Further, Reactive Oxygen Species (ROS) are critical players in BER development which, combined with unbalanced Ca2+ concentrations, greatly affect the severity of the disorder. The availability of a high-quality reference tomato genome as well as the whole genome resequencing of many accessions has recently permitted the genetic dissection of BER in segregating populations derived from crosses between cultivated tomato accessions. This has led to the identification of five loci contributing to BER from several studies. The eventual cloning of the genes contributing to BER would result in a deeper understanding of the molecular bases of the disorder. This will undoubtedly create crop improvement strategies for tomato as well as many other vegetables that suffer from BER.Blossom-end rot (BER) is a devastating physiological disorder affecting vegetable production worldwide. Extensive research into the physiological aspects of the disorder has demonstrated that the underlying causes of BER are associated with perturbed calcium (Ca Vegetable production is challenged by a range of biotic and abiotic factors, often resulting in a substantial loss of the produce in each growing cycle. As the population is growing, the world is facing increasing demands for a stable food supply grown on agricultural lands across the globe. Unfortunately, abiotic stresses are becoming increasingly more prevalent especially in light of climate change. Climate-related changes, which are exemplified by extreme air and water temperature, increased frequency and intensity of rainfall, intense hurricanes and so forth are becoming more prevalent , pepper (Capsicum annuum L.), watermelon (Citrullus lanatus (Thunb.) and eggplant (Solanum melongena L.)  and distal (low) end of the fruit is correlated to the appearance of BER such that the higher the difference, the higher incidence of BER , encoding a functional Ca2+/H+ antiporter in tomato. sCAX1 encodes a N-terminal truncated version of the full-length gene that does not contain its regulatory region and therefore is constitutively active. When sCAX1 is expressed in tomato, 100% of the fruit exhibited BER symptoms  increased water-soluble Ca2+ concentration in tomato fruits resulting in less electrolyte leakage and less BER , Solyc07g064170 (PE1), Solyc07g064180 (PME2.1), Solyc06g051960 (LES.9028) and Solyc03g083360 (Les.10790) , magnesium (Mg+), sodium (Na+) and ammonium (NH4+) have a negative impact on the uptake of divalent cation Ca2+, thereby increasing BER incidence  may also influence BER incidence. Fruits that were collected from a BER-resistant accession showed a high correlation between B+ and Ca2+ concentration in the distal part of each fruit whereas the susceptible accession showed no correlation between the two elements  are reported to accelerate fruit growth and cause an increase in BER  increases fruit weight by increasing the cell size  that carry the derived allele of CSR showed significantly higher BER incidence compared to FW11.3 NILs that carry the wild type allele, indicating that FW11.3/CSR may have a role in BER development  affecting Ca2+ concentration and growth rate in the early stages of fruit development. Additionally, further use of these IL8–3 lines revelated that many Ca2+-transport-related genes such as cation exchanger (CAX), Ca2+-ATPase, Ca2+-channel and Na+/Ca2+exchanger were differentially expressed between M82 and IL8–3 ten days after flowering but none of these genes mapped to location of IL8–3 on chr08  and S. lycopersicum var. lycopersicum (SLL), four loci were identified: BER3.1 and BER3.2 on chr03, BER4.1 on chr04 and BER11.1 on chr11 (Topcu et al. BER3.2 and BER11.1 were further finemapped to 1.58 and 1.13 Mb respectively, whereas BER11.1 was also mapped in another population derived from SLL cv Ailsa Craig and SLL cv Kentucky Beefsteak (Prinzenberg et al. BER3.2 is likely corresponding to the fruit weight gene FW3.2/KLUH which was segregating in one of the populations (Topcu et al. FW3.2/KLUH (Fig. Due to the low genetic diversity between closely related tomato accessions, the genetic basis of BER in populations derived from crosses among cultivars was hampered by the lack of molecular markers until recently. With the advent of the full genome sequence of tomato (Tomato Genome Consortium 2+ homeostasis and ROS accumulation perform critical roles in the development of the disorder. Together, they affect membrane stability and cell wall properties as to the degree of pectin methylation and hence BER appearance. Because the combination of environmental stress and nutritional factors affect the incidence of BER greatly, this disorder is often difficult to manage in field and greenhouse growth conditions in many agricultural settings. Going forward, growers will need to remain vigilant and pursue proper field management practices such as mulching, effective water drainage, proper irrigation systems, balanced fertilizer applications, and soil reclamation, which is the removal of salt from the root zone (Machado and Serralheiro The research on BER has led to the findings that the interplay of Ca


Following the arrival of rabbit haemorrhagic disease virus 2 (RHDV2) in Australia, average rabbit population abundances were reduced by 60% between 2014 and 2018 based on monitoring data acquired from 18 sites across Australia. During this period, as the seropositivity to RHDV2 increased, concurrent decreases were observed in the seroprevalence of both the previously circulating RHDV1 and RCVA, a benign endemic rabbit calicivirus. However, the detection of substantial RHDV1 seropositivity in juvenile rabbits suggested that infections were continuing to occur, ruling out the rapid extinction of this variant. Here we investigate whether the co-circulation of two pathogenic RHDV variants was sustained after 2018 and whether the initially observed impact on rabbit abundance was still maintained. We monitored rabbit abundance and seropositivity to RHDV2, RHDV1 and RCVA at six of the initial eighteen sites until the summer of 2022. We observed sustained suppression of rabbit abundance at five of the six sites, with the average population reduction across all six sites being 64%. Across all sites, average RHDV2 seroprevalence remained high, reaching 60–70% in adult rabbits and 30–40% in juvenile rabbits. In contrast, average RHDV1 seroprevalence declined to <3% in adult rabbits and 5–6% in juvenile rabbits. Although seropositivity continued to be detected in a low number of juvenile rabbits, it is unlikely that RHDV1 strains now play a major role in the regulation of rabbit abundance. In contrast, RCVA seropositivity appears to be reaching an equilibrium with that of RHDV2, with RCVA seroprevalence in the preceding quarter having a strong negative effect on RHDV2 seroprevalence and vice versa, suggesting ongoing co-circulation of these variants. These findings highlight the complex interactions between different calicivirus variants in free-living rabbit populations and demonstrate the changes in interactions over the course of the RHDV2 epizootic as it has moved towards endemicity. While it is encouraging from an Australian perspective to see sustained suppression of rabbit populations in the eight years following the arrival of RHDV2, it is likely that rabbit populations will eventually recover, as has been observed with previous rabbit pathogens. Rabbit haemorrhagic disease virus 2 (RHDV2) emerged as a novel rabbit calicivirus in 2010 in Europe ,4,5,6.Oryctolagus cuniculus) [Lepus sp.) and cottontails (Sylvilagus sp.) [Similar to the previously known RHDV of rabbits (termed RHDV1), recent isolates of RHDV2 are highly virulent, causing infectious hepatitis with high case fatality rates in susceptible rabbits (niculus) . Howevergus sp.) ,9,10, angus sp.) ,12. Furtgus sp.) . TogetheThe impact of RHDV2 has been substantial. For domestic and farmed rabbits, strict biosecurity measures were the only effective protection against RHDV2 until specific vaccines were produced . For lagIn Australia, RHDV2 was first reported in May 2015 , but retAnalyses of the data from the national rabbit monitoring network until 2018 revealed that the arrival and subsequent spread of RHDV2 resulted in an average reduction in the abundance of wild rabbits by 60% , with imFollowing the conclusion of the study by Ramsey et al. 2020) , rabbit 020 , rabMonitoring of rabbit abundance post-2018 was undertaken at six of the original 18 national monitoring network sites, Mirrabooka (NSW), Gudgenby (ACT), Coorong and Scobie (SA), and Nelsons and Drummonds (WA) . MonitorFollowing the completion of spotlight counts at each site, serum samples were collected from up to 20 shot rabbits from areas in the vicinity of each transect. Serum samples collected post-2018 were undertaken at five of the same six monitoring sites; no serum samples were obtained from the Coorong site in SA. Serum samples were screened by a series of enzyme-linked immunosorbent assays (ELISAs) to determine the presence of RHDV1 or RHDV2 antibodies as well as antibodies to RCVA, as described previously . Due to An equivalent dynamic state-space model to that detailed in Ramsey et al. (2020)  was fittre model .yitk ~ gulation .(2)ln . Estimat. (2020) .k at site t with k . The parameters t−1 on a variant at time t [i and time t  to examine trends in age-specific seroprevalence for the period following the arrival of RHDV2 .(5)nakit time t . Using m. (2020) .t4 priors were specified for all standard deviation parameters, and weakly informative The state-space model (Equations (1)–(4)) as well as the serological models (Equations (5) and (6)) were fitted to the data using the Bayesian Markov chain Monte Carlo (MCMC) software Stan . Weakly-matrices , settingThe convergence of the MCMC algorithms was assessed using the scale-reduction diagnostic of Brooks & Gelman  and by vPopulation trajectories of rabbit abundance for the six sites ranged between one and 495 rabbits per spotlight km . The higAverage trends in seroprevalence from the five sites with post-2018 serum samples revealed that RHDV1 seroprevalence continued to decline, especially in adult rabbits . HoweverEstimates of the interactions between variants (Equation (6)) indicate that RCVA seroprevalence in the previous quarter had a strong negative effect on RHDV2 seroprevalence, while the reciprocal effect of RHDV2 on RCVA seroprevalence was also evident . This diAnalysis of additional spotlight monitoring and serological data from rabbit populations at 6 of the original 18 long-term monitoring sites has revealed additional insights into the interactions and effects of RCVA/RHDV1/RHDV2 infections on rabbit populations. Rabbit populations at all monitored sites, with the exception of Scobie, were suppressed compared to pre-RHDV2 arrival populations, with the average population reduction across all six sites being 64%. As predicted, the serological data show that RHDV2 continues to be the dominant calicivirus variant in rabbit populations with a seroprevalence of 60–70% in adult rabbits, consistent with findings from recent molecular epidemiology and serology studies ,35. AlthThe seroprevalence of RHDV1 continued to decline at monitored sites, approaching 0% seroprevalence in adult rabbits. Hence, it is unlikely that RHDV1 strains now play a major role in the regulation of rabbit abundance. Nevertheless, some residual infections in juvenile rabbits suggest that the RHDV1 variants continue to occur in some areas, especially in South Australia. However, this conclusion should be tempered by the low number of juvenile rabbits subject to serological testing, which was evidenced by the high uncertainty in prevalence estimates for RHDV1, especially in the Western Australian sites. It also needs to be noted that the RHDV-K5 virus continues to be deliberately released across the continent , which mWhile it is encouraging from an Australian perspective to see the initial suppression of wild rabbit populations by RHDV2 sustained eight years after its initial emergence, previous experiences with myxoma virus and RHDV1 biological control have shown that rabbit populations invariably start to recover ,42. This


Beta-lactams are an important family of antibiotics used to treat infections and are commonly used in critically ill patients. Optimal use of these drugs in the intensive care unit (ICU) is important because of the serious complications from sepsis. Target beta-lactam antibiotic exposures may be chosen using fundamental principles of beta-lactam activity derived from pre-clinical and clinical studies, although the debate regarding optimal beta-lactam exposure targets is ongoing. Attainment of target exposures in the ICU requires overcoming significant pharmacokinetic (PK) and pharmacodynamic (PD) challenges. For beta-lactam drugs, the use of therapeutic drug monitoring (TDM) to confirm if the desired exposure targets are achieved has shown promise, but further data are required to determine if improvement in infection-related outcomes can be achieved. Additionally, beta-lactam TDM may be useful where a relationship exists between supratherapeutic antibiotic exposure and drug adverse effects. An ideal beta-lactam TDM service should endeavor to efficiently sample and report results in identified at-risk patients in a timely manner. Consensus beta-lactam PK/PD targets associated with optimal patient outcomes are lacking and should be a focus for future research. Streptococcus pyogenes, writing “Higher levels at the focus of infection represent largely waste penicillin, and lower levels have little if any therapeutic effect”  Database). Trough concentrations within the target range were significantly higher in the intervention arm, with real-time TDM more than doubling the odds of meeting PK/PD targets; albeit without benefit in infection-related outcomes. Rates of clinical success were very high in both groups (92% intervention group vs. 97% control group) and MICs of identified pathogens were low (mean piperacillin MIC 2 mg/L and mean meropenem MIC 0.125 mg/L).Following this, Fournier et al. published results from their RCT conducted between 2013 and 2016, including 38 patients from a burns ICU . PatientPseudomonas aeruginosa). The primary outcome was assessed using the mean daily sequential organ failure assessment (SOFA) score. In total, 249 patients were included . Attainment of PK/PD targets improved with TDM , without significant benefit in SOFA scores, mortality or clinical or microbiological cure. Despite the improvement in target attainment, no difference in median piperacillin concentrations was seen. Mortality was linked to piperacillin concentrations; patients with the highest mortality had piperacillin concentrations over 96 mg/L, with a 28-day mortality rate over 4-fold higher than those with a piperacillin concentration between 32 and 64 mg/L . This may be reflective of worse outcomes in patients with severe sepsis and subsequent kidney impairment.More recently, the TARGET trial was conducted between 2017 and 2019 in patients with severe sepsis and septic shock . After eThe DOLPHIN trial was undertaken in eight sites across the Netherlands between 2018 and 2021 and included 388 patients prescribed either beta-lactam antibiotics or ciprofloxacin . PatientAs yet, none of the RCTs have shown a difference in patient outcomes associated with TDM; however, failure to improve target attainment rates, use of surrogate MICs instead of actual MICs, broad inclusion criteria, small patient numbers and delay in TDM are significant limitations that will need to be addressed in future RCTs.Although largely considered an antibiotic class with a wide therapeutic index, beta-lactams can cause a variety of exposure-related adverse drug reactions, including cytopenia, neurotoxicity, hepatotoxicity and nephrotoxicity. There is emerging evidence that some commonly used beta-lactam antibiotics may have concentration thresholds that are associated with an increased risk of toxicity see . IdiosynBenzylpenicillin dose and duration of treatment have been associated with neutropenia, potentially via immunologic or direct toxicity to cells, though the exact mechanism is unknown . High doDuration of therapy has also been noted as a risk factor for the development of neutropenia with other penicillins . One expp = 0.02) ) and meropenem  [Imani et al. reported higher trough concentrations in patients who developed nephrotoxicity as opposed to those who did not for piperacillin  . Logisti< 0.01]) , weakeniClostridioides difficile-associated colitis, but an exposure–response relationship has not been described [Beta-lactam antibiotics can also cause allergy, thrombocytopenia and escribed .Although beta-lactam TDM is primarily considered for ensuring adequate concentrations and drug effectiveness, monitoring for exposure-related toxicity is increasingly being recognised as a valuable role of TDM. For patients with organ impairment, or for beta-lactam antibiotics with a documented exposure–toxicity relationship , regular clinical assessment and use of TDM should be considered to minimise drug toxicity.An ideal beta-lactam TDM service in the ICU will involve a balance of resource management, appropriate patient selection, timeliness of sampling and reporting of results, along with expert interpretation and dose modification see . OngoingAccess to real-time microbiology and MICs to direct TDM targets is ideal; however, patients often may not have an identified causative organism within 48 h of treatment initiation, and many laboratories do not routinely report MICs ,102. FurTDM results require clinical interpretation. Clinician-led dose modification has been shown to increase the proportion of patients who reach PK/PD targets, albeit with significant inter-clinician variability ,106. UsiGiven their widespread use in ICUs, optimising beta-lactam antibiotic dosing in critically ill patients remains important . A robusGlobally, beta-lactam TDM is becoming more prominent in the ICU . Researca priori prediction algorithms in dosing software packages may be a useful approach in the ICU [In settings where TDM is unavailable, model-informed precision dosing using  the ICU . HoweverMore data are needed before the value of beta-lactam TDM in the ICU can be understood. Currently, its role consists of ensuring adequate concentrations for effectiveness  and checking that toxicity thresholds are not being exceeded. Coupled with the rapid turnaround of results, the use of TDM early in beta-lactam therapy may provide the greatest clinical benefit for critically ill patients.Guidelines for TDM should identify patients most likely to benefit from TDM and report targets for clinical effectiveness and toxicity. Every TDM result requires clinical interpretation and action; improved accuracy and uptake of dosing software will be valuable when designing new dosing regimens.Further research to robustly define beta-lactam PK/PD targets based on improvements in clinical outcomes is imperative to ensure that a meaningful assessment of TDM-based interventions can be undertaken.


Here we report that CA1 pyramidal neuron-specific conditional BK knock-out (cKO) mice display normal locomotor and anxiety behavior. They do, however, exhibit impaired memory acquisition and retrieval in the Morris Water Maze (MWM) when compared to littermate controls (CTRL). In line with cognitive impairment in vivo, electrical and chemical long-term potentiation (LTP) in cKO brain slices were impaired in vitro. We further used a genetically encoded fluorescent K+ biosensor and a Ca2+-sensitive probe to observe cultured hippocampal neurons during chemical LTP (cLTP) induction. cLTP massively reduced intracellular K+ concentration ([K+]i) while elevating L-Type Ca2+ channel- and NMDA receptor-dependent Ca2+ oscillation frequencies. Both, [K+]i decrease and Ca2+ oscillation frequency increase were absent after pharmacological BK inhibition or in cells lacking BK. Our data suggest that L-Type- and NMDAR-dependent BK-mediated K+ outflow significantly contributes to hippocampal LTP, as well as learning and memory.Mutations of large conductance CaThe online version contains supplementary material available at 10.1007/s00018-023-05016-y. KCNMA1, the human gene encoding the pore forming α subunit of the large-conductance calcium ion (Ca2+)- and voltage-activated potassium ion (K+) channel BK  are linked to a growing number of clinically relevant phenotypes. Besides movement disorders and epileptic seizures, these mutations are accompanied by cognitive impairments like developmental delay and intellectual disability i were performed as previously described i levels, was calculated from the corrected values of the YFP images to those of CFP. First 5 min of basal FRET ratio signal were used for normalization.FRET-based recordings of i levels, was calculated from the corrected values of the images at 340 nm to those taken at 380 nm. Only spikes with at least 10% of the height of the glutamate peak were considered in the analysis.Cad before . 9 DIV n4(3)) i in both genotypes .To examine in detail how BK supports hippocampal LTP and, in turn, hippocampus-dependent memory formation, we next performed live cell imaging of dissociated hippocampal neuronal cultures generated from homozygous BKEPII 1.0 , 36  i is necessary for BK activation, BK channel activity also supports Ca2+ entry by rapidly repolarizing the plasma membrane to return voltage-gated Na+ or Ca2+ channels into an activatable state to support fast AP firing rates [2+-sensitive dye Fura-2-AM  indicates equivalent basal [Ca2+]i in both genotypes .Although high local i, but not sufficiently to support LTP. Only the simultaneous activity of both Ca2+ sources raises global [Ca2+]i sufficiently to trigger LTP and to efficiently activate BK channels.How could BK influence hippocampal synaptic plasticity in the form of LTP? LTP induction depends on postsynaptic Caing Fig. A. Concom+]i Fig. C. We assTCC Fig. F and G. tivation , 9–11. Itivation , 20. In LTP Fig. D and E, rol Fig. F–H. It ion of BK  or due ton of BK . Alterna2+ entry activates BK channels, we were surprised to discover that BK activity, in contrast to SK activity [2+ oscillations  pyramidal neurons in the mouse auditory cortex [2+ spikes mediated by NMDAR [2+ channels [2+ accumulation may be prevented, so that fewer synapses or dendrites perish [2+ spikes to optimize temporal coding and signal summation, which might facilitate coincidence detection and thus also the occurrence of LTP [So far, we assumed neurons to act as single functional units despite their well-known morphological subdivision into different compartments. An increasing body of evidence, however, suggests specialized functions of dendritic compartments, particularly in respect to synaptic plasticity –87. Feary cortex . Paradoxy cortex , which cby NMDAR  and voltchannels . This cochannels . This cochannels . Next, es perish . Lastly,e of LTP .2+ entry through both NMDAR and LTCC in postsynaptic membranes. BK-mediated K+ outflow, in turn, amplifies Ca2+ spiking frequency, which facilitates Ca2+ entry in support of the Ca2+-mediated mechanisms ultimately leading to AMPAR phosphorylation and LTP expression.Based on the presented data we propose a mechanism by which BK is activated by CaSupplementary file1 (PDF 1470 KB)Below is the link to the electronic supplementary material.


Locally restricted and short-lived regions of Ca2+ increase, called Ca2+ microdomains, constitute building blocks that are differentially arranged to create cellular Ca2+ signatures controlling physiological responses. Here, we focus on Ca2+ microdomains occurring in restricted cytosolic spaces between the plasma membrane and the endoplasmic reticulum, called endoplasmic reticulum-plasma membrane junctions. In T cells, these microdomains have been finely characterized. Enough quantitative data are thus available to develop detailed computational models of junctional Ca2+ dynamics. Simulations are able to predict the characteristics of Ca2+ increases at the level of single channels and in junctions of different spatial configurations, in response to various signaling molecules. Thanks to the synergy between experimental observations and computational modeling, a unified description of the molecular mechanisms that create Ca2+ microdomains in the first seconds of T cell stimulation is emerging.Cellular Ca ATP-driven Ca2+ pumps, such as sarco-/endoplasmic reticular Ca2+ ATPase (SERCA) and plasma membrane Ca2+ ATPase (PMCA), build up such Ca2+ gradients allowing for rapid Ca2+ release from organellar Ca2+ stores and/or Ca2+ entry across the plasma membrane upon cell stimulation.Cand fungi , 2. Ca2+2+ signaling in the form of an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) throughout the cytosol, most often extending into the nucleus, was first described in the 1980s , termed ‘sparks’ or d-myo-inositol 1,4,5-trisphosphate receptors (IP3R), termed ‘puffs’. Smaller fundamental Ca2+ signal events that relate to Ca2+ release from single RYR, termed ‘quarks’ or IP3R, termed ‘blips’ were also described  that provide privileged platforms for Ca2+ transfer from the ER into mitochondria . The latter mechanism relies on the stromal interaction molecules (STIM1 and/or STIM2) that are ER Ca2+ sensors regulating the activity of the Orai1 PM Ca2+ channels. Thus, upon local depletion of the ER Ca2+ store, Ca2+ dissociates from STIM, which allows recruitment of Orai1 to the junction and their gating (2+ release activated channels (CRAC)”. The ER-PM junctions are areas in the cytosol where the ER gets really close to the PM, with a depth of approximately 15 nm and an extension of ~200 nm  or PM (Orai1) Ca2+ channels, but the detailed interplay between these channels, including their molecular effectors, remain to be fully characterized.Local Ca2+ toolbox molecules, e.g. second messenger forming and degrading enzymes, Ca2+ mobilizing second messengers and their receptors, Ca2+ channels, Ca2+ storing organelles, and Ca2+ pumps are often ubiquitously expressed, the specific configuration of a particular cell type determines the exact mechanism of both local and global Ca2+ signaling. Thus, in this review, we will concentrate on a particular cell type that has been extensively characterized regarding mechanisms involved in the formation of Ca2+ microdomains, mammalian T-lymphocytes. The main molecules involved are listed in 2+ microdomains at the ER-PM junctions have been extensively studied in these cells. Despite disadvantages of this cell model, e.g. small diameter of about 6-7 μm and a large nucleus as compared to the cytosol, as well as spherical shape with many stacked z-layers that potentially result in image blurring, development of a high-resolution Ca2+ imaging method combined with image refining by off-line deconvolution resulted in spatio-temporal resolution of 368 nm and 25 ms (Although most of the Cand 25 ms . Furthernd 25 ms .2+ microdomains in T cells were identified: (i) adhesion-dependent Ca2+ microdomains (ADCM) evoked by integrin-mediated IP3 signaling and subsequent SOCE (2+ microdomains evoked by TCR/CD3/CD28 stimulation (TDCM), proceeding via nicotinic acid adenine dinucleotide phosphate (NAADP) formation by dual NADPH oxidase 2 (DUOX2), and activation of RYR1 via the NAADP receptor hematological and neurological expressed 1-like protein (HN1L)/jupiter microtubule associated homolog 2 (JPT2). Hereafter, ADCM and TDCM will refer to the spatially restricted and short-lived Ca2+ increases occurring in the so-called microdomains. TDMC are amplified by SOCE  proteins . FAK is thought to be a key component of the integrin-mediated signaling cascade , which evokes Ca2+ signals through ryanodine receptor type 3  IP3 evoked Ca2+ release between approx. 30s and 10 to 15 min  cyt 60 min . SOCE viiewed in .2+ dynamics and localized Ca2+ events. These observations however raise numerous questions about the molecular mechanisms involved in the foundation of Ca2+ microdomains. Some of these questions are best addressed by computational modelling, which is made possible thanks to an impressive quantitative characterization of the properties of ER-PM Ca2+ microdomains. In particular, models allow to predict the number and spatial arrangement of the channels inside the junction, which remains inaccessible with current experimental techniques.As detailed above, current techniques in microscopy allow to analyze both global Ca2+ microdomains due to SOCE. In a series of pioneering studies  and in the extracellular medium. Given that the latter can be seen as an infinite source of Ca2+, its concentration can be considered as constant. Thus, Ca2+ concentrations in the junction and in the sub-PM ER compartment represent key variables. Because they dynamically regulate the junctional Ca2+ concentration, buffers should in principle be taken into account. Most models however ignore the effect of buffers because of the short time and length scales involved in microdomain formation. Because of the high Ca2+ concentrations and the small volume of the junction, local Ca2+ buffers are expected to be saturated (2+ diffusion is assumed to occur in an unbuffered cytosol (cD = 220 μm2s-1). When considering the relation between cell adhesion/stimulation and Ca2+ signaling, IP3 and NAADP should also be involved because they modify ER and junctional Ca2+ by affecting the opening states of the IP3Rs and RYRs, respectively. Although NAADP mobilizes lysosomal Ca2+ , istions in . The distions in , 69, 70.5 SERCA’s spread over the entire surface area of the ER, which would also correspond to ~10 pumps per ER-PM junction assuming a homogenous distribution. However, model outcomes are practically independent of the numbers of SERCA’s that are found to exert little influence on the spatio-temporal dynamics of microdomain formation. In line with this finding, SERCAs were not included in the first studies of Ca2+ changes at the immunological synapse , or by a stochastic reaction-diffusion scheme such as, for example, the Gillespie’s algorithm  and secondly, of 65 nm (non-clustered), as shown in 2+ changes in the junction. When clustered, junctional Ca2+ concentration rises from an initial value of 100 nM to 7 μM in the center of the junction, while this increase is limited to 4 μM in the non-clustered configuration (D of SERCA pumps (~300 nM). Orai channels are assumed to remain open with a constant current of 2.1 fA  was considered, to account for molecular crowding in the lumen and for the tortuosity of the tubular ER network . Following stimulation, NAADP signaling activates RYR1 and releases Ca2+ from the ER to the cytosol  or by early NAADP signaling (TDCM) display similar characteristics in terms of amplitude and spatial extent because of the change in STIM isoform involved. Moreover, the model predicts that the flux at the mouth of the RYR1 or IP3R, which determines the extent of local Ca2+ depletion, rapidly becomes similar despite the largely different conductance of these channels, due to the slow Ca2+ diffusion in the ER.In a similar way, Gil et al.  investig cytosol . Very re cytosol ; however cytosol . This su cytosol  considermyocytes , one rec3R. STIM2 recruits STIM1 close to the junction under such low stimulation conditions (ADCM) 3R. STIM2chniques , 43, 48 ytoplasm , 93. Theytoplasm . It need2+ microdomains, this synergy has allowed to predict the number and spatial arrangement of Orai channels in the junction (2+ increase and the spatial spread of Ca2+ microdomains (2+ channels surrounding the junction, a comparison between simulation results and high-resolution Ca2+ imaging uncovered the main role played by ER local depletion induced by weak IP3 or NAADP signaling in triggering the formation of adhesion-dependent and TCR/CD3-dependent Ca2+ microdomains in T cells (2+ signaling, which could emphasize the key role played by the PMCA’s and the mitochondria in the T cell Ca2+ dynamics beyond the first seconds after stimulation (2+ signaling pathway fine-tunes T cell immune responses.Computational modeling is increasingly used in cell biology to optimize the information that can be gained from the ever-improving techniques in imaging microscopy. In the field of ER-PM Cajunction , 61, andodomains . When co T cells , 64. In mulation . DespiteAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Abnormal or acquired automaticity is thought to initiate ventricular arrhythmias associated with myocardial ischemia. Ca2+ flux from mitochondria can influence automaticity, and lysosomes also release Ca2+. Therefore, we tested whether lysosomal Ca2+ flux could influence automaticity. We studied ventricular human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs), hiPSC 3D engineered heart tissues (EHTs), and ventricular cardiomyocytes isolated from infarcted mice. Preventing lysosomal Ca2+ cycling reduced automaticity in hiPSC-CMs. Consistent with a lysosomal role in automaticity, activating the transient receptor potential mucolipin channel (TRPML1) enhanced automaticity, and two channel antagonists reduced spontaneous activity. Activation or inhibition of lysosomal transcription factor EB (TFEB) increased or decreased total lysosomes and automaticity, respectively. In adult ischemic cardiomyocytes and hiPSC 3D EHTs, reducing lysosomal Ca2+ release also inhibited automaticity. Finally, TRPML1 was up-regulated in cardiomyopathic patients with ventricular tachycardia (VT) compared with those without VT. In summary, lysosomal Ca2+ handling modulates abnormal automaticity, and reducing lysosomal Ca2+ release may be a clinical strategy for preventing ventricular arrhythmias.Automaticity involves Ca A lysosomal Ca2+ release channel was correlated with arrhythmic risk in humans, and inhibition of lysosomal Ca2+ release prevented arrhythmias in isolated hearts, suggesting that lysosomal Ca2+ release may contribute to arrhythmic risk.Abnormal ventricular automaticity occurs in injured myocardium and is thought to be one mechanism of enhanced arrhythmic risk. Enhanced and reduced lysosomal Ca2+ cycling systems, the membrane and the intracellular Ca2+ clocks (2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptor type 2 (RyR2). SR Ca2+ release has its effect by increasing a depolarizing current mediated by the Na+/Ca2+ exchanger (NCX) (K1) (f pacemaker current (3R)  . Other iCX) (K1) , the If  current , and theent (3R) .2+ cycling is central to automaticity, we tested whether mitochondrial Ca2+ cycling could play a role in automaticity. We demonstrated a role for mitochondrial Ca2+ cycling in abnormal automaticity in ventricular CMs, including mouse embryonic stem cell (ESC)–derived ventricular-like CMs, human-induced pluripotent stem cell (hiPSC)–derived ventricular-like CMs (hiPSC-CMs), and ischemic adult mouse ventricular CMs   . Mitochorial NCX .2+ release may contribute to lysosomal Ca2+ loading  and protons are exchanged for Ca2+ by an H+/Ca2+ exchanger in CMs (2+ concentration in lysosomes is significantly higher than that in the cytosol (∼500 µmol/L in lysosomes) (2+ release is mediated by a type 2 two-pore channel (TPC2) and a transient receptor potential mucolipin channel (TRPML1)  , 22. Lys(TRPML1) . The Ca2membrane , 23. Lysmembrane .2+ flux can modulate ventricular automaticity.The spontaneous electrical activity of hiPSC-CMs provides an opportunity for a more in-depth examination of the ion currents that drive ventricular automaticity . Therefo2+ cycling is central to automaticity and TRPML1 is considered to be the principal Ca2+ release channel in the lysosome , while the TRPML1-specific antagonists (P < 0.01). The effect of blocking another lysosome Ca2+ release channel (TPC2) by 5 µmol/L Ned-19 on beating rate was similar to that of inhibition of TRPML1 . Inhibiting the V-ATPase prevents proton flux into lysosomes, reduces the H+/Ca2+ exchanger activity, and depletes lysosomal Ca2+ stores (P < 0.01), supporting the role of lysosomes in automaticity. As expected for a small Ca2+ release, pharmacological manipulation of lysosomal Ca2+ release did not influence basal Ca2+ or cellular Ca2+ transients, except ML-SI1 which reduced the amplitude of the Ca2+ transients by 41% for unclear reasons . On the other hand, the mRNA expression of TRPML1 was significantly decreased after TRPML1 siRNA transfection . Up-regulation of TRPML1 did not substantially accelerate SR Ca2+ release or the spontaneous beating in hiPSC-CMs, presumably because TRPML1 overexpression alone did not increase lysosomal Ca2+ release . Despite this, increasing TRPML1 channel activity by the TRPML1 agonist ML-SA1 could increase automaticity, while TRPML1 inhibition reduced automaticity  CMs .To suggest human relevance of our findings, ventricular TRPML1 was measured in cardiomyopathic heart failure (HF) patients with and without reported ventricular arrhythmias. The protein level of TRPML1 in HF patients with ventricular tachycardia (VT) was increased when compared with those subjects without VT   . In the channels . The Ca2membrane , 23. TRP and ROS , 30. TPC channel . TRPML1 lysosome . The eneactivity . Therefo2+ release and Ca2+ influx  were purchased from FUJIFILM Cellular Dynamics, Inc.  and differentiated for 30 days. Cell plating and maintenance were carried out according to the protocol in the manufacturer's instructions. To reduce the variability of the beating rate, CMs with the lowest quartile beating rates in the same culture conditions were selected for comparison, and the experiments were repeated in at least three different differentiation CM batches. Ventricular hiPSC-derived CMs were selected by action potential (AP) characteristics. Ventricular-like APs had maximum diastolic potentials close to those of human CMs . MI was induced in 11- or 12-week-old mice  by coronary artery occlusion . Mice we2+ transient measurements.Ventricular CMs were isolated as we described before , 36. CMs2+ transients in hiPSC-CMs  loaded for 20 min followed with 20 min of de-esterification was employed to measure cytoplasmic CaiPSC-CMs . Ca2+ tr2+ release measurements, Fluo-4 was loaded as above. For imaging, the time-lapse mode with a frame interval of 3 s was used. In TRPML1-CFP overexpressing hiPSC-CMs, cells with CFP fluorescence were chosen. Their Fluo-4 fluorescence images and traces were recorded from these cells. To measure the small lysosomal Ca2+ release, we focused on only the Fluo-4 fluorescence.For lysosomal TRPML1 Ca2 was added to pipette solution to keep free [Ca2+]i ≈ 50 nmol/L. Junction potentials were compensated when membrane potentials were calculated. APs were recorded before and after chemical compounds were applied for about 2 min at room temperature.AP was recorded as we described before . A totalTo silence TRPML1, specific TRPML1 siRNA and scrambled siRNA were purchased from Santa Cruz Biotechnology, Inc. . TRPML1-CFP was a gift from Craig Montell . Cells were transfected by TransIT-siQUEST Transfection Reagent  for 72 h with 50 nM siRNA and by FuGene6 transfection reagent  with 1 μg DNA. TRPML1 mRNA was measured after 72 h. qPCR was performed using PowerUp SYBR Green Master Mix (ThermoFisher Scientific). The primer set is as follows: hMCOLN1_F-CGG ACT GCT ATA CCT TCA GCG T; hMCOLN1_R-GGT GCT TAC ACT CCT GGA TGT G, and hGAPDH_F-GAA GGT GAA GGT CGG AGT CAA C; hGAPDH_R-CAG AGT TAA AAG CAG CCC TGG T. Primers were purchased from Integrated DNA Technologies .CMs were fixed with 4% formaldehyde for 15 min at room temperature. Then, they were rinsed three times in 1XPBS for 5 min each followed with blocking for 60 min. After aspirating the blocking solution, primary antibodies were incubated overnight at 4°C and rinsed three times in 1XPBS for 5 min each. Primary antibodies were TRPML1 , LAMP1 , and TFEB . CMs were incubated in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer (1:500) for 1–2 h and rinsed three times in 1XPBS for 5 min each while being protected from light. Images were acquired by confocal microscope  with FluoView FV31S-SW software.Western blotting and left ventricular cardiac tissues samples preparation were performed as we described before . The ant2+ sparks were recorded from the same batch of CMs before and after chemical compounds were applied for about 2 min at room temperature.The method was described previously . Ca2+ spThe EHT negative mold was printed on a Stratasys J750 PolyJet 3D Printer using digital ABS plastic. The ABS mold was thoroughly cleaned, oxygen plasma treated for 10 min , and treated with silane vapors for 24 h in a desiccator vacuum. Sylgard 184 PDMS precursor was mixed at a 1:10 mass ratio with PDMS curing agent and cast onto the ABS negative mold. The curing PDMS was degassed for 1 h at room temperature and cured overnight at 50°C. The resultant PDMS mold contained 12 rectangular EHT culture wells, and each EHT well contained 2 posts. The PDMS mold was further cured at 50°C for 1 week. The mold was then cut into six two-mold squares that were sonicated in 70% ethanol for 30 min and autoclaved at 121°C for 25 min.w/v% Pluronic F-127 for 30 min at room temperature. The hiPSCs were differentiated into CMs using small molecule WNT modulation. Briefly, the hiPSC  were plated at a cell number of 0.5 × 106 per well of a Matrigel (Corning)–coated 12-well plate. The cells were grown in mTesR1 (Stemcell) for 2–3 days until they reached 100% confluency. On day 0 of differentiation, the hiPSC were treated with 6.5 μM CHIR99021 (Sigma Aldrich) for 48 h following treatment with 7.5 μM IWP2 (Tocris) for 48 h in RPMI supplemented with 1X B27 minus insulin supplement (RPMI/B27/−ins). After IWP2 treatment, on day 4 of differentiation, the media was replaced with fresh RPMI/B27/−ins. Starting on day 6 until day 13, the media was replaced every 48 h with RPMI supplemented with 1X B27 supplement (RPMI/B27/+ins). On day 13, the hiPSC-CMs, along with other contaminating cells, were dissociated with 0.25% Trypsin and split at a 1:2 ratio onto new Matrigel-coated plates. The cells were left to recover for 48 h, and then, they were lactate purified using DMEM (−)glucose (Gibco) that was supplemented with 4 mM sodium l-lactate (Sigma Aldrich) for 4 days. The purified hiPSC-CMs were recovered in RPMI/B27/−ins until day 22 after differentiation initiation. Neonatal human dermal fibroblasts  were expanded and maintained in DMEM-high glucose with 20% EmbryoMAX FBS (Millipore). At day 22 of hiPSC-CM differentiation, the purified hiPSC-CMs were dissociated in 0.25% Trypsin-EDTA and combined with nhDF that were singularized in Accutase (Millipore). The hiPSC-CMs and nHDF were combined at a 3:1 ratio and a density of 10 × 106 cells/mL in fibrin gel mixture containing 20 mg/mL fibrinogen from human plasma, RPMI/B27/+ins with 20 μM Rock Inhibitor (VWR), and 100 U/mL thrombin from human plasma (Sigma) at a 6:3:1 ratio. Each EHT culture well was seeded with 100 μL of fibrin gel cell suspension, which immediately formed a gel at room temperature. The EHTs were then cultured in RPMI/B27/+ins with 10 μM Rock Inhibitor and 20 μg/mL aprotinin for 24 h. Then, the EHTs were given fresh RPMI/B27/+ins with 20 μg/mL aprotinin every 2 days until assessment on day 16.The sterile two EHT well PDMS squares were transferred into a sterile petri dish and oxygen plasma treated for 2 min. The molds were then transferred to a 12-well plate and treated with 0.5 2+ transients in EHTs as we used before  were used to record the Cad before . A slicet-test was employed for statistical analysis. Bonferroni correction was used in one-way and two-way ANOVA tests for multiple comparisons. A value of P < 0.05 was considered statistically significant. OriginPro 2017 SR2  was used for statistical analysis.Data are shown as the mean ± SEM. As noted in the text, the Mann–Whitney test or paired or two-sample pgad174_Supplementary_DataClick here for additional data file.


AML is a heterogenous disease caused by different mutations. We have previously shown that each mutational sub-type develops its specific gene regulatory network (GRN) with transcription factors interacting with multiple gene modules, many of which are transcription factor genes themselves. Here we hypothesized that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We tested this hypothesis using FLT3-ITD mutated AML as a model and conducted an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict identifying crucial regulatory modules required for AML but not normal cellular growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD AML and that its removal leads to GRN collapse and cell death. Due to the differing effects of these mutations, there is a drive to identify druggable targets for each distinct AML subtype to tailor treatment to the individual and reduce the need for intensive chemotherapy which is often not tolerated by elderly patients.Cancer occurs when mutations in signalling genes, transcription factors (TFs) and epigenetic regulators cause a block in the normal program of differentiation and an increase in proliferation. Acute Myeloid Leukaemia (AML) is no exception and, due to the clonal nature of the disease, different driver mutations of the cancer cause distinct patterns of gene regulation in AML cells3. The FLT3-ITD mutation occurs on the background of other mutations, mostly in epigenetic regulators such as DNMT3 or TET2, or as a result of clonal evolution5. For reasons that are as yet unknown, this mutation often occurs together with a mutation in nucleophosmin (NPM1). Less than 60% of FLT3-ITD AML patients over 60 year-olds reach complete remission and patients often relapse within a year with an overall relapse rate of 77%6. For this reason, therapy efforts have focussed on the development of improved FLT3-specific inhibitors, resulting in the approval of Gilteritinib for use in a clinical setting. However, relapse still frequently occurs after treatment with these inhibitors, often due to activating mutations in other signalling genes7. The identification of other druggable targets in FLT3-ITD AML is therefore necessary to improve the outcome of patients. To this end, various efforts have been conducted to employ genome-wide RNAi or CRSPR/Cas9 screens which highlighted numerous targets9. However, a caveat of such screens is often that they come up with a large number of targets, many of which are also important for normal cells, and often require follow-up experiments to identify therapeutic windows for their inhibition. To home in on the true AML subtype-specific targets, it is therefore necessary to elucidate the fine details of the molecular mechanisms driving AML growth and maintenance of each sub-type.AML with an internal tandem duplication of the FLT3 receptor (FLT3-ITD+ AML) which converts a ligand responsive receptor into a constitutively active molecule is a highly aggressive AML subtype which is frequently refractory to first line therapy1. To construct GRNs, we then integrated transcriptomic (RNA-Seq), Hi-C and digital footprinting data based on high resolution DNaseI-seq experiments2. The comparison between the GRNs of normal and malignant cells identified TF families showing FLT3-ITD+ AML subtype-specific interactions with their targets which could be attributed to aberrant expression of genes promoting AML survival. For several of these TFs, such as the AP-1 TF family we could indeed show that they were required for the maintenance of AML but not normal cells. For example, inhibiting AP-1 activity completely blocked tumorigenesis in two different patient-derived xenograft (PDX) mouse models2. However, the molecular details of how specific TFs contribute to AML development and their interaction with genes encoding other TFs is largely unclear.Differential gene expression and thus cellular identity are controlled by the regulatory interactions between transcription factors (TFs) and their target genes which form extensive interacting gene regulatory networks (GRNs). We have previously shown that AML blast cells with FLT3-ITD and FLT3-ITD/NPM1 mutations display a specific chromatin signature distinct from healthy CD34+ cellsTo answer these questions, we generated a refined GRN for FLT3-ITD and FLT3-ITD/NPM1 AML. Based on this analysis, we designed a targeted shRNA screen to interrogate the contribution of specific TF network nodes and their down-stream targets to AML establishment and maintenance. These analyses highlight a crucial role of several different TFs including RUNX1 in FLT3-ITD pathology and identify drug responsive AML-subtype-specific and overlapping regulatory modules. Taken together, our data show that identifying AML-subtype specific GRNs is predictive for genes required for AML maintenance.10. Our previously constructed gene regulatory networks for AML with FLT3-ITD and FLT3-ITD/NPM1 genotypes highlighted interactions between TF families and distal elements demonstrating that each AML-sub-type displayed a specific gene regulatory phenotype distinct from that of normal cells. However, to demonstrate the full regulatory relevance of the GRN and its downstream targets we developed a novel computational method to produce a GRN that contained the full set of regulatory interactions between all cis regulatory elements including promoters as depicted schematically in 2 as determined by digital footprinting11. Such regions were assigned to promotors using previously determined promoter-capture HiC (CHiC) interactions from a FLT3-ITD+ AML sample or the nearest gene (both within 200kb of the promoter)2. RNA-seq was used to identify genes specifically upregulated in the FLT3-ITD+ AML subtype compared to healthy CD34+ cells . These data were plotted as a GRN showing specific interactions between TF families and their target genes in FLT3-ITD+ AML, which each TF interacting with its own specific gene modules of down-stream target genes. These modules can be AML-specific, shared or specific only to normal cells . We have previously shown that FLT3-ITD and FLT3-ITD/NPM1 cluster together2 and form very similar AML-specific GRNs , suggesting that such genes are regulated by more than one factor. We also observed cross-regulation between modules as exemplified by FOXC1 being part of the NFI module (highlighted). Note that FOXC1 and NFIX are examples of TFs which are aberrantly expressed as compared to healthy cells16. FOXC1 is truly mis-expressed, and NFIX plays a role in hematopoiesis and HSC survival18 but is strongly up-regulated in FLT3-ITD/NPM1 cells. Their expression is therefore a part of the aberrant FLT3-ITD/NPM1 leukemic phenotype.Since TFs and other regulators of gene expression are part of an interacting network, targeting such molecules in a clinical setting requires careful examination of the effects of perturbation on their down-stream targets and how GRNs shift in the absence of specific TFs. We therefore connected the above-described transcription factors to the wider patient derived GRN by using digital footprinting and (where available) ChIP analyses to identify AML-specific and shared TF modules. These analyses were conducted for individual TFs  and the previously studied TF families AP-1, FOXC1, NFIX) which were linked to down-stream genes specifically up-regulated in FLT3-ITD/NPM1 cells as outlined in To examine the degree by which nodes are shared between AML and normal cells we calculated their similarities as shown in Taken together, this analysis showed that FLT3-ITD specifically expressed genes are regulated by specific and overlapping sets of TF modules.DUSP5, FOXC1 and EGR1 in healthy CD34+ PBSCs by transducing a mini-library of lentivirally expressed shRNAs together with a non-targeting control (NTC), where no suppression of growth was observed  and C/EBP family members can physically interact to drive macrophage differentiation21 and our data show them to co-regulate a number of genes. In this context it is interesting to note that one the motifs enriched in open chromatin regions lost after dnC/EBP expression is a C/EBP/AP-1 composite motif  from FLT3-ITD/NPM1 patients suggested an important role for this TF in this AML-sub-type as well25. To ensure that our digital footprint-based module construction was valid, we validated binding sites by comparing them to previous ChIP-Seq experiments in FLT3-ITD/NPM1 patients and cell lines1  which disrupts the interaction between the RUNX DNA binding domain and its binding partner CBFβ27. We first used a proximity ligation assay to determine the optimal time point to detect a complete dissociation of RUNX1::CBFβi  which is an important regulator of SRC kinases in blood cells29. The analysis of up-regulated genes after CBFβi showed a strong enrichment of genes organised in the overlapping NFI, AP-1 and RUNX modules scoring in our screen. This included again DUSP6 but also the genes encoding the Zn++ finger TFs KLF2 and KLF6 which have been shown to be repressed in AML cells and which are associated with myeloid differentiation30.Similar to the depletion of RUNX1 by shRNA mediated knock-down, the removal of RUNX1 from the genome led to not just a loss but also the gain of open chromatin regions and changes in gene expression  includine gained , indicate gained , Fig S6Ee gained  showed tandidate 28. Down-In our final set of experiments, we asked the question of which cell type in primary AML cells and which genes were most affected by CBFβi. To this end, we cultured primary FLT3-ITD AML cells in the presence and absence of CBFβi and conducted single cell (sc) RNA-Seq experiments. Untreated cultures contained a mixture of early precursor cells, myeloid and erythroid cells of variable differentiation stages . The anaIn summary, our experiments show that targeting an important TF comprising a highly connected node within an AML sub-type-specific GRN such as RUNX1 leads to a profound impact on other TF modules which rewires the GRN of AML cells, leading to a cell cycle block and eventually, cell death.2 leads to the identification of genes whose expression is vital for the maintenance of malignant cells. The hit-rate for our screen was very high, and we found numerous TF encoding genes which were important for the growth of AML, but not normal cells. Moreover, the identification of the down-stream targets of such TFs highlighted a complex web of interactions between genes which were essential for FLT3-ITD/NPM1 AML growth. This and our previous studies show that the idea of an AML sub-type specific GRN that maintains AML cells is not only true for groups affected by mutations of gene regulatory molecules themselves, but also for those with signalling mutations who generate chronic growth signals, such as the FLT3-ITD. Decades of clinical trials have tried to target aberrant signalling and have failed due to the development of drug resistance. Moreover, such treatments only affected fast growing cells, and not quiescent leukemic stem cells. Based on these data and those of others32, we believe that the most viable way to eliminate malignant cells is to identify the factors crucial for tumour maintenance and reprogram cells into either normal cells that differentiate or change their GRNs into an unstable, non-viable state by identifying TFs that are essential for AML maintenance.The present study shows how identification of an aberrant AML sub-type specific GRN based on the data collected by Assi et al.,33 or are aberrantly over- expressed 16 In addition, these sub-type specifically deregulated TFs form regulatory modules that contain multiple signalling genes that are up-regulated compared to healthy cells, such as the DUSPs or TNF-superfamily members (See 35. Such interactions can be aberrant as exemplified by the formation of AML-specific protein complexes containing FOXC1 and RUNX136Our GRN analyses inform us as to which factors and genes to target. As schematically outlined in bers See –F. Genes37. It takes even longer until cells differentiate or die, and it is this feature which is relevant in a clinical setting. Our data highlight the molecular reason for this finding by showing that an AML-specific GRN sustaining leukemic growth is stabilised by multiple modules which cooperate in driving AML sub-type specific expression. The removal of one essential module leads to a rewiring of others, as exemplified by our detailed studies of the RUNX1 module. Here, the result is a profound cell cycle block followed by eventual cell death suggesting an inability of RUNX1-depleted cells to further rewire their GRN into a stable state and which could be the basis of therapy. A recent study applying a degron technology to target IKZF2 in MLL-driven AML38 demonstrates that such a strategy is indeed feasible.Recent experiments coupling degrons to oncoproteins or normal transcription factors have shown that only a small number of genes immediately respond to the degradation of one factor, and that it takes a number of hours until the whole GRN responds and changes the expression of multiple genesA detailed description of the methods can be found in Supplement 1


However, the role of K+ transport in PTP regulation is unclear. Here, we studied the role of K+ and other monovalent cations in the regulation of the PTP opening in an in vitro model. The registration of the PTP opening, membrane potential, Ca2+-retention capacity, matrix pH, and K+ transport was performed using standard spectral and electrode techniques. We found that the presence of all cations tested in the medium  strongly stimulated the PTP opening compared with sucrose. Several possible reasons for this were examined: the effect of ionic strength, the influx of cations through selective and non-selective channels and exchangers, the suppression of Ca2+/H+ exchange, and the influx of anions. The data obtained indicate that the mechanism of PTP stimulation by cations includes the suppression of K+/H+ exchange and acidification of the matrix, which facilitates the influx of phosphate. Thus, the K+/H+ exchanger and the phosphate carrier together with selective K+ channels compose a PTP regulatory triad, which might operate in vivo.The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K In particular, the role of the K+ transport in the induction of the PTP by Ca2+ is not clear. It was suggested that the activation of K+ transport causes mild mitochondrial uncoupling, which decreases the accumulation of Ca2+ by mitochondria and the generation of ROS and, thus, negatively regulates the PTP [The opening of the PTP in mitochondria is a key event in the initiation of cell death and injury to organs and tissues in various pathological conditions, in particular, prolonged ischemia followed by reperfusion ,2. It istioning) ,7,8,9,10itioning ,7,8,9,10 the PTP ,15,16,17+ transport in isolated mitochondria deserve further clarification [+ fluxes were modulated by known activators and inhibitors of K+ channels, which have additional targets in mitochondria and exert effects on the PTP opening independent of K+ transport. For instance, the activator of mKATP channel diazoxide inhibits succinate dehydrogenase [+ transport-independent stimulation of PTP opening by glibenclamide was also reported [+ entry into mitochondria by low doses of the K+ ionophore valinomycin on Ca2+-retention capacity (CRC)  are controversial [+ ions in the incubation medium on the PTP opening was not studied in detail previously.However, the data on the regulation of the PTP by modulation of Kfication ,21,22,23rogenase ,25, whilrogenase , importareported . On the oversial ,28. More2+ and some other divalent cations. In turn, the concentration of free Ca2+ in the mitochondrial matrix can be modulated by the matrix pH and the concentrations of inorganic phosphate (Pi) and adenine nucleotides [m) may be an additional PTP regulator since depolarization is known to stimulate PTP opening [There are at least four classes of the regulators of PTP state, each of which can be affected by numerous modulators A. The fileotides ,32,33,34leotides ,37,38,39leotides ,44,45,46leotides ,50,51,52leotides ,54,55,56leotides ,58,59. T opening ,61,62,63+ entry into mitochondria can affect the state of at least three out of four regulators. Indeed, K+ entry can change the matrix pH due to the K+/H+ exchange [+ entry may affect ROS production and NAD(P)H/NAD(P) ratio [+ entry can decrease the ΔΨm and the gating potential of the PTP [+ channels have been found in mitochondria confirms the important role of K+ transport in mitochondrial physiology [2+  ratio ,13,67,68 the PTP ,67. The ysiology ,70. It ilogy [2+ B.+ in the regulation of the PTP in an in vitro model, to compare the effects of K+ and other monovalent cations, and to elucidate the mechanisms of this regulation.Therefore, the goal of this study was to examine the role of KATP channels are opened [2+ to the mitochondria should lead to an opening of Ca2+-activated and voltage-gated K+ channels, the latter due to transient membrane depolarization [+ inward transport through any channel type is essential for mitochondrial protection, organelles will be more resistant to Ca2+-induced PTP opening in the K+-containing medium. 2+-dependent high-amplitude mitochondrial swelling (A) and the dissipation of ΔΨm (C), as well as the results of the measurements of CRC (B) in the mitochondria incubated in KCl-based medium (KCl-BM), NaCl-based medium (NaCl-BM), and sucrose-based medium (S-BM). Recordings in all media were started after approximately 2-min of incubation of the mitochondria with the respiratory substrate  for the initial equilibration of ion and solute concentrations. As follows from the figure, the longest time between the addition of Ca2+  and the initiation of mitochondrial swelling (A) and the dissipation of ΔΨm (C) was observed in S-BM. In KCl- and NaCl-BM, the swelling and ΔΨm loss were initiated four to eight times faster, indicating a lower resistance of the mitochondria to PTP opening in cation-containing media.Previously, it was shown that in isolated mitochondria in the absence of added ATP, mKe opened . The addrization . It is lm can be due to both PTP opening and the electrophoretic influx of cations through the activated cation channels (regardless of their selectivity). To examine the latter possibility, we added 600- and 3350-Da polyethylene glycols (PEGs) to swollen mitochondria and observed either transient or sustainable mitochondrial shrinkage, respectively. These data clearly indicate that the inner mitochondrial membrane (IMM) contains opened pores permeable for solutes of molecular weight higher than 600 but lower than 3350 Da, i.e., the PTP. In accordance with a slower PTP opening, the highest CRC (B) was observed in S-BM (232 ± 26 vs. 144.7 ± 28.6 (KCl-BM) and 146 ± 24.4 nmol∙min−1∙mg prot−1 (NaCl-BM)). Thus, the presence of K+ or Na+ ions in the incubation medium did not postpone but considerably stimulated the PTP opening.Mitochondrial swelling and the dissipation of ΔΨThere are several possible reasons for the stimulation of PTP opening in the presence of salts: the effect of the ionic strength, the triggering of the PTP opening by the influx of solutes into the matrix, and the effect of the salts on the PTP regulation independently of the influx of cations or anions. All of these reasons were experimentally examined.The ionic strength of media may affect the interaction of charged groups of proteins of a PTP complex and membrane phospholipids, thus stimulating the pore opening. We measured the rate of PTP opening in the media containing different concentrations of KCl, NaCl, LiCl, and choline-Cl (ChCl) in S-BM . The fin1/2) were as low as 4.4 ± 1.5 and 5.9 ± 1.8 mM, respectively. The concentrations of LiCl and NaCl required for a 50% reduction of sucrose protection were somewhat higher at 20.5 ± 3.2 and 23.6 ± 7.3 mM, respectively. Thus, the PTP opening can be triggered by various cations. Nevertheless, K+ and Ch+ are three to five times more efficient as triggers than Na+ and Li+. The low concentrations of salts required for PTP stimulation count rather in favor of mechanisms involving ion channels and exchangers. Moreover, the fact that an increase in ionic strength from 15 to 19–20 mM reduces the protection by a factor of two (KCl and ChCl) implies the existence of too sharp a threshold in the dependence of ionic interactions on the ionic strength of the medium. Thus, these data show that the contribution of the ionic strength to the stimulation of PTP opening is very unlikely. Then, we explored whether the stimulation of PTP opening in the presence of salts is a consequence of the electrophoretic influx of cations via any mechanism.+ channels on the PTP opening in KCl-, NaCl-, and S-BM. 1/2 = 0.04–5 μM), and an activator of mKATP channel (K1/2 = 0.5–0.8 μM), respectively [Ca channel, K1/2 ≤ 100 nM [1.3 channel, K1/2 = 3 nM [1.1, Kv1.2, Kv1.4, and Kv1.7 channels, though with lower efficiency, we assessed its effect in the 30–1000-nM range in separate experiments (+ or both (A–F). Thus, the entry of cations into the mitochondria through selective K+ channels seems to have minimum influence on PTP induction.Potassium was the strongest PTP trigger among the cations tested. Thus, we examined the effect of potent and selective modulators of Kectively ,72); the≤ 100 nM ), psora 2 = 3 nM ), and oferiments . Since a2+ initially transforms the PTP into a K+/Na+-channel and stimulates the inward cation flow, which triggers full PTP opening. If this is true, CsA should decrease the efficiency of PTP triggering by cations. Therefore, we studied the effect of 1 µM CsA on the Ca2+-dependent swelling in K+/Na+-containing media and S-BM  and can prevent Ca2+ cycling between cytosolic and matrix sides of the IMM via the MCU and the Ca2+/H+ antiporter (TMBIM5), the data obtained are in favor of this mechanism but not the mechanism involving the non-selective cation channel.One more way for the electrophoretic entry of cations into the matrix of energized mitochondria to trigger the PTP opening is the hypothetical ruthenium red (RR)- and Mg channel . Though 2+- and Mg2+-ionophore A23187, which bypasses the MCU. In these conditions, the entry of monovalent cations should be suppressed, which, in turn, must eliminate the difference in the swelling rate between mitochondria in S-BM and salt-based media. As follows from 2+ into the matrix through bypassing the MCU suppresses the ability of RR to inhibit mitochondrial swelling. One can conclude that the hypothetic non-selective cation channel does not contribute to the PTP triggering by monovalent cations.In order to further explore the role of the non-selective cation channel in the PTP triggering, we applied a model of de-energized mitochondria supplemented with Ca50 ~ 25 µM) and Ro5-4864 (IC50 ~34 µM), on the Ca2+-dependent swelling in different media , namely propranolol /H+ exchanger  [2+/H+ antiporter (TMBIM5) [2+/Na+ exchanger (SLC8b1/NCLX) is important for mitochondrial Ca2+ homeostasis [+/H+ exchanger can regulate PTP opening in de-energized liver mitochondria [+ exchangers accelerates the PTP opening in energized mitochondria.A mechanism underlying the higher susceptibility of the mitochondria to PTP opening in cation-containing media may involve exchanger-mediated ionic transport. Mitochondria contain systems capable of transporting Ky Letm1) , choliney Letm1) , and Ca2(TMBIM5) . Moreoveeostasis . It was chondria . Therefo+/H+ exchanger is sensitive to Mg2+, quinine, and its enantiomer quinidine. Both quinine and quinidine at a concentration of 0.5 mM cause almost complete inhibition of the carrier [i~33–40 µM) [2+ present in the cytosol in near-millimolar concentrations is an important modulator of the PTP, we evaluated the effect of the inhibitors of the K+/H+ exchanger in intact energized mitochondria in different media supplemented with Mg2+. The rate of Ca2+-dependent (PTP) and -independent swelling was titrated by increasing the concentration of quinidine  ,88,89. Suinidine . +, Na+, Pi, glutamate, malate) to netto entry. In NaCl-BM, quinidine was a more potent swelling inducer than in other media. In ChCl-BM, the inhibitor at low concentrations was rather protective, indicating that Ch+ is transported to the matrix almost exclusively via the quinidine-sensitive Ch+/H+ exchanger [2+-dependent swelling (PTP) was relatively weakly modulated by quinidine in all cation-containing media , except ChCl-BM, quinidine dose-dependently stimulated PTP-independent swelling in the presence of EGTA A,C,E,G. xchanger ,89. Quinng media D,F,H. In+/H+ exchanger on Ca2+-dependent swelling was two-fold: an increase in the initial swelling but a decrease in the total swelling amplitude in long-term experiments due to the elimination of visible cooperativity (B). A reason for this effect will be revealed in the following sections. Nevertheless, the data obtained clearly demonstrate that cations do not trigger PTP opening via entry through the K+/H+ and Ch+/H+ exchangers. In S-BM, the effect of the suppression of the K2+/Na+ exchanger (SLC8b1/NCLX) CGP-37157 on PTP opening in different media. As the carrier is abundant in heart mitochondria but absent in liver mitochondria, the effect of its inhibitor was expectedly negligible  exchanger(s) when the Ca2+ influx via MCU is blocked  was added to the medium to assess the total amount of Ca2+ accumulated by mitochondria. 2+ extrusion from mitochondria. On the contrary, the minimum rate of Ca2+ release was observed in S-BM. In ChCl-BM it was almost the same as in S-BM. In other media, it was approximately 2.0 (KCl-BM), 1.6 (NaCl-BM), and 2.7 times higher (LiCl-BM) than in S-BM. Thus, the rate of Ca2+ release in various media is not the main factor that determines the sensitivity of mitochondria to PTP induction in the presence of salts.The inhibition of PTP opening by RR is most likely due to the Ca blocked . One couon media . The rel+/OH−-dependent symporters and antiporters [i and glutamate. Therefore, if exchanger-mediated cation entry into the matrix is essential for the triggering of the PTP, the latter will open more readily at an alkaline external pH. Conversely, if the PTP opening is activated by the entry of anions, the acidic external pH will stimulate the pore opening. The data presented in 2+-dependent PTP opening occurred most rapidly at pH 6.8 and most slowly at pH 7.6. Hence, conditions that hamper the cation entry through any H+-dependent antiporter but promote the acidification of the matrix via the antiporters, or Pi and glutamate carriers, strongly stimulate PTP opening [The pH of incubation media is an efficient regulator of the activity of all Hiporters ,90,91,922+-Pi complexes [i and substrates. We assumed that cations in the media change the matrix pH in a way of facilitating the PTP opening. Therefore, we measured matrix pH in mitochondria incubated in S-, KCl-, NaCl-, and ChCl-BM in the presence and absence of Ca2+, the inhibitor of the respiratory chain antimycin, and an uncoupler . The addition of Ca2+ to the media caused the alkalization of the matrix in S-BM and suppressed acidification in saline media. In the presence of CsA, the Ca2+ effect was less pronounced. In all media, the lowest pH of the matrix was observed in the presence of antimycin and the uncoupler. Approximately in the first 20 min of incubation in the absence of Ca2+-dependent swelling, which indicates its operation in the mode of Ca2+ injection in exchange for the matrix H+. In the presence of salts, A23187 inhibited PTP opening because a more alkaline matrix pH facilitates the extrusion of Ca2+ in exchange for external H+.The effect of the addition of A23187 to energized mitochondria confirms these observations . In S-BM+/H+ exchanger . Thus, monovalent cations regulate the PTP opening by modulating the activity of the K+/H+ exchanger which, in turn, regulates the matrix pH and hence the Pi transport into the matrix.The only reason for the difference in the matrix pH in S- and KCl-/NaCl-/LiCl-based media is the different mode of operation of the Kxchanger A. In ChC± 3.7 μM B,C. In Ndent one B. We sugswelling D, which + channels suppress PTP-dependent mitochondrial dysfunction and cell death [+ channels, or their knockout, stimulate both mitochondrial dysfunction and cell death [+ transport should affect PTP opening in isolated mitochondria in the same way. Therefore, the idea of this work was to examine how the presence of K+ in the incubation medium affects the susceptibility of mitochondria to PTP opening, to compare the effect with the effects of other monovalent cations, and elucidate mechanisms underlying the effects revealed. In in vivo systems, ischemia/reperfusion causes PTP-dependent cell death ,2. Activll death ,7,8,9,10ll death ,7,8,9,10+ in the incubation media would protect isolated mitochondria from the Ca2+-dependent PTP opening, while other cations, namely Na+, Li+, and Ch+ would not. Against our expectations, the opening of the large (600 < P < 3350 Da) pore (PTP) was dramatically accelerated, and Ca2+-retention capacity was reduced in all cation-containing media tested in comparison with the cation-free S-BM  on PTP opening in K+-, Na+- and sucrose-containing media demonstrated that it is weak and unspecific  occurred simultaneously /H+ [+/H+ exchanger CTL1/SLC44a1 [+/H+ and Ch+/H+ exchangers for the stimulation of PTP by cations. One more possible mechanism of stimulation of PTP opening by cations of the medium involves the activity of some cation exchangers. Indeed, in de-energized liver mitochondria, the activation of the Ke matrix . Therefo Li+)/H+  and Ch+//SLC44a1  in the s/SLC44a1 ,88,89 af2+/H+ exchanger can be a potent inhibitor of PTP opening, at least when MCU is blocked. Hence, the rate of Ca2+ extrusion should inversely correlate with the rate of PTP opening. We hypothesized that cations in the media suppress the Ca2+ pumping out and accelerate the pore opening. However, this assumption has not received experimental confirmation /H+ exchanger. It should be activated in S-BM and suppressed  in the presence of salts. In ChCl-BM, the exchanger presumably works in the same mode as in S-BM, but the acidification of the matrix is prevented by a competing process of alkalinization via the Ch+/H+ exchanger. The data obtained supports this assumption ), quinine (145904), pinacidil monohydrate (P154), diazoxide (D9035), cgp-37157 (220005), and superoxide dismutase from bovine erythrocytes (S7571) were purchased from the Sigma-Aldrich Corporation . Ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA)  was obtained from PanReac AppliChem ITW Reagents . Tetramethylrhodamine, methyl ester (TMRM), Calcein-AM, BCECF-AM -5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester, (B1170)), Fluo-5F-AM (F14222) were obtained from Thermofisher . Coomassie (Bradford) Protein Quantitative Assay Kit was obtained from Wuhan Servicebio Technology Co. . Other chemicals were of analytical grade and were purchased from local suppliers. Bovine serum albumin (BSA) (A2153), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (C2920), Cyclosporin A (30024), digitonin (D141), 4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid (HEPES) (H3375), rhodamine 123 (R8004), rotenone (R8875), sucrose (S7903), succinate (S3674), SF-6847/Tyrphostin A9 (T182), Trizma Base (93352), glybenclamide (G0639), psora-4 (P9872), quinidine (Q3625), iberiotoxin , national requirements for the care and use of laboratory animals, and protocol 9/2020 of 17 February 2020 approved by the Commission on Biological Safety and Bioethics at the ITEB RAS. Adult male Wistar rats were decapitated after anesthesia with COrocedure . The homrocedure .3PO4, 2 mM MgCl2, and, as stated, either 5 mM succinate plus 2.5 µM rotenone or 5 mM glutamate plus 5 mM malate. The osmolarity of the solutions was adjusted to isotonic values with either 120 mM KCl  or 120 mM NaCl , or 120 mM choline chloride , or 120 mM LiCl , or 260 mM sucrose .Measurements were performed at 37 °C in isotonic (~300 mOsm) media, which contained 20 mM sucrose, 10 mM HEPES (pH adjusted to 7.3 with Trizma Base), 2 mM H3PO4/MgCl2 medium with the addition of the indicated concentrations of salts and necessary amounts of sucrose to establish osmolarity. Media containing different concentrations of salts were prepared using a basic sucrose/HEPES/HIn several experiments, media of different pH were applied. For this, the pH value of stock media was adjusted to 7.6 with Trizma Base. Just before the beginning of the recording, 1.05 and 2.35 µL of 400 mM HCl were added to the wells of 96-well plates with 100 µL of incubation media in order to shift the pH to 7.3 and 6.8, respectively.2+. There are three experimental approaches to induce Ca2+ overload: a gradual infusion of Ca2+ solution to mitochondrial suspension [2+ in pulse(s) giving low final concentrations followed by prolonged incubation [2+ in pulse(s) giving high final concentrations followed by short incubation [2+ concentrations that far exceed those observed in any known physiological and pathophysiological states and can overcome any endogenous mitochondrial protective mechanism. Therefore, we applied a compromise experimental model: Ca2+ in concentrations close to the highest local concentrations near MAM-contacts (contacts of membranes of mitochondria and endoplasmic reticulum) [m, and the release of preliminarily accumulated Ca2+.The induction of PTP opening occurs due to mitochondrial overload with Caspension , the addcubation ,56, and cubation . The firticulum)  and a ph2+ concentrations [2+-dependent PTP opening was determined by two parameters: the half-maximum swelling time (HMST), which reflects the time when PTP opening occurs in half of the mitochondrial population [A540 in mitochondrial suspensions using a 96-well plate reader Infinite 200 Tecan . Other details are given in the figures and figure legends.Swelling is a dynamic process, which can be partly reversible in salt-based media in the presence of relatively low Catrations . The suspulation ; and them across the IMM was measured using the ΔΨm-sensitive fluorescent dye rhodamine 123 and a plate reader Infinite 200, Tecan . Incubation media contained respiratory substrates, 330 nM rhodamine 123, and, where indicated, 1 mM EGTA and 1 µM CsA. Other additions (Ca2+ and 500 nM FCCP) were made in the course of recordings. In order to calibrate the fluorescent signal, each experimental series contained samples with a cocktail of respiratory inhibitors and ionophores, which disrupt ionic gradients across the IMM , and valinomycin (25 ng/mL)) [m was calculated using the Nernst equation, assuming that the matrix volume is equal to 1 µL/mg protein (liver mitochondria) and that the fluorescence of rhodamine 123 is directly proportional to the concentration in solution and is totally quenched upon accumulation in the mitochondria.Additionally, ΔΨ ng/mL)) ,106. In 2+ uptake and release were recorded in a temperature-controlled electrode chamber using a Ca2+-electrode  connected to the computerized recording system Record 4 . The electrode was calibrated with known amounts of Ca2+ at the beginning of each experimental series. The CRC of the mitochondria was defined as the amount of Ca2+ taken up by the mitochondria in small pulses before the Ca2+ release. Other experimental details are given in figure legends.Mitochondrial CaV3) and state 4 respiration rates (V4), 200–500 μM ADP was added to mitochondria respiring in the presence of substrates. The respiratory control coefficients were calculated as the ratio of respiration rates in state3/state4. The P/O ratio was calculated as the amount of completely phosphorylated ADP per number of oxygen atoms consumed.The mitochondrial respiration was measured using an Oroboros Oxygraph-2k device . Mitochondria (1 mg/mL) were incubated at 25 °C in incubation media supplemented with 5 mM glutamate and 5 mM malate. In order to assess state 3  connected to the computerized recording system Record 4 . The electrode was calibrated with known amounts of KCl at the beginning of each experimental series. Other experimental details are given in figure legends.The release of mitochondrial Kg for 10 min and washed twice. The final pellet was suspended in 0.1 mL of the same medium without BCECF-AM and kept on ice until use. The fluorescence of BCECF trapped in the matrix was measured in a plate reader Infinite 200, Tecan . A BCECF signal was calibrated either using a range of buffers of known pH (6.8–7.6) and equilibrating the matrix and external pH using alamethicin (40 µg/mg prot.) or by the addition of known amounts of Trizma-Base and HCl solutions to mitochondria incubated with 300 µM Ca2+.The matrix pH of liver mitochondria was estimated as described previously , with sop) values were derived by the Student’s t-test.The data shown represent the means ± standard error of means (S.E.M.) or are the means of at least three experiments. Statistical probability (