Arrhythmias are clinically prevalent with a high mortality rate. They impose a huge economic burden, thereby substantially affecting the quality of life. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) is a new type of hypoglycemic drug, which can regulate blood glucose level safely and effectively. Additionally, it reduces the occurrence and progression of heart failure and cardiovascular events significantly. Recently, studies have found that SGLT2i can alleviate the occurrence and progression of cardiac arrhythmias; however, the exact mechanism remains unclear. In this review, we aimed to discuss and summarize new literature on different modes in which SGLT2i ameliorates the occurrence and development of cardiac arrhythmias. Arrhythmia is a common cardiovascular disease (CVD) generally associated with organic heart diseases. Different types of arrhythmias are known where bradyarrhythmias and tachyarrhythmias are the most common. The development of pacemakers has proven to be a boon for patients with bradyarrhythmias. However, the onset of tachyarrhythmia is usually acute, and some tachyarrhythmias, such as atrial fibrillation (AF), ventricular tachycardia, and ventricular fibrillation (VF), increase the risk of disability and death . Althougp < 0.0001) (p < 0.005) and reduce the duration of action potential (Sodium-glucose cotransporter 2 inhibitors (SGLT2is) are a new class of hypoglycemic drug that includes empagliflozin (EMPA), dapagliflozin (DAPA), canagliflozin (CANA), sotagliflozin, and ertugliflozin. Unlike traditional hypoglycemic drugs, this class does not rely on improving insulin secretion or resistance. However, it reduces the renal threshold for glucose and increases its excretion in the urine by inhibiting sodium-glucose cotransporter 2 of the proximal renal tubules from active reabsorption of glucose, thus lowering blood glucose . This an 0.0001) . The DAP 0.0001) . Recentl 0.0001) . In mice 0.0001) . EMPA waotential . Additiootential . In diabotential . It alsootential . However+ and Ca2+ overload, caused by the Na+ and Ca2+ channel remodeling, and the disorder of K+ currents, play important roles in the occurrence and maintenance of arrhythmias.Arrhythmia is closely related to cardiac electrical remodeling. In 1995, Wijffels et al. found that in the goat model of AF created by implanting a pacemaker, the atrial effective refractory period gradually shortened with the extension of atrial pacing time. This proved that cardiac electrical remodeling is involved in the process of AF and that cardiac electrical remodeling caused AF to persist (AF-induced AF) . Ion chaINa-Late) is formed by a small amount of slow inactivation or reopening of sodium channels during myocardial repolarization, persisting in the repolarization phase of the action potential, especially the plateau phase, and is important for regulating the duration of action potentials (INa). When INa-Late increases, the increased Na+ influx causes Na+ overload in cardiomyocytes (INa-Late could ameliorate arrhythmias (+-H+ exchanger (NHE) also plays an important role in the occurrence and maintenance of arrhythmias of cardiomyocyte membrane are activated in the depolarization phase. Thus, a small amount of Ca2+ enters into the cytoplasm and binds to Ca2+-binding sites in the sarcoplasmic reticulum (SR) membrane. This is followed by opening of Ca2+-releasing channels ryanodine receptors 2 (RYR2) in SR leading to generation of the Ca2+ spark. This results in an increase of intracellular Ca2+ to over one hundred times instantly and is referred to as calcium-induced calcium release (CICR) (2+ sparks are the basis for excitation-contraction coupling in cardiomyocytes (+-Ca2+ exchanger (NCX) is a type of membrane protein involved in bidirectional transport (3 Na+ for 1 Ca2+) and is regulated by a transmembrane Na+ gradient. In the case of Na+ overload, NCX reverse transport leads to cytoplasmic Ca2+ increase, eventually leading to Ca2+ overload. Increased Ca2+ levels can induce CICR, enhance the excitability of cardiomyocytes, and increase the possibility of arrhythmias  is the basis of Ca2+ leakage from the SR in cardiomyocytes of patients with arrhythmias (INa-Late influences Ca2+ homeostasis in cardiomyocytes by activating CaMKII (2+ ATPase 2a (SERCA2a), a calcium pump on SR, plays an important role in maintaining intracellular Ca2+ homeostasis in cardiomyocytes  . Ca2+ spmyocytes . Na+-Ca2hythmias . Ca2+ lehythmias . Studiesg CaMKII . In addimyocytes . During hythmias .+ currents are the main current in the cardiomyocytes’ repolarization phase, they have been confirmed to be related to arrhythmias, including the transient outward K+ current (Ito), the delayed-rectifier K+ current (IK), the inward rectifier K+ current (IK1), and the ATPsensitive K+ current (KATP)  . The risk of sudden cardiac death may be enhanced by the decreasing of IK , thus exerting profound effects on cardiac excitability and arrhythmogenesis. Studies reported IK1 is closely with ventricular arrhythmias after myocardial infarction (The Kt (KATP) . The Itoon (APD) . Cardiomntenance . And theng of IK . IK1 is farction .+ overload in cardiomyocytes. Philippaert found that EMPA, DAPA, and CANA had no effect on peak INa in the cardiomyocytes of an HF mouse model, but could significantly inhibit INa-Late, which is a potent selective inhibitor of INa-Late. Furthermore, EMPA reduced the incidence of spontaneous calcium transients induced by INa-Late  exposures than in the metabolic syndrome rats’ cardiomyocytes  with the overexpression of TNF-α are prone to spontaneous atrial arrhythmias. Compared with control atrial myocytes, female and male TNF1.6 myocytes displayed a significant reduction in the amplitude, the +dCai2+/dtmax and -d Cai2+/dtmax of the Cai2+ transient, and prolongation of the TD50%  induction and generation of oxidant species. But the ICaL didn’t change  in ventricular myocytes, and the effect of IL-1β on NSE was weakened in cardiomyocytes from mice with genetic CaMKII inhibition by cardiac-specific expression of autocamtide 3 inhibitory peptide . ATP production is achieved by the mitochondrial membrane potential (ΔΨ). ΔΨ is a criterion for evaluating the function of mitochondria, which can generate the necessary proton propulsion for ATP generation and release the energy required to phosphorylate ADP to ATP . Mitochohythmias . Overloahythmias . Mitochohythmias . PGC-1α hythmias , alleviahythmias .The mitochondrial respiratory chain is a major component of the reactive oxygen species (ROS) production in the body. NADPH oxidase, xanthine oxidase, and uncoupled endothelial nitric oxide synthase (eNOS) have been identified as the main sources of ROS. Under physiological conditions, ROS is produced in small amounts and can be removed by the antioxidant system over time. Under pathological conditions, ROS production is increased. When a large amount of ROS cannot be removed by the antioxidant system in time, oxidative stress occurs, resulting in the degeneration of proteins, lipids, and nucleic acids, and the normal structure and function of cells is destroyed . When miin vitro  activity. It can also be induced by separation of the Beclin protein complex from Bcl-2 . AutophaSGLT2i can ameliorate autophagy by upregulating mitochondrial autophagy-related protein Bnip3 mRNA levels, thereby increasing Bnip3 protein levels and preventing a decrease in the number of autophagy vacuoles in diabetic myocardial infarction model mice . EMPA anSGLT2i are a new type of hypoglycemic drug with good safety and tolerance. It can regulate blood glucose levels safely and effectively, thereby reducing the occurrence and progression of heart failure and cardiovascular events significantly. Studies have confirmed that SGLT2i ameliorates arrhythmia, but the specific mechanism remains unclear. It may ameliorate the occurrence and development of arrhythmias by improving cardiac electrical remodeling, mitochondrial dysfunction, cardiac structural remodeling, inflammation, oxidative stress, and autophagy. However, there is a lack of relevant studies to directly prove that SGLT2i can improve arrhythmias by improving the above-mentioned factors. A clinical trial to explore the related mechanisms of SGLT2i in ameliorating arrhythmias is required. This can aid in understanding the mechanisms in play and provide effective guidance for the drug treatment of arrhythmias in clinical practice.


The taste bud is a tightly packed cell population, wherein extracellular Ca2+ is expected to fluctuate markedly due to the electrical activity of taste cells. It is currently unclear whether the Ca2+ entry-driven synapse in type III cells could be reliable enough at unsteady extracellular Ca2. Here we assayed depolarization-induced Ca2+ signals and associated serotonin release in isolated type III cells at varied extracellular Ca2+. It turned out that the same depolarizing stimulus elicited invariant Ca2+ signals in type III cells irrespective of bath Ca2+ varied within 0.5–5 mM. The serotonin release from type III cells was assayed with the biosensor approach by using HEK-293 cells co-expressing the recombinant 5-HT4 receptor and genetically encoded cAMP sensor Pink Flamindo. Consistently with the weak Ca2+ dependence of intracellular Ca2+ transients produced by VG Ca2+ entry, depolarization-triggered serotonin secretion varied negligibly with bath Ca2+. The evidence implicated the extracellular Ca2+-sensing receptor in mediating the negative feedback mechanism that regulates VG Ca2+ entry and levels off serotonin release in type III cells at deviating Ca2+ in the extracellular medium.Type III taste cells are the only taste bud cells which express voltage-gated (VG) Ca The echannels ,8,12,13.Cav 2.1) . To all ably ATP ,16.2+, deviate markedly from stationary levels, as the electrical activity of taste cells redistributes ions between the spacious cell cytosol and the small extracellular space. In the case of Ca2+ homeostasis, one more significant factor is the high buffering capacity of the cell cytoplasm, which is commonly characterized by the ratio β = Δ(bound Ca2+)/Δ(free Ca2+). Among N Ca2+ ions translocated from the extracellular medium into the cytosol, most should be bound, and only N/(1 + β) ions remain free. With the effective extracellular and intracellular volumes Vout and Vin, respectively, the translocation of N Ca2+ ions into the cytosol would decrease extracellular Ca2+ concentration by ΔCout = N/Vout, while free intracellular Ca2+ would be increased by ΔCin = N/((1 + β) Vin). Given that β > 100 in apparently all assayed cells [in/Vout > 102, ΔCout/ΔCin =(1 + β) Vin/Vout > 104. This estimate suggests that the 100 nM increase in cytosolic Ca2+ mediated by Ca2+ entry into a taste cell should be accompanied by the local drop in extracellular Ca2+ of about 1 mM. If so, the question arises of whether the Ca2+ entry-driven synapse in type III cells could be reliable enough while extracellular Ca2+ is unsteady to a high extent. Here, we addressed this issue by assaying depolarization-elicited Ca2+ signals in isolated type III taste cells and associated serotonin release at varied Ca2+ in the bath.In the taste bud, cells are packed very densely ,17. The ed cells  and that2, 1 CaCl2, 10 glucose, 20 HEPES-NaOH (pH 7.8). Once injected, a tongue was incubated in the oxygenated low-Ca2+ solution (in mM): 130 NaCl, 10 KCl, 0.7 CaCl2, 1.1 MgCl2, 1 EGTA, 1 EDTA , 10 HEPES-NaOH (pH 7.4), 10 glucose) for 30–35 min. After the treatment, a lingual epithelium was peeled off from the underlying muscle, pinned serosal side up in a dish covered with Sylgard resin, and then it was incubated in the Ca-free solution for 10–30 min. The isolated epithelium was kept at room temperature in a solution , 10 glucose, 5 Na-pyruvate) for 4–6 h. To obtain individual taste buds, those were removed from a CV papilla by gentle suction using a fire-polished pipette with an opening of 80–100 μm. Taste cells were isolated by using suction pipettes with tips of 50–70 μm. The obtained cellular material was then expelled into an electrophysiological or photometric chamber.All experimental protocols were in accordance with local regulatory bodies and the European Communities Council Directive (2010/63/EU) and approved by the Commission on Biosafety and Bioethics . Taste cells and taste buds were isolated largely from mouse circumvallate papillae and sometimes from foliate papillae as described previously . Briefly2, 0.1 EGTA, 10 HEPES-CsOH (pH 7.2), 400 µg/mL amphotericin B. The basic bath solution included (mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES-NaOH (pH 7.4), 10 glucose. When needed, 140 mM NaCl + 5 mM KCl was replaced with 75 mM NaCl + 70 mM KCl and/or CaCl2 was added at a concentration of 0.5, 1, or 5 mM. The recording chamber of nearly 75 μL has been described previously [Ion currents were recorded, filtered, and analyzed using an Axopatch 200 B amplifier, Digidata 1440A, and pClamp 10 software . External solutions were delivered by a gravity-driven perfusion system at a rate of 0.1 mL/s. Cells were typically polarized by serial voltage pulses of appropriate duration at a 10 mV step. Generally, the perforated patch approach was used, and recording pipettes were filled with (mM): 140 CsCl, 1 MgCl2+ dye Fluo-8 at room temperature (23–25 °C) by adding Fluo-8AM (4 μM)  and Pluronic (0.02%)  to the bath. As being prone to quick rundown, isolated taste cells were loaded for 15 min, rinsed with the bath solution several times, and immediately assayed. HEK-293 cells were loaded for 20 min, rinsed several times, and kept in the bath solution for 30 min prior to recordings.Isolated cells were plated onto a photometric chamber of nearly 150 μL volume. The last was a disposable coverslip  with an attached ellipsoidal resin wall. The chamber bottom was coated with Cell-Tak  ensuring sufficient cell adhesion. Attached cells were loaded with the Ca2+ from the resting level in individual Fluo-8-loaded cells were quantified by the ratio ΔF/F0, where ΔF = F − F0, F is the instant intensity of cell fluorescence, F0 is the intensity of cell fluorescence obtained in the very beginning of a recording and averaged over a 20 s interval. All chemicals were applied by the complete replacement of the bath solution in a 150 μL photometric chamber for nearly 2 s using a perfusion system driven by gravity.Experiments were carried out using an inverted fluorescent microscope Axiovert 135 equipped with an objective Plan NeoFluar 20×/0.75 , a digital ECCD camera LucaR , and a hand-made computer-controllable epi-illuminator with a set of light-emitting diodes, particularly allowing for the excitation of Fluo-8 at 480 ± 10 nm. The Fluo-8 emission was collected at 535 ± 25 nm. Serial fluorescent images were captured every second and analyzed using Imaging Workbench 6 software . Deviations of cytosolic CaThe used salts, buffers, spermine, and serotonin were purchased from Sigma-Aldrich ; NPS-4123 and NSP R-568 were from Tocris Bioscience .4 coupled to adenylyl cyclase and genetically encoded cAMP sensor Pink Flamindo, served as a serotonin biosensor. The receptor 5-HT4 was cloned from the mouse brain .v/v) fetal bovine serum , glutamine (1%), and the antibiotic gentamicin (100 μg/mL) (Invitrogen). Cells were grown in 12-well culture plates in a humidified atmosphere (5% CO2/95% O2) at 37 °C. To induce expression of the 5-HT4 receptor and Pink Flamindo sensor, HEK-293 cells were transfected with two appropriate plasmid vectors. Transfected cells were then subjected to selection to obtain cellular monoclones, which stably expressed 5-HT4 and Pink Flamindo, thus being responsive to nanomolar serotonin with cAMP transients detectable by Pink Flamindo fluorescence .HEK-293 cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)  containing 10%  papillae and examined with the perforated patch-clamp technique and Canditions ,23. In pchannels ,8,12. Wiogically , while tthe bath B. The suansients C. Consisath Ca2+ B, these t −10 mV D.2+ currents on extracellular Ca2+. The value of this current at a particular voltage was calculated as a difference between integral VG currents recorded in the control and in the presence of 50 μM Cd2+. Note that Ca2+ ions entering through VG Ca2+ channels into the cytosol can affect their activity through several negative feedback mechanisms [2+ currents on bath Ca2+, taste cells were loaded with the fast Ca2+ chelator BAPTA by preincubating them in the presence of the permeable precursor BAPTA-AM (10 μM) for 10 min. It is noteworthy that VG Ca2+ currents in taste cells were usually prone to rapid rundown within 5–10 min. Fortunately, this interfering phenomenon could be slowed down sufficiently by incubating cells with the AC activator forskolin (0.1 μM), which elevated intracellular cAMP and that maintained the activity of VG Ca2+ channels [2+ currents within 20 min at least, and these stabilized cells were further used to examine the dependence of VG Ca2+ entry on extracellular Ca2+ varied within 0.5–10 mM.Next, we analyzed the dependence of VG Cachanisms . To prevchannels . As a re2+ currents, and among them, 49 cells (69%) exhibited VG Ca2+ currents being unexpectedly subtly affected by the increase in bath Ca2+ from 1 to 2 mM  and (2) give the following dependence of the integral Ca2+ current on external Ca2+:Imax = imaxN, C is the concentration of bath Ca2+, and C1/2 is the half-effect concentration. Equation (3) describes the monotonic saturating curve without any plateau, provided that the open probability is independent of Ca2+. Since here cytosolic Ca2+ was strongly buffered with BAPTA, which should have canceled the effects of intracellular Ca2+ on Ca2+ channel gating, the plateau in the experimental curve i=imaal curve B suggest2+ and Ca2+ transients elicited by VG Ca2+ entry in type III cells. Being preloaded with Fluo-8, cells were patch clamped to identify type III cells with sufficiently active VG Ca2+ channels. Given that short depolarization elicited usually small and poorly resolved Ca2+ signals  always initiated much stronger Ca2+ signals compared to depolarizing voltage  and generated Ca2+ transients that were similar in a particular cell but varied by response lag and value, in terms of ΔF/F, from cell to cell  as a primary mechanism underlying the step-like dose dependence of agonist responses [3- and ryanodine receptors [2B receptor responded to serotonin with Ca2+ transients in an “all-or-nothing” manner  [4/PF-positive cells and found several of them to generate well-resolved cAMP transients in response to nanomolar serotonin applied shortly    fraction of isolated type III taste cells liberated serotonin reproducibly on three consecutive applications of 70 mM KCl, the minimal series necessary for the conclusive recordings   A. The tan sensor B, which n sensor E.2+  [2+. If so, CASR activation/inhibition should have impaired/facilitated serotonin secretion.We pondered on which machinery could stabilize neurotransmission in type III cells at varied Caocytosis ,42,43,44hreshold A [35,36,nal Ca2+ B,C. Howe voltage B, follow current A. This oath Ca2+ C,D. Thisble Ca2+ B,C. Giver (CASR) , this GP2+ transients and associated serotonin release in type III cells. In particular, we probed some CASR ligands, including the natural agonist spermine and orthosteric synthetic agonist NPS R-568, as well as the CASR antagonist NPS-2143 [50 ≈ 0.3–0.5 μM [+ currents (n = 8) (50 ≈ 0.04 μM [+ and outward currents in type II cells (n = 6) by 6–14%  . These oby 6–14% . Althougn = 9) dialyzed with 140 mM CsCl, 3 mM spermine reversibly inhibited VG Ca2+ currents by 41 ± 3% at −10 mV and outward, presumably anionic, currents by 13 ± 2% at 50 mV   C,D.2+ transients elicited by the same depolarization of type III cells at different bath Ca2+  (n = 48), but this small effect was poorly reversible  A. NSP-21versible D. Thus, n = 15) were pretreated with 3 mM spermine, their stimulation with KCl triggered biosensor responses that were reduced by 48 ± 16% on average compared to the control  that could provide the appropriate regulation of the neurotransmission in type III cells. Among possible scenarios, we considered the CICR mechanism, which has been implicated in the regulation of neurotransmitter exocytosis in a variety of different cells ,43,44. Ilar Ca2+ B,C. Howe voltage C, and thux value B,D. Thistuations C,D. ThisII cells , this GProcesses .q/11, Gi/o, G12/13, and Gs proteins and β-arrestins to a variety of effectors and signaling pathways. Ubiquitously engaged are adenylyl cyclase producing cAMP and phospholipase C (PLC) hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [Reportedly, CASR is involved in the regulation of neuronal excitability, neurotransmission, and synaptic plasticity ,57. It wol (DAG) ,60.2+ channels. The gating of these channels is particularly modulated by direct interaction with G proteins, Ca2+/calmodulin, lipids, and fatty acids as well as by phosphorylation mediated by a number of protein kinases, including PKA, PKC, and CaMK regulated by cAMP, Ca2+/DAG, and Ca2+/calmodulin, respectively [2+ influx via VG Ca2+ channels but also by controlling the level of PIP2 that serves as a hub for proteins, which govern the turnover of presynaptic vesicles [Among intracellular molecules involved in CASR signaling, many are documented as regulators of VG Caectively ,62,63,64vesicles ,66,67. Ivesicles ,69,70. T2+ channels is a complicated issue because VG Ca2+ currents in type III cells are usually small under physiological conditions (2+ in the bath, VG Ca2+ currents were enhanced by nearly three times compared to the normal conditions (2+. In addition, characteristic of CASR-mediated responsivity of cells to extracellular Ca2+ are high cooperativity and EC50 ≈ 2.5 mM [2+ should have elicited maximal CASR activity in type III cells, thus saturating a regulatory circuit downstream of CASR and making its sensitivity to inhibitors of effectors involved presumably abnormal. In any way, a particular compound could be considered as a probe for the CASR-mediated circuit only if its effect on cell functions, VG Ca2+ currents in the given case, would be influenced by CASR ligands. We, therefore, inferred that the detailing of the signaling circuit, which couples CASR to VG Ca2+ entry and, more generally, to serotonin release, would require special efforts, including multiple recordings from sufficiently robust taste cells occurring rarely. As highly complicated and laborious, the appropriate experiments were not carried out in this work but suggested for the future.The analysis of the CASR coupling to VG Canditions A. Althounditions B, type I≈ 2.5 mM ,71. Cons2+ in the extracellular medium. VG Ca2+ channels appear to be under the control of a negative feedback that regulates the neurotransmitter exocytosis in an extracellular Ca2+-dependent manner. Given, however, that VG Ca2+ currents were invariant in the relatively narrow range of bath Ca2+ concentrations of 1–2 mM (2+ (To conclude, our overall findings strongly argue for the existence of a CASR- mediated circuit that stabilizes serotonin secretion in type III cells at fluctuating Caf 1–2 mM B, the CA2 mM (2+ . The inv


SCGN is expressed largely in pancreatic β-cells, certain parts of the brain, and also in neuroendocrine tissues. The expression of SCGN is altered in several diseases, such as diabetes, cancers, and neurodegenerative disorders; however, the precise associations that closely link SCGN expression to such pathophysiologies are not known. In this work, we report that SCGN is an early responder to cellular stress, and SCGN expression is temporally upregulated by oxidative stress and heat shock. We show the overexpression of SCGN efficiently prevents cells from heat shock and oxidative damage. We further demonstrate that in the presence of Ca2+, SCGN efficiently prevents the aggregation of a broad range of model proteins in vitro. Small-angle X-ray scattering (BioSAXS) studies further reveal that Ca2+ induces the conversion of a closed compact apo-SCGN conformation into an open extended holo-SCGN conformation via multistate intermediates, consistent with the augmentation of chaperone activity of SCGN. Furthermore, isothermal titration calorimetry establishes that Ca2+ enables SCGN to bind α-synuclein and insulin, two target proteins of SCGN. Altogether, our data not only demonstrate that SCGN is a Ca2+-dependent generic molecular chaperone involved in protein homeostasis with broad substrate specificity but also elucidate the origin of its altered expression in several cancers. We describe a plausible mechanism of how perturbations in Ca2+ homeostasis and/or deregulated SCGN expression would hasten the process of protein misfolding, which is a feature of many aggregation-based proteinopathies.Secretagogin (SCGN) is a three-domain hexa-EF-hand Ca It is hecretion , 2, 3. Secretion , 5, 6, necretion , 8, 9, cecretion , 11, 12,ecretion . The conecretion , 17, 18.2+ sensor . After steadily increasing for 3 h postheat shock treatment, a gradual decline in Scgn expression was observed till 6 h. The expression of inducible heat shock protein 70 (Hsp70), which was used as a positive control was found to be maximum at 6 h which coincides with the previous reports . This was compared with glucose-regulated protein (Grp78) levels, a known marker for oxidative stress . Oxidative stress induced by rotenone treatment resulted in an increase in SCGN protein levels for up to 9 h, followed by a significant reduction in the protein levels (E and F). Consistent with the transcript level, GRP78 protein level was found to be upregulated 6 h post rotenone treatment. The transient increase in transcript levels of SCGN during the initial short-term recovery phase after heat shock or oxidative stress is followed by rapid transcript degradation (A and B). Likewise, the SCGN protein levels elevate during the early response to both heat shock and oxidative stress, but during the late response to oxidative stress, SCGN is maintained at a lower steady-state level (D and F). This indicates that SCGN is an early responder to stress.To check if the change in transcript level also translates to a change in SCGN protein levels, we examined SCGN levels after heat shock and rotenone treatment. A noticeable increase in SCGN levels, similar to that seen in HSP70 expression, was seen until 6 h of recovery after heat shock treatment , C and Dn levels , E and Fradation , A and Bte level , D and F2+ sensor protein, all the experiments described above were performed either in the presence of EDTA or Ca2+. As expected, the sample turbidity of the substrates alone  rapidly increases with time and eventually reaches a maximum saturation represented as a plateau phase (A–H). BSA, used as a control, was merely effective or even displayed slightly enhanced aggregation (with ADH and MDH) but did not reduce aggregation, which is consistent with earlier reports (2+ (A–D). In the presence of Ca2+, the aggregation of substrates was suppressed substantially as compared to that observed in the absence of Ca2+ (E–H). While SCGN could reduce the aggregation in the absence of Ca2+, the efficacy of chaperone action of SCGN was increased significantly in the presence of Ca2+ (80–90%) (I–L). In the case of ADH, about 50% protection was observed in the presence of EDTA, which increased to 90% in the presence of Ca2+ (I). At a 1:2 M ratio of SCGN and substrates, SCGN could reduce scattering  to ∼40%, while at a ratio of 2:1, the inhibition of aggregation was more than 80%, suggesting dose-dependent inhibition of aggregation by SCGN (I–L). These results suggest that Ca2+ drastically increases the anti-aggregation activity of SCGN and might be a prerequisite for efficient chaperone action of SCGN in the cellular milieu.The stress-inducible expression of SCGN points toward a possible chaperone-like activity of SCGN. To establish this, we investigated if SCGN would act as an antiaggregant against various model proteins: alcohol dehydrogenase (ADH), citrate synthase (CS), lysozyme, and malate dehydrogenase (MDH). While the aggregation of the heat-labile enzymes ADH, CS, and MDH was induced by heating them at 41.5, 45, and 50 °C respectively, the aggregation of lysozyme was induced chemically by incubation with 20 mM DTT , 32, 33.au phase , A–H. BS reports . Howeverorts  I–L. In t of Ca2+ I. At a 1 by SCGN I–L. ThesA). Moreover, SCGN overexpression further increased luciferase activity up to 80% after 4 h of recovery, suggesting the role of SCGN in refolding and regaining luciferase activity. The control and GFP-transfected cells did not show any increase in luciferase activity.Having established that SCGN possesses strong anti-aggregation properties toward general protein substrates, we explored the chaperone activity of SCGN in the cellular milieu by luciferase assay. Taking advantage of the fact that luciferase loses its activity when exposed to heat shock, we checked if SCGN overexpression would rescue luciferase from heat-induced loss of activity. HEK-293T cells were cotransfected with a pGL3 vector containing luciferase construct and eGFP(N3)-SCGN construct. SCGN overexpression was validated in HEK-293T cell line before performing the experiment using RT-PCR and Western blot . To ruleFig. 3in vitro and ex vivo, we assessed if SCGN action is similar to that of an HSP aiding cell survival during cellular stress. Cell death was induced by treating HEK-293T cells with increasing concentrations of rotenone. Cell viability was examined by MTT assay in the rotenone-treated cells with or without SCGN overexpression (B). In contrast, SCGN overexpressing cells were up to 35% more viable than the control cells, demonstrating that SCGN plays a role in rescuing from cell death.Under stress, the expression of HSPs is increased to alleviate protein misfolding and cell death. As SCGN expression was temporally altered by heat and oxidative stress and SCGN exhibits chaperone activity both pression . A substpression B. In con2+ sensor protein that exhibits Ca2+-dependent molecular chaperone activity, we next analyzed the effect of Ca2+ on the structural rearrangement of SCGN using small-angle X-ray scattering (SAXS). To ensure sample purity and homogeneity, a quality check of purified samples was performed before SAXS experiments by SDS-PAGE and gel filtration analysis (A and B). The homodispersity of the SCGN samples was also ensured by dynamic light scattering analyses prior to SAXS (C). SAXS data of protein solutions in the range of 5 to 17 mg/ml concentrations and of the respective matched buffers as blank controls were collected. Data obtained with increasing concentrations of SCGN exhibit a profile characteristic of a compact globular molecule as depicted from the log I(q) versus log q plot (A). The Kratky plot confirms a well-folded conformation and exhibits only subtle differences between apo, Ca2+-, and Mg2+-bound SCGN (B). At low q values, a plot of ln(I(q)) versus q2 is linear and independent of protein concentration indicating the homogeneity of the protein sample as observed in the Guinier plot (A). The Guinier Rg calculated from the Guinier plot correlates well with distance distribution function (P(r)) Rg and CRYSOL Rg calculated from the crystal structures (PDB ID: 2be4 and 6jlh) of Danio rerio SCGN (B). The protein samples remained unaffected post-X-ray exposure as confirmed with SDS-PAGE analyses of post-SAXS samples (A). This was further corroborated with MALDI analysis of SCGN samples before and after X-ray exposure. The m/z peak corresponding to a molecular weight of 33 kDa (monomeric SCGN) did not alter (B and C).Since SCGN is a Caanalysis , A and B to SAXS C. SAXS dg q plot A. The Krund SCGN B. At lowier plot A. The Gurio SCGN B. The pr samples A. This wot alter , B and Cg) of apo-protein was found to be 23.4 Å, which increases drastically to 37.7 Å upon the addition of Ca2+. Interestingly, at a higher protein concentration (13.5 mg/ml), the radius of gyration in the presence of Ca2+ increased to 47.5 Å. On the other hand, even the saturated level of Mg2+ (8 mM) did not induce any change in Rg value (23.7 Å) with increasing concentrations, suggesting a Ca2+-specific structural rearrangement .At protein concentrations of ∼5 mg/ml, the radius of gyration , the Rg value of apo-SCGN was 22.8 Å, which was slightly increased to 25.6 Å in oxidizing conditions (in the presence of 100 μM H2O2). SCGN exists as a monomer in reducing conditions, while in oxidizing conditions, the protein tends to oligomerize. However, the effect of Ca2+ was more pronounced in oxidizing conditions. At low protein concentrations of holo-SCGN under oxidizing conditions, the Rg value was increased to 37.4 Å, and at higher protein concentrations, the Rg value was further increased to 46.9 Å. However, the Rg value of holo-SCGN in reducing conditions was increased only up to 33.3 Å, suggesting that the increase of Rg is not due to disulfide-mediated dimerization but is a consequence of Ca2+ binding  in reducing conditions is increased to 92 Å (from 75 Å for apo-SCGN), while in oxidizing conditions, Dmax increased considerably to 184 Å (from 106 Å for apo-SCGN). This supports our earlier data which showed that SCGN forms oligomers in a concentration-dependent manner under oxidizing conditions, while the addition of DTT abrogates oligomerization . On the other hand, Ca2+-bound SCGN in the presence of DTT remains a monomer that adopts a partially open conformation with the two terminal domains flanking away from the central region. Thus, intracellular Ca2+ concentration and cellular redox status would enable SCGN to adopt multiple conformations resulting in diverse substrate binding, a characteristic of a molecular chaperone.It is also important to note that the Drization , 36. Whig values B. On the2+ binding causes major rearrangements with the relocation of both terminal domains and the formation of an open elongated structure, which is largely an extended arrangement of the domains. To reveal the progression of intermediate events, structural changes in SCGN (7 mg/ml) were monitored by SAXS by the sequential addition of Ca2+. The Rg value of the apo-SCGN (23.2 Å) did not change significantly when titrated with Ca2+ up to 100 μM Ca2+ but reduced marginally to 22.7 Å at 150 μM Ca2+  and Dmax (93 Å) was noted, signaling the initiation of a conformational transition. At this Ca2+ concentration, the V-shape geometry of apo-SCGN turns toward a U-shape, as seen in the GASBOR model, whereas one of the terminal domains is already flanked away as seen in the DAMMIF model (2+ concentrations (250 μM [Ca2+] for DAMMIF model and 400 μM [Ca2+] for GASBOR model), the protein begins to open up, as displayed by the movement of one domain being pushed away from the center of the protein. Finally, at saturated Ca2+ concentrations (1 mM Ca2+), the Rg (31 Å) and Dmax (123 Å) values increased, indicating the opening of the molecule into an extended conformation  and open (holo-SCGN) conformations of SCGN, we decided to dissect the dynamics of these conformational changes. As shown above, Ca μM Ca2+ . At 200 IF model , and S7.ormation . In othe2+ binding, we analyzed the SAXS data using the MULTI-FOXS program, which provides population-weighted ensembles fitting to a SAXS profile of the protein in solution  (A). The model built from the SAXS data of the SCGN at saturated Ca2+ concentration fits well with the crystal structure of Ca2+-bound D. rerio SCGN (PDB ID: 6jlh) (B). A substantial expansion of SCGN molecule from about 65 Å in the apo form to 82 Å in Ca2+-bound form is observed indicating the adoption of an open conformation exerted by Ca2+ binding to the molecule (A and B). The models of apo- and Ca2+-bound SCGN overlap only in the C-terminal domain, whereas Ca2+ causes a large shift of the N-terminal domain along with the central region (C). The intermediate state during this transition captured at 250 μM [Ca2+] is a mixed ensemble of three randomly selected ensembles with several different orientations that do not overlap at all, demonstrating the high flexibility of this intermediate state (D). This is further corroborated by a sharp increase in the Rg value  and Ca2ID: 6jlh ) from D.D: 2be4) A. The moD: 6jlh) B. A subsmolecule , A and Bl region C. The inte state D. This i of SCGN . Unlike din D28K . We thus2+ transforms SCGN from closed to open conformation. To investigate how this process impacts the interaction of SCGN with client proteins, we selected α-synuclein, an intrinsically unstructured protein prone to aggregation and implicated in proteinopathy, and insulin known to interact with SCGN (2+ follows an endothermic process and data best fit to one-set of binding site model (A). The binding of SCGN to α-synuclein is an enthalpically unfavorable but entropically favorable process with enthalpy change (ΔH) being 1.24 cal/mol and entropy change (ΔS) being 435 cal/mol/deg. The dissociation constant (Kd) for binding of α-synuclein to SCGN is 110 μM, indicating a weak binding. We further confirmed it by monitoring the stability of SCGN–α-synuclein complex using analytical gel filtration. We found that SCGN does not form a stable complex with α-synuclein suggesting a weak interaction with the substrate (2+-free SCGN binds insulin with an affinity of (Kd) ∼213 μM which, in the presence of Ca2+, increases up to ∼73 μM (C and D). These results demonstrate that Ca2+ modulates the binding positively. Our results align with the finding that the binding of target proteins to chaperones is weak which enables target proteins to dissociate easily from the complex to be available to form stronger interactions of the folded state , suggesting that binding of SCGN and α-synuclein takes place only when Ca2+ is present. This highlights the indispensable role of Ca2+ in mediating structural changes in SCGN, which allows substrate binding.It is seen above that Caith SCGN , 23. Thete model A. The biubstrate . The into ∼73 μM , C and Ded state . On the t change B, sugges2+ leading to the creation of a binding site for substrates led us to assign the role of each domain, if any, in imparting chaperone activity. To this end, we prepared two deletion constructs of SCGN: C-terminal deleted (ΔCTD) and N-terminal deleted (ΔNTD) constructs. The recombinant ΔCTD (20.4 kDa) and ΔNTD (19.1 kDa) proteins having the central domain in common were tested for chaperone activity. The choice of selection of two domains out of the three was to have stable protein preparations. While both proteins possess chaperone activity, there was a variation in the ability of ΔCTD (20.4 kDa) and ΔNTD (19.1 kDa) to suppress aggregation at identical protein to substrate ratios, depending on the model substrate. For example, in the case of CS, ΔCTD was more efficient than ΔNTD in precluding aggregation, while in the case of lysozyme, ADH, and MDH, ΔNTD was more efficient , SCGN could act as a Ca2+ buffer, while in the cytosol’s reducing environment, it might act as a Ca2+ sensor  aggregation of a wide range of proteins  process , 51. Ano+ sensor , 36, 52.We further checked if SCGN could mediate its chaperone functions in a cellular scenario. We found that SCGN overexpression prevents the loss of luciferase activity in HEK-293T cells. Our results further bolster the earlier published results of SCGN-mediated inhibition of fibrillogenesis of insulin and α-synuclein , 23. Bas2+ and elucidate why deregulated SCGN levels accelerate protein misfolding disorders. Reduced levels of SCGN in certain tissues, as noted in protein folding diseases  protein misfolding diseases, such as Parkinson’s and Alzheimer’s disease. A reduction in SCGN expression, or even a perturbation in Ca2+ homeostasis, would consequently affect SCGN’s propensity to function as a chaperone, which would hasten the aggregation of the concerned proteins, accelerating the onset of disease.We now identify chaperone activity as one of the crucial functions of SCGN vis-a-vis Cadiseases , 9, 24, 2+ in the augmentation of SCGN’s chaperone activity. BioSAXS has provided us with an innovative perspective on the Ca2+-dependent folding mechanism of SCGN in unprecedented detail. The pair distribution curve (the P(r) function), the Guinier plot, Io/C value, and concentration-dependent monitoring of Rg values are good quality control checks, and the current work appropriately accounted for them. Monitoring the ensembles of structures during the sequential addition of Ca2+ has allowed us to display the presence of a conformational switch of SCGN. Ca2+ binding triggers the drifting of both terminal domains away from the central domain, creating an open groove, enough to accommodate the binding of a broad range of substrates. The ends of both terminal domains appear to move away from each other, with the connecting residues—between NTD and the central domain (residues 94–100) and between the central domain and CTD (residues 173–178)—acting like flexible hinges (computed using the HingeProt program) (2+ bound). It is to be noted that a similar Ca2+-dependent elongation of the structure is reported in gelsolin, a Ca2+-binding protein  . This dr protein , which i binding . Thus, o2+-dependent chaperone and its role in stress physiology has helped resolve this enigma. This study enables the extraction of important insights into the multifunctional nature of SCGN and lays a foundation for future therapeutic discoveries for neurodegenerative diseases exploiting the SCGN model.To summarize, the present study describes a previously unidentified function of SCGN wherein it not only efficiently chaperones various protein substrates but also helps in maintaining a healthy proteome in cells during conditions of stress. The interplay between stress, neurodegenerative diseases, and SCGN expression was unclear for a long time. The importance of SCGN as a CaAlcohol dehydrogenase lyophilized powder sourced from baker’s yeast was obtained from SRL. CS and MDH sourced from the porcine heart (Sigma), and lysozyme from chicken egg white (Sigma) were used for chaperone assays. The antibodies used in these experiments are anti-SCGN bs-, anti-Hsp70 , anti-GRP78, anti-β-Actin , and HRP conjugated secondary antibody .E. coli BL21-DE3 cells was induced with 1 mM IPTG at A600. of 0.6 and incubated at 25 °C for 10 h postinduction as reported previously  were incubated at respective temperatures with constant stirring and monitored at the abovementioned wavelengths. The reaction mixtures of each substrate with the deletion constructs of SCGN (ΔNTD and ΔCTD) were also incubated at temperatures as mentioned above, and absorbance was monitored at 500 nm, 450 nm, 465 nm, and 360 nm wavelengths for CS, MDH, ADH, and lysozyme, respectively. Experiments were performed with varying stoichiometric ratios of SCGN to substrates . All the experiments were performed in triplicates, and the obtained data were analyzed and plotted using Origin Lab (2019b version) and GraphPad Prism 8. The percent change in chaperone activity of SCGN is calculated using,Substrate is the absorbance at a respective wavelength for the substrates used , and ASCGN + Substrate is the highest absorbance value obtained with substrate and SCGN.Aggregation kinetics was monitored in time-dependent mode on a Lambda 35 UV spectrophotometer (PerkinElmer) equipped with a water bath or on a Hitachi Fluorescence Spectrophotometer F-7000 connected to a temperature controller (TCC100). Thermal aggregation of ADH (1.3 μM), MDH (1.5 μM), or CS (2 μM) solutions in 50 mM Hepes, 100 mM KCl, pH 7.5, was induced at 41.5 °C, 50 °C, and 45 °C respectively, and monitored at 465 nm, 450 nm, and 500 nm, respectively , 32, 33.2 incubator. Luciferase construct in basic pGL3 vector [procured from Promega luciferase assay system kit ], pEGFP(N3)-SCGN plasmid, and SCGN-eGFPN3 constructs were used for transient overexpression in HEK-293T cells. Transfection of cells was performed with Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's protocol.RIN-5F and HEK-293T cells were cultured in RPMI and DMEM media, respectively, supplemented with 10% fetal bovine serum  and maintained at 37 °C in a 5% CORIN-5F cells (0.2 million) were seeded in a T-25 flask. After 24 h, the cells were subjected to heat shock at 42 °C for 45 min. Cells without heat shock were used as control. Samples in triplicate were collected at 0, 1, 2, 3, 4, 5, and 6 h of recovery from heat shock. The cells were either lysed in RIPA buffer for Western blotting or in Trizol reagent for qRT-PCR analysis.5 cells/well. The next day, the cells were incubated with 1 μM rotenone (Sigma), and the samples were collected at 3, 6, 9, 12, 18, and 24 h. Untreated cells were maintained as control. Cells were lysed in RIPA buffer for Western blotting and in Trizol reagent for RNA preparation for qRT-PCR. Each experiment was performed in triplicate.RIN-5F cells were seeded in a 6-well plate at a density of 2 × 10Protein in cell lysate was quantified using a Takara BCA protein estimation kit. For each sample, 35 μg total protein was loaded on 12% SDS PAGE gel and transferred onto a nitrocellulose membrane at 100 V for 2 h. After blocking with 5% BSA for 2 h, blots were incubated overnight at 4 °C with either anti-SCGN antibody (1:4000) or anti-HSP70 antibody (1:2000) or anti-GRP78 (1:2000) or β-Actin . The next day, the blots were washed with TBST  four times at 10 min intervals. Subsequently, the blots were incubated with anti-mouse or anti-rabbit secondary antibody conjugated with HRP  for an hour. Post incubation, the blots were washed four times every 10 min with TBST and developed using the chemiluminescence method with a Bio-Rad ECL substrate kit. The obtained bands were analyzed using Image J software and normalized with β-actin.-ΔΔct as a mean of at least three technical replicates. Primers for the target genes used for qRT-PCR are listed in Supplementary Information . mRNA expression levels were quantified by qRT-PCR on an ABI Prism 7900 HT (Applied Biosystems) using the SYBR Green detection system (Applied Biosystems). The reactions were performed according to the following conditions: initial hold at 95 °C for 30 s, followed by 40 cycles of amplification at 95 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. At least three technical replicates were prepared in each group of samples. β-actin/RPL11 or TBP was used as a reference control. The relative mRNA expression was calculated using the 2ormation .2 incubator. Subsequently, MTT was removed, 100 μl of DMSO was added to dissolve MTT crystals and incubated for 30 min at 37 °C. Post incubation, the absorbance was recorded at 540 nm in an EnSpire Multimode plate reader and for% cell viability was calculated in the percentage of untreated control cells. Each group consisted of eight biological replicates.HEK-293T cells transfected with either pEFGP(N3) or pEGFP(N3)-SCGN constructs were seeded at a cell density of 15,000 cells/well in a 96-well plate. After 24 h, cells were incubated with increasing concentrations of rotenone (Sigma)  for 24 h. Cells without any treatment and cells treated with 5% Triton X-100 were employed as controls. After 24 h of incubation, the cells were washed twice with PBS. Thereafter, 0.5 mg/ml of MTT was added to serum-free DMEM and incubated for 4 h at 37 °C in a CO6) seeded in 6-well plates were transfected using lipofectamine LTX reagent (Invitrogen) by following the manufacturer’s protocol. Transfection was performed in incomplete media with firefly luciferase pGL3 vector . Either only luciferase or luciferase with pEGFP(N3)-SCGN construct were cotransfected into cells. Luciferase and pEGFP(N3) vector cotransfection were used as controls. The transfection medium was replaced by DMEM complete media after 6 h. After 36 h of transfection, cells were incubated with 20 μg/ml cycloheximide for 30 min to suppress the expression of other chaperones. Subsequently, cells were subjected to heat shock at 42 °C for 45 min. Untreated cells were used as control. The luciferase activity for one set of cells was measured immediately after heat shock treatment, while for another set of cells after 4 h of recovery using EnSpire Multimode Plate Reader for reading time of 5 s with an initial delay time of 2 s in luminescence mode. All experiments were performed in triplicates. The percentage luciferase activity is calculated considering without heat shock (WHS) as a control for each group using the formula.HEK-293T cells (0.2 × 102O2) with multiple concentrations of apo-SCGN ranging from 5 mg/ml to 15 mg/ml and in the presence of either 8 mM Ca2+ or 8 mM Mg2+. The protein solutions were prepared in 50 mM Tris, pH 7.5, 100 mM KCl buffer. The matched buffer for each sample was collected from flow through and used as a buffer blank.SAXS data were collected as mentioned earlier on a Rigaku BioSAXS 2000 system at a wavelength of 1.54178 Å with 2D Kratky collimation, equipped with Rigaku HyPix-3000 hybrid pixel array detector attached to MicroMax 007 HF generator, operated at 40 kV and 30 mA . SAXS ex2+ titration (from 50 μM to 1 mM) with SCGN concentration at 7 mg/ml (with 5 mM DTT). It was an appropriate concentration at which the quality of data was good . The sample and its matched buffer were exposed to X-ray for 60 min. The data were processed with the Automatic Data Analysis Pipeline (AAP), which is based on the ATSAS suite (version 2.7) and ATSAS online (Version 3.7) (Second set of experiments included Caion 3.7) . The Gui2+-bound structure (PDB ID: 6jlh) of D. rerio SCGN.All SAXS models were constructed using DAMMIN and GASBOR online tools , 62. Mul2+. Insulin binding to SCGN in the absence/presence of Ca2+ were performed on a Microcal VP-ITC instrument at 30 °C in Chelex-purified 50 mM Tris, pH 7.5, 100 mM KCl. All experiments were performed using 1 mM insulin in the syringe and 30 μM SCGN. For Ca2+ free titrations, 100 μM EGTA was added both to SCGN and insulin, while for Ca2+ saturated titrations, 3 mM Ca2+ was added both to SCGN and insulin. The graphs obtained were buffer subtracted by titrating into α-synuclein or insulin with buffer under similar conditions. Data fittings were performed with the help of Origin 8 software.The energetics of SCGN binding to substrates (α-synuclein and insulin) were studied at 30 °C on an ITC-200 instrument. SCGN samples (60 μM in the cell) and 600 μM α-synuclein (in the syringe) were prepared in Chelex-purified 50 mM Tris buffer, pH 7.5, and 100 mM KCl in the presence of 2 mM CaDynamic light scattering measurements of SCGN (13 mg/ml) samples prepared in buffer  were performed on an SZ-100 Horiba instrument SCGN (13 mg/ml) at room temperature with 20 accumulations each time. A buffer blank reading was used for buffer subtraction. Prior to analyses, the protein samples were centrifuged at 15,000 rpm for 15 min.SCGN samples (7 mg/ml) to be analyzed were mixed with equal volumes of a sinapinic acid matrix (1:1). The 2 μl mixed sample was loaded onto a MALDI plate and allowed to air dry. The MALDI plate was placed in a mass spectrometer to acquire the spectra. The m/z values were analyzed using the software MASCOT.p-value of <0.05 was considered statistically significant between different test conditions determined by a two-sided Student’s t test for western and qRT-PCR experiments. Data from luciferase and MTT assay were analyzed, and p-value (<0.05 significance) was calculated by two-way ANOVA with Bonferroni multiple comparison test.All data were analyzed using GraphPad Prism 8 and expressed as mean ± standard deviation. A All data generated and analyzed during this study are included in this article.This article contains The authors declare that they have no conflict of interest with the content of this article.


ATRA has greatly improved the treatment of APL. Since 1,25-dihydroxyvitamin D  is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs was widely accepted. However, early clinical trials in which cancer patients were treated either with 1,25D or with analogs did not lead to conclusive results. Recent results have shown that AML types with certain mutations, such as isocitrate dehydrogenase (IDH) mutations, may be the right targets for differentiation therapy using 1,25D, due to upregulation of vitamin D receptor (VDR) pathway.Acute myeloid leukemia (AML) is an aggressive and often fatal hematopoietic malignancy. A very attractive way to treat myeloid leukemia, called “differentiation therapy”, was proposed when in vitro studies showed that some compounds are capable of inducing differentiation of AML cell lines. One of the differentiation-inducing agents, all- Acute myeloid leukemia (AML) is a malignancy of the myeloid blood lineage, characterized by the rapid growth of abnormal cells (blasts) in the bone marrow. The blast cells overgrow bone marrow, preventing normal blood cell production, and expanding to circulation, where they are unable to function properly. Since leukocytes produced in bone marrow belong to the immune system, every AML is accompanied by an immune deficiency resulting in vulnerability to infections. In addition, inability to produce appropriate amounts of red blood cells and platelets results in anemia and bleeding .The primary goal in the treatment of AML is an elimination of leukemic blasts. However, chemotherapy blocks not only the proliferation of blasts, but also the proliferation of immune cells, an essential step in immune cells’ activation. Therefore, chemotherapy-induced immunodeficiency adds to leukemia-induced immunodeficiency .AML is a relatively rare disease which constitutes about 1% of all malignancies. It is a disease common in elderly people and very rare in children, with about 25% of cases diagnosed among adults aged 65–74 years and 34% among these aged 75 and older . This aberration leads to the fusion between the promyelocytic leukemia gene  in differentiation therapy originated from a study published in 1981, where mouse myeloid leukemia cells exposed in culture to 1,25D were induced to differentiate into functional macrophages . Wh. WhFGFRsFGFR family contains five genes, out of which four encode transmembrane tyrosine kinase receptors that exist in multiple splicing variants. Binding of the ligand to FGFRs results in a dimerization of these receptors and transphosphorylation of their tyrosine kinase domains [VDR expression.The  domains . As a re domains . It has  domains ,93. In c domains . Our unpAML is a disease of the elderly, and the proportion of older people is increasing steadily in modern societies. The current estimate of the probability of developing cancer is one in two for people born after 1960 , and des


Aim: The main goal of this study was to elucidate at the transcript level the tyrosine kinase expression profiles of primary leukemia cells from mixed lineage leukemia 1 gene rearranged (KMT2A/MLL-R+) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients.Methods: We evaluated protein tyrosine kinase (PTK) gene expression profiles of primary leukemic cells in KMT2A/MLL-R+ AML and ALL patients using publicly available archived datasets. Results: Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified. Conclusion: These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias. Rearrangements (r) of the KMT2A/MLL gene have been reported in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)-14. AML and ALL patients with KMT2A/MLL-R+ leukemia have a poor prognosis with disappointing event-free survival (EFS) and overall survival (OS) outcomes on contemporary treatment regimens due to relapses caused by cancer drug-resistant clones-19. Therefore, more effective frontline as well as salvage treatments for MLL-R+ acute leukemias are urgently needed. The lysine [K]-methyltransferase 2A (KMT2A)/mixed-lineage leukemia 1 (KMT2A/MLL) gene on chromosome 11 encodes a 431-kDa protein involved in the regulation of transcription+ AML and ALL as potential therapeutic targets to overcome cancer drug resistance. We compared the transcriptomes of primary leukemic cells from KMT2A/MLL-R+vs. KMT2A/MLL-R- AML and ALL patients with an emphasis on the relative gene expression levels of for 21 PTK, including ERBB1, FGR, FLT3, FYN, HCK, JAK2, LCK, LYN, MERTK, SRC, BLK, BMX, BTK, ERBB2, ERBB3, JAK1, JAK3, PTK2, SYK, TEC, TYK2. Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family member TYK2, and the SRC family PTK HCK and FGR were differentially amplified. These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias. The main goal of the present study was to evaluate the protein tyrosine kinase (PTK) profiles of primary leukemic cells in patients with KMT2A/MLL-R. We also used a working database derived from archived datasets including data on primary leukemia cells from 542 adult patients with AML (GSE13159), and 279 pediatric patients with AML  along with the 74 normal/non-leukemic control bone marrow samples (GSE13159) in our comparative gene expression analyses, as described. Probeset level normalization procedures were used as previously reported. A recently published working database, including data on primary leukemia cells from 201 adult patients with B-ALL (GSE13159), 119 pediatric patients with B-ALL (GSE11877 and GSE13351) and 97 infants with B-ALL (GSE68720), as well as 74 normal/non-leukemic control bone marrow samples (GSE13159), was built with previously archived datasets from the NCBI repository and used in our comparative gene expression analyses, as previously described in detail+ pediatric AML patients (N = 48 from GSE17855) with those of primary leukemia cells from 189 FLT3-ITD- pediatric AML patients (GSE17855) as well as normal hematopoietic cells from 74 non-leukemic control bone marrow samples (GSE13159). Forty-seven patients from the KMT2A/MLL-R- “other” group of pediatric AML patients were FLT3-ITD+, including 18 patients with cytogenetically normal AML, one patient with inv AML, 12 patients with t AML, 3 patients with t, 9 patients with AML and other cytogenetic features, and 4 patients who had AML with unknown cytogenetic features. One pediatric patient with KMT2A/MLL-R+ AML was FLT3-ITD+. This double mutant (FLT3-ITD/MLL-R) case was removed from the analysis of FLT3-ITD+  versus KMT2A/MLL-R+ pediatric AML patients . We also compared the gene expression profiles of primary leukemia cells from FLT3-ITD+ AML harbored NPM1 or CEBPA mutations. Within the KMT2A/MLL-R- (“other”) group of KMT2A/MLL-R+ AML pediatric AML patients, 17 harbored NPM1 mutations (NPM1+) and 16 patients had CEBPA mutations (CEBPA+) (GSE17855). We investigated the gene expression profiles from primary leukemia cells from 71 KMT2A/MLL-R- pediatric AML patients who were FLT3-ITD+, NPM1+ or CEBPA+  versus 88 cases of KMT2A/MLL-R+ . One patient with both FLT3-ITD and MLL-R was removed from this comparison.No pediatric patients with KMT2A/MLL-RERBB1, FGR, FLT3, FYN, HCK, JAK2, LCK, LYN, MERTK, SRC, BLK, BMX, BTK, ERBB2, ERBB3, JAK1, JAK3, PTK2, SYK, TEC and TYK2. Standard statistical methods, including mixed model ANOVAs, and hierarchical clustering method were employed, as reported-24. Our analyses for ALL and AML focused on the expression levels (interrogated with 64 probesets) of the following 21 PTK genes: N = 80), children (N = 25) and adults (N = 70) with KMT2A/MLL-R+ B-ALL vs. normal hematopoietic cells from healthy volunteers (N = 74). Notably, FMS-like tyrosine kinase 3(FLT3) expression in infant KMT2A/MLL-R+ ALL cases was 16.09-fold higher than in normal hematopoietic cells in non-leukemic control bone marrow samples  . The genes for several additional PTK showed augmented expression in KMT2A/MLL-R+ infant ALL cells, including TEC, SRC, BLK, JAK2, BTK, PTK (3 probesets), SYK and JAK1 (2 probesets) . As the second most upregulated gene, the gene for BLK was expressed at a 6.77-fold higher level in infant KMT2A/MLL-R+ ALL cells than in normal hematopoietic cells  [Supplementary Table 1]. Similarly, FLT3 wasexpressed at a 22.38-fold higher level in pediatric KMT2A/MLL-R+ ALL cells than in normal hematopoietic cells  . As in infant leukemia cells, BLK was the second most upregulated gene in pediatric KMT2A/MLL-R+ ALL cells showing a 4.38-fold higher expression level than in normal hematopoietic cells . The genes for several additional PTK showed augmented expression in pediatric KMT2A/MLL-R+ ALL cells, including PTK2, TEC, BTK, and SYK . Similarly, FLT3 and BLK expression in leukemic cells from adult patients with KMT2A/MLL-R+ ALL were 21.00-fold  and 21.79-fold , respectively, higher than in normal hematopoietic cells . We first examined PTK gene expression profiles of primary leukemic cells from infants . In infants [Supplementary Table 4], children [Supplementary Table 5] and adults [Supplementary Table 6] with KMT2A/MLL-R+ ALL, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK1, as well as several SRC family PTK, including BLK, were differentially and significantly amplified. In KMT2A/MLL-R+ infant ALL cells, FLT3_206674_at was the most significantly upregulated probeset  followed by BLK_206255_at  when compared to infant ALL cells with germline/wildtype KMT2A/MLL gene [Supplementary Table 4]. Likewise, in KMT2A/MLL-R+ pediatric ALL cells, FLT3_206674_at was the most significantly upregulated probeset  followed by BLK_206255_at  when compared to pediatric ALL cells without KMT2A/MLL rearrangements [Supplementary Table 5]. Similarly, in KMT2A/MLL-R+ adult ALL cells, FLT3_206674_at was the most significantly upregulated probeset  followed by BLK_206255_at  when compared to leukemic cells from adult ALL patients without KMT2A/MLL rearrangements [Supplementary Table 6]. We next compared the PTK gene expression profiles of leukemic cells from each KMT2A/MLL-R+ AML vs. normal hematopoietic cells from healthy volunteers (N = 74). In comparison to normal hematopoietic cells, KMT2A/MLL-R+ pediatric as well as adult AML cells were characterized by amplified expression of FLT3 gene. FLT3_206674_at was the most significantly upregulated probeset  followed by TEC_206301_at  .We examined the PTK gene expression profiles of primary leukemic blasts from 89 children and 38 adults with KMT2A/MLL-R+ AML cells to the PTK gene expression profiles of AML cells without KMT2A/MLL rearrangements from the corresponding control AML patients in the “other” categories . In pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified [Supplementary Table 9]. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified [Supplementary Table 10]. We next compared the PTK gene expression profiles of leukemic cells from pediatric and adult KMT2A/MLL-R+ subset (N = 48) among the pediatric AML patients exhibited upregulated FLT3 expression when compared to non-leukemic control samples (N = 74)  as well as FLT3-ITD- pediatric AML cases (N = 189) . Comparing 189 cases of FLT3-ITD- samples with 48 cases of FLT3-ITD+ pediatric AML samples (GSE17855) exhibited 13 differentially expressed probesets, of which four probesets were significantly upregulated in pediatric FLT3-ITD+ cases. FLT3_206674_at was the most significantly upregulated probeset  followed by BTK_205504_at  and TEC_206301_at  .The FLT3-ITD+ pediatric AML cases with 47 FLT3-ITD+ pediatric AML cases showed 31 differentially expressed probesets, of which 24 probesets were significantly upregulated in pediatric KMT2A/MLL-R+ cases. FGR_208438_s_at was the most significantly upregulated transcript  followed by SYK_207540_s_at  and JAK1_1552611_a_at . ERBB1_1565483_at was the most significantly downregulated transcript in KMT2A/MLL-R+ cases  followed by ERBB1_1565484_x_at  and MERTK_211912_at  A comparison of the 88 KMT2A/MLL-R+ pediatric AML cases with 71 cases of FLT3-ITD+/NPM1+/CEBPA+ pediatric AML cases exhibited 34 differentially expressed probesets, of which 26 probesets were significantly upregulated in the KMT2A/ MLL-R+ subset. FGR_208438_s_at was the most significantly upregulated probeset  followed by SYK_207540_s_at  and HCK_208018_s_at . ERBB1_1565483_at was the most significantly downregulated transcript in KMT2A/MLL-R+ cases  followed by ERBB1_1565484_x_at  and MERTK_211912_at  .A comparison of the 88 KMT2A/MLL-R+ AML and B-ALL patients, especially those with relapsed or refractory leukemia, is disappointingly poor after contemporary treatments-8,10-14. New treatment strategies are urgently needed for both KMT2A/MLL-R+ AML and KMT2A/MLL-R+ ALL. Therefore, a large panel of drugs are being evaluated as potential therapeutic agents against KMT2A/MLL-R+ AML and ALL, including inhibitors of Menin-MLL1 interaction-28. The present study demonstrates that PTK inhibitors (PTKi), especially inhibitors of FLT3, may have clinical impact potential as therapeutic agents against KMT2A/MLL-R+ B-ALL as well as AML. Further, inhibitors of SRC family PTK may be clinically useful against KMT2A/MLL-R+ B-ALL and inhibitors of BTK, SYK, and JAK family PTK may be clinically useful against KMT2A/MLL-R+ AML. If our observations in this small series are confirmed in a larger series of leukemia patients, preferably with corresponding proteomics/phosphoproteomics data, proof-of-concept studies with FDA-approved PTKi would be warranted to further evaluate the clinical potential of PTK targeting in KMT2A/MLL-R+ ALL and AML. The outcome of KMT2A/MLL-R-31. The incorporation of PTKi into the standard of care has caused a paradigm shift in the treatment of CML (PTKi: ABL1 and SRC inhibitors), CLL (PTKi: BTK inhibitors), Ph+ B-ALL (PTKi: ABL1 and SRC inhibitors), and AML with FLT3 mutations (PTKi: FLT3 inhibitors),30. There is growing consensus regarding their evolving role in Ph-like B-ALL  and pre-B ALL with t (PTKi: BTK and SRC inhibitors)-53 [+ B-ALL was associated with a very poor outcome in both children and adults, with less than 20% long-term survival until the introduction of PTKi capable of inhibiting the oncogenic BCR-ABL tyrosine kinase-48 [+ B-ALL are no longer considered to have a poor prognosis. The standard of care is being optimized by developing novel combinations of PTKi such as dasatinib and ponatinib with bispecific antibodies such as blinatumomab, CAR-T cells, and antibody-drug conjugates and using less toxic chemotherapy regimens-48. Likewise, FLT3 inhibitors have contributed to improved outcomes in AML patients with a FLT3 mutation. FLT3 is a receptor tyrosine kinase that plays an important role in normal lymphohematopoiesis. Leukemic cells from AML patients abundantly express FLT3 and many AML patients have activating mutations of FLT3, including internal tandem duplication mutations (FLT3-ITD) and kinase domain activation loop mutations (FLT3-ALM),51. FLT3 mutations are rare in ALL, but a high-level expression of FLT3 was also reported in MLL-AF4 positive ALL patients and showed poor prognostic value,53. Our study significantly expands the knowledge regarding FLT3 expression in ALL as well as AML by demonstrating amplified expression levels in infants, children, and adults with KMT2A/MLL-R+ B-ALL and children with KMT2A/MLL-R+ AML. Several PTKi have been approved for the treatment of AML with FLT3 mutations, including midostaurin and gilteritinib [+ ALL. It is noteworthy that Dovitinib, a multi-functional PTKI with FLT3 inhibitory activity, was reported to exhibit nanomolar in vitro activity against KMT2A/MLL-R+ ALL cells. PTK play a critical role in normal lymphohematopoiesis, and they have also been implicated as leukemogenic oncoproteins in the development of acute and chronic leukemiastors)-53 . Notablyinase-48 . Due to eritinib . Our res+ pediatric AML cells. Except for GSE17855, the archived datasets used in the present study did not contain information about the FLT3 status of the respective leukemia cases. Additional KMT2A/MLL-R+ patients whose leukemia cells do not have amplified FLT3 gene expression but an activating FLT3 mutation like FLT3-ITD may also benefit from the use of FLT3 inhibitors. Analysis of the GSE17855 contributed to our finding that in pediatric patients with KMT2A/MLL-R+ AML, the gene expression level for FLT3 is differentially amplified. It is noteworthy that only one patient with KMT2A/MLL-R+ AML had FLT3-ITD whereas 47 patients from the KMT2A/MLL-R negative “other” group of pediatric AML patients were FLT3-ITD+, including patients with cytogenetically normal AML (N = 18); AML with inv (N = 1); AML with t (N = 12); AML with t (N = 3); AML with other cytogenetic features (N = 9); and AML with unknown cytogenetic features (N = 4). Therefore, our results should not be interpreted to suggest that FLT3 inhibitors would be preferentially active in pediatric AML patients with KMT2A/MLL rearrangements, as many cases without KMT2A/MLL rearrangements whose leukemia cells are FLT3-ITD+ may also benefit from FLT3 inhibitors. Some PTK, such as FLT3, participate in immune suppression mediated by leukemia cells, which may promote the immune escape of leukemic clones. Therefore, their inhibition with PTKi may partially contribute to favorable treatment outcomes by lifting the immune suppression. For example, the PTKi sorafenib has been shown to abrogate the transcriptional downregulation of interferon regulatory factor 7 (IRF7), which resulted in an augmented CD8+CD107a+IFN-γ+ T cell response in mouse models of FLT-ITD+ AML. Mathew et al. proposed that sorafenib may therefore exhibit immune-mediated anti-leukemic activity in FLT3-ITD mutant AML. One of the intriguing findings of our study relates to the amplified expression of the gene for FLT3 in KMT2A/MLL-R-31. FDA-approved SRC kinase inhibitors have become part of the standard of care in the treatment of CML as well as Ph+ ALL-48 [+ B-ALL. The gene for BLK was expressed at a 6.77-fold higher level in infant KMT2A/MLL-R+ ALL cells, 4.38-fold higher level in pediatric KMT2A/MLL-R+ ALL cells, and 21.79-fold higher level in adult KMT2A/MLL-R+ ALL cells than in normal hematopoietic cells. Notably, the expression of the genes for BLK, HCK and LYN in primary leukemic cells from KMT2A/MLL-R+ ALL patients was differentially and significantly amplified regardless of the age group compared to the expression levels in leukemic cells from B-ALL patients without KMT2A/MLL gene rearrangements. Likewise, the genes of several SRC family PTK were differentially upregulated in pediatric and adult KMT2A/MLL-R+ AML cells. The availability of FDA-approved potent inhibitors of SRC family PTK such as ponatinib, bosutinib, and dasatinib [+ leukemias in proof-of-concept clinical trials. The insights and lessons learned in the clinical development of these PTKi as precision medicines against Ph+ ALL should facilitate their development as potential precision medicines against KMT2A/MLL-R+ ALL as well. The SRC kinase family includes several cytoplasmic PTK, including BLK, HCK, FGR, LYN, FYN, and LCK, which have important regulatory functions for signal transduction pathways related to survival, proliferation, and apoptosis of leukemic cells+ ALL-48 . In the asatinib  provides+ acute leukemias. These results extend our earlier studies regarding upregulation of the JAK-STAT pathways in infant B-ALL,57 and upregulation of SYK expression in pediatric B-ALL-62. The availability of FDA-approved potent inhibitors of SYK and JAK provides the opportunity to evaluate their clinical efficacy in KMT2A/MLL-R+ acute leukemias with overexpression of their respective targets. A significant portion of high-risk acute leukemia patients, especially those with KMT2A/MLL-R+ ALL or AML relapse after being treated on contemporary chemotherapy protocols due to cancer drug resistance of their leukemic clones, and the survival outcome of available salvage regimens is disappointing due to the short nature and poor quality of second remissions. We previously reported that the JAK/STAT signaling pathway is constitutively active in infant pro-B ALL cells and treatment with a JAK3 inhibitor or a pan-JAK kinase inhibitor effectively triggered their apoptosis,57. JAK targeting with a small molecule inhibitor may be a viable strategy to overcome cancer drug resistance in KMT2A/MLL-R+ B-ALL cells. Constitutively active JAK2-STAT5 signaling has been shown to be associated with increased surface PD-L1 expression due to amplified PD-L1 promoter activity in myeloproliferative neoplasms, which may facilitate PD-L1-mediated immune escape,64. JAK2 inhibition reduces the PD-L1 expression levels and may therefore mitigate this risk. Additional insights from the present study relate to the upregulation of SYK and JAK expression in pediatric and adult KMT2A/MLL-R-62. Our earlier studies have identified high-level SYK expression as a likely contributor to cancer drug resistance and relapse in B-ALL,61. Upregulation of SYK expression was associated with significant upregulation of at least one of the STAT3 target genes. Inhibition of SYK caused apoptotic death in primary leukemia cells from B-ALL patients that are resistant to chemotherapy. Notably, a nanomedicine candidate containing the SYK-inhibiting small molecule compound 1,4-bis(9-O-dihydroquinidyl) phthalazine/hydroquinidine1,4-phathalazinediyldiether (C61) was capable of destroying > 99.9% of clonogenic B-ALL cells in vivo and thereby improved the event-free survival outcome of SCID mice challenged with otherwise invariably fatal doses of human leukemic B-cell precursors in each of three different xenograft models of chemotherapy-resistant human B-ALL. Taken together with these earlier observations, the amplified expression of SYK in the poor prognosis KMT2A/MLL-R+ ALL patients prompts the hypothesis that SYKi may help overcome the cancer drug resistance of relapse clones and thereby provide the foundation for more effective multi-modality treatment regimens for KMT2A/MLL-R+ acute leukemias.SYK has been discovered to regulate the cancer drug resistance-related anti-apoptotic STAT3, NF-κB as well as PI-3K-AKT-mTOR pathways


However, present information on the functions of Ca2+ in plant gravitropism was obtained mainly on coleoptiles, hypocotyls, and petioles, little is known about the dynamic changes of Ca2+ during root gravitropism. In the present study, the transgenic Arabidopsis thaliana R-GECO1 was placed horizontally and subsequently vertically on a refitted Leica SP8 laser scanning confocal microscopy with a vertical stage. Real-time observations indicated that gravistimulation induced not only an increase in the Ca2+ concentration, but also an accelerated occurrence of Ca2+ sparks in the root cap, especially in the lower side of the lateral root cap, indicating a strong tie between Ca2+ dynamics and gravistimulation during the early stage of root gravity response.Gravitropism is an important strategy for the adaptation of plants to the changing environment. Previous reports indicated that Ca For example, using Ca2+ fluorescent indicators or transgenic Ca2+-reporter systems, increases in Ca2+ concentration in response to gravistimulation were already demonstrated in multiple plant species.15–17 Besides, pharmacological treatments with extra cellular Ca2+ chelatorsor, Ca2+-channel blockers, as well as inhibitors of calmodulin or Ca2+/calmodulin dependent protein kinases, inevitably led to altered root gravitropism.19 All these data indicated that Ca2+ might play a vital role in plant gravitropic response.As an intracellular second messenger, Ca2+ dynamics happened, let alone to clarify which phase of gravitropism was affected by Ca2+ dynamics. Especially, plant bending growth results from gravity-induced asymmetric cell elongation,8 conventional cell live-imaging technologies, including laser scanning confocal microscopy, are incompetent for synchronous visualization and quantification of the information between the upper and lower sides of statocytes after gravity stimulation. Hence, real-time observations on intracellular changes of signaling molecules, such as Ca2+ and pH, with a vertical-stage microscope are very promising.20–23Unfortunately, previous studies were carried out mainly on coleoptiles, hypocotyls, and/or petioles, and only a few data were obtained from roots. Besides, due to limited imaging technologies, the temporal and spatial resolution of previous researches was difficult to define the specific cell types where Ca2+ reporters, genetically encoded indicators (GECIs) are appropriate tools to study spatiotemporal information of Ca2+ dynamics in living cells.24–26 R-GECOs are red emitting reporters generated by replacing the cpGFP with cpmApple. Among these reporters, R-GECO1 is an intensity-based Ca2+ reporter capable of monitoring Ca2+ dynamics in plants.26 To further understand gravity-induced dynamic changes of Ca2+ in the root, seeds of transgenic Arabidopsis thaliana R-GECO1 were cultured in 1/2 Murashige & Skoog (MS) with 1% sucrose for 5 days. Seedlings were positioned on the glass bottom of a Petri dish, covered with fresh culture medium containing 1/2 MS, and placed vertically in an artificial climate box for 30 min. Then, the Petri dish was placed on a Leica SP8 laser scanning confocal microscopy refitted with a vertical stage and a 40× oil objective (NA 1.3). All samples were excited with 561 nm and fluorescence emission was detected between 570 and 640 nm using a HyD detector. Laser power and channel setting were kept identical for all samples to make the results comparable. Firstly, time-series images were obtained with root grew vertically down for 15 min. Subsequently, the Petri dish was placed horizontally and time-series images were obtained for additional 60 min. The results indicated that bright fluorescence signals were easily detected in the whole root cap , which was significantly higher than that of vertically placed roots . Similarly, the mean fluorescence intensity of the cells in the lower side of the lateral root cap was significantly increased from 9603 ± 1684 to 11467 ± 2079  by gravity stimulation. However, no marked change in the mean fluorescence intensity was detected in the upper side of the lateral root cap cells . These data are consistent with previous reports that gravistimulation induced a specific Ca2+ increase in the horizontally oriented coleoptiles, hypocotyl, and petiole.17 Thus, we might here further speculate that the increase in the concentration of Ca2+ was a universal phenomenon existing in various plant organs responding to gravistimulation.Starch-filled amyloplasts within the columella cells sedimenting in the direction of gravity were believed to trigger the generation of a signal that causes asymmetric growth.detected . In fact2+ sparks in the root cap (p > 0.05), an accelerated occurrence of Ca2+ sparks in the lower side of the lateral root cap were indeed observed, especially within 15 min after the onset of the gravistimulation , while the data in the lower and upper sides of the lateral root caps were about 6.28 ± 2.76 and 1.39 ± 0.89 per 10 min (n = 13), respectively, indicating that gravity stimulation resulted in an asymmetrical increase in the frequencies of Ca2+ sparks in the lateral root caps (p < 0.001). With the lapse of time, the frequency of Ca2+ sparks in the lower sides of the lateral root caps reduced gradually . This phenomenon might be due to the fact that the columella cells are located inside the root cap,30 and Ca2+ dynamics, especially in the inner layers of the columella cells, are relatively difficult to be detected. Given that the presentation time for Arabidopsis roots is approx. 0.4 min,31 and that it would take approx. 0.5 min for us to rotate the vertically positioned sample by 90 degrees, another possibility that cannot be excluded is that, due to the limitations of the technique used, our data might have missed earlier signal changes occurring in the root columella cells.On the other hand, though the mean CaCeratopteris richardii.32 Gravistimulation-induced Ca2+ waves moved from the root tip toward the elongation zone on the lower side of the root were also observed in roots of A. thaliana.15 In the present study, each Ca2+ spark occurred strictly within a single cell. No obvious signal to transmit from one cell to neighboring cells was observed (2+ sparks occured.Transcellular calcium currents were regarded as the earliest changes directed by gravity in Spores of the fern 33 In the present, an increase in Ca2+ concentration, as well as the Ca2+ sparks, can be detected within 1 min after gravistimulation, suggesting that Ca2+ dynamics were indeed induced by gravistimulation (2+ plays a role during root gravitropism. Especially, it was still impossible to confirm whether Ca2+ was the initial signal or the transmission signal of the root in response to a gravity stimulus. Perhaps, gravity stimulation somehow induced the changes in the Ca2+ concentration, as well as the frequencies of Ca2+ sparks, which in consequence triggered the relocalization of PINs, such as PIN3 and PIN7, and an auxin asymmetry essential for gravitropic bending between the upper and lower side of the root.34–36 On the other hand, auxin can somehow induce Ca2+ signals,37–39 which in turn promotes differential apoplastic pH changes at both sides of the gravistimulated root,15 resulting in root curvature soon after the onset of the gravistimulation. Anyhow, Ca2+ dynamics in the root cap might be one of the most important signaling in the early phase of the tropic response.Since root gravitropic response began quickly after the onset of a gravity stimulus, the initial phase of gravitropism cannot rely on newly synthesized proteins and, therefore must be non-genomic.Click here for additional data file.


In acute myeloid leukemia (AML) many patients experience relapse, despite the achievement of morphological complete remission; therefore, conventional morphologic criteria are currently considered inadequate for assessing the quality of the response after treatment. Quantification of measurable residual disease (MRD) has been established as a strong prognostic marker in AML and patients that test MRD negative have lower relapse rates and better survival than those who test positive. Different techniques, varying in their sensitivity and applicability to patients, are available for the measurement of MRD and their use as a guide for selecting the most optimal post-remission therapy is an area of active investigation. Although still controversial, MRD prognostic value promises to support drug development serving as a surrogate biomarker, potentially useful for accelerating the regulatory approval of new agents. In this review, we will critically examine the methods used to detect MRD and its potential role as a study endpoint. Acute myeloid leukemia (AML) includes a heterogeneous group of diseases driven by recurrent mutation, chromosomal aberrations and/or altered signaling pathways . ConventFor over 60 years, cytomorphology has been the standard method for assessing the presence of residual leukemia after treatment. Morphological CR is defined as less than 5% bone marrow blast and no Auer rods in normocellular bone marrow, the absence of extramedullary leukemia, neutrophils greater than 1.000/μL and platelets greater than 100.000/μL . HoweverThe aim of this review is to illustrate the current techniques for assessing MRD with their performance and the possible use of MRD measurement as surrogate efficacy-response biomarker to accelerate drugs development/approval in AML. The Food and Drug Administration (FDA) defines biomarkers as “a characteristic that is objectively measured and evaluated as an indication of normal biologic or pathological processes, or pharmacologic responses to a therapeutic intervention ”. IdentiNPM1 or TP53 mutations, have well-defined prognostic and predictive significance, whereas others, such as FLT3 or IDH1/2 mutations, are the target of new therapies. However, molecular/cytogenetic markers are still insufficient for predicting the individual risk of relapse in AML patients because they do not take into account the extreme biological heterogeneity of leukemic cells as well as the individual patient’s response to treatment. MRD measurement, which summarizes all of these interactions, would be the preferred biomarker for defining patient outcome. Indeed, numerous studies have shown that MRD has prognostic value. Patients in remission who have been tested MRD positive have a higher cumulative incidence rate of relapse and, in many cases, a shorter relapse-free survival (RFS) and/or overall survival (OS) than similarly treated MRD negative patients. The strong correlation between detectable MRD and poor patient outcomes has been confirmed at different timepoints throughout the course of intensive AML therapy, including after one or two courses of induction chemotherapy, after post-remission therapy, and both before and after ASCT [In the AML setting the capability to predict outcomes and long-term prognosis is gradually improving as genetic characterization and molecular definitions evolve. As a result, a growing number of molecular and chromosomal biomarkers are being progressively incorporated into AML classification schemes and clinical practice . Some ester ASCT . Increaster ASCT . Similarter ASCT . Finallyter ASCT . Similarter ASCT . These fter ASCT .Different techniques to assess MRD in AML are currently being used and their potential application is under continuous investigation ,17. ConvCBF) positive AML and AML with NPM1 mutations should be monitored by molecular methods (RT-qPCR) while all other AML subgroup should undergo MPFC testing [These methods are complementary to each other, and the ELN guidelines recommend their combined use when possible . The cur testing . NGS tes testing ,16. MPFC MRD−3–10−5 [The increasing use of MPFC in MRD measurement is due to its wide applicability (>90% of AML), rapidity, specificity and ability to distinguish viable cells from bone marrow debris and dead cells. By using ten-color panels, the current sensitivity limit is put around 10−3–10−5 ,18. The −3–10−5 ,20. The −3–10−5 ,22. Diff−3–10−5 ,18,23,24−3–10−5 , while i−3–10−5 . It is a−3–10−5 . One of −3–10−5 . Approxi−3–10−5 . Unpredi−3–10−5 . Both no−3–10−5 ,29. In t−3–10−5 .From a technical point of view, the role of appropriate sampling plays a crucial role. The poor quality of the bone marrow sample and contamination from peripheral blood are causes of invalidation of the flow cytometric analysis, altering the evaluation of the real value of MRD. Furthermore, the background noise generated by the regenerating marrow, with the presence of myeloid precursors, can results in an unreliable specificity of the method.b.Molecular Biology-based ApproachesPML-RARα [RUNX1/RUNX1T1 [CBFB-MYH11 [NPM1 [RT-qPCR allows MRD detection in patients with molecular targets that are specific and stable over the treatment course such as PML-RARα ,32, RUNX/RUNX1T1 , CBFB-MYFB-MYH11  and muta11 [NPM1 ,36. The 11 [NPM1 . This ap11 [NPM1 . This teNPM1, IDH1 and IDH2. DdPCR is a newer high-throughput technology that works like conventional PCR, except that it splits DNA samples into thousands of separate reaction chambers. The term ‘droplet’ derives from the method used: a water-oil emulsion that divides the samples into 20.000 droplets. PCR amplification occurs in each droplet, and the wells that contain the target DNA are read as ‘positive’ while the remaining are recorded as negative. DdPCR provides the means to count the absolute number of DNA or RNA molecules in each sample, thus being a technology characterized by high sensitivity and specificity. Unlike RT-qPCR, it is able to amplify target genes, without a standard reference curve. Despite the higher sensitivity compared with traditional RT-qPCR (up to tenfold) and precision, the major pitfall of DdPCR is that for each mutation a specific assay needs to be developed in the same gene . TherefoDNMT3A, TET2 and ASXL1 do not contribute to CHIP and then do not correlate with an increased risk of relapse, again favoring misinterpretations of MRD [Next-generation sequencing (NGS) technologies provide the opportunity to study a large number of somatic mutations in one single experiment. The possibility of analyzing many molecular abnormalities at the same time, including cytogenetically normal cases, makes NGS an attractive method for MRD evaluation in AML, especially if one considers that the wide intra-clonal heterogeneity often makes the leukemic clone a moving target . Howevers of MRD ,41. FinaRegulatory approval of a drug requires evidence of clinical benefit. In clinical trials for AML, survival estimates such as OS, disease-free survival (DFS) and event-free survival (EFS) are standard endpoints, the meaningful improvement of which is accepted as evidence of a clinical benefit of the experimental drug . When thCurrent clinical trials using MRD status as a primary endpoint are reported in The last few years have been characterized by the introduction of new agents/therapies for AML. However, the duration of clinical trials for providing patients with the access to new molecules is still too long. Although MRD is not officially recognized as a surrogate endpoint, some recent phase I/II trials include CR-MRD as a primary endpoint of their study . Using M