Conversely, the incidence of GVHD directly correlates with the graft-versus-leukemia (GVL) effect and achieving disease remission. This observation is likely due to the donor T cells targeting antigens that are shared between the AML blasts/stem cells and normal tissue. Despite decades of laboratory and clinical research, there remains a paucity of antigens that are known to be associated with GVHD and/or GVL. Yet, separating GVHD from GVL is critical to the success of future targeted therapies for AML.Acute myeloid leukemia (AML) remains an incurable malignancy. Although the incidence of AML is approximately 20,000 cases annually, the 5-year survival rate remains low at nearly 30%. There are a number of promising therapies for AML currently under development, especially in the relapsed and refractory settings. However, to date, there have been very few breakthroughs in the treatment of relapsed or refractory AML. Allogeneic hematopoietic stem cell transplant  remains a cornerstone therapy for patients with AML, especially patients with aggressive disease. However, allo-HSCT carries a high risk of mortality and morbidity due to the toxicities associated with conditioning regimens used prior to infusing the donor cells, and graft-Our data, along with studies from a number of other investigators, have identified myeloid azurophil granules as a source for immunogenic antigens in AML –3. PR1, n = 42), MDS (n = 11) or CML (n = 13) [n = 30) and grade 2 (n = 4) injection-site reactions. In addition, there was no worsening of GVHD in any of the 16 patients who had received allo-HSCT prior to vaccination, and none of the patients developed anti-P3 antibodies (cANCA), an important observation considering that PR1 peptide is derived from P3. An immunological response (defined as a ≥2-fold increase in the frequency of PR1-CTL) was seen in 53% of patients (n = 35), did not correlate with the PR1 peptide vaccine dose or the number of vaccinations received, and occurred soon after the receipt of the vaccination (by 3 weeks in 89% of the patients). Importantly, having < 5% bone marrow blasts (P = 0.01) and younger age (P = 0.03) correlated with achieving an immunologic response (IR).We reported results from a phase I/II dose escalation clinical trial investigating the toxicity, as well as clinical and immunologic responses following PR1-peptide vaccine in 66 patients with AML n = 2, MDS . Thirdly, the data highlight the safety of targeting PR1 in hematologic malignancies, an observation that is highly significant for the future of PR1- targeting approaches since the PR1 epitope is expressed by normal hematopoietic cells, although at a much lower level [These data are critical for vaccine therapy for AML, as well for PR1-targeting approaches. First, we showed that an immune response can be elicited in patients with AML. This is an important finding since the AML microenvironment has been reported to be immunosuppressive due to quantitative and qualitative factors. Nevertheless, the correlation between lower AML burden and achieving an IR highlights a window where vaccinations may be effectively administered to patients to elicit anti-AML immunity. Second, our data point to the effectiveness of targeting the leukemia-associated antigen, PR1. In our study , PR1-CTL


When Ca2+ was low, diarrhea occurred; when Ca2+ levels normalized with replacement, diarrhea stopped. Based on this and other observations, a broader question arises as to whether the Ca2+ lost in diarrhea should be replaced promptly in these patients.Diarrhea causes monovalent and divalent ion losses that can influence clinical outcome. Unlike the losses of monovalent ions, such as Na Withfication . As a rediarrhea . This Zn2+. When blood (serum) Ca2+ was low, diarrhea occurred; when Ca2+ levels normalized with replacement, diarrhea quickly stopped. In light of this and other observations, we propose that, similar to Zn2+ deficiency, the loss of Ca2+ without prompt replacement may compromise the diarrhea-protective capability of Ca2+ and CaSR in the gut and lead to severe, protracted, and recurrent diarrhea.In this communication, we report a malnourished child with an immune-mediated enteropathy. Despite adequate immunotherapy, he developed episodes of “breakthrough” diarrhea with concurrent hypocalcemia. Interestingly, during these disease flare-up episodes, diarrhea symptoms inversely fluctuated with levels of blood CaZ = −0.56, weight Z = −2.25, BMI Z = −2.85) child with hypothermia (temperature 97.5 °F), hypotension (blood pressure 83/54 mmHg), tachycardia (pulse 103 beats per minute), dry mucus membranes, sunken orbits, temporal wasting, and a protuberant but otherwise benign abdomen. His laboratory values are shown in Figures The patient is a 6-year-old African-American male with autoimmune enteropathy  who was hospitalized for worsening non-bloody, watery diarrhea, severe malnutrition, and hypocalcemia. Initially, he responded well to glucocorticosteroid monotherapy. However, for the 6 months prior to this admission, social issues gradually led to interruption of this therapy with recurrence of diarrhea , and worsening hypokalemia and metabolic acidosis. He was transferred to the intensive care unit for a closer monitoring and given three consecutive doses of q 3-h intravenous calcium chloride , in addition to intravenous fluids and other electrolyte replacements. Remarkably, as his serum-ionized Ca2+ normalized, his diarrhea resolved   in the + Figure , as welldiarrhea .Considering the chronic nature of the diarrhea prior to admission and the absence of gastrointestinal infection, our patient’s diarrhea was most likely related to the underlying autoimmune enteropathy. Indeed, the diarrhea responded well to appropriate immunotherapy while calcium levels were normal. However, it recurred with hypocalcemia and again resolved with normalization of this element’s serum levels. The direct correlation between calcium concentration and stool volume Figure  strongly2+ metabolism. First, he has malnutrition. It is common for children with malnutrition to have a negative calcium balance. Second, this child had a history of enteropathy that could impair Ca2+ absorption and induce Ca2+ loss. Third, he also has milk-dairy intolerance and was consuming a dairy-free diet. The long restriction of Ca2+-rich dairy intake could lead his Ca2+ metabolic balance to a further negative direction, precipitating hypocalcemia and diarrhea.There are multiple reasons that our patient would have a negative balance of Ca2+ is required for the maintenance of epithelial tight junction integrity, a critical determinant of intestinal barrier function and inflammatory diarrhea . By contrast, while oral replacement raises serum Ca2+ relatively slowly, it produces local therapeutic anti-diarrheal (and other) actions and helps restore Ca2+ balance without causing much unwanted systemic adverse effects. Given this, the oral route is considered both safer and more physiological. Whenever clinically allowed, prompt switching from intravenous to oral supplementation of Ca2+ is recommended.Of note, the localization of CaSR in the gut has been described on both the apical and the basolateral sides of the plasma membrane of the enterocyte , and rec0 values –9. It is2+ and CaSR in both secretory and inflammatory diarrheal conditions, we suggest that Ca2 stool losses should be routinely replaced as is currently done for Na+, K+, Cl−, 2+. As summarized in Table Given the wealth of accumulating data on the role and importance of CaThe written consent for publication was obtained from the child’s parent.This study was carried out in accordance with the recommendations of the University of Florida Health Center’s IRB Guidelines and Privacy Rules about case reports, and it has been reviewed by UF IRB with written informed consent from the guardian of the studied subject.SXC conceptualized and designed the work, JF and SXC collected and analyzed the data, RG, MM, GLB, CDJ, and SXC interpreted the data, JF and SXC drafted and RG, MM, GLB, and CDJ revised this manuscript, and all authors approved the final version and agreed to be accountable for the content of this work.The authors declare that the submitted work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cultural forms are constrained by cognitive biases, and writing is thought to have evolved to fit basic visual preferences, but little is known about the history and mechanisms of that evolution. Cognitive constraints have been documented for the topology of script features, but not for their orientation. Orientation anisotropy in human vision, as revealed by the oblique effect, suggests that cardinal  orientations, being easier to process, should be overrepresented in letters. As this study of 116 scripts shows, the orientation of strokes inside written characters massively favors cardinal directions, and it is organized in such a way as to make letter recognition easier: Cardinal and oblique strokes tend not to mix, and mirror symmetry is anisotropic, favoring vertical over horizontal symmetry. Phylogenetic analyses and recently invented scripts show that cultural evolution over the last three millennia cannot be the sole cause of these effects. Their shape can instruct us about two issues central to the field of cultural evolution: the influence of cognitive constraints on cultural forms  symmetry Mach, . InfantsThe contribution of cultural evolution to these three forms of legibility  was measured in two ways. One consists in asking whether extinct scripts are less legible than the ones currently in use . A script's legibility and its survival may share similar causal underpinnings. Demography is a plausible candidate. Widely spoken languages have been claimed to be easier to learn than less spoken ones . There was also high agreement between cardinality ratings produced for Unicode versions of a script and for manuscript versions . The scripts were coded by reporting, for each letter, the number of horizontal (distant from the 180° axis by less than 10°), vertical (less than 10° away from 90°), and oblique lines , and averaging this value over all letters in the script (SM 2.2). Cardinal orientations as defined here thus cover 22% of the compass (80/360°). For each script, the average number of cardinal lines per letter  was computed by two raters, who agreed on their measurement , and “mixed” letters (like K) less so by 20 percentage points. Absolute agreement between raters was acceptable , like the agreement between Unicode and manuscript sources . One script, Thaana, had to be excluded because none of the letters in it have more than one straight line.For each script, a “separation index” was computed. It measured the extent to which cardinal and oblique lines are mixed between letters or separated. That index is positive for scripts like the Latin script, which have more pure cardinals  and pure obliques  than chance would predict Fig. . For eac2.3n = 82 scripts), as well as agreement between Unicode and manuscript sources .For each script, two coders counted the number of horizontally or vertically mirror‐symmetrical letters (excluding other types of symmetry). The prevalence of vertical‐symmetrical letters among those mirror‐symmetrical letters was computed for all the scripts that featured more than three usable letters for one coder at least (this cut‐off point was pre‐registered before the measures were taken). The prevalence of vertical symmetry could be computed for 82 scripts from all seven families. Inter‐rater agreement was high  was coded using the authoritative literature. This live/extinct variable was then used as predictor in a nested regression analysis. The scripts were grouped into seven families based on the classifications most common in the literature (SM 2.3.), as a way of keeping Galton's problem in check . Cardinality is above the random threshold (22%) in all seven families of scripts, and in 10 of 14 idiosyncratic scripts (modern inventions that cannot be traced back to one single ancestor) . The null model for cardinality has an intercept value of 61% . “Pure” letters like E or W are more prevalent than chance would predict, by 8 percentage points on average. The separation index is positive in all seven families and in 11 of 14 idiosyncratic scripts. It is weak in India and South‐East Asia, presumably because these scripts, which used to be written on flexible leaves, tend to avoid angular shapes for motor reasons . This tendency ran in the opposite direction as predicted, extinct scripts having a higher separation index compared to live ones. The null model estimates the separation index at +8%, clearly above the chance value of 0 . Vertical symmetry dominates horizontal symmetry in all seven script families, and in 8 of the 12 idiosyncratic scripts for which it could be measured. One outlier was identified: the Old Persian cuneiform syllabary, which has no vertical‐symmetrical letters at all. Removing it does not change the results of this paper. The second model that included each script's status (live or extinct) was less informative than the null model (AIC = −18 vs. −19 for the null model) and included no significant effect for being a live script as opposed to an extinct one . The null model estimates vertical symmetry prevalence at 70.6%  were identified (SM 2.4.). For each of the three features of interest , a distinct linear mixed model was built to predict the direction of changes in these ancestor‐descendant pairs of scripts. For each pair, the ancestor's value on the measure of interest was subtracted from the descendant's value. These differentials were grouped by ancestry: Scripts sharing the same ancestors were nested together. For each of the three features, the best‐fit linear mixed model showed an intercept value that did not significantly depart from zero, and in two cases, I found some support for the null hypothesis .3.6df = 65, t = −0.17, p = .87). Descendants show no tendency to be more cardinal than their ancestors. This lack of a general trend does not make cardinality a highly heritable trait: Descendant cardinality correlates with ancestor cardinality to a weak degree only . A Bayesian one‐sample t test was conducted to see whether the data supported the hypothesis that descendants do not, on average, decrease or increase their cardinality compared to their ancestor. It found moderate support for the null (BF = 3.7) , suggesting no tendency for scripts to expand the separation of oblique and cardinal strokes between letters. Here again, descendant scripts tend not to inherit their ancestor's separation index: the correlation between ancestor and descendant is undetectable . A Bayesian t test similar to the one conducted for cardinality found moderate support for the null hypothesis (BF = 4.7).The null model estimates descendant–ancestor differences in separation index at −0.2% , indicating only a weak increase with time. Vertical symmetry dominance in ancestor scripts was not found to correlate with their descendants’ . Given the small sample size, the data do not support the null hypothesis (BF = 1.8).The null model estimates descendant–ancestor differentials on this trait at +3.8%, , and the Bayes factor indicates moderate support for the null hypothesis (BFs between 3.5 and 4.9).As a post hoc analysis (at the editor's invitation), I replicated this result on three additional features of scripts (see SM 2.7). One I considered to be cognitively appealing: the proportion of horizontally or vertically symmetrical letters in the script . It fails to explain why our three features show in most idiosyncratic scripts, many of which have no clear pictographic past. It does not square with the fact that legibility does not decline when new scripts originate from their ancestors. It is also worth noting that none of the three features documented here are heritable to an important degree. This failure of cultural inheritance to preserve basic visual features like cardinality contrasts with high fidelity in other domains. Direction of reading  is a case in point. It is highly preserved, with more than 95% of scripts staying in line with their ancestor . Thus, something beyond mere inertia seems to be sustaining our three features: They keep being reinvented, with little contribution from cultural inheritance.An evolutionary scenario is often deemed more likely. In this view, writing systems would have been selected by cultural evolution to fit cognitive constraints. Two indicators of cultural evolution dynamics were examined here: selective extinction, and transformations between ancestor scripts and descendants. Selective extinction shows a small but significant effect for cardinality, not for the other two features. No clear indication was found that scripts branching out from older scripts rate more highly on the aspects of legibility examined here. To be precise, the data provided moderate support for the null hypothesis in three of four relevant cases , not for the fourth one . The dataset is not lacking in historical depth: It covers at least three millennia—most of the recorded history of writing. How could this apparent weakness of evolutionary trends be explained?The shape of letters is subject to two competing dynamics of attraction: motor constraints and visual constraints. Writers should try to minimize the effort involved in producing letters, but in so doing they risk a diminution in legibility. Faster hand‐writers produce less legible letters , and that they changed little since their invention. It has been claimed that the first writing systems were developed by small communities of specialists over short time spans, perhaps no more than one generation Houston, . This isOnline Supplementary Material 1. The complete dataset on all the “ISO 15924” scripts included in the study is accessible at this address: https://osf.io/8hgr5/.Online Supplementary Material 2. Methodological appendix. https://osf.io/8hgr5/.Click here for additional data file.


However, the interaction between abiotic stress-induced [Ca2+]i increases and biotic stress-induced [Ca2+]i increases is still not clear. Employing an aequorin-based Ca2+ imaging assay, in this work, we investigated the [Ca2+]i changes in response to flg22, Pep1, and NaCl treatments in Arabidopsis thaliana. We observed an additive effect on the [Ca2+]i increase which induced by flg22, Pep1, and NaCl. Our results indicate that biotic and abiotic stresses may activate different Ca2+ permeable channels. Further, calcium signal induced by biotic and abiotic stresses was independent in terms of spatial and temporal patterning.To survive, plants must respond rapidly and effectively to various stress factors, including biotic and abiotic stresses. Salinity stress triggers the increase of cytosolic free Ca In the natural environment, plants have to continuously cope with various stress factors, such as salt, drought, attacks of herbivorous insects, and invasion of microbial pathogens. To survive, plants should respond rapidly and effectively to each stressor. Recent studies revealed that about 10 million hectares of agricultural land are abandoned every year due to high salinity . Plant d2+) signaling has been implicated in regulating many perspectives of plant growth and responses to the environment  that lasts for approximately 2 min  or damage-associated molecular patterns (DAMPs) is a rapid change of [Ca2+]i and concomitant membrane depolarization (Arabidopsis so far are EF-Tu/EFR (elongation factor thermo unstable receptor) and flagellin/FLS2 (flagellin-sensitive 2), with the peptides elf18 and flg22, respectively, functioning as the elicitor-active PAMPs  is a topic, which is also worthy of investigation.From recent studies, we know that both biotic and abiotic stresses can trigger a rapid increase in cytosolic CaArabidopsis. We found that the increases of [Ca2+]i induced by both stimuli were higher than those induced by a single stress, suggesting that biotic and abiotic stresses have an additive effect on [Ca2+]i. We also found that flg22-induced [Ca2+]i increases may inhibit both PAMP- and DAMP-activated [Ca2+]i channels via a feedback mechanism, but not abiotic-activated [Ca2+]i channels. These results suggest that the responses involve in both inhibitory feedback mechanisms, as well as an interaction between the stimuli-mediated Ca2+ signaling pathways.In this study, we systematically investigated and analyzed the relationship and interaction between biotic and abiotic stresses in Arabidopsis thaliana ecotype Col-0 constitutively expressing intracellular Ca2+ indicator aequorin (pMAQ2) is a gift from M. Knight and the principles of how the active aequorin is formed can be found in Arabidopsis plants were grown in 150 mm × 15 mm round Petri dishes in half-strength Murashige and Skoog salts , supplemented with 1.5% (w/v) sucrose (Sigma), and 0.8% (w/v) agar (Becton Dickinson) adjusted to pH 6.0 with KOH in controlled an environmental room at 21 ± 2°C. The fluency rate of white light was ∼110 μmol m-2 s-1. The photoperiods were 16 h light/8 h dark cycles. Seeds were sterilized with 2.5% plant preservative mixture (Caisson Laboratories) and stratified at 4°C for 3 days in the dark, and then transferred to the growth room.Arabidopsis thaliana plants expressing cytosolic apoaequorin were used for [Ca2+]i measurements (in vivo by spraying seedlings with 3.3 mL of 10 μM coelenterazine (from Prolume) per Petri dish followed by incubation at 22°C in the dark for 8 h. Treatments and aequorin luminescence imaging were performed at room temperature using a ChemiPro HT system, which includes a cryogenically cooled and back-illuminated charge-coupled device (CCD) camera, liquid nitrogen autofiller, camera controller, and computer-equipped WinView/32 software (Roper Scientific) as described previously (2 in 10% (v/v) ethanol and recorded for 5 min until values were within 1% of the highest discharge value  was added into Petri dish in the dark, and luminescence was recorded. For changes in bath solution, a four-channel peristaltic pump  was used to perfuse Petri dish with water as indicated in the figures. Then, additional stress treatment was applied by adding 100 mL solution into Petri dish.For stress treatments, Petri dishes were placed individually into the ChemiPro HT chamber and luminescence images were started 80 s prior the treatment and taken at 20 s intervals or 7 min continuously. The treatment solution (100 mL) at 1 μM concentrations of flg22 and Pep1 , which w2+ concentration ([Ca2+]i) can be monitored by the bioluminescent Ca2+-binding protein aequorin in vivo  at any measurement point is normalized to the total remaining aequorin . The magnitudes of [Ca2+]i increases were found to be dependent on the concentration of flg22, with a higher concentration of flg22 evoking a greater increase in [Ca2+]i. The flg22 concentration required for a half-maximal response  was 1 μM, which was chosen as an optimum concentration for the subsequent analysis of interaction with Pep1- and NaCl-induced increases in [Ca2+]i. Next, we determined the temporal dynamics of flg22-induced [Ca2+]i increases under the imposed experimental conditions as a control for further comparison . We found that [Ca2+]i increased immediately after the application of 1 μM flg22, reached a peak at about 120 s, and then declined gradually . Imaging aequorin bioluminescence for less than 20 s resulted in images with a low signal-noise ratio in our system. Thus, the temporal resolution was set at about 20 s, which was sufficient for the current study. At about 200 s, the [Ca2+]i was reduced to a new resting level. Similarly, we analyzed the increases in [Ca2+]i in response to Pep1. Seedlings were treated with different concentrations of Pep1 from 0 to 4 μM, and [Ca2+]i was analyzed. As expected, Pep1 induced increases in [Ca2+]i in a dose-dependent manner . The [Ca2+]i increases recorded after single treatments were consistent with the results described above . The Pep1 concentration required to achieve a half-maximal response was around 1 μM, with the magnitude of [Ca2+]i similar to that induced by 1 μM flg22. We then determined the temporal dynamics of the [Ca2+]i increase induced by 1 μM Pep1. Following treatment with 1 μM Pep1, the [Ca2+]i increased and reached a peak at 140 s , then it took another 250 s for the [Ca2+]i to reach a new basal level. In overall, it seems that the increases of [Ca2+]i occur faster in response to flg22 than Pep1, but reset to a resting level 400 s after the treatment.To determine whether the mechanisms behind the increases of [Caaequorin . Plants  ± 21 nM . As expe2+]i signaling, plants were treated either with the same or different stimulus. When the Arabidopsis seedlings were treated with 1 μM flg22, the level of [Ca2+]i increased quickly to reach a peak, then decreased to the new resting level after 400 s , as described in Figure 1A. A subtle increase in [Ca2+]i could be detected in seedlings after they had been washed with deionized water at around 500 s . Next, flg22 was added again, which resulted in a very minor increase in [Ca2+]i. After 800 s, it decayed to a level similar to the previous resting level. Compared with the first flg22 treatment, which led to a large [Ca2+]i increase, the second flg22 treatment resulted in a [Ca2+]i increase that was only a fraction of the size of the first [Ca2+]i increase. This observation suggests that the flg22-activated Ca2+ permeable channel may be desensitized or adapted by some unknown signaling elements upstream. To test whether the desensitization or adaptation occurs, we can (after waiting for 3 h) detect a normal [Ca2+]i increase in response to flg22. This result suggests that desensitization of the channel is likely to happen, which agrees with the results reported by Heese’s group . Interestingly, the peak of [Ca2+]i induced by 1 μM Pep1 was clearly greater than that of 1 μM flg22 (P < 0.001). After 900 s, the [Ca2+]i decreased to a new basal level . The lower inhibition of the Pep1-induced [Ca2+]i increase compared with the increase induced by the initial flg22 treatment suggests that the initial high level of [Ca2+]i, which resulted from the flg22 treatment, inhibited flg22 to a greater extent than Pep1 . By analogy, we used Pep1 as the first stimulus to treat the seedlings, and then analyzed the second treatment using flg22 or Pep1. When the second Pep1 was added to the Petri dish, following the first Pep1 treatment and water washing step at around 540 s the [Ca2+]i level stabilized to a point similar to previous resting levels . The column chart has also been used to clearly show the results, as in Figures 2C,F. However, when we used 1 μM flg22 to replace Pep1 at 600 s, the peak value was smaller but significantly higher than that induced by the second Pep1 treatment . Similarly, our results suggested that the high [Ca2+]i resulting from the initial Pep1 activation inhibited the MAMP-induced [Ca2+]i to a greater degree than the PAMP-induced [Ca2+]i .To further characterize the potential interaction between the different biotic stress stimuli-triggered [Ca’s group . Subsequ2+]i increases via different channels. It is of great importance to further characterize the interaction between biotic and abiotic stress stimuli-triggered [Ca2+]i signaling. For such a purpose, we treated the plants with both the biotic and abiotic stimulus. When the Arabidopsis seedlings were treated with 1 μM flg22, the level of [Ca2+]i increased rapidly to reach a peak, and decreased to the new resting level after 400 s. A subtle increase in [Ca2+]i could be detected in the seedlings after they were washed in deionized water at around 500 s . Next, NaCl was added, which caused a sharp increase in [Ca2+]i . The [Ca2+]i then decayed from 700 s to a level similar to previous resting level. Compared with the single NaCl treatment, which led to a large increase in [Ca2+]i increase , the NaCl treatment after the flg22 stimulus still resulted in an increase in [Ca2+]i that was similar to a single NaCl-induced increase in [Ca2+]i. This observation suggests that there may be no interaction between the NaCl-activated Ca2+ permeable channel (NaC) and flg22-activated Ca2+ permeable channel. To verify this hypothesis, we treated the seedlings with 200 mM NaCl. The [Ca2+]i increased quickly to reach a peak and then decreased to the new resting level after 150 s . Next, we added 1 μM of flg22, which caused an increase in [Ca2+]i similar to the effect with a single flg22 treatment. Subsequently, we used a DAMP elicitor Pep1 instead of flg22 to determine whether a DAMP-induced [Ca2+]i increase can affect an NaC. To clearly show the results, column chart were used in Figures 3B,D. As expected, it appeared that there was no interaction between the NaC and Pep1-activated Ca2+ permeable channel . Based on this study, our results suggest that abiotic and biotic stress stimuli-activated Ca2+ permeable channels may be completely independent of each other.Based on our study, we know that different biotic stresses may induce [Ca2+]i increases triggered by biotic and abiotic stresses, Arabidopsis seedlings were treated with 1 μM flg22, 1 μM Pep1, or 200 mM NaCl separately, or any two of these three elicitors. The [Ca2+]i increases recorded after single treatments were consistent with the results described above. When plants were treated with 1 μM flg22 and 1 μM Pep1 together, the peaks of [Ca2+]i were slightly larger than those induced by each individual stimulus . However, when plants were treated with 200 mM NaCl together with 1 μM flg22 or 1 μM Pep1, the peaks of [Ca2+]i were larger than those induced by each individual stimulus, showing an additive effect . To further analyze the difference in [Ca2+]i increases in response to both biotic and abiotic stresses, we calculate the [Ca2+]i to clearly illustrate the data. As shown in Figure 5B, compared with individual treatment with Pep1, plants treated with flg22 and Pep1 together show only a slight increase in [Ca2+]i. However, compared with individual treatment with NaCl, plants treated with flg22 and NaCl together, or Pep1 and NaCl together, show an increase in [Ca2+]i that is almost equal to that shown with the combined treatment . These results suggest that the flg22-induced and Pep1-induced [Ca2+]i increases may operate through similar Ca2+ permeable channels, though biotic stress-induced [Ca2+]i increases may occur as independent events. In other words, biotic and abiotic stresses may activate different Ca2+ permeable channels.To investigate thoroughly the relationship and interaction between [Ca2+]i. Treated either by flg22 with NaCl or Pep1 with NaCl, the increase of [Ca2+]i could indicate that biotic and abiotic stresses activate different Ca2+ permeable channels. While it is not clear whether the treatment of NaCl will affect the calcium signal process and peak value, which are caused by the flg22/Pep1 treatment. To answer this question, in our experiments, seedlings were treated either by 1 μM flg22 along with 200 mM NaCl or 1 μM Pep1 along with 200 mM NaCl. The results are shown in Figure 6. The shaded area in Figures 6A,D illustrates the difference between the co-treated plants and NaCl-only treated plants. The subtracted shaded area reveals that the dynamic process and peak value of [Ca2+]i achieved by the treatment of using both flg22 and Pep1 are similar to those delivered by using the flg22 or Pep1 alone . These results indicate that biotic and abiotic stresses induce the increase of [Ca2+]i through different Ca2+ permeable channels. Such results are consistent with our prediction.The co-treatment of biotic and abiotic stresses triggers an additive effect on the increase of [Ca2+]i in response to various abiotic and biotic stresses  in plants have been a topic of much interest over the past two decades  and concomitant membrane depolarization follows MAMP/DAMP perception, the interaction and interrelationship between many early MAMP/DAMP signaling components in Arabidopsis are not well understood. The lessening of the [Ca2+]i increases induced by both MAMPs and DAMPs observed in this study  suggests that the feedback inhibitory mechanism could inactivate the stimulus-activated Ca2+ permeable channels. Briefly speaking, elevated [Ca2+]i will inhibit the ion channels in plants. We speculate that this phenomenon may be similar to the depolarization process of receptor ion channels typically seen in animals .It is well known that recognition of PAMPs or DAMPs by PRRs leads to a first line of inducible defenses that restrict microbial propagation in multicellular organisms . Althoug animals . One par animals . However2+]i than using either flg22 or Pep1 alone , indicating that flg22 and Pep1 may, in part, share Ca2+ permeable channels flg22-C and Pep1-C . The flg22-C and Pep1-C are likely regulated by feedback inhibition , considering their desensitization seen in this study  as well as in previous reports. We demonstrated that repetitive flg22 treatments failed to trigger repetitive [Ca2+]i increases . This indicates that the flg22-C cannot be activated repetitively within a short period of time—that is, flg22-C is possibly desensitized. We can therefore deduce that a feedback inhibition may be involved in the desensitization process . Upon flg22 treatment, the flg22-C opens, leading to a localized increase in [Ca2+]i, flg22-C [Ca2+]i microdomain/puff. The flg22-C [Ca2+]i, in turn, signals the channel to close, which prevents further [Ca2+]i increases and allows the basal [Ca2+]i to be reset via Ca2+ pumps. Such feedback inhibition avoids any excessive increase in [Ca2+]i, which could be highly deleterious to plant cells. The same phenomenon was also observed with the activation of Pep1-C . Clearly, the most significant effect was observed after the initial treatment by flg22-C or Pep1-C, when the rate of [Ca2+]i increases induced by both flg22-C and Pep1-C decreased . It is most probably that localized flg22-C [Ca2+]i and Pep1-C [Ca2+]i merge to form a obviously global [Ca2+]i, the feedback of which then inhibits both flg22-C and Pep1-C . In contrast, flg22 or Pep1, together with NaCl, induced greater increases in [Ca2+]i than using flg22 or Pep1 alone, leading us to conclude that there is no interrelationship between the NaCs. It should be noted that our study does not prove that flg22-C, Pep1-C, and NaC are localized in discrete and different microdomains, instead illustrates that flg22-C, Pep1-C, and NaC may differ and interact via [Ca2+]i microdomains.flg22 and Pep1 induced slightly increases of [Ca2+]i increases differ, but they all belong to the same pattern. In plants, biotic stresses that include PAMP- and DAMP-induced [Ca2+]i increases are similar in spatial and temporal patterning. Thus, we treated plants with flg22 together with Pep1, and found that the [Ca2+]i peaks were slightly larger than those induced by each stimulus alone, showing an enhanced signaling mechanism . This is similar to NaCl- and H2O2-induced [Ca2+]i increases . These results suggest that biotic and abiotic stresses may activate different Ca2+ permeable channels. In Figure 6, we present the calcium signal oscillation curve within the same image. We noticed that the calcium signal induced by biotic and abiotic stresses was independent in terms of spatial and temporal patterning. This study further demonstrates that early signaling is relatively independent to biotic and abiotic stresses.To some extent, PAMP- and DAMP-induced i signatures as well as other downstream events could be analyzed further once their molecular nature being revealed.Plants resist biotic and abiotic stresses by triggering two different sets of calcium signaling pathways. This is of great significance to the plant’s survival strategies. It will be important for future research to analyze the pharmacological properties of these putative CaSZ, Z-MP, and Z-HJ conceived and designed the experiments. X-QC, SZ, Y-YY, and YY performed the experiments. SZ, X-QC, and Z-MP analyzed the data. L-PK contributed reagents/materials/analysis tools. SZ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ca2+ flux into mitochondria is mediated by the mitochondrial calcium uniporter (MCU) channel whereas Ca2+ export is often through an electrogenic Na+–Ca2+ exchanger. Here, we report remarkable functional versatility in mitochondrial Na+–Ca2+ exchange under conditions where mitochondria are depolarised. Following physiological stimulation of cell-surface receptors, mitochondrial Na+–Ca2+ exchange initially operates in reverse mode, transporting cytosolic Ca2+ into the matrix. As matrix Ca2+ rises, the exchanger reverts to its forward mode state, extruding Ca2+. Transitions between reverse and forward modes generate repetitive oscillations in matrix Ca2+. We further show that reverse mode Na+–Ca2+ activity is regulated by the mitochondrial fusion protein mitofusin 2. Our results demonstrate that reversible switching between transport modes of an ion exchanger molecule generates functionally relevant oscillations in the levels of the universal Ca2+ messenger within an organelle.Mitochondrial Ca 2+ homoeostasis is tightly regulated and export of Ca2+ is mediated by an Na+Ca2+ exchanger. Here authors show that in depolarised mitochondria the exchanger initially operates in reverse mode, transporting cytosolic Ca2+ into the matrix before it reverts to its forward mode state.Mitochondrial Ca The porous outer mitochondrial membrane is freely permeable to Ca2+ but the inner membrane is not and therefore requires transporters to shuttle Ca2+ between the cytosol and mitochondrial matrix2. A major route for mitochondrial Ca2+ uptake is through the mitochondrial Ca2+ uniporter (MCU), a highly Ca2+-selective low conductance ion channel4. MCU is part of a larger complex involving regulators MICU1 and MICU2, MCUR1 and EMRE5. Flux through the MCU complex is determined by the prevailing electrochemical Ca2+ gradient6, with a major factor being the large electrical driving force that arises from the negative potential (~−200 mV) across the inner mitochondrial membrane.Mitochondrial Ca2+ transporters that extrude Ca2+ from the matrix have also been characterised at a molecular level and include Letm1 (leucine zipper-EF-hand-containing transmembrane protein 1)7 and mitochondrial Na+–Ca2+ exchange (NCLX)8. Letm1 is a Ca2+/2H+ electroneutral antiporter whereas NCLX is thought to be electrogenic9, although the precise Na+:Ca2+ stoichiometry is unclear10. In one study where the relative contributions of Letm1 and NCLX to mitochondrial Ca2+ export was investigated, NCLX was found to play the dominant role11.Ca12. The precise architecture of mitochondria is important for cell viability, growth, proliferation and signalling13. Mitochondrial fusion is regulated by dynamin-related protein (Drp1) where outer and inner mitochondrial membrane fusion depend on mitofusin 1 and mitofusin 2, and OPA1, respectively12. Mitofusin 2 is also found on the endoplasmic/sarcoplasmic reticulum surface and is therefore thought to act as a physical tether bringing mitochondria and endoplasmic/sarcoplasmic reticulum together at specialised regions called mitochondrial associated membranes14. Close apposition of the two organelles allows for rapid and effective local Ca2+ signalling16. Ca2+ release from the endoplasmic reticulum by the Ca2+-releasing second messenger inositol trisphosphate (InsP3) leads to a high local Ca2+ signal that can be transported into mitochondria by the MCU. The rise in matrix Ca2+ stimulates rate-limiting enzymes in the Krebs cycle17, resulting in accelerated ATP production. In the heart, for example, mitochondrial fusion dynamics depends on contractile activity18. In cardiac myocytes, shuttling of Ca2+ released from the sarcoplasmic reticulum by ryanodine receptors into mitochondria drives rapid bioenergetic responses that are important for cardiac function19.Mitochondria are dynamic organelles, undergoing fusion and fission with the capacity to form reticular networks3 (ref. 20). Low concentrations of agonist, which are thought to mimic physiologically relevant doses, typically evoke oscillations in cytosolic Ca2+. The oscillations arise from regenerative Ca2+ release from the endoplasmic reticulum by InsP3-gated Ca2+ channels followed by store-operated Ca2+ entry20. Previous work has shown cytosolic Ca2+ oscillations following stimulation of native cysteinyl leukotriene type I receptors in mast cells with the natural agonist leukotriene C4 are propagated rapidly and faithfully into mitochondria to generate oscillations in matrix Ca2+ (ref. 21). Knockdown of the MCU or mitochondrial depolarisation, which impairs Ca2+ flux through the MCU, suppressed mitochondrial Ca2+ uptake21. Loss of mitochondrial Ca2+ buffering resulted in rundown of cytosolic Ca2+ oscillations, which arose through enhanced Ca2+-dependent inactivation of InsP3 receptors.Stimulation of Gq protein-coupled receptors activate phospholipase C to generate InsP2+ oscillations are sustained by Ca2+ entry through store-operated Ca2+ channels, which refill the endoplasmic reticulum with Ca2+ following InsP3-evoked Ca2+ release20. In mast cells and T lymphocytes, mitochondrial Ca2+ uptake sustains Ca2+ entry by reducing Ca2+-dependent slow inactivation of the store-operated Ca2+ channels23. In addition, mitochondria also regulate the redistribution of STIM1, a molecule necessary for the activation of store-operated Ca2+ channels, from the endoplasmic reticulum to the plasma membrane24. In mast cells, inhibition of store-operated Ca2+ influx following mitochondrial depolarisation can be rescued by knockdown of the mitochondrial fusion protein mitofusin 2 (ref. 24). In this study, we show that mitochondrial Ca2+ oscillations induced by leukotriene receptor stimulation that are lost following mitochondrial depolarisation can be rescued when mitofusin 2 levels are reduced. We find that the mitochondrial Ca2+ oscillations under these conditions arise from mitochondrial Na+–Ca2+ exchange, operating sequentially in reverse and forward transport modes. Our data show that the same transport molecule can both raise and lower matrix Ca2+ in response to receptor stimulation. More generally, our results reveal that an ion transporter can compensate for impaired activity of an ion channel and thereby sustain functionally relevant Ca2+ signals within an organelle.Cytosolic Ca4 (LTC4) increases the levels of the second messenger InsP3, which triggers oscillations in cytosolic Ca2+  depolarised and remained low after stimulation with LTC29. However, the rescue of cytosolic Ca2+ oscillations that was achieved by knockdown of mitofusin 2 in cells with depolarised mitochondria  and some responded by generating 1 or more small matrix Ca2+ spikes (25%). Ouabain had no inhibitory effect when cells were stimulated with agonist in the absence of FCCP and oligomycin , a couple of small Ca2+ oscillations developed followed by a quiescent period and then one or two oscillations reappeared several tens of seconds later  of FCCP-treated cells overexpressing Na+–Ca2+ exchange but no responses (21/21 cells) were observed when the catalytic mutant was expressed instead  to intact cells. In cells in which we reduced both MCU and mitofusin 2 expression and then depolarised mitochondria, oscillations in matrix Ca2+ to leukotriene receptor stimulation were rescued only if matrix Ca2+ had been increased slightly by ionomycin prior to agonist exposure  NasP3 Fig.  and rema157 Fig. . Collect2+ to 10 μM led to a large rise in matrix Ca2+ and this was suppressed by ruthenium red  and a whole mitoplast current of ~800 pA, suggesting a single mitoplast expresses ~500 channels6. Therefore we simulated matrix Ca2+ oscillations for between 1 and 10,000 exchangers. The number of exchangers in Fig. 2+ buffering capacity of 100 . Reverse mode Na+–Ca2+ exchanger activity required a small increase in matrix Ca2+. This was accomplished by the very low flux through the MCU in depolarised mitochondria or after artificial elevation of matrix Ca2+ by a low dose of ionomycin. Whether matrix Ca2+ directly binds to the exchanger or its effects are mediated by an intermediary mechanism is currently under investigation.The mechanism whereby mitofusin 2 regulates reverse mode but not forward mode is currently unclear but does not require mitochondrial fusion because knockdown of OPA1 did not replicate the effects of reduction in mitofusin 2. Mitofusin 2 is in the outer mitochondrial membrane and Na+–Ca2+ exchanger, alternating between transport modes generates oscillations in Ca2+ within the mitochondrial matrix and these faithfully reflect the cytosolic Ca2+ oscillations that trigger exchanger activity. Although the MCU is the main route for mitochondrial Ca2+ uptake, our data reveal that the exchanger can, under certain conditions, provide an alternative route for Ca2+ entry. More generally, our data show that forward and reverse mode activities of an ion transporter can substitute for a Ca2+ channel in generating Ca2+ signals evoked by a physiologically relevant agonist.The mitochondrial Na2) in Dulbecco’s modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine and penicillin–streptomycin. RBL cells were transfected with the AMAXA system, using nucleofector cell line kit V solution  and program T-30 were used. Transfection of HEK293 cells was achieved using the lipofectamine method35. For transfection of one dish (10 cm), 50 μl of Opti-MEM was mixed with 4 μl of Lipofectamine 2000  in one eppendorf tube and 50 μl of Opti-MEM was mixed with the required amount of DNA or siRNA in another eppendorf tube. The components were gently mixed by pipetting and after 5 min all the components were mixed together. This transfection mixture was then incubated at room temperature for 20 min to generate lipoplexes for transfection. Thereafter, 1 ml of cell culture medium was added to the transfection mixture, which was then pipetted into the dish. After 1 h incubation at 37 °C and 5% CO2, the transfection mixture containing medium was replaced by 2 ml fresh medium. Cells were cultured for 24–48 h in an incubator and then used for experiments.Rat basophilic leukaemia (RBL-1) and HEK293 cells were purchased from ATCC and were cultured  for 40 min at room temperature in the dark and then washed several times in standard external solution of composition (in mM): NaCl 145, KCl 2.8, CaCl2 2, MgCl2 2, d-glucose 10, HEPES 10, pH 7.4 with NaOH. After a 15 min de-esterification period, cells were alternately excited at 356 and 380 nm (20-ms exposures) and emission collected >505 nm. Images were acquired every 2 s. Ca2+ signals are plotted as R, which denotes the 356/380 nm ratio.Cytosolic Ca2 2, MgCl2 2, d-glucose 10, HEPES 10, pH 7.4 with NaOH. Cells were alternately excited at 490 and 440 nm (20-ms exposures) and images were acquired every 5 s. The 490/440 nm ratio was calculated and pH signals are plotted as R/R0, where R0 denotes the resting ratio.Cells were incubated with BCECF-AM (5 μg per ml) for 30 min at room temperature in the dark and then washed three times in standard external solution of composition (in mM): NaCl 145, KCl 2.8, CaCl2+ was measured using the genetically encoded ratiometric pericam protein21. Following transfection, with pericam, recordings commenced 24 h later using the TILL Photonics system. Cells were illuminated alternatively at 430 and 488 nm (20 ms exposures) at 0.5 Hz and the emitted light was filtered at >510 nm.Matrix CaCells were loaded with TMRE (50 nM) in standard external solution for 30 min in the dark, followed by several washes in external solution. Cells were excited at 545 nm and emitted light was collected at >560 nm.2PO4, 10 mM HEPES, 1 mM MgCl2, 1 mM Na-pyruvate, 2 mM Mg-ATP, 50 μM EGTA (pH 7.2). Low Na+-based intracellular medium contained 129 mM KCl, 2 mM KH2PO4, 10 mM HEPES, 1 mM MgCl2, 1 mM Na-pyruate, 2 mM Mg-ATP, 50 μM EGTA (pH 7.2). Li+-based intracellular medium contained: 120 mM KCl, 9 mM LiCl 2 mM KH2PO4, 10 mM HEPES, 1 mM MgCl2, 1 mM Na+-pyruvate, 2 mM Mg-ATP, 50 μM EGTA (pH 7.2). In a separate series of experiments, the exposure time for digitionin was determined experimentally by tracking the loss of the fura 2 fluorescence signal at 356 nm from the cytosol after loading with fura 2-AM, as described for cytosolic Ca2+ measurements. Intracellular solutions with different Ca2+ concentrations  were perfused onto the cells as indicated in the text.Cells expressing pericam were permeabilised by exposure to 5 μM digitonin for 5 min in an intracellular medium containing: 120 mM KCl, 9 mM NaCl, 2 mM KHTotal cell lysates were separated on 10% denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) plus 0.1% Tween 20 (PBST) buffer for 1 h at room temperature, were washed with PBST three times and then incubated with primary antibody overnight at 4 °C. ERK2 antibody was from Santa Cruz Biotechnology (Cat. No. sc-154) and was used at a dilution of 1:5000. The antibodies against mitofusin 2 and OPA1 were from Santa Cruz Biotechnology (Cat. No. sc-100560) and BD Transduction Laboratories (Cat. No. 612606) respectively, and used at dilutions of 1:250 and 1:1000. NCLX antibody was kindly provided by Prof. Israel Sekler and was used at 1:1000–1:2000 dilution. The membranes were then washed with PBST again and incubated with 1:2500 dilutions of peroxidase-linked anti-rabbit IgG from Santa Cruz Biotechnology (Cat. No. sc-2004) or anti mouse IgG from BD Bioscience (Cat. No. 554002) for 1 h at room temperature. After washing with PBST, the bands were detected by an enhanced chemiluminescence plus western blotting detection system (Amersham Biosciences). Blots were analysed by UN Scan software.2O, the Master Mix (Applied Biosystems) and Probe & Primer Mix (Applied Biosystems). The mRNA levels of MCU were normalised to GAPDH. Data were analysed by using ABI7000 System Software.After treatment, cells were washed with PBS and total RNA was extracted by using an RNeasy Mini Kit (Qiagen). RNA was quantified spectrophotometrically by absorbance at 260 nm. Total RNA (1 µg) was reverse-transcribed using the iScriptTM cDNA Synthesis Kit (Bio-Rad), according to the manufacturer’s instructions. Following cDNA synthesis, PCR amplification was then performed using BIOX-ACTTM. ShortDNAPolymerase (Bioline) with primers specific for the detection of c-fos and beta-actin  were syn2+-dependent gene expression. At 24–36 h after transfection with the GFP plasmid, cells were stimulated with LTC4 and the % of cells expressing GFP subsequently quantified ~24 h later per field of view35. Cells were stimulated with LTC4 in culture medium for 8 min and then medium was changed (to remove agonist) and cells were then maintained in the incubator for ~24 h prior to detection of GFP. FCCP/oligomycin was applied 5 min prior to LTC4 exposure and was washed out with agonist after 8 min stimulation.GFP under an NFAT promoter  was used as a reporter of CaTM 5193986). siRNA against human mitofusin 2 was from Origene (Cat No: SR306670). siRNA against OPA1 was from Origene (Cat No: SR505373). siRNA against MCU was from Origene (Cat No: SR508660). siRNA against the rat mitochondrial Na+–Ca2+ exchanger was from Invitrogen (AACGGCCACUCAACUGUCU) and human mitochondrial Na+–Ca2+ exchanger was from Origene (Cat No: SR312772). Sequences are listed in the siRNA against rat mitofusin 2 was from Invitrogen  we model the change in concentration of free mitochondrial matrix Ca2+ ([Ca2+]mito) due to a given number of NCX in the mitochondrial membrane. The mitochondria are modelled as having a volume of Vmito = 1.31 × 10−15 l . We model the mitochondrial matrix as a ‘well mixed’ single compartment, so the change in total mitochondrial [Ca2+]mito_total is given byNNCX is the total number of NCXes in the mitochondrial membrane, and NA is Avogadro’s constant. [Ca2+]mito is then given byGiven a known concentration of Ca2+]cyt is taken from our fluorescence measurements of cytosolic Ca2+ and scaled logarithmically such that the low concentration is 100 nM and the high concentration is either 1.5 or 15 μM as 10 mM. We do not model changes in sodium concentrations as these levels are far above those for [Ca2+]mito_total and are assumed to be roughly constant. Mitochondrial membrane potential was set to −20 mV, and temperature to 298 K. The initial condition for [Ca2+]mito_total was set to steady state before beginning the simulation. The code to run the simulations shown in Fig. [Cat-test or by analysis of variance (ANOVA) for multiple groups. Differences were considered statistically significant at values of p < 0.05.Results are presented as mean ± SEM. Data were compared using Student’s Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


De novo and acquired drug resistance and subsequent relapse remain major challenges in acute lymphoblastic leukemia (ALL). We previously identified that pevonedistat , a first-in-class inhibitor of NEDD8 activating enzyme (NAE), elicits ER stress and has potent in vitro and in vivo efficacy against ALL. However, in pevonedistat-treated ALL cell lines, we found consistent activation of the pro-survival MEK/ERK pathway, which has been associated with relapse and poor outcome in ALL. We uncovered that inhibition of the MEK/ERK pathway in vitro and in vivo sensitized ALL cells to pevonedistat. The observed synergistic apoptotic effect appears to be mediated by inhibition of the MEK/ERK pro-survival cascade leading to de-repression of the pro-apoptotic BIM protein. Mechanistically, Ca2+ influx via the Ca2+-release-activated Ca2+ (CRAC) channel induced protein kinase C β2 (PKC-β2) was responsible for activation of the MEK/ERK pathway in pevonedistat-treated ALL cells. Sequestration of Ca2+ using BAPTA-AM or blockage of store-operated Ca2+ entry (SOCE) using BTP-2 both attenuated the compensatory activation of MEK/ERK signaling in pevonedistat-treated ALL cells. Pevonedistat significantly altered the expression of Orai1 and stromal interaction molecule 1 (STIM1), resulting in significantly decreased STIM1 protein levels relative to Orai1. Further, we identified eIF2α as an important post-transcriptional regulator of STIM1, suggesting that pevonedistat-induced eIF2α de-phosphorylation selectively down-regulates translation of STIM1 mRNA. Consequently, our data suggest that pevonedistat potentially activates SOCE and promotes Ca2+ influx leading to activation of the MEK/ERK pathway by altering the stoichiometric Orai1:STIM1 ratio and inducing ER stress in ALL cells. Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and adolescents and has an overall 5-year event-free survival (EFS) of 80%, whereas adults with ALL have a 5-year EFS rate under 50% , 2. For in vitro and in vivo anti-leukemic effects . Ta. Ta2+ mo+ influx . Indeed,2+ influx by migrating to ER-plasma membrane appositions (puncta) to interact and open Orai1 [Having established the role of CRAC and its pore-forming subunit Orai1 in pevonedistat-induced calcium influx and MEK/ERK activation in ALL, we examined the mechanistic interaction between Orai1 and the ER-membrane protein STIM1, which orchestrates Caen Orai1 . It has en Orai1 , 30. Ind2+ influx, albeit other known mechanisms such as the induction of ER stress per se may also play a role in this cascade.To investigate the regulatory role of the Orai1:STIM1 ratio on the activation of MEK/ERK signaling in pevonedistat-treated ALL cells, we examined the effects of down-regulating STIM1 on p-ERK1/2 activation by pevonedistat. Figure The fact that pevonedistat induced UPR-mediated cell death via de-phosphorylation of p-eIF2α (Ser51) in ALL cells, altering the UPR regulation of protein translation , promptewww.ncbi.nlm.nih.gov/refseq) for putative 5’-uORF regulatory sequences within human, mouse and rat STIM1 genes, and identified only one 5’-uORF conserved sequence among these three species  [in vivo inhibitory effects of pevonedistat on the NEDDylation of cullins, and activation of p-ERK1/2 in the NALM6-Luc cells harvested from mice treated with pevonedistat ± SEL, and these findings were comparable to the in vitro data obtained with ALL cell lines and primary ALL cells [in vivo mimics the more extensive analysis conducted in vitro using ALL cell line models and patient-derived samples, highlighting the potential clinical relevance of our findings.The LM6-Luc) . As prevLM6-Luc) , followiLL cells . Therefo2+ release-activated Ca2+ (CRAC) channel [2+ via activation of the store-operated Ca2+ entry (SOCE) leading to augmentation of the CRAC-mediated Ca2+ influx, and activation of the PKC/MEK/ERK signaling cascade. This is evidenced by direct measurement of intracellular calcium using the Ca2+ binding Fluo-8 AM dye, and the CRAC inhibitor BTP-2 which clearly demonstrated that pevonedistat increased Ca2+ influx in ALL cells, and that activation of p-ERK1/2 was significantly reduced in presence of the Ca2+ chelator BAPTA-AM or the PKC inhibitor enzastaurin. Furthermore, Ca2+ influx was shown to be triggered by changes in the stoichiometric ratio between the CRAC’s pore-forming subunit Orai1 and the Ca2+ sensor protein STIM1 [2+ homeostasis regulation as a second messenger in ALL cells’ response to stress conditions.This study identified the Ca channel  as the min STIM1 , 16 [29,in STIM1 . Our datin STIM1 , 36, 37.2+ influx [2+ regulation in pevonedistat-treated ALL cells, we showed that in NALM6 cells overexpressing Orai1-YFP, the fluorescent signal was distributed evenly in the plasma membrane with undetectable intracellular localization, whereas in the pevonedistat-treated NALM6-Orai1-YFP expressing cells the signal was clustered in the plasma membrane with minimal intracellular localization. These findings are consistent with pevonedistat-induced alteration of the Orai1:STIM1 ratio leading to CRAC activation and increased Ca2+ influx, a critical event required for activation of the PKC/MEK/ERK pathway. Consequently, we propose that de-phosphorylation of p-eIF2α by pevonedistat will not only lead to ER stress/UPR-mediated cell death via increased protein translation in ALL as previously reported [2+ influx to subsequently activate the compensatory survival PKC/MEK/ERK pathway as depicted in the proposed model , the UP folding . However2+ influx in pevonedistat-treated ALL cells is manifested by the downstream activation of the pro-survival MEK/ERK pathway. Recently, Zhou et al. [2+ influx, and that co-targeting the NEDD8 conjugation pathway plus the MEK/ERK pathway led to in vitro [in vivo synergistic cell death . Successful treatment strategies for ALL continue to require the use of multi-agent regimens, and given that multi-agent therapy is still important in patients with relapse, a favored strategy for the introduction of new agents into ALL therapy has been to incorporate promising new agents into established multi-agent platforms, such as vincristine, prednisone (frequently replaced by dexamethasone in practice), L-asparaginase, and doxorubicin (VPLD). An example of this strategy is the successful incorporation of bortezomib into VPLD therapy for ALL recently reported by Messinger et al. [in vitro and in vivo synergy between pevonedistat and dexamethasone in ALL [Our pre-clinical data show significant synergistic effect of pevonedistat and SEL in ALL, and suggest a clear path for future translation into the clinic for both children and adults with ALL. Indeed, we are initiating a clinical trial of pevonedistat in combination with reinduction chemotherapy for adolescents and young adults with refractory/relapsed ALL , supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cellgro) and antibiotics , was used to culture the ALL cell lines CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL) at 37 °C with 5% CO2. Stably transfected NALM6 cells expressing the luciferase gene (NALM6-Luc) were generated as described . PevonedCell viability and cell death were measured with trypan blue exclusion using the Vi-CELL XR analyzer . Synergism of combinational drug treatments was determined by calculating the combination index (CI) using CalcuSyn Version 2.0  as described . StatistStable NALM6 cells expressing shRNAs against CHOP, ERO1, STIM1, Orai1, and BIM were generated using lentiviral particles obtained from Santa Cruz Biotechnology  and transduced by spinoculation as described . Stably Proteins were extracted using 1X RIPA buffer supplemented with protease/phosphatase inhibitor cocktail , and concentration determined using the BCA protein assay kit . Immunoblots (western blot) were performed as previously described . Primary2+ was measured using the Fluo-8 No Wash Ca2+ Assay Kit , a method previously validated by others [2+ measurement using microscopic immunofluorescence, cells were treated with BTP-2 (10 µM) or pevonedistat (400 nM) either alone or in combination (pevonedistat + BTP-2) for 24 h, washed twice with HHBS, and centrifuged at 1000 rpm for 5 min. Fluorescent cell images were captured using a Leica immunofluorescence microscope with a 10× magnification.Intracellular Cay others -52. BrieNALM6-Orai1-YFP expressing cells were treated with vehicle (DMSO) or pevonedistat (400 nM) for 24 h, washed with PBS, and transferred onto coverslips. Then, plates were sealed using nail polish. Images were captured using a Leica SP5 spectral confocal inverted microscope with a 60× magnification, and analyzed using Leica LAS X software.ALL cells engraftment in NSG mice, bioluminescent imaging and leukemia burdens were carried out as previously described . Once le


Long noncoding RNAs (lncRNAs) have emerged as a class of pivotal regulators of gene expression. Recent studies have shown that lncRNAs contribute to the initiation, maintenance, and development of acute myeloid leukemia (AML). In this review, we summarize the current knowledge of the lncRNAs that play critical roles in AML. We first briefly describe the characteristics of lncRNAs, and then focus on their regulatory roles in AML, including the modulation of differentiation, proliferation, cell cycle, and apoptosis. We further emphasize the action of lncRNAs during leukemogenesis by describing how they interact with RNA, protein and chromatin DNA to exert their functions. We also highlight an urgent need to investigate the mechanisms by which lncRNAs contribute to the pathogenesis of AML. Finally, we discuss the prognostic value of lncRNAs in AML patients. Acute myeloid leukemia (AML) is a group of hematopoietic malignancies with various genetic abnormalities, including chromosomal translocations and/or somatic mutations, which are mainly responsible for the abnormal proliferation, differentiation or survival of myeloid progenitors , 2. ExteRecently, long noncoding RNAs (lncRNAs) have emerged as a novel class of pivotal regulators of gene expression and has received increasing attention in the field of AML. LncRNAs are operationally defined as RNA larger than 200 base pairs that appear to lack coding potential. They participate in various cellular processes, like inflammatory response , neuronaThe latest studies have demonstrated that lncRNAs contribute to many critical signaling pathways in AML development and therapy. We summarize the reported AML-related lncRNAs and their roles in Table LncRNAs exert important roles in myeloid differentiation and can respond to differentiation induction therapy. At present, two lncRNAs, HOTAIRM1 and NEAT1 are known to regulate the differentiation of AML cells. HOTAIRM1 is a myeloid-specific long non-coding transcript, which is transcribed from the locus between HOXA1 and HOXA2 genes. HOTAIRM1 regulates myeloid differentiation genes such as CD11b and CD18, and its knockdown impairs all-trans retinoic acid (ATRA)-induced granulocytic differentiation . This woLncRNAs also exert effects on proliferation, cell cycle and apoptosis in AML cells. Such lncRNAs tend to be expressed more widely than those myeloid-specific transcripts that regulate differentiation. A typical example is lncRNA PVT1, which can promote the proliferation of AML cells . The oncRecently, the discovery of a novel class of lncRNAs piques the curiosity of scientists and potentially ignites their efforts to characterize them: fusion transcripts between coding genes and lncRNAs. In an AML patient sample and an AML-derived HL-60 cell line, it is verified that NSMCE2 rearrangement gives rise to two novel chimeric genes, PVT1-NSMCE2 and CCDC26-NSMCE2 . AlthougLncRNAs exert their functions through multiple ways Fig. .Fig. 1LnFirst, lncRNAs can interact with other RNA molecules, e.g. microRNAs and mRNAs. The lncRNAs that can act by competing with endogenous RNA to sequester microRNAs include BGL3, CCAT-1, HOTAIR and HOTAIRM1. BGL3 functions as a competitive endogenous RNA for binding miR-17, miR-93, miR-20a, miR-20b, miR-106a and miR-106b to cross-regulate PTEN expression . CCAT-1 Second, lncRNAs can bind protein partners to form a complex. For instance, a proposed mechanism for UCA1 action in proliferation regulation is to sequester hnRNP I, which is a positive translation regulator of p27 protein. Consistent with this hypothesis, it has been shown that UCA1 has a moderate binding capacity to hnRNP I and that UCA1 knock-down leads to an increased expression of p27 protein in AML cells . FurtherCurrently, orchestrating the chromosomal looping is a seldom-observed yet critical aspect regarding the action of lncRNAs in AML. Two lncRNAs have been reported to mediate the long-range regulation. Interestingly, both lncRNAs are transcribed from the loci that produce critical protein regulators. LncRNA RUNXOR is derived from the locus of transcription factor RUNX1 (AML1). It is 216 kb long and covers the entire locus of RUNX1, an important transcription factor in AML . The 3′ However, apart from the cases mentioned above, the mechanisms of lncRNA action in AML are still largely unknown. Further studies are required to investigate the interactome of lncRNAs, and to determine the causal relationship between the direct physical interaction and the resultant phenotypic changes. For example, although HOTAIRM1 and NEAT1 are found to be required for ATRA-induced myeloid differentiation in AML cells, the mechanism of how they exactly exert their functions is poorly known.Emerging studies have indicated that lncRNAs specifically expressed during myelopoiesis are under the direct control of hematopoietic transcription factors. C/EBPα (CCAAT/enhancer-binding protein-α) is one of key regulators in myelopoiesis. A genome-wide study on investigating C/EBPα-regulated lncRNAs has revealed that 930 lncRNAs, including 600 up-regulated and 330 down-regulated, are regulated by C/EBPα . At the PU.1 is another master regulator in myeloid differentiation. Most recently, we have found that the myeloid differentiation lncRNA HOTAIRM1 is a direct target of PU.1 . The up-R132 mutated patient samples have up-regulated DLEU2 as well as down-regulated RP11-147N17.1. RUNX1-mutated patients have up-regulated vault RNA 1-1 (VTRNA1-1) [In addition to the direct regulation by transcription factors, lncRNA expression is closely associated with recurrent mutations in AML , indicatTRNA1-1) . WhetherPresently, mechanisms behind the dysregulation of lncRNAs in AML are far less clear than their biological functions. For example, previous studies have shown the involvement of PU.1-AS and EGO in myelopoiesis , 22, butLncRNAs are associated with AML clinical features and outcomes. A recent study has shown that lncRNAs can be used to predict treatment response and outcome in older patients with cytogenetically normal AML. Garzon et al. have investigated the associations of lncRNA expression with clinical characteristics, recurrent mutations, and outcome in 148 cytogenetically normal older (age >60 years) AML patients and built a prognostic score based on the expression values of 48 lncRNAs that can been used to for outcome prediction . Future LncRNAs are promising candidates for cancer therapy, especially considering some of them are tissue-specific drivers of cancer. Preclinical researches have implicated the efficacy of lncRNA-targeted therapeutics in certain types of cancer. In the MMTV-PyMT breast cancer mouse model, Malat1 exerts oncogenic activity by enhancing cell proliferation and tumor metastasis, and serves as a potential target for treatment . PerturbIt becomes increasingly clear that lncRNAs play critical regulatory roles in AML. However, there are as yet unidentified lncRNAs and undiscovered machineries involved in AML. The road is still long before we fully understand their disease relevance and biological significance in AML. Genome-wide studies, in parallel with careful and in-depth investigations of individual lncRNAs, will greatly advance our understanding of how lncRNAs are involved in the initiation, maintenance and development of AML. Furthermore, the nature of the interactions between lncRNAs and other molecules, to a large extent, remains a mystery in AML. Resolving such issues requires the implementation of methodologies such as chromatin isolation by RNA purification (ChIRP) , capture


Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC). A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca2+- and GTPγS-induced contractions in β-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca2+ signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca2+]i), inhibited a stimulus-induced increase in [Ca2+]i, and inhibited a cyclopiazonic acid (CPA)-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca2+]i. Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca2+ mobilization pathway, possibly involving CPA-dependent Ca2+ entry via an AMPK-independent pathway. That MET activated AMPK without causing arterial relaxation suggests that AMPK activation does not necessarily cause VSM relaxation.Although recent studies reveal that activation of the metabolic and Ca Notably, MET, which caused an increase in AMPK-pT172 in EA , had no effect on contractions induced by any stimulus examined , and SIM that did not elevate AMPK-pT172  caused inhibition of contractions induced by all of the stimuli tested .To compare the relaxant effects of A-769662 with MET and SIM, EA was contracted in the presence and absence of 30 μM MET , in β-escin-permeabilized MA, 100 μM A-769662 increased the potency and maximum efficacy of a Ca2+ CRC . For comparison, BBR and SIM were also examined. In intact MA, 30 μM BBR, like 100 μM A-769662, reduced the potency of a PE CRC, and 30 μM SIM dramatically reduced the maximum efficacy . However, neither BBR nor SIM had an effect on a Ca2+ CRC in permeabilized MA . Together, these data indicate that when the cytosolic Ca2+ level is “clamped” at discrete levels (Ca2+-CRC in permeabilized muscle), A-769662, BBR and SIM did not cause VSM relaxation, suggesting that the relaxation of non-permeabilized (plasma membrane-intact) muscle by these agents was due to an alteration in Ca2+ regulation at the plasma membrane and not to signaling pathways down-stream from Ca2+, such as MLCK, or independent of Ca2+, such as ROCK.Chemical permeabilization abolishes plasma membrane Ca2+-induced contractions in permeabilized MA as expected . To further test the hypothesis that the A-769662-induced relaxation of contraction was not due to an inhibition of signaling systems downstream from, or independent of, Ca2+, permeabilized MA was contracted submaximally with Ca2+ at pCa 6.25, then contracted further by addition of GPTγS  to cause Ca2+ sensitization by activation of ROCK. A-769662 had no effect on the contraction induced by Ca2+  compared to control  or that induced by GTPγS  compared to control . As expected, TFP strongly inhibited the Ca2+-induced contraction , and the ROCK inhibitor H-1077 depressed the contraction induced by GTPγS . Moreover, A-769662 had no effect on the basal phosphorylation levels of MLC , rhoA , cofilin , and MYPT1 , and did not reduce the degree of active rhoA  in FA. Likewise, A-769662 did not cause an increase in PLB-pS16 in FA as did FSK, a positive control . Together, these data support the previous experiments suggesting that A-769662 did not cause inhibition of contraction by inhibiting MLCK or ROCK.As a positive control, the calmodulin and thus MLCK blocker TFP strongly inhibited Ca2+ regulation. To test this hypothesis, the effect of A-769662 on cytosolic free Ca2+ was examined in fura-2-loaded artery. Concomitant with its ability to inhibit contraction , 30 μM A-769662 inhibited the ability of a submaximum concentration of PE to induce a strong increase in [Ca2+]i . This was true both for RA  and MA . For a comparison, SIM also caused a strong inhibition of PE-induced contraction and increase in [Ca2+]i, whereas MET did not . Notably, A-769662 and SIM, and not MET, produced an immediate reduction in the basal level of [Ca2+]i . The strong reduction in force during a second contraction  that did not correlate with a reduction in [Ca2+]i  in MA was likely due to receptor-activation induced desensitization   , 1999.2+ mobilization. To determine whether inhibition of store-operated Ca2+ entry played a role in A-769662-induced relaxation, the ability of A-769662 to relax a CPA-induced contraction in FA was assessed. Inhibition of the SERCA pump by CPA causes the release of sarcoplasmic reticulum Ca2+ stores which, in turn, activates Ca2+ entry through store-operated Ca2+ channels located in the plasma membrane. In FA, 10 μM CPA caused an initial strong contraction that declined to a weaker steady-state response . A-769662 added during the tonic phase of a CPA-induced contraction (∼30 after CPA addition) caused an immediate relaxation , reducing the steady-state response at 90 min from ∼25% of the peak contraction to ∼10% . By comparison, the selective store-operated Ca2+ channel inhibitor YM-58483   and reduced the steady-state response to ∼-10% . That is, YM-58483 reduced force to a value less than the resting value, indicating that FA displayed a small degree of active tone. Together, these data suggest that A-769662 caused relaxation, in part, by immediate inhibition of store-operated Ca2+ channels.Data from the fura-2-loaded artery experiments suggest that A-769662 caused relaxation by inhibiting Caa, BTP2)  also cau2+]i, our results do not support the hypothesis that A-769662 causes contraction inhibition by activation of AMPK, or that AMPK activation is both necessary and sufficient to inhibit contraction. Rather, our working model is that A-769662, like SIM  and small (MA) rabbit muscular visceral arteries, 30 and 100 μM A-769662 reduced the basal level of i and stimulus-induced increases in [Ca2+]i which, in turn, reduced the strength of VSM contraction. Although contraction inhibition was produced in both large and small musculocutaneous and visceral arteries, the degree of inhibition displayed a dependency on both artery- and stimulus-type. Specifically, A-769662 strongly inhibited contractions induced by Ang II and U-46619 in all arteries examined, modestly inhibited contractions induced by PE in visceral arteries, and did not inhibit contractions induced by PE in musculocutaneous arteries. KCl-induced contractions were inhibited weakly in the larger FA and RA, and not inhibited in the smaller EA and MA. Thus, the effect of A-769662 may be dependent on the expression levels in different artery segments of a specific signaling systems regulating [Ca2+]i. Although A-769662 did not cause an increase in arterial AMPK-pT172, it did cause an increase in phosphorylation of the AMPK substrate ACC. Thus, although our study supports a model by which A-769662 caused contraction inhibition by an AMPK-independent mechanism, our data cannot rule out the possibility that A-769662 enhanced AMPK activity independently of an increase in AMPK-pT172 (2+ mobilization.In summary, based on the observations that an increase in AMPK-pT172 did not necessarily cause VSM relaxation, and that A-769662 did not increase AMPK-pT172 above the basal level, our working model is that A-769662 may have acted independently of an increase in AMPK-pT172 to inhibit basal [CaPK-pT172 , and thaYH was responsible for assisting in protocol development, data acquisition and analysis and in assisting in the drafting of the article. CS, GC, BS, and AM were responsible for data acquisition. RB was responsible for revising the article for intellectual content. PR was responsible for project concept, protocol development, data analysis and interpretation, drafting the article and revising the article for intellectual content, and for final approval of the completed article.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.


Investigation of the interactions between the cTnI mutants and WT cardiac troponin C (cTnC) or WT cardiac troponin T (cTnT) showed that all the mutations investigated, except R204G, affected either or both cTnI:cTnT and cTnI:cTnC interactions. The R204H mutation affected both cTnI:cTnT and cTnI:cTnC interactions while the R204C mutation affected only the cTnI:cTnC interaction. These results suggest that different mutations at the same site on cTnI could have varying effects on thin filament interactions. A mutation in fast skeletal TnI  also significantly increased Ca2+ sensitivity of force development (ΔpCa50 = 0.16). Our studies indicate that known cTnI mutations associated with poor prognosis (R204C and R204H) exhibit large increases in Ca2+ sensitivity of force development. Therefore, other R204 mutations that cause similar increases in Ca2+ sensitivity are also likely to have poor prognoses.Mutations in human cardiac troponin I (cTnI) have been associated with restrictive, dilated, and hypertrophic cardiomyopathies. The most commonly occurring residue on cTnI associated with familial hypertrophic cardiomyopathy (FHC) is arginine (R), which is also the most common residue at which multiple mutations occur. Two FHC mutations are known to occur at cTnI arginine 204, R204C and R204H, and both are associated with poor clinical prognosis. The R204H mutation has also been associated with restrictive cardiomyopathy (RCM). To characterize the effects of different mutations at the same residue (R204) on the physiological function of cTnI, six mutations at R204  were investigated in skinned fiber studies. Skinned fiber studies showed that all tested mutations at R204 caused significant increases in Ca Familial hypertrophic cardiomyopathy (FHC) is a genetically heterogeneous autosomal dominant inherited heart disease of the myocardium that results in a high incidence of sudden cardiac death and has been linked to mutations in genes coding for at least 10 sarcomeric proteins. These mutations have been found in the genes for: α-myosin heavy chain  complex  R204H mutation have a high incidence of sudden death  that are linked to FHC  in fsTnI was shown to be associated with distal arthrogryposis type 2B   refers to a group of disorders characterized by multiple congenital contractures of the hands/wrists and feet/ankles, and affected individuals typically have a triangular shaped face, a small mouth, a prominent chin and positional foot deformities .The cTnI R204 residue is well conserved in different animals suggesting that this residue is evolutionarily important. A comparison of the primary amino acid sequence of cTnI and fast skeletal Troponin I (fsTnI) shows that R204 residue corresponds to the R174 residue of fsTnI. It is hypothesized that changes at the fsTnI R174 residue will show similar increases in Ca2+ sensitivity of force development. The different effects of these mutants on the interaction between Tn subunits and the maximal force suggest that the change in Ca2+ sensitivity of force development may be a major determinant for the poor prognosis of patients with the cTnI R204C, R204H, and fsTnI R174Q mutations.Investigation of six cTnI R204 residues as well as the fsTnI R174Q mutation showed that all the mutations investigated increased the Ca2 and loaded onto an affinity column having covalently bound HcTnC. Pure HcTnI and HcTnImutants were eluted with a gradient of 0–3 mM EDTA and 1–6 M urea. The purity of the TnI proteins was determined by SDS-PAGE as previously described . A column with cardiac TnC immobilized onto CNBr-activated Sepharose-4B (2 mg TnC/ml agarose) was prepared as described by Pharmacia LKB Biotechnology. The dialyzed TnI was loaded onto the TnC-agarose column equilibrated with TnC affinity buffer. The bound TnI was eluted with TnC elution buffer containing 6 M urea and 3 mM EDTA . The eluates were run on SDS gels and purified TnI pooled.The purification of fsTnI was similar to previously described  containing 10−8 M Ca2+, 5 mM Mg2+, 7 mM EGTA, 20 mM MgATP, 20 mM creatine phosphate, and 15 U/mL creatine phosphokinase, pH 7.0, at an ionic strength of 150 mM at 4°C for 24 h. These skinned muscle preparations were dissected into small bundles  and were stored at –20°C in the same solution without triton X-100 before use.Cardiac skinned muscle fibers were prepared following a common laboratory procedure published by Zhang et al.  Zhang e,b. Fresh2+ concentration (10−4 M). To determine the Ca2+ dependence of force development, the contraction of the skinned fibers was tested in solutions of intermediate concentrations of Ca2+. The Ca2+ dependence of force was determined before and after performing the displacement and reconstitution protocols that are described below. To remove the endogenous Tn complex, the TnT displacement method was used  in pCa 8 solution for ~1.5 h at room temperature. To determine the Ca2+ dependence of force development, the contraction of skinned fibers was tested in solutions with intermediate concentrations of Ca2+ (from pCa 8 to pCa 4). The Ca2+ dependence was determined before and after treatment of the skinned fibers with displacement and reconstitution solutions. The Ca2+ dependence data were analyzed using the Hill equation : relative force (%) = [Ca2+]n/([Ca2+]n+[pCa50]n), where pCa50 is the pCa of a solution in which 50% of the change is produced, and n is the Hill coefficient.The skinned fiber preparation was mounted with stainless steel clips on a force transducer and was immersed in the contracting solution to measure initial force before treatment. The contraction solution (pCa 4) had the same composition as the relaxation buffer except for the increased Ca−8 M [Ca2+], 1 mM [Mg2+], 7 mM EGTA, 5 mM [MgATp2+], 20 mM imidazole, pH 7.0, 20 mM creatinine phosphate, and 15 units/ml creatinine phosphokinase, ionic strength = 150 mM. To determine the Ca2+ sensitivity of force development, the fibers were gradually exposed to the solutions of increasing Ca2+ concentrations, from pCa 8 to pCa 4. To displace the endogenous Tn complex from the fibers they were incubated in a solution containing 250 mM KCI, 20 mM MOPS, pH 6.2, 5 mM MgCl2, 5 mM EGTA, 0.5 mM dithiothreitol, and 1.6–1.8 mg/ml fsTnT, for 1 h at room temperature. A fresh fsTnT protein was applied to the fibers for another 1 h incubation. This was to increase the efficiency of the endogenous Tn displacement from the fibers. Displaced fibers were then washed with the same solution without the protein (10 min at room temperature) and tested for Ca2+-unregulated force that developed due to the absence of the endogenous TnI and TnC. The Ca2+ regulation of steady-state force was restored with a preformed fsTnI·fsTnC complex. The reconstitution with the fsTnI:fsTnC complex (25 μM) was performed in the pCa 8 solution for ~1.5 h at room temperature, or long enough for the force to reach a stable level. Control fibers were run in parallel and treated with the same solutions minus the proteins. The final Ca2+-sensitivity of force development was determined after fsTnI·fsTnC reconstitution and the data were analyzed with the Hill equation.Skeletal muscle fiber bundles from rabbit psoas muscle were mounted on a force transducer and treated with the pCa 8 relaxing solution containing 1% Triton X-100 for ~ 1 h. The composition of the pCa 8 solution was 10Protein:protein interactions were measured using the Checkmate Mammalian Two-Hybrid System (Promega) as previously described . Values of P < 0.05 were considered statistically significant.All data are presented as mean ± S.D. Comparisons of mammalian-two hybrid data and maximal force data were carried out using one way ANOVA. Unpaired Student's Figure 2+ unregulated force established after treatment with HcTnT and also to determine if the proteins were able to fully reconstitute the skinned fibers by forming a functional Tn complex. Wild-type HcTnI·HcTnC complex resulted in complete inhibition of Ca2+ unregulated force.To determine the effect of these mutations (R204C and R204H), as well as other potential cTnI mutations, R204G, R204P, R204Q, and R204W, cardiac skinned fiber calcium-force measurements were carried out. We employed a well-established method in our lab  to 0.37 (R204G)  Figure . The mut) Figure . RecoverRenilla reniformis luciferase under the control of the SV40 promoter, which allows the normalization for differences in transfection efficiency. The troponin subunits were cloned into pBIND and pACT Vectors to generate fusion proteins with the DNA-binding domain of GAL4 and the activation domain of VP16, respectively. Association of the DNA-binding domain and the transcriptional activation domain results in transcriptional activation of the firefly luciferase reporter gene and an increase in firefly luciferase expression when compared to the negative controls. Mutations in interacting proteins that disrupt or significantly reduce the interactions between the two proteins being investigated would result in reduced association between the DNA-binding domain and the transcriptional activation domain resulting in less firefly luciferase expression.To determine the effect of the R204 mutations on cTnI: cTnT and cTnI: cTnC interactions, the mammalian two-hybrid luciferase assay was utilized. In the CheckMate mammalian two-hybrid system utilized, the pACT vector contains the herpes simplex virus VP16 activation domain upstream of the cloning region, while the pBIND vector contains the yeast GAL4 DNA-binding domain upstream of the cloning region. The pBIND Vector also expresses The cTnI R204P and R204H mutations showed the weakest interactions with cTnT when compared to wild-type cTnI Figure . The cTn2+ (pCa 8.0) after fsTnT displacement is a measure of the extent of displacement of endogenous fsTnI (referred to as the Ca2+ unregulated force). In the absence of fsTnI, fibers were unable to relax, as the inhibitory activity of cTnI is deficient. This measurement of Ca2+ unregulated force was utilized to ensure that all the skinned fibers are displaced to the same extent. All the fibers selected for these studies had a Ca2+ unregulated force of >95%.Rabbit fsTnI was studied in place of HfsTnI because it is the best characterized fast skeletal muscle system and the C-terminal half of rabbit fsTnI shows 100% homology with HfsTnI. The homology between rabbit fsTnI and HfsTnI is 98.9%. Under the conditions utilized, essentially all of the endogenous Tn complex was displaced following treatment with fsTnT. The amount of force development in the presence of very low concentration of Ca2+ sensitivity of force development  increased the Cat Figure , Table 22+-sensitivity of force development  were investigated in skinned fiber studies. All of the cTnI R204 mutations investigated displayed increased Ca2+ sensitivity of force development when compared to WT cTnI (ΔpCa50 = 0.22–0.37), similar to what is observed for most FHC mutations. These results suggest that the R204 residue is important in cTnI function and that increased Ca2+ sensitivity may be a major factor in HCM. The increase in myofilament sensitivity to Ca2+ is important since reduction in the Ca2+ sensitivity was found to prevent the development of HCM , developed a phenotype of stunned myocardium . These two mutations also showed increased maximal force compared to WT cTnI. R204W also showed increased maximal force. The R204H mutation was found to be associated with HCM in an Australian family and is associated with a poor prognosis  to its binding partners is disrupted pending measurement of actual affinity constants. Interestingly, while the R204C mutation weakened the interaction of TnI with TnC, it did not significantly affect the interaction of TnI with TnT. However, the cTnI R204H mutation disrupted the interaction between cTnI and cTnT as well as between cTnI and cTnC, which is consistent with a previous report using a similar mammalian two hybrid assay . This difference in Ca2+ sensitivity changes may be due to the size of the tryptophan residue, which is the largest amino acid and closer to the size of arginine than proline or glycine  and no change in maximal force   and get heart tissue or some skeletal muscle for research from these animals that have already been sacrificed.AG: design of work, experiments, acquisition, interpretation of data, drafting of work, final approval, agreement to be accountable for all aspects of work. SN: acquisition, interpretation of data, revising, final approval, agreement to be accountable for all aspects of work. RS: acquisition, revising, final approval, agreement to be accountable for all aspects of work. SD: acquisition, revising, final approval, agreement to be accountable for all aspects of work. ZC: interpretation of data, revising, final approval, agreement to be accountable for all aspects of work.This research was supported by a UC Davis funds and a Hellman Fellowship (AG).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HSH and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.