This review summarizes how millisecond Ca2+ dynamics activate multiple protein cascades for control of the release site replenishment with release-ready SVs that underlie presynaptic short-term plasticity.An action potential (AP) triggers neurotransmitter release from synaptic vesicles (SVs) docking to a specialized release site of presynaptic plasma membrane, the active zone (AZ). The AP simultaneously controls the release site replenishment with SV for sustainable synaptic transmission in response to incoming neuronal signals. Although many studies have suggested that the replenishment time is relatively slow, recent studies exploring high speed resolution have revealed SV dynamics with milliseconds timescale after an AP. Accurate regulation is conferred by proteins sensing Ca A. A64]. AThe questions, how many APs can activate myosin IIB and how long does it take place, can be answered by applying repetitive AP. APs at 10 Hz revealed that the time window of myosin IIB action is short, within hundreds of milliseconds B 64]. F. F64]. FThe site where myosin acts is predictable as that myosin IIB and VI contribute to local SV mobilization to the release site from two pieces of evidence. With the myosin IIB or VI knockdown, (1) number of release-ready SVs (≈50 SVs) was similar to that of control . Knockdown of synaptotagmin 7 with its siRNA shows potentiated depression of paired EPSP similar extent to that with dynamin 3 knockdown. Double knockdown of synaptotagmin 7 and dynamin 3 did not show a further additive decrease in paired EPSP seen with synaptotagmin 7 or dynamin 3 knockdown. Notably, the double knockdown induced failure of paired EPSP seen in 50% of paired-AP application with the interval 20 ms.As described above, in rat sympathetic presynaptic neurons, dynamin 3 is involved in ultrafast endocytosis within 20 ms of an AP. For the dynamin 3-mediated ultrafast endocytosis, synaptotagmin 7 acts mainly as the CaOn the contrary, in response to repetitive neuronal activity, during and after a burst of APs, the dynamin 1-mediated fast vesicle membrane recycling C guarant2+ drives docked vesicles into an undocked state, leading to synaptic depression. In fact, in cultured sympathetic neurons, paired-AP depression with the interval of 15 ms is reduced by fast Ca2+ chelation with BAPTA [2+ sensor synaptotagmin 1 likely mediate this transient docking [2+-sensor molecules.Watanabe and co-workers suggested that time-dependent SV dynamics of undocking and docking at the AZ triggered by a single AP may underlie presynaptic short-term plasticity . AP induth BAPTA . In hippth BAPTA . Ca2+ se docking , suggest2+ sensor molecules which bind to raised Ca2+ after a single AP. Synaptotagmin 1, 2, and 9 serve for the synchronous transmitter release [2+ via its C2A domain [2+ sensor protein DOC2 [The time-dependent SV dynamics described above is regulated by Ca release ,103,104,A domain  mediatesA domain . In addiA domain , and SNAA domain , and a Cein DOC2 , are impein DOC2 . Synaptoein DOC2 , in addi2+-dependent phosphorylation of synaptic proteins. Long-term depression in cerebellar [2+/CaM-activated protein phosphatase. In addition, low-frequency depression of transmitter release caused by stimulation at 0.2 Hz, at crayfish phasic synapses, requires presynaptic phosphatase calcineurin activated by Ca2+/calpain [Synaptic depression is often related to Carebellar  and hipprebellar , for exa/calpain . 2+ buffers such as calbindin, parvalbumin, and related Ca2+-binding proteins l control synaptic strength [2+ homeostasis in the presynaptic termina [2+ buffer [2+ buffer [2+-dependent regulatory proteins may provide a means of cell-type-specific regulation of short-term synaptic plasticity that reflects SV states in the AZ.Castrength ,114,115  termina . Calbind+ buffer , alters + buffer , whereas+ buffer , control+ buffer . Differe2+ sensor synaptotagmin 1, 7, and 9 action and Ca2+ buffers. Modulation of Ca2+ current by presynaptic Ca2+ channel regulation, responding to millisecond Ca2+ dynamics after neuronal firing, is also an important mechanism for presynaptic short-term plasticity [2+ currents [2+ currents mediate synchronous and asynchronous SV fusion, at most fast synapses [2+ channels controls neurotransmitter release efficacy. The Ca2+ channel proteins consists of a pore-forming α1 subunit associated with β, α2-δ and possibly γ subunits [2+-dependent regulation of synaptic transmission [Presynaptic short-term plasticity may be a combination of multi-molecular mechanisms, in addition to the C2-domain Caasticity . In prescurrents  are acticurrents . P/Q-typsynapses ,125,126.subunits . The α1 smission ,125,128.2+ channel α1 subunits encoded by ten distinct genes in mammals are divided into three subfamilies [V2 subfamily members CaV2.1, CaV2.2, and CaV2.3 channels conduct Ca2+ currents classified as P/Q-type, N-type, and R-type, respectively [V2 channels are expressed diversely. At the calyx of Held synapse, for example, a combination of P/Q- and N-type presynaptic currents in young mice show activity-dependent facilitation [2+ current facilitation and synaptic facilitation are lost by CaV2.1 channel deletion [2+ currents do not support synaptic facilitation and are less efficient for synaptic transmission [2+ channels for modulation of synaptic transmission and synaptic plasticity. Cafamilies ,129,130.ectively ,129,130.litation ,132. Thedeletion ,132,133.V2.1 channel mediates facilitation of the presynaptic Ca2+ current, leading to synaptic facilitation [2+ currents increase and then decrease due to the channel modulation by Ca2+ elevation [2+ elevation the CaV2.1 channel bind to CaM [2+-binding proteins, calcium-binding protein 1 (CaBP1), visinin-like protein-2 (VILIP-2) [2+-binding motifs binds to the α1 subunit at the IQ-like motif (IM) and the nearby CaM-binding domain (CBD) in the C-terminal [2+-sensor proteins CaBP1, VILIP-2, and NCS-1 modulate CaV2.1 channel activity. CaBP1, highly expressed in the brain and retina [V2.1 channel inactivation [2+-dependent CaV2.1 channel facilitation but inhibits Ca2+-dependent inactivation [2+-dependent CaV2.1 channel inactivation [The Calitation ,132,133.levation . With Cad to CaM ,136,137 VILIP-2) ,139,140 VILIP-2) . CaM witd retina , bindingtivation , bindingtivation . 2+-sensors-mediated presynaptic CaV2.1 channel modulation was explored by expressing the α1 subunit derived from the brain CaV2.1 channel that functionally generates P/Q type currents with other endogenous subunits in long-term cultured sympathetic neurons [V2.2 channels mediate synaptic transmission [V2.1 channel activity interacting with Ca2+ bound CaM (Ca2+/CaM) and changes paired EPSP [2+ binding sites of CaM, N and C lobes, sense differently increase in Ca2+ concentration. The N-lobe sensing rapid and higher increase in Ca2+ concentration [2+ concentration binds to the IM. Thus, pairs of APs induced synaptic depression and facilitation with varied stimulation intervals (2+ elevation after the first AP causes temporal regulation of the CaV2.1 channel activity mediated by Ca2+/CaM, resulting in a change in the efficacy of SV fusion triggered by following AP (The physiological role of these Ca neurons  where Casmission ,145. A sred EPSP . Ca2+ bintration  binds tontervals A. AP witowing AP B. 2+/CaM-dependent CaV2.1 channel regulation. Ca2+/CaM binding to the CBD controls negatively the SV fusion efficacy, whereas Ca2+/CaM binding to the IM controls it positively. APs burst with a higher frequency over 20 Hz shapes the time course of short-term presynaptic plasticity by Ca2+/CaM-mediated CaV2.1 channel inactivation (APs burst with short interval induces presynaptic short-term plasticity with the Cativation C, sugges2+ channel and creates a steep gradient of Ca2+ elevation in the AZ. Within the transient Ca2+ elevation, each Ca2+-sensor proteins, having a different affinity and binding speed to Ca2+ [V2.1 channel modulation. Ca2+ concentrations for CaM, CaBP1 and VILIP-2 binding to Ca2+ are 5–10 μM, 2.5 μM and ~1 μM, respectively [V2.1 channel activity shortly after Ca2+ entry and lasts 100 ms (2+-binding proteins on the CaV2.1 channel causes various short-term plasticity at different synapses [V2.1 channel activity by each Ca2+-sensor would substantially control transmitter release efficacy in response to non-stop neuronal signaling [Presynaptic AP opens a Ca to Ca2+ , determiectively . After as 100 ms A,B, whilsynapses . Thus, aignaling .2+ dynamics after an AP control SV states in the AZ, not only in the synchronous fusion, but also undocking, redocking, and asynchronous fusion states [2+ dynamics [2+-sensor molecules, including AZ proteins complex, motor proteins, and endocytic proteins [2+ dynamics also control presynaptic Ca2+ channels activity, by Ca2+ bound CaM and neuron-specific Ca2+-binding proteins [2+ elevation with incoming APs. These multiple protein cascades activated by a single AP should be activated during and after bursts of APs, resulting in presynaptic short-term plasticity. In conclusion, release-ready SVs in the AZ are controlled by Ca2+ dynamics with each AP, and the SV fusion efficacy changes sustainably in response to neuronal activity.SV fusion takes place within milliseconds after the firing of an AP . SV dynan states . The unddynamics  that actproteins ,64,85 (Fproteins ,148,151,


Background: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in adults. Bone marrow angiogenesis is crucial for pathogenesis of leukemia, and increasing bone marrow Mean Vascular Density (MVD) and level of angiogenesis factors are seen in patients with AML. Higher level of bone marrow MVD is associated with poor prognosis of AML according to previous studies. The present study aimed to compare bone marrow MVD in AML patients and controls and evaluate the relation between bone marrow MVD and number of residual blast cells after AML treatment.Materials and Methods: This study is a longitudinal study on AML patients who were admitted to Omid hospital. The bone marrow biopsies of patients with AML and patients with normal diagnosis –as control group- were taken from archives of pathology laboratory. Immunohistochemistry staining was used for all specimens by using thrombomodulin markers for calculating MVD. Flow cytometry findings of AML patients were assessed for percent of minimal residual disease (MRD) after AML treatment in AML patients group. Results: In this study, 27 AML patients and 24 healthy individuals with mean age of 40.92±15.13 years were evaluated, of whom 56.86% were male. The mean bone marrow MVD was significantly higher in AML patients than controls. The mean bone marrow MVD was significantly higher in males and there was insignificant reverse correlation between bone marrow MVD and MRD. About 59.3% of AML patients had response to treatment and there was no significant relationship between MVD and response to treatment. Conclusion: Bone marrow MVD was higher in AML patients than controls and there was no remarkable relationship between bone marrow MVD and MRD and response to treatment. Acute myeloid leukemia (AML) is most prevalent in adults, and one of the most prevalent cause of death in the United States. With a low survival rate, although leukemia is considered a treatable disease these days, it is rarely treated2. The exact pathogenesis of leukemia and factors modulating cell proliferation in leukemia have not yet been fully known3.  Leukemia is a hematological neoplasm induced by uncontrolled proliferation of immature leukocytes4. Bone marrow angiogenesis is the formation of new blood vessels, playing an important role in pathophysiology of hematologic malignancies 5, 6. The mean bone marrow micro-vascular density (MVD), recently recognized as a prognostic value7, shows neoplastic cell angiogenesis capable of predicting prognosis and invasion of disease. Increasing bone marrow MVD and level of angiogenesis factors seen in patients with different types of hematological malignancies like AML, was associated with the severity of leukemia 8,9. Decrease in bone marrow MVD and angiogenesis factors were seen after AML remissions10. There is evidence that higher levels of bone marrow MVD is associated with poor prognosis of AML9.Bone marrow angiogenesis is crucial to pathogenesis of leukemia11,12.On the other hand, one of the best ways to assess the chemotherapeutic response and risk of relapse in acute leukemia patients is the minimal residual disease (MRD) measurement. Nowadays, multicolor flow cytometry is considered a specific and sensitive method to investigate MRDPrevious studies evaluating bone marrow MVD in patients with AML and comparing it with the controls, as well as studies evaluating the relationship between angiogenesis and response to treatment are, to our knowledge, limited. Accordingly, this study aims to compare the bone marrow MVD in AML patients with the controls and to evaluate the relationship between bone marrow MVD and the number of residual blast cells after AML treatment in Isfahan. th day flow cytometry findings of specimens about the proportion of minimal residual disease (MRD) by multicolor flow-cytometry (four colors) considering the aberrant expression of markers after AML treatment. Where the 28th day flow cytometry specimens were diluted, the specimens were excluded from the study. The bone marrow biopsies of patients with normal diagnosis in paraffin-wax blocks were taken from the archives of pathology laboratory and treated as control groups. The bone marrow biopsies of the control groups were from the patients with anemia or vitamin B12 deficiency.  This study is longitudinal, conducted on new cases of AML patients admitted to Omid Hospital affiliated to Isfahan University of Medical Sciences (IUMS) between 2013 and 2017. The bone marrow biopsies of patients with AML processed with paraffin-wax blocks were taken for thrombomodulin immunostaining to evaluate angiogenesis. The inclusion criteria for these specimens were as follows: 1) presence of adequate tissue on paraffin-wax block for immunohistochemistry (IHC) staining and 2) presence of the 28All the biopsy specimens were firstly fixed in paraformaldehyde, and then embedded in paraffin and decalcified with EDTA. Hematoxylin and eosin-stained slides were evaluated by a skillful pathologist to confirm diagnosis. 4 µm-thickness sections of bone marrow biopsy specimens were used for IHC staining to identify the endothelial cells with antihuman thrombomodulin antibody (Anti-Thrombomodulin antibody [141C01] (ab33513), abcam®). All the specimens were evaluated by two pathologists in blinded manner using a light microscope as follows: first, at ×100 magnification, three fields with a maximum number of vessels were selected as hot spots. Then, at ×400 magnification, the field was set to cover the maximum number of vessels within the hot spots. Next, the vessels were counted in all the hotspots. Each single cell or cell accumulation with or without lumen formation previously colored with IHC staining was treated as blood vessel and the mean number of the cells from the three locations was reported as bone marrow MVD. Blast Counting and Response to ChemotherapyThe data were extracted from the patients' medical records as follows: age, gender, type of disease (M3 or non-M3 AML patients), and flow cytometry (FCM) findings about the amount of the minimal residual disease (MRD) after induction chemotherapy in AML patients group based on antigen aberrancy or leukemia associated with the phenotype compared with the normal or regenerating marrow on the first presentation. Four-color FCM along with Dako and Exbio Company antibodies were performed on the bone marrow aspirates, with the panel comprising eight tubes and up to 800,000 events per tube acquired. The MRD was considered as the population showing the deviation from the normal expression of antigens. Statistics analysisThe data were input to SPSS 22  and then analyzed. To report the quantitative and qualitative data, the mean ± standard deviation and number or proportions were used, respectively. To analyze the data from the independent t-test, Chi-square and Pearson Correlation Coefficient tests were used. A two-sided α-level of 0.05 was used to assess the statistical significance. The study was approved by the Ethics Committee of IUMS.  In this study, 27 AML patients and 24 healthy individuals were evaluated. The mean age of the participants in the AML group and controls were 40.89±16.35 and 40.96±13.76 years, respectively (P =0.99). About 70.37% (n=19) of the patients in the AML group and 79.16% (n=19) of the patients in the controls were under 50 (P =0.47). About 66.7% (n=18) in the AML group and 45.8% (n=11) in the control group were male . In the AML group, 29.5% (n=7) had M3 type of AML and 74.1% (n=20) had non-M3 type of AML. After induction chemotherapy, morphological assessment of bone marrow aspiration smears revealed that 71/4 % of patients achieved AML remission and 29/6 % of them had blast more than 5%. The mean bone marrow MVD was significantly higher in the AML patients than in the controls . The mean bone marrow MVD was higher in patients with M3 type of AML than in those with non-M3 types of AML, but it was not significant . There was no significant relationship between the mean bone marrow MVD and age (P =0.7). The mean bone marrow MVD was significantly higher in males than in females  .As shown in None of the patients showed a blast of under 0.1%; about 33/3% (n=9) of them showed a blast range of 0.1-1 %, and finally about 66/7% (n=18) showed a blast of over 1%. There was no significant relationship between the MRD and age, gender and type of AML . The mean MVD was 32.27±11.23 and 27.5±10.59 in patients with a blast of over and under 1%, respectively (P =0.27). This study evaluated the mean bone marrow MVD in AML patients and controls. The mean between the bone marrow MVD and MRD showed no significant relationship between the bone marrow MVD and MRD, a higher bone marrow MVD in AML patients as compared to the control group and males as compared to females. The mean MRD was significantly higher in M3 types of AML.10. Another study showed a higher rate of angiogenesis in patients with AML than in the controls, and like this study, there was no significant difference in the angiogenesis in the different types of AML13. Another study conducted on 30 AML patients and 30 healthy individuals for evaluation of the bone marrow MVD in these two groups reported that the mean bone marrow MVD was higher in AML patients14. This study showed a higher bone marrow MVD in males than in females. However, other similar studies did not show a significant relationship between angiogenesis and gender15. It is probable that the higher bone marrow MVD in males in this study was due to the higher number of male patients in AML group. The mean bone marrow MVD was higher in AML patients than in the controls in this study. There are similar studies that have evaluated angiogenesis in AML patients. Padro et al. showed that the mean angiogenesis was 2-3 fold higher in patients with AML than in the controls and the patients with complete remission of diseases had lower rates of angiogenesis than those with partial remissions. This study used MRD as a factor for evaluating the response to treatment, but it did not evaluate the relationship between angiogenesis and MRD7. Another study reported that the increased level of bone marrow MVD was associated with poorer prognosis of disease in AML patients, but it did not use MRD9 for prognosis evaluation. Chemotherapy in patients with AML inhibits angiogenesis inducing endothelial cell apoptosis16. It also reduces residual blast cells. Further, there was a hypothesis that there might be a relationship between angiogenesis and MRD that was not supported in this study.This study, like previous studies, showed no significant relationship between the bone marrow MVD and MRD or response to treatment. One study on AML patients having evaluated the bone marrow MVD before, during, and after AML treatment, reported that there was no significant relationship between the bone marrow MVD and disease relapses, but this study did not use MRD as a factor for evaluating the response to treatment and relapsesFor further evaluation of the relationship between angiogenesis and MRD, more studies are needed with greater sample sizes, including other angiogenesis factors excluding bone marrow MVD. 17. Since 1990, using a medication like all-trans-retinoic acid in chemotherapies has significantly increased the survival in m3 AML patients with 20% decrease in the relapse rate18. There is some evidence that modern treatments can cause complete remission in M3 patients19. This study showed lower MRD in M3 AML patients than in non-M3 AML patients, which may be the result of different types of treatment. Recent advances in the treatment of patients with the M3 type of AML showed a remarkable increase in the treatment outcomesThis study has its strengths and limitations. One strength of this study was calculating the bone marrow MVD in patients as an angiogenesis factor, used widely in different previous studies. Another strength was measuring the blast number using multicolor flow cytometry, which is sensitive and specific method to evaluate MRD. On the other hand, this study suffers from limitations. One limitation of this study was the relatively small sample size, which may restrict the generalization of results to the general population. Another limitation was failure to use serum angiogenesis factors evaluated along with the bone marrow MVD like previous studies. Further studies are needed with larger sample sizes, including other angiogenesis factors or molecular parameters to evaluate the exact relationship between angiogenesis and MRD.  In conclusion, the bone marrow MVD was higher in AML patients than in the controls and there was no remarkable relationship between the bone marrow MVD, MRD, and response to treatment.


The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration.Mitochondrial Ca Recent research has focused on the role of mitochondria in dynamically regulating intracellular and steady-state free matrix Ca2+ ([mCa2+]ss) in response to increases in cytosolic Ca2+  occurs mainly through the voltage-dependent anion channel (VDAC), which allows movement of a variety of ions, including both influx and efflux of Ca2+  occurs mainly through the mitochondrial Ca2+ uniporter (MCU). MCU allows a selective influx of Ca2+ down its electrochemical gradient and is blocked by ruthenium red (RuR) and Ru360  is well-characterized as the dominant Ca2+ efflux mechanism in excitable cells  is less studied and consequently, less understood. Early work indicated that a Na+-independent Ca2+ efflux (NICE) exists in both excitable and non-excitable tissues , compared to cardiac mitochondria   .Letm1 gene that is proposed to encode the CHE protein of the same name has provided some insights into the putative structure, function and mechanism of the NICE (+/H+ exchanger (KHE) in yeast mitochondria  depending on the mitochondrial microenvironment  injury and provide a greater reserve capacity to modulate Ca2+ transport across the IMM.Previous studies have reported that CHE activity is more robust in hepatic mitochondria than in cardiac mitochondria, where the contribution of CHE to Caly small . Yet, exly small . In conts efflux . Furtheronly 80% , suggest2+ and/or pH modulate Ca2+ efflux via the CHE and intra-mitochondrial Ca2+ in isolated rat heart mitochondria. The results show that a basal NICE in cardiac mitochondria increases with extra-matrix acidification, indicating that H+ is a driving force for the exchanger. Furthermore, the rate of Ca2+ efflux was modulated by total matrix Ca2+ (mCa2+), indicating that Ca2+ efflux via CHE is modulated by both Ca2+ buffering and the [mCa2+]ss. In permeabilized H9c2 cells, the mitochondrial Ca2+ flux was modulated by targeted knockdown of LETM1. The results suggest that contribution of CHE in regulating matrix Ca2+ may be closely aligned with the Ca2+ retention capacity (CRC) of the mitochondria as well as changes in the extra-matrix pH.To delineate the key modulators of CHE activity in cardiac mitochondria and the conditions under which CHE activity is enhanced, we (1) investigated the contribution of a NICE via CHE under basal conditions and (2) determined how changes in extra-matrix CaAll animal use was approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC).All reagents and chemicals were purchased from Sigma-Aldrich  unless specified otherwise. Fluorescent dyes Fura-4F pentapotassium salt and Tetra Methyl Rhodamine (TMRM) were purchased from Life Technologies . CGP37157 and Cyclosporine A (CsA) were purchased from Tocris Bioscience .2PO4, 5 mM MOPS, 1 mM EGTA, 5 U/ml of protease (Bacillus licheniformis), and 0.1% BSA, with pH adjusted to 7.15 with KOH), the suspension was homogenized for 15 s. After further addition of 17 ml isolation buffer, the suspension was again homogenized for 15 s. Mitochondria were isolated using differential centrifugation as described before . Fresh isolation buffer was added to the mitochondrial suspension to obtain the final desired concentration of 12.5 mg protein/ml.Hearts were harvested from Sprague Dawley rats , anesthetized intraperitoneally with Inactin and heparin and sacrificed. After mincing the tissue in ice-cold isolation buffer  were energized with 10 mM freshly prepared K+-succinate (pH 7.15) substrate, followed by addition of 250 μM freshly prepared ADP. RCI was calculated by dividing the state 3 respiration slope (after adding ADP) by the state 4 respiration slope . The RCI of the mitochondria used in experiments ranged between 3 and 7. These values are consistent with reported RCI values for rat cardiac mitochondria energized with Complex II substrate . Isolated mitochondria (0.5 mg/ml) added to experimental buffer  mixed with experimental buffer and using a cuvette-based spectrophotometer . Fura-4F was dissolved in DMSO. For all protocols, 960 μl of experimental buffer  containing Fura-4F (1 μM) was added to a cuvette. 40 μl of mitochondrial suspension was then added (0.5 mg/ml) to the experimental buffer to yield a final EGTA concentration of 40 μM in solution. At 1 min, mitochondria were energized with 10 μl of freshly prepared 1M K+-succinate (pH = 7.15). Succinate was used as the substrate, since the CRC of cardiac mitochondria has been found to be greater when respiring on complex II substrate compared to that when respiring on complex I substrate O) was calculated from a previously obtained fluorescence calibration assay by serial dilution of TMRM dye in experimental buffer. This value was subtracted from the initial amount of TMRM in the cuvette (before addition of mitochondria) to obtain the amount of TMRM associated with mitochondria ([TMRM]T). All concentration values were converted to a per mg basis. The concentration of free TMRM in the matrix ([TMRM]M) was calculated from the following equation:Fluorescent Ratios (573 nm/546 nm) were measured and converted to estimated ΔΨT in (nmol/mg), (TMRM)M and (TMRM)O in (μl/mg), Ki and Ko are partition coefficients in (μl/mg).Units are: [TMRM]m was calculated using the Nernst equationΔΨm was plotted against the fluorescent ratio measured before centrifugation to obtain the final calibration curve  and Ko (5 μl/mg) were estimated such that ΔΨm = ∼−180 mV in fully charged mitochondria when FCCP = 0 μM. The values of Ko and Ki were similar to the values of α (4.727) and ß (0.3799) obtained by Values for K2. Seventy to eighty percent confluent cells were transfected with 15 nM of either universal negative control siRNA or a pool of 3 LETM1-targeted siRNA duplexes  using the Lipofectamine RNAiMAX transfection reagent  and used for experiments 48 h post-transfection. Rat LETM1 siRNA sense sequences were: GAGGAAAUGGCCCUGAAGA, CUGUAUCACGAGAUCCCUA and CGGAUUUGUGCAG ACCUCU.H9c2 cells  were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% (vol/vol) FBS and 5% 100 U/ml Penicillin-Streptomycin mixture at 37°C and 5% COLETM1 were measured by quantitative real-time PCR (qRT-PCR) using the SYBR Mix ExTaq . GAPDH was used as an endogenous control to normalize gene expression levels. The primers used for the analysis are: Rat LETM1 5′–ATC CCTACATCATTGCTCATACTG–3′ (forward) and 5′–CCTC CTTTGCCACAATTTCTG–3′ (reverse); Rat GAPDH 5′–ATG ACTCTACCCACGGCAAG –3′ (forward) and 5′–GGAA GATGGTGATGGGTTTC–3′ (reverse). Relative expression was calculated using the ΔΔCT method.Total RNA was extracted from the siRNA-treated H9c2 cells using Trizol reagent . 1 μg of total RNA was converted to cDNA using random primers and SuperScript III First-Strand Synthesis System . mRNA levels of H9c2 cells were lysed in RIPA lysis buffer , supplemented with 1 mM PMSF and 1% protease inhibitor cocktail (Sigma-Aldrich). Lysates were incubated on ice for 30 min followed by centrifugation at 17,000 g for 15 min at 4°C. The supernatant was collected and quantified using the bicinchoninic acid assay (Thermo Fisher Scientific). Proteins were resolved on 4–20% mini-PROTEAN TGX gels (BioRad) and transferred to nitrocellulose membranes . The membranes were blotted with the following antibodies: Letm1 , NCLX , MCU  and GAPDH . GAPDH was used as the loading control. The LI-COR infrared fluorescent mouse (925–68020) and rabbit (925–32211) secondary antibodies were used for visualization by a LI-COR Odyssey scanner. Band quantification (densitometry) was performed using ImageJ and Origin 8 software .2+-free Tyrode buffer (mM): 132 NaCl, 5 KCl, 10 HEPES, 5 Glucose, 1 MgCl2, pH = 7.4 (NaOH). For permeabilization, cells (1 × 106 cells per experiment) were transferred to Buffer A (mM): 250 Sucrose, 100 HEPES, pH = 7.3 and incubated on ice for 10 min with 30 μg/ml digitonin (Sigma-Aldrich) in the presence of 20 μM EGTA and 1/200 EDTA-free protease inhibitor cocktail (Roche cOmplete via Sigma-Aldrich). The reaction was neutralized by addition of Buffer A and after centrifugation , the cells were resuspended in Na+-free experimental buffer. Experiments were performed in the presence of 1 μM Fura-4F penta potassium salt (to measure cytosolic Ca2+), 20 μM EGTA, 5 μM thapsigargin (to inhibit Ca2+ release from the sarcoplasmic reticulum) and 1M K+-Succinate (to energize complex II in mitochondria). As before, mitochondrial Ca2+ handling was observed by repetitive application of 5 μM CaCl2 boluses. Ca2+ efflux was revealed by the application of 1 μM Ru360 and the contribution of NCLX was eliminated by application of 2 μM CGP37157 (CGP) in the presence of Ru360.Freshly dissociated wild-type (WT) or LETM1-siRNA treated (siLETM1) H9c2 cells were suspended in Ca2+]c (d[Ca2+]c/dt) was analyzed for the 4th CaCl2 bolus for the traces shown in 2+]c/dt was estimated as the slope “m” (in units of RFU/s) of the linear equation, y = m*x + c, used to fit the [Ca2+]c response over 120 s following the application of the 4th CaCl2 bolus. For siLETM1, d[Ca2+]c/dt was estimated as the decay rate “τ” (in units of 1/s) of the single-exponential equation, y = y0 + a*exp(−x*τ), used to fit the [Ca2+]c response over 120 s following the application of the 4th CaCl2 bolus.Rate of change of ss by increasing Ca2+ buffering .We next tested whether agents that regulate Cauffering  and in amCa2+]ss . Both agmCa2+]ss . Figure than CsA . Interes2+ , eventually reaching a lower [mCa2+]ss than Ru360, but the same [mCa2+]ss as in control  nor decreased the eventual [mCa2+]ss compared to control or Ru360 alone  may be involved in Ca2+ uptake . The observed depolarization also correlated with the times at which the rate of Ca2+ efflux via the CHE was increased as revealed in the presence of Ru360  or LETM1 siRNA-treated (siLETM1) H9c2 cells energized with 10 mM K-Suc  reveals a Ca2+ efflux that is modulated by changes in matrix free Ca2+, Ca2+ buffering and pH, (2) increases the rate of Ca2+ efflux as well as free matrix Ca2+. (3) CHE activity is associated with depolarization of the mitochondrial membrane potential. (4) Ca2+ dynamics in H9c2 cells is modified in the absence of LETM1.The novel findings of this study are that in cardiac mitochondria, inhibition of MCU-mediated Ca2+ uptake was balanced by Ca2+ efflux mechanisms to maintain [mCa2+]ss, since inhibition of MCU-dependent Ca2+ uptake by ruthenium red (RuR) or Ru360 had no effect on ΔΨm , leading to more Ca2+ to bind to CHE. On the other hand, processes that increase Ca2+ buffering or decrease the matrix free Ca2+ (through reduced Ca2+ uptake or greater efflux via NCLX) would decrease Ca2+ efflux via the CHE by making less Ca2+ accessible to bind to CHE. Such a scheme would explain the effect of ADP on the Ca2+ efflux rate. Under conditions where matrix Ca2+ is maintained at a steady state, if ADP sequesters more Ca2+, less free Ca2+ would be available to bind to CHE, leading to decreased activity of the exchanger and subsequently, reduced Ca2+ efflux. The reverse would be true when, at near buffering capacity, less Ca2+ would be buffered and more Ca2+ would be available to bind to CHE and increase the rate of Ca2+ efflux. Additional experiments would be needed to confirm this notion.Our observations lead us to propose a novel three-way dynamic relationship between CHE activation, Cared Ca2+ . Accordi2+ buffering and delay in mPTP opening by CsA and ADP suggests that, the mechanisms that regulate CHE activity will also be similarly impacted by tissue-specific and species-specific differences.It is also to be noted that in our study, which uses mitochondria from rat heart, ADP is more effective in delaying mPTP opening, whereas 2+ efflux , on the other hand, did not have an effect, which may be attributed to the fact that the extra-matrix pH was similar to previously reported values of matrix pH (∼ 7.6)  depolarized the IMM and increased the rate of Ca+ efflux , 7. In ecidic pH . This is (∼ 7.6) , so thatrt chain . It suggrt chain . Howeverrloading . The rep2+, including matrix free Ca2+ and Ca2+ buffering. The results point to a previously unattributed unique involvement of CHE with the Ca2+ handling capacity of mitochondria. Understanding how CHE activity is associated with mitochondrial Ca2+ handling will have implications for pathophysiological conditions such as ischemia/reperfusion injury.The current study shows that the CHE activity is stimulated in response to two triggers: (1) a pH gradient across the IMM and (2) changes in matrix CaAll datasets generated for this study are included in the article/The animal study was reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.W-MK, AC, and LG designed the initial study. LG, GN, and JM performed the experiments, analyzed the data, and prepared the figures. LG, GN, JM, DS, AC, and W-MK were involved in critical interpretation and discussion of the results, and wrote and edited the manuscript. All authors have read and approved the final version of 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.


Embedding these particles in the hydrated cement resulted in the formation of calcite crystals after a hardening time of 10 days, which were not seen in controls, indicating that the particles dissolve and then transform into calcite. While there was no significant repair in the controls without ACC, almost complete closure of the cracks was observed after a 10 days healing period in the ACC-supplemented samples. Nanoindentation measurements on the self-healed crack surfaces showed a similar or slightly higher elasticity at a lower hardness compared to non-cracked surfaces. Our results demonstrate that bioinspired approaches, like the use of polyP-stabilized ACC shown here, can significantly improve the repair capacity of Portland cement.There is a strong interest in cement additives that are able to prevent or mitigate the adverse effects of cracks in concrete that cause corrosion of the reinforcement. Inorganic polyphosphate (polyP), a natural polymer that is synthesized by bacteria, even those on cement/concrete, can increase the resistance of concrete to progressive damage from micro-cracking. Here we use a novel bioinspired strategy based on polyP-stabilized amorphous calcium carbonate (ACC) to give this material self-healing properties. Portland cement was supplemented with ACC nanoparticles which were stabilized with 10% ( Concrete has proven to be an excellent building material since a few thousand years, like in the Ancient Rome or in the German city of Trier, the Roman “Augusta Treverorum”, with the Basilica of Constantine which was built at the beginning of the 4th century . Even thCement, the binder in the concrete, is a powdery substance, composed of calcined lime and clay as the major ingredients . Portlan3) on the crack surface [3)2] coming from the water and reacting with calcium hydroxide [Ca(OH)2] from concrete or from the calcium hydroxide reacting directly with CO2 from the air . Upon hydration reaction, calcium hydroxide is also liberated during the process. The XRD spectrum of cement powder shows strong signals in the XRD pattern, especially around the 2θ (degrees) of 30 which is attributed to alite and belite crystals  a and C2S . Notablen the pH .Force/displacement curves were obtained from the compression testing studies both with “hCEM” and with “hCEM•ACCP10” . AltogetThe breaking load of the Na–polyP/amorphous calcium carbonate (“ACCP10”) doped cement samples was determined and compared with the properties of the “hCEM” control by using the 3-point bend system. The samples were kept for 10 days at room temperature (20 °C). After putting onto the platform of the machine, the samples were exposed to increasing loads until a failure of the materials was detectable. For the cement control, the result of the respective images is depicted in For the “hCEM”-enriched cement, “hCEM•ACCP10” (aged for 10 days), the fracture behavior is strikingly different. At the beginning of the measurement, again an intact block is visible A’. AfterThe cement samples, either “ACCP10”-free, “hCEM” or cement containing “ACCP10” nanoparticles (“hCEM•ACCP10”) were selected. The samples were aged for 10 days. Microcracks were inserted into 10 days aged cement blocks by shock freeze–warming cycles. The samples, comprising microcracks between ≈15 and ≈66 µm, were submersed in water up to 3 mm below the top edge of the blocks and continued to be left for additional 10 days.Light microscopic inspection revealed that the cracks induced in the samples showed an irregular progression pattern, following the micrograins within the cement. After treatment in water for additional 10 days the “hCEM” samples were inspected again. Although it is likely that a significant amount of unhydrated cement is still present in the “ACCP10”-free “hCEM” sample after this period of time, no significant change in the morphology of the cracks was seen, if compared to the one at the beginning of the 10 days period A–D. In cThe change of the surface morphology of the cracked specimen (“hCEM”) as well as of a polyP-doped cement “hCEM•ACCP10” sample was analyzed with a surface structure imaging surface ; represen = 48), compared with the self-healed crack area with 0.44 ± 0.12 GPa (n = 48). Interestingly, the self-healed crack surface area is slightly, but not significantly, more elastic, with a reduced Young’s modulus of 31.28 ± 7.97 GPa (n = 48) compared to the values measured for the adjacent non-cracked surface areas .Nanoindentation measurements were performed at the surface plains of “ACCP10” containing cement samples, “hCEM•ACCP10”, both at repaired  and at adjacent non-cracked sites. Representative load/displacement curves for both sites are shown in The material filled into cracks that had been introduced into “ACCP10” containing cement samples, “hCEM•ACCP10”, was analyzed both by ESEM and by XRD . The sam3  (cubic and orthorhombic), 12.6% ferrit [Ca22O5], 1.9% K2SO4, 0.5% MgO, 0.3% CaO, 1.5% Ca(OH)2, 2.6% CaSO4, 2.7% CaSO4•0.5 H2O, 0.8% CaCO3, 0.4% SiO2, 0.6% CaMg(CO3)2.Na–polyPhosphate (Na–polyP) with an average chain length of 40 phosphate  and a Na2CO3 solution  at an equimolar concentration ratio between Ca2+ and CO32−. In brief, 20 mL of 0.1 M NaOH was added to 1.05 g Na2CO3 and then diluted with 30 mL of deionized water. This solution was added to 50 mL of water containing 1.47 g CaCl2•2H2O. The CaCO3 precipitate was rinsed with acetone to dry the solid material. In one series of experiments a sample of the precipitate was taken after a short, 30 min precipitation process and inspected by electron microscopy.Amorphous calcium carbonate (ACC) nanoparticles (ACC-NP) were prepared as previously described ,30 by di3, Na–polyP was added during the precipitation process. In turn, 10% (w/w) of the polymer (0.1 g Na–polyP) was immediately added to the CaCl2•2H2O/Na2CO3 solution, prior to the CaCO3 precipitation. The suspension obtained was filtrated, washed with acetone and dried at room temperature. The samples were termed “ACCP10”.To stabilize the amorphous phase of the CaCOThe unhydrated cement was termed “CEM”. The CEM I 42.5 R cement, 100 g, was mixed with 38 g of distilled water. This sample of hydrated cement was termed “hCEM“. In order to ensure a homogenous distribution of the solid particles in the cement, a longer blending time than usually used  with 30 In separated series of experiments, the cement was supplemented with “ACCP10” as follows: CEM I 42.5 R (100 g) was supplemented with 1 g “ACCP10” and subsequently reacted with 38 g of water; and termed “hCEM•ACCP10”.The pH in the cement slurry, after addition of water, remained at ≈12.5.3) at room temperature (20 °C). Then the samples were transferred to room humidity (at 20 °C) for 1 day and used for the experiments.The cement samples were prepared in 5 cm × 2.5 cm × 2.5 cm plastic molds and allowed, if not mentioned otherwise, to harden for 10 days prior to analysis; during this period the specimens remained in a humidity chamber (humidity: ~20 g/mElectron microscopy was performed either with a scanning electron microscope (SEM), a HITACHI SU8000 electron microscope  or with an environmental scanning electron microscope (ESEM), using an ESEM XL-30 apparatus , as described . Light mFourier transformed infrared spectroscopy (FTIR) was performed with ground cement material using a micro-mill and applying an ATR -FTIR spectroscope/Varian 660-IR spectrometer , fitted with a Golden Gate ATR unit , as described .X-ray powder diffraction (XRD) of dried powder samples were analyzed with a D8 Advance A25 diffractometer  connected with a monochromatic Cu-Kα radiation .w/w) polyP-stabilized ACC (“ACCP10”) was assessed by means of compression testing at the following time-points after mixing: 5 h, 1 day, 2 days and 4 days (curing in a humidity chamber at 20 °C as mentioned above). The tests were performed using a “MultiTest 2.5-xt Force Testing System” equipped with a 2500 N Load Cell unit . The cement samples were loaded in longitudinal direction with a loading speed of 5 mm/min using a 14 mm load-button. For this, loads of 2.5 N (5 h), 25 N (1 day) or 250 N (2 and 4 days) were applied to the samples and kept stable for 60 s. Subsequently, an unloading period (0 N) followed for 300 s. The force–displacement-time data were continuously recorded at a frequency of 50 Hz using the Emperor XT Force software .The hardening of the cement material with and without the addition of 1% , following a previously described procedure . The staFollowing a previously described protocol , the samE = 1143 GPa. All calculations were performed with the Nano-Test Platform Four V.40.08 software package .For these studies, the nanoindentation approach was applied, since this technique is useful for the determination of local mechanical properties, e.g., surface hardness and the local Young’s modulus. The instrument NanoTest Vantage System  was applied, equipped with a Berkovich diamond indenter ,36. For p < 0.05 were considered statistically significant.The values reported are the average ± standard deviations. Statistical analyses were performed with the one-way ANOVA test, by using SigmaStat 3.5 software . Values of i units (“ACCP10”), substantially increases the self-healing property of Portland cement (“hCEM”). The reaction mechanism of this additive in the cement is quite unique (i [2+ is known to accumulate in cement and to leach out from this material [i generated by the ALP, will readily precipitate with Ca2+ to Ca-phosphate at alkaline conditions [32−, which will also immediately turn to a crystalline phase in the presence of Ca2+ [The present study shows that addition of the bioinspired amorphous calcium carbonate, ACC, stabilized with Na–polyP of a chain length of 40 Pe unique . Prior tnique (i ,38. Ca2+material . In turnnditions . The sec of Ca2+ . This pr


Mimosa pudica L., possesses the motor organ pulvinus at the junction of the leaflet-rachilla, rachilla-petiole, and petiole-stem, and upon mechanical stimulation, this organ immediately closes the leaflets and moves the petiole. Previous electrophysiological studies have demonstrated that a long-distance and rapid electrical signal propagates through M. pudica in response to mechanical stimulation. Furthermore, the spatial and temporal patterns of the action potential in the pulvinar motor cells were found to be closely correlated with rapid movements. In this review, we summarize findings from past research and discuss the mechanisms underlying long-distance signal transduction in M. pudica. We also propose a model in which the action potential, followed by water flux  in the pulvinar motor cells is a critical step to enable rapid movement.As sessile organisms, plants do not possess the nerves and muscles that facilitate movement in most animals. However, several plant species can move quickly in response to various stimuli . One such plant species,  Unlike animals, plants are often regarded as immotile organisms, as they do not normally make movements that can be easily observed by humans. However, in nature, plants can slowly displace their bodies in response to various stimuli. Such types of slow movements (minutes to hours) are widely seen in plants as they adapt to various environmental pressures. For instance, many plant organs can sense changes in the sunlight direction and bend their bodies towards it accordingly, a phenomenon called phototropism . FurtherMimosa pudica quickly folds its leaves. Many studies have documented the propagation of stimulus information induced by mechanical stimuli  in response to external stimuli; for example, in response to mechanical stimulation, the sensitive leguminous plant i e.g., ,5,6,7,8),6,7,8MimM. pudica is mechanically stimulated , it drops and leaflets are folded upward  was removed (described in cyt) and contractile movements cyt. This latter mechanism has not yet been elucidated, but the knowledge of Ca2+ dynamics in nyctinastic movements may be useful to explain the role of [Ca2+]cyt in the rapid movement of M. pudica. Red light irradiation, which causes phytochrome-mediated nyctinastic leaf closure, generated transient [Ca2+]cyt increases in motor cells of Robinia pseudoacacia and this Ca2+ could be partly derived from the cell wall cyt increase may activate Ca2+-dependent Cl− channels, followed by turgor loss during nyctinastic slow movement (and possibly the rapid movement) of M. pudica.Using microscopic and pharmacological techniques, the potential relationship between the rapid movement of tor cell . Previou2+ store . Further to fold . The eff pulvini . Moreovemovement ,33. Whenell wall . Therefochannels , or a [CM. pudica [2+-dependent manner in vitro [2+]cyt in motor cells might regulate both ion fluxes and actin depolymerization during movement. Meanwhile, phalloidin, an inhibitor for actin depolymerization, retarded the increase in [Ca2+]cyt in the motor cell protoplast, the actin filament might also regulate changes in [Ca2+]cyt cyt . On top Ca2+]cyt . InhibitCa2+]cyt  while seM. pudica, which have different molecular structures for each plant species cyt increase activates Ca2+-dependent Cl− channels to depolarize the plasma membrane and voltage-dependent K+ channels transfer K+ into the extracellular region. Alternatively, the central vacuole releases its sap with small molecules , reducing the motor cell volume. In the recovery phase, the effused substances and ions are actively transported into the motor cell and water is then osmotically pulled back into the cell. The ion uptake appears to be coupled to the H+ gradient produced by the activity of H+-ATPases [+ through inward-rectifier K+ channels, which are activated by H+ pump-driven plasma membrane hyperpolarization [2+ could be taken back into a tannin vacuole and apoplast assisted by Ca2+ pump, as the Ca2+ pump inhibitors delay the recovery phase of movement [Although the aforementioned studies were largely based on experiments examining the primary pulvinus, the findings can clarify the mechanism of rapid bending initiated in the tertiary pulvinus. This led to the creation of a turgor loss model driving pulvinar movements . In this-ATPases ,39. It irization  or otherrization . The cenrization . Cytosolmovement . Beside M. pudica is stimulated, it generates a stimulus-induced signal propagating toward distant organs. Early studies examining this phenomenon monitored the pulvinar movement as a visible indicator of signal transmission [M. spegazzinii (a sensitive relative of M. pudica). As applying the stem extract solution to the cut end of the shoot generated signal propagation, he concluded that this conduction was a result of stimulating chemicals released from wounded tissues through water transported in the xylem vessels  reporteribed in ). This p by Snow . Since tM. pudica possesses three types of signals in mechanosensory transduction as follows: (1) an action potential, (2) a variation potential, and (3) a rapid unknown signal (quicker than the previous ones). The variation potential is an electrical signal triggered by wounding  [Houwink  conducteand cut) . Propagaand cut) . Therefoand cut) . The rapand cut) . The mecand cut) . In thisM. pudica and has a steep spike-like waveform. Houwink [M. pudica were examined. These studies reported that excitable cells in petioles were parenchyma cells in the protoxylem and parenchyma or companion cells in the phloem [M. pudica [− concentration [2+-free extracellular fluid were smaller than those in the Ca2+-containing solution [2+ and Cl− both contribute to action potential generation in M. pudica, but that further analysis is required to obtain conclusive evidence.The action potential is the most commonly observed signal in  Houwink  referred Houwink . When pa Houwink ,7. When e phloem ,43. Usine phloem . These v. pudica ,43,44. S. pudica  attemptentration . SubsequM. pudica had been studied in detail and is widely thought to be one of the mechanisms of stimulus transmission. However, this hypothesis is not yet fully accepted. In a study where a part of the leaf is burned, changes in the leaf thickness of others, more distant leaves emerged almost immediately [As discussed, the action potential of ediately . This reediately .M. pudica. While a large part of the underlying mechanisms is yet to be determined, the contribution of several ions and cytoskeleton playing a role in these events has been suggested. Most studies investigating mechanical signaling in M. pudica are old, and until now, research on this species has largely relied on microscopic, pharmacological, electrophysiological, and histochemical techniques or their combined methods. Generally, legumes are supposed to be difficult to transform [M. pudica, unlike other plants . As such, genetic approaches have rarely been used for research on M. pudica. However, recently a stable transformation method using Agrobacterium was developed [M. pudica, as various intra and extracellular signals are visualized in real-time in Arabidopsis and Nicotiana species [M. pudica.In this review, we conclude that electrical signals, in particular action potentials, are likely to be important for the rapid movement and long-distance signaling in ransform  and thereveloped , enablin species ,48,49. T


We present a new investigation of the habitability of the Milky Way bulge, that expands previous studies on the Galactic Habitable Zone. We discuss existing knowledge on the abundance of planets in the bulge, metallicity and the possible frequency of rocky planets, orbital stability and encounters, and the possibility of planets around the central supermassive black hole. We focus on two aspects that can present substantial differences with respect to the environment in the disk: (i) the ionizing radiation environment, due to the presence of the central black hole and to the highest rate of supernovae explosions and (ii) the efficiency of putative lithopanspermia mechanism for the diffusion of life between stellar systems. We use analytical models of the star density in the bulge to provide estimates of the rate of catastrophic events and of the diffusion timescales for life over interstellar distances. With the growing number of exoplanets discovered in the past two decades, the question of what factors make an environment conducive to life has received increasing attention. In addition to exploring the question of habitability in a planetary context, many studies have investigated it from a broader perspective, with the purpose of identifying locations in the Milky Way where the chance of finding habitable worlds is higher.In analogy with the habitable zone around a star , the regSo far, little attention has been devoted to the habitability of the innermost region of the galaxy. Generally, locations closer to the Galactic center have been judged unfavorably in terms of habitability. This is mainly due to the highest rate of potentially hazardous astrophysical events  and to the increasing content of heavier elements . The latter is thought to cause an overproduction of gas giant planets and hamper the development of terrestrial planets . HoweverIn addition to these general considerations, most previous studies on the GHZ have skipped altogether the investigation of regions within roughly 2 kpc of the Galactic center. In this article, we focus our attention precisely on the habitability of such innermost regions, and especially on the Galactic bulge. First, we review what is currently known about the possible presence of planets around stars in the bulge. Next, we explore the factors that have been deemed relevant in previous studies of Galactic habitability, and we investigate their importance in the bulge. We then focus on the radiation environment in the bulge, arising from the presence of the central supermassive black hole and from supernovae explosions. Finally, we investigate the efficiency of the lithopanspermia mechanism as a possible way to exchange biological material between stellar systems.The abundance and distribution of planets in the Galactic bulge is still mostly unconstrained. Radial velocity and transit observations are biased toward nearby planetary systems and provide only sparse indication on bulge planets. Results from the SWEEPS transiting survey ,16 suggeMicrolensing events, although rare, can extend the search for planetary candidates to larger distances, since they do not depend on the flux of the host star. Microlensing provided the first possible evidence of a super-Earth in the Galactic bulge  and of tIt must be noticed that the bulge is expected to be an older component of the Galaxy, with a predominantly old stellar population . ObservaA relevant factor in most studies of galactic habitability is the metallicity of the host stars. Theoretical models suggest that terrestrial planets form only above a threshold in metallicity of order ar value . Observaar value ,29,30. Oallicity . This haZ values ranging from −1.0 to 0.4, peaking at solar metallicity. Such a varying mean metallicity is unusual for just one stellar population. Instead, the idea of a bimodal stellar population in the bulge has gained consensus [However, there is evidence that the metallicity of the Galactic bulge behaves differently from that of the disc. With the development of multi-object spectrographs and more thorough observations in many fields of the bulge (not just the low-extinction regions), it has become apparent that the bulge is not simply made of old metal-rich stars. Observations of the bulge revealed a broad metallicity distribution  with Z vonsensus . Recent onsensus  seem to onsensus . Higher These considerations provisionally suggest that metallicity in the bulge is not incompatible with the presence of rocky habitable planets. In itself, the abundance of heavier elements does not seem to be a showstopper to Galactic habitability even at distances smaller than 2.5 kpc from the center.A separate question, more directly related to habitability, is whether planetary orbital configurations can remain stable for extended periods  in a dense stellar environment. Early studies suggest that habitable planets are unlikely in long-lived stellar clusters, as they can be expelled from their host system . Others hborhood ,37. A rehborhood . WhetherA specific treatment has been reserved to the so called S-stars nearest to the Galactic center, with regard to their interaction with the supermassive black hole Sgr A*: even in such extreme case, it is not evident that the orbit of planets around such stars would be significantly affected ,40,41,42We can tentatively assume that stellar encounters, although more frequent, do not present special concerns in the bulge with respect to the disk. Anyway, we point out that assessing the habitability conditions in the bulge is not limited to planets in bound orbits around a host star: it would be relevant also for possible rogue, free-floating planets that may host life, for example below an icy surface ,44.Stars are not the only astrophysical objects surrounded by disks of gas and dust, and they might not be the only sites of planet formation. Such disks are also often found surrounding supermassive black holes (SMBH). According to the unified model of AGNs ,46, gas ormation  around lThe center of the Milky Way hosts an SMBH , that is currently in a quiescent phase. Simulations show  that somThe thermodynamics of exoplanets around SMBH, in principle, do not exclude the existence of life based on known biology. As shown in , hypotheIf rocky planets can exist around stars in the Galactic bulge, the dominant hindrance to habitability would be the presence of higher levels of ionizing radiation in the inner Galactic regions with respect to the outer disc. We then examine the consequences of the two main aspects that contribute to the radiation environment in the bulge: the presence of the SMBH in the Galactic center and the occurrence of transient radiation outpouring from supernovae (SN) explosions. While, in principle, gamma ray bursts (GRB) could also be included as a potential source of transient radiation events, their contribution is most relevant in the outskirts of the galaxy, as they are preferentially observed at low metallicities ,50. TherD from the galactic center over a time Several studies have investigated the impact of the SMBH at the center of the Milky Way on Galactic habitability ,12,13,14 time Δt :(1)Mlostc center ).The biological consequences of the irradiation from Sgr A* during its active phase are hard to assess, as they depend not just on the radiation tolerance of specific biota, but also on the amount of screening provided by the residual atmosphere. In the case of a totally exposed planetary surface, the level of ionizing radiation during the active phase of Sgr A* could prove lethal for unicellular radiation-resistant organisms up to 1 kpc from the Galactic center . DiffereWhile severe atmospheric loss can undoubtedly affect the habitability of Earth-like planets in the bulge, it must be kept in mind that the the active phase of the SMBH occurred during the initial stage of the Galaxy formation and it is expected to have lasted far less than a Gyr. Therefore the estimates above do not apply to planets that formed at later times. Moreover, as pointed out in , rocky pA separate concern arising from the presence of a SMBH is related to effects that can last or repeat themselves over the entire galactic history, e.g., from charged particle winds or tidal disruption events. The impact of such effects on habitability is still poorly understood and would certainly deserve further investigation.By far, the most important deterrent to the habitability of the inner region of the Galaxy emerging from previous studies is the highest density of stars, resulting in increasing risk of nearby supernovae (SN) explosions.The most probable occurrence of SN is expected to be from Type II: these are the endpoint of stars with masses above  [R≤8 pc . This es [R≤8 pc . However≤0.04 pc .x and y axes lie in the galactic plane, with the x axis forming an angle No detailed analysis of the impact of SN explosions on planets in the bulge has been performed so far. Some insight on the issue can be obtained by adopting an analytic model of the star density in the Galaxy and estimating the expected number of nearby SN at any given location. The density of stars in the bulge can be modeled as a triaxial Gaussian profile :(2)nBPSN,⊙We can then estimate the SN rate within a spherical volume at any location by rescaling with the appropriate star density:R corresponding to mass extinction (8 pc) and complete sterilization (0.04 pc). It is apparent that, also at the relatively high stellar densities in the bulge, the probability that a nearby SN explosion completely sterilizes a planet is quite low: the expected number of such events per Gyr, at locations closer than 2 kpc from the Galactic center, is ∼This is shown in  per Gyr .R). This is shown in We can estimate the average interval between events as erflares . The intA caveat worth mentioning is that we treated the stars as stationary, while their velocity dispersion in the bulge can be substantially higher than in the disk. We also point out that our model does not incorporate a self-consistent treatment of star formation in the bulge. In this respect, we note that if we assume that star formation for the bulge happened in a single burst, as in inside-out formation scenarios, then most stars that could potentially become SNII did it over the first few tens million years from the formation of the Milky Way. This would leave plenty of time for planets to evolve conditions suitable for life at later epochs. Thus, the results presented here should probably be seen as a worst-case scenario.t can be modeled as One potential advantage of the high density of stellar systems in the bulge might be the possibility of transferring active biological material over interstellar distances. Rocks containing microorganisms could be expelled from a life-bearing planet and be captured at later times by habitable planets elsewhere, possibly seeding the new location with life—a mechanism called ‘lithopanspermia’ ,57. The val time . There iThe probability that an ejected rock can be captured by another terrestrial planet in a different stellar system has been first estimated for the solar neighborhood and deemed too small to be relevant as a life-spreading mechanism . Howeverv is the relative velocity of rocks with respect to the stars, An initial estimate can be obtained by adopting the model outlined in ,61 for t0.05 AU2 . Here we0−4 from . We note-section ,64. For [σc from  would reThe number density of life-bearing rocks per year can be assumed to be proportional to the star density,  γ∼15/yr . Then, tThis represents the evolution timescale of the fraction of inhabited planetary systems in the bulge, and is shown in We have attempted a new assessment of the habitability of the inner 2 kpc regions (bulge) of the Milky Way. Although the actual frequency of terrestrial planets in the bulge is currently still mostly unknown, there is no compelling argument for ruling out the possibility that it is comparable to what is found in the disk region.The main concern for the presence of suitable conditions for life in the bulge is the radiation environment from transient sources and the central SMBH. The latter has been addressed in previous works and recognized as a strong source of ionizing radiation during its active phase, possibly leading to detrimental effects for habitability over the full extent of the bulge. In the present paper, we have evaluated the rate of SN explosions in the bulge using simple analytical models and found that, although much higher than in the disk, it is not likely to result in the complete eradication of life on inhabited planets. SN explosions, however, can cause mass extinction events with a frequency of ine life . It is tWe have also shown that lithopanspermia scenarios would be more efficient in the bulge with respect to the solar neighborhood. If indeed the spreading of life between stellar systems is possible, then the highest rate of catastrophic events might be balanced by the possibility that life can migrate quickly to safer locations. Any attempt to model the possible distribution of inhabited planets in the inner region of the galaxy should therefore take this aspect into account. This would provide an additional motivation for a more thorough investigation of the bulge habitability.An interesting aspect that we did not address in this paper would be to establish a measure for the overall propensity to life in the bulge, based on detailed Monte Carlo simulations of the habitability/sterilization of stellar systems, combined with models of life appearance and recovery. Intuitively, one can foresee a trade-off between the higher risk of catastrophic events in the bulge and the greater abundance of stellar systems—with the latter increasing the potential number of locations that can host life. As shown in ,9, there


Different Ca2+ channels, pumps, or exchangers regulate variations in the duration and levels of intracellular Ca2+, which may be transient or sustained. These changes are then decoded by an elaborate toolkit of Ca2+-sensors, which translate Ca2+ signal to intracellular operational cell machinery, thereby regulating numerous Ca2+-dependent physiological processes. Alterations to Ca2+ homoeostasis and signaling are often deleterious and are associated with certain pathological states, including cancer. Altered Ca2+ transmission has been implicated in a variety of processes fundamental for the uncontrolled proliferation and invasiveness of tumor cells and other processes important for cancer progression, such as the development of resistance to cancer therapies. Here, we review what is known about Ca2+ signaling and how this fundamental second messenger regulates life and death decisions in the context of cancer, with particular attention directed to cell proliferation, apoptosis, and autophagy. We also explore the intersections of Ca2+ and the therapeutic targeting of cancer cells, summarizing the therapeutic opportunities for Ca2+ signal modulators to improve the effectiveness of current anticancer therapies.Calcium (Ca Indeed, there is a 20,000-fold gradient between outside (about 1.2 mM) and inside (approximately 10–100 nM) of cells. Moreover, in the mitochondria and in the nucleus, the concentrations of Ca2+ are similar to those in the cytoplasm. In the endoplasmic reticulum (ER), considered the main intracellular Ca2+ store, the [Ca2+] ranges between 100 and 800 μM [2+ levels show that lysosomes present an internal [Ca2+] of about ≈500 μM  and regulates the propagation of intracellular Ca2+ changes that are fundamental to intracellularly transmitted biological information and important physiologic processes, including metabolism, cell proliferation and death, protein phosphorylation, gene transcription, neurotransmission, contraction, and secretion [2+]i can increase more than twofold at the micromolar level. Different channels situated in the plasma membrane (PM) induce the influx of extracellular Ca2+ into the cells. Among these channels, the most important are transient receptor potential channels (TRPC) i has adverse effects for the cells. Therefore, different channels, pumps, and buffering systems reestablish low [Ca2+]i. The reuptake of Ca2+ into the ER lumen is allowed by the activity of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), which pumps Ca2+ into the ER with a stoichiometry of 2:1 Ca2+/ATP and by the secretory protein calcium ATPase (SPCA), which transports Ca2+into the Golgi apparatus i increases during the G1/S phase transition c levels and the simultaneous activation of pro-survival autophagy in cancer cells. Consistently, addition of intracellular Ca2+ chelators prevented autophagic activation and induced cell death. Different downstream effectors were proposed to regulate Ca2+-dependent autophagy. For example, it was suggested that increases in [Ca2+]c determined activation and phosphorylation of protein kinase Cθ stimulating LC3-II conversion and autophagy [2+]c was also associated to activation of the ERK pathway, which activates mitochondrial depolarization, autophagy, and apoptosis c promotes activation of CAMK2, which, in turn, activates autophagy through the regulation of the AMPK/mTOR pathway [2+]c dynamics are regulated at ER levels by BCL-2 protein, which lower [Ca2+]ER levels, thus reducing the Ca2+ leak from ER [Autophagic dynamics in cancer are modulated by Caic death . ORAI1 isurvival . Additio agents) ,117 and  agents)  provoke utophagy . Rapid epoptosis . However pathway . Importa from ER .2+ mobilization from the ER can also increase autophagic flux. In this scenario, IP3Rs are the primary regulator. Lithium and L-690330 stimulate autophagy by reducing the levels of IP3 and inositol and consequent IP3Rs activities [2+ dynamics and promotes autophagy [2+-dependent mitochondrial activation. Correct IP3R-mediated Ca2+ transfer between ER and mitochondria supports the tricarboxylic acid cycle and consequent ATP production. This signal determines the inhibition of the energy sensor of the ATP/AMP ratio, AMPK, which is also the most investigated positive regulator of autophagic induction mechanism: AMPK-ULK1-mTOR [2+ from the ER to mitochondria with a subsequent decrease in ATP production, which determines the activation of the AMPK pathway, thereby promoting pro-survival autophagy [In contrast, inhibition of Cativities . Consistutophagy . InteresLK1-mTOR . Recentlutophagy . Similarutophagy   for the treatment of various solid tumors [2+, inducing the apoptosis in prostate and bladder cancer cells, respectively [TRPCs are classified into different subfamilies  and are implicated in different diseases, including cancer . Alteratma cells ,129. Thema cells . In addid tumors . This cld tumors , no receectively . Finallyectively . Interesectively .2+-dependent cellular processes, including cell proliferation, survival, and differentiation [2+ channels and are frequently altered in different cancer types. The upregulation of T-type Ca2+ channels has been mainly observed in prostate, breast, and ovarian cancers; however, it has also been found in melanoma, retinoblastoma, glioma, glioblastoma, hepatocellular, colon, and esophageal cancers cells [2+ channels by using the channel blocker mibefradil reduced esophageal and colon cancer cell proliferation by upregulating p53. Moreover, this inhibitor induced the apoptosis in glioblastoma cells and ovarian cancer cells. Furthermore, the administration of mibefradil or NNC-55-096, another T-type Ca2+ channel blocker, decreased tumor growth in xenograft models of glioblastoma and ovarian cancers [VGGCs are classified in high voltage activated (HVA) and low voltage activated (LVA) channels according to their pharmacological and electrophysiological profiles. They regulate various Cantiation . LVA arers cells . The pha cancers .2+ mobilization by the activation of IP3Rs and SOCE channels [Purinergic P2 receptors are classified in two subfamilies named P2X and P2Y. P2X receptors, activated by ATP, are ligand-gated nonselective cation channels formed by homotrimeric or heterotrimeric complexes of seven different subunits (P2X1–7). The P2Y receptor category, comprising eight members , may be activated by ATP, ADP, UTP, UDP, and UDP-glucose. In particular, P2Y2, P2Y4, and P2Y6 receptors are coupled to Gq proteins and their stimulation leads to Cachannels . In cancchannels . Furtherchannels . Among Pchannels . Many anchannels . Consistchannels , meanwhichannels .2+-sensors are molecular SOCE components. Currently, in mammalian cells, three isoforms of ORAI  and two isoforms of STIM (STIM1 and STIM2) have been identified. Remodeling of Ca2+ signals due to SOCE dysregulation may cause various diseases, including cancer. In fact, activating ORAI1 mutations were found in different types of cancer, including colorectal, stomach, and uterine cancers [2+ channels expressed in prostate and colorectal cancers but not in healthy tissue [ORAI channels and STIM Ca cancers . Moreove cancers . Altered cancers . Neverthy tissue ,140.3+ and Gd3+; however, these channel blockers, as well as SKF-96365, are not SOCE-specific inhibitors; therefore, treatment with these compounds may cause side effects. In this regard, DPB-162AE and DPB-163AE, derivatives of 2-APB, have been developed and are potent SOCE inhibitors capable of inhibiting SOC channels without affecting IP3Rs activity. In addition, RO2959, which inhibits the ORAI1-mediated current, may represent an important therapeutic tool since it is able to selectively increase the ORAI1 channel [2+ influx. These mTOR inhibitors are being tested in different clinical trials as anticancer therapy [2+ influx was also found to be crucial for activating the cell death induced by the anti-CD20 monoclonal antibody GA101/obinutuzumab in non-Hodgkin lymphoma and primary B-cell chronic lymphocytic leukemia cells. Moreover, in addition to ORAI1-dependent Ca2+ influx, in this study, it was demonstrated that GA101 determines intracellular Ca2+ elevation by provoking Ca2+ release from lysosomes [The first described inhibitor for ORAI1 channel was SKF-96365, which is able to reduce the growth and migration of breast cancer cells . SOCE ch channel . Another channel . As thes therapy . ORAI1-dysosomes .IP3Rs consist of three isoforms  that are activated by the generation of intracellular IP3. The most isoform of IP3 receptor isoforms involved in carcinogenesis is IP3R3. The dysfunction of this receptor was found in clear cell renal cell carcinoma cells and in colorectal and ovarian cancer cell lines, where this receptor exerted proliferative and antiapoptotic effects . Further2+-dependent cell proliferation [Altered expression or mutations of SERCA isoforms (SERCA2 and SERCA3) was observed in several cancer types, including colon, gastric, lung, and prostate carcinoma ,146,147.feration . In addiferation ,150. In feration ,152. How2+ levels [2+ levels by inhibiting PMCA in prostate cancer cells. Unfortunately, the function of the latter compounds is exerted by the activation of IP3Rs; therefore, they cannot be considered PMCA-specific inhibitors [In the last years, many compounds able to inhibit SERCA for cancer treatment have been produced. Mipsagargin (G-202), a thapsigargin derivate, has been tested on different solid tumors, including prostate cancers, glioblastoma, kidney, and hepatocellular carcinoma, in phase I and II clinical trials. The results obtained in phase I demonstrate that mipsagargin displays a favorable pharmacokinetic profile and acceptable tolerability. Furthermore, significant disease stabilization was observed, suggesting possible antitumor activity . The res+ levels . Furtherhibitors ,157.Alterations in the MCU complex expression/function were found in different cancer types. High expression levels of MCU were detected in colorectal, ovarian, pancreatic, stomach, and prostate cancers, while genetic mutations were observed mainly in prostate, breast, and uterine cancers. Genetic modifications linked to cancer development were also detected in the other components of the MCU complex . Based o2+-permeable pore located on the outer membrane of mitochondrion, where it regulates the flux of ions and metabolites from cytosol to mitochondria and vice versa. Three isoforms for this channel, VDAC1, VDAC2, and VDAC3, have been identified in mammalian cells [2+ fluxes, it is speculated that VDACs may be involved in the control of cell proliferation and apoptosis; therefore, these channels may affect the fate of cancer cells. VDAC1 is upregulated in a variety of human cancer cell lines, while VDAC2 is overexpressed in melanoma, mesothelioma, and thyroid cancer cells [Another important mitochondrial protein is VDAC; this pore is a nonselective Caan cells . Based oer cells . The tarer cells .2+ signaling is extensively involved in these molecular processes, it is not surprising that an anticancer agent may indirectly modulate Ca2+ dynamics in cancer cells. Various studies demonstrated that chemotherapeutic agents modulate intracellular Ca2+ levels. For example, 5-fluorouracil (5FU) is an approved anticancer treatment for several cancer types. It has been observed that 5FU mediates in hepatocarcinoma cell death by diminishing Ca2+ influx. Indeed, 5FU administration decreased ORAI1 levels and induced autophagic cell death by inhibiting PI3K/AKT/mTOR pathway [2+ amounts to a level necessary to activate calmodulin, which, in turn, phosphorylated p53 to trigger apoptosis [2+-mediated apoptosis [2+ transport. However, in this case, Ca2+ dynamics were associated with chemoresistance. Indeed, both inhibition of TRPCs and Ca2+ chelation increased the sensitivity of human leukemia cells to dexamethasone [2+ signaling. Accordingly, these drugs induced the persistent release of Ca2+ from intracellular stores, provoking mitochondrial Ca2+ accumulation and apoptosis. Interestingly, doxorubicin also promoted the binding of p53 to SERCA in the ER. In this state, p53 increased Ca2+ transmission between the ER and mitochondria to induce apoptosis [2+ oscillations that affect neuronal Ca2+ sensor 1 proteins, leading to Ca2+ release from the ER via an IP3R3-dependent pathway [2+ concentration and activating the apoptotic pathway in a p53-dependent pathway [2+ signaling in tumors, particularly 4-aminoquinoline antimalarial compounds chloroquine (CQ) and hydroxychloroquine (HCQ). Currently, more than 30 clinical studies are evaluating the antitumor efficacy of CQ and HCQ. Publications reporting the clinical trial results are encouraging. Indeed, most of these investigations describe positive and/or partial effects of CQ and HCQ in reducing tumor growth alone or in combination with the conventional therapies used for several cancer types [2+ accumulation by inhibiting the IP3Rs-dependent ER Ca2+ release and the Ca2+ influx mediated by TRPCs, ORAI, and STIM channels [2+ signaling to modulate the immunological response [2+ levels that were necessary to activate p38, NF-kB, and TFEB to reprogram the TAM phenotype [2+ homeostasis also in solid tumors associated with PML absence or downregulation. Loss of PML conferred resistance to chemotherapies due to a reduction in ER-mitochondria Ca2+ transmission that activates autophagy and establishes a metabolic advantage for the cancer cells. As a consequence of blocking autophagy with the specific inhibitors , the apoptotic process was rescued in vitro and in vivo [2+ release, and activation of apoptosis. Interestingly, specific MCU inhibitors or MCU silencing abrogated CQ-dependent effects, thus confirming the importance of mitochondrial Ca2+ overload for cell death induction [2+ mobilization in glioma cells that had previously been rendered sensitive to anticancer therapy [2+ uptake and ATP production and diminishing cancer cell proliferation and migration [2+ signaling, which improves the chemotaxis of T cells by increasing intracellular Ca2+ influx [2+ signaling inhibits PDL1 and PDL2 expression [2+ flux is abolished when T cells express high levels of PD-1 [2+ transmission. However, a greater understanding of the steps involving Ca2+ signaling during cancer immunotherapy is required.Proliferation, invasiveness, cell death, neovascularization, gene transcription, protein production, and phosphorylation/dephosphorylation events are some of the numerous targets of anticancer compounds. Given that Ca pathway . In contpoptosis . Similarpoptosis . Additioethasone ,165. Stupoptosis . Taxane  pathway . Photody pathway . Finallyer types . It has channels . This efresponse . This hyhenotype . CQ exer in vivo . Similarnduction . A reduc therapy ; in addiigration . Anotherigration . PD-1 bi+ influx . Furtherpression  and that of PD-1 . Therefo2+ is a ubiquitous signal that contributes to the control of diverse cellular functions. Uncontrolled remodeling of Ca2+ flux contributes to severe pathophysiology processes and often intersects key aspects of cancer progression, such as tumor proliferation, malignant transformation, escape from cell death, and resistance to anticancer agents. Accumulating preclinical and clinical evidence supports the relationship between Ca2+ and cancer, indicating that Ca2+ signaling is a reliable target for novel anticancer treatments. As summarized in this review, defects in Ca2+ channels/transporters/pumps are typical features of cancerous cells and confer low sensitivity to cell death inducers, thus sustaining the tumor growth and metastasis. Hence, pharmacological modulation of these proteins may be a reliable approach to restore the effectiveness of current cancer treatment regimes (2+-regulators, it is important to consider other critical aspects. Targeting these processes is difficult, and most importantly, the tumor environment presents substantial cellular heterogeneity in which only a subset of cancer cells should be targeted. Recent investigations have made progress in overcoming these problems. For example, by encapsulating pumps and channel agonists in lipid nanocapsules, it is possible to efficiently modulate the activities of these pumps and channels and the related cellular processes [2+ modulators can be solely directed to the cancer cell population [2+ in cancer, this area of study is still in its infancy, with a number of studies starting to explore and highlight the importance of these interconnections. Removing the remaining gap in our knowledge on the intersections between Ca2+ and cancer will help researchers better understand the multiple molecular mechanisms that affect tumor development, maintenance, and metastasis and help clinicians design and develop new-generation drugs with the final aim of breaking all the defense barriers of cancer.Cellular Ca regimes . Howeverrocesses . In addipulation ,180. Fur