfaction and intent to stay in the nursing profession.Despite the findings, this study contributes to the body of knowledge for the nursing profession related to mentoring relationships, nurses' career satisfaction, and intent to stay in the nursing profession. Nurses responded favorably about their careers in nursing and the importance of mentoring relationships which is promising for the profession of nursing. Further theory development and research are needed to add to the body of knowledge about the outcomes of mentoring related to career satisfaction and intent to stay in nursing.


Crystal structures suggest that activation of SERCA occurs when the cytoplasmic head-piece, in an open (E1) conformation stabilized by Ca2+, undergoes a large-scale open-to-closed (E1 to E2) transition that is induced by ATP binding. However, spectroscopic measurements in solution suggest that these structural states (E1 and E2) are not tightly coupled to biochemical states (defined by bound ligands); the closed E2 state predominates even in the absence of ATP, in both the presence and absence of Ca2+. How is this loose coupling consistent with the high efficiency of energy transduction in the Ca2+-ATPase? To provide insight into this question, we performed long (500 ns) all-atom MD simulations starting from the open crystal structure, including a lipid bilayer and water. In both the presence and absence of Ca2+, we observed a large-scale open-to-closed conformational transition within 400 ns, supporting the weak coupling between structural and biochemical states. However, upon closer inspection, it is clear that Ca2+ is necessary and sufficient for SERCA to reach the precise geometrical arrangement necessary for activation of ATP hydrolysis. Contrary to suggestions from crystal structures, but in agreement with solution spectroscopy, the presence of ATP is not required for this activating transition. Principal component analysis showed that Ca2+ reshapes the free energy landscape of SERCA to create a path between the open conformation and the activated closed conformation. Thus the malleability of the free energy landscape is essential for SERCA efficiency, ensuring that ATP hydrolysis is tightly coupled to Ca2+ transport. These results demonstrate the importance of real-time dynamics in the formation of catalytically competent conformations of SERCA, with broad implications for understanding enzymatic catalysis in atomic detail.We have performed molecular dynamics (MD) simulations to elucidate, in atomic detail, the mechanism by which the sarcoplasmic reticulum Ca Closely related SERCA isoforms are also responsible for pumping Ca2+ into the endoplasmic reticulum of virtually all non-muscle cells, potentiating a myriad of Ca2+-dependent cellular activation processes. Structurally, SERCA is formed by four functional domains: nucleotide-binding (N), phosphorylation (P), actuator (A) and transmembrane (TM) 2+ ions and one molecule of ATP to spatially distant sites in SERCA. The Ca2+-binding sites are located within the TM domain, while the N domain contains the ATP-binding site. The key step in catalysis is a reorganization of the N, P and A domains that positions the γ phosphate of ATP near Asp351 (P domain), facilitating the formation of the phosphoenzyme intermediate SERCA, a member of the P-type ATPase family, is an integral membrane protein responsible for the active transport of Ca2+-bound SERCA is quite flexible, populating open, partially closed and closed conformations  is arranged in an open conformation, with the ATP-binding and phosphorylation sites separated by more than 2.5 nm rmations Fig. 1, 2+2+-affinity conformation (E2) to a high-Ca2+-affinity one (E1) is not driven by Ca2+, but by the ionization of residues Glu309 and Glu771 of the Ca2+-binding site; yet in the absence of Ca2+, SERCA cannot undergo the conformational transitions required for catalytic activation Furthermore, biochemical characterization of the effect of calcium and ATP in the activation of SERCA showed that ATP is hydrolyzed only in the presence of Ca2+-bound SERCA in the open geometry represent a functional conformation? Does Ca2+-induced activation originate from conformational selection or an induced fit mechanism? Why is calcium binding so indispensable for SERCA activation, especially for ATP hydrolysis and phosphate transfer to SERCA? To address these questions, we have performed all-atom MD simulations of SERCA in the presence and absence of calcium, starting from the E1 state crystal structure of SERCA in the open conformation.Despite the wealth of experimental data, major issues need to be resolved at the atomic level: Does the crystal structure of Ca2+ and Apo), starting from the crystal structure of the open conformation . This crystal structure was chosen for two reasons: (a) because experimental evidence indicates that changes in protonation of SERCA, and not Ca2+ binding, drive the transition from E2 to E1 states 2+ in SERCA activation. As described in 2+ was removed from the Apo system. 2+ and Apo), following a 10-ns equilibration period. Both simulations show substantial rearrangements of the cytosolic headpiece, producing conformations that are substantially different from that observed in the crystal structure of open SERCA  are quite closed  and Lys515 (N domain)  state are 4.5 and 4.9 nm, respectively, in close agreement with interdomain distances measured experimentally at equilibrium by FRET 2+: Ca2+-bound SERCA can sustain the open conformation for 300 ns (2+ or Apo) but that E1 is significantly more stabilized in the presence of Ca2+. 2+ on the conformational transitions of SERCA.To analyze these conformational transitions more quantitatively, we plotted the interdomain C domain) Fig. 2c.2+-bound SERCA, where a distance increase of ∼0.5 nm, producing the same distance as in the Apo case, was observed between 429 and 460 ns , suggesting that discrete closed-to-open transitions of the cytosolic headpiece of Ca2+-bound SERCA can occur on the nanosecond time scale.Upon closure, the interdomain distance remains fairly stable (ΔR≤0.1 nm), except for one brief interval in the trajectory of Ca2+ and Apo), the RMSD of the TM domain is very small, with maximum values of 0.23 nm, indicating a clear lack of large-scale structural changes in the sub-microsecond time scale. In contrast, all three domains of the cytosolic headpiece  show large changes in RMSD relative to the initial structure in both trajectories  are clearly greater for this domain than for others. The RMSD time course for the N domain  for each functional domain of SERCA Fig. 3. 2+ is bound to SERCA, even in the absence of ATP  structural transition is only loosely coupled to the biochemical transitions of ligand binding. The simulated trajectories appear to show that a complete open-to-closed transition is observed whether or not Cae of ATP Fig. 2c.2+ and AMPPCP . The arrangement of the A domain observed in the crystal structure has been proposed to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites While the Ca2+ and ATP), but (b) only in the presence of Ca2+ is SERCA capable of reaching an active conformation via an induced-fit mechanism that positions the N and P domains in a conformation that can facilitate γ-phosphate transfer from ATP to Asp351  the large-scale open-to-closed structural transition in SERCA is only loosely coupled to the biochemical transitions defined by the binding of ligands   Fig. 7b.2+-bound SERCA were reported In a recent paper, MD simulations of Ca2+. Although this large-scale structural transition natively occurs in SERCA and is not tightly coupled to Ca2+ or ATP binding, Ca2+ is necessary and sufficient for SERCA to reach the precise geometrical arrangement necessary for ATP hydrolysis. Ca2+ plays a crucial role in activating SERCA by reshaping its free energy landscape, which creates a path between the open conformation and the active closed conformation. Based on the observed Ca2+-dependent changes in the free energy landscape, we speculate that the allosteric activation of SERCA does not rely on a specific network of interactions between the Ca2+-binding site and the headpiece, but on the malleability of the free energy landscape within SERCA. This thermodynamic tuning effectively connects dynamics with function in SERCA, thus ensuring that ATP hydrolysis is tightly coupled to Ca2+ transport.MD simulations revealed a large-scale open-to-closed conformational transition of full-length SERCA in the presence and absence of CaAlthough X-ray crystallography has proven to be a powerful tool in understanding the atomic structure of SERCA, this study supports the conclusion, based on previous FRET experiments 2+ (PDB: 1su4) as a starting point for the simulations of Ca2+-bound and Apo SERCA. For the simulation of Ca2+-bound SERCA, we used the crystal structure as is, keeping all ions and water molecules. A similar approach was used to simulate Apo SERCA, except for the removal of Ca2+ from the structure; furthermore, we adjusted the ionization states of the acidic residues of Ca2+-binding sites to mimic the E1 state of SERCA z-axis. Finally, Na+ and Cl− ions were added to the system to neutralize the charge of the system and to produce an ion concentration of approximately 150 mM. Both Ca2+-bound and Apo SERCA systems contained ∼270,000 atoms. CHARMM22 force field topologies and parameters with CMAP correction We used the crystal structure of SERCA 1a with two bound Ca−1 Å−2). Each simulation was continued for 500 ns without any restraints.MD simulations were performed by using the program NAMD 2.7 2+ (PDB: 1vfp). Docking simulations were carried out using the Lamarckian Genetic Algorithm, with an initial population of 100 individuals, a maximum number of 10,000,000 energy evaluations and a maximum number of 50,000 generations. Docking simulations consisted of 100 independent runs. Resulting orientations lying within 0.15 nm in the RMSD were clustered together, and the best-ranked orientation was used for analysis.Docking simulations of AMPPCP to the ATP-binding site were performed with AutoDock 4.2 Figure S12+-bound SERCA. The transition was detected in the trajectory between 430 and 450 ns, and it is characterized by an increase in the in Cα-Cα distance between residues Met1 (A domain) and Lys515 (N domain). This distance is shown in the cartoons as a black arrow.Spontaneous closed-to-open transitions of Ca(TIF)Click here for additional data file.Figure S22+-bound SERCA. Backbone superimposition of the structure of Ca2+-bound SERCA at the end of the simulation (solid ribbons) and the crystal structure of SERCA bound to Ca2+ and AMPPCP (transparent ribbons). While N and P domains move into positions similar to those of the crystal structure, this is not true for the A domain. The oval indicates the A-N interdomain contact observed in the crystal structure but not in the MD simulation, and the arrow indicates the direction of the expected movement of the A-domain.Comparison of the headpiece arrangement between MD simulation and crystal structure of Ca(TIF)Click here for additional data file.


Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca2+-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca2+ responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca2+ mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca2+ mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca2+ mobilization, particularly antigen-mediated Ca2+ release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion. T. gondii has evolved to be an extremely successful obligate intracellular parasite. It parasitizes a multitude of mammalian and avian species as intermediate hosts. In felines, which serve as the definitive host, sexual reproduction results in shedding of highly infectious oocysts. The Center for Disease Control and Prevention estimates one fifth of the US. human population is latently infected with T. gondii. In most hosts, infection is long-lived but asymptomatic. Nevertheless, under certain conditions, such as in immuno-compromised individuals, acute toxoplasmosis poses serious health risks  activation, both of which are necessary for stimulated granule exocytosis -dependent Ca2+ mobilization. Additional experiments revealed that PLCγ activation by Syk tyrosine kinase is inhibited by Toxoplasma infection. Finally, we found that inhibition of degranulation prevails under conditions in which inhibition of actin polymerization prevents parasite invasion. Collectively, these results support a model in which T. gondii inhibits FcεRI receptor signaling during invasion by releasing a factor that inhibits Syk mediated activation of PLCγ, and thus interferes with hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messengers IP3 and diacylglycerol (DAG) important for Ca2+ mobilization and degranulation.In the present study we demonstrate that CaIndo-1-AM and Fluo-4-AM were purchased from Invitrogen Corp. 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide, cytochalasin D, FITC-dextran, and thapsigargin were purchased from Sigma-Aldrich. Unless otherwise noted, all other tissue culture reagents were purchased from Invitrogen, and all other chemicals were purchased from Sigma-Aldrich. Anti-DNP IgE was purified as described previously  were purchased from either The Jackson Laboratory  or Taconic Farms  and were maintained in the Transgenic Mouse Core Facility at the Cornell University College of Veterinary Medicine, accredited by American Association of Accreditation of Laboratory Animal Care. Mouse neutrophils were isolated by percoll gradient purification as described previously . All efforts were made to minimize animal suffering during the course of these studies.in vitro via passage through human foreskin fibroblast cultures in DMEM with FCS (1%), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Fibroblast media). For experiments, tachyzoites were harvested and passed through a 3 μm track-etched membrane filter (Whatman) to remove fibroblast debris. Infections were performed at a multiplicity of infection (MOI) of 10:1 unless otherwise indicated and were synchronized by brief centrifugation (200 × g for 4 min).RH, PTG, CTG, Veg, and RH-tomato strains of parasites were used in this study. The RH-tomato strain, stably expressing tomato fluorescent protein, was generated by Dr. B. Striepen . All tachyzoites were maintained 5/well and incubated overnight. The next day, cells were washed with fibroblast media and parasites were introduced as described above. For some experiments, 1 μM cytochalasin D was added during infection to prevent invasion. Following infection, cells were washed three times with buffered saline solution  and β-hexosaminidase release in response to DNP-BSA was assessed as described previously , 200 nM thapsigargin or 10 μM fMLP was carried out in tachyzoite infected cells using indo-1 as a Ca2+ indicator dye as described previously . Briefly, code was written to track the location and average fluorescence intensity of the green channel (Fluo-4) within a circular region of interest (ROI) in the cytoplasm of each cell. These measurements were plotted with respect to time, and the number of oscillations for each ROI, reflected by increases in fluorescence intensity, were enumerated.Analysis of Ca5/ml) on # 1.5 coverslips or in 35 mm glass bottom dishes (MatTek). After overnight culture, cells were transfected with either PH- PLCδ-EGFP  for 10 min at room temperature. Excess fixative was quenched by 10 mg/ml BSA in PBS with 0.01% sodium azide. Fixed cells were imaged on a Leica SP2 confocal system. Line scan analysis of equatorial cross sections using ImageJ (NIH) was performed using average fluorescence values to determine the ratio of the PH domain at the plasma membrane to that in the cytoplasm . All bar graphs display mean ± SEM unless otherwise noted. Statistical significance was determined by One-Way ANOVA  followed by Tukey's post test. Level of significance is denoted as follows: T. gondii is known to actively modulate signaling in the immune cells it infects , II (PTG), or III (CTG or VEG) tachyzoites. After 1 h we assessed the capacity of the infected cells to degranulate in response to multivalent antigen, DNP-BSA. In a bulk assay we found that acute d Figure . Under td Figure .T. gondii inhibits mast cell degranulation, and this inhibition directly correlates with infection.We used RH-tomato parasites in live imaging experiments to assess degranulation by FITC-dextran release Figure . In thes2+ release from ER stores is mediated by binding of IP3 to its receptors in the ER membrane. This event triggers coupling of the ER-localized Ca2+ sensor, STIM1, and the plasma membrane Ca2+ channel Orai1, resulting in additional Ca2+ influx from extracellular space, a process known as store operated Ca2+ entry (SOCE). These events are important downstream steps in the pathway that leads to degranulation in mast cells , resulting in hydrolysis of PIP Figures .2, it must be recruited to the plasma membrane and phosphorylated at specific tyrosine residues. Western blotting with anti-pY1217-PLCγ2 shows that antigen-stimulated phosphorylation at this residue is reduced in all infected samples . As shown in Figure Our single cell CaParasite entry depends primarily on the parasite actin cytoskeleton . Our data collectively suggest a model in which one or more T. gondii-derived proteins act directly to reduce the activity of PLCγ by inhibiting Syk activation, thereby reducing the levels of IP3 and inhibiting subsequent signaling steps . Nevertheless, ROP protein early release, relation to virulence, and immunomodulatory capabilities make these proteins attractive candidates for the Toxoplasma secreted factor responsible for the reduction in Ca2+ mobilization (Ong et al., Furthermore, our results suggest that a secreted product from Toxoplasma inhibits mast cell immune responses, this is not contradictory to reports that mast cell responses contribute to the primary host response to Toxoplasma in vivo (Ferreira et al., in vitro. Furthermore, our findings that PLCβ-mediated neutrophil responses are inhibited by Toxoplasma infection (Figure 2+ responses (Haroon et al., T. gondii to subvert normal Ca2+ signaling. In future studies it will be important to assess whether inhibition of Ca2+ signaling is manifest in other immune cells infected by T. gondii, as well as the mechanisms utilized. Collectively, our results indicate that T. gondii targets Syk-dependent PLCγ activation as one mechanism to interfere with immune signaling that depends on Ca2+ mobilization.We also note that while our results indicate that n Figure , as are 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 aim of this study was to examine atrial myocyte contractile function and Ca2+ handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction.There is limited knowledge about atrial myocyte Ca2+ amplitude, decreased SR Ca2+ content, and slower Ca2+ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca2+ ATPase activity, increased Na+/Ca2+-exchanger activity and increased diastolic Ca2+ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca2+ handling in HF.Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca2+ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca Reduced Ca2+ transients, slowed transient decay, increased diastolic Ca2+, reduced sarcoplasmic reticulum (SR) Ca2+ stores, reduced function of the SR Ca2+ ATPase (SERCA) 2a, increased Na+/Ca2+ exchanger (NCX) activity and increased diastolic SR Ca2+ leak are commonly observed in cardiomyocytes from failing hearts 2+ handling in atrial myocytes from failing hearts. Disruption of the rhythmic beating of atrial myocytes can lead to life-threatening conditions such as atrial fibrillation (AF), the most common cardiac arrhythmia in clinical practice 2+ handling and changes in Ca2+ regulatory proteins was observed 2+ handling and atrial myocytes contractile dysfunction was also observed in goats with dilated atrias Impaired cardiomyocyte contractility and Ca2+ handling in rats with post-infarction HF 2+ handling in HF. To address this issue we compared contractile function and Ca2+ handling in atrial myocytes of sham-operated rats and rats with post-infarction HF, and studied the effects of aerobic interval training.Exercise training has been reported to improve left ventricular function after post-infarction HF in patients The Norwegian council for Animal Research approved the study, which was in accordance with Guide for the Care and Use of Laboratory Animals .2 30% N2O mixture. After left thoracotomy and pericardium opening, the descending artery was ligated with a polyester suture . Sham operated rats were subjected to the same surgical procedures except the ligation. Buprenorfin (0.04 mg/kg) was injected subcutaneous during the surgery and repeated 8 hours after surgery for relieving the pain. After 1 and 4 weeks, MI operated rats were examined by echocardiography to determine the extents of MI. Previous echocardiography examinations of MI rats in our laboratory have shown that infarct size is a strong predictor for remodeling and HF where infarct size >40% confirmed HF progression VO2max), rats ran on a customized treadmill in a metabolic chamber, as previously described VO2max and 2 min at 50% of VO2max for 60 min/day, 5 days/week, for 8 weeks. VO2max was determined every second week to account for the improved training status, then the workload was adjusted to keep high intensity intervals at 85%–90% of VO2max.Female Sprague Dawley rats were randomized to either sham operation or myocardial infarction (MI) surgery. MI was induced by ligation of the left coronary artery, as previously described 2 30% N2O mixture while the heart was removed and left atria was selected for cell experiments. The estimated infarct size from echocardiography was confirmed in the excised heart as previously described After the intervention period, the rats were sacrificed during anesthesia; breathing 5% isoflurane in a closed chamber, thereafter breathing 1.5% isoflurane in a 70% O2, 0.33 mM NaH2PO4, 22 mM glucose, 50 µU/ml bovine insulin , 25 mM HEPESNaOH (pH = 7.4)) with 0.4 mM EGTA and immediately canulated through aorta and retrogradely perfused  with isolation buffer containing 0.4 mM EGTA for 2–4 min. Then the hearts were perfused with the enzyme solution containing isolation buffer supplemented with 0.048 mM CaCl2 and 1 mg/ml collagenase . From the digested hearts (10–15 minutes perfusion) left atria were removed, cut into 3–5 pieces, and further digested by gentle stirring for 5–10 min in fresh enzyme solution until myocytes appeared. Tissue chunks were then transferred to isolation buffer containing 0.096 mM CaCl2 and 10 mg/ml 0.1% bovine serum albumin, cut into as small pieces as possible and mechanically agitated with a pipette. The cell suspension was centrifuged at 40×g for 2 minutes in a 15 ml plastic centrifuge tube, the supernatant was gently removed and the cell pellet was resuspended in 2 ml of isolation buffer with 0.026 mM CaCl2.Left atrial cells from rat were isolated using a modified mouse model protocol 2+ measurements cells were resuspended in HEPES solution  containing 1.8 mM CaCl2. Fura-2/AM-loaded  cardiomyocytes were field stimulated by bipolar electrical pulses at 2 Hz on an inverted epifluorescence microscope  with a 40×1.3NA oil-immersion objective. 8–10 cardiomyocytes were analyzed for each measurement. Cell shortening was measured by video-based sarcomere spacing  and intracellular Ca2+ concentration was measured by counting 510 nm emission with a photomultiplier tube  after exciting with alternating 340 and 380 nm wavelengths (340/380F ratio) . Total SR Ca2+ content was measured by assessing peak Ca2+ amplitude after rapidly applying Caffeine (10 mM) to the perfusion solution by a pipette placed directly above the cardiomyocyte. The application of caffeine was performed immediately after stopping the electrical stimulation in normal HEPES solution. Diastolic Ca2+ cycling was quantified with the use of rate constants of Ca2+ removal during twitch-induced stimulation, sustained caffeine stimulation in physiological HEPES solution and sustained caffeine stimulation in a solution containing 0Na+/0Ca2+. During a regular twitch-induced Ca2+-transient in normal physiological HEPES solution, Ca2+ is removed by the SERCA2a, NCX, and the plasma membrane Ca2+ ATPase (PMCA), and the rate constant of Ca2+ decline in this situation (Ktw) can be described as the sum of the rate constants associated with each efflux mechanism. During caffeine-induced Ca2+-transients, the contribution from SERCA2a is abolished, and the decay rate constant thus depends only upon NCX and PMCA. To derive the rate constant of NCX (KNCX), the rate constant of Ca2+ removal during caffeine-induced Ca2+ transients in a solution containing 0Na+/0Ca2+ was recorded and subtracted from the rate constant in the presence of these ions (Caffeine+regular HEPES) SERCA2a = Ktw – KNCX, and the relative contribution by SERCA2a is KSERCA2a/KTW.For Ca2+ leak was assessed using a modified protocol after Shannon et al 2+ handling during twitch-induced transients in HEPES 1.8 mM Ca2+ solution, the electrical field stimulation was ceased and quiescent cardiomyocytes were perfused with a physiological solution containing 0 Na+/0Ca2+. The 0Na+/0Ca2+ solution prevents the Na+ - Ca2+ exchange, which is the primary Ca2+-influx and efflux mechanism at rest. After recording the Ca2+ level in the quiescent cardiomyocyte for 40 seconds we rapidly switched to Caffeine +0Na+/0Ca2+. When Ca2+ levels were close to baseline, caffeine perfusion was replaced by HEPES perfusion and cardiomyocytes were stimulated at 1 Hz until stable Ca2+ transients were restored (approximately 20 seconds). Subsequently, perfusion was switched to a 0Na+/0Ca2++Tetracaine (1 mM) solution whereupon diastolic Ca2+ level were measured for another 40 seconds, again followed by a 0Na+/0Ca2++caffeine-induced transient. The quantitative difference in diastolic Ca2+ level between the 0Na+/0Ca2+ solution with and without tetracaine represents the absolute SR Ca2+ leak, since 0Na+/0Ca2+ prevents the NCX Ca2+ influx and efflux across the sarcolemma and tetracaine inhibits Ca2+ movement across RyR2. Diastolic SR Ca2+ leak was quantified in relation to total SR Ca2+ content.Diastolic SR Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) on diastolic SR Ca2+ leak from the RyR2, the CaMKII inhibitor  and PKA inhibitor  were used. Pre-incubation of cardiomyocytes with AIP/H-89 started 1 hour prior to experiments. During the experimental protocol, the inhibitors were added to the perfusion solution to avoid potential washout of inhibitors.To determine the influence of CaData are presented as mean ± SD. One-way ANOVA with Bonferroni post-hoc test adjusted for multiple comparisons was used to identify statistical differences between the groups. P<0.05 was considered statistical significant.2max was 7.6% lower in rats with HF compared to sham (p<0.05). Aerobic interval training improved VO2max by 41.0%, which was higher than that observed in inactive sham-rats , but it remained lower (p<0.05) than that observed in sham (2+-load was 48% lower (p<0.001) in inactive HF-rats vs. sham, and increased by 78% (p<0.05) after aerobic interval training, but still remained 15% lower (p<0.05) than that observed in sham .In line with contractile function, atrial myocyte Cap<0.001, . Aerobic in sham . Similar in sham . In line2+ level was higher in atrial myocytes from inactive HF-rats compared to sham .The intracellular diastolic Cap<0.001, . AerobicThe activity of SERCA2a was 26.8% lower in atrial myocytes from HF-rats compared to sham p<0.001, . Aerobic2+ leak in atrial myocytes from HF-rats compared to that observed in sham rats , but the Ca2+ leak was still significantly higher compared to sham rats (p<0.05). Pharmacological inhibition of CaMKII by AIP reduced Ca2+ leak in atrial myocytes from inactive and exercise trained HF-rats to that observed in sham  and diastolic Ca2+ leak over the RyR2 2+ leak over the RyRs explain the reduced SR Ca2+ content. The observed impairment in atrial Ca2+ handling resembles previously reported HF-induced changes in ventricular cardiomyocytes Reduced fractional shortening and prolonged time to diastolic re-lengthening in HF-rats were paralleled by reduced Ca2+ leak in HF-rats while H89 that inhibits PKA had no effect. This suggests that Ca2+ release channel dysfunction is caused by CaMKII-dependent RyR2 hyperphosphorylation. This is consistent with data from several HF models, in which CaMKII-dependent increase in RyR2 phosphorylation was associated with higher ventricular myocyte SR Ca2+ leak 2+ leak 2+ content was shown in two goat models of atrial dysfunction 2+ content was associated with a reduced reuptake of Ca2+ into SR by SERCA2a in addition to increased SR Ca2+ leak during diastole. Furthermore, despite not directly assessed in our study, the data from goat showing CaMKII mediated RyR2 hyperphosphorylation as mediator of SR Ca2+ leak 2+ leak by inhibiting CaMKII with AIP. On the other hand, in contrast to the Ca2+ handling abnormalities we identified, Yeh et al 2+ load, associated with increased Ca2+ entry (ICaL) and increased CaMKII phosphorylation of phospholamban (PLB) in a rapid ventricular tachypacing induced dog HF model. In its dephosphorylated state PLB inhibits SERCA2a. Phosphorylation by CaMKII (or PKA) dissociates PLB from SERCA2a increasing Ca2+ uptake into SR 2+ fluxes are significant different between dogs and rats  2+ loading from that model is not unexpected, since rapid pacing of the heart normally would cause this effect of increased Ca2+ loading of the cardiomyocyte 2+ removal by SERCA2a in the present study, though we did not measure the phosphorylation level of molecular determinants. Being as how earlier studies on goats have shown that PLB phosphorylation was reduced 2+ uptake in the present study.In the current study, the CaMKII inhibitor AIP decreased the SR Ca2+ homeostasis in HF may be a predisposing factor to atrial arrhythmic activity 2+ leak from SR has been shown to be an important contributor to arrhythmogenesis 2+ uptake as well as SR Ca2+ leak through RyR2 contributes to accumulation of diastolic Ca2+2+ concentration enhances the NCX activity and inward Na+ current during Ca2+ extrusion promoting delayed afterdepolarizations (DADs) and triggered activity 2+ accumulation may induce diastolic dysfunction by activation of contractile proteins, preventing full relaxation of myofilaments 2+ release from SR 2+ handling abnormalities in this model may be of main importance for development of AF.Alterations in CaWe demonstrate here for the first time that aerobic interval training improved atrial myocyte contractile function in HF-rats. It has previously been shown that such training programs improve contractile function in healthy animals 2+ handling after exercise in HF-rats explains the observed increase in myocyte contractility. The normalized SERCA2a activity, reduced NCX activity, and reduced SR Ca2+ leak explains the increased Ca2+ amplitude and SR Ca2+ content, thereby contributing to the observed improvement in contractile function. Similarly, the normalized ventricular contractility in trained HF-rats was explained by increased rates of rise and decay of the Ca2+ transient associated with normalized NCX and SERCA2a 2+ removal from the outer plasma membrane to the SR and the consequent decrease in Na+ current through the NCX constrain the ability to produce DADs, and likely decrease the susceptibility to triggered activity and arrhythmias.We suggest that the improved atrial myocyte Ca2+ leak in the present study. This explanation is in line with observation of increased phosphorylation level of CaMKII in diabetic cardiomyocytes showing abnormally high Ca2+ leak 2+ leak. The reduced Ca2+ leak in the present study is suggested to be explained by training-induced dephosphorylation of CaMKII. In several HF models, inhibition of CaMKII has been shown to abolish SR Ca2+ leak As discussed above, we suggest that increased CaMKII activity in HF explains the increased diastolic Ca2+ handling, whereas aerobic interval training can restore the dysfunction via improved Ca2+ handling. In future studies this model can help us to elucidate important mechanisms behind HF as a substrate that promotes AF, and also the effect of training as a treatment.This study increases our understanding of how atrial myocyte contractile dysfunction in post-infarction HF is associated with major impairment in Ca


In this study, we focused on the [Ca2+]i and its control mechanisms in process of hydrogen peroxide (H2O2)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca2+ indicator to detect [Ca2+]i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca2+]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca2+]i, which appeared only at 0.5 h after βE2 application; c) increased [Ca2+]i under 100 µM H2O2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca2+ stores; d) importantly, the transiently increased [Ca2+]i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca2+ channels (L-VGCC), but the increased [Ca2+]i induced by 100 µM H2O2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H2O2, which was also associated with its transient [Ca2+]i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.Intracellular calcium concentration ([Ca Accumulating evidence suggests that both the excessive elevation of [Ca2+]i and the loss of [Ca2+]i are crucial for degenerative diseases [2+]i leads to the inappropriate activation of Ca2+-dependent processes, which are normally inactive or operate at low Ca2+ levels, thus causing metabolic derangements that ultimately lead to cell death i promotes cell proliferation, whereas relatively high [Ca2+]i leads to increased mitochondrial Ca2+ and accounts for the release of pro-apoptotic factors resulting in cell death i increases in degenerative disorders of CNS [2+]i reduction and deficiency have also been studied and shown to play a role in degenerative disorders of CNS [2+]i may occur or Ca2+ may be reduced in specific cellular compartments and increased in other compartments i [2+]i reduction [2+]i assessment. Estrogen is an antioxidant that exerts various role by itself or by regulating intracellular signaling pathways , and it eostasis . Neverth [Ca2+]i –22, but eduction ,24. Thes2O2-induced apoptosis of retinal cells in vitro and inhibited light-induced photoreceptor apoptosis in vivo, suggesting that βE2 has retinal protective properties [2+]i in apoptosis and anti-apoptosis in our study model remain unknown. In this study, we detected the [Ca2+]i of primary cultured Sprague-Dawley (SD) rat retinal cells treated with different concentrations of H2O2 or βE2 and at different time points after H2O2 or βE2 treatment. Next, we measured [Ca2+]i under βE2 and H2O2 co-treatment, and we explored the controlling mechanisms of [Ca2+]i. Consequently, we found that treatment with 100 μM H2O2 led to primary cultured SD rat retinal cell injury and apoptosis, while treatment with 10 μM βE2 played a protective role. Both completely different roles were mediated by increasing the [Ca2+]i, which occurred at the early stage of apoptosis and at 0.5 h after βE2 treatment. Furthermore, both of the increased [Ca2+]i under completely opposite conditions were partially due to extracellular [Ca2+]i. Importantly, the transient [Ca2+]i increase induced by βE2 was gated by the L-type voltage-gated Ca2+ channels (L-VGCC) and phosphatidylinositol-3-kinase (PI3K) was involved, but it was not involved in the H2O2-induced [Ca2+]i increase.Several recent reports have shown that both estrogen receptor (ER) subtypes, ERα and ERβ, are present in the retina ,26. Evidoperties ,29. HoweSD rats  were housed in a controlled environment in a specific pathogen-free animal center. The temperature was maintained at 24±2°C, the humidity was 52±10% and fresh air was circulated continuously. All of the procedures used in the experiments were approved by the Institutional Animal Ethics Committee, Medical School of Xi’an Jiaotong University (permission No. 2009-12) and conformed to accepted ethical standards of the Animals in Research and the Association for Research in Vision and Ophthalmology statement for the use of animals in vision and ophthalmic research. 2O2 was purchased from Xi’an Pure Chemical Industries . Fetal Bovine Serum (FBS) and phenol red free 1:1 DMEM/F-12 were obtained from Hyclone . Poly-lysine, βE2, Hoechst 333342 dye and nifedipine, an L-VGCC blocker, were purchased from Sigma . We used 95% ethanol as the solvent to make the βE2 stock solution at a concentration of 1x10-2 M. Fluo-3 AM, an indicator of intracellular Ca2+ levels, was purchased from Biotium . We used Dimethylsulfoxide (DMSO) as the solvent for making 5 mM Fluo-3 AM stock solution and 20% Pluronic F-127 (5900) (offered by Biotium) in DMSO to facilitate AM ester solubilization. Trypsin, DMSO, 3--2, 5-diphenyltetrazolium bromide (MTT) and ethylene glycol tetraacetic acid (EGTA), an extracellular Ca2+ chelator, were purchased from Amresco . LY294002, a PI3K inhibitor, was purchased from Cayman . The Annexin V-FITC Apoptosis Assay Kit and bicinchoninic acid (BCA) Protein Assay Kit were purchased from Zhuhai Joincare Bioscience Ltd , and radio immunoprecipitation assay (RIPA) buffer was purchased from Biotech . Anti-p-Akt and anti-Akt antibodies were purchased from Cell Signaling , and Anti-β-actin antibody was purchased from Santa Cruz Biotechnology .H6 cells/ml and plated onto 24-well or 6-well plates (Corning Costar) with 1 ml or 3 ml of cell suspension per well. Before culturing, all the plates were coated with poly-lysine (0.1 mg/ml) and maintained in a humid incubator overnight. Next, we washed the plates three times with sterile double distilled water (ddH2O), once with D-Hanks balanced salt solution, and then with 200 μl of medium, which provided a pre-environment for cell growth. The cells were cultured at 37°C in a 5% CO2 atmosphere until they were used at 4-6 days in vitro, during which the medium was replaced according to the cell growth and metabolism conditions.We cultured primary retinal cells referencing other’s study  and maki2O2 and/or βE2 were added by bath application. Overall, 1 M H2O2 was prepared from 30% H2O2 dissolved in sterile cool PBS and was diluted with the medium to 10 mM. Next, the 10 mM H2O2 was diluted with the essential medium gradually to 200-25 μM, and 0 μM was regarded as the control. The 0.5-100 μM βE2 was prepared from the 1x10-2 M βE2 stock solution with the medium and was added to the cultures. We considered 0 μM as the control. The βE2 stock solution was dissolved in 95% ethanol, and a small amount of ethanol was present in the medium (<1%), but it had no effect on the primary cultured SD rat retinal cells i, different concentrations of EGTA were added directly to the medium 1 hr before the application of 100 μM H2O2 for 2 hrs or 10 μM βE2 for 0.5 hrs to chelate the extracellular Ca2+. Under the co-application, we pre-treated cells with 10 μM βE2 treatment for 0.5 hrs before the application of 100 μM H2O2 for 2 hrs. To conduct the channel experiments and the mechanism study, the cultures were pre-conditioned for 2 hrs by nifedipine or for 0.5 hrs by LY294002 before the other treatments.After the cultures were maintained for 4-6 days in vitro, Hal cells . Except 2, and the wells with no cells were used as blank controls. The medium was then removed, and DMSO was added to solubilize the colored formazan crystal product. The absorbance was determined at 490 nm on a Measurement Photometric multi-well plate reader  with Skanlt RE for Mass 2.2 software after the plates were agitated at 37°C for 10 mins. All absorbance values were subtracted by the blank value, and the untreated cultures were considered as the control group. The mean cell viability for each condition was determined by averaging at least quadruplicate values, the fold change relative to the control was calculated, and the control values were normalized to 1. All experiments were performed using 3-5 separate experiments to confirm reproducibility. To determine the cell viability of the primary cultured SD rat retinal cells, we performed an MTT assay. MTT was applied to the cultures at a final concentration of 0.5 mg/ml for 4 hrs at 37°C in 5% CO2O2 for 0-24 hrs, apoptosis was assayed by Annexin V/Propidium Iodide (PI) staining and Hoechst 33342 staining. For Annexin V/PI staining, the cells were collected, centrifuged at 1000 rpm for 5 mins, suspended and diluted with 1×binding buffer (Annexin V-FITC Apoptosis Assay Kit) to 5×105 cells/ml. The 500 μl suspension was loaded with 5 μl Annexin V-FIFC and 10 μl PI for 15 mins. After incubated in the dark at room temperature, the cells were analyzed within one hour with a ﬂow cytometer . For Hoechst 33342 staining, 40 μl of suspension was dropped onto the slide, ﬁxed in 4% paraformaldehyde in PBS at room temperature for 20 mins and stained with 2 μg/ml Hoechst 33342 dye in the dark for 10 mins. The samples were then observed under a ﬂuorescence microscope  with ﬂuorescence excitation at 340 nm and emission at 510 nm. The cells with condensed DNA were counted as apoptotic cells, and the average apoptotic cells of each field were calculated. The sample fields with approximately 100 cells were randomly selected, and each sample was evaluated. The cells in 3-5 random fields/cultures were scored, and the counts were based on at least four separate cultures in each treatment condition.After exposure to 100 μM H2+]i detection was performed by FACS analysis i alteration. To confirm the reproducibility, all experiments were performed at least 3-5 times with separate cultures.  i in our study model and to dynamically observe the [Ca2+]i alteration during apoptosis under a modest treatment condition, we performed the following experiments. First, cell viability and the [Ca2+]i were assayed simultaneously at 2 h after treatment with different concentrations of H2O2. As shown in 2O2 decreased cell viability  and a slight decrease in cell viability; however, the 100 µM H2O2 treatment for 24 hrs caused a remarkable decrease in cell viability (P<0.001), but no significant alteration was discovered in [Ca2+]i  of βE2 are insufficient to increase cell viability and higher concentrations (100 µM) of βE2 are toxic for retinal cells. Interestingly, cell viability was significantly increased at 0.5-24 h after the application of 10 μM βE2  receptor, the a-amino-3-hydroxy-5-methyl-4-isoxa-zole propionic acid (AMPA) receptor and VGCC i increase occurs at the early stage of apoptosis but not during the later stages of this process. Moreover, the increased [Ca2+]i induced by H2O2 is partially caused by extracellular stores.Recent evidence indicates that i due to H2O2. Inconsistent with our hypothesis, we found that 10 μM βE2 played a protective role by immediately sharpening but not restoring the increased [Ca2+]i induced by H2O2. Furthermore, up to 2-5 mM doses of EGTA significantly attenuated the sharpening effect of βE2, indicating that this effect may be caused by a large Ca2+ transient influx. Many studies have proposed that L-VGCC plays an important role in the protective process in CNS, including retina i through one or more relevant Ca2+ channels and signaling pathways. Excitedly, we discovered that the retinal protective role of βE2 through potentiating Ca2+ influx is controlled by L-VGCC and mediated by PI3K pathway.As for the mechanisms involved in βE2 retinal protection in our model, we speculated that βE2 resisted Hg retina –22,43. Iotection –46. The otection –50. βE2 channels , and preon cells . Therefo2O2 injury and βE2 protection are mediated by increasing the [Ca2+]i sourced from extracellular Ca2+ influx. These findings can be explained by the following ideas. First, Ca2+ exerts a biphasic effect on cellular growth, and a modest increase in [Ca2+]i promotes cell proliferation, whereas relatively high [Ca2+]i leads to increased mitochondrial Ca2+ and accounts for the release of pro-apoptotic factors resulting in cell death [2+]i is tolerated and may be needed to modulate biological functions, but the sustained increase in [Ca2+]i leads to various degrees of cell damage until cell death. Third, under the two treatment conditions, the increased [Ca2+]i may be due to different channels, and Ca2+ inﬂux through different routes may perform different biological functions [2+ loads are toxic when entering via the NMDA channels but not when entering via the VGCC [2+]i increase induced by H2O2 is harmful, but a transient [Ca2+]i increase induced by βE2 for only 0.5 hrs is protective. Furthermore, the favorable [Ca2+]i increase due to βE2 was gated by L-VGCC and was mediated by the PI3K pathway, but the harmful [Ca2+]i increase caused by H2O2 was not gated by L-VGCC or mediated by the PI3K pathway. Perplexedly, the results in our present study showed that both Hll death ,9. Seconunctions . For exathe VGCC . Our pre2+]i induced by the 100 μM H2O2-induced injury  [2O2-induced apoptosis of primary cultured retinal cells represents the pathogenesis of multiple retinal degenerative diseases. Certainly, in our future studies, we will do some research using in vivo model to obtain results that are more closely applicable to in vivo conditions.The data from our present study were based on a primary mixed cell culture of retinal cell population. The in vitro model is widely used for studying the pathogenesis of diseases. The primary retinal cell culture began in the late 1950’s. Today, it is routinely used for studies and remains the most widely-used form of retinal cell culture . Moreovees (ROS) ,13. Ther