An early consequence of ageing in humans is a decline in vision. Usually, this is limited to presbyopia starting at the age of forty. However, at later age, more severe age-related eye disease can occur, possibly resulting in blindness. Proper corneal epithelium maintenance is indispensable for corneal clarity and good vision quality. A major culprit of blindness associated with corneal epithelium defects is the formation of corneal scars. These scars are the outcome of a wound healing process initiated by re-epithelialisation and further driven by activated keratocytes, which help to re-establish the integrity of the tissue but fail to restore optical properties.2+ signalling, bioenergetics and cell survival. Thus, given the crucial role of ER-mitochondrial Ca2+ signalling in health and disease, CISD2 is uniquely positioned to impact cellular fitness. However, the exact mechanisms by which CISD2 influences intracellular Ca2+-transport systems remain unclear.A protein key to healthy ageing in mammalian organisms is CDGSH Iron Sulfur Domain 2 (CISD2) Vice versa, CISD2-deficient mice displayed corneal epithelium deficits due to a vicious cycle of inflammation and continuous loss of epithelial cells, further aggravated by impaired renewal and differentiation of epithelial cells from CLSC. Comparing young versus old mice, the authors revealed a continuous proliferation of CLSCs compensating for the increased loss of epithelial cells in young CISD2-knockout mice, eventually resulting in CLSC depletion and accumulation of corneal epithelium damage. Next, the authors revealed that corneal epithelial cells lacking CISD2 displayed higher basal cytosolic Ca2+ levels, resulting in augmented calcineurin activity and increased expression of genes driven by nuclear factor of activated T-cells (NF-AT), a transcription factor activated by calcineurin. Together with reduced mitochondrial metabolism and increased ROS production, loss of CISD2 impaired epithelial cell migration and wound healing. Interestingly, cyclosporin A (CsA) and FK506, two calcineurin inhibitors of independent classes, rescued these features and normalised basal cytosolic Ca2+ levels, thereby suppressing NF-AT signalling. Not only did CsA restore corneal functionality in CISD2-deficient mouse models, it improved clinical outcomes of patients with corneal defects due to severe dry eye disease. Here, the authors enrolled five patients with dry eye disease, who were topically treated with a more concentrated CsA solution compared to the standard (0.1% instead of 0.05%). Clinical analysis revealed improved corneal epithelium integrity and functionality, reducing dry eye characteristics. Hence, this study advocates revisiting the clinical treatment regimens of patients with dry eyes by augmenting the CsA concentration in topic applications. Although a modest increase from 0.05% to 0.1% already had a beneficial outcome on the corneal functionality, CsA concentrations up to 2% are considered safe for such treatments 2+ signalling in human health, as also reduced Ca2+ signalling have been implicated in dry eye disease. Indeed, mice lacking IP3 receptors isoforms 2 (IP3R2) and 3 (IP3R3) display dry eye disease 3R2 and IP3R3-protein levels and a concomitant decrease in IP3R-mediated Ca2+ release Inspired by their previous findings in CISD2-deficient mice suffering from blindness due to corneal damage Many questions remain, representing exciting future research avenues.2+-transport systems and thus enables adequate intracellular Ca2+ homeostasis and dynamics in corneal epithelial cells. These additional CISD2 targets may provide additional strategies to normalise Ca2+ signalling in the cornea, possibly including pumps such as Ca2+ sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs) and intracellular Ca2+-release channels such as IP3Rs and ryanodine receptors (RyRs) (reviewed in 2+ dynamics. This further supports the concept that, beyond transcriptional programs, hyperactive calcineurin can directly modulate the properties of Ca2+-transport systems.It remains unclear how CISD2 sustains the function of intracellular CaThe underlying mechanisms downregulating CISD2-protein levels remain to be established. Additionally, it is not clear whether only CISD2 proteins or also other proteins are declined in dry eye diseases. Such insights may provide novel opportunities to elevate CISD2 levels, thereby boosting the repair of corneal epithelium defects. This might be feasible, as studies focusing on spinal cord injuries demonstrated that curcumin could counteract injury-induced downregulation of CISD2 and subsequent inflammation 2+ signalling dysregulation might underlie commonalities in the pathogenesis and disease outcomes upon loss of function of WFS1 or CISD2 Finally, Wolfram syndrome type 1 and 2 are caused by mutations in WFS1 and CISD2, respectively, and are both characterised by optical atrophy, diabetes type 1 and deafness. It is also clear that their converging role in CaGB conceived this invited commentary with further input from FS and JL. All authors contributed to the writing.None of the authors have any competing interests to declare.


TP53, is associated with abysmal survival outcomes in acute myeloid leukemia (AML). Although it is the most commonly mutated gene in cancer, its occurrence is observed in only 5–10% of de novo AML, and in 30% of therapy related AML (t-AML). TP53 mutation serves as a prognostic marker of poor response to standard-of-care chemotherapy, particularly in t-AML and AML with complex cytogenetics. In light of a poor response to traditional chemotherapy and only a modest improvement in outcome with hypomethylation-based interventions, allogenic stem cell transplant is routinely recommended in these cases, albeit with a response that is often short lived. Despite being frequently mutated across the cancer spectrum, progress and enthusiasm for the development of p53 targeted therapeutic interventions is lacking and to date there is no approved drug that mitigates the effects of TP53 mutation. There is a mounting body of evidence indicating that p53 mutants differ in functionality and form from typical AML cases and subsequently display inconsistent responses to therapy at the cellular level. Understanding this pathobiological activity is imperative to the development of effective therapeutic strategies. This review aims to provide a comprehensive understanding of the effects of TP53 on the hematopoietic system, to describe its varying degree of functionality in tumor suppression, and to illustrate the need for the adoption of personalized therapeutic strategies to target distinct classes of the p53 mutation in AML management.Mutation of the tumor suppressor gene,  TP53 gene and its protein was first described in 1979 and has since taken center stage due to an avalanche of research after mutations in this gene were identified as the common denominator in more than 50% of human cancers. TP53 mutation associated acute myeloid leukemia represents a distinct molecular cohort with a notorious reputation for uniformly poor prognosis . P5. P5TP53 TP53 gene is largely a stress response protein with functions ranging from apoptosis to cell cycle control. Therefore, a mutation in the TP53 gene pronounces the effects of oncogenes leading to the uncontrolled proliferation of tumor cells. These mutations are known to give rise to a deleterious gain of function (GOF), a loss of function (LOF) or a non-mutational dysfunction or inactivation of the p53 protein ..69].TP53 mutation resulting in loss or alteration of function is seen in approximately 8% of de novo AML. However, these genetic variants are present in 30% of t-AML cases and in patients with complex karyotype, especially in the elderly, they are encountered in around 70% of subjects. Nevertheless, prognosis and overall survival is poor in all subgroups [TP53 mutated leukemia as an unfavorable group. Overall survival for patients with TP53 mutation and complex cytogenetics  is worse in comparison to TP53 WT and complex karyotype or TP53 mutated with no complex cytogenetics [ubgroups ,112,113.genetics .TP53 mutations as part of CHIP have been observed although mutations in DNMT3A, TET2 and ASXL1 are more common compared to TP53 [TP53 in a normal population through prospective study, subjects that harbored a detectable VAF of 1% or more in TP53 or IDH1/2 mutation eventually developed AML. RUNX1 had the shortest latency to develop AML (1.5 years vs. 9.6 years without mutation). Of note, the RUNX1 study was limited by small sample size (only 3 patients), hence more studies are needed to validate this finding. Other mutations with increased odds of developing AML included DNMT3A, TET2, and spliceosome-related genes SRSF2, SF3B1 and U2AF1 [TP53 and IDH mutations, wherein the odds to develop AML was independent of VAF [Presence of  to TP53 ,63,114. TP53 and PPM1D clones were strongly enriched during and after induction chemotherapy. This finding does not necessarily represent the initiation of these mutations but is rather considered to be secondary to preferential expansion of candidate mutations that were present in small frequencies prior to cytotoxic chemotherapy [Mutated otherapy ,115.TP53 mutation is commonly associated with complex karyotype, chromothripsis, chromosomal arm losses , cytogenetic alterations to copy number (aneuploidy) or some combination of these features [TP53 mutations are not associated with low risk aberrations like t (8:21) or inv 16 [TP53 mutations were associated with trisomy of chromosome 8, gain of chromosome 17 (11.2), 14 (q32.3), 16p (11.2–11.3) and deletion of chromosome 12 (p12.3) [TP53 mutated AML is unknown, the frequent observation of chromosome 5, 7 and 17 aneuploidy with mutated TP53 advocate for their involvement in the transition from CHIP to AML [features ,66,116. r inv 16 . An anal (p12.3) ,117. AltP to AML .TP53 mutations are less often linked with alterations in the RAS pathway (4%), FLT3 (6%) or NPM1 (8%), [TP53 mutation. If associated with a founding clone, TP53 mutation usually coexists with transcription factors  or epigenetic genes  or can be seen as a subclone in the process of clonal evolution triggered by a poly comb pathway of SF3B1, SRSF2 or a signaling mechanism involving JAK2, RAS, FLT3, PTPN11, or BCOR [ pathway %, FLT3 (M1 (8%),  and to aM1 (8%), ,112,116. or BCOR ,61,114.TP53 mutation in AML. In the study TP53 mutations were divided by their VAF into subgroups of >40%, 20–40%, and <20%. VAF of <20%, although considered sub-clonal, had a negative impact on overall survival (OS), complete remission rate and event free survival rates. However, a lower VAF was associated with fewer chromosomal losses and complex karyotype [TP53 can be associated with diploid cytogenetics but usually the VAF is lower compared to non-diploid cytogenetics.Recent studies have shown the importance of VAF to prognosticate the role of sub-clonal aryotype . Similararyotype ,120. TP5TP53 VAF of >40% was independently correlated with worse relapse free survival and OS [Short et al. further validated these findings by demonstrating that AML with mutated l and OS .Furthermore, Sallman et al. describe the striking relationship between an increase in VAF% and worsening karyotype complexity, which again correlated with worsening OS .TP53 mutation clonal evolution, which is generally associated with resistance to lenalidomide and transformation to AML. As per the world health organization (W) 2016, monitoring for the appearance of clonal evolution or increase in the clone of TP53 predicts disease progression [About 20% of patients with del 5q have gression ,124,125.TP53 genome instability varies depending on whether it is a monoallelic TP53 hit per single gene mutation or biallelic/multiple hits. Rate of transformation to AML from MDS and overall survival was worse with multiple TP53 hits in comparison to monoallelic TP53 hits- which was very similar to the TP53 WT [The extent of  TP53 WT .TP53 mutated AML with loss of TP53 allele displays a lack of chemotherapy induced apoptosis and subsequently a poor response rate (RR), disease-free survival (DFS) and OS while being independent of cytogenetic aberration [TP53 mutations, however, it has not been able to deliver a durable response or clear subclones of TP53 mutations [TP53 mutations compared to WT TP53 (100 vs. 41%) [TP53 AML to DAC is currently uncertain. TP53-mutated AML patients receiving a monthly regimen of 5-day DAC had an overall response rate of 62% [Therapy with standard chemotherapy in utations ,128. Nevvs. 41%) . The undTP53 mutated status. Intriguingly, response rate did not differ depending on baseline VAF. Four patients had a VAF ≥20% at the time of remission suggesting the presence of TP53 mutation preleukemic clones rather than solely myeloblasts. The two patients that had the longest remission duration had no detectable TP53 mutations present in the remission bone marrow sample [TP53 mutations have poor outcomes but recently sub-clones were noted to have equally poor outcomes [Short et al. assessed the efficacy of a 5-day DAC schedule compared to a 10-day regimen in a randomized phase II, open label, single center trial. There was no difference in outcome between the two DAC schedules for those with w sample . It is woutcomes .TP53 mutated cells are more sensitive to hypomethylating agents in comparison to cytotoxic agents, providing the impetus for a combination with bcl-2 inhibitor- Venetoclax (VEN) AML study. In patients harboring TP53 mutations, the complete response (CR) + complete remission with incomplete blood count recovery (CRi) rate was 47% while the median OS was 7.2 months. Compared to historical standards of 28% CR rates, the combination displayed promising efficacy. VEN mediated apoptosis appeared TP53 independent [ependent ,132,133.TP53 perturbation. Hence, on disrupting TP53 function within an AML cell line by using CRISPR/Cas9, DiNardo et al. were able to demonstrate an association between TP53 loss and resistance to VEN, hypomethylating agent (HMA) and cytarabine (ARA-C) as a single agent or in combination [TP53 and BAX [One of the primary and adaptive forms of resistance to VEN-based combination therapy is activated bi-allelic bination . Similar and BAX ,136.TP53 mutation and complex cytogenetics portend inferior OS in patients with MDS. However, outcomes in patients with complex cytogenetics and WT TP53 are not poor, solidifying the fact TP53 mutation is a critical driver for poor overall survival [Allo-SCT with survival .TP53 mutated AML, necessitating the dire need for newer therapeutic options. The primary objective of newer therapies is to restore normal TP53 function by either degrading or inactivating mutant p53 or reviving WT p53. (Relapses and primary resistance still occur in  WT p53. .TP53 was considered undruggable. Eprenetapopt (APR-246), a PRIMA-1 analog, is a novel, first-in-class, small molecule therapeutic agent that binds covalently to cysteine residues in mutant p53 protein and induces a conformational shift to a WT-like structure.For the longest time TP53 expression and in WT TP53 cells by inducing heightened oxidative stress resulting in cell apoptosis [TP53 mutation. Median duration of response was 6.5 months and median time to response was 2 months. Of the 45 patients evaluated, overall response rate was 88% and 53% achieved CR [This enables p53 binding to a specific DNA sequence leading to reactivation of pro-apoptotic function by enhancement of target gene transcription ,139. APRpoptosis  Due to aieved CR .TP53 VAF clearance correlated with CR in both the studies. On the basis of these encouraging results, APR246 received fast track and orphan drug designation from FDA in April 2020. Results of the randomized phase 3 study of APR-246 and AZA compared to azacytidine alone (NCT03745716) is awaiting full report.A European phase 2 study with a similar cohort of HR-MDS and AML showed an overall response rate of 75% and CR of 56% . TP53 VATP53 mutated myeloid malignancies (NCT04214860) and being assessed in combination with azacitidine following allo-SCT as maintenance therapy in TP53 mutated AML/MDS (NCT03931291).APR346 is being investigated in a phase I study in combination with AZA/VEN for newly diagnosed TP53 mutated MDS (NCT04638309). The synergy observed between APR-246 and AZA is hypothesized to be related to down regulation of FLT3 pathway [APR-548, an orally bioavailable derivative of APR-246, is currently being developed to be used in a phase 1 study with AZA in  pathway .TP53 mutational status [TP53 activity, targeting degradation via ubiquitination. In AML, MDM2 is often overexpressed, hence cells may have low functional TP53 activity without TP53 deletion/mutation. Nutlin-3a is a small molecular inhibitor of the MDM2/p53 interaction, thus this therapeutic provides increased p53 levels secondary to reduced p53 destruction, consequently upregulating apoptosis [p53 pathway dysfunction is highly prevalent in acute myeloid leukemia independent of l status  MDM2 andpoptosis .TP53 stabilization, cell cycle arrest and apoptosis were produced in a dose dependent fashion [TP53 mutational status alone did not correlate with CR, although increased expression of MDM2 among HSPC and leukemia blasts was consistent with CR [RG7388/idasanutlin is an orally bioavailable MDM2 inhibitor which was investigated in the placebo controlled, double blind phase III MIRROS study in combination with cytarabine in the management of relapsed refractory (r/r) AML. Unfortunately, the study had to terminate early as it did not meet primary end point of improved survival in comparison to ARA-C alone . TP53 st fashion . TP53 mu with CR ,148.Idasanutlin is currently in clinical trials in combination with Venetoclax in r/r AML (NCT02670044 and NCT04029688). The preliminary results were presented at the 2019 American Society of Hematology annual meeting, and the CR/Cri was reported in 11 of 49 patients . It is iTP53 binding pocket on the surface of MDM2 [AMG 232 is a structurally distinct MDM2 inhibitor and interacts with the glycine self-region of the  of MDM2 . AMG 232TP53 mutations.Of the 30 patients evaluated, four achieved a morphological leukemia-free state. Unfortunately, no response was noted in patients with AML possesses high expression of MDMX. Current MDMD2 inhibitors provide a limited effect on other members of the MDM family, including MDMX, which decreases their utility in AML treatment . ALRN-69TP53 mutated AML patients (n = 9) resulting in a CR/Cri of 78% and 44%, respectively and negative MRD in 57% of the responders, with a median follow up of 6.9 months. Median duration and survival were not reached [Cell surface CD47 interacts with its corresponding receptor on macrophages to inhibit the phagocytosis of normal, healthy cells. CD47 seems to be over expressed in myeloid malignancies, and overexpression of CD47 in myeloid leukemia increases its pathogenicity by allowing for tumor evasion of macrophages. Inhibition of CD47 induces engulfment of leukemic cells . Magroli reached .TP53 via 26S proteasome and upregulate WT TP53 functions, thereby inactivating proliferating leukemia cells and promoting apoptosis [TP53 with minimal effects on WT p53 and DNA contact mutants [Arsenic trioxide has been shown to inactivate poptosis ,157. Fur mutants .TP53 on normal HSC self-renewal. The impact of TP53 mutations on CH is indisputable. Improved characterization of the role of TP53 in normal hematopoiesis will lead to a more complete understanding of TP53 mutations in the propagation of CH and leukemogenesis. Pathways leading to TP53 dysregulation represent a common element in non-TP53 mutated AML, hence leveraging WT TP53 in unmutated AML serves as a corner stone in the design of anti-AML therapeutic strategies.There is growing evidence of age-related mutations and CH in the HSC and the influence of TP53 and mutated TP53 within tumor cells can be therapeutically targeted, which has led to the use of novel therapies like, APR-456 which has reported to restore transcriptional activity in mutant p53 or unfolded WT p53, leading to apoptosis or the promising result of anti CD47monoclonal antibody magrolimab in TP53 mutated AML. Recently, it has become understood that unaltered TP53 mutations in AML are distinctive, although rare, and confer dismal responses to chemotherapeutic agents with very poor outcomes.This approach is of great precedence, as TP53 pathway activators may yield immense contributions to the management of AML, a pathology associated with a very high risk of therapy failure.Hence, research focused on the exploitation of


Directional cell migration requires the polarization of both signaling and structural elements. This polarization is reflected in various Ca2+ signaling pathways that impinge on cell movement. In particular, store-operated Ca2+ entry (SOCE) plays important roles in regulating cell movement at both the front and rear of migrating cells. SOCE represents a predominant Ca2+ influx pathway in non-excitable cells, which are the primary migrating cells in multicellular organisms. In this review, we summarize the role of Ca2+ signaling in cell migration with a focus on SOCE and its diverse functions in migrating cells and cancer metastasis. SOCE has been implicated in regulating focal adhesion turnover in a polarized fashion and the mechanisms involved are beginning to be elucidated. However, SOCE is also involved is other aspects of cell migration with a less well-defined mechanistic understanding. Therefore, much remains to be learned regarding the role and regulation of SOCE in migrating cells.Ca In this review, we briefly outline the involvement of Ca2+ signaling in cell migration while focusing on the role of store-operated Ca2+ entry (SOCE). Cell migration is essential for the development of multicellular organisms and is critical for many physiological processes, including organ development, morphogenesis, tissue repair and homeostasis, immune response and wound healing . It is a2+ dependent assembly and disassembly cycles [Cell migration is a complex coordinated process that incorporates many cellular components and responds to a plethora of environmental cues. Typically, those environmental signals guide the directional migration of cells . For a cy cycles ,6, as wi2+/calmodulin-activated myosin-light chain kinase regulate actomyosin fibers contraction that cause the retraction of the trailing end of the cell and its net forward movement [The actin cytoskeleton is vital in the regulation of focal adhesions as well as in generating the forces required for cell migration. In that context, actin dynamics are regulated by small GTPases, including Rac1, Cdc42 and RhoA . Rac regmovement ,9. Cdc42movement ,11.Therefore, the actin cytoskeleton through spatially controlled cycles of polymerization and depolymerization supports membrane deformation and force generation that are required for cell movement. In addition to the actin fibers that crisscross the cell, cells form a shell of cortical actin bundles around their periphery. Cortical actin directly interacts with the PM through the ERM proteins , that associate with PtdIns  P2 at the cell membrane through their FERM domain at one end and with actin at the other end, thus linking the PM to the actin cytoskeleton . In addi2+ transients that underlie cellular responses can be mediated either by Ca2+ release from intracellular Ca2+ stores (primarily the ER), or Ca2+ entry from the extracellular environment [2+ transients are typically generated in response to activation of cell surface receptors (G-protein or tyrosine-kinase coupled receptors) leading to stimulation of PLCs that hydrolyze PI  P2 at the cell membrane producing the second messengers IP3 and DAG. IP3 diffuses readily and binds IP3 receptors (IP3R) on the ER membrane, which are ligand gated cation channels, thus releasing Ca2+ from ER stores. DAG is a lipid second messenger that diffuses within the plasma membrane (PM) and activate PKC, thus expanding the signaling modalities. DAG and Ca2+ combine to activate PKC and by extension its downstream targets to code for specific cellular responses. Interestingly, many receptors activate this signal transduction cascade with disparate cellular responses ranging from cell migration to division. Therefore, somehow Ca2+ signals produced through the PLC-IP3 pathway must encode specificity to direct a particular cellular response. Much of the specificity is encoded in the spatial and temporal properties of the Ca2+ signals produced [2+ release from stores in response to agonists, but also, a subsequent Ca2+ influx from the extracellular space. Ca2+ influx can be mediated through receptor-operated cation channels of the TRP family, which can be activated by DAG or its metabolites among other diverse stimuli [2+ release from stores results in Ca2+ store depletion leading to activation of store-operated calcium entry (SOCE) [Cytoplasmic Caironment . Physiolproduced . Such sp stimuli . Alternay (SOCE) . SOCE pl2+ signaling in migrating cells is polarized. This was first reported three decades ago as an ascending front to rear Ca2+ gradient across the cell (higher Ca2+ at the rear compared to the front) [2+ ATPase (PMCA) to the front of migrating cells resulting in enhanced Ca2+ extrusion out of the cell [2+ influx through voltage-gated L-type Ca2+channels at the trailing edge, thus supporting increased Ca2+ concentration at the back of migrating cells [2+ channel activities are not fully understood. As is the case with the actin cytoskeleton and its modulators, Cae front) ,23  activation and allow small local Ca2+ pulses to effectively stimulate actino-myosin contraction [Furthermore, localized Ca channel ,27 ; and a sterile α-motif (SAM) that is essential for STIM dimerization [Structurally, STIM1 is an ER transmembrane protein with an ER luminal region encompassing a canonical EF-hand motif that senses ER Carization ,48,49,50rization ,53,54,552+ store levels with STIM2 sensing mild store depletion to maintain basal Ca2+ homeostasis, whereas STIM1 senses significant store depletion to activate SOCE [2+ entry [2+ concentration upon minimal to moderate store depletion, while STIM1 is considered the major ER Ca2+ sensor for strong store depletion [2+ influx mediated by STIM2 in NFAT activation [Vertebrate genomes code for two STIM isoforms: STIM1 and STIM2, both of which are enriched in the ER membrane. Despite the high sequence conservation and the structural homology between STIM1 and STIM2, they differentially sense ER Caate SOCE ,57,58. Tate SOCE . Further2+ entry ,61. Hencepletion . Overexptivation . These etivation ,65. 2+ selective channel at the PM with 4 transmembrane domains and cytosolic N- and C-termini [2+ ATPase SPCA2 to elicit constitutive SOCE and enhance the carcinogenesis process in human breast cancer [STIM1 which exists as a dimer at rest adopts an open activated confirmation after store depletion that exposes the SOAR/CAD domain, which binds to the Orai1 channel and gates it open . Orai1 i-termini . Vertebr-termini ,68,69. T-termini . Typicalt cancer ,72. There is significant interest in the literature in the role of SOCE in cancer progression and metastasis with implication on the role of STIM1 and Orai1 in cell migration. This was first assessed in the context of breast cancer where STIM1 and Orai1 were shown to be important for breast cancer cell migration and metastasis to the lung using xenograft mouse models . This stIn the context of cell migration STIM1 knockdown was shown to accelerate sheet cell migration in human umbilical vein endothelial cells (HUVEC) and its overexpression results in decreased migration . In cont2+-calmodulin binding IQ motif [The above studies show a role for SOCE in modulating FA turnover. This was recently extended in a study focused on the role of the Arf family of small GTPases in cell migration. The GTPase activity of Arfs is modulated by guanine nucleotide exchange factors (GEF) and GTPase activating proteins (GAP), which increase nucleotide exchange or GTP hydrolysis, respectively. One of these Arf GEFs is IQSec1, which contains a CaIQ motif . IQSec1 IQ motif . Knockdo2+ gradient in migrating cells with low Ca2+ at the front and the role of SOCE in FA disassembly at the rear. It was proposed that the SOCE machinery at the front of migrating cells is important to replenish Ca2+ stores depleted following Ca2+ transients mediated by TRPM7 with the associated Ca2+-induced Ca2+ release through IP3 receptors [2+ stores in the front of a migrating cell without inducing a broad cytoplasmic Ca2+ rise. This argues for the involvement of Ca2+ tunneling, which allows for Ca2+ flowing through SOCE channels to fuel IP3-dependent Ca2+ release without inducing a cytoplasmic Ca2+ rise [2+ tunneling is a tightly coupled Ca2+ signaling modality that allows for Ca2+ flowing through SOCE channels to be taken up into the ER through the activity of the sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) and then released through open IP3 receptors [3 receptors localize to the leading edge of migrating acinar cells. Intriguingly, IP3 receptors surround individual focal adhesions and IP3-dependent Ca2+ release enhances FA size. Furthermore, inhibition of either SOCE or IP3-dependent Ca2+ release inhibits migration [2+ flowing through Orai channels at the leading edge of migrating cells is tunneled through IP3 receptors to FA to strengthen nascent adhesion. These studies argue that the modulation of FA by SOCE is polarized and diagonally opposed at the front and rear of migrating cells, but functionally culminates in supporting cell migration. At the leading edge of migrating cells SOCE strengthens FA and enhances cell migration, whereas at the trailing end SOCE results in FA disassembly, which is also required for cell migration . Intereseceptors . This isa2+ rise ,101,102.eceptors   were mutated to alanines reduced cell migration . Further2+ homeostasis has been repeatedly associated with cancer progression, which requires cellular migration to mediate metastasis. Ca2+ in the extracellular space is between 1–2 mM, in the cytosol ~100 nM and in the endoplasmic reticulum (ER) ranges between 100–800 µM [2+ homeostasis, including channels, transporters, receptors, downstream effectors and buffering proteins [2+ levels or decrease ER Ca2+ have been associated with cancers and changes in the expression of specific Ca2+ pumps or channels have been documented in several tumor types [Interestingly, disruption of Ca0–800 µM . Severalproteins . Disruptor types . This afor types ,112,113.2+ channels  have been associated with different cancer types such as melanoma, colon, prostate, and pancreatic cancers [Expression and function of members of the TRP channel superfamily, including TRPC1, TRPC6, TRPV1, TRPV2, TRPV6, TRPM7 and TRPM8 have been implicated in tumor growth and migration ,122,123. cancers . For ins cancers . In anot cancers . Further cancers ,118. 2+ signaling pathways store operated Ca2+ entry (SOCE) has also been implicated in cancer metastasis and tumor cell migration as will be discussed in further details below. This is consistent with the notion that Ca2+ influx plays an essential role in the tumorigenesis and metastasis [In addition, to these Catastasis ,128. Cancer metastasis is considered the end stage of the progression of any tumor ,130,131.2+ and proton transport, which are important for cell migration. Among Ca2+ influx pathways, SOCE has been repeatedly implicated in the migration and proliferation of many cancer types such as cervical cancer, breast cancer and melanoma [2+ content [In addition to cytoskeletal remodeling, signaling cascades, ion channels and transporters have also been implicated in the metastatic cascade . Such trmelanoma ,76,117.  content ,134,135. content ,121,122, content ,137. As summarized in Clinically breast tumors with high expression of STIM1 and low expression of STIM2 were associated with poorer prognosis . High le2+ influx. This finding argues that STIM2 upon complete loss of STIM1 can support Ca2+ influx to modulate cell migration [As is the case in cancer cells, modulation of the expression of STIM1/Orai1 has differential effects in other non-cancerous cell lines . Knockdoigration .2+ levels at protrusion sites in migrating cells, thus, enhancing the reorganization of the cytoskeleton network and supporting both lamellipodia and filopodia formation [Finally, knockdown of STIM1 in MEFs resulted in a significant reduction in the number of invadopodia/podosomes . Invadopormation . Moreoveormation .Collectively these studies show that the expression of STIM1 has differential effects in different cancers and sometimes as is the case for melanomas in the same cancer type. In most cases STIM1 downregulation inhibits metastasis, but there are clear examples that are difficult to ignore where STIM1 downregulation enhances metastasis and cell migration. In particular, in the example of the unbiased ribozyme screen using a weak melanoma cell line that identified STIM1 as a tumor suppressor . Based oSOCE may also be involved in the epithelial to mesenchymal transition (EMT). In colorectal cancer cell lines STIM1 knockdown resulted in increased expression of E-cadherin and β-catenin and decrease in the level of vimentin and fibronectin . β-caten2+ levels, activates NFAT1, which in turn induces TGF-β1 expression to promote EMT and enhance cell migration and metastasis of breast cancer cells. The STIM1 effect on cell migration was also attributed to NFATc1 activation in both osteosarcoma and gastric cancer cells [STIM2 was also implicated in breast cancer metastasis through supporting EMT . STIM2 ker cells ,88. Addier cells . The lin2+ signaling is intimately connected to the regulation of cell migration and regulates both adhesion to the ECM as well as cytoskeletal remodeling. The Ca2+ signaling machinery is polarized in migrating cells and this polarization has functional consequences on migration. In particular, SOCE has differential effects at the front and rear of migrating cells in terms of regulation of focal adhesion dynamics. Furthermore, modulation of either STIM1, STIM2 or Orai1 expression may be associated with remodeling of the cell’s transcription program given the well-documented induction of NFAT through calcineurin activation downstream of SOCE [Ca of SOCE . This ma2+ entry through SOCE. Furthermore, SOCE does not function independently in modulating Ca2+ signaling dynamics in migrating cells. Hence, changes in the expression levels of SOCE components may be countered differentially in terms of modulating the activity/expression of other Ca2+ signaling components involved in cell migration . In turn these Ca2+ channels/transporters would affect Ca2+ dynamics and accordingly differentially modulate the actin cytoskeleton and focal adhesions with functional consequences on cell migration. Therefore, a comprehensive understanding of the role of SOCE in cell migration will require more in-depth mechanistic studies on multiple cell types to better define not only the molecular regulation of SOCE in the polarized migrating cell, but also to define the SOCE downstream effectors in the context of cell migration.The effect of modulating SOCE in terms of cell migration and tumor metastasis is tissue and cell type dependent with sometimes opposing effects. For example, STIM1 was originally isolated as a tumor suppressor and was validated as such in an independent screen ,43,125,


Superresolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit; however, most of the techniques are hampered by the need for fluorescent labels. Nonlinear label-free techniques such as second-harmonic generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. We use the photonic nanojet (PNJ) phenomena to achieve SR-SHG. A resolution of  HoweverTo realize label-free SR-SHG imaging, we have employed the photonic nanojet (PNJ) phenomena, as it does not rely on fluorescent switching/blinking and is simple to implement. A PNJ is a localized focus of light with an intensity cross section narrower than the diffraction limit that is able to propagate over multiple wavelengths . InitialThe focal spot of a PNJ, when generated using a microsphere, is dependent on multiple parameters, including sphere size, imaging wavelength, and the refractive index contrast with the surrounding medium . For bioSHG interrogates the second-order polarizability of materials and detects noncentrosymmetric structures. It is widely used to image fibrillar collagen in bioimaging . SHG is Here we present the first demonstration of SR-SHG microscopy. We use PNJs generated using a microsphere to achieve SR-SHG imaging in the far field and demonstrate a route to extended field of view of superresolved SHG imaging. We investigate the origin of SR imaging using PNJs highlighting a critical near-field component. Most importantly, we find that polarization properties of the excitation are maintained in a PNJ. This unexpected finding crucially enabled polarization-resolved SR-SHG (pSR-SHG), which further enhances resolution. In an unprecedented and highly relevant application, we show that nanoscopic anisotropy analysis enabled by pSR-SHG allows the detection of underlying nanoscale changes in collagen during the development of lung fibrosis. Overall, our work heralds the role that label-free SR techniques can play to unravel underlying biological phenomena and improve our understanding of disease mechanisms.2.Supplement 1, Fig. S1A and S1B). In the context of SHG from single collagen fibers, it has been shown that with higher excitation power, sensitivity to smaller fiber diameters is achieved [Supplement 1, Fig. S1(C–E)). Since SNR is inherently linked to resolution, further experiments were conducted at the highest excitation power possible  within the damage threshold of the sample to obtain the highest resolution within the imaging conditions.Achieving SR-SHG imaging can have applications in understanding nanoscale biology and evolution of SHG-active materials, such as in disease processes. Here, we used the PNJ phenomena to achieve SR-SHG . The SHGachieved . We cleaSupplement 1, Fig. S2) and a lung tissue section . We demonstrated that PNJ-assisted SHG imaging allowed the distinction of individual fibers and their directionality, an observation that was not possible with conventional diffraction-limited imaging. A typical collagen fiber is between 12 and 500 nm (age-dependent) in diameter, as determined previously by electron microscopy [We acquired images of the same location of the sample without and with PNJ excitation of SHG to investigate the extent of improvement over conventional diffraction-limited SHG. A clear improvement in resolution is apparent in SHG signals from barium titanate nanocrystals  , whereasSupplement 1, Fig. S4; smaller spheres can achieve better resolution due to their narrower PNJ [Supplement 1, Fig. S5) the slits can be well resolved, satisfying the Rayleigh criterion for slits (81% of peak) [In order to quantitatively determine the resolution limit for SR-SHG, a patterned silicon sample was fabricated of five individual cuts etched on a thin silicon membrane using a focused ion beam. The width of each cut is 100 nm, while the spacing between them is 125 nm (py (TEM) . Figure ower PNJ . As showof peak) ,38. Thisof peak) . In our of peak) . This coDespite imaging occurring in the far field, it has been proposed that the SR effect of PNJs has a strong near-field component ,42. To fAs the PNJ moves closer to the sphere surface, the components of near-field components increase, resulting in a reduced spot size . The sma results .Supplement 1, Fig. S4). Therefore, if taken with an array of spheres, the image from neighboring spheres can overlap and hence needs to be optimized. With a 60 µm diameter sphere, a focal position of While SR is clearly demonstrated with PNJ-assisted SHG, a potential limitation is the restriction of the FOV given by one sphere. f images . The cloite FOVs –45 and cSupplement 1, Fig. S6C). A similar result is obtained with two closely spaced barium titanate crystals . Moreover, this dependence has been used previously to achieve SR using SHG labels [Having shown that SR-SHG is possible using the PNJ approach and that the FOV can be expanded, we sought to investigate ways to achieve ultrahigh resolution and develop a technique that can be used for distinction at the nanoscale level. Hence, we examined the property that in SHG, signals are dependent on the relative orientation of harmonophores versus the polarization of the excitation . FurtherG labels . To demoG labels . HoweverG labels –9. This Supplement 1, Fig. S7). The same location taken from a sample containing collagen fibrils  was imaged. Polarization-dependent changes can be measured by polarization anisotropy; e images  that shoSupplement 1, Fig. S8) that in this model, the collagen ultrastructure changes significantly following treatment with lysyl-oxidase/lysyl-oxidase-like (LOX/LOXL) inhibitor b-aminoproprionitrile (BAPN), which inhibits cross-linking of collagen fibers [Supplement 1, Fig. S9). The polarization anisotropy measured from diffraction-limited images is unchanged . However, SR-SHG indicated that the mean fiber widths for control and BAPN-treated samples were 119 and 158 nm, respectively. This increase is in agreement with electron microscopy analysis . These widths are much narrower than the Supplement 1, Fig. S9), Supplement 1, Fig. S10). Thus, the PNJ is able to probe a greater number of fibers in the axial direction while maintaining subdiffraction-limited lateral resolution, increasing sensitivity to SHG polarization anisotropy and, by extension, their changes as a result of drug treatment or disease.Diffraction-limited as well as PNJ-assisted SHG images of sections from a tissue spheroid model of the fibrotic lung disease, idiopathic pulmonary fibrosis (IPF) were acquired . We haven fibers . Figure n fibers , and hernchanged . SR-SHG irection . The axibased on . We attrCurrently, imaging of such nanoscale changes in collagen is carried out by electron microscopy (EM)  and occa3.SR-SHG is able to improve the resolution of a conventional SHG microscope and facilitate label-free SR imaging in a simple and cost-effective way. In this work, we showed both qualitatively and quantitatively that PNJ-assisted SR-SHG can be achieved. We demonstrated a A.1.SHG imaging was performed on a home-built multiphoton imaging system  consisti2.Spheres made of barium titanate glass (BTG), (refractive index of 1.9) were used. Their high refractive index contrast allowed them to be used with water  Act 1986 set out by the UK Home Office. Female C57BL/6 mice aged between 4 and 6 months were culled via 5.Dewaxed formalin-fixed paraffin-embedded human lung tissue sections (5 µm) were from tissue surplus to clinical requirements from lung biopsies of patients with a subsequent diagnosis of idiopathic pulmonary fibrosis (IPF) under the approval of the Southampton and South West Hampshire and the Mid and South Buckinghamshire Local Research Ethics Committees (Ref. 07/H0607/73). All subjects gave written informed consent.6.Details of spheroid growth have been described in detail previously . Briefly7.As previously described , for TEM8.Silicon membranes were commercially sourced  -based numerical software package to investigate light propagation through the barium titanate microsphere (refractive index was assumed to be 10.Polarization anisotropy images were created using a home written Fiji macro in which the key steps are as follows: A difference image was generated by (11.Collagen fiber analysis was performed using CT-FIRE V1.3. SHG intensity images were converted to 8-bit format before being batch-processed using default parameters. Four parameters were measured to describe fiber morphology: width, length, straightness, and angle.


There is increasing awareness of the benefits of formal mentorship programmes in radiology. In the context of the COVID 19 pandemic which impacted on education, professional engagement and networking within the wider radiological community, the European Society of Head and Neck Radiology (ESHNR) decided to develop a formal mentoring programme. The ESHNR mentoring initiative is novel in its scope, whereby European and international members of a subspecialty radiology society are matched into mentor–mentee pairings to disseminate good practice, knowledge and ideas. The purpose of this report is to describe the motivations, planning, challenges and early experience of the ESHNR mentoring programme together with initial feedback from the scheme.The development of the programme and iterative modifications during the first phase of the scheme are described. The programme has enrolled 33 mentors and 27 mentees with international representation and 24 mentor–mentee pairs have participated in 2.6 (mean) meetings. The experience and benefits reported by the participating ESHNR members (mentees and mentors) were evaluated by a questionnaire at six months following the start of the programme. There were 80% of mentors and 88% of mentees who strongly agreed that the mentoring programme was rewarding rather than an obligation, and all participants reported that they would recommend the scheme to colleagues.A formal mentoring programme has been established for an international subspecialty radiology society. The early experience is encouraging and suggests that it is both useful and sustainable. Our experiences may be of benefit to other subspecialty societies considering a mentoring programme. There is increasing awareness of the benefits of formal mentoring in radiology.The development of an international subspecialty radiology society mentoring programme is described.Initial feedback found it was rewarding for the individuals and the society.“Mentoring” is the action of advising or training another person, especially a less experienced colleague . Whilst The European Society of Head and Neck Radiology (ESHNR) was founded in January 1987 in order to advance knowledge, stimulate interest, develop methods and foster science in head and neck radiology. It performs a range of roles including teaching and research and encourages interaction between radiologists with an interest in this field. There are 686 members (as of 16/8/21) with widespread international representation from Europe and beyond. There is an executive committee of 14 fellows and administrative support is provided through the European Society of Radiology (ESR) office. In view of the society’s objectives and the cessation of normal functions during the COVID 19 pandemic, the concept of a ESHNR mentoring scheme for its members was proposed as an extension of its activities in 2020. The purpose of this report is to describe the motivations, planning, challenges, iterative adaptations and early experience of the mentoring programme, and to document some early outcomes and feedback. The report is structured to incorporate Standards for Quality Improvement Reporting Excellence in Education (SQUIRE-EDU) guidelines.In view of the accumulating evidence for the positive effects of mentoring in academic radiology and other settings , 14–24, Firstly, the addition of a mentoring programme to the society’s range of functions and activities could raise the profile of the ESHNR, promote head and neck radiology, attract new members and expand, as well as rejuvenate the society. By directly engaging with the mentors and mentees, it was anticipated that there would be an improved sense of inclusivity, overcoming generational differences between members and facilitating both relationships and networking within the society. By enhancing the connection of members with the society, it hoped to create equal opportunities for them to contribute and become educational and research leaders within the ESHNR. The international spread of ESHNR members would enable the programme to disseminate good practice across the geographically diverse society and to help equalise opportunities and access. The common interest in head and neck radiology would also allow the mentors and mentees to focus on the particular challenges and issues most relevant to the subspecialty.Secondly, the ESHNR was keen to help and enhance the careers of its younger members, to increase their opportunities and to give them confidence to progress as head and neck radiologists. Given the diversity of the society, this was felt to be most relevant where members had less access to any local mentoring infrastructure or career support. The executive committee of the ESHNR perceived there to be a wide variation in the availability of local mentoring resources for head and neck radiologists. In particular, it was recognised that early years consultants, who are practicing independently for the first time, may be overlooked. It was expected that their needs could be addressed by careful matching with a skilled and experienced mentor, who was familiar with the clinical, professional and personal challenges of the head and neck radiologist. In addition, the ability of the society to provide mentors from outside the mentee’s institution was felt to be advantageous, since it would avoid any conflicts of interest with regards to productivity and resources.Finally, the ESHNR also recognised that the mentor–mentee programme should be a fulfilling two-way process and should enhance the professional life of the mentor as well. The concept of reverse mentoring is well established . It was The aspirations of the ESHNR mentoring programme were brought into stark focus by the consequences of the COVID 19 pandemic, whose radical impact on education and professional engagement is well recognised. Interaction with peers and networking through face-to-face ESHNR annual meetings or through European School of Radiology visiting fellowships was no longer possible, and the opportunities for developing short-term informal mentoring relationships were reduced. There was perceived to be a potential for a sense of isolation and career attrition amongst ESHNR members, which the society had an opportunity to address. In addition, all are familiar with the synchronous rapid development of online meetings and remote collaborations during this period . The facIt was hoped that the mentoring scheme would provide benefit to the individual mentors and mentees as well as the ESHNR society and would be enriched by the diversity of participants and matching. The operational objectives of the formal mentoring programme were to bring together ESHNR members in appropriate mentor–mentee matchings, to prepare them for the mentor–mentee relationship and to guide them through the one-year mentorship period. The structure and processes were expected to evolve on the basis of feedback, and it was anticipated the programme would grow. The ESHNR mentoring programme was not designed to replace but to complement any existing local mentoring strategies available. Of note, the mentoring process was not primarily intended to be a training tool, with the mentee-mentor pairings being encouraged to explore non-clinical areas  in an open discussion that should include feedback from both parties. The key components to the programme were envisaged to be information on the intentions of the mentoring, administration of the programme, vetting of applicants, timely mentor–mentee matching, training resources, clear expectations and advice on conducting meetings, a defined duration of the programme and a process of feedback Table .Table 1KThe journey commenced in May 2020 with the idea being proposed to the ESHNR executive committee by the senior author (S.R.). The planning started in earnest in June 2020 with the formation of a core team (S.C./S.R.) and with administrative support from the ESHNR office. Over the following months, a series of mentee/mentor requirements, processes, documents and website content was developed. To be eligible for the mentoring programme, mentees were required to be ESHNR members who were in at least their fourth year of training or in their early years at a consultant level, whilst mentors were expected to have been in independent head and neck radiology practice for at least five years. Additional desirable qualities for the mentor included an interest in research and teaching as well as previous experience in mentoring, whilst prospective mentees were encouraged to question their specific needs prior to applying. Engagement in the programme was expected to be three years for mentors and one year for mentees .www.eshnr.eu/mentoring)  Fig.  compriseng) Fig.  (5) engaSpecific documents and website content were formulated in order to support the process. Formal engagement letters and enrollment letters were supplemented by a series of website links to support self-directed mentoring training. A letter to the head of training/head of department explained the programme and how it should be seen as additional to any local mentoring process. Feedback forms were designed to determine whether the aims of the mentoring scheme had been achieved. Website content outlined the drivers and goals of the mentoring programme, the requirements of applicants, details of the matching process, expectations concerning the frequency and number of meetings, and advice on maintaining a successful mentor–mentee relationship.As part of our “pre-launch checks” there were some specific concerns and decisions which warrant further explanation Table .Table 3“The mentoring programme was launched in December 2020. Some initial challenges pertained to the imbalance in the recruitment between mentors and mentees, erroneous contact information, mentees disengaging from the programme and enquiries concerning the role and format of the mentoring process.Firstly, initial recruitment of mentors did not keep pace with that of mentees. This was addressed by a round of personal approaches to prospective mentors, with emphasis on engaging from different countries in order to expand the geographic spread and those who were peri-retirement in order to vary the ages of the applicants. Secondly, human error in completing and transcribing the application forms prevented contact between several mentor/mentee pairings. It was subsequently decided that two different email contacts would be requested to aid communications. Finally, some mentees decided to discontinue the programme prior to any meetings due to changes in circumstance or misunderstanding the purpose of the mentoring process. In response to this and ongoing enquiries for information, the core team created and disseminated a “top ten tips for mentoring” to foster a successful mentoring relationship and to address some misconceptions Table . An earlOver the first six months, 33 mentors  and 27 mentees  enrolled in the ESHNR mentoring programme. The mentoring experience and motivations of the applicants were documented Table . Mentorsn = 6), Austria (n = 4), Germany (n = 4) and Italy (n = 3)) and nine from outside Europe . The mentors had a mean of 17.5 years’ experience (range 5–34) in head and neck radiology. There were 19 mentees from Europe (including United Kingdom (n = 6), and Italy (n = 4)), and eight from outside Europe . There were 23 mentees who were early years consultants and four mentees who were trainees. There were 27 mentor and mentee pairings initially matched. The mean interval from mentee application to mentoring engagement letters being sent was 57.6 days (range 51–68) for the first 14 pairs and 9.2 days (range 0–34) for the next 13 pairs. At six months following the start of the programme, one mentee had withdrawn from the program.There were 24 mentors from Europe . Five respondents were yet to commence mentoring meetings at 6 months into the programme of which two had been unable to contact their mentor/mentee. Hence there were 27 (10 mentors and 17 mentees) respondents who were actively involved in mentoring meetings and who completed the questionnaire. These were anonymised for analysis and the results are shown in Fig. The mean number of mentoring meetings taking place in the first six months of the programme was 2.6 (range 1–6). Although there was some emphasis on specific head and neck radiology topics, protocols and techniques , a number of more general issues were reported to have been discussed .As an example of the range of mentoring discussions, one of the authors documented the mentoring discussion during the initial four mentoring meetings Table . SpecifiThe questionnaire feedback was generally positive from both groups, particularly the mentees Fig. . The majWith respect to whether the mentoring programme is meeting its aims for the individuals and the society, the 6-month feedback Fig.  indicateHowever, improvements could be made in terms of preparing mentors and mentees and ensuring that their responsibilities are clearly established. This may also be reflected in the paucity of discussion about topics other than head and neck radiology, which may be addressed by education on the importance of these elements in the mentoring process. Although website material was made available and e-mail bulletins  were employed, there may be a need for more resources  or active interventions such as mentor online meetings or mentoring sessions and workshops at ESHNR meetings. However, soft mentoring skills are a feature of medical practice in most settings, and it is felt that the society should not be responsible for formal mentoring training.A mixed qualitative and quantitative approach is usually employed to evaluate mentoring programmes , and ourTo our knowledge, this is the first mentoring initiative to be formally organised by an international subspecialty radiology society. Its strengths are in its attempt to harness the unique qualities of the ESHNR, by bringing together the benefits of international diversity with a common interest and focus on head and neck radiology. Although the context differs to previous experiences of mentoring in radiology practice , 14–24, Although there is relatively little on-going demand on resources once the website, documentation and organisational infrastructure are in place, it is appreciated that the core team, mentors and mentees have already saturated working schedules, which limits time for these activities. The cost of programme administration has been incorporated within pre-existing arrangements. The programme is still in an early phase, and it is not possible to determine whether the benefits outweigh the resources involved, which will become evident with further evaluation and feedback following completion of the first-year cycle.There is currently little evidence base for the positive impact of mentoring schemes in medicine, and the emphasis has previously been on academic medicine. A systematic review found four studies describing the importance of mentorship –31 and eSeveral potential challenges to the ESHNR mentoring programme have been identified, and these may influence outcomes. Some of these are contextual, such as the international differences in institutional frameworks, accreditation and working environments possibly impeding discussions, although the potential to learn and understand the variation may be seen as an advantage. Similarly, although the programme was generally conducted in the English language, there were language constraints in some cases, which required the pairing of mentors and mentees from the same country. Other challenges are more generic, for instance finding adequate time for the organisation and preparation for mentoring within busy schedules. It may be possible to address this by the recruitment of peri-retirement mentors who will have tremendous experience and more time. In the future, differing approaches to the matching process may also be considered, such as assembling a wider “mentorship team” to give different perspectives or to give the mentees some input . FurtherWe have documented the process of establishing a mentoring programme for an international subspecialty society, together with an early feedback survey of mentors and mentees. The early experience is encouraging and suggests that it is both useful and sustainable. There is further scope for the dissemination of mentoring resources and mentoring fora, and the prospect of face-to-face mentoring lectures, discussions and social meetings in the post-COVID 19 era is awaited with anticipation. The mentoring programme will attempt to adapt and evolve on the basis of regular feedback, and its success will also be judged on its retention and growth of mentors and mentees. Finally, our strategy may be generalizable to other international subspecialist radiology and other medical societies.


Genetically heterogeneous disorder acute myeloid leukemia (AML) is marked by recurring mutations in FLT3. Current FLT3 inhibitors and other emerging inhibitors have helped in the improvement of the quality of standard of care therapies; however, the overall survival of the patients remains static. This is due to numerous mutations in FLT3, which causes resistance against these FLT3 inhibitors. For effective treatment of AML patients, alternative approaches are required to overcome this resistance. Here, we will summarize the biomarkers for FLT3 inhibitors in AML, as well as the alternative measures to overcome resistance to the current therapies.Acute myeloid leukemia is a disease characterized by uncontrolled proliferation of clonal myeloid blast cells that are incapable of maturation to leukocytes. AML is the most common leukemia in adults and remains a highly fatal disease with a five-year survival rate of 24%. More than 50% of AML patients have mutations in the FLT3 gene, rendering FLT3 an attractive target for small-molecule inhibition. Currently, there are several FLT3 inhibitors in the clinic, and others remain in clinical trials. However, these inhibitors face challenges due to lack of efficacy against several FLT3 mutants. Therefore, the identification of biomarkers is vital to stratify AML patients and target AML patient population with a particular FLT3 mutation. Additionally, there is an unmet need to identify alternative approaches to combat the resistance to FLT3 inhibitors. Here, we summarize the current knowledge on the utilization of diagnostic, prognostic, predictive, and pharmacodynamic biomarkers for FLT3-mutated AML. The resistance mechanisms to various FLT3 inhibitors and alternative approaches to combat this resistance are also discussed and presented. Acute myeloid leukemia (AML) is a hematological malignancy that accounts for the most common leukemia that occurs in adults. In the US, there were 20,240 cases and 11,400 deaths due to AML in 2021. The five-year relative survival rate for AML patients is 29.5% .AML is characterized by clonal proliferation of poorly differentiated cells of hematologic origin. These cells are genetically altered with recurrent deletions, amplifications, point mutations, and rearrangements ,5.The human flt3 (FMS-like tyrosine kinase 3) gene is located on chromosome 13q12 and has 24 exons . It encoThe extracellular region contains a binding domain with a high affinity for its ligand (FLT3 ligand or FL). FL is expressed by most tissues, including the spleen, thymus, and bone marrow; however, the highest expression is seen in peripheral blood mononuclear cells .Once the FL binds to FLT3, it induces receptor dimerization and conformational changes. Subsequently, FLT3 autophosphorylation activates intracellular signaling cascades that control cell proliferation, differentiation, and survival ,9,10. Thmut), which is detected in 25% of patients, and point mutations in the tyrosine kinase domain (FLT3-TKDmut) that are detected in 7–10% of the patients [mut potently activates STAT5, which activates cyclin D1, c-myc, and Pim-2; the activation of these proteins results in the accelerated growth of leukemic cells [mut consists mainly of missense point mutations, deletions, or insertions in the tyrosine kinase domain of FLT3. The most frequent point mutations are primarily seen in the activation loop in amino acid residues D835, I836, Y842, and some in the TKD1, including the residues N676 and F691 [The most common mutations in FLT3 include FLT3 internal tandem duplication  can be utilized as diagnostic biomarkers for AML. A study identified six serum miRNAs  which were specifically upregulated in the serum of AML patients using a next-generation sequencing approach ,44.Four miRNAs  were used for the diagnosis of AML with 97% accuracy and analyzed using RT-PCR. miR-142-3p and miR-29a can also be used as diagnostic biomarkers for AML . InteresThese studies suggest that miRNAs from serum or blood samples can be effective diagnostic biomarkers for AML patients.There have been multiple FLT3 inhibitors in clinical trials, but predictive biomarkers remain undiscovered. In a recent study aimed at identifying gene expression changes associated with FLT3 mutation in AML patients, the transcriptomic patterns of six different cohorts of AML patients were analyzed, and a FLT3-mutation-like pattern was highly enriched in NPM1 and DNMT3A mutants. In addition, FLT3-like patterns consisted of numerous homeobox (HOX) genes .Based on the FLT3 mutations, companion diagnostics were generated that tested for a predictive biomarker . These tOne such FDA-approved companion diagnostic test was the LeukoStrat CDx FLT3 mutation assay. This is a PCR-based, in vitro diagnostic test that detects ITD and TKD mutations D835 and I836 from the genomic DNA extracted from the mononuclear cells from peripheral blood or bone marrow aspirates of AML-diagnosed patients .This test was used with FLT3 inhibitors, including midostaurin, gilteritinib, and quizartinib .Similarly, co-occurrence of mutations in FLT3 with national comprehensive cancer network (NCCN)-listed gene mutations were used as predictive biomarkers ,53. Co-omut AML patients is higher in patients with mutations in NPM1 or DNMT3A and particularly in those with both genes mutated [Another study identified that the response to gilteritinib and crenolanib among relapsed FLT3 mutated ,59. When mutated . Additio mutated .FLT3 is widely overexpressed and the most frequently mutated gene in both pediatric and adult patients with AML . Higher + AML, the levels of FL fall to undetectable levels. It was observed that, after the induction of chemotherapy, FL levels are restored in patients with complete remission but not in patients with refractory disease. FL levels were measured in a randomized study with lestaurtinib where it was seen that patients achieving complete remission (CR) had higher FL levels after the completion of the therapy followed by a normal range after recovery. However, patients with refractory disease had a transient increase in FL levels followed by rapid depletion [FLT3 ligand (FL) is detectable during homeostasis and is increased in hypoplasia. FL is markedly elevated upon the depletion of the hematopoietic stem or progenitor cells. However, in FLT3epletion . Thus, FThe presence or absence of specific gene mutations can be utilized to classify AML patients and determine their prognosis. The NCCN AML prognostic stratification system listed FLT3, NPM1, CEBPA, IDH1/2, DNMT3A (DNA methyltransferase 3A), KIT, TP53 (tumor suppressor 53), RUNX1, and ASXL1 (ASXL transcription factor) gene mutations for the classification of the AML patient population ,66. Mutamut) occur in the JM domain internal tandem duplications, FLT3-ITDmut, or in the tyrosine kinase domain, FLT3-TKDmut. FLT3-ITDmut, are in-frame mutations consisting of duplications of 3–400 base pairs which lead to an elongated JM. This results in constitutive activation of the FLT3 receptor and the downstream signaling  p = 0.0008) and OS (p = 0.004) [The prognostic value of FLT3-ITD is determined by various factors, including the allele ratio (AR), ITD length, karyotype, insertion site, and co-mutations (NPM1) ,70. AR i= 0.004) . However= 0.004) .mut, in young adult AML patients [mut, the OS and relapse risk were comparable between FLT3 wild-type and FLT3-ITDmut (AR < 0.5), but worse when AR ≥ 0.5 [mut patients had an increased risk of relapse and inferior OS, regardless of the AR. The European Leukemia Net (ELN) guidelines categorize FLT3-ITDmut AML into three categories: favorable (NPM1mut with FLT3 wild-type or NPM1mut with FLT3-ITD AR < 0.5), intermediate (NPM1mut with FLT3-ITD AR > 0.5 or NPM1WT with FLT3-ITD AR < 0.5), and adverse (NPM1WT with FLT3-ITD AR > 0.5) [Favorable relapse risk and OS was seen with the occurrence of co-mutations NPM1, along with FLT3patients . In patiAR ≥ 0.5 . Among pR > 0.5) . AlthougR > 0.5) .mut AR remains obscure. However FLT3-TKDmut has a high incidence in co-occurrence with mutations in NPM1, CEBPA, and NRAS [FLT3-TKD mutations have prognostic value in the overall AML patient population, but the impact of FLT3-TKDand NRAS . These Tand NRAS .Since FLT3 mutations lead to dysregulation of cell proliferation pathways, inhibiting FLT3 signaling using small molecule inhibitors is a viable strategy for AML patients .FLT3 inhibitors can be classified into type I and type II based on their mechanism of interaction with FLT3 . Type I FLT3 inhibitors can also be classified into first generation and second generation based on their specificity for FLT3 . First-gmut AML patients [Currently, there are only three FDA-approved FLT3 inhibitors, sorafenib, midostaurin, and gilteritinib, for use in the U.S. Of these, only two are approved for AML indication: midostaurin, along with chemotherapy, and gilteritinib . Midostapatients .Several other FLT3 inhibitors are in the early and late stages of clinical development. Quizartinib is the most potent and selective type II inhibitor and crenolanib is a potent type I inhibitor in the late stages of clinical development .Biomarkers that predict the efficacy of FLT3 inhibitors have important applications in clinical care. Activation of FLT3 triggers the phosphorylation and activation of downstream signal transduction pathways including PI3K/AKT/mTOR, RAS/RAF/MAPK and JAK/STAT . AutophoAnother study identified that the expression of immune checkpoint markers CD155 and CD112  was specifically downregulated upon treatment with gilteritinib and quizartinib in FLT3-mutated cell models. Thus, CD155 and CD112 have the potential to serve as PD biomarkers for FLT3-ITD AML patients .Although FLT3 inhibitors show response in AML patients, the duration of this response is short-lived due to primary and acquired resistance. The most common mechanism of acquired resistance in patients is due to on-target mutations in the tyrosine kinase domain. F691L and D835 are frequently occurring FLT3 gatekeeper mutations. These mutations hinder the drug binding which results in an active kinase conformation unfavorable to interaction with FLT3 inhibitors ,106. ThiIn addition, FLT3-ITD mutations contribute to resistance to the FLT3 inhibitors. This is because FLT3-WT is sensitive to FLT3 ligand and resistant to FLT3 inhibitors. FLT3-ITD has a WT sequence; therefore, it contributes to the resistance ,112,113.It was also observed that high levels of FL in the bone marrow microenvironment during induction and consolidation therapy can lead to activation of the FLT3-MAPK pathway and provide a survival signal to the blast cells even in the presence of FLT3 inhibitors . Some prAcquired resistance due to non-overlapping, secondary mutations is caused by different FLT3 inhibitors. In-vitro-based studies demonstrated that SU5614 produced TK2 changes in D835 exclusively; however, midostaurin produced mutations in TK1 at N676. In addition, sorafenib produced resistant mutations in TK1 (F691L) and TK2 (Y842). These mutations led to different drug responses. While TK2 mutations were sensitive to midostaurin, sunitinib, and sorafenib, TK1 mutations had a differential response to SU5614, sorafenib, and sunitinib but impaired response to midostaurin .Additional resistance mechanisms were seen where FLT3-TKI resistant cells became FLT3-independent due to the activation of parallel signaling pathways, including Ras/MEK/MAPK and PI3K/Akt, which compensate for cell survival signals when FLT3 is inhibited. Additionally, activating mutations in the Ras/MAPK pathway, including NRAS, PTPN11, KRAS, and CBL, were of common occurrence in gilteritinib and crenolanib resistance ,117,118.Given that FLT3 inhibitors present limited efficacy due to the reasons mentioned above, alternate approaches are required to cure FLT3-AML patients. One of the approaches is the use of combination therapies of FLT3 inhibitors with other agents to enhance their efficacy and identify synergistic drug combinations. A combination of sorafenib with vorinostat (histone deacetylase inhibitor) was seen to be effective against FLT3 AML in an early-phase clinical trial . AdditioSome studies used anti-FLT3 monoclonal antibody (mAb) treatment for FLT3-TKI-resistant clones, but it was found to be a cytotoxic ,129,130.Additionally, chimeric antigen receptor (CAR) T cells are an emerging novel therapeutic approach to target FLT3 AML. CD8+ and CD4+ T-cells expressing an FLT3-specific chimeric antigen receptor (CAR) were efficacious in vitro. Treatment with crenolanib enhanced the surface expression of FLT3 on FLT3-ITD AML cells which led to increased recognition by FLT3-CAR T cells in preclinical studies .FLT3 inhibitors show promising efficacies in progressive and relapsed AML, but the duration of the clinical response is short. Biomarkers are of great importance in predicting the biological behavior of AML, as well as monitoring the efficacy of FLT3 inhibitors in patients. The expression of biomarkers can be used to predict the disease activity in real time. They can also serve as a guide for administering a particular FLT3 inhibitor to achieve CR and high OS.Additionally, to overcome the resistance to FLT3 inhibitors caused by different mechanisms, combination therapies of FLT3 inhibitors with other targeted agents or immunotherapeutic approaches are additional areas of investigation.


More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Cav blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca2+]cyt although the mechanism is poorly known. The chanzyme TRPM7 , the release of intracellular Ca2+ pools, and Ca2+ influx by ORAI channels have been associated with the increase in [Ca2+]cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil’s effect on [Ca2+]cyt. To address these questions, we monitored changes in [Ca2+]cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca2+]cyt at concentrations above 10 μM (IC50 around 50 μM) and a fast Ca2+ increase capacity at 100 μM. We found that inhibiting IP3 receptors, depleting the ER-Ca2+ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca2+]cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca2+ influx by store-operated Ca2+ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP3R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (CaThe online version contains supplementary material available at 10.1186/s43556-021-00037-0. Patch-clamp studies revealed that mibefradil has an IC50 around 0.3–2.7 μM for Ca2+, Na+, and K+ voltage-dependent currents in HEK-293 cells cyt although the molecular mechanism involved is unclear [v block [2+ channel TRPM7  that is permeable to Ca2+ [2+]cyt that was abolished by perfusing with the TRPM7 antagonist NS8593 [2+]cyt elicited by mibefradil is the release of Ca2+ from intracellular stores cyt via STIM1 and ORAI-1 [2+]cyt and its impact on the CRAC channels is important to better understand the broader biological effects of this drug.Physiologically, the release of Cand (IP3) . DAG and 3.1–3.3 , 23. Thepathways . Electropathways . This ma+ influx . Additiod ORAI-1 . Thus, a2+]cyt in vitro. Using the murine epithelial cells LS8 and ALC, and the human HEK-293 cells that are a generalized cell model widely used in signal transduction research [2+]cyt in all cell lines. We found that this effect was reduced or abolished by inhibiting IP3R or PLC, or by depleting the ER-Ca2+ stores. Mibefradil also triggered SOCE, likely as a result of its role in activating the PLC pathway. Taken together, our findings suggest that the application of mibefradil at micromolar (≥10 μM) concentrations elevates [Ca2+]cyt, highlighting its effects on the PLC pathway and the ER-Ca2+ released via the IP3R as potential targets thus providing novel insights into the effects of mibefradil.Here, we have investigated the cellular targets and pathways associated with the transient application of mibefradil and its effect on cyt in LS8 and ALC cells, although it caused a small elevation (∼30 nM) in HEK-293 cells . Taken together, these data suggest that TRPM7 channels are not the main target of mibefradil although these channels may have a small contribution. Our results, together with previous reports [2+]cyt when used above 10 μM, with a maximum [Ca2+]cyt increase at 100 μM. Thus, we used 100 μM mibefradil to study its effects on Ca2+ transients and possible molecular targets.To determine the maximum capacity of Cacurves 0.–100 μM i 1.2–3.3 , had no  reports –20, 29, 3 signaling we first investigated if mibefradil affected the IP3 content in cells. We lysed cells (1.106 cells/mL) of each type to measure the IP3 levels before and after mibefradil stimulation (100 μM for 3–4 min)  was the most highly expressed in LS8 and ALC cells but type 2 (ITPR2) was the most abundant mRNA in HEK-293 cells  cyt  and the PLC inhibitor U73122 (5 μM). In the presence of 2 mM Ca2+ solution, m-3M3FBS elicited a significant increase in [Ca2+]cyt in all three cell lines, with the highest increase in the Ca2+ peak area observed in HEK-293 cells , ALC (~ 54%), and HEK-293 (~ 35%) cells  and secondary (δ) PLC isoforms. We found that the mRNA encoding PLC-γ1 was the most highly expressed in LS8 and HEK-293 cells, whereas in ALC cells, the β-γ isoforms were dominant Fig. S. Next, tlls Fig. a-b. Aftecyt Fig. c. Simila, ALC ~ 5%, and HE, ALC ~ 5%, and HE2+ from ER stores and this ER-Ca2+ efflux is a common step in the activation of SOCE cyt, we monitored [Ca2+]cyt transients before and after re-addition of 2 mM extracellular Ca2+ in cells with replete ER-Ca2+ stores. We showed that this had only a minimal effect in rising [Ca2+]cyt (≤ 20 nM)   Fig. S. By contcells ~ 10–400 nM cells ~ 10–400 nM 2+ is a key ubiquitous intracellular messenger coupling membrane-mediated processes to downstream cellular responses cyt  by dosing 25–350 mg/day of mibefradil in four doses/day for 7–17 consecutive days . This st2+]cyt in mammalian spermatozoa [2+]cyt by unknown mechanisms [2+ channel ORAI [2+]cyt, we have investigated a numbers of potential cellular targets and have studied their effects on Ca2+ uptake by SOCE mediated by the ORAI channels. We show that the transient perfusion of mibefradil elevates [Ca2+]cyt in all cell lines tested  with a concentration-dependent manner and LogEC50 around 45–50 μM. Its efficiency to elevate [Ca2+]cyt was fully reversed by perfusion of a solution without mibefradil  increased [Carmatozoa . Similarchanisms . Mibefranel ORAI . Given t0 μM incr2+]cyt by activation of multiples second messengers and downstream pathways cyt triggered by mibefradil [2+]cyt in HEK-293 cells overexpressing TRPM7 channels and that the TRPM7 antagonist NS8593 reduced the effects of mibefradil cyt (∼30 nM) in all cell lines  increased ∼2–6 fold the total IP3 content in cells  concentrations, mibefradil was reported to have no effect on the ER-Ca2+, basal Ca2+ levels, or on the frequency of Ca2+ oscillations [2+]cyt at micromolar concentrations (10–100 uM) was independent of extracellular Ca2+ influx [2+ stores. We thus tested if altering intracellular Ca2+ by blocking IP3R with Xestospongin C modified the effects of mibefradil on [Ca2+]cyt. In the presence of the IP3R inhibitor, the effects of mibefradil were markedly reduced  caused the activation of IP3R [3 prevented a rise in [Ca2+]cyt elicited by mibefradil cyt homeostasis cyt  and double (ORAI1/ORAI2) knockdowns by CRISPR/Cas9, originally reported in cyt measurements were performed as previously described cyt, according to the manufacturer’s specifications, as previously described cyt.cyt, were determined in normal or Ca2+-free Ringer’s solution supplemented with EGTA . The role of IP3R was analyzed by pretreatment with the selective IP3R inhibitor Xestospongin C  for 20 min c transients versus time under the stimulus duration using Origin Pro 8 software version 8.08 . The kinetics of the rate of Ca2+ activation were calculated in each trace and fitted by the GraphPad Prism software using the one-phase association eq. *(1 - exp.(−K*(X-X0)))). Concentration-effect (0.1 μM to 100 μM) curves for mibefradil were constructed using sigmoidal log (agonist) vs. response - variable slope equation fitted by the GraphPad Prism software and parameter of the LogEC50 (apparent affinity) was calculated. Data represent the mean ± SEM of the minimum of three independent experiments. The total number of cells used is indicated. Differences between the means of the group data that fit a normal distribution were analyzed using a two-tailed unpaired Student’s t-test variance or by one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test. The limit of significance was established at *p < 0.05; **p < 0.01; ***p < 0.001.All data, mathematical analyses, and graphs were analyzed and/or generated using the GraphPad Prism software version 9.0 , as previously described . The ∆ CAdditional file 1: Figure S1. Effects of Ringer’s solution and mibefradil (50–100 μM) on background autofluorescence. Figure S2. Effects of the two consecutive mibefradil (50 μM) stimulus on [Ca2+]cyt transients in HEK-293 cells under regular extracellular Ca2+ levels (2 mM Ca2+). Figure S3. TRMP7 expression and activation by naltriben. Figure S4. Alternative stimulation with mibefradil (100 μM) and ATP (100 μM) under Ca2 + −free Ringer’s solution in ALC cells. Figure S5. Expression of PLC isoforms. Figure S6. Effects of the Ca2 + −free Ringer’s solution replacement for 2 mM extracellular Ca2+ in LS8, ALC, and HEK-293 cells. Figure S7. Optical density (OD) values of standard IP3 concentration. Table S1. Mouse primers sequences for qRT-PCR used in LS8 and ALC cells. Table S2. Human primers sequences for qRT-PCR used in HEK-293 cells.


In parallel with these electrical and Ca2+ oscillations are oscillations in metabolism, and the periods of all of these oscillatory processes are similar. A key question that remains unresolved is whether the electrical oscillations are responsible for the metabolic oscillations via the effects of Ca2+, or whether the metabolic oscillations are responsible for the electrical oscillations due to the effects of ATP on ATP-sensitive ion channels? Mathematical modeling is a useful tool for addressing this and related questions as modeling can aid in the design of well-focused experiments that can test the predictions of particular models and subsequently be used to improve the models in an iterative fashion. In this article, we discuss a recent mathematical model, the Integrated Oscillator Model (IOM), that was the product of many years of development. We use the model to demonstrate that the relationship between calcium and metabolism in beta cells is symbiotic: in some contexts, the electrical oscillations drive the metabolic oscillations, while in other contexts it is the opposite. We provide new insights regarding these results and illustrate that what might at first appear to be contradictory data are actually compatible when viewed holistically with the IOM.Insulin is secreted in a pulsatile pattern, with important physiological ramifications. In pancreatic β-cells, which are the cells that synthesize insulin, insulin exocytosis is elicited by pulses of elevated intracellular Ca Pulses in vivo insulin oscillations and in vitro calcium oscillations from the same mouse and show that the frequencies are well correlated Hntration , mitochontration , and thentration , 2016.active phase) followed by a period of membrane repolarization (the silent phase), repeated periodically. Since the electrical impulses are produced by Ca2+ influx through voltage-dependent Ca2+ channels (Ca2+ channels), the intracellular Ca2+ concentration is higher during each burst active phase and lower during each silent phase. These periodic rises in Ca2+ evoke exocytosis of insulin-containing vesicles during each active phase  channels], or (indirectly) on ATP-sensitive K+ channels [K(ATP) channels]  channel activity  oscillate with similar periods, the question of “who is driving bursting?” has yet to be answered. Are Ca2+ oscillations driving metabolic oscillations, or vice versa? Mathematical modeling provides a valuable tool for addressing this question, as we demonstrate herein. Models can incorporate different oscillation mechanisms that yield differential predictions. One model, the Integrated Oscillator Model (IOM)  , incorpohttps://www.math.fsu.edu/∼bertram/software/islet/.We used the Integrated Oscillator Model (IOM) to simulate a representative β-cell in a well-coupled islet. JPDH) is given byIn this model, the flux through the pyruvate dehydrogenase (PDH) reaction  reaction, and NADHm and NADm are dinucleotides of mitochondria. The GPDH reaction rate (JGPDH) is given bywhere 2+ concentration of the mitochondria. In the IOM, the K(ATP) current (IK(ATP)) is described bywith gK(ATP) is the maximum conductance, V is the plasma membrane potential, VK is the K+ Nernst potential, and o∞ is the fraction of activated K(ATP) channels,where 3– = 0.135 ADP, and ATP4– = 0.05 ATP, while the parameters ktt, ktd and kdd represent the dissociation constants for ATP, ADP, and MgADP, respectively.Here, MgADP = 0.165 ADP, ADPktt) from 1 to 2μM. The application of KCl, which induces cell depolarization, was simulated by increasing the K+ Nernst potential (VK) from –75 to −70mV.We simulated the addition of the K(ATP) channel activator diazoxide (Dz) by increasing the dissociation constants for ATP  and occur out of the normal glucose range (likely due to heterogeneity) and, in islets, asynchronous . See the2+ signaling, and membrane electrical activity. We describe these pathways in some detail below, but begin with an overview illustrated in The transduction of glucose input to insulin output in β-cells is complex, and can be thought of in terms of feedforward and feedback pathways implemented through the metabolism of glucose , intracellular Ca+ current, resulting in cell depolarization. This depolarization opens voltage-dependent Ca2+ channels and allows Ca2+ influx, producing electrical impulses and increasing the intracellular Ca2+ concentration  channels and thereby reduces net outward K2+, as well as Ca2+ entering organelles such as the endoplasmic reticulum (ER) and mitochondria, in turn affects metabolism through several pathways , speeding up metabolism .  led to the conclusion that metabolic oscillations are not intrinsic (AMOs), but must be driven by the Ca2+ oscillations (PMOs) H were made to test this prediction  . As our ediction , and it ediction , a more 2+ was clamped were AMOs H, FBP, or ATP/ADP remains to be done. This is a particularly tricky experiment since, from an analysis of the model, subthreshold AMOs do not always occur. That is, the result will likely vary from islet to islet, so that many trials might be needed to firmly establish that subthreshold AMOs occur. Indeed, this could be why subthreshold Ca2+ oscillations is the strongest evidence to date that β-cells have intrinsic metabolic oscillations. Compound oscillations consist of fast V bursts (or Ca2+ oscillations) that are grouped into slower episodes. This type of oscillatory pattern has been observed since the earliest reports of islet electrical activity (2+ imaging (2+ (2+ onto K(Ca) channels, while intrinsic slow metabolic oscillations group the fast oscillations into slower episodes  during bursting. The inner membrane potential is very important as it provides the electrochemical driving force needed for mitochondrial ATP synthesis. In one study, the measurement of △ψm using the fluorescent dye rhodamine-1,2,3 showed that △ψmdepolarized during each burst active phase , while if the electrical effect of the Ca2+ flux dominates, then △ψm will depolarize during the active phase (right panels).In 2+ influx during the active phase from △ψmhyperpolarization to depolarization  channels, which are found in cardiac myocytes , smooth 2+ on ATP production and consumption through hydrolysis, dehydrogenases, and the mitochondrial Ca2+ uniporter. Oscillations in the Ca2+ concentration produce oscillations in ATP that are carried forward to the K(ATP) conductance, which in turn, influences the cell membrane potential. The loop is closed by the influence of the membrane potential on voltage-dependent Ca2+ channels, which determine the cytosolic Ca2+ concentration through Ca2+ influx. This feedback loop is responsible for slow bursting oscillations associated with PMOs in the IOM, and is represented as a large loop for the slow oscillations. While the loop is still active during fast and compound oscillations and slow oscillations with AMOs, we propose that it plays a lesser role in either, so we depict it as being small in the diagram.2+ on K(Ca) ion channels. Activation of these channels has a hyperpolarizing effect on the cell’s membrane potential, which in turn closes V-dependent Ca2+ channels, reducing the cytosolic Ca2+ concentration. We propose that while this loop has only a minor role to play in slow bursting, it is the main driver of fast bursting, including fast bursting that occurs during each episode of compound bursting.The second loop (loop II) describes the action of cytosolic Ca2+ oscillations, which affect the cell’s ATP level, which in turn affects PFK since ATP is an allosteric inhibitor of PFK  describes the positive feedback effect of FBP on PFK, which produces intrinsic glycolytic oscillations (AMOs). These glycolytic oscillations result in oscillations in ATP, which affect the membrane potential again through K(ATP) channels. The electrical bursting results in Car of PFK . Loop II2+ subsystems of β-cells.It is our belief that the vast majority of observed phenomena regarding oscillations in β-cell activity in mouse islets, where most of the research on islet oscillations has been performed, can be understood from the standpoint of these three canonical feedback loops. Because all three are incorporated into the IOM, the model is a useful tool for determining the conditions that must hold to explain various phenomena. This article focused on a few specific behaviors that can be interpreted with the use of the model; some others have been the focus of prior reports (see IM performed mathematical simulations and analysis. PF provided conceptual advice on the model. AS, LS, and RB provided resources and supervision. All authors contributed to writing 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mitochondria are the vital organelles in heart tissue. Mitochondrial Ca2+ homeostasis is particularly important for maintaining the physiological and pathological mechanisms of the heart. Mitochondrial Ca2+ homeostasis plays a key role in the regulation of cardiac energy metabolism, mechanisms of death, oxygen free radical production, and autophagy. The imbalance of mitochondrial Ca2+ balance is closely associated with cardiac remodeling. The mitochondrial Ca2+ uniporter (mtCU) protein complex is responsible for the uptake and release of mitochondrial Ca2+ and regulation of Ca2+ homeostasis in mitochondria and consequently, in cells. This review summarizes the mechanisms of mitochondrial Ca2+ homeostasis in physiological and pathological cardiac remodeling and the regulatory effects of the mitochondrial calcium regulatory complex on cardiac energy metabolism, cell death, and autophagy, and also provides the theoretical basis for mitochondrial Ca2+ as a novel target for the treatment of cardiovascular diseases.Mitochondria are the sites of oxidative metabolism in eukaryotes where the metabolites of sugars, fats, and amino acids are oxidized to harvest energy. Notably, mitochondria store Ca However, the importance of mitochondrial Ca2+ buffering capacity, the kinetics of mitochondrial Ca2+ uptake/release, and the molecular mechanism of [Ca2+]m-mediated ATP and ROS production remain controversial.High cytoplasmic Ca+ uptake , and somntration . Althougeostasis ,133. VDAchondria ,41,134.  limited , and VDAthe cell . Furtherate Ca2+ ,137. Vasscovered . A year  (MICU1) , which r and NCX , which mnium red ,41. In tscovered ,143,144.nium red . In mammnium red . Recentlreported ,147. Theting MCU . The Ca2xchanger . The uptr sodium . When inmyocytes . Additioocardium  and flow via NCX . Increas2+ influx through mitochondrial Ca2+ channels and transporters m and plays an important role in regulating mitochondrial Ca2+ homeostasis c) changes to the MCU complex and activates mitochondrial in situ Ca2+ uptake [2+] sensor on the matrix side m uptake rate, increased mtROS, and enhanced cell death induced by [Ca2+]m overload c has been shown to be increased in failing guinea pig hearts relative to [Na+]c in normal hearts [2+ outflow and reduces [Ca2+]m in failing hearts ), suggesting that mitochondrial Ca2+ overload is a key determinant of heart failure  or substantially affect cardiac function under basic conditions [The increased incidence of malignant arrhythmias can contribute to heart failure, diabetic cardiomyopathy, senile cardiac insufficiency, and hereditary diseases . Abnormadiseases . Garcia diseases . Thus, tnditions . The incnditions .2+ transport and Ca2+-induced membrane permeability transition, inhibit the activation of the MAPK/JNK pathway, inhibit foam-cell formation, and reduce the progression of atherosclerosis [Studies have shown that drugs targeting mitochondria interfere with mitochondrial Caclerosis . CsA is clerosis . In a Phclerosis ,279.2+ regulation. These peptides primarily include Ca2+ channels and pumps that regulate ER localization and small peptides or proteins that regulate mitochondrial Ca2+ channels, affecting Ca2+ transport from ER to mitochondria  as a polypeptide has been shown to inhibit IP3R-mediated Ca2+ release and protect cells from pro-apoptotic Ca2+ transfer to mitochondria [2+-induced Ca2+ release via Ca2+ influx. Further studies have shown that a short fragment of the cytosolic II–III loop of the DHPR, called peptide A, induces RyR1-mediated Ca2+ release [2+ uptake by directly or proximally acting on VDAC and MCU. Short peptides from the N-terminal of VDAC1 and LP4  may significantly inhibit mitochondrial Ca2+ uptake and lung cancer cell migration by blocking the interaction between VDAC1 and Bcl-XL, McL-1 [2+ levels, and this event was associated with VDAC1 oligomerization, cytochrome c release, and apoptotic cell death. Peptides derived from accessory protein sequences of VDAC1 show potential therapeutic applications [2+ uptake into mitochondria, making cells more resistant to the pro-apoptotic release of Ca2+ from the MAM-targeted ER [2+ signaling, such as ischemia-reperfusion injury [2+ flux or homeostasis. The fungus-derived cyclic peptide cyclosporine A (CsA) desensitizes mPTP to Ca2+ and inhibits pore opening. In recent years, an increasing number of CypD-selective and non-immunosuppressive derivatives of CsA  have been developed as promising cardioprotective agents, as their ability to reduce the harmful effects of acute myocardial infarction has been observed in different models [2+ retention in diabetic animals, reduce oxidative damage of heart tissue, and improve the glucose utilization rate [2+ permeable hemichannels, contributing to a mitochondrial Ca2+ overload and loss of energy and ion gradients, leading to cell death [2+ overload and infarct size during cardiac ischemia-reperfusion [2+ crosstalk that deserve further study. In conclusion, preclinical data using decoy or regulatory peptides acting on major Ca2+ channels in the ER-mitochondria will be needed to facilitate the rapid development of these tools into practical therapies.It is promising to develop therapeutic peptides targeting mitochondrial Cachondria . Anti-apchondria . An IP3Rchondria . The mec release . The lip release . Some pe release . CalcinsL, McL-1 , or HKI.ications . Furthergeted ER . Owing tn injury , Alzheimn injury , and Parn injury . Studiesn injury . The rect models . Anothert models . Studiesion rate . More reion rate . Furtherll death ,296. RNYerfusion . In addi