Aims: The underlying mechanism of diabetic enteropathy, a common complication of type 1 diabetes, remains unclear. Store-operated Ca2+ entry (SOCE) is a ubiquitous type of Ca2+ influx involved in various cellular functions. Here, we show that SOCE-related stromal interaction molecule 1 (STIM1) and Orai1 participate in inappropriate cellular Ca2+ homeostasis, augmenting agonist-induced small intestinal smooth muscle contraction and small bowel transit speed in a mouse model of type 1 diabetes.Methods and Results: We used small interfering (si)RNA to suppress STIM1 and Orai1 proteins, and employed intracellular Ca2+, small intestinal contraction and intestinal transit speed measurement to investigate the functional change. We found that SOCE activity and Orai1 and STIM1 expression levels of small intestinal smooth muscle were significantly increased in cells cultured in high glucose medium or in diabetic mice. Gastrointestinal transit speed and SOCE-mediated contractions were markedly increased in diabetic mice; Knocking down Orai1 or STIM1 with siRNA rescued both alterations in diabetic mice. However, the Orai1-large conductance Ca2+-activated K+ (BKCa) channel interaction was decreased in diabetic mice, and suppressing Orai1 expression or inhibiting the BKCa channel increased agonist-induced small intestinal contractions in normal mice.Conclusion: We concluded that the increased SOCE caused by excessive STIM1 and Orai1 expression and decreased Orai1-BKCa interaction augmented small intestinal smooth muscle contraction and accelerated small bowel transit speed in diabetic mice. This finding demonstrates a pathological role for SOCE in diabetic enteropathy and provides a potential therapeutic target for diabetic enteropathy. Diabeti2+ plays a critical role in numerous physiological functions. Store-operated Ca2+ entry (SOCE) is a ubiquitous mechanism of Ca2+ influx in many cell types (2+ sensor located in the lumen of the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR). Upon Ca2+ depletion of the ER/SR, STIM1 aggregates and translocates to ER-plasma membrane junction regions and then activates Orai1, which is an ion channel formed by four transmembrane spanning protein subunits (3) and diacylglycerol (DAG) production, IP3 acts on IP3 receptors, causing Ca2+ release from the ER/SR and depletion of internal Ca2+ stores. Growing evidence indicates an association of altered SOCE with various diabetic complications. Enhanced SOCE in glomerular mesangial cells  to investigate changes of SOCE in a high glucose environment Ca channel (APC-107) primary antibody was purchased from Alomone Lab. Specific small interfering (si)RNA for mouse STIM1 (5′-UACAGUGGCUCAUUACGUA-3′) and Orai1 (5′- GCCAUAAGACGGACCGGCA-3′) (Thapsigargin (TG), carbachol (CCh), and adenosine triphosphate (ATP) were obtained from Calbiochem. Iberiotoxin (IbTX) and streptozotocin (STZ) were purchased from Sigma-Aldrich. Anti-STIM1 (sc-68897) and anti-Orai1 (sc-68895) primary antibodies were purchased from Santa Cruz Biotechnology. The anti-BKGGCA-3′)  and scraMUS-M1, a mouse SISM cell line, was purchased from the Kunming Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. Only sub-passage 3–10 MUS-M1 cells were used in the present study. For the group with normal glucose (NG) levels, the culture medium contained 5.6 mM glucose and 20 mM α-mannitol as an osmotic control. In the group exposed to high glucose (HG), the medium contained 25 mM glucose . The med2+ concentration ([Ca2+]i) was measured as described previously (5 per well on glass coverslips in 12-well culture plates as described above. Cells were loaded with a fluorescent Ca2+ ion indicator Fluo-8/AM (6 μmol/L) and 0.02% pluronic F-127 at 37°C for 30 min. The Ca2+ stores in MUS-M1 cells were depleted using 2 μmol/L TG or 100 μmol/L CCh for 10 min in Ca2+-free saline solution, which contained (in mmol/L) 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 0.2 EGTA, and 5 HEPES at pH 7.4. The Ca2+ influx was initiated by applying 2 mmol/L extracellular Ca2+. The fluorescence signal was recorded using a Nikon fluorescence microscope with 488-nm excitation and 515-nm long pass emission wavelengths. Changes in the peak value of cytosolic [Ca2+]i were displayed as the ratio of fluorescence intensities relative to the baseline intensity before the application of extracellular Ca2+ (F1/F0). Each data point is an average of 20–30 cells.The cytosolic Caeviously . BrieflyWestern blotting and co-immunoprecipitation assays were performed as described in our previous study . ProteinAll animal experiments were performed according to the guidelines presented in NIH publication no. 8523 and approved by the Experimentation Ethics Committee of Anhui Medical University. Five-week-old Kunming mice  were provided by the Experimental Animal Center of Anhui Medical University and randomly divided into two groups: STZ and age-matched controls. Both groups were fed regular chow and given free access to tap water. STZ (6 mg/mL) was dissolved in 50 mM sodium citrate buffer (pH 4.4) before the injection. As previously described, mice in the STZ group were injected with STZ (40 mg/kg) intraperitoneally for five consecutive days to induce type 1 diabetes . Mice inin vivo via i.p. injections. With our approach, 80% of the animals injected with siRNA met this criterion and were analyzed as described.Diabetic mice were randomly separated into three groups. Each mouse received two injections every 3 days of Orai1 siRNA, STIM1 siRNA, or scrambled control siRNA. Based on previous studies , all siRad libitum), a charcoal meal marker  was freshly prepared by dispersion. The mixture was administered orally to assess upper gastrointestinal transit speed. After 20 min, the mice were euthanized using an overdose of CO2 gas, and the intestines were immediately isolated. The distance traveled by the marker was measured (in centimeters). The results of the intestinal transit speed experiment are shown as a percentage of the total length of the mouse small intestine, from the pylorus to the terminal ileum.The small intestinal transit speed experiment was performed as described elsewhere . Briefly2 gas. The small intestine was quickly dissected and placed in Krebs-Henseleit solution containing (in mmol/L) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.2 NaHCO3, and 11.1 glucose, pH 7.4 at room temperature. The peripheral adipose tissue was removed. The small intestine was cut into segments 2 cm in length. One end of the tissue was fastened to a hook on the bottom of a glass organ bath. The other end was connected to an isometric force transducer, which was connected to an amplifier. The bubbling O2 should mix the solution. The tension signal was recorded and analyzed by a data acquisition and analysis system . The segments were placed in 5 mL organ baths containing Krebs solution at 37°C and continuously bubbled with a gas mixture of 95% O2 and 5% CO2 to maintain pH at 7.4. After equilibrating for 60 min with 1 g passive tension, the small intestine segments were activated by two applications of high-K+ solution, containing 60 mmol/L K+, prepared by replacing NaCl with an equimolar amount of KCl. Next, the contractile response to CCh (100 μmol/L) or Ca2+ (2 mmol/L) was determined. Detailed group treatments are described in the section “Results.” For the IbTX group, IbTX (50 nmol/L) was added to Krebs-Henseleit solution for 20 min pretreatment.Small intestine segment preparation and tension measurements were performed as described elsewhere . Brieflyt-test was used to analyze the difference between two groups with SigmaPlot 12.5 software. A value of P < 0.05 was considered statistically significant.Data are presented as means ± SEM. Student’s unpaired 2+ entry is a crucial Ca2+ signal that initiates many cellular biological processes (in vitro show enhanced SOCE (2+ imaging and measured the change in [Ca2+]i evoked by SOCE in NG- and HG-cultured MUS-M1 cells. The Ca2+ store in MUS-M1 cells was depleted by 2 μM TG (a Ca2+ pump inhibitor in the ER) and 100 μM CCh (a muscarinic receptor activator). SOCE was initiated by the re-addition of 2 mM Ca2+ using a classic “Ca2+ added back” protocol. MUS-M1 cells were cultured in NG and HG media for 1, 3, and 7 days. The SOCE evoked by TG and CCh was significantly increased in HG-cultured cells at 3 and 7 days, but not at 1 day, compared with that in NG-cultured cells at 1, 3, and 7 days  MUS-M1 cells . Re-adding 2 mM Ca2+ induced longitudinal contractions of the small intestine segment. Because VDCCs were inhibited by verapamil, any contraction induced by re-adding Ca2+ would be due mainly to the SOCE-mediated Ca2+ influx. These results indicated that SOCE-mediated small intestine segment contractions were significantly increased in diabetic mice compared with those in control mice , western blotting analysis showed that Orai1 and STIM1 expression levels were significantly reduced in the SISM of diabetic mice . Our con2+ store .Small intestinal longitudinal contractions are correlated with gastrointestinal transit speed . Therefo2+-activated K+ channels  to form signal complexes regulating smooth muscle contraction  and Orai1 siRNA transfection  suppressing Orai1 expression or inhibiting BKCa channel activity increased agonist-induced small intestine contractions. Thus, we demonstrated that SOCE activity and SOCE-mediated contraction were significantly increased in SISM cells cultured in HG medium or in mice with type 1 diabetes and that altered SOCE activity and Orai1-BKCa channel interactions might contribute to accelerated gastrointestinal transit speed.Here, we examined SOCE in SISM cells cultured in HG medium or in a mouse model of type 1 diabetes. Our major findings are as follows: (1) SOCE activity and Orai1 and STIM1 expression levels in SISM cells were significantly increased in HG culture conditions or in diabetic mice compared with those in NG culture conditions or age-matched control mice, respectively; (2) gastrointestinal transit speed and SOCE-mediated contractions were markedly increased in diabetic mice; (3) siRNA knockdown of Orai1 or STIM1 rescued the augmented gastrointestinal transit speed and SOCE-mediated contractions in diabetic mice; (4) Orai1 pulled down the BK2+ homeostasis of muscle cells disrupts contractile function and gastrointestinal motility, and SOCE disorders may lead to many dysfunctions, ranging from immunodeficiency to myopathy and vascular diseases  pacemaker activity. Besides, colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions (2+ stores by SOCE may also be important for mediation and shaping of Ca2+ signals in ICC-SM. Therefore, increased SOCE activity and Ca2+ mobilization in SISM, ICC cells and/or even nervous system may all contribute to the accelerated gastrointestinal transit speed and diabetes-associated diarrhea because in body Orai1 and STIM1 siRNAs may affect many cell types involving gastrointestinal transit speed. However, which cell is more important and why the gastrointestinal transit speed was shown to be faster in some studies but delayed in others in animal or patients with type 1 diabetes is unknown. Perhaps factors such as the stage of type 1 diabetes, meals, degree of blood glucose, etc., may differentially affect gastrointestinal transit speed. This issue should be addressed in future studies.Numerous previous studies have shown that the alteration of gastrointestinal tract motility in type 1 diabetes is controversial. Several studies have reported that gastrointestinal transit speed is faster in patients with type 1 diabetes than in controls , but othractions . Mainten2+ entry regulates various cellular functions, including cell proliferation, contraction, and secretion (+-induced small intestine contractions between diabetic and age-matched control mice and found no significant difference between them. Our HE staining results also showed that the SISM layer thickness was not altered in diabetic mice. Therefore, our results exclude the effect of proliferation or hypertrophy of SISM cells on enhanced small intestine transit speed.Store-operated Caecretion . In typeecretion . In addiecretion . To excl2+ homeostasis regulation and Ca2+-mediated signal transduction are complicated. Although we demonstrated that the SOCE-mediated Ca2+ signal was increased in the SISM cells of mice with type 1 diabetes, another group found that Ca2+-activated Cl– channels also participate in glucose-induced [Ca2+]i increase in small intestine cells (2+ homeostasis. Therefore, our finding may be only one of the pathways involved in type 1 diabetes pathology.In live cells, Cane cells . In diabWe demonstrated that SOCE activity was significantly increased in SISM cells cultured in HG medium or in mice with type 1 diabetes. Upregulated SOCE promoted small intestinal muscle contractions and gastrointestinal transit speed, which may be among the factors inducing diabetic diarrhea. Therefore, the SOCE pathway likely represents a potential therapeutic target for diabetic diarrhea.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Ethics Committee of Anhui Medical University.FD and JG performed the experiments, analyzed the data, and co-wrote the manuscript. YW, TJ, HC, and YH conducted the experiments and analyzed the data. XX and JD designed a part of the study and helped to interpret the part of the data. BS and QZ conceived the idea, designed and supervised the study, analyzed the data, and revised the manuscript. All authors reviewed the report and approved the final version.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.


Aβ can disrupt Ca2+ signaling through several mechanisms, by increasing the influx of Ca2+ from the extracellular space and by activating its release from intracellular stores. Here, we review the different molecular mechanisms and receptors involved in calcium dysregulation in AD and possible therapeutic strategies for improving the treatment.Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder that is characterized by amyloid β-protein deposition in senile plaques, neurofibrillary tangles consisting of abnormally phosphorylated tau protein, and neuronal loss leading to cognitive decline and dementia. Despite extensive research, the exact mechanisms underlying AD remain unknown and effective treatment is not available. Many hypotheses have been proposed to explain AD pathophysiology; however, there is general consensus that the abnormal aggregation of the amyloid β peptide (Aβ) is the initial event triggering a pathogenic cascade of degenerating events in cholinergic neurons. The dysregulation of calcium homeostasis has been studied considerably to clarify the mechanisms of neurodegeneration induced by Aβ. Intracellular calcium acts as a second messenger and plays a key role in the regulation of neuronal functions, such as neural growth and differentiation, action potential, and synaptic plasticity. The calcium hypothesis of AD posits that activation of the amyloidogenic pathway affects neuronal Ca Alzheimer’s disease (AD) is the most common cause of age-related neurodegenerative disease, which is characterized by progressive memory loss, cognitive dysfunction, language disorders, and personality changes . While tAlthough extracellular Aβ plaques and NFTs in the brain are hallmarks of AD, the multiple mechanisms related to the disease are still unclear. The amyloid cascade was considered for a long time as a dominant model for AD pathogenesis . AccordiThere are two major forms of AD: the sporadic or late-onset form (SAD), the most common one, and the familial or early-onset form (FAD), representing less than 5% of the cases . AlthougThe Aβ peptide is generated from the endosomal proteolytic cleavage of the APP by γ-secretase and β-site APP cleaving enzyme 1 (BACE1), which catalyzes the rate-limiting step . During The tau protein is an axonal microtubule-associated protein that is distributed in the axons of neurons and is one of the main components of the cytoskeleton . Tau is A dominant theory for the incidence of AD is the “amyloid hypothesis”, which describes a complex sequence of pathogenic events responsible for neurodegeneration ,58,59,60According to the “tau hypothesis”, the intracellular NFTs, mainly composed of abnormally phosphorylated and aggregated tau proteins, are considered as the principal effectors of neuronal loss and memory impairment in AD via the impairment of axonal transport. In contrast to the view that there is no particular interaction between Aβ and tau, recent experimental and clinical evidence supports a strong Aβ–tau synergy . Indeed,+) entry into neurons is essential for the propagation of action potentials, whereas Ca2+ signaling is involved in neurotransmitter release, synaptic plasticity, gene expression and other important neuronal functions  is slow and small for mitochondria that are distant from the microdomains. The alteration of MAMs has been linked to pathological conditions, such as cancer, neurodegenerative diseases, and metabolic syndromes . I. I3 and ar space . Furthero the ER . Restinge (PMCA)  and the er (NCX) . Mitochoeostasis . Indeed,x (MCUC) ,124, ther (NCLX) , the Ca2tiporter , the voltiporter . Mitocho2+ rises . The sit2+ rises . MAM is 2+ rises . It is ification . The incyndromes .2+ signaling pathways responsible for cognition. According to this hypothesis, the depolarization of aged neurons causes the influx of Ca2+ from the extracellular space and excitotoxicity. Other studies instead demonstrated that neuronal aging is associated with the alteration of neuronal Ca2+ extrusion, leading to old neurons being more vulnerable [2+ dyshomeostasis has been reported in both peripheral and central neurons during the aging process [The so-called “Calcium hypothesis” was first postulated by Khachaturian in 1989  followinlnerable ,137,138. process ,138 as w process ,140, inf process ,142  in the hippocampal cell membranes of AD models, causing the influx of Ca2+ [2+ via the RyR caused the increase in cytoplasmic Ca2+. This overloading caused the activation of β-secretase and thus an increased Aβ production and aggregation [Although there is a variety of experiential evidence, the amyloid channel hypothesis still remains controversial. Indeed, numerous mechanisms can be responsible for the Aβ interaction with neuronal membranes causing the disruption of Caeability ,158. Sevof VGCCs , the downe level ,138 and ganelles ,138. The of Ca2+ . In addi of Ca2+ . The inhregation .2+ influx from the extracellular space across these Aβ pore-channels [2+ channels in neuronal cells. In particular, blocking Ca2+ influx was found to reduce the neurotoxicity of Aβ oligomers and the levels of insoluble Aβ1–40 and Aβ1–42 in the hippocampus of AD transgenic mice [2+ promotes the activation of CaN and that of the phosphatases, including PP1, which is involved in long-term depression (LTD) [2+/CaM complex activates the CaMKII, playing an important role in memory formation and synaptic plasticity. Taking into account that many kinases can be activated by Ca2+, the dyshomeostasis of Ca2+ can increase tau phosphorylation [2+ overload, causing the dephosphorylation of CaMKIV and CREB by activating CaN [2+ can also activate JAK2-STAT1 signaling, leading to the binding of STAT1 to NMDARs, and thus inhibits the transcription of specific GAS elements [2+, as well as in the ER and mitochondrial Ca2+ signaling. Indeed, mutations in these proteins affect Ca2+ homeostasis [In the past few years, in vitro studies demonstrated that the Aβ peptide formed cation-selective pores into the plasma membrane, thus causing Cachannels ,163,164.channels . Followinic mice . The incon (LTD) . CaN canon (LTD) ,169. Therylation ,171,172.ting CaN . The incelements ; the incelements . All of eostasis .2+ entry in postsynaptic neurons disrupts the intracellular homeostasis and induces an increase in ROS production. Aβ has been reported to bind to NMDA and AMPA glutamate receptors [2+-permeable. Furthermore, Aβ can influence VGCCs and IP3R [2+ increase [2+ transport and is known to play a decisive role in Ca2+-induced apoptosis, was overexpressed in AD models compared to controls [An altered expression of calcium-binding (CBP) and calcium sensing proteins can modify the calcium-buffering capacity, causing the oversensitivity of neurons to glutamate released in the extracellular space ,176,177.eceptors , as welleceptors , and alland IP3R . Numerouand IP3R . In partand IP3R , or induand IP3R  and in rand IP3R . The effand IP3R . Indeed,increase ,185,186 increase ,188 and/increase , or by cincrease . Importaincrease  has beenincrease ,193, to increase . The synincrease . Hippocaincrease . In partcontrols . The percontrols . We havecontrols . In partcontrols . Memanticontrols ,198. How2+ from the ER, thus promoting the activation of the unfolded protein response (UPR) and ER stress [2+ homeostasis indirectly by the increase in Ca2+ influx from the extracellular space, which in turn triggers Ca2+ release from intracellular stores [2+, such as RyR and I3PR [2+ dyshomeostasis causes abnormal Aβ production and neuronal apoptosis [3R was found to modulate Ca2+ homeostasis in AD [As reported above, Aβ peptides are able to induce a high release of CaR stress ,200. Notr stores , or direand I3PR ,206,207.poptosis ,209,210.is in AD  and its is in AD ,212.2+ signaling [2+ to prevent Ca2+ overload into the cytosol, activating the Ca2+-dependent mitochondrial matrix dehydrogenase to produce ATP. However, this disruption of mitochondrial Ca2+ regulation affects energy production and oxidative stress, resulting in mitochondrial dysfunction [2+ influx into the mitochondria induces mitochondrial outer membrane permeabilization and the subsequent release of pro-apoptotic factors into the cytoplasm, including cytochrome C and apoptosis-inducing factor, which activate apoptosis cell death [2+ homeostasis. In addition, synaptic mitochondria are more sensitive to Ca2+ dyshomeostasis with respect to non-synaptic mitochondria [2+-dependent CaN and suppressing nuclear CaMKIV/CREB signaling, thus revealing a new mechanism by which tau can induce synaptic toxicity [2+ uptake [2+ transfer from the ER to mitochondria [2+ levels in neuronal mitochondria of transgenic mice appeared only after plaque deposition and before neural death [2+ overload involves toxic extracellular Aβ oligomers and requires the mitochondrial Ca2+ uniporter. The authors propose a novel potential therapeutic target for AD through the blocking of the mitochondrial Ca2+ uniporter that was found to reduce mitochondrial Ca2+ overload [Mitochondria play a key role in the modulation of intracellular Caignaling . Indeed,function . In partll death  (Figure ll death . Studiesll death , morpholll death , the imbll death  and the ll death . Synapsechondria . During chondria . CaMKII chondria . The acctoxicity . In vitr+ uptake ,225 and chondria  in cultual death . This mioverload .2+ levels in the ER caused the leakage of Ca2+ into the cytoplasmic compartment, affecting the mitochondrial calcium homeostasis [Various studies have observed that the increase in Caeostasis ,228. Receostasis . As alreeostasis . Thus, s2+ homeostasis is an early event in the cascade of neuronal alterations underlying the cytotoxicity induced by misfolded Aβ aggregates and hyperphosphorylated tau. However, so far, no common consensus has been reached on the molecular mechanisms of neuronal Ca2+ overload, causing the remodeling of signaling pathways with excitotoxicity and memory dysfunction in AD. The effects of Aβ on NMDARs have attracted considerable interest as these ligand-gated channels are involved in synaptic plasticity and LTP. Importantly, the over-activation of GluN2B NMDAR subunits induced by Aβ has been correlated to ER stress and to the depolarization and dysfunction of mitochondria. Recently, ER and mitochondrial Ca2+ dyshomeostasis have also been proposed as early causative events in AD.Many studies have revealed that the perturbation of Ca2+ alterations caused by Aβ extracellular deposits could be non-specific, involving just lipid membrane components, or specific by membrane receptors and other cell surface proteins. There is strong evidence of a key role for PrPc, associated with lipid rafts, as a receptor for Aβ oligomers, resulting in the activation of a Fyn-mediated complex signaling cascade, leading to tau phosphorylation and loss of Ca2+ homeostasis. However, the data reported in several other studies support the idea that Aβ oligomers can interact with membranes through direct binding to GM1. This then results in the disruption of lipid bilayers, the alteration of their permeability and the misfunction of raft-associated Ca2+ channels, leading to Ca2+ influx into cells. These findings do not necessarily contradict the view that PrPc behaves as a receptor of a class of Aβ oligomers. Considering the existence of many structurally distinct conformers, different Aβ aggregates could interact with PrPc and GM1 with different affinities. It is increasingly evident that toxicity is not a feature that is inherent to a given type of misfolded protein oligomer, but is instead a property that emerges from the complex interplay between the structural features of oligomers and the lipid composition of the neuronal membranes. Trodusquemine, a natural product in the aminosterol class, was recently shown to prevent Aβ toxicity by displacyng the aggregates from the cell membranes, suggesting that molecules that interact directly with cell membranes, rather than binding oligomeric aggregate themselves, could represent a useful approach in the treatment of AD.The main unsolved issue is whether the neuronal Ca2+ perturbation in AD pathophysiology needs further investigations for the development of targeted therapies for AD. Memantine, an NMDAR antagonist approved in 2002 as a therapeutic drug in moderate to severe AD, appears to be promising in AD therapy, together with other potential drugs targeting NMDARs and PrPc. However, according to the latest evidence that both Aβ and tau pathologies have synergistic effects, the most efficacious approach to slow AD may be to combine anti-Aβ and anti-tau therapies.Overall, the question of the molecular basis of Ca


Its inhibition has formed the basis for control of human disease but remains largely unexplored for plant disease. This study investigated the role of Ca2+ signaling on motility and chemotaxis of Spongospora subterranea zoospores, responsible for root infections leading to potato root and tuber disease. Cytosolic Ca2+ flux inhibition with Ca2+ antagonists were found to alter zoospore swimming patterns and constrain zoospore chemotaxis, root attachment and zoosporangia infection. LaCl3 and GdCl3, both Ca2+ channel blockers, at concentrations ≥ 50 μM showed complete inhibition of zoospore chemotaxis, root attachment and zoosporangia root infection. The Ca2+ chelator EGTA, showed efficient chemotaxis inhibition but had relatively less effect on root attachment. Conversely the calmodulin antagonist trifluoperazine had lesser effect on zoospore chemotaxis but showed strong inhibition of zoospore root attachment. Amiloride hydrochloride had a significant inhibitory effect on chemotaxis, root attachment, and zoosporangia root infection with dose rates ≥ 150 μM. As expected, zoospore attachment was directly associated with root infection and zoosporangia development. These results highlight the fundamental role of Ca2+ signaling in zoospore chemotaxis and disease establishment. Their efficient interruption may provide durable and practical control of Phytomyxea soilborne diseases in the field.Ca Plasmodiophora brassicae and Spongospora subterranea which causes clubroot in crucifers  the only molecule known to serve as a universal messenger in all eukaryotes  concentrations at 20,000-fold less compared to the extracellular matrix by pumping Ca2+ into endoplasmic reticulum or out of the cell  in April 2018. Tubers were washed under running water to remove adhering soil and then air dried overnight at room temperature. The contents of individual lesions were scraped from the tuber surface with a scalpel blade minimizing removal of potato tissues, and the removed material oven-dried at 35°C for 7 days. Dried inocula was stored at room temperature for 2 months before use. To obtain zoospores, multiple 15 ml centrifuge tubes containing 100 mg of dried inoculum in 5 ml of deionized water were incubated for 3 months at room temperature. Any tubes found with excessive bacterial contamination were discarded. Zoospore identity was confirmed by microscopic examination of morphological features  and typical helical swimming patterns of the zoospores in water  chloride (LaCl3) and gadolinium (III) chloride (GdCl3), ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), trifluoperazine (TFP), and amiloride hydrochloride. Various concentrations of these compounds were tested for their effects on zoospore flagella movement. One microliter zoospore suspension was added to 5 μl of each test compound at 50–250 μM in a taxis chamber, mixed well and allowed 10 min to acclimatize before observing for zoospore flagella movement at 400 × using light microscope . This was repeated for TFP at 5–25 μM, and EGTA at 1,000–2,000 μM. Each trial was replicated five times. Chemical doses that caused a cessation of flagella movement were classified as lethal and excluded from subsequent trials.A range of compounds which interfere with Ca3 and GdCl3, both Ca2+ channel blockers  was aliquoted, to which 50 μL of each Ca2+ antagonist at each tested concentration, or water only (control) was added. Tubes were gently vortexed and allowed to stand for 10 min. Glutamine (0.01 M), a known chemotaxis attractant for S. subterranea zoospores (2+ antagonists and incubated for 24 h in the dark at room temperature (20 ± 2°C). Each treatment was replicated six times. Following incubation, the tubes were removed from the zoospore suspension, the lower 10 μL of the solution within the microcapillary ejected onto a microscope slide and the total number of zoospores that had migrated to and entered each microcapillary tube was manually counted using light microscopy at 400 × magnification  scanning the entire sample.The Cary assay  by integoospores , was intoospores . Additio2+ imaging and Microelectrode Ion Flux Measurement (MIFE) were also explored for the determination of the direct role of Ca2+ in S. subterranea zoospore motility and chemotaxis  added to the outer margins of the antagonist solution to prevent direct contact of the zoospore suspension with the root pieces and a cover slip carefully added. To prevent the drying, the prepared slide was placed on moistened Whatman filter paper  within a Petri dish, to create high humidity in the chamber. Each Petri dish constituted a replicate with each treatment replicated six times. The set up was incubated for 12 h in a dark cabinet at room temperature (20 ± 2°C). Following incubation, the root pieces were carefully removed with forceps, gently rinsed in sterile deionized water to remove unattached zoospores, mounted on a microscope slide, and observed by light microscopy at 400 × magnification . The number of zoospores attached to the root and root hairs were counted.Root tissues were excised from 6-week-old axenic tissue-cultured plantlets of potato cv. Iwa, a variety highly susceptible to 2+ antagonist at each concentration were prepared using Hoagland’s nutrient solution, important to support plant hydroponic growth and a known stimulant of S. subterranea zoospore germination as a diluent (c. 8 zoospores/μl) ensuring a ready and continual supply of zoospores over the course of the experiment. Each bottle was covered with aluminum foil to exclude excessive light and a the roots of a 6-week-old potato tissue-culture plantlet (cv. Iwa) was inserted into each bottle. The bottles were incubated in a plant growth chamber  set at 18°C, 90% relative humidity, 10,800 Lux light intensity, with a 14/10 h photoperiod for 8 weeks. The solutions were topped-up with 4 ml of Hoagland’s nutrient solution at 4 weeks.Solutions of each Ca diluent . Ten ml S. subterranea zoosporangia within root epidermal cells and root hairs using a 0–5 rating scale  to a microcentrifuge tube to which 3 μl of zoospore suspension (c. 11 zoospores/μl) was added, the solutions then vortexed for 5 s and allowed to rest for a further 10 min. After this period, 3 μl of each treated zoospore suspension was pipetted into a taxis chamber . The chamber was covered with a cover slip and observed under light microscope  at 400 × magnification. An individual zoospore was brought into focus and native videos of the swimming behavior of the zoospores were recorded at 59.94 fps via video-microscopy using a Nikon D850 camera  fixed in position to the trinocular port of the microscope set in phase contrast mode as described by Deionized water (control), CaThe native video files were standardized to 30 s whilst the quality was digitally optimized by cropping and sharpening images with Adobe Photoshop CC 2019  to enhance image contrast to enable the tracking software to accurately follow zoospore movements. The recorded swimming behavior of the zoospores were tracked using a computer-vision tracking software, idTracker, version 2.1 . The zoospores’ spatial positions, XY, were captured frame by frame and aligned with their temporal positions, Z, indicated by the frame number, for a 3D pattern analysis. A 3D trajectory plot from the data was reconstructed using the data analysis software Origin (Pro), Version 2018 .viz instantaneous speed , instantaneous acceleration , motility rate, exploration rate [(Number of Areas)/(Number of Explored Areas (from Exploration)], and total distance traveled . 3D and 2D trajectory for each video were examined and compared among treatments to determine the impact on zoospore swimming pattern.2D analysis of the same videos were done using the computer-vision tracking application, ToxTrac, version 2.84  with autP-value showed difference at 5% significance level, mean separation using LSD was done for all experiments except the root infection experiment where differences in infection intensity between treatments were tested using ordinal linear regression. This was done using R statistical language framework v 4.0  or R statistical language framework v 4.0 . Where trk v 4.0 . A post s v1.2.3  and Tuke2+ antagonists treatments affected zoospore flagella movement in a dose dependent manner. The concentration that caused a cessation of active flagella movement varied among the compounds. In TFP zoospore flagella remained motile at 5 μM, but this movement ceased at concentrations ≥ 10 μM. Similarly, in amiloride hydrochloride zoospore flagella remained motile at 200 μM but ceased movement in concentrations ≥ 250 μM. In both LaCl3 and GaCl3 flagella motility was sustained at ≤ 150 μM but not at concentrations ≥ 200 μM. In EGTA flagella motility ceased only when concentration was increased to ≥ 1,200 μM.CaP ≤ 0.05) diminished by all the Ca2+ antagonists at most of the tested concentrations when compared to the control  and 200 μM (80.7%) concentrations but not at 100 μM (2+ chelator EGTA significantly reduced chemotaxis at 500 μM (78.0%) and 1 mM (94.9%) concentrations but not at 100 μM. Migration of zoospores into the microcapillary tube was significantly inhibited by all concentrations of the Ca2+ channel blockers LaCl3 and GdCl3, all showing complete inhibition except 50 μM LaCl3  by all Ca2+ antagonist treatments at all concentrations when compared to the control  and LaCl3  treatments zoospore motility was not observed.The mean number of zoospores found attached to root segments  was sign control . Amiloriof GdCl3 . It was 2+ antagonists significantly (P ≤ 0.05) diminished the prevalence  and thus data was combined for each root. All Caevalence , 3 of zoevalence . TFP rednd GdCl3 .2+ antagonists significantly affected the motility of S. subterranea zoospores in most measured parameters. Examples of native video files are provided in 3 (2.1 μm/s) were significantly slower than the control. Zoospore motility completely ceased when treated with LaCl3 at 100 and 150 μM and GdCl3 at 50, 100, and 150 μM.All Ca2) which showed a significant increase in IA compared to the control  of zoospores was not significantly affected by any treatment except trifluoperazine at 2 μM  in this study were found to have antagonistic effects on zoospore chemotactic attraction to glutamine, root attachment and root infection in a dose dependent manner.Many Carganisms . These arganisms  and neutrganisms . However3 or GdCl3 treated zoospores at 50–150 μM concentration in this study is presumed to be due to their Ca2+ channel blocking activity. Other studies also found lanthanides such as La3+ and Gd3+ significantly constrain chemotaxis (2+ channels in the plasma membrane and membranes of intracellular Ca2+ stores, the lanthanides limit the availability of free cytosolic Ca2+, the movement of which is required to phosphorylate ATP and drive motility and chemotaxis (2+ and translate cytosolic Ca2+ transients associated with environmental stimuli also play crucial role in chemotactic response (Dictyostelium chemotaxis toward cAMP and folic acid.The cessation of chemotaxis of LaClemotaxis . It was emotaxis . Conversresponse . Inhibitresponse ] functio2+ to impact on cytosol free Ca2+. In Dictyostelium discoideum, 2+ concentration, however, 10 mM was enough to reduce the intracellular concentration significantly. 2+ medium, plasma membrane Ca2+ ATPases pump Ca2+ out of the cell, to deplete the intracellular stores. It is this Ca2+ depletion that results in diminished chemotaxis. Inhibition of chemotaxis by amiloride hydrochloride such as we found with S. subterranea zoospores has been previously observed in the chemotactic response of neutrophils to N- formyl- methionyl- leucylphenylalanin (2+ fluxes by blocking Na+-Ca2+ exchange (2+.The addition of EGTA generally reduced chemotaxis except at 100 μM which was not significantly different to the controls. It is conceivable that at 100 μM, EGTA was unable to chelate sufficient extracellular Caylalanin . Amiloriexchange  to limit2+ signaling in zoospore attachment, although recent evidence points to the regulatory role of Ca2+ in cell adhesion of pathogenic bacteria (3 (2+ signaling on adhesion. At the organism level, adhesion and encystment of the unicellular protist Phytophthora zoospore is reported to be mediated by the secretion of proteins (2+ signaling and adhesive protein secretion remains unknown. However, based on the dose dependent response of Ca2+ antagonist treatment on S. subterranea zoospore root attachment, it is conceivable to presume a Ca2+ signal interference in the adhesive secretion cascade. This may have led to interference with chemotaxis and root attachment. The prevalence of zoosporangia root infection is directly associated with the level of zoospore attachment, and thus the similarity of response of antagonists to these pathogen-host interactions is expected. Similar observation was made by P. capsici zoospores to wounded pepper roots.Little is known of the physiological role of Cabacteria . The inhteria (3  providedproteins . The rel2+ concentration with a Ca2+ antagonist led to impairment of swimming behavior and chemotaxis, we can assume that for S. subterranea zoospores, swimming behavior is critical to chemotaxis and the regulation of Ca2+ concentration is central to it. However, the optimum Ca2+ concentration for swimming and chemotaxis is yet to be determined.Given delimiting cytosolic Ca2+ antagonist. These changes in qualitative swimming behavior mirrored the changes in quantifiable behaviors. Ca2+ flux inhibitor or amiloride treatments changed the S. subterranea zoospore swimming pattern into more oscillatory movement which alternated frequently with helical and zig-zag patterns. In contrast, with Phytophthora aphanidermatum, 2+ chelator EGTA, did not alter the helical swimming patterns observed in the control, rather it reduced the density of the loops and thus diminished the distance traveled.Directly linked to basic quantitative swimming behavior, is swimming trajectory. We found the normal extended helical movement punctuated with changes in direction  was affeP. aphanidermatum (Achlya spp., 5 μM trifluoperazine was sufficient to cause instant cessation of motility (2+ imposed by the various antagonist treatments. Since Ca2+ is the most important intracellular regulator for modulating flagella movement (2+ antagonist classes is beyond the scope of this study, the inherent differences in mechanism with which they achieve Ca2+ antagonism could be responsible for the differences in qualitative and quantitative swimming behaviors.With the calmodulin antagonist, trifluoperazine, zoospore movement pattern appeared more convoluted, combining multiple swimming patterns perhaps indicating a state of confusion which culminated in frequent changes of direction at 2 μM. As the concentration increased the level of agitation reduced along with slowing down in speed. The characteristic alternation of acceleration from high to low and back to high, gave a distinct hourglass swimming pattern at 5 μM. Trifluoperazine also induced slow and spiral movements in dermatum , but in motility . Variousmotility . The obsmovement  a limitamovement . Though P. aphanidermatum where perturbation of the typical helical pattern into circular or straight-line movement with Ca2+ antagonists was associated with cessation of chemotaxis (Overall, the further swimming pattern deviated from the helical trajectory observed in the controls, the greater the inhibition of chemotaxis, root attachment and zoosporangia root infection. This is consistent with observation in emotaxis .S. subterranea zoospores appear to be dependent on Ca2+ signaling. It is an important prerequisite for zoospore root attachment and zoosporangia root infection. Ca2+ channel blockers, chelators, flux inhibitors and calmodulin antagonists are all effective at inhibiting zoospore chemotaxis and reducing S. subterranea root attachment and root infection, but channel blockers, LaCl3 and GdCl3 at ≥ 50 μM provides the optimum inhibition. Ca2+ signaling affects both qualitative and quantitative swimming behaviors just as it affects chemotaxis, thus the rate of chemotaxis is proportional to the quantifiable zoospore swimming behaviors, notably speed, acceleration, and distance. Zoospores with helical swimming patterns, traveled longer distances at higher speed and acceleration and were the most chemotactically active. Detraction from the helical pattern to more oscillatory, twisted, or meandering patterns with the application of Ca2+ antagonists reduce all quantifiable swimming behaviors as well as chemotaxis. It remains, however, necessary to determine the effect of Ca2+ antagonists on the intracellular load of Ca2+ to confirm that Ca2+ antagonists operate by limiting the cytosolic concentration of Ca2+. The evidence above suggests Ca2+ antagonist as a potential treatment for limiting S. subterranea root infection.Chemotaxis of The original contributions presented in the study are included in the article/JA, RT, TT, and CW designed the experiments. JA performed the experiments, analyzed the data, and prepared the original draft. CW, RT, and TT reviewed and edited the manuscript. CW was recipient of financial support for the project. All authors provided critical feedback to the article and approved the final version.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. 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It used established quantifications of sarcomere and T-SR anatomy (radial diameter 2+ influx densities,influxes, 2+ signals following muscle fibre depolarization. Predicted steady-state T-SR junctional edge [Ca2+], [Ca2+]edge, matched reported corresponding experimental cytosolic [Ca2+] elevations given diffusional boundary efflux2+ diffusion coefficients predicted [Ca2+]edge upon experimental [Ca2+] upon Ca2+ release through their entire test voltage range. The resulting model consistently predicted elevated steady-state T-SR junctional ~ µM-[Ca2+] elevations radially declining from maxima at the T-SR junction centre along the entire axial T-SR distance. These [Ca2+] heterogeneities persisted through 104- and fivefold, variations in D and w around, and fivefold reductions in d below, control values, and through reported resting muscle cytosolic [Ca2+] values, whilst preserving the flux conservation (2+] T-SR microdomains that could regulate cytosolic and membrane signalling molecules including calmodulin and RyR, These findings directly fulfil recent experimental predictions invoking such Ca2+ microdomains in observed regulatory effects upon Na+ channel function, in a mechanism potentially occurring in similar restricted intracellular spaces in other cell types.A finite element analysis modelled diffusional generation of steady-state Ca These include both excitable  and non-excitable, including thrombocyte, cell types8. These intracellular membranes often form appositions with surface membrane with proximities (< 10–30 nm) permitting direct protein–protein/lipid interaction10 though not accommodating entire organelles. Their intervening electron-dense cytosol could also reflect local concentrations of proteins, lipids or ions. Electron microscopic sections can reveal parallel alignments extending over ~ 100–400 nm distances without fusion of the component membranes potentially offering restricted diffusion spaces permitting ion, including Ca2+, accumulation and microdomain formation.Intracellular endoplasmic or sarcoplasmic reticular (SR) membrane systems gating store Ca+ channel mediated action potentials are conducted into the cellular interior at regular intervals along the muscle length through electrically continuous transverse (T-) tubular membranes. At specific regions, these come geometrically close (~ 12 nm) to, whilst remaining electrically isolated from, terminal cisternal membranes of the SR Ca2+ store. The resulting T-SR triad and dyad junctions are strategic to excitation–contraction coupling13. In cardiac muscle, tubular membrane Ca2+ influx through voltage-activated dihydropyridine receptors  activates SR Ca2+ release by cardiac-type SR RyR214. In skeletal muscle, Cav1.1 DHPR1 conformational changes themselves allosterically activate directly coupled RyR115.In skeletal and cardiac muscle, following surface membrane propagation, Na2+ then diffuses into the cytosol activating nearby myofilaments and consequent muscle contraction, prior to its SR Ca2+-ATPase mediated re-sequestration at longitudinal as opposed to T-SR membrane sites. However, recent evidence implicates Ca2+ in further regulatory functions involving T and SR membrane proteins. Skeletal and cardiac muscle Na+ channels (Nav1.4 and Nav1.5) possess potential Ca2+ and calmodulin (CaM) binding modulatory sites16. Here Ca2+ can bind directly to the Nav C-terminal domain at one or more of its EF-like hand motifs17, or indirectly, through Ca2+/CaM binding, to its isoleucine-glutamine (IQ) domain region following initial Ca2+ binding to EF-hand motifs on CaM19 or to a site between Nav domains III and IV20. Voltage-gated Na+ channels additionally contain sites phosphorylatable by Ca2+/CaM-regulated CaM kinase II (CaMKII)21, and by protein kinase C22. In vitro single-cell patch clamp studies reported that elevating [Ca2+]i to ~ 2 µM by rapid Ca2+ photo-release or overspill from neighbouring Ca2+ channels, reduced Na+ current, INa, in Nav1.4-transfected HEK293 cells and skeletal muscle cell lines. These effects were abrogated by intracellular BAPTA23, or mutations involving either the CaM Ca2+-binding-EF hands or the Nav1.4 C-terminal IQ domain24.Released Ca2+ signalling25. Activation or inhibition of SR Ca2+ release then respectively reduced or increased Na+ current, INa. The latter were accomplished by direct pharmacological RyR1 activation by caffeine or 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate (8-CPT), or RyR1 inhibition by dantrolene. Dantrolene pretreatment further abrogated the downregulatory effects of caffeine or 8-CPT on INa27. Murine cardiac muscle showed similar effects when SR Ca2+ release was enhanced either by 8-CPT challenge, or with the RyR2-P2328S genetic modification29. In contrast, cyclopiazonic acid (CPA) increased skeletal muscle INa, while preserving its time courses, steady-state half-maximum voltages and steepness factors. CPA pre-treatment also abrogated the effects of caffeine30. Yet previous Ca2+ fluorescence studies performed in rat soleus and oesophageal striated muscle using fluo-3-AM and fura-PE3-AM had reported that both caffeine and CPA increased bulk cytosolic [Ca2+]34. However, in contrast to modifying RyR-mediated SR Ca2+ release, CPA inhibits SR Ca2+-ATPase (SERCA)-mediated cytosolic Ca2+ re-uptake32. The consequent store Ca2+ depletion would then be expected to reduce, rather than increase, RyR1-mediated Ca2+ influx into the T-SR junction30.Intact native murine skeletal muscle fibres were also studied by a loose patch clamping technique that minimized perturbations in intracellular Ca2+ release took place into a microdomain in the vicinity of both the SR RyR1 and the T-tubular membrane Nav1.4 and that the local elevation in Ca2+ concentration, [Ca2+]TSR, would then modify Nav1.4 function30. Such a hypothesis would predict contrasting increases and decreases in local microdomain [Ca2+] following caffeine, and dantrolene or CPA challenge. The narrow, ~ 12 nm T-SR junctions that could form spaces with restricted intracellular diffusion close to the RyR1 Ca2+-release sites might be implicated in such microdomain formation. These could result in changes in local in vivo [Ca2+]TSR, distinct from those of [Ca2+]i in the remaining bulk cytosol. This could explain the contrasting actions of RyR1 agonists and RyR1 or SERCA antagonists on INa through correspondingly contrasting effects on local Ca2+ or Ca2+/CaM levels, to which the Nav1.4 would be directly or indirectly exposed, despite their similar effects on bulk cytosolic [Ca2+]i34.These paradoxical findings prompted suggestions that RyR1-mediated Ca2+]TSR microdomains possibly using fluorescent Ca2+ indicator methods need to address the small dimensions and dispersed nature of the T-SR compartment37. The present complementary approach applies diffusional modelling techniques38 to explore the physical parameters permitting accumulation or depletion of released SR Ca2+ within the T-SR junction. It demonstrated that established anatomical and physiological features related to skeletal muscle excitation–contraction coupling are physically compatible with generation of significant Ca2+ microdomains in both activated and resting muscle fibres. We then discuss their possible physiological effects both in myocyte T-SR junctions and in similar membrane appositions in other cell types.Direct experimental explorations for such  distributions resulting from diffusional processes using the computational T-SR parameters listed in Table 2+ release influx densities into each individual T-SR junction, Jinflux, used in the MATLAB program PDE Toolbox, could be related to previously reported45 experimental initial rates of SR Ca2+ release d[Ca2+]/dt. The latter would give a Ca2+ flux into the sarcomere cytosolic volume V of The modelling process first sought three-dimensional (3D) reconstructions of radial steady state [Ca45 reported a value of d[Ca2+]/dt = 180 μmol/(dm3 s). The Ca2+ then diffuses through the T-SR space down its resulting concentration gradients with the diffusion constant D = 4.0 × 107 nm2/s previously reported for amphibian skeletal muscle48. The Ca2+ finally leaves the T-SR junction space effluxing into surrounding cytosol at the edge of the T-SR junction across diffusional area πdw at a rate driven by [Ca2+]TSRedge. This proved close and proportional to reported experimental peak cytosolic Ca2+ concentration, [Ca2+]max at the explored 0 mV test voltage with the use of an exit length value λ = 9.2 nm45.Antipyrylazo III absorbances in amphibian skeletal muscle fibres subject to voltage clamp steps from a − 90 mV resting to a 0 mV test membrane potential2+ fluxes upon [Ca2+]TSRedge could be determined using the predicted number of T-SR junctions in unit muscle volume,An overall rate constant describing the dependence of the summed Ca2+ efflux into unit muscle volume is then:The total Ca2+]TSRedge is then.The constant of proportionality describing this linear dependence on  through the radial geometry of the T-SR junction. The false colour maps reconstruct perspective steady-state [Ca2+] at the (A) tubular and (B) SR membrane faces, and (C) en face [Ca2+] at the tubular membrane face of the T-SR junction. Between elements where a solution has not been calculated, the result is interpolated automatically according to the coarseness of the mesh. These maps show a significant [Ca2+] heterogeneity or microdomain across the radial axis resulting in a > fivefold difference between centre and edge T-SR junction [Ca2+]. These resulting Ca2+ microdomains extended the entire T-SR distance mapping [Ca2+] gradients onto the tubular membrane surface. The colour map . The solution from the node closest to selected points representing the edge and centre of the geometry, and half-way between these was plotted against time through the time trajectory of the computation  and SR membranes (superscript ‘SR’), and halfway along their axial distance (superscript ‘TSR’). [Ca2+] declined from its maximum values with radial distance from the T-SR junction centre (subscripts below: ‘centre’) towards the T-SR edge (subscript ‘edge’) with similar readouts close to the T-tubular (subscript: ‘T’) and SR membranes (subscript: ‘SR’), and half-way (subscript: ‘50%’) along their axial distance.Firstly, all the test voltages were associated with T-SR junction Cains Fig. . Figure 2+ microdomains arising from the range of Jinflux values and corresponding test voltages showed that these were similar in form, consistent with the linearity expected of diffusional processes. This emerged from comparing ratios between [Ca2+] at the T-SR centre with those at the edge and halfway between (50%) at the T-tubular and SR membranes, and within the T-SR space. Thus [Ca2+]T50%/[Ca2+]Tcentre, [Ca2+]TSR50%/[Ca2+]TSRcentre, and [Ca2+]SR50%/[Ca2+]SRcentre values were all close to 79% and [Ca2+]Tedge/[Ca2+]Tcentre, [Ca2+]TSRedge[Ca2+]TSRcentre and [Ca2+]SRedge/[ Ca2+]SRcentre were all close to 14%. Similarly, the radial distances from the T-SR centre over which [Ca2+] fell by 50% of its centre-edge range were all 77.67, 77.90 and 77.76 nm whether close to the T-tubular membrane, the SR membrane and halfway between these respectively.Secondly, quantifications of CaJinflux values corresponding to the increasing experimental d[Ca2+]/dt with progressive depolarization correspondingly predicted increased steady state [Ca2+] whether at the centre, 50% from or at the T-SR edge, or close to T-tubular or SR membranes or between these. They produced substantially higher, ~ 10’s of µM [Ca2+] values at the T-SR centre than the ~ µM [Ca2+] values at the edge.Thirdly, progressively larger Jinflux at differing test voltages and [Ca2+]edge closely matched the corresponding reported experimental relationship between d[Ca2+]/dt and [Ca2+]max. Figure Jinflux values gave [Ca2+]edge values similar to those of the corresponding reported experimental [Ca2+]max45. Figure 2+]edge, and [Ca2+]max with close to unity gradient and zero intercept. Furthermore, combining the Ca2+ influx and efflux equality condition, Φinflux = Φefflux, and the efflux equation  converged monotonically to steady state [Ca2+] measurable at selected radial distances from the T-SR junction centre yielding (B) uniform radial [Ca2+] gradients along the T-SR distance visualized as (C) radial [Ca2+] gradients extending the full axial T-SR junction distance . Furthermore, radial distances from the T-SR centre over which [Ca2+] fell by 50% of its centre-edge range, whether close to the T-tubular membrane, the SR membrane and half way between these respectively remained similar . [Ca2+] levels through the T-SR junction fell proportionally with 10- and 100-fold Jinflux reductions: [Ca2+]Tcentre fell from ~ 0.43 μM to 0.043 μM and 0.0043 μM respectively, as expected from the relationship established in Fig. This yielded data points replicating the predicted linear dependence between 2+ microdomains robustly persisted with wide variations involving previously experimentally reported Ca2+ diffusion constants, D, and T-SR junction geometries represented by their axial distances w, and diameters, d. These further computations varied each parameter in turn holding the remaining variables constant under conditions of fixed Jinflux.Skeletal muscle T-SR junction properties vary with physiological conditions, both within and between muscle fibres, individual muscles and muscle types. Furthermore, such surface-cytoplasmic membrane appositions also occur in and vary amongst other cell types. Nevertheless, the Ca2+ diffusion coefficient D = 4.0 × 107 nm2/s48. Higher reported values reach 5.2 × 108 nm2/s in other cell types50 (cf:54) and 1 × 109 nm2s at infinite dilution in vitro56 . Plotting log10[Ca2+] values at the centre, edge of the T-SR junction, and halfway between these against log10(D) all yielded similar linear plots reflecting the constant radial [Ca2+] profiles shown by the Ca2+ microdomains  agreeing with the steady state Nevertheless, even the highest w = 12 nm to 6.6 nm in hypertonic extracellular solutions58. They increase to 20.15 nm and 29.60 nm with fatiguing low-frequency intermittent stimulation59 and exposure to hypotonic solutions60 respectively. Computational solutions modelling these variations in w at constant Jinflux, D and d  then fell from 0.763 to 0.748 whereas Xedge/(T-SR radius) increased from 0.765 to 0.793. Finally, both steady state centre and edge [Ca2+], whether at the T-tubular or SR membranes, or within the T-SR junction, decreased. Thus, [Ca2+]Tcentre and [Ca2+]Tedge fell from 43.965 and 6.190 to 8.428 and 1.081 respectively. Nevertheless, even the lowest values of [Ca2+]Tedge resulted in µM-[ Ca2+] changes. Finally, [Ca2+]edge inversely depended upon w  approximated predictions from parameter values in the steady state conservation condition –11 mol/m2.In addition, radial distances for Tcentre and [Ca2+]Tedge fell from 21.876 and 3.025 to 1.047 and 0.473 respectively. Thus, even a d = 40 nm diameter produced µM-[Ca2+] microdomain differences. [Ca2+]edge positively correlated with d, with slopes between 1.41 × 104 to 1.35 × 104 mol/m4 in agreement with steady state flux conservation, giving 4 mol/m4 -mediated Ca2+ fluxes modulating Nav1.4 function even in resting skeletal muscle30. Available Fura-2 studies suggest resting cytosolic [Ca2+] between 0.06 and 0.14 μM61. The highest reported, Fluo-3, studies reported up to 0.30 μM63 and the lowest Ca2+-sensitive microelectrode measurements, 0.038 μM64 , and X’centre, X’50%, and X’rim values  all identical to corresponding values at higher Jinflux. Finally, the [Ca2+]Tcentre,, [Ca2+]TSRcentre and [Ca2+]SRcentre approached 1 µM [Ca2+] concentrations, giving 0.268, 0.270 and 0.274 at the lowest and 0.990, 0.995 and 1.010 µM at the highest resting [Ca2+].Finally, the recent reports that had suggested an existence of T-SR junction Cange Fig. A indicatles Fig. B,C, rati2+ microdomains in skeletal muscle. These had implicated RyR-mediated SR Ca2+ release in modulating tubular Nav1.4 function in both activated and resting skeletal muscle30. The spatial [Ca2+] data arrays obtained from the model of activated  solutions of basic Fick diffusion equations. Their boundary conditions first included a RyR-mediated Ca2+ release into the T-SR space by a uniform Ca2+ influx density Jinflux across its SR face. Subsequent Ca2+ diffusion with a diffusion coefficient established from previous experimental reports from amphibian myoplasm was then mapped through the radially symmetric T-SR junctional space. The second, efflux, boundary condition at the edge of the modelled junction was similarly described by a first order [Ca2+]-dependent process into a well-stirred cytosolic space of infinitely large volume. The latter formalism further matched previous reported eventual first order steady state SERCA-mediated resequestration of the released cytosolic Ca2+49.2+]45. At the influx boundary, Ca2+ influx densities Jinflux for each voltage were determined directly from the corresponding reported maximum rates of SR Ca2+ release, d[Ca2+]/dt, and the model geometrical properties. At the efflux boundary, the resulting [Ca2+] at the edge of the T-SR junction, [Ca2+]edge, was first matched to the corresponding experimental maximum cytosolic concentration [Ca2+]max, by optimising the single free parameter giving exit length λ = 9.2 nm under conditions of full activation by a test step to 0 mV. This assumed the quantities were proportional and close to each other. Both the latter approximations were then further tested in subsequent explorations of varying Jinflux through different test voltages. In all events, further corrections for any discrepancies arising from a small [Ca2+]edge > [Ca2+]max would tend to enhance rather than reduce the computed [Ca2+] magnitudes.The boundary conditions simulated conditions both of full and of graded activation by previously reported experimental voltage clamp steps from resting to both 0 mV and varying test potentials and the resulting alterations in cytosolic [CaThe resulting T-SR model predicted Ca2+microdomains fulfilling the previous suggestions30. The computational solutions following step impositions of Jinflux converged to give steady state T-SR junctional Ca2+ microdomains. Their heatmap representations demonstrated radial [Ca2+] gradients extending the entire axial T-SR junction distance. The microdomains were quantified radially at the centre, at distances halfway to, and at the edge of the T-SR junction, in axial regions close to the T-tubular and SR membranes and in the intervening T-SR junction space. Furthermore, varying Jinflux to reflect previously experimentally reported d[Ca2+]/dt, obtained at varying test voltages, at a constant λ value, gave predicted voltage dependences of [Ca2+]edge closely approximating those of the corresponding experimental [Ca2+]max. The accordingly linear, [Ca2+]edge–[Ca2+]max relationship had unity gradient and zero intercept. Furthermore, the [Ca2+]edge values themselves depended linearly on the corresponding Ca2+ influx Φinflux terms with a gradient fulfilling predictions from the geometrical terms in the equation for the corresponding Ca2+ efflux.Jinflux conditions consistently generated T-SR junction Ca2+ microdomains containing physiologically significant, ~ μM-[Ca2+], heterogeneities graded with imposed depolarization. These could locally elevate [Ca2+] from ~ 0.3–0.4 µM at activation threshold, to 17–20 µM at full activation, relative to the remaining bulk extra-junctional cytosolic [Ca2+]. These microdomains all extended through the entire axial T-SR distance. They showed similar normalised [Ca2+] profiles with radial distance, in which [Ca2+] declined > fivefold from its maximum in the centre to the edge of the T-SR junction. The spatial [Ca2+] dependences persisted with proportionately reduced [Ca2+] even with 10 and 100-fold Jinflux reductions below threshold levels for observed Ca2+ release45. These modelling studies adopting established quantifications for baseline T-SR and sarcomere membrane structure, and diffusion coefficient values, D could thus replicate reported physiological d[Ca2+]/dt and [Ca2+]max in activated amphibian skeletal muscle.All these microdomain formation and characteristics were robust through systematic tests at constant Jinflux that varied Ca2+diffusion coefficient, D, T-SR distance, w, and T-SR diameter, d, in turn, holding the remaining variables constant. These tests further made it possible to survey the relative importance of diffusional or geometric properties to microdomain formation and characteristics.Detailed characteristics of such membrane appositions vary amongst muscle or cell types, and with physiological and physical conditions. Nevertheless, D within the T-SR space could reflect Ca2+ buffering capacities, κ = (Δ[bound Ca]/Δ[free Ca])), of its contained immobile and mobile buffers, and the diffusion coefficient Dmobile of the mobile buffer. Assuming the rapid buffer approximation, the resulting steady state D is related to the free diffusion coefficient DCa2+ by the expression67:First, alterations in Immobile buffer could reflect fixed Ca2+ binding sites including negatively charged membrane bilayer phospholipid groups68 and Ca2+-binding domains in Ca2+ dependent ion channel, cytoskeletal, transport motor and membrane-associated Ca2+ binding kinase proteins69. This would generate local, steady state equilibria between Ca2+ binding and Ca2+ diffusion: depleted Ca2+-free immobile buffer cannot be replaced by diffusion from remote sites. Immobile buffer would then leave steady-state Ca2+ microdomains unaffected67. Whilst its action could alter the [Ca2+] kinetics, our modelled step impositions of Ca2+ influxes increased T-SR free [Ca2+] to steady state values over ~ 0.2 ms exponential timecourses. These were 1–2 orders of magnitude shorter than those of experimentally observed Ca2+ transients70. In contrast, mobile buffer could influence D to extents dependent upon Dmobile. Our computations explored a wide range of conditions extending from limiting maximal D values at infinite Ca2+ dilution without buffer55 then progressively reducing D to values yielding T-SR [Ca2+] values approaching and exceeding SR [Ca2+]. They included physiologically realistic values corresponding to known in vivo Ca2+ buffering capacities of ~50 and ~100 in the respective absence and presence of 1 mM MgATP71, and empirical in vivo skeletal muscle values48.2+] heterogeneities despite large increases or decreases in diffusion coefficient D relative to established skeletal myocyte cytosolic values (D = 4.0 × 107 nm2/s)48, including values reported either in vitro56 or in other cell types54. On the one hand, even the highest reported, in vitro, D = 1 × 109 nm2/s, value corresponding to infinite dilution55 persistently yielded [Ca2+] microdomains approaching μM-[Ca2+] at the T-SR junction centre. On the other, the electron-densities within T-SR junctions could reflect local protein, lipid or ion concentrations reducing D44. Here, progressive tenfold decreases down to D = 4.0 × 105 nm2/s predicted ~ mM hypothetical [Ca2+] actually approaching reported in situ free SR [Ca2+] . These correspondingly slowed the formation time courses of such microdomains. Nevertheless, in the steady state, through this entire explored D range, Ca2+ microdomains persisted with unchanged radial [Ca2+] profiles extending the full distance between T and SR membranes confirming the linearity condition in this model system. [Ca2+]edge varied inversely with D, giving a dependence and proportionality constant that matched predictions of the T-SR junctional model.These explorations demonstrated persistent  nonuniformities themselves varying along the axial distance between SR and T-tubular membranes, to extents increasing with increasing w. Nevertheless, [Ca2+]edge varied with w, through an inverse relationship with proportionality constant matching predictions of the T-SR junction model. Thirdly, successive reductions of T-SR junctional diameters, from d = 220 nm to d = 40 nm, similarly disrupted Ca2+ microdomain heatmaps again giving tapering plume-like forms at the smallest diameters. These were quantified as increased radial non-uniformities and marked axial [Ca2+] differences between regions close to the SR and T-tubular membranes and the intervening space along the axial T-SR distance. Falls in [Ca2+] with radial distance from the T-SR junction centre and [Ca2+] at the SR relative to the T-tubular membranes became less marked with decreasing d. Finally [Ca2+]edge increased with d as expected from the T-SR junction model. Nevertheless, through both these latter modifications in T-SR junction geometry, despite their altered spatial characteristics, the ~ μM-[Ca2+] heterogeneities between their periphery and centre persisted even with more than 100% increases in T-SR distance or 75% reductions in T-SR diameter from control values derived from established morphometric data.Secondly, whilst averaging 2+ in Ca2+ microdomain generation in resting in addition to activated muscle30. Accordingly, the T-SR junction model was extended further to investigate formation and properties of Ca2+microdomains in resting myocytes. This employed background Jinflux values calculated from previously reported cytosolic [Ca2+] values in resting muscle. These previous Fura-red fluorescence studies had suggested experimental resting [Ca2+] ranging between 0.060 to 0.140 μmol/dm3 (ref. 61). We further explored further reduced resting [Ca2+] limits of ~ 0.038 μmol/dm3 from Ca2+-sensitive microelectrode studies64 and possible higher 0.300 μmol/dm3 limits63. The resulting T-SR modelling continued to predict Ca2+ microdomains with their characteristic spatial characteristics. Furthermore, microdomain [Ca2+] levels at the T-SR centre, whether close to the T-tubular or SR membranes or in the intervening space, approached or attained ~ μM-[Ca2+].The previous experimental reports had also invoked background, RyR-mediated influxes of Cav1.4 exposed to successively greater levels of T-SR junction microdomain [Ca2+] in both activated and resting muscle. Successively greater proportions of activated T-tubular membrane became exposed to successively higher, 0.1 to 10 µM [Ca2+] with increasing depolarization. In addition, significant proportions of even resting T-tubular membrane remained exposed to significant, ~ 0.5 µM [Ca2+]. These findings therefore provide a physical basis for the previous suggestions implicating Ca2+ microdomain formation in observed modifications in Nav1.4 function30. Ca2+-CaM binding takes place with ~ µM [Ca2+] dissociation constants73. Feedback µM-[Ca2+] levels arising from SR Ca2+ release could therefore potentially modify both skeletal30 and cardiac muscle29 Nav1.4 or Nav1.5 through direct or indirect, Ca2+-calmodulin (Ca2+-CaM) mediated, actions on their C-terminal domains24. Such concentrations further match the photorelease-induced 1–2 µM cytosolic [Ca2+] elevations previously reported to modify Nav1.4 function23.The present findings taken together could be used to reconstruct the proportions of T-tubular membrane area and therefore of resident Na2+] could inhibit tubular Nav1.4 function following normal sustained activity60 and contribute to particular clinical skeletal myopathies74. A myotonic hyperexcitability disorder disrupting Ca2+-mediated inhibition of Nav1.4 function has been associated with Nav1.4 C-terminal EF hand-like domain mutations76. A condition associated with increased myotube diameters and resting [Ca2+]i, and decreased RyR1-mediated Ca2+ release reflecting possible abnormalities in triad junction formation and maintenance is associated with another, junctophilin (JP2), mutation77. In murine cardiac muscle, Nav1.5 inhibition followed increased SR Ca2+ release following pharmacological challenge29 and in RyR2-P2328S variants modelling the pro-arrhythmogenic catecholaminergic polymorphic ventricular tachycardia80. In these examples, the underlying in vivo source of microdomain Ca2+ would likely remain the RyR-mediated Ca2+ release modelled here, as opposed to T-tubular Cav1.1 or Cav1.2 channel mediated entry of extracellular Ca2+. Thus, early Ca2+ skeletal muscle voltage clamp currents, ICaf (~ 25 µA/cm2)81 and later cell attached patch clamped cardiomyocyte ICaL  one and two orders of magnitude lower than the corresponding RyR-mediated Jinflux, at comparable test voltages. The larger skeletal muscle late ICa  evolves over time courses (hundreds of ms) more prolonged than excitation contraction coupling and shows rapid off kinetics on action potential repolarization84.In skeletal muscle, elevated T-SR junctional microdomain [Ca2+] could also regulate other signalling biomolecules. They are involved in a bell-shaped in vitro open probability relationship for single channel RyR activation and inhibition14. Here, cardiac and neuronal, RyR2 and RyR3 are more Ca2+-sensitive than RyR1 but all are activated over the ~ 1 µM [Ca2+] predicted in the present analysis86. However, under their respective in vivo physiological [ATP] and [Mg2+] conditions, cardiac87 but not skeletal muscle88 RyR activation involves Ca2+-induced Ca2+ release. Skeletal muscle RyR activation instead involves direct allosteric coupling with either T-tubular Cav1.1-L-type Ca2+ channel voltage sensors15 or possibly other adjacent SR RyRs themselves coupled to such Cav1.190. ~ µM Ca2+-CaM may also exert other cytosolic effects as on glyceraldehyde 3-phosphate dehydrogenase91 or itself provide local signaling domains36.Microdomain µM- heterogeneities in the skeletal muscle T-SR-junction. Its findings might next prompt investigations of structure and function in these further examples. These might be complemented by direct experimental fluorescent Ca2+ indicator [Ca2+]TSR measurements37 were these to be able to address the small dimensions and dispersed nature of the microcompartments concerned.Closely apposed membranes potentially mediating localized Ca2+] patterns were computationally examined in a model T-SR junction. This involved replicating the geometry within and through which the diffusional processes occurred, pre-processing through meshing and definition of physical conditions including loads, initial and boundary conditions, generation of system solutions and post-processing of the results . It was implemented on an IBM compatible computer ; GPU: ASUS STRIX GeForce GTX970; installed RAM: 16 GB, running Windows  10, Home 64-bit version 1909).Diffusional mechanics and its consequent temporal and spatial Ca+ patternThe underlying T-SR junction geometry was reconstructed virtually for use in a finite element analysis solving partial differential equations (PDEs) for the resulting diffusional processes with their accompanying boundary conditions (BCs) (See Supplementary File for software archive). The finite element method (FEM) described the complex geometry as a collection of subdomains (elements) by superimposing upon this geometry a mesh of tetrahedral elements joined at their vertices (nodes) and edges. The subdivision accurately represents this complex geometry, permits inclusion of dissimilar material properties, and provides a straightforward representation of the total solution whilst capturing local, microdomain, effects.38. The original PDE problem is accordingly represented within each element with simpler equations approximating the solution to the original equations. Stationary linear problems whose coefficients are independent of the solution or its gradient yield a linear system of equations. In this case our PDE is time-dependent and hence the system of simpler equations is a set of ordinary differential equations (ODEs) then passed onto MATLAB solvers for numerical integration for solution. The FEM approximates the solution by minimizing the associated error function, automatically finding the linear combination of basis functions closest to the solution u. The FEM could therefore capture both concentration differences local to the T-tubular and SR membranes, and across the entire modelled geometry.Specified BCs provided values of the field and related variables, in the present case, the normal derivatives of the field variable in the form of a Neumann-type BC. The FEM equations are formulated such that at the nodal connections, the value of the field variable at any connection is the same for each element connected to the node. Solutions at the edges of each adjacent element are therefore equal, ensuring continuity of field variables between elements, avoiding physically unacceptable gaps or voids in the solutionhttps://www.mathworks.com/products/pde.html). PDE Toolbox also provides the ability to automatically mesh the T-SR junction geometry, providing a basis for solving the diffusion PDE, and stores the solution as matrices amenable to various methods of presentation and post-processing of data within MATLAB. The PDE toolbox is designed to solve equations of the form:m, b, h, f and g can be functions of spatial position, the solution u, or its spatial gradient. In a diffusive system, this generalised problem reduces to the first order equation:b set to unity, where c represents the diffusion coefficient D, and h represents the boundary flux term for the Neumann condition  within MATLAB. This provides functions for solving structural mechanics, heat transfer and general PDEs using the FEM (This entirely theoretical study did not involve animal procedures.Supplementary Information.


The MAPK-interacting kinases 1 and 2 (MNK1/2) have generated increasing interest as therapeutic targets for acute myeloid leukemia (AML). We evaluated the therapeutic potential of the highly-selective MNK1/2 inhibitor Tomivosertib on AML cells. Tomivosertib was highly effective at blocking eIF4E phosphorylation on serine 209 in AML cells. Such inhibitory effects correlated with dose-dependent suppression of cellular viability and leukemic progenitor colony formation. Moreover, combination of Tomivosertib and Venetoclax resulted in synergistic anti-leukemic responses in AML cell lines. Mass spectrometry studies identified novel putative MNK1/2 interactors, while in parallel studies we demonstrated that MNK2 - RAPTOR - mTOR complexes are not disrupted by Tomivosertib. Overall, these findings demonstrate that Tomivosertib exhibits potent anti-leukemic properties on AML cells and support the development of clinical translational efforts involving the use of this drug, alone or in combination with other therapies for the treatment of AML. Acute myeloid leukemia (AML) is the second most common form of leukemia in adults, and has a very poor overall survival rate , 2. For The mitogen-activated protein kinase (MAPK) signaling pathway is a major regulatory cascade, activated in 70–80% of AML patients , 14. MAP50) values of 2.4 nM and 1 nM; and has demonstrated potent anti-neoplastic effects against several cell lines and tumor models including diffuse large B-cell lymphoma (DLBCL), breast cancer, and lung cancer [in vitro.Tomivosertib, also known as eFT-508, is a potent, highly selective and orally bioavailable MNK1 and MNK2 inhibitor that is currently under investigation in Phase 1/2 clinical trials for the treatment of patients with advanced solid tumors and lymphomas . This drg cancer . HoweverInitially, we determined whether Tomivosertib blocks phosphorylation of the MNK effector eIF4E in AML cells. U937, MV411 and MM6 cells were treated with increasing doses of Tomivosertib for a period of 1 and 4 hours and eIF4E phosphorylation was assessed. Treatment with Tomivosertib abrogated the phosphorylation of eIF4E on serine 209 at a concentration of 0.1 μM, supporting potent inhibition of MNK1/2 activity in AML cells –1C. To dIn consequent studies, we investigated the antileukemic activity of a combined treatment of Tomivosertib with Venetoclax in AML cell lines. Venetoclax is a novel BCL-2 inhibitor that was recently approved by the FDA for the treatment of elderly patients with AML due to its significant clinical activity in combination with cytarabine or hypomethylating agents , 44. HowTo identify novel binding partners of MNK1/2, liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on protein-MNK1/2 complexes immunoprecipitated from 293T cells. We found that 16 proteins interacted with MNK1, 10 proteins interacted with MNK2 and 24 proteins interacted with both MNK1 and MNK2 . PathwayAmong the different proteins, RAPTOR and mTOR were identified as interacting partners of MNK2 in the LC-MS/MS studies . We firs7G) cap or prevent the interaction of eIF4E with other proteins in the cap-binding complex [in vitro and in vivo [Protein synthesis is commonly dysregulated in solid tumors and hematological malignancies , 48. A k complex , 49–55.  complex , 25, 56. complex –60; and  complex ; this st in vivo , 32–37.In the current study, we assessed the anti-leukemic effects of Tomivosertib , a highlOur data also demonstrate that the combination of Tomivosertib with Venetoclax enhances the anti-leukemic responses in mutant p53-expressing AML cell lines. The combination of these two drugs resulted in synergistic suppression of cellular viability and CFU-L growth in U937 and KG-1 cells. It has recently been proposed that AML cells can develop resistance to BH3 mimetics, such as Venetoclax, through increased levels of the anti-apoptotic protein MCL-1 via regulation of MCL-1 expression and stability , 63, 64.It is well-established that MNK1/2 drives mRNA translation through phosphorylation of its thoroughly-characterized substrate eIF4E –17. PhosViewed altogether, these studies indicate that MNK1/2 inhibition would most likely be a successful strategy in only a subset of AML patients. In future studies it will be crucial to ascertain what pathways are responsible for sensitivity to MNK inhibitors. These studies will help to identify potential regulatory programs through which MNK1/2 modulates cell signaling pathways critical for leukemic cell survival and may lead to the development of novel therapeutic interventions for AML.Tomivosertib (eFT-508) and Venetoclax (ABT-199) were purchased from TargetMol. All compounds were dissolved in dimethyl sulfoxide (DMSO) and used at the indicated doses.All cells were cultured as previously described –73, 77 aClonogenic assays in methylcellulose were conducted as in previous studies –73, 77. Cell viability assays were performed and analyzed as previously reported using WST-1 reagent (Roche) –75, 77.Cells were treated with either DMSO (vehicle-control) or Tomivosertib at the indicated doses and time points and processed for immunoblotting using an enhanced chemiluminescence method as described previously –75, 77. MNK1 and MNK2a cDNAs were subcloned from pMX-puro  into pcDNA3.1-FLAG plasmid. pRK5-HA-RAPTOR  was purcCells were treated with either DMSO (vehicle-control) or Tomivosertib at the indicated doses and time points. Samples were processed and immunoprecipitation was performed using anti-FLAG-M2 agarose conjugated beads (Sigma-Aldrich) or anti-HA Sepharose conjugated beads  as previously described .Samples were prepared and processed by LC-MS/MS analysis as previously reported . ImmunopThis was performed as previously described using the Metascape database , 78.t-test was used to assess differences between two groups. Differences were considered statistically significant when p values were less than 0.05. All statistical analyses were performed using Prism GraphPad 6.0.All experiments were performed in triplicate and repeated at least twice; variations about mean were presented as standard error. One-way ANOVA was used to compare more than two groups followed by Tukey’s multiple comparison test. Student’s